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Editorial Board

C. Bryant Division of Biochemistry and Molecular Biology, The Austra-


lian National University, Canberra, ACT 0200, Australia

M. Coluzzi Director, Istituto di Parassitologia, Universid Delgi Studi di


Roma “La Sapienza”, P. le A. Moro 5 , 00185 Roma, Italy

C. Combes Laboratoire de Biologie Animale, UniversitC de Perpignan,


Centre de Biologie et d’Ecologie Tropicale et MBditerranCenne, Avenue
de Villeneuve, 66860 Perpignan Cedex, France

S.L. James Chief, Parasitology and Tropical Diseases Branch, Division of


Microbiology and Infectious Diseases, National Institute for Allergy and
Infectious Diseases, Bethesda, MD 20892-7630, USA

W.H.R. Lumsden 16A Merchiston Crescent, Edinburgh EHlO 5AX, UK

Lord Soulsby of Swaffham Prior Department of Clinical Veterinary


Medicine, University of Cambridge, Madingley Road, Cambridge CB3
OES, UK

K. Tanabe Laboratory of Biology, Osaka Institute of Technology, 5-16-1


Ohmiya, Asahi-Ku, Osaka 535, Japan

K.S. Warrent Comprehensive Medical Systems, Inc., 461 Fifth Avenue,


New York, NY 10017, USA

P. Wenk Falkenweg 69, D-72076 Tubingen, Germany

t Deceased.
Advances in
PARASITOLOGY
Edited by

J.R. BAKER
Royal Society of Tropical Medicine and Hygiene,
London, England

R. MULLER
International Institute of Parasitology,
St Albans, England
and

D. ROLLINSON
The Natural History Museum,
London, England

VOLUME 39

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CONTRIBUTORS TO VOLUME 39

S.M.A. BROWN, Infection and Immunity, Institute of Biomedical and Life


Sciences, Joseph Black Building, University of Glasgow, Glasgow, GI2
8QQ, UK
G.H. COOMBS, Infection and Immunity, Institute of Biomedical and Life
Sciences, Joseph Black Building, University of Glasgow, Glasgow, GI2
8QQ, UK
P.S. COULSON, Department of Biology, The University of York, PO Box No.
373, York, YO1 5YW
H. DENTON,Infection and Immunity, Institute of Biomedical and Life
Sciences, Joseph Black Building, University of Glasgow, Glasgow,
GI2 8QQ, UK
C.A. FACER,Department of Haematology, St Bartholomew's and The
Royal London School of Medicine and Dentistry, Turner Street, London
E l 2AD, UK
A.M. JOHNSON, Molecular Parasitology Unit, Faculty of Science,
University of Technology, Sydney, GPO Box 123, Broadway, New South
Wales 2007, Australia
J.M. KELLY, Department of Medical Parasitology, London School of
Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK
M. TANNER, The Swiss Tropical Institute, Socinstrasse 57, CH-4002, Basel,
Switzerland
A.M. TENTER, Institut fur Parasitologie, Tierarztliche Hochschule
Hannover, Bunteweg 17, 30559 Hannover, Germany
K.-W. THONG,Discovery Biology I, Pfizer Central Research, Sandwich,
Kent, CT13 9NJ, UK
This volume commences with a review on malarial vaccines by Christine Facer at
St Bartholomew’s and the Royal London School of Medicine and Dentistry and
Marcel Tanner from the Swiss Tropical Institute, Basel.
This is a subject that has been in the news for the last twenty years with a
vaccine ‘just around the corner’ for most of that time. However, as is clear from the
title, the whole subject has now progressed from a mainly research topic to one of
practical application. This review is very timely because of the progressive spread
of resistance to all current antimalarial drugs and the recent conclusion of three
major clinical trials of vaccines against this most important of parasitic diseases.
The authors cogently argue the need for a vaccine and describe the strategies and
assessments of the success of field trials. Various types of vaccine have been
suggested and investigated. he-erythrocytic vaccines have the capability of pre-
venting infection. Immunization with attenuated live sporozoites has given excel-
lent results in animal models but despite much research, vaccine trials based on
sporozoite proteins have not lived up to expectations. A vaccine targeting the
asexual blood stages is likely to ameliorate rather than prevent infection and should
be of particular use in children living in highly endemic areas. A novel synthetic
polymeric vaccine (SFY66) is being extensively tested against populations in many
parts of the world and the authors include the very latest findings in their review of
this exciting and promising development. Specific merozoite surface proteins are
also being investigated experimentally. The authors conclude that multivalent and
multiantigen plasmodia1 DNA vaccines are likely to prove most promising for the
future.
The volume continues with two chapters also devoted to members of the Api-
complexa, though not the Plasmodiidae - the coccidia. Astrid Tenter, from the
Hannover Tieriirztliche Hachschule in Germany, and Alan Johnson, from the Uni-
versity of Technology in Sydney, Australia, discuss the phylogeny of the so-called
tissue cyst-forming coccidia in the family Sarcocystidae.
After considering the ‘traditional’ taxonomic characters such as morphology and
life cycle, the authors go on to discuss the newer ‘molecular’ techniques, including
nucleotide sequencing and sequence alignment of small subunit ribosomal RNA.
They discuss in some detail how these data may be used to construct phylogenetic
‘trees’, and conclude that the members of the Sarcocystidae are indeed a mono-
phyletic group. Inter-relationships within the family are, however, rather more
controversial, although the major genera themselves also appear to be valid mono-
phyletic groupings.

vi i
...
Vlll PREFACE

Following this discussion, there is a full treatment by Graham Coombs and his
colleagues at the University of Glasgow, UK, of the biochemistry of the coccidia in
general, including the tissue cyst-forming species of Toxoplasma and Sarcocystis
and the monoxenous genera Eimeria and Cryptosporidium. The chapter considers
the metabolic processes concerned with energy production, proteins and amino
acids, polyamines, purines, pyrimidines, nucleic acids and lipids. There are also
sections on cultivation in vitro, parasite-host cell interactions, and anticoccidial
drugs. The authors conclude by emphasizing that much still remains to be eluci-
dated about the biochemistry of these perhaps rather neglected but increasingly
important parasites.
Advances in our understanding of molecular biology have provided opportu-
nities for the genetic manipulation of parasites. This is a new and challenging area
of research which is currently attracting a great deal of attention from parasitol-
ogists. John Kelly from the London School of Hygiene and Tropical Medicine, who
has been directly involved with many recent research developments, provides an
authoritative overview of progress in genetic transformation of parasitic protozoa.
In this context transformation is considered as the stable acquisition by a cell of a
new gene(s) by uptake of foreign DNA. The first successful stable transformation
of a parasitic protozoan was achieved with Leishmania, and in this chapter parti-
cular attention is given to work with the trypanosomatids Leishmania, Trypano-
soma brucei and Trypanosoma cruzi. Rapid progress has led to research on gene
targeting, functional analysis of genes and analysis of gene expression. One area of
particular interest has been the application of genetic manipulation to elucidate
mechanisms of drug resistance. Other sections deal with the development of
transfection techniques for Toxoplasma and Plasmodium and the intestinal proto-
zoa Entamoeba histolytica and Giardia lamblia.
The final chapter in this volume is a detailed and important contribution by
Patricia Coulson from the University of York, UK, concerning radiation-attenuated
vaccines against schistosomes in animal models. A vaccine against schistosomiasis
is not yet available and control of the disease depends upon many factors such as
health education, snail control and chemotherapy. The need for a vaccine is well
recognized and, despite considerable progress in recent years, much still needs to be
done to understand the immunological response of the human host and the ability of
the parasite to survive within the host. The irradiated vaccine model, which is
unlikely to be suitable for use in humans, can provide a useful example for the
development of a recombinant antigen vaccine. This chapter provides a compre-
hensive account of work associated with radiation-attenuated vaccines, the induc-
tion of immunity and immune effector mechanisms, and concludes with ideas
concerning future research directions.

JOHN BAKER
RALPH MULLER
DAVID ROLLINSON
Clinical Trials of Malaria Vaccines:
Progress and Prospects

Christine A . Facer’ and Marcel Tanner2

‘Department of Haematology. St Bartholomew’s and


The Royal London School of Medicine and Dentistry.
Turner Street. London. E l 2AD. UK and
’The Swiss Tropical Institute. Socinstrasse 57.
PO Box CH.4002. Basel. Switzerland

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. The need for a vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2. Aims of a vaccine and immunological constraints .................... 5
1.3. Phases in the evaluation of a malaria vaccine ....................... 7
1.4. Assessing vaccine efficacy: trial end points ......................... 9
1.5. Synthetic peptides for vaccine inclusion require carriers
and adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.6. New generation nucleic acid (DNA) vaccines ....................... 13
1.7. Strategies for the development of a malaria vaccine . . . . . . . . . . . . . . . . .15
2. Pre-erythrocytic (Infection-blocking) Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.1. The circumsporozoite (or CS) protein and T-cell epitopes . . . . . . . . . . . . . .18
2.2. Cytotoxic T lymphocytes (CTL) for vaccine design . . . . . . . . . . . . . . . . . . . 23
2.3. SSP-PITRAP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4. Liver stage antigen 1 (LSA-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3. Asexual Blood-stage Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.1. Role of antibodies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2. The problem of parasite antigenic diversity ........................ 29
3.3. MSA-1 and MSA-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4. RESA/Pfl55 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.5. Other target antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.6. The first chemically synthesized vaccine against P. falciparurn: SPf66 .... 37
3.7. Immune responses to SPf66 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

ADVANCES IN PARASITOLOGY VOL 39 Copyright 0 1997 Academic Press Limited


ISBN 0-12-031739-7 AN rights of reproduction in any form reserved
2 C.A. FACER AND M. TANNER

4. Asexual Stage ’Disease-modifying’ Vaccines. .......................... 46


4.1. ‘Anti-toxic‘ vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.2. A vaccine to prevent cerebral malaria?. ........................... 50
5. Multi-stage and Multi-antigen DNA Vaccines. .......................... 55
6. TheFuture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

1. INTRODUCTION

The traveller telephoned to ask where he could obtain the malaria vaccine
that he had read about in the national press. Hopefully a reality in the near
future, but this was April 1993 and a report on the successful outcome of
the first human field trials with the SPf66 malaria vaccine in South America
had just been published (Valero et al., 1993). This was certainly a mile-
stone in the history of parasitology and moved the scientific and medical
communities into a mixed wave of admiration, scepticism and criticism.
Not unusual for malaria vaccines, the evolution of which has been long (yet
not so long as Jenner’s smallpox vaccine which had to wait two centuries
before proof of success), convoluted and not without controversy.
Control of malaria represents one of the world’s greatest health chal-
lenges and never before has a malaria vaccine been more urgently required.
Worldwide, the number of cases is rising at a rate of 5% annually. Increas-
ing mosquito resistance to insecticides is a contributor to this effect, as is
parasite multi-drug resistance. Chloroquine-resistant parasites, which first
appeared in South-East Asia and Latin America simultaneously in the early
1960s, now affect almost every country where the disease is endemic. The
current malaria statistics are frightening, with an estimated 300-500 mil-
lion people infected of whom 2.3 million die every year (Sturchler, 1989;
WHO, 1995). The global impact of malaria is estimated as 35 million
DALYs (Disability Adjusted Life-Years; World Bank, 1993), primarily a
result of infection with Plasmodium falciparum.
As the most important parasitic disease in the world, it is disproportio-
nately prevalent in Africa where approximately 90% of the world’s malaria
sufferers are sub-Saharan Africans (WHO, 1995). This has hindered the
drive to develop new and novel antimalarial drugs as the pharmaceutical
industry sees little return in terms of profit in a market confined to the needs
of impoverished populations in the developing world. Similarly, the world
of commerce is unlikely to underwrite the expensive and long-term clinical
trials required for the development of a malaria vaccine. Fortunately,
however, enlightened governments and charities have been forthcoming
with funds.
CLINICAL TRIALS OF MALARIA VACCINES 3

This review aims to bring the reader an updated state-of-the-art appraisal


of clinical malaria vaccines which is timely given the recent conclusion of
three major clinical trials with a P. falciparum vaccine in three different
countries, and the design and first trials of malaria DNA vaccines. We
assess the need for and aims of a vaccine and the relative merits of the
types of vaccine being developed. Vaccination of the individual as well as
vaccination of communities within endemic areas is discussed, as is the
economic impact of the latter. We deliberately do not review all the animal
studies and the relevance of animal models for deciding on the design of
field trials, as this has been reviewed recently elsewhere (Hoffman, 1996).
Furthermore, our focus is primarily on vaccines for P. falciparum malaria
because of the obvious importance of this species in terms of its associated
morbidity, mortality and drug resistance.
We also consider vaccine design for anti-infection, anti-disease and
disease-modifying strategies to control malaria with detailed reference
only to human trials. Vaccines designed to prevent parasite transmission
(transmission-blocking vaccines) have not yet reached clinical trials and
are thus not included in this review.

1.1. The Need for a Vaccine

Vaccines have probably prevented more infectious disease than any other
medical intervention except sanitation, and represent the most cost-effec-
tive approach to control. However, within the discipline of infectious
agents, human parasites stand alone in the absence of any commercially
available vaccine. A human malaria vaccine promises to correct this situa-
tion. The earliest attempt to immunize humans dates back to 1936 (Boyd
and Kitchen, 1936). Following this, vaccination attempts made to improve
immunogenicity by including an adjuvant were successful in that they
achieved partial protection of monkeys immunized with killed blood stage
parasites (Freund et al., 1948). A long-standing difficulty in the develop-
ment of a malaria vaccine has been the lack of a small animal model for
studying P. falciparum, the only appropriate model being P. falciparum in
Aotus monkeys. This together with the increasing conservation of non-
human primates and escalating costs of monkeys for experimentation, has
resulted in some delay in the development of a malaria vaccine.
During 1975, the United Nations, the World Bank and the World Health
Organization jointly advertised funding to cover the development of a
malaria vaccine and this led to a concerted effort and competition between
research groups. Sadly, despite promises of a ‘vaccine around the corner’
and a ‘vaccine within the next 5 years’, it was not forthcoming. Not until
1987 that is, when news of the first Patarroyo SPf66 human vaccine trials in
4 C.A. FACER AND M. TANNER

South America were published and the next crucial step was made (Patar-
royo et aZ., 1987, 1988).
Yet 9 years on in 1996, and at an estimated total cost of all malaria
vaccine development of around 700 million dollars, SF‘f66 continues to
undergo efficacy trials. While it is not the vaccine that will reach routine
application, it is the one that, through attracting controversy, is stimulating
vaccine development. However, at present the most realistic of optimists
will not provide a date when they envisage that a commercially produced
malaria vaccine will be available.
For whom is a malaria vaccine being designed? Malaria vaccines can be
envisaged for protection of non-immune travellers and military personnel
who are transiently exposed to risk as compared to infants and children
living in malarious areas. In order to prevent the death of some 1.2 million
children every year in Africa alone, a malaria vaccine would need to be
‘therapeutic’ in that its aim would best be to prevent life-threatening
disease (in individuals with pre-existing blood-stage infections) whilst
allowing the development of natural immunity through repeated re-
infection. This, of course, requires intimate knowledge of what consti-
tutes ‘malarial disease’. In endemic regions, since the majority of the
morbidity and mortality occurs in young children (Greenwood et aZ.
(1987) estimate that one quarter of African children aged 1 - 4 years die
from malaria), they should be the primary targets for vaccination which
should occur as soon as possible after birth.
The practical importance as well as intellectual appeal of a malaria
vaccine is without question. A vaccine would side step the problems of
multi-drug resistance, it would be more convenient than regular prophy-
laxis and would have the advantage of causing few, if any, side effects.
Last, but not least, the vaccine might synergize with existing drugs and
other tools of control such as bednets (Tanner et al., 1995). In any case, for
most endemic areas, a vaccine is not believed to be the only tool but should
become part of integrated control strategies.
There are attendant intellectual concerns as with any other vaccine:

1. What proportion of the population should be immunized? Is eradication


the goal (considered unlikely for malaria, but feasible in distinct epide-
miological settings such as parts of China) or partial control of the
disease with reduction or elimination of clinical symptoms? In the
design of a malaria vaccine there is the need to consider whether the
vaccine is aimed at preventing infection or disease and the differing
circumstances in which such vaccines might be applied.
2. What is the best age to immunize against malaria? As most morbidity
and mortality is experienced in infants, then the earlier the better.
CLINICAL TRIALS OF MALARIA VACCINES 5

3. Will immunization result in the selection of resistant (and more viru-


lent?) strains?
4. Will vaccination (particularly transmission-blocking vaccines) in the
long term reduce transmission to the extent that it may actually increase
the incidence of severe disease in those infected by altering the pattern
of semi-immunity (a recent Kenyan study linked low natural transmis-
sion with a greater incidence of severe malaria; Mbogo et al., 1995)?

Thus, there are many basic and important questions that require addressing
right from the planning of the first clinical trials.

1.2. Aims of a Vaccine and Immunological Constraints

To understand what a vaccine is trying to achieve, one must consider what


constitutes the natural development of immunity to malaria in endemic
countries. Even today we do not have one laboratory assay or surrogate
marker that provides a measure of protective immunity, and which would
help to select vaccine candidates for further trials and help to predict a
successful outcome in those trials. What we do know is that resistance to
malaria develops slowly and in young children there is high parasitaemia,
morbidity and mortality (dependent on the endemic setting, particularly
exposure levels; Snow et al., 1994) and it is not until later childhood that
clinical symptoms decline together with parasite levels (Marsh, 1992).
Older children, nevertheless, often carry a substantial parasite load yet
are asymptomatic with no accompanying fever or other adverse symp-
toms (Figure l), a state known as ‘clinical tolerance’ or ‘anti-toxic
(disease-modifying) immunity’ (see Section 4). Adults rarely have a high
parasitaemia and appear to have acquired a degree of non-sterile immunity.
These features of malaria immunity could have several possible expla-
nations. The biology of the malaria parasite is complex, with multiple
developmental stages within the host resulting in the production of a
multiplicity of parasite-derived molecules of varying antigenicity. Effec-
tive immunity to malaria may reflect the long period required to be infected
with a large number of antigenically diverse parasite populations; that is,
an individual would need to encounter the locally transmitted repertoire of
‘strains’ (Gupta and Day, 1994). In this scenario, a child who dies of
malaria despite previous parasitization, would have encountered a new
(and virulent?) ‘strain’ although eventually there must be some degree of
cross-protection. The complex interaction between genetic diversity of host
and of parasite (which encompasses the perception of virulent parasites
which cause disease) is currently a topic of considerable interest although
6 C.A. FACER AND M. TANNER

y NINFECTED

I+
No ymptoms

INFECTED AND ASYMPTOMATIC


No symptoms but may have hepatosplenomegaly
and mild anaemia
Frequently a high parasitaemia

I f
UNCOMPLICATED MALARIA
Fever, headache, shivers, rigors, vomiting,
diarrhoea and cough

E MALARIA

I Fever, altered consciousness, convulsions,coma,


severe anaemia, respiratory distress
T

Figure I The clinical spectrum of manifestations of P.fulcipurum malaria. It is


uncertain whether uncomplicated malaria invariably leads to severe disease or
whether severe malaria is caused by virulent parasites.

it lies beyond the bounds of this review other than to highlight its
importance.
The concept of therapeutic vaccines has stimulated considerable interest
and debate within and outside the subject of malaria. It is regarded with
caution by many researchers who believe it impossible for a vaccine to
improve on the natural immune response, a dogma that has been challenged
for more than a century. Yet the immune system can be supercharged by a
vaccine even after infection as shown recently, for example, in people with
herpes, leprosy or leishmaniasis. The final choice of how many and which
parasite epitopes should be included in a vaccine is a formidable challenge.
Serum from malaria-immune adults identifies reacting malaria antigens,
making the assumption that the best antigens for vaccine inclusion are
those that stimulate good humoral responses. However, this approach
might not identify the best immunogens for a T-cell (helper and cytotoxic)
mediated immunity (Table 1). In addition, the immunodominant epitopes
might not be the ones eliciting the most effective immune responses.
CLINICAL TRIALS OF MALARIA VACCINES 7

Table 1 Ideal requirements of a malaria vaccine


A malaria vaccine needs to:

Include covalently linked T-cell (helper and cytotoxic and B-cell epitopes. Pro-
blem of antigenic polymorphism (sexual reproduction leads to maintenance of and
increase in heterozygosity). Definition of ‘common, cross-reacting epitopes’ .

Include T-cell epitope(s) recognized by individuals of diverse genetic makeup to


overcome the problem of genetic control of the immune reponse (HLA haplotype
associated unresponsiveness).

Avoid induction of mutation in vaccine targets andlor selection of ‘virulent’


parasites.

Ensure natural boosting.

Lack suppressor T-cell epitopes.

Be formulated (dose, carrier, adjuvants) in a way that induces an appropriate


immune response and avoids suppressive effects.

Be assessed for the epidemiological implications of its coverage; i.e. for its
effectiveness in different epidemological settings.

Indeed they may have an adverse effect by stimulating ‘non-protective’


immune responses, the so-called smokescreen effect. The malaria parasite
has evolved elaborate mechanisms to evade host immune responses, and a
successful vaccine should include those antigens, mainly the well-con-
served epitopes, against which an immune response is directed. It is
currently assumed that these epitopes do not dominate immune responses
elicited by natural exposure.

1.3. Phases in the Evaluation of a Malaria Vaccine

The properties of a potential vaccine are likely to be assessed in vitro


and/or experimentally in animal models. Should any products at this
stage, termed phase 0 (Figure 2), show any promise then they may be
administered to human volunteers in clinical trials (phase I) to assess
safety and immunogenicity. This step is followed by first efficacy trials
among highly selected volunteers (phase IIa [non-immune] and phase IIb
[immune]). Phase IIb trials provide a means of establishing a logical basis
for judging the adequacy of vaccine potency and this is thus an important
component of a vaccine trial. Phase 1Ia and IIb trials start as phase I trials
but following evidence for safety and immunogenicity, artificial challenge
8 C.A. FACER AND M. TANNER

Animal models Safety, immunogenicity,


tolerability,efficacy
Precllnlcal

Figure 2 Clinical trials of malaria vaccines: standard evaluation phases.

(by malaria-infected mosquitoes) of vaccinated individuals is included.


Efficacy is established by an assessment of protection against parasitaemia
or disease due to malaria in comparison with a control group of unvacci-
nated individuals. If, after these studies, there is evidence of protective
efficacy, then field trials to test the vaccine under conditions of natural
challenge begin (phase 111). Both phases I1 and I11 are run under strict
conditions as double-blind randomized controlled trials. Trials with subunit
sporozoite and asexual stage malaria vaccines are currently at phase 111.
Finally, phase IV studies, conducted after a vaccine has been deployed,
represent attempts to evaluate the effectiveness and, finally, the public
health impact of the vaccine. Phase IV trials are usually programme-based
operations and may no longer follow the strict criteria of randomized
controlled trials.
Although the primary objective of malaria vaccines (other than transmis-
sion-blockingvaccines) is protecting the individual, they may also provide the
additional benefit (if distributed to a large proportion of the population and
give good protection) of making an impact on malaria transmission within the
community, an effect which can only be monitored by phase IV trials.
CLINICAL TRIALS OF MALARIA VACCINES 9

Field trials in malarious areas should be conducted with the availability


of background information on the epidemiology of malaria in that area.
Information should also be sought on the use of antimalarial drugs and
the prevalence of human genetic factors such as haemoglobinopathy and
other red cell disorders that affect host responses. Finally, field trials also
need linking to the existing health services and should be performed
within communities where there is a well-established relationship with
individuals involved in trial design and execution (Tanner et al., 1993;
Alonso et al., 1995).

1.4. Assessing Vaccine Efficacy: Trial End Points

In any vaccine trial it is essential to have clear-cut end points against which
the success of the vaccine is assessed. Possible end points include asymp-
tomatic infection through to incidence of either mild disease or severe
disease and death (malaria-specific or all causes; see Figure 1). For these
to become acceptable as outcome variables to allow comparison between
trials, it is important that there are agreed approaches that are feasible and
repeatable, standardized definitions of what is meant by mild and severe
disease and criteria established for the identification of possible malaria
deaths. Recent discussions have clearly outlined these difficulties (Alonso
et al., 1995).
The ultimate objective of a malaria vaccine in sub-Saharan Africa is
reduction in mortality and life-threatening malaria. However, as both are
relatively rare events, very large numbers of individuals must be included
in prospective trials in order to detect as effect on these end points. There-
fore most trials must evaluate the efficacy of candidate vaccines against
uncomplicated malaria. Is fever alone a good indicator of uncomplicated
malaria? There are many causes of fever in children in the tropics of which
malaria is one. The relationship between parasite density and the risk of
fever suggests that setting specific parasite density cut-offs should be used
to distinguish malaria from other causes of fever with high specificity at the
individual level (Alonso et al., 1995).

1.5. Synthetic Peptides for Vaccine Inclusion Require Carriers and


Adjuvants

The use of chemically defined synthetic peptides (often polymerized and


known as multiple antigen peptide systems) for vaccination represents a
novel approach with advantages over existing approaches (Table 2). The
short peptide (up to 30 mer) epitopes, because they are chemically defined,
Table 2 Advantages and disadvantages of different malaria vaccine products.
Advantages Disadvantages
Purified malaria Peptides in natural configuration May contain sequences which induce pathology
proteins Glycosylated Parasite culture in large quantities required
Purification costly and time consuming
Recombinant Immunogenic sequences can be produced May include epitopes that may mask protective
proteins Glycosylated (if produced in eucaryotic expression responses and/or induce pathology
systems) Possible contaminants
Cheap
Synthetic Immunogenic linear and cyclized defined sequences Sequences not in natural configuration therefore B-cell
peptides Production of sequences normally ‘hidden’ from stimulation may be inadequate
immune response in natural protein Good T-cell stimulation but MHC restriction
Cheap and easy bulk production Decreased antibody levels and affinity
Stable at ambient temperature Stimulate predominantly humoral antibody response
No infectious agent present without appropriate adjuvants
Provides opportunity for delayed release mechanisms Creation of artificial epitopes
Can be designed to stimulate appropriate immune
responses
Attenuated Stimulates best immune response Production of sufficiently large quantities very difficult
Good T-cell response (CD4+ and CDB+ cytotoxic) Costly in bulk
Ethical problems in association with the use of live
attenuated organisms in humans
Parasite antigenic variation
Reversion to virulence
Limited shelf-life and refrigeration

MHC, major histocompatibility.


CLINICAL TRIALS OF MALARIA VACCINES 11

do not contain any infectious material and hence are devoid of biohazard
risk. A further bonus is that they can be rapidly synthesized in unlimited
quantities. On the other hand, one concern surrounding the use of synthetic
peptides for vaccines is that if included in linear form they may fail to
induce antibodies reacting with the cognate protein, a finding recently
reported for a synthetic sporozoite peptide (see Section 2). This is because
linear peptides may not adopt the conformation of the corresponding region
in the native molecule (Fries et al., 1992a). Various strategies have been
used to induce ‘native-like’ conformation in peptide immunogens, for
example cyclization of peptides whereby the two ends of the peptide are
joined by amino groups. The resulting cyclized peptide now lacks the
‘terminal epitopes’ that frequently elicit the formation of useless antibody
(Etlinger and Trzeciak, 1993). However, cyclization does not always
improve the level of conformational mimicry between peptide and intact
protein and should not be construed as a panacea for increasing reactive
antigenicity of exposed loops of protein.
To be efficacious synthetic vaccines must include epitopes capable of
inducing T-cell effector and T-cell helper activity. The lack of this essen-
tial requirement may be, in part, responsible for the limited efficacy of the
early sporozoite subunit vaccines in clinical trials which focused primarily
on B-cell epitopes (Millet et al., 1992). The malaria vaccines in production
today, in order to bypass this problem, contain an adjuvant and a carrier.
Both potentiate immune responses; the adjuvant by stimulating a higher
and more sustained antibody response (by providing a depot of antigen
with subsequent slow release, macrophage activation and activation of T
helper cells), the carrier by circumventing major histocompatibility com-
plex (MHC) restriction in T-cell epitopes contained within the vaccine.
Recognition of carrier epitopes by specific T helper cells will ultimately
induce B lymphocytes to make antibodies against those B-cell epitopes that
are covalently coupled to the carrier. Adjuvants currently licensed for
human use are few. Some, such as alum, are unfortunately poor stimulators
of cellular effector mechanisms, whilst potent stimulators such as Freund’s
complete adjuvant, are too reactogenic.
Novel adjuvants are being investigated (Hui, 1994). For example, lipo-
somes, concentric lipid layers enclosing the malaria antigens and mono-
phosphoryl lipid, proved to be safe and effective in inducing high titres of
anti-sporozoite antibodies in human volunteers (Fries et al. 1992a).
ISCOMS (immunostimulating complexes), large spherical structures where
immunogens are presented as multimers in a matrix of the adjuvant Quil-A,
have been shown to enhance the immunogenicity in rabbits of a malaria
fusion protein comprising the C-terminal repeat subunit (EENV) of the P.
falciparum blood-stage antigen Pf155 or RESA (ring erythrocyte-asso-
ciated antigen; Sjolander et af., 1993). Other advantages of ISCOMS are
12 C.A. FACER AND M. TANNER

priming of class I restricted CD8+ cytotoxic T cells and immunogenicity


when delivered orally. The new adjuvant DETOX (which consists of the
cell wall skeleton of Mycobacterium phlei, monophosphoryl lipid-A and
squalene) significantly increased immunogenicity to the recombinant P.
falciparum circumsporozoite protein (R3* NSIsl) compared to the same
antigen adsorbed to alum (Rickman et al., 1991). Whether this increased
immune responsiveness is associated with protection against challenge has
yet to be assessed.
Immune enhancement has also been achieved using cytokines as adju-
vants. Interleukin-1 (IL-1) has been found to enhance the immune response
to a variety of antigens and IL-la or interferon gamma (IFNy), when given
with an experimental malaria vaccine in mice, significantly increased
protection against a challenge P. yoelii infection by upregulating T-cell
help for antibody production (Heath and Playfair, 1992). An increased
immune response to many antigens also occurs with co-administration
with interleukin-2 (IL-2) but has yet to be evaluated as a component of a
malaria vaccine (Heath and Playfair, 1992).
In addition to an adjuvant, synthetic peptides often require a carrier mole-
cule for immunogenicity. To be recognized by T lymphocytes, epitopes must
be presented in the context of MHC molecules. Many epitopes only bind to a
limited spectrum of MHC haplotypes. Thus within a given population, a
defined malaria epitope is recognized only by those individuals carrying
the ‘correct’ MHC ‘make-up’. This MHC-linked regulation of T-cell recog-
nition can be circumvented in some cases by the chemical linking of a carrier
molecule (e.g. keyhole limpet haemocyanin, bovine serum albumin, teta-
nus toxoid) to the malaria B-cell epitope. The carrier protein provides a
source of T-cell epitopes expressed on the surface of antigen-presenting
cells and can thereby induce carrier-specific T-cell help that is necessary
for optimal B-cell priming. Both tetanus and diphtheria toxoids have been
included as carriers in malaria vaccines (Herrington et al., 1987; Rama-
samy et a1.,1995).
Epitope-specific suppression is one problem with the use of carrier
molecules. Here, antibodies produced to the carrier may interfere with
the induction of an effective antibody response to the parasite epitope.
The ideal carrier is therefore one that fails to induce a significant anti-
body response to itself, an effect well demonstrated in mice primed with
tetanus toxoid and then immunized with a synthetic malaria peptide
conjugated to the tetanus toxin (residues 73-93). There was no epitope
suppression, only an enhanced antibody response to the malaria peptide
(Etlinger and Knorr, 1991).
Another problem with the use of carriers is related to the structure of the
MHC molecule. Since only a single peptide can bind in its heterodimeric
CLINICAL TRIALS OF MALARIA VACCINES 13

groove, competition between T-cell epitopes for ‘best fit’ results. This
could be a concern where vaccines comprise multiple epitopes.
A carrier epitope taken from the same protein as the ‘protective’ epitope
would be a convenient design strategy. Algorithms can ‘screen’ areas of
native protein adjacent to the candidate vaccine sequence and predict
probable T-cell epitopes. However, the inability to predict the MHC range
to which a given epitope will bind is a problem to be faced.
One solution to overcoming antigen recognition would be to find a
‘promiscuous’, malaria T-cell epitope, that is a molecule capable of react-
ing with T cells irrespective of the MHC background. In fact such an
epitope exists in the carboxy terminus of the circumsporozoite (CS) protein
of P.falciparum. This region (amino acid residues 378-398), when added
to T-cell clones from non-exposed Caucasians of varying HLA-DR status,
induced in all of them a MHC-restricted proliferation (Singaglia et al.,
1988). This particular peptide, however, was not recognized by Gambians
(Good et al., 1988a, b) and in only 13% of Australians who had lived in
Papua New Guinea (Zevering et al., 1990). The search for other universal
epitopes on malaria antigens must continue since this strategy does repre-
sent a promising way forward. Their use would mean that a malaria vaccine
would protect many populations irrespective of their MHC haplotype. The
one drawback is that while universal T-cell epitopes are potentially active
with any B-cell epitope conjugated to them, strong adjuvants are some-
times required for the induction of a long-lasting high titred antibody
response (Del Giudice, 1992).
The possibility of a malaria vaccine designed to induce generation of
cytotoxic T lymphocyte (CTL) effectors is attracting attention as advances
in molecular immunogenetics allows analysis of motifs on human HLA
antigens which bind malaria peptides (see Section 2). However, this pre-
sents a daunting approach since, at present, very few peptide, subunit or
recombinant vaccines have been shown to induce CTL against any foreign
antigen in humans. Improved delivery systems are currently under inves-
tigation to rectify this situation including the intramuscular injection of
parasite DNA, shown to be most effective in inducing CTL activity in
animal models (Hoffman, et al., 1995).

1.6. New Generation Nucleic Acid (DNA) Vaccines

A revolutionary new approach in vaccinology is the development of DNA


vaccines (Krishnan et al., 1995; Waine and McManus, 1995; McDonnell
and Askari, 1996). Simply put, this novel vaccine technology involves
taking a parasite gene that encodes for a vaccine target protein and incor-
porating it into plasmids. When these engineered plasmids (called naked
14 C.A. FACER AND M. TANNER

plasmid DNA) are injected intramuscularly into the recipient, the muscle
cells, having successfully incorporated the foreign gene, begin producing
(for prolonged periods of time) the target protein, presumably in the exact
or similar conformation as found in the native form.
Delivery of foreign genes to mammalian somatic cells has still to be
perfected. In addition to delivery using plasmid DNA as described above,
others have used DNA cornplexed with specific protein carriers, attached to
cationically charged molecules such as liposomes or calcium salts, or
incorporated into live attenuated carriers such as vaccinia virus.
DNA vaccines have distinct advantages although, by their very nature,
attendant potential disadvantages (Table 3) when compared with recombi-
nant or subunit vaccines. One clear beneficial effect is that DNA vaccina-
tion stimulates all arms of the immune response including cytotoxic T
cells. The parasite protein, because it originates from inside the host cell,
is processed by the MHC class I pathway. Class I molecules carry peptide
fragments of the parasite protein to the cell surface where they stimulate
CD8+ cytotoxic T lymphocytes. By contrast, standard vaccine antigens are
taken up by phagocytosis and processed through the MHC class I1 system
which primarily stimulates antibody responses. Nevertheless, experience to
date with DNA vaccines indicates that, despite this theory, some DNA
vaccines (e.g. the Schistosomu mansoni Sm23 DNA vaccine) fail to elicit a
CTL response stimulating only THO and TH1 responses (D. Ham, personal
communication).

Table 3 Nucleic acid (DNA) vaccines


Advantages :

expression of antigens in their native form, improved processing and presentation


to. the immune system
induction of cytotoxic T cells (in addition, protective antibody and CD4+
responses in the same individual)
easy to produce and purify
easy to modify and combine vaccines
induction of long-lived immunity
possibility of reducing the number of doses because of prolonged antigen
expression

Disadvantages:

introduced (foreign) DNA may become incorporated into host chromosomes and
subsequent potential for a transformation event (DNA triplex formation)
introduced DNA may become incorporated into germ line cells
0 the DNA may stimulate anti-DNA antibodies
unexpected and untoward consequences of the persistent expression of a foreign
antigen therefore difficult to proceed to clinical trials
CLINICAL TRIALS OF MALARIA VACCINES 15

One unusual feature of the immune response to plasmid-encoded vectors


is that the antibody response rises slowly, not reaching peak levels until
10-12 weeks after immunization. In addition, although the immune
response to genetic immunization is weak compared with that induced
by traditional vaccines, it is exceptionally long lasting with antibody and
cytotoxic T lymphocytes remaining at plateau levels for the lifetime of
animals inoculated with plasmids; an extremely attractive feature for any
vaccine (Ertl and Xiang, 1996).
Despite these advantages, DNA immunization is faced with some
major hurdles before it can become a clinical reality (Robertson, 1994;
Krishnan et al., 1995). One concern is the unknown consequences of
long-term persistence of plasmid DNA and foreign gene expression in the
recipient which make it difficult for clinical trials and subsequent regis-
tration to meet the ethical requirements. The unwanted side effects might
include induction of anti-DNA antibodies, the induction of tolerance,
anaphylaxis, hyperimmunity, autoimmunity, and autoaggression (where
a strong CTL response results in destruction of the expressing and
surrounding cells). These speculations are hypothetical but nevertheless
feasible and need to be kept in mind since if manifest, their reversal
would be difficult. Another theoretical concern is the possibility of the
foreign DNA being incorporated into the host genome resulting in a
transformation event. In such a situation, foreign DNA introduced into
a host cell can bind to host DNA to form ‘triplex’ DNA which results in
mutations in the absence of a tethered mutagen (Wang et al., 1996).
Muscle cells are normally chosen as targets for DNA injection since
they are postmitotic, making it more unlikely that the foreign DNA would
be integrated. One option for the future may be the use of mRNA which,
being transient, will not cause insertional mutagenesis.
Nucleic acid vaccines have been used successfully in experimental ani-
mal models predominantly in relation to virus infections (Donnelly et al.,
1995). DNA vaccines against malaria have only very recently been designed
with encouraging immunization results in rodents and monkeys. These
promising and exciting developments that will lead to the first human trials
in 1997 (Fricker, 1996) are discussed fully later (see Section 5).

1.7. Strategies for the Development of a Malaria Vaccine

There are four main strategies in the development of a malaria vaccine: (i)
blocking the sporozoite from invading or developing within hepatocytes
(anti-infection);(ii) blocking merozoite invasion of red cells and inhibiting
development of schizonts (anti-disease or asexual stage); (iii) blocking the
adverse pathology-inducing effects of cytokines and parasite sequestration
16 C.A. FACER AND M. TANNER

(disease-modifying); and (iv) developing an ‘altruistic’ vaccine that would


block human-mosquito transmission by immunizing against the sexual
stages or gametes (transmission blocking). The anti-gamete vaccine would
not protect the individual but would help prevent the spread of disease
within a population.
The effective vaccine is envisaged as a cocktail of the above, being a
multi-stage, multi-epitope, multi-valent, multi-immune hybrid vaccine that
will stimulate arms of the immune response against several stages of the
life cycle at one time (Bathurst et al., 1993). The recent observations that
some immunogenic epitopes are expressed in more than one stage (Table 4)
raises important considerations affecting the design of antimalarial
vaccines.

2. PRE-ERYTHROCYTIC (INFECTION-BLOCKING) VACCINES

he-erythrocytic immunity is an all-or-none phenomenon and infection-


blocking vaccines need to be one hundred per cent effective. If a single
sporozoite or intrahepatic parasite escapes, clinical malaria ensues.
Two general types of pre-erythrocytic vaccines have been developed;
vaccines that induce antibodies to sporozoites and those that induce
immune T cells to parasitized hepatocytes. In both cases the end result is
to prevent infection and reduce malaria transmission and both are envi-
saged for use in areas of low to moderate transmission and stability.
There is no doubt that immunization with irradiation-attenuated (non-
pathogenic) sporozoites has given excellent protective immunity in animal
models and in humans against P. fulciparurn and P. v i v a (Clyde et al.,
1975). In the human studies, volunteers were immunized by repeated bites
from irradiated P. falciparum-infected mosquitoes, a process that renders
sporozoites non-infectious but does not prevent their invasion into hepa-
tocytes. They are unable to divide and remain (at least in the mouse model)
for unknown periods as uninucleate intrahepatic bodies (Ramsey et al.,
1982). Protection against subsequent sporozoite challenge was complete
following challenge with the same parasite ‘strain’. Both the intravenous
route of inoculation and live parasites (dead sporozoites gave little or no
protection) .were essential requirements for success. The features of the
protection so achieved include a short-lived sterile immunity that is species
and stage specific and which requires repeated boosts or relies on contin-
uous natural boosting.
Despite the success of experimental irradiated sporozoite vaccines in
human volunteers, there remains the anomaly that although individuals
living for many years in malarious areas receive many infectious mosquito
Table 4 Proteins and immunogenic epitopes identified on P. falciparum pre-erythrocytic stages
Protein Immunogenic Responding
where found Size (aa) Function epitope lymphocytes Comments

cs 412 Gliding NANP (conserved) B cells Antibodies


Sporozoite surface Attachmenthvasion into protective in humans but
Parasitized hepatocyte (minor quantities) hepatocytes insufficient alone
(RGD sequence in Region II) CTL induction (in mice)
even though liver cells not invaded
Gene incorporated into multi-stage
DNA vaccine (NYVAC - H 7 )
Polymorphic regions T cells
CD4+ helper
CD8+ cytotoxic
TRAP or SSP2 574 RGD sequence on SZ for T cells Shares RGD sequence with region I1
Sporozoite surface attachmend invasion of CD8+ CTL of CS protein
Parasitized hepatocyte surface? hepatocytes (class 1 HLA Gene incorporated into multi- stage
Blood schizont (minor quantities) restricted) DNA vaccine (NYVAC - H7)
Region between aa B cells Highly polymorphic
2 6 3 4 4 contains Ahs inhibit SZ invasion into
B-cell epitope(s) hepatocytes
LSA- 1 200 Unknown Epitopes LS6 and T cells Gene incorporated into multi-stage
Surface of parasitized hepatocytes LS8 bind to HLA CD8+ CTL DNA vaccine (NYVAC - Pf7)
Release on ~ p t ~ofr hepatic
e schizont B53 and HLA-B35
Hsl6 157 Unknown No CTL epitope B cells Not detectable in asexual erythrocytic
Sporozoite surface stages
Parasitized hepatocyte No repetitive sequences
Macrogamete surface
STARP 604 Unknown Many tandem repeats B cells Actively produced in liver stage of
Sporozoite surface (uneven distribution) - immunogenic? CD8+ CS protein
Parasitized hepatocyte (cytoplasmic) CTL
Asexual blood stage (rings only)

Several other pre-E stage antigens have been identified: this list is not fully comprehensive. Pf,Plasmodium falciparum; aa, amino
acids; Abs, antibodies; CTL, cytotoxic T lymphocytes; SZ, sporozoite.
18 C.A. FACER AND M. TANNER

bites, they do not develop a sterile immunity. One explanation for this is
the presumed presence of low levels of protective effector cytotoxic T
lymphocytes that could be enhanced by an appropriate CTL-generating
vaccine (Hill et al., 1992).
The several disadvantages associated with attenuated vaccines (see
Table 2), in particular, practical difficulties in producing en m a n e suffi-
cient numbers of sporozoites or infected mosquitoes for irradiation, has
led to the search for immunogenic epitopes on the sporozoite that could
be produced either by recombinant DNA technology or by chemical
synthesis. In addition, the identification of genes encoding proteins shared
by the liver stages should allow incorporation of those genes into the new
DNA vaccines (see Section 5).
A conservative estimate of the number of different proteins synthesized
by asexual stage parasites is > 2000, the genes for only a small proportion
of these having been cloned. What among these proteins are the important
vaccine candidates? (for summary, see Table 4 and Figure 6). Most have
been chosen using either a rational or an empirical approach. In the former,
one selects specific antigens that will have certain predetermined properties
and that will elicit a specific response (e.g. the choice of antigens which
elicit antibodies that react with the surface of merozoites and prevent
merozoite re-invasion of red cells). In order that all variants of natural
parasites will be susceptible to the elicited antibodies, it is essential that the
immunogen is antigenically conserved. In the alternative empirical
approach to vaccine development, antigens are selected solely on their
ability to protect an animal in a vaccine trial without the necessity of
characterizing the properties of that malaria antigen in other assays. The
SPf66 vaccine is an example of this approach (see Section 3).

2.1. The Circumsporozoite (or CSI Protein and T-cell epitopes

The sporozoite is coated with a protein known as the circumsporozoite


(or CS) protein (Figure 3a) which has probably received more attention
than all the other cloned and sequenced pre-erythrocytic antigens put
together. It comprises 412 amino acids (in P. falcipamm 7G8 clone)
divided into three main regions: an amino terminus containing the signal;
a major central component notable for the large multiple repeated units of
four amino acids, asparagine, alanine, asparagine, proline (NANP in
single letter code) plus repeats of asparagine, valine, aspartic acid, proline
(NVDP); and a carboxy terminus containing an anchor sequence (Dame
et al., 1984).
In P. falciparum (but not P. vivax), the repeats are well conserved
although sporozoites of different ‘strains’ have a variable number of
CLINICAL TRIALS OF MALARIA VACCINES 19

Figure 3(a) Schematic representation of P.falciparurn CS protein with region I1


sequences. These sequences are a conserved group of amino acids showing homol-
ogy with other proteins, some with an adhesive function. FU is also conserved. The
starred area over Th3R is variant and is involved in CD8 CTL recognition (mod-
ified from Sinnis et al., 1994). (b) Schematic representation of PfSSP-2/TRAP and
position of the non-variable epitopes, BH1 (3-11) and A6 (214-233) inducing
protective CD8+ CTL in mice. The region spanning amino acids 250-258 shares an
RGD sequence (WSPCSVTCG) with the CS protein (modified from Wizel et al.,
1994).
20 C.A. FACER AND M. TANNER

repeats (Weber and Hockmeyer, 1985). In malaria-exposed communities


there is a slow development of antibody to these repeats (Tanner et al.,
1986; Del Guidice et al., 1987). Nevertheless, the observation that they
form a highly immunodominant B-cell epitope (Zavala et al., 1987) led to
their incorporation in synthetic (Ballou et al., 1987) and recombinant
(Herrington et al., 1987; Vreden et a1.,1991; Sturchler et al., 1992)
vaccines.
In phase 0 trials in rodent models, antibodies to these repeats mediated
protection against sporozoite-induced infections (Nussenzweig and Nus-
senzweig, 1989). This encouraging start stimulated various human trials
which included the repeats in a vaccine (Fries et al., 1992; Herrington et
al., 1992). However, the outcome has been disappointing with equivocal
results and poor correlation with antibody titres to repeats and functional in
vitro studies with these antibodies (inhibition of sporozoite invasion into
hepatocytes) thus highlighting the problem of predicting a favourable out-
come based on an animal model. Other phase I1 and III trials with a
recombinant (FSV- 1 or R32tet32) or synthetic ([NANP]3-TT) vaccine
(Ballou et al., 1987), both using alum as adjuvant, similarly gave poor
efficacy.
In view of the limited success of these two vaccines, there have been
attempts to improve immunogenicity (in terms of antibody titre to repeats)
using adjuvants more potent than alum. The use of [NANPI3* fused to a
portion of the influenza virus administered with monophosphoryl lipid A as
adjuvant, produced levels of antibody to the repeat sequence one order of
magnitude higher than those adsorbed to the same recombinant peptide
adsorbed to alum (Rickman et al., 1991) but still the vaccine failed to
protect on challenge in Phase I1 trials in Thai adults (G. Sadoff, personal
communication).
The importance of adjuvants as immune potentiators in any vaccine
formulation for inducing high antibody titres and protection is highlighted
in a very recent breakthrough in the development of sporozoite vaccines.
The Walter Reed Army Institute of Research in conjunction with Smith-
Kline Beecham Biologicals have evaluated three formulations of their
vaccine RTS,S (Stoute et al., 1997). In phase IIa human volunteer trials,
vaccine formulation SBAS2 (vaccine 3) achieved the best result to date of
any sub-unit malaria vaccine in humans protecting six of seven volunteers
following challenge with infected mosquitoes (85% efficacy, RR 0.14; 95%
CI 0.02-0.88; p < 0.005).
RTS,S is a hybrid recombinant vaccine consisting of two polypeptides
that, on their simultaneous synthesis in yeast, form composite particulate
structures. One of the polypeptides (RTS) corresponds to the central tan-
dem repeats and carboxy-terminus epitopes of the P. falciparum (strain
3D7) CS protein (amino acids 207-395, see Figure 3a) that is fused to
CLINICAL TRIALS OF MALARIA VACCINES 21

HBsAg (hepatitis B antigen). The other polypeptide (S) corresponds to


HBsAg. One formulation, SBAS2 (vaccine 3), comprises RTS,S in an oil-
in-water emulsion plus the immune stimulants monophosphoryl lipid A and
QS21. Volunteers received three doses of 50 pgkg intramuscularly and
were then challenged three weeks after the final dose with five infected
mosquitoes.
The mechanism behind the protection afforded by RTS,S is uncertain.
The vaccine with its central tandem repeat epitopes and carboxy-terminal
epitopes provides targets for both antibody and cellular responses. Anti-
body responses to the repeats tended to be highest in the protected volun-
teers (those receiving SBAS2 or vaccine 3 formulation) suggesting that
humoral immunity has an important protective role. However, high anti-
body responses to repeats were also seen in a group receiving a different
vaccine formulation (SBAS3 or vaccine 2) who were not all protected,
suggesting that antibody responses alone were insufficient to confer pro-
tection. There was no evidence for cytolytic T cells. A strong IFNy response
to RTS,S in some subjects might indicate a role for CD4+ T cell responses
and this cytokine in eliminating liver stage parasites (see Section 2.2).
It will be important to determine the efficacy of this vaccine in endemic
areas where the level of transmission is high and there are multiple parasite
strains. Plans are now in place for phase IIb and phase I11 trials with RTS,S
in Africa. As many African children have already received hepatitis B
vaccination, one questions what effect this pre-immunity might have on
the anti-sporozoite response to the hybrid malaria vaccine. In addition, will
the short-lived vaccine-induced anti-sporozoite antibody responses be
maintained by natural boosting from infected mosquitoes?
Failure of anti-NANP antibodies to protect completely may relate not to
low antibody titre but to the fact that some individuals appear to produce
antibodies that are non-reactive with intact sporozoites (Etlinger et al.,
1988). A likely contributor to the formation of epitopes giving rise to
pathogen-unreactive antibodies is that the terminal proline of [NANPI3
in linear form, is not a terminal residue in the native protein (which is
predicted to have helical conformation). A recent novel vaccine in mice
found that by cyclizing [NANPI3, (c[NANPI3) it was possible to eliminate
the generation of non-parasite reactive anti-peptide antibodies (Etlinger
and Trzeciak, 1993). This model now requires further evaluation with
regards to its relevance in humans.
Initial experiments using human monoclonal and polyclonal antibodies
directed to P. fulcipurum pre-erythrocytic stage antigens had previously
shown that multiple non-CS antigens were present on the sporozoite sur-
face and/or in liver stages (Fidock et al., 1994). The screening of a h gtl 1
genomic expression library of P. fulciparum with sera from malaria-
exposed individuals under continuous malaria prophylaxis has identified
22 C.A. FACER AND M. TANNER

several other pre-erythrocytic antigens such as liver-stage antigen- 1 (LSA-


1) and sporozoite surface threonine- and asparagine-rich protein (STARP)
(see Table 4 and below). The presence in STARP of tandem repeats of
oligopeptide sequences (showing size polymorphism between different
parasite populations as for the CS protein) is a characteristic of many P.
falciparum antigens in which the repeats often contain major B-cell
epitopes.
Malaria vaccine design is beginning to move away from strategies aimed
at stimulating antibody to one focusing on cell-mediated immune responses
to pre-erythrocytic antigens (see Tables 1 and 6). It is obvious from the
many animal experiments cited in the literature, that cellular immunity is a
critical feature in protection against sporozoite-induced malaria. Thus, the
current approach is to look elsewhere on the CS and other molecules for
sequences that may represent T-helper or T-cytotoxic epitopes (Doolan and
Good, 1992). Of considerable interest are two regions (Regions I and 11)
which are highly conserved in all mammalian species of Plasmodium (see
Figure 3a).
Conservation of an epitope is interpreted as defining an essential func-
tion for that epitope. Several lines of evidence indicate that the initial event
involved in invasion of hepatocytes by sporozoites is recognition of the
COOH terminus of CS region I1 by heparan sulphate (a protein-
polysaccharide conjugate which contributes to cell-cell interactions) pre-
sent on the membrane of hepatocytes (Frevert et al., 1993). Interestingly,
the conserved region 11, naturally poorly immunogenic and therefore a
good vaccine target, has an RGD motif (Arg-Gly-Asp) similar to that
found on several host proteins such as thrombospondin, some complement
proteins (properdin, C6) and another recently described P. fakiparum
protein known as thrombospondin-related anonymous protein (TRAP) or
sporozoite surface protein 2 (SSP-2) (Robson et al., 1990). Although the
function of the motif on both CS and SSP-2 proteins is unknown, it may
have cell adhesive properties in common with the mammalian proteins and
thus be involved in hepatocyte invasion by sporozoites. A multiple-antigen
peptide that mimics the hepatocyte-binding ligand and inhibits CS binding
to a hepatoma cell line (HepG2 cells) was recently described (Sinnis et al.,
1994). In addition, invasion of hepatocytes is likely to involve other com-
plex molecular interactions perhaps with Region I of CS protein (Holling-
dale et al., 1993). Despite potential adverse pathological effects resulting
from cross-reactivity with host proteins such as thrombospondin, CS vac-
cines containing non-repeats, including Region 11, are now undergoing
clinical trials.
Other regions on the CS molecule are polymorphic and contain human
T-cell epitopes (Hill et al., 1992; Zevering et al., 1994). Extensive sequen-
cing of the CS protein from many strains of P. fakiparum has characterized
CLINICAL TRIALS OF MALARIA VACCINES 23

two regions of extensive polymorphism known as Th2R and Th3R posi-


tioned near the COOH terminus which contain helper T-cell epitopes (see
Figure 3a).
Recognition of these epitopes in human populations is genetically
restricted by the major histocompatibility locus (MHC) a factor which
may contribute to their poor immunogenicity. In a recent field study in
malaria-exposed Thais, memory CD4+ T cells from all volunteers
responded to a panel of Th2R (119) and Th3R (98) variants with minimal
cross-reactivity between variants (Zevering et uf., 1994). Thus CD4+ T
cells are capable of discriminating variant natural sequences (essential if T
cells are to exert biological pressure for parasite diversity) even although
some only differed by one or a few amino acids. Such selection by CD4+ T
cells would be expected to occur in the liver and would be reliant on
hepatocytes expressing CS peptide(s) in association with MHC class I1
molecules. Since immunodominant polymorphic sites are desirable for
inclusion into a subunit vaccine, strategies to overcome this immune eva-
sion facilitated by natural polymorphism must be developed.

2.2. Cytotoxic T Lymphocytes (CTL) for Vaccine Design

The early studies of experimental rodent malaria revealed quite clearly that
MHC class I restricted CTL could provide protection against sporozoite
challenge (Romero et af., 1989; Rodriguez et af., 1991) and lyse-infected
hepatocytes in vitro (Hoffman et al, 1989; Weiss et u f . , 1990).
It was also possible to reverse immunity to sporozoite challenge in mice
by experimental depletion of CD8+ cells coupled with neutralization of
interferon gamma (IFNy) (Schofield et a f . , 1987). MHC restriction in CTL
epitopes occurs since, in two congenic strains of mice (expressing between
them five different MHC class I molecules) vaccinated with attenuated
sporozoites, only a single CS epitope (polymorphic) was recognized
(Kumar et u f . , 1989). These original animal experiments provided consid-
erable impetus to attempt to identify CTL against P. fulciparum in humans
and a current review outlines various approaches to the design of a CTL-
inducing malaria vaccine (Lalvani et af., 1994).
The various lines of evidence for protective CTL responses, potential
epitopes and information pertaining to the generation of CTL in vivo, are
summarized in Table 5 . There are now three lines of evidence implicating
the involvement of CTL in protection against P. falciparum in humans.
First, HLA class I restricted CTL responses have been identified in Africans
repeatedly exposed to P. fufciparum sporozoites (Hill et uf., 1991, 1992,
Lalvani et al., 1994, 1996). Although only low levels of precursor CTL
were present in the circulation, these cells could provide some useful
24 C.A. FACER A N D M. TANNER

Table 5 A P. falciparum pre-erythrocyte vaccine inducing CTL responses


CTL and protection
0 In rodent models: CD8+ depletion enhances parasitaemia; anti-IFNy reverses
sterile immunity to SZ challenge; adoptive transfer of ‘immune’ CD8+ cells
protects.
Presence of HLA-restricted CTL response in vitro with cells from malaria exposed
individuals.
HLA-B53 association with resistance to severe malaria (conserved motifs present
in CS protein, SSP-2/TRAP, LSA-1 and Pfsl6 which binds to HLA-B53).
Extensive polymorphism in CS protein: no synonymous nucleotide changes in
Th2R and Th3R regions suggesting selection by immune pressure.
Protection of experimental animals following DNA vaccination with plasmids
containing genes for CS protein and liver stage proteins is dependent on CTL.
However, no evidence that human CTL kill parasitized hepatocytes. Protection in
vivo may be in part, due to local production (by Kupffer cells) of cytokines
stimulated by activated CD8+ cells, in particular IFNy.

CTL epitopes on parasite


HLA polymorphism implies many epitopes.
Epitope identification difficult due to low levels of circulating CTL.
Epitopes found on LSA-1, SSP-2 (amino acids 214-233 and 3-1 1) and STARP -
also found on blood stages.
0 Peptide Ls6 from LSA-1 recognized by HLA-B53 CTL and Ls8 by HLA-B35.

Generation of CTL in vivo


Antigen-presenting cells which have phagocytosed SZ or blood stages.
Expression of epitopes on surface of the parasitized hepatocyte.
Release of antigens (e.g. LSA-1) following rupture of hepatic schizont.

Boosting CTL by vaccination


Animal models: use of recombinant vaccinia, influenza and Salmonella or DNA
injection induce protective CTL (in absence of antibody).
Recombinant particles (yeast-derived virus-like particles or Ty-VLPs).
0 Protective CTL must be boosted.

degree of protection. It was suggested that individuals did not develop


strong CTL responses in the Gambia because they experienced only a very
low antigenic load following infection (Aidoo et al., 1995). However,
recent studies in Tanzania, an area of high perennial transmission with
an entomological innoculation rate 10-50 times higher than found in the
Gambia, also showed only low to moderate CTL responses (Lalvani et al.,
1996). The low levels of CTL questions their specificity. For example, the
CTL may have arisen as a result of cross-reacting epitopes of other organ-
isms, as has been demonstrated with ‘malaria-specific’ CD4+ T cells
(Good, 1995). Clearly, if these responses are real, then vaccination must
CLINICAL TRIALS OF MALARIA VACCINES 25

be able to boost the numbers to prevent parasite development within


infected hepatocytes (see below) and thus protect from malaria.
Secondly, CTL responses to a conserved P. falciparum epitope has been
identified for the HLA class I antigen, HLA B53, itself associated with
resistance to severe malaria in African children (Hill et al., 1992).
Finally, the pattern of polymorphism of pre-erythrocytic antigens sug-
gests that CTL may be protective in genetically variable human popula-
tions. Within the two regions of extensive polymorphism found in the CS
protein (Th2R and Th3R; see Figure 3a), amino acid changes are encoded
by all of the nucleotide substitutions and no silent (synonymous) changes
have been found, arguing strongly that these polymorphisms are a result of
immune selection pressure (Good et a1.,1988a). This immune selection is
likely to involve CTL even though these regions also encode T-helper-cell
epitopes. Variation in Th2R and Th3R can lead to loss of CTL recognition.
Thus the most common allele of the P. falciparum CS protein in the Th3R
region (cp27) fails to bind to HLA-35 (the most frequent HLA allele in
Gambians) because of a single amino acid change from the cp26 epitope
(Lalvani et al., 1994).
For many years immunologists have believed that CTLs functioned
solely by killing (lysing) target cells. However, a new report indicates
that frequently the number of targets in a host is in excess of the total
number of killer T lymphocytes, and that cytokines produced by T cells
also play a vital role in controlling intracellular pathogens (Crawford,
1996). Thus the cytokines IFNy and TNFa, stimulated liver cells to
synthesize more of these two cytokines together with reactive nitrogen
intermediates which, in turn, inhibited development of hepatitis B within
infected hepatocytes. It is possible that the high TNFa and IFNy levels in
malaria may be having a similar inhibitory effect on malaria parasites
within liver cells (Schofield et al., 1987; Section 2.1). One necessary
feature of a pre-erythrocytic malaria vaccine is thus likely to be the
potential to stimulate high concentrations of IFNy in the liver.
The news that a single subcutaneous injection of 10 pgkg recombinant
human IL-12 afforded complete protection to sporozoite challenged mice
and monkeys is remarkable (Hoffman et al., 1997). Protection in these
animals was associated with high plasma levels of IFNy, presumably a
result of IL-12 stimulation of T cells. Because IFNy does not have direct
anti-parasite activity, it is likely that elimination or inhibition of parasites
is a result of the nitric oxide generated in infected hepatocytes by this
cytokine.
The likelihood that CTL act as effectors in protection against malaria has
stimulated two main groups into designing and developing a vaccine that
will promote CTL production. A new DNA vaccine which incorporates
seven different malaria genes (mainly pre-erythrocytic) has been produced
26 C.A. FACER AND M. TANNER

and is about to enter phase I1 trials (see Section 5 ) . Another group has
adopted an HLA approach to the design of a CTL vaccine, the aim of which
is to identify the most appropriate malaria CTL-stimulating epitopes in
view of the inevitable human HLA class I restriction of CTL responses
(Lalvani et al., 1994; Aidoo et al., 1995).
Using the reverse immunogenetic method, whereby short P. fakiparum
peptides (8-10 amino acids) are synthesized to match the known charac-
teristics of peptides binding in the groove of the particular HLA class I
molecules, 12 CTL epitopes have been identified in naturally exposed
Gambians, in four P. fakiparum pre-erythrocytic antigens: CS protein,
SSP-2, LSA-1 and STARP (Aidoo et al., 1995). Fortunately, the extent
of polymorphism detected in these epitopes appears limited. The HLA
types used for epitope identification, HLA-B53, HLA-B35, HLA-B8,
HLA-B17, HLA-B7 and HLA-A2 (both HLA-A2.1 and HLA-A2.2) were
chosen because of their high frequencies (70-75%) in Caucasians and
Gambians.
The next step is to develop a sufficiently sensitive in vitro efficacy assay
that will measure the CTL-induced killing of P. fakiparum within hepa-
tocytes. Primary pre-erythrocytic stage peptide-specific and MHC-
restricted CTL clones are now being generated from malaria-naive
individuals for analysis of cytotoxicity against freshly isolated and
malaria-parasitized human hepatocytes in which sporozoite invasion and
multiplication occurs (multiplication does not occur in the commonly used
hepatoma cell line) allowing the surface expression of malaria antigens on
these cells.
Finally, Hill and colleagues (1992) plan to incorporate P. fakiparum
CTL-stimulating epitopes into yeast-derived virus-like particles (Ty-VLR)
and to use this construct as a vaccine. Preliminary experiments have shown
that Ty-VLR in mice induce impressive levels of CTL to various foreign
epitopes. With this vaccine approach however, there remains the potential
problem of having to include in the vaccine P. fakiparum CTL epitopes for
all the HLA types within a given population.

2.3. SSP-PITRAP

The ideal pre-erythrocytic vaccine thus needs to be polyvalent and to


include specific CD8+ CTL epitopes combined with CD4+ T-helper
epitopes from the same protein to allow boosting following exposure to
the parasite, as well as additional sporozoitefliver stage antigens such as
SSP-2.
SSP-2 (sporozoite surface protein 2) or TRAP (thrombospondin-related
anonymous protein) is a recently described second sporozoite and early
CLINICAL TRIALS OF MALARIA VACCINES 27

liver stage protein (Charoenvit et al., 1987; Knusmith et al., 1991) which is
also expressed in the asexual erythrocytic stages (Robson et al, 1990). It
joins the CS protein (P. falciparum, P. yoelii and P. berghei) and the P .
falciparum liver stage antigen 1 (LSA-1) as the third plasmodia1 protein
described that has been cloned and sequenced and to which CD8+ CTL
have been generated.
SSP-2 in P. falciparum (see Figure 3b) shares a RGD(Arg-Gly-Asp)-
binding motif with the CS protein implying perhaps a function in hepato-
cyte recognition, attachment and invasion (Cowan et al., 1992). SSP-2 has
potential as a component of a human malaria vaccine (Knusmith et al.,
1991) since it has, at least in the experimental mouse models, the ability to
generate protective CD8+ CTL (Wizel et al., 1994). The CTL appear to
recognize SSP-2 peptides with class I HLA molecules on the surface of
infected hepatocytes.
One constraint for inclusion into a vaccine, as with most malaria anti-
gens, is the high degree of polymorphism within this protein (Robson et al.,
1990), although two regions, peptides A6 and BH-1 (see Figure 3b) which
generated CTL, appear conserved (Wizel et al., 1994). In a trial with a
small number of exposed Gambians of HLA-B35 and B53 status, 23
synthetic SSP-2 peptides failed to induce CTL responses in vitro, although
peptide BH-1 was not included (Hill et al., 1992).
These studies have now been extended to include a larger number of
Gambian donors and several CD8+ CTL clones have been established
which are specific for peptides on SSP-2 and LSA-1 (see below) despite
the difficulty in producing such clones because of low precursor frequency
in the peripheral circulation and cytokine requirements in culture (A.V.S.
Hill, personal communication). Two conserved peptides (tr42 and tr43)
located in region A6 of SSP-2 are recognized by a Gambian HLA type,
HLA-B8 (Aidoo et al., 1995).
TRAP, in addition to stimulating a CTL response, also stimulates the
production of anti-TRAP antibodies in malaria-exposed individuals whose
epitope specificity and possible association with protection is currently
being assessed in an age-related study in Mali and the Gambia (Scarselli
et al., 1993; K. Robson, personal communication).

2.4. Liver Stage Antigen 1 (LSA-1)

A third pre-erythrocytic stage antigen that has vaccine potential is the


200 kDa liver stage antigen- 1 (LSA- 1) (Zhu and Hollingdale, 1991;Fidock
et al., 1994), a protein localized in the parasitophorous vacuole space of
liver stage parasites. It is composed of a large central repetitive region
28 C.A. FACER AND M. TANNER

(conserved) and two flanking short non-repetitive and variable N- and C-


terminal regions (Yang kt al., 1995).
This antigen is of considerable interest in vaccine development because
two conserved epitopes, Ls6 and Ls8, bind to HLA-B53 and HLA-B35
respectively (Aidoo et al., 1995). However, generation in vitro of CTL
clones from HLA-B53- and HLA-B35-positive donors by restimulation
with these peptides was not possible, presumably because of the low
frequencies of precursor CTL.
Likewise, the recently described sporozoite surface threonine- and
asparagine-rich protein (STARP) (Fidock et al., 1994) (see Table 4)
has also been found to contain CTL epitopes (Lalvani et al., 1994; Aidoo
et al., 1995). In fact, of all the pre-erythrocytic stage antigens so far
described in detail, only the smallest, Pfsl6, a novel protein of P.
falciparum located on the surface of sporozoites and gametes (Moelans
et al., 1991), has failed to reveal any CTL epitopes.
The search continues for other P. falciparum-associated antigens that
might contain immunogenic epitopes recognized in association with class I
(CD8+ cytotoxic cells) and perhaps class I1 (CD4+ cytotoxic cells) HLA
antigens which may provide protection and thus be suitable as vaccine
candidates.
In summary, the future pre-erythrocytic vaccine is likely to contain
several parasite epitopes: one B cell specific (NANP), one specific for
CD4+ helper and CD8+ (and CD4+) cytotoxic T cells, and invariant
epitopes for universal MHC binding.

3. ASEXUAL BLOOD-STAGE VACCINES

Vaccines targeting the asexual blood stages are not expected to prevent
blood-stage infection but rather to control parasite growth. Natural immu-
nity would develop while the vaccine prevented life-threatening disease. It
is an approach that is suited to areas of high malaria transmission where the
priority target is young children, a high-risk group for malaria morbidity
and mortality.
The largest human trials of an asexual blood stage vaccine have been
achieved with SPf66 (see Section 3.6). The vaccine includes several
antigens, the exact parasite location and function of which remain
obscure.
CLINICAL TRIALS OF MALARIA VACCINES 29

3.1. Role of Antibodies?

Antibodies to asexual blood-stage antigens are undoubtedly important in


the control of blood parasitaemia and possibly clinical symptoms. This is
clearly demonstrated by the many experiments in animal models and by the
protection afforded to children receiving passive immunotherapy with
pooled ‘immune’ immunoglobulin from adults living within the same
endemic area (McGregor, 1964). Some of the parasite target antigens of
this immunoglobulin are likely to be shared between geographically remote
strains of P. fulcipurum since an African adult IgG pool, given intrave-
nously to Thai malaria patients, produced a clearance of parasites and
symptoms which was as fast or faster than that achievable with effective
antimalarial drugs (Sabchareon et al., 1991).
Malaria antibodies have thus formed the basis for a number of criteria
used for selecting parasite antigens for examination as potential candidates
for inclusion into a vaccine that would stimulate humoral in addition to
cellular responses. Antigens have been selected because of their surface
location and consequent accessibilty to antibody-mediated attack (e.g.
merozoite surface antigens). The correlations between antibody specifici-
ties present in immune sera and protection (reduction in parasitaemia and
clinical symptoms) have also been employed as has the effect of mono-
clonal and polyclonal antibodies in functional in vitro assays, for example
inhibition of merozoite invasion of red cells, intraerythrocyte parasite
maturation, and preventiodreversal of cytoadherence of trophozoites and
schizonts to endothelial cells.
Using one or more of these criteria, more antigens than for any other
stage of the life cycle have been identified, sequenced and proposed as
vaccine targets. In this review we are able to highlight only a few of these,
placing particular emphasis on what are considered the most promising
following phase 0 and phase I vaccine trials.

3.2. The Problem of Parasite Antigenic Diversity

One characteristic feature expressed predominantly during the erythrocytic


cycle of P. fulciparum is extensive genetic plasticity and consequent anti-
genic diversity, which remains a concern for malaria vaccine development
(Anders, 1991). A major cause of this diversity is the expression of
different repetitive sequences in allelic forms of several antigens including
the heat stable, water soluble, ‘S’ antigens and two merozoite surface
antigens, MSA-1 (also known as merozoite surface protein- 1, MSP- 1)
and MSA-2 (also known as merozoite surface protein-2, MSP-2) (see
below). In addition, diversity can also arise from simple point mutations,
30 C.A. FACER AND M. TANNER

intragenic recombination (MSA-1) and antigenic variation (PfEMP- 1). The


bias towards mutations causing an amino acid replacement (non-synony-
mous) rather than to silent mutations (synonymous) suggests positive
selection for these mutations presumably because they enable parasites
to evade anti-parasitic immune responses (McConkey et al., 1990)
although others have argued against this (Amot, 1989).
In view of the capacity of malaria parasites to mutate the structures of
potential vaccine target antigens, asexual stage vaccines are likely to be
multi-component, thereby minimizing the opportunity for parasite avoid-
ance of vaccine-elicitated immune responses.
Several antigens have been identified as suitable candidates for inclusion
into an asexual stage vaccine and these are listed in Table 6. Merozoite
surface proteins, MSA-1 and MSA-2, which may function in the invasion
of erythrocytes, are prime targets. Antibodies to a further merozoite surface
protein, MSA-3, have recently been described as co-operating with blood
monocytes in antibody-dependent killing of blood stage P. fa lciparum
(Oeuvray et al., 1994).

3.3. MSA-1 and MSA-2

The precursor molecule of MSA- 1, the most intensely studied asexual


stage antigen, undergoes a two-step processing; (i) a primary event gives
rise to major fragments (including 83 kDa, 36 kDa and 41 kDa proteins)
found on the merozoite surface at schizont rupture; and (ii) a secondary
event in which the 41 kDa protein is proteolytically cleaved to give two
products, a 33 kDa protein (which is shed as a complex with other
MSA-1 fragments) and a smaller, cysteine-rich 19 kDa fragment (which
remains on the merozoite and is carried along with it into the invaded
erythrocyte) (Cooper, 1993). Interestingly, this 19 kDa fragment consists
of two epidermal growth factor (EGF) domains (Blackman et al., 1994).
These appear to be the target of invasion-inhibiting mouse monoclonal
antibodies that recognize the first EGF domain (Chappel and Holder,
1993). Conversely, although affinity purified IgG from malaria immune
donors recognized the same epitope it failed to inhibit merozoite invasion
unlike the mouse monoclonals. The antigenicity of the EGF domain is
conformation dependent since reduction of the disulfide bonds abolishes
immunogenicity (Spetzler et al, 1994). EGF-like structures, widely dis-
tributed in the animal kingdom, are not always associated with growth
promotion and many seem to be involved in cell-cell interactions
(Cooper, 1993).
CLINICAL TRIALS OF MALARIA VACCINES 31

Vaccination experiments have been attempted in experimental animal


models using a recombinant 100 amino acid EGF domain plus alum as
adjuvant (GST-MSP19). In mice, this construct proved immunogenic with
the integrity of a double domain vital to this immunogenicity (the two
EGF domains given individually or together were ineffective). In contrast,
the vaccine failed to protect Aotus monkeys on challenge with homologous
and heterologous P. falciparum although antibodies were made to the
construct (A.A. Holder, personal communication). One reason for this
anomaly, which might also explain the results with human IgG from
malaria immune individuals described above, could be the production of
other MSA- l-specific blocking antibodies that either inhibit processing of
MSA-1 or, by means of steric hindrance, block the binding of antibodies to
the EGF epitope.
The complete amino acid sequence of the polymorphic MSA-1 mole-
cule is now documented for an impressively large number of geographi-
cal strains of P. falciparum (Miller et al., 1993) and this clearly
demonstrates blocks of variable, constant and dimorphic regions (Figure
4).Both cellular and humoral immunity to MSA-1 have been implicated.
Mice immunized with MSA-1 of P. yoelii were protected but their sera
failed to transfer protection to naive mice (Freeman and Holder, 1983)
suggesting cellular immunity. In another study, monkeys immunized with
P. falciparum MSA- 1 had antibodies that blocked merozoite invasion of
red cells and may have affected parasite growth by traversing the con-
troversial ‘parasitophorous duct’ (Pouvelle et al., 1991). Passive transfer
of anti-MSA-1 antibody also confers protection in vivo (Lew et al.,
1990). Regions of the molecule thought to be important include the C-
terminal 19 kDa domain described above, and the N-terminal domain
(Riley et al., 1992).
Immunization of Aotus and Saimiri monkeys with the whole MSA-1
molecule provided complete protection against asexual parasite challenge
(Deans et al., 1988) although recombinant polypeptides, representing
MSA- 1 conserved regions, induced only partial protection. In another
study, fusion of a recombinant conserved MSA-1 fragment to a universal
P. falciparum CS protein T-cell epitope induced partial protection in Aotus
monkeys that was not correlated with antibody titre to the construct but,
interestingly, correlated with levels of serum IFNy in the vaccinated ani-
mals (Herrera et al., 1992), a cytokine which is known to arrest maturation
of P. falciparum erythrocytic schizonts in vitro (Orago and Facer, 1993).
The conclusion was that protection was T-cell mediated through the release
of this cytokine.
The synthetic peptide polymer, SPf66, which has now undergone exten-
sive human trials in South America (see Section 3.6), contains sequences
from three merozoite proteins, one of which is MSA-1.
Table 6 Target antigens for asexual vaccine development
Size
Target antigen (ma) Location Function and other comments Reference

(a) Merozoite
MSA-1 (MSP-1, g ~ 1 9 5 ) 195 Merozoite surface Red cell invasion. Many vaccine studies. Holder ( 1993)
Protection against homologous challenge in
Aotus. Gene incorporated into multi-stage
DNA vaccine (NWAC-F'f7)
MSA-2 (MSP-2) 45 Merozoite surface Function unknown. Phase I trial underway Epping et ul. (1988)
MSA-3 (MSP-3) 48 Merozoite surface Function unknown. Conserved repeats. Oeuvray et al. (1994)
Induces protective cytophilic antibodies
RESA 155 Dense granules Stabilization of rbc spectrin. Found in culture Perkins (1992)
supernatants. Weak protection in Aotus
vaccine trial
EBA-175 175 Micronemestapical Red cell invasion. EBA peptide 4 binds to Sim (1995)
end sialic acid on glycophorin A
AMA- 1 83 Rhoptry organelle Red cell invasion? Found in culture superna- Thomas et al. (1 990)
tant. Gene incorporated into multi-stage
DNA vaccine (NYVAC-Pf7)
QF3 (RAP-112) 80142 Rhoptry organelle Red cell invasion? Impressive protection in Crowther et al. (1990)
Saimiri monkeys
SPf66 (a peptide polymer) 5000 Merozoite proteins Immunogenic in human
Plus (NANPh vaccine trials. Protection not
universal
(b) Infected erythrocyte membrane surface
PEMP- 1 250-400 PRBC surface Ligand for cytoadherence to endothelial cells Howard and
Extensive antigenic variation Gilladoga (1989)
Gene cloned (var genes). Important role in Baruch et al. (1995)
cerebral malaria
PfHRP-2 65-75 PRBC surface Histidine-rich Rock et al. (1987)
Protein very stable (identified in Egyptian
mummies)
Rosettin 22-28 PRBC surface Binds to unifected rbc which rosette around Helmby el al. (1993)
PRBC. Gene not cloned. May be involved
in cerebral malaria
Ag332 2000 PRBC surface Monoclonal blocks binding PRBC to Mattei et al. (1992)
endothelial cells. Parts of gene cloned

(c) Soluble antigens


PfHRP-2 65-75 Secreted into Function unknown. Protein very stable. Aotus Rock et al. (1987)
plasma from PRBC monkeys partially protected from live
ch allenge
Ag7 ? Released on rupture Function unknown. May cause TNF release Jacobson et al. (1993)
of PRBC and fever
Ag2 (SERP) ? Released on rupture Function unknown. May cause TNF release Jacobson et al. (1993)
of PRBC and fever
SERA 110 Released on rupture Function unknown. Immunogenic and Delplace et al. (1987)
of PRBC protective in monkeys. Gene incorporated
into multi-stage DNA vaccine
(NYVAC-Pf7)
34 C.A. FACER AND M. TANNER

EGF motifs

t
Repeats(b1ock 2)
f

83kDa 28-30kOa 36kDa 42kDa

19kDa

Signal sequence semi-conserved (dimorphic) regions

variable regions conserved regions

Figure 4 Schematic representation of MSA-1 from the WellcomeLiverpool


West African strain of P. falciparum showing processing products (modified
from Riley et al., 1992).

The second well-characterized merozoite surface protein is MSA-2. With


a size variation between P. falciparum strains of between 45 kDa and
55 kDa, it contains a central repeat region flanked either side by conserved
domains (McBride et al., 1985). Monoclonal antibodies against MSA-2,
like those generated against MSA-1, inhibit parasite growth in vitro parti-
cularly those monoclonals reacting with the sequence Ser-Thr-Asp-Ser
(single letter code: STNS) which occurs twice in the molecule (Clark et al.,
1989). Successful vaccination experiments, as yet only performed in mice
(P. chabaudi), show the potential of MSA-2, particularly its conserved
sequences, for future vaccine development. MSA-2, in combination with
RESA and pl90 of MSA- 1, has now undergone safety and immunogenicity
studies (phase I and I1 trials) in male adults in Australia and Papua New
Guinea (PNG). Provided the results are satisfactory, this combination
vaccine will undergo phase I11 efficacy testing among children in PNG
(Genton et al., 1997).
One concern frequently raised is whether malaria vaccines will alter
parasite diversity (multiplicity) in vaccine recipients. MSA- 1 and MSA-2,
both highly polymorphic proteins, can be grouped into two or three allelic
families respectively. The alleles, detected by polymerase chain reaction
(PCR) (for MSA-1) and PCR followed by restriction fragment length
CLINICAL TRIALS OF MALARIA VACCINES 35

polymorphism (RFLP) (for MSA-2), represent useful markers in the study


of P . falciparum diversity within individuals living in malaria endemic
areas (0.Puijalon, personal communication; Paul et al., 1995; Beck et al.,
1996). Multiplicity of infections has been linked to age, presence or
absence of symptoms, parasite density and haemoglobinopathy (Table 7;
0. Puijalon, personal communication; Beck et al., 1996). Interestingly, a
high multiplicity of infections appears to protect against clinical attacks of
malaria in Tanzanian children (Beck et al., 1996). Thus children with
asymptomatic infections have a significantly higher mean level of infec-
tions compared to those with clinical infections using a highly sensitive
technique to detect and differentiate MSA-2 alleles. In contrast, perhaps as
a result of technical constraint, no difference was detected in multiplicity
of infections as adjudged by MSA-1 specific allele priming.
Furthermore, parasites with MSA-2 FC27-like genotypes were more
likely to be found in clinical cases (and interestingly, in individuals with

Table 7 Observations on multiplicity of infections using MSA-1 and MSA-2


allele frequencies
Alleles Feature Result from PCR
MSA- 1 Asymptomatic or symptomatic No difference in multiplicity"
Vaccination with SPf66 (1-5 No change in multiplicity"
year olds)
MSA-2 Asymptomatic Increased multiplicity of infections
compared with clinical cases",
Have fewer FC27-like infections'
Vaccination with SPf66 ( 1-5 Statistically significant reduction in
year olds) multiplicity in asymptomatic
vaccinees compared with
asymptomatics given placebo (but
not between symptomatic recipients
of vaccine or placebo)"
High parasite density Increased multiplicityb
Associated with strain FC27-like
infections in Tanzania"
Age (c 15 years) Younger the child, greater the
multiplicityb
HbASd Fewer cases with strain FC27 infectionsb

" Beck et al. (1996).


0.Puijalon (personal communication).
Englebrecht et al. (1995).
sickle cell heterozygotes.
36 C.A. FACER AND M. TANNER

haemoglobinopathy) than were parasites with 3D7-like genotypes (0.


Puijalon, personal communication; Babiker et al., 1995; Engelbrecht et
al., 1995). The implication of this is that FC27-like parasites may be
more virulent than other genotypes and that children with sickle cell
haemoglobin are in some way ‘resistant’ to the virulent effects of this
infection. Continuing field studies in Tanzania, in conjunction with
several SPf66 vaccine trials, indicate that multiplicity of infections in
asymptomatic vaccines is reduced (see later under SPf66 vaccine; Beck
et al., 1996).

3.4. RESA/Pfl55

The vaccine candidature of other antigens is less clear. RESNPfl55 is an


antigen that is released from the apical organelles of the merozoite and
transferred to the erythrocyte membrane at the time of merozoite invasion
(Anders and Brown, 1990). The protein contains two immunogenic oligo-
peptide repeat regions at its carboxy terminus (five copies of Glu-Glu-
Asn-Val-Glu-His-Asp-Ala and 30 copies of Glu-Glu-Asn-Val). These
repeats are conserved among different geographical strains of P. falci-
parum and contain B- and T-cell epitopes in humans, although a study
of the T-cell response to this antigen in West Africa failed to reveal defined
MHC class I1 associations (Troye-Blomberg et al., 1991).
Since anti-RESA monoclonals have been shown to inhibit merozoite
invasion in vitro, it has been assumed that RESA functions somehow in
this process. However RESA is not expressed in a line of P. falciparum
isolate FCR3 which invades and grows well in vitro. In addition, vacci-
nation of monkeys with synthetic or recombinant repeats or with recom-
binants expressing full-length RESA has failed to induce protection
(Collins et al., 1991; Pye et al., 1991). A new function for RESA has
recently been proposed. The region between the two repeats contains a
domain that binds to spectrin in the red cell cytoskeleton, an association
which stabilizes an otherwise unstable parasitized red cell. The implica-
tion is that RESA, by modifying the erythrocyte membrane, favours the
survival of this abnormal cell in the circulation of the infected host (Da
Silva et al., 1994).

3.5. Other Target Antigens

The merozoite rhoptry antigens (QF3 complex, RAP-1/2 and apical mem-
brane antigen-1 [AMA-11, Crowther et al., 1990) and a microneme protein,
erythrocyte binding antigen-175 (EBA-175; Sim, 1995), are discharged on
CLINICAL TRIALS OF MALARIA VACCINES 37

to the red cell surface by the invading merozoite. Specific monoclonals


inhibit P . falciparum merozoite invasion in vitro and Saimiri monkeys
immunized with P. falciparum RAP112 (Ridley et al., 1990) or P . fragile
AMA- 1 (Howard and Pasloske, 1993) were protected against blood-stage
challenge.

3.6. The First Chemically Synthesized Vaccine Against


P. fakiparum: SPf66

Much attention has been focused on the novel synthetic polymeric blood-
stage vaccine, SPf66, designed and developed by Dr Manuel Patarroyo in
Bogota, Colombia, following reports that the vaccine induced significant
protection against blood-stage challenge in Aotus monkeys (Patarroyo et
al., 1987) and in a small group of army volunteers (Patarroyo et al., 1988).
In the latter phase I trial, infection was not prevented but the three volun-
teers had mild infections with a steady decrease in parasite counts (all
parasitaemias were c 0.5%) and total recovery 21 days following
challenge.
Following these encouraging results, extensive phase I11 trials in Latin
America, most of them in Colombia, were performed with the first report of
the vaccine’s success in a randomized double-blind placebo-controlled trial
in 1993 (Valero et al., 1993). This represented a crucial milestone in
malaria vaccine development and provided the first evidence that immu-
nization with this synthetic peptide polymer has the potential of reducing
the risk of clinical malaria in populations under natural exposure to P .
falciparum. Its other attractions were a comparatively low estimated cost
(around 25 US cents for the three doses), stability and ease of administration.
What peptides are included in the construct, how were they chosen and
what is the vaccine’s efficacy under different degrees of malaria trans-
mission?
Patarroyo and his colleagues first identified, with the aid of natural
malaria immune sera, several proteins (and then several constituent pep-
tides) of P. falciparum which provided protection against experimental
infection in monkeys. Three of the most promising peptides, representing
sequences from three P. falciparum blood-stage antigens, formed the basis
of the vaccine. The peptides 35.1 and 55.1 were based on partial sequences
from two, as yet unidentified, P. falciparum molecules. The third peptide,
83.1, corresponds to the highly conserved region 45-53 of MSA- 1. The
construct also includes two NANP sequences from the B-cell immunodo-
minant repeat of the CS protein which apparently are added to create hair-
pin bends in the sequence rather than for their immunogenicity. The
sequence, shown in Figure 5, is artificial notably because, in order to
38 C.A. FACER AND M. TANNER

Spf 55.1

Asp-GIu-Leu-GIu-Ala-GIu-Thr-GIn-Asn-Val-Tyr-Ala-Ala

Tyr-Ser-Leu-Phe-Glnl-t Met-Val-Leu

Tyr-GIy-Gly-Pro-Ala-Asn-Lys-Lys-Asn-Ala-GIy

Structure of monomer unit in the polvmer svnthetic


vaccine SPf66

-
Cys-Gly- (SPf 55.1) - Pro-Asn-Ala-Asn-Pro (SPf 83.1) Pro-Asn- -
Ala- Asn-Pro- ( SPf 35.1) -Cys
Figure 5 The synthetic SPf66 vaccine construct and the amino acid sequences of
the peptides contained within it. The peptide numbering corresponds to the mole-
cular weight of the malaria protein from which the sequence is derived. Peptide
83.1 is an 11 amino acid synthetic peptide corresponding to residues 43-53 of
MSA-1 (a conserved epitope in the majority of P. falciparum isolates) and contains
the red cell binding motif Lys-Glu-Lys (KEK, shown boxed). One Cys residue is
added at end of the molecule to permit polymerization via formation of disulphide
bridges between pairs of Cys residues on oxidation (Patarroyo et al., 1987).

permit polymerization of the 50 kDa construct (achieved by oxidation),


cysteine residues are added at each end. Some concern was expressed at the
time that polymerization might lead to batch to batch variation during
production with subsequent altered immunogenicity. However, these fears
have been dispelled following the demonstration that immunization of
rabbits with batches produced in Colombia and the USA gave similar
results. The final formulation involves adsorption of the polymer to alu-
minium hydroxide as adjuvant (Lopez et al., 1994).
The safety and immunogenicity of SPf66 has been well established in a
series of studies, first in Latin America (Patarroyo et al., 1992), and then in
Africa where the population has been heavily exposed to malaria prior to
CLINICAL TRIALS OF MALARIA VACCINES 39

vaccination (Teuscher et al., 1994). The maximum immunogenicity is


achieved following a long course of vaccination with 2 mg (1 mg for
children) of the construct subcutaneously on days 0, 30 and 180 (Patarroyo
et al., 1992).
Adjuvant vaccines are normally administered intramuscularly for full
effect. To date, all trials with SPf66 have been performed with the vaccine
given subcutaneously. This might explain the reported occurrence of local
skin reactions at the site of vaccination? If SPf66 was administered intra-
muscularly would immunogenicity be altered? An attempt to answer these
questions is currently underway in Colombia and in Kenya. The Kenyan
phase I/IIa SPf66/GMP trial in 90 adults will compare slun reactions and
immunogenicity (both humoral and cellular) between volunteers receiving
the vaccine either subcutaneously or intramuscularly (P. Duffy, personal
communication).
In Latin American countries (Colombia, Venezuela and Ecuador) almost
27 000 individuals, including large numbers of children, have now
received the vaccine. Some of the earlier trials in Colombia were criticized
on the basis of design flaws (they were not randomized double-blind
placebo-controlled) and therefore did not permit a definite conclusion to
be reached concerning the levels of protective efficacy achieved. However,
the results from correctly designed trials in Colombia (Table 8) demon-
strated an overall (all ages) estimated efficacy (against first clinical epi-
sodes of P . falciparum malaria) of 38.8% and a higher 77.2% efficacy in
children under 5 years of age (Valero et al., 1993). There was no difference
in the density of parasitaemia between the two groups. This is now being
followed with two additional independent phase I11 trials in Colombia.
Another independent trial in Ecuador confirmed the effectiveness of the
Colombian SPf66 trials and showed an overall estimated protective effi-
cacy of 66.6% although the 95% confidence interval was wide, from -2.7
to 89.3% (Sempertegui et al., 1994).
One picture that emerged from the Colombian trials is that not all
immunized subjects respond to the vaccine and there is evidence of genetic
restriction associated with T-cell receptor (TcR) variability (Murillo et al.,
1992). Low responsiveness was seen in 20-25% of recipients associated
with positivity for HLA DR4 class I1 antigens. The following explanation
for this was proposed by Patarroyo. A motif found in the conserved region
(amino acids 45-53) of the 83 kDa peptide, tyrosine and neighbouring
lysine-glutamic acid-lysine (single letter code: Y . . . KEK), binds to the
red cell and may be used by the merozoite during the process of invasion.
An examination of TcR Vp gene coding sequences in poor responders (who
were Vp-10) suggested that they shared Y . . . KEK motifs which were
absent in the responders (who were Vp-8). Thus, this crucial epitope passed
unrecognized.
40 C.A. FACER AND M. TANNER

Table 8 Efficacy of human trials with SPf66


Age of
Efficacy" % volunteer at Trial
Country (95% CI) first dose designb Reference
Colombia 33.6 (19/76) All ages DB/RCT Valero et al. (1993)
(La Tola) 77.2 (20/94) < 5 years
67.0 (39182) > 45 years
Venezuela 55.0 (21/75) 2 11 years No RCT Noya et al. (1994)
Ecuador 67.0 (-2/90) 2 1 year DB/RCT Sempertegui et al.
( 1994)
Tanzania 31.0 (0/52) 1-5 years DB/RCT Alonso et al. (1994)
The Gambia 8.0 (-18/29) 6-11 months DB/RCT D'Alessandro et al.
-9.0 (-33/14) 2-15 years (1995)
Thailand 21.0 (-9/55) c 6 years DB/RCT Nosten et al. (1996)
-24.0 (-58/6) 2 6 years
Colombia (Rio 36.0 (9/54)' 1-86 years DB/RCT Valero et al. (1996)
Rosario)
Tanzania Results awaited 1 month DB/RCT Spanish- Swiss-
1998 Tanzanian trial
team

" Vaccine efficacy based on overall incidence rates of first clinical episodes of
malaria (P. falciparum) in vaccine and placebo groups.
DB/RCT (double-blind, randomized, placebo-controlled trial conditions).
' Efficacy 22 months after last vaccine dose.

The acid test for the vaccine has been its performance in African and
other countries where the transmission of malaria is several orders of
magnitude greater than in South America. Three such trials recently com-
pleted are a Swiss/Spanish sponsored and led independent phase I11 trial in
Tanzania, another in the Gambia initiated by the British Medical Research
Council, and one run by the US Walter Reed Army Institute of Research
(WRAIR) in Thailand (Ballou et al., 1995). All batches of SPf66 used were
produced under good manufacturing practice (GMP) conditions and given
Food Drug Administration Approval (FDA) approval or European Com-
mission licences, for the Thailand and African human trials, respectively.
One requirement specified in the GMP conditions is that each batch of
vaccine produced must be biochemically analysed for evaluation of the
content of peptide monomers and polymers. The potential for the hybrid
peptide to cyclize during or after production is also being considered an
important issue since such peptides may be superior to linear forms in
CLINICAL TRIALS OF MALARIA VACCINES 41

inducing ‘parasite-reactive’ antibodies (for discussion, see Section 2). The


potential batch-to-batch variation in this respect highlights the importance
that any vaccine trial should be performed with a single vaccine batch
having a defined structural analysis.
The results of these SPf66 trials performed outside Latin America are
now published (Alonso et al., 1994; Tanner er al., 1995; D’Alessandro et
al., 1995; Nosten et al., 1996). One trial in Tanzania involved 586 children
aged from 1 to 5 years and was carried out in an area of intense perennial
malaria transmission. The vaccine was partially effective giving an esti-
mated vaccine efficacy (against first episodes of malaria) of 31% with a
wide 95% confidence interval of 0-52. It was safe, immunogenic and
capable of modifying the risk of clinical malaria among children exposed
to natural infection. However, it did not reduce the incidence of anaemia, a
major cause of morbidity in African children with malaria. This may be
because severe malarial anaemia is seen predominantly in much younger
children (aged 6 months to 2 years; Snow et al., 1994) than those included
in this trial. A vaccine trial focusing solely on this younger age group might
provide an explanation for this situation (see below). Although the trial was
not intended to establish the impact of vaccination on severe malaria or
death, it is of interest that of the six deaths recorded in the study only one
was a vaccinee.
The incidence of P. falciparum infections was similar in SPf66 and
control groups clearly indicating that the vaccine does not induce anti-
sporozoite immunity. An increase in antibodies to merozoites suggests that
the anti-SPf66 antibodies raised after immunization with the vaccine recog-
nize native epitopes of P. falciparum merozoites. The vaccine had an anti-
parasite effect in this trial in that reduced parasite densities were noted after
the third vaccine dose.
The long-term efficacy of the vaccine in children has yet to be estab-
lished. However, we do know, from an extended follow-up period of 18
months after the third vaccine dose, that the estimated vaccine efficacy for
all clinical episodes is 25% (95% CI = 1 4 4 % ; P = 0.044), suggesting that
vaccine-induced protection does not wane at least within this period of
follow-up (Alonso et al., 1996).
One important implication of the Tanzanian vaccine results is that
whatever the mechanism(s) mediating protection, they must be effective
against many strains since the best estimates of efficacy in both South
America and Africa are considerably higher than the prevalence of any one
strain of parasite (Babiker el al., 1994).
In contrast to the Tanzanian trials, SPf66 did not protect young Gam-
bian children against the overall incidence of infection or malaria attacks
nor first attacks of clinical malaria (D’Alessandro et al., 1995). Analysis
of vaccine efficacy performed on 547 children indicated that SPf66
42 C.A. FACER AND M. TANNER

vaccination was associated with a protective efficacy against the overall


incidence of clinical episodes of malaria of only 3% (95% CI = -24 to 24;
see Table 8). The reason why this vaccine trial failed to protect in the
Gambia is unknown, but may relate to the period of time of follow-up after
the last vaccine dose. In this respect, it is interesting to note that in the
Tanzanian trial a decrease in the incidence of clinical episodes of malaria
was only observed 3 months after the third vaccine dose - the approx-
imate length of the malaria transmission season in the Gambia. One
assumption from this could be that for the vaccine to be protective, a
period of > 3 months of natural exposure to and infection by malaria is
required. Despite the vaccine’s failure to protect against clinical episodes
of malaria (and a failure to prevent a fall in haematocrit), all vaccinated
children produced high titres of antibody to SPf66, unlike those children in
the control group (all features of the Tanzanian trial).
The outcome of the recently published vaccine trial in Thailand (Nosten
et al., 1996) is similar to that of the Gambian study (d’Alessandro et al.,
1995). SPf66 failed to protect 610 Karen children aged 2 to 15 years
(overall vaccine efficacy -9%; CI -33 to +14). These negative results
must now be considered in the context of previously reported efficacy trials
that indicated the potential of SPf66. Many reasons may account for this
variation ranging from: (i) different levels of endemicity (estimated ento-
mological inoculation rate of 0.3/year for Thailand and 300/year for
Tanzania); (ii) levels of pre-existing immunity (each Karen child experi-
ences only one P. falcipamm infection every 2-3 years); (iii) age of study
participants (two-thirds of the Karen vaccines were age 6 years or older); to
(iv) case definitions and their specificity. However, most explanations are
difficult to reconcile with the controlled Colombian studies showing partial
efficacy (La Tola: Valero er al., 1993; Rio Rosario: Valero et al., 1996)
where many conditions such as exposure (and consequently levels of back-
ground immunity) are comparable with the Thailand situation, except for
the broader range of the Colombian vaccinees. The Thai trial team con-
cluded that ‘. . . in context of the negative results of the Gambian trial and
the borderline efficacy reported in Tanzania, there appears to be little
justification for further trials of this vaccine’.
A meta-analysis of all double blindhandomized, placebo-controlled
(DB/RCT) trials to date (but excluding the results of the Thai trials which
were not available at the time) has been performed by the independent
Cochrane Review Group. This has shown that SPf66 reduces the risk of
first or only episodes by 27% (99% CI: 13-38%) and the total number of
malaria episodes by 38% (99% CI: 2549%). These conclusions therefore
reject the null hypothesis that the vaccine has no effect (the addition of the
Thai results will ‘pull down’ this efficacy but will not reverse the pattern of
efficacy).
CLINICAL TRIALS OF MALARIA VACCINES 43

Despite the Gambian and Thai trial results, the WHO is supporting
further trials in Tanzania. The second series of Tanzanian trials designed
to examine the effect of vaccinating children at a very young age, which
might provide better results, started in February 1996 and addresses two
essential questions. First, does the reduction in the risk of clinical malaria
as shown in the first trials translate into similar reductions against all
episodes of clinical malaria, particularly life-threatening malaria morbid-
ity? Second, as more than half of all malaria-related morbidity and mor-
tality in Kilombero takes place before the age of 1 year, can SPf66
vaccination be delivered following the existing EPI (extended programme
of immunization) schedules and schemes?
In a first step, a randomized, double-blind, placebo-controlled study of
the safety, immunogenicity, potential interactions with EPI vaccines and
efficacy against clinical episodes of malaria (phase YIII trial) is being
carried out among 1000 infants with the three doses of SPf66 (or placebo)
being administered at age 1, 2 and 7 months (Alonso and Tanner, 1995).
The study will have sufficient power to detect a 25% reduction in the
incidence of malaria episodes. Providing the results confirm the safety
and minimum estimated efficacy of 25%, then a further phase I11 trial (to
be completed in 1999) will be conducted among 6000 children under 2
years of age in the same region. This second series of trials is a con-
sequence of the initial trial in Tanzania (Alonso et d . , 1994) and will
conclude the debate on the real potential of SPf66 to reduce the risk of
clinical malaria in highly endemic areas.

3.7. Immune Responses to SPf66

SPf66 is immunogenic and stimulates the production of specific antibodies


(Alonso et al., 1994; Teuscher et aZ., 1994). Recent analyses of the humoral
immune response in relation to protection suggest that the SPf66-specific
antibodies as well as those against the individual peptides, can partly
explain the efficacy observed in the Tanzania trial (Alonso et al., 1996).
Cell-mediated immune responses and serum factors such as cytokines are
likely to be important. Vaccine-stimulated non-specific immunity may also
play a part, for example macrophages, y6 T cells and natural killer (NK)
cells, all of which have a role in immunity to malaria (Orago and Facer,
1991; Langhorne, 1996). Studies looking at changes in the number and
function of these cells and the cytokines that they produce in protected
vaccinees may provide some clues.
Another interpretation is that the vaccine, besides the antibody effect,
induces or upregulates immune responses that inhibit merozoite invasion of
red cells. Focusing on the amino acid sequence of P. fakiparum MSA-1,
44 C.A. FACER AND M. TANNER

known to be involved in invasion of red cells, a battery of small peptides


has been synthesized representing selected constant, variable and
dimorphic regions of the molecule (Table 9; M.E Patarroyo, personal
communication). These peptides have been tested for ability to bind to
red cells (using a sensitive competitive radiolabelled peptide binding assay)
and for antigenicity (using sera from both malaria exposed individuals and
from experimentally infected Aotus monkeys). An interesting picture
emerged. Those MSA-1 sequences that are known to vary between P.
falciparurn strains induced a good antibody response but failed to bind to
red cells (human and Aotus) and the opposite was true for the constant
regions. The highly conserved regions may not change because they are not
exposed to immune pressure and/or lack of change may relate to con-
straints in variation because of functional requirements of the molecule.
The same peptides that bound to red cells also blocked merozoite invasion
in v i m . Other synthetic P. fakiparum peptides derived from sequences of
the proteins GPB (glycophorin-binding protein) and EBA- 175 (erythro-
cyte-binding protein- 175), produced a similar picture; the constant regions
contained one or more red cell binding motifs. As one of these universal
motifs, Y . . . KEK (Tyr . . . Lys, Glu, Lys) is present on the 83.1 peptide
contained within SPf66 (see Figure 5), then this may explain the absence of
strain specificity that has emerged from the Tanzanian vaccine trial. It may
also explain the indication of protection from P.vivax in the Venezuelan
Las Majadas trial since KEK is a motif found in the Pv200 protein involved

Table 9 Red cell binding motifs identified on P fakiparum and P . vivax


merozoite antigens
Number per pro- Constant/ Detectable
Protein Motif" tein molecule variable antibodyb
P. fakiparum
MSA- 1 KEK 3 C NoC
LKKL (?) 4 C No
GBP VERRNA~ Tandem R variesb No
EBA- 175 KEW 4 C No

P. v i v a
Duffy K E W 2 C No
Pv200 LKKL 4 C No
KEK 1 C No

" One letter abbreviations for amino acids.


In human sera from malaria-exposed individuals.
But antibodies to the KEK motif present in the serum from SPf66 vaccinees.
CLINICAL TRIALS OF MALARIA VACCINES 45

in invasion in this species (Table 9). Interestingly, polyclonal sera from


SPf66-vaccinees recognize the KEK motif within the peptide 83.1 and
protection was associated with high anti-KEK titres (Molano et al.,
1992). It has been suggested that the dimorphic regions of MSA-1 may
be involved in red cell invasion of genetic variants of the red cell
membrane, for example the negroid S-s-U+ erythrocytes which lack
glycophorin B and show reduced susceptibilty to invasion in vitro by
P. falciparum merozoites (Facer, 1983).
Other motifs have been identified and a new construct containing these
motifs is presently being tested for vaccine potential in Aotus monkeys in
Colombia (M. Patarroyo, personal communication).
At present, given the partial efficacies reported from Latin America, the
marginal and partial efficacy in Tanzania, and the negative results from the
Gambia and Thailand, the vaccine remains a powerful stimulator of con-
troversy (Maurice, 1995). Issues at the centre of this controversy include
the vaccine’s performance in clinical trials and the origin of peptides 35.1
and 55.1. The fairly low efficacies resulting from these trials (for compar-
ison the use of insecticide-impregnated bed-nets in the Gambia provides
between 20 and 63% protection from clinical malaria) have questioned the
usefulness of the vaccine. SPf66 is now being developed further with a
rational design aimed at blocking both sporozoite invasion of liver cells
and merozoite re-invasion of red cells. This, together with the use of novel
adjuvants that are superior to alum, might lead to improved second-gen-
eration synthetic vaccines.
It remains to be seen whether the pressure of any vaccine will select
resistant parasites, a situation thought unlikely but possible. Indeed, there is
evidence from an experimental animal model that an apparently invariant
malaria asexual stage antigen can undergo vaccine-induced variation
(David et al., 1985; Klotz et al., 1987). To circumvent this important
possibility, future asexual vaccines may include the addition of antigens
from the sexual stages which will block the transmission of any vaccine-
resistant parasites that may arise.
Another issue that has just emerged is that vaccination with SPf66 can
reduce the multiplicity of P. falciparum infections in Tanzanian children
with asymptomatic malaria (Beck et al., 1996; see Section 3.3 and Table
5). Since high-multiplicity infections appear to protect against clinical
attacks of malaria, vaccination might reduce this effect; that is, reduce
the rate of accumulation of parasite genotype repertoire thereby possibly
effecting the state of semi-immunity - an interesting and important issue
requiring further investigation.
46 C.A. FACER AND M. TANNER

4. ASEXUAL STAGE ‘DISEASE-MODIFYING‘ VACCINES

‘Fever is probably caused by a little toxin which escapes from each parasite
. . .’ (Ross,1911). A hallmark of all malaria infections is fever which
occurs when schizonts rupture, an event recognized as early as 1889 by
Golgi (see Kean et al., 1978). There is now compelling evidence that much
of the pathophysiology of malaria, including fever, is mediated by proin-
flammatory cytokines (TNFa, IL- 1 and IL-6) released from macrophages,
monocytes and T cells activated by malaria ‘endotoxins’ originating from
ruptured schizonts (Figure 6).
TNF is a prime candidate for fever induction and this is supported by
experiments showing that injection of TNF alone can elicit many of the
clinical symptoms of acute malaria, such as hyperexia, hypotension, pul-
monary oedema and diffuse intravascular coagulation (Beutler and Cerami,
1988). Another feature of malaria caused by P. falciparum in humans and
linked to high TNF production, is cerebral malaria (CM, see below), a
relatively rare yet frequently fatal event occurring in about 1% of
infections (Marsh, 1992).
Any vaccine intervention which could reduce the morbidity (‘anti-toxic’
vaccine) and mortality (vaccine to prevent or reverse CM) would be a
useful adjunct to existing vaccines. Disease-modifying vaccines would
alleviate symptoms without imposing a lethal pressure on the parasite.
The target antigens may not be critical for parasite survival and would
ideally have the advantage of not being countered by parasite immune
evasion mechanisms. By reducing childhood mortality, disease-modifying
vaccines would extend life until naturally acquired anti-parasite immunity
develops at a later stage.

4.1. ‘Anti-toxic’ Vaccines

It has been recognized for many years that children living within malaria-
endemic regions develop what is known as ‘anti-toxic’ immunity, manifested
as a progressive reduction in disease severity, following repeated malarial
attacks and occurring several years before parasitaemias begin to fall.
Thus, it is common to find 5-10 year old African children who are
asymptomatic yet have levels of parasitaemia that would be associated
with fever and disease symptoms in a non-exposed malaria-naive indivi-
dual. As fever is density dependent, that is fever only occurs when para-
sitaemia reaches a certain level known as the fever threshold (Kitchen,
1949), it is obvious that these children must have acquired an altered
Figure 6 Erythrocytic cycle of P. falciparum showing expression and/or release of antigens proposed for inclusion into anti-
parasite and anti-disease vaccines.
48 C.A. FACER AND M. TANNER

(higher) threshold. Defining the mechanism behind this phenomenon would


pave the way towards the development of an ‘anti-toxic’ vaccine.
At schizogony, large amounts of parasitized red blood cell (PRBC)-
derived antigens (lipids, glycolipids, glycoproteins) are released into the
circulation over a short period of time (notable in P. falciparum infections
because of high parasitaemias). These include the malaria ‘endotoxins’
which act on host cells to induce secretion of a variety of factors including
cytokines responsible for clinical symptoms. Biochemical identification
and inclusion of these components into a vaccine might lead to their
immune-mediated removal so reducing morbidity (Playfair et al., 1990).
What is the nature of these malaria ‘endotoxins’ and how do they exert
pathophysiological effects? Several investigators have demonstrated that
malaria culture supernatants (P. yoelii and P. falciparum) and PRBC
lysates, contain substances thought to correspond to the malaria ‘endotox-
ins’ that induce fever. They are capable of inducing TNFa secretion from
macrophages both in vitro and in vivo (Taverne et al., 1990a; Playfair et
al., 1990). When these active supernatants are injected into mice, they
induce high titres of blocking antibody which cross-react between different
species of plasmodia and are measurable in vitro (by their inhibition of
TNF release by supernatants) and in vivo (by their inhibition of mortality in
mice pretreated with D-galactosamine) (Taverne et al., 1990b). This block-
ing antibody to parasite culture supernatants was predominantly IgM, was
short-lived and could not be boosted - all features suggestive of a T-
independent stimulating antigen. Mice vaccinated with malaria ‘toxins’
survived challenge although they developed an interesting picture of a
prolonged and high parasitaemia (a picture similar to that seen in asympto-
matic African children with high parasitaemias, see above) which finally
declined over a period of weeks (J.H.L. Playfair, personal communication).
Some progress has been made towards characterization of the chemical
nature of the triggering molecules although the exact structure has not yet
been determined. A number of different malaria antigens have been
reported to stimulate TNF production in vitro including RESA, MSA-1,
MSA-2 and the soluble antigen complex known as Ag7 (Kwiatkowski and
Bate, 1995). The malaria ‘endotoxin’ has high activity (as active as lipo-
polysaccharide), low abundance (approximately 10’’ parasites are required
to produce 1 yg of active material) and is a proteolipid comprising a
15 kDa protein, an ester-linked fatty acid group and incorporating phos-
phatidylinositol (PI) (D. Kwiatowski, personal communication). Antisera
raised in mice against commercially available PI coupled with a carrier,
contained high titres of IgG anti-PI which effectively inhibited malaria
‘toxin’-induced TNF release by macrophages (Bate et al., 1991). Further-
more, immunized mice did not develop hypoglycaemia as did control mice
when challenged with parasite toxic antigens (Bate et al., 1993). Interest-
CLINICAL TRIALS OF MALARIA VACCINES 49

ingly, markedly raised levels of anti-phospholipid antibodies (IgG and


IgM) are found in patients with falciparum and vivax malaria, a proportion
of which are specific for PI (Facer and Agiostratidou, 1994). It is possible
that these are produced in response to PI-containing malaria ‘endotoxins’
and are capable of complexing with these substances thereby inhibiting
TNF release. IgM to PI (but not other phospholipids) derived from the
serum of two Caucasian malaria patients was found to be capable of
inhibiting TNF induction by a variety of P. falciparum isolates (Bate and
Kwiatkowski, 1994).
A detailed biochemical analysis of two P. falciparum merozoite surface
antigens, MSA-1 and MSA-2 (see Table 7), indicates that the TNF-indu-
cing properties of the proteins relates to the glycosylphosphatidylinositol
(GPI) anchor that holds the proteins to the merozoite surface (Schofield and
Hackett, 1993) and is, presumably, released by the parasite in a soluble
form into culture supernantants. The purified GPI stimulates secretion of
high levels of TNF and IL-1 by macrophages and induces hypoglycaemia
through an insulin-mimetic pathway (Schofield et al., 1993) thus confirm-
ing the earlier observations by Bate and colleagues.
Similar ‘endotoxins’ from the other species of human malaria parasites
have not been described (essentially because continuous in vitro culture of
these, necessary as a source of antigens, has been unsuccessful). However
‘endotoxins’ are presumed to exist because of the classical paroxysms of
fever that follow infection. It is possible that P. v i v a , P. mulariae and P.
ovule produce ‘endotoxins’ that are quantitatively or qualitatively (more
potent) greater than that produced by P. falciparum since fever in malaria
caused by these species develops at a much lower parasitaemia (rarely
above 1-2% infected red cells). Should the chemical nature of these
‘endotoxins’ be identical or similar to that of P. falciparum, then a vaccine
strategy aimed at modifying disease may be effective in reducing morbidity
caused by all four species.
Implications that this may be the case have arisen from a study of
children, homozygous for a-thalassaemia ( - a k ) ,living on the remote
Pacific island of Vanuatu who rarely suffer from severe malaria even
though they are repeatedly infected with P. falciparum. A closer study
revealed that malaria attacks in children under the age of 2 years were most
frequently caused by P. v i v a whereas 5- and 6-year-old children were
experiencing P. falciparum infections but with little clinical disease. One
explanation for these observations is that there is cross-reactivity of anti-
toxic immunity between the two species of Plasmodium and that early
exposure to P. v i v a lessens severity of a later falciparum infection
(Williams et al., 1996).
Finally, the short-lived, T-independent nature of the natural immune
response to the PI-containing ‘endotoxins’ indicates that any anti- ‘toxic’
50 C.A. FACER AND M. TANNER

or disease-modifying vaccine would require a carrier and an adjuvant to


induce a memory response. Continued immunity would then be maintained
by the persistence of a low level of infection over a long period of time, a
situation known as ‘premunition’.Such premunition would, in theory, reduce
the risk of severe disease but not effectively protect against superinfection.
These features may explain asymptomatic P. fulcipurum infections in the
presence of high parasitaemias in school-age African children. The short-lived
nature of anti-toxic immunity might also explain the rapid loss of acquired
protection against the clinical malaria seen in African malaria immune adults
who are away from endemic regions for periods greater than 6 months.

4.2. A Vaccine to Prevent Cerebral Malaria?

By inducing TNF and other cytokines, malaria-derived ‘endotoxins’ may


also contribute to the pathogenesis of cerebral malaria (CM), one of the
most life-threatening and dramatic consequences of infection with P.
fulcipurum. It is not the object of this review to provide a detailed descrip-
tion of the clinical and molecular events involved in CM and the reader is
referred to excellent articles on this subject (Howard and Gilladoga, 1989;
Hommel, 1993; Berendt ef ul., 1994). Instead, we highlight here recent
research advances which have led to a greater understanding of the
mechanisms of CM, a result of which might enable design of an appro-
priate protective vaccine.
The primary event in the pathogenesis of CM is thought to be the
withdrawal of trophozoite- and schizont-infected erythrocytes from the
peripheral circulation and their subsequent cytoadherence to the capillary
endothelium (in the brain and other sites) by means of expression of
malaria antigens on the surface of the parasitized red cell and subsequent
specific ligand-receptor interactions (Figure 6). Known as sequestration, it
is unique to P. fakiparum and is thought to have evolved to enable the
parasite to avoid traversing the spleen thereby escaping the local immune
responses and removal of red cell inclusions (‘pitting’) that is a function of
that organ. All P. fulcipurum field isolates undergo this process of seques-
tration. Although this trick allows the parasite to escape splenic death, it
now becomes vulnerable to immune attack because of the surface-
expressed antigens. To mitigate this, P. fulcipurum uses antigenic variation
of these parasite-encoded molecules (see below).
A cascade of events is involved in cerebral sequestration and each step
may be modulated by a combination of host and parasite factors. As
mentioned earlier, CM is a relatively rare outcome of malaria infection.
What then determines whether an infected individual will develop CM?
The answer is likely to be multifactorial and various parameters appear to
CLINICAL TRIALS OF MALARIA VACCINES 51

-
CIDR domain of
300 400aa : some Hydrophobic
-
homology with EBA 175
transmembrane
segment

I
t
1-
Conserved 1 - 3 variable domains
I
II
head structure Conserved
acid terminal
segment
(anchor to rbc
cytoskeleton?)

Figure 7 Diagrammatic representation of the conserved and variable regions of


a molecule encoded by var genes. CIDR is a cysteine-rich interdomain region.
DBL-D1, D2,D3 and D4 are domains homologous to the P. v i v a and P. knowlesi
Duffy antigen-binding proteins. D B L - 2 4 undergo shuffling and deletion (after Su
et al., 1995).

be particularly important: (i) the capacity of the parasite population carried


by an individual at any point in time, to express erythrocyte-associated
antigens involved in cytoadherence to endothelial cells; (ii) the capacity of
the parasite to release ‘endotoxins’ resulting in excessive TNF production
by the host; (iii) linked to this, homozygosity in the host for the TNF2
allele, a variant allele associated with increased TNFa transcription; (iv)
genetically determined differences in host endothelial cell receptors for
cytoadherence and their susceptibilty to upregulation by cytokines; and (v)
host ‘premunition’ and immunity.
It is well established that growth of the parasite within the red cell is
accompanied by a significant number of alterations to the PRBC membrane
which include incorporation of exported malaria antigens. Thus far, at least
two components are known to be involved in adhesion to endothelial cells.
One constitutes electron-dense protrusions on the surface PRBC known as
‘knobs’ (Udeinya et al., 1981) the major constituent of which is the knob-
associated histidine-rich protein (KAHFW; Kilejian, 1979). The gene has
been cloned and mapped to chromosome 2 (the parasite has 14 chromo-
somes) (Pologe and Ravetch, 1986). Knobs are not essential for receptor-
specific recognition but are thought necessary for facilitating close contact
with the endothelial cell.
Various endothelial cell molecules are used as ligands for the cytoad-
herence of P. fakiparum PRBC including the membrane glycoprotein
52 C.A. FACER AND M. TANNER

CD36 (Ockenhouse and Chulay, 1988), the multifunctional glycoprotein


thrombospondin (Roberts et al., 1985), chrondroitin sulfate A (Fried and
Duffy, 1996) and molecules upregulated by cytokines (including TNF)
such as intercellular adhesion molecule 1 (ICAM-1; Berendt et al.,
1989), E-selectin and vascular adhesion molecule 1 (VCAM-1; Ocken-
house et al., 1992), and possibly P-selectin (CD62P; Facer and Theodor-
idou, 1994). Different P. falciparum isolates are thought to have variable
cytoadherent characteristics to these ligands (although all appear to share
binding to CD36 and thrombospondin) and the existence of diversity in
cytoadherence has led to the suggestion that parasite isolates with a high
cytoadherence phenotype might be associated with a greater frequency of
cerebral malaria. However, the final conclusion drawn from a number of
studies is that highly cytoadherent parasites do not induce severe disease
and clinical outcome cannot be predicted from this one parasite feature
(Marsh et al., 1988).
The prime candidate for a cytoadherent parasite ligand is the P. falci-
parum erythrocyte membrane protein 1 (PfEMP-1; see Figure 6 and Table
6), a trypsin-sensitive protein found on the surface of PRBC which is
highly polymorphic and exhibits extensive antigenic variation (Roberts et
al., 1992; Borst et al., 1995). These are all unwanted features for a vaccine
candidate, yet given that cytoadherence contributes to the pathogenesis of
CM, and that the incidence of CM in children in endemic countries declines
with increasing age, then specific natural immunity must develop to those
antigens involved. As a consequence PfEMP-1 is being closely researched
for possible inclusion into a vaccine.
PfEMP-1 (200-350 kDa) is an antigenically diverse protein which can
be immunoprecipitated and PRBC agglutinated, by human polyclonal
malaria immune sera. Its mechanism of anchorage in the red cell membrane
is unknown, although its extraction in sodium dodecyl sulfate (SDS) but
not Triton X-100, suggests linkage to the erythrocyte cytoskeleton (Aley
et al., 1984).
The virulence of P. falciparum has been attributed to its capacity to
express variant forms of PfEMP-1 which can arise with an extraordinarily
high frequency of 2.4% per generation. Both antigenic variation and bind-
ing to the endothelium are functions of the same molecule (PfEMP-1) since
switches in antigenic type are accompanied by a change in PfEMP-1
molecular mass and loss of ability to bind to ICAM-1 (Roberts e f al.,
1992). However, up to very recently, there was no proof that the variant
antigen genes encoded those parasite ligands responsible for cytoadher-
ence, and attempts at cloning the gene for PfEMP-1 proved unsuccessful.
This major barrier to the understanding of molecular mechanisms behind
sequestration and the development of a malaria vaccine to prevent CM may
now be behind us. During the processing and mapping of a 300 kb segment
CLINICAL TRIALS OF MALARIA VACCINES 53

on chromosome 7 linked to chloroquine resistance in a P. falciparum cross,


a large family of genes, named var genes, has been identified (Su et al.,
1995). These var genes encode PfEMP-1 proteins and Southern blot ana-
lysis indicates that they may be present on most or all of P. falciparum
chromosomes, although their expression in situ remains unknown. Alto-
gether there are between 50 and 150 var genes corresponding to 6% of the
malaria genome.
A characteristic of var genes is that they contain multiple domains
homologous to region I1 of the erythrocyte-binding proteins of P. v i v a
(Duffy binding protein; DAPB) and P. falciparum (EBA-175) even though
these two species bind to different red cell receptors (Duffy blood group
antigen and glycophorin-A, respectively). Although the deduced protein
sequences of the var genes are highly diverse, all contain common struc-
tural features and conserved motifs (see Figure 7). The two to four domains
known as Duffy-binding-like (DBL) cysteine-rich domains are capable of
mediating binding to the various endothelial cell receptors (CD36, ICAM-
1, etc.) in a manner that may relate to the number of domains expressed
(Smith et al., 1995), and some (DBL-1 and DBL-3) are known to interact
with uninfected red cells during rosetting (see below; C. Newbold, personal
communication).
What evidence is there to show that var genes do encode proteins on the
PRBC surface? Baruch and colleagues (1995) have just cloned a gene from
the Malayan Camp strain of P. falciparum by screening an expression
library with antibody raised to the PRBC surface. Sequencing showed
that this gene was indeed a member of the var gene family and antibody
to the cloned gene product immunoprecipitated PfEMP- 1. Interestingly, the
antisera reacted over the knobs which, as mentioned earlier, are the points
of contact with the endothelium.
The central role of PEMP-1 in the pathogenesis of CM has led to the
suggestion that it be included in a malaria vaccine. To make this a viable
proposition, an antigenically conserved portion of the molecule (for exam-
ple the head domain seen in Figure 7) must be identified that would elicit
antibodies capable of blocking adherence of all P. falciparum variants
(there may be millions of PEMP-1 genes in the world’s P. falciparum
population) to endothelial cells.
The phenomenon of rosetting, whereby normal red cells bind to PRBC
containing trophozoites and schizonts via a 22-28 kDa parasite molecule
called Rosettin (see Table 6; Wahlgren et al., 1989; Scholander ef al.,
1996), is the only adherence phenotype that has been associated with
severe disease (CM) in some studies. It is possible that inclusion of the
molecule into a vaccine might elicit antibodies that would prevent rosetting
in vivo and reduce vaso-occlusive events that are thought to result from this
phenomenon.
54 C.A. FACER AND M. TANNER

CM might also be determined, in part, by a genetic variability within the


infecting P. fakiparum population to induce cytokine production by the
host. Children with CM have significantly higher plasma levels of TNFa
than those with mild malaria even though the levels of parasitaemia are
similar (Kwiatkowski et al., 1990). However, anti-TNF monoclonal anti-
bodies, whilst reducing fever, do not prevent or reverse CM (Kwiatkowski
et al., 1993). Nevertheless, in support of the TNF theory are recent data
showing variation between P . fakiparum isolates in their ability to induce
TNF (Allan et al., 1993). Whether this relates to a quantitative or qualita-
tive variation in the TNF-inducing ‘endotoxin’ remains to be seen.
A major clinical study of Gambian children with malaria has recently
provided one explanation as to why cerebral malaria occurs in only a small
proportion of individuals infected with P. fakiparum and points to a
genetic polymorphism relating to TNFa (McGuire et al., 1994). TNF
responsiveness is controlled by variable genetic elements within the
MHC and in humans there are two allelic forms of the TNF gene referred
to as TNFl and TNF2. The TNF2 allele is associated with higher con-
stitutive and inducible levels of TNF than the TNFl allele. Gambian
children homozygous for the TNF2 allele were found to have a relative
risk, RR, of 4.0 for cerebral malaria and 7.7 for death or neurological
sequelae due to cerebral malaria and also had higher serum levels of TNFa
than did children with mild malaria. The percentage of children found to be
homozygous for the TNF2 allele was 4.5% in all cerebral malaria cases and
this increased to 8.1% in children who had died or who had sequelae,
compared to 1.2% in mild malaria controls. The disease association was
independent of HLA class I and class I1 variation. The maintenance of the
TNF2 allele at a gene frequency of 0.16 implies that in this population the
increased risk of cerebral malaria in homozygotes is counterbalanced by
some biological advantage, perhaps relating to the general anti-microbial
activity of TNFa. Another allele, TNF-A, associated with high TNF pro-
duction (and negatively associated with the TNF-2 allele), has recently
been found in a high percentage of Kenyan and Gambian children with
severe malarial anaemia (RR 2.9 in Kenyans) and with cerebral malaria
(RR 1.9 in Gambians; E.E. Coleman, personal communication).
In conclusion, whilst there remain many gaps in our knowledge of the
processes that lead to severe malaria, both host and parasite components,
nevertheless the idea of a disease-modifying vaccine coupled with an anti-
parasite vaccine is a novel and exciting one. Most antigens currently on
trial, both pre-erythrocytic and blood stage, display a degree of MHC-
related variabilty in immunogenicity and antigenic polymorphism. An
anti-‘toxic’ vaccine would not be subject to these constraints and could
perform more consistently. However, this would not apply to a vaccine
against the highly polymorphic PfEMP1, although prevention of upregula-
CLINICAL TRIALS OF MALARIA VACCINES 55

tion of the respective endothelial ligands with an anti- ‘toxic’ vaccine might
modify the degree or site of sequestration.

5. MULTI-STAGE AND MULTI-ANTIGEN DNA VACCINES

Promising results have emerged from recent attempts to vaccinate experi-


mental animals with plasmodia1 DNA (Hedstrom et al., 1994). Indeed the
results are so encouraging that injection of DNA looks to be the way
forward for the future of malaria vaccines.
As a first step in the production of a multi-stage and multi-antigen
vaccine, Hoffman and colleagues have constructed a plasmid DNA vaccine
that includes the gene encoding the P. yoelii CS protein (PyCSP; Hoffman
et al., 1995). Its efficacy was demonstrated by the fact that vaccinated mice
produced higher levels of antibody and cytotoxic T lymphocytes against
the construct than did mice immunized with irradiated sporozoites. Three
doses of only 2.5 pg of PyCSP plasmid DNA provided protection which
was dependent on the presence of CD8+ lymphocytes (deletion of the latter
abrogated protection). The degree of protection was found to vary (19-
75%) between mouse strains indicating considerable genetic restriction in
immune responses to PyCSP. The inclusion of other genes in the plasmid
DNA increased the protection (S. Hoffman, personal communication).
HLA diversity in humans is expected to cause similar variations in
immune responsiveness as different peptides derived from the malaria-
encoding plasmids bind differently to HLAs expressed on the recipient’s
cells. Just how many plasmids may eventually be required to confer
complete protection is not known, although it is thought that if eight
HLA types are targeted, then 95% of the population should be covered
(Fricker, 1996).
A multi-stage P. fakiparum DNA vaccine, NYVAC-Pfl, has just been
developed and is in the early stages of phase 0 trials (S. Hoffman, personal
communication). This new vaccine candidate employs a live attenuated
vaccinia vector ( 107-108) which contains the genes for seven different
malaria antigens: CS protein, SSP-2 (TRAP), LSA-1, MSA-1, AMA-1,
SERA and Pfs25 (a gamete antigen only exposed in the mosquito gut).
This selection of antigens is based on proteins expressed on the infected
hepatocyte and co-expressed on blood stages (lymphocytes from humans
vaccinated with irradiated sporozoites proliferated in vitro on addition of
the blood stage antigens MSP-1 and MSP-2).
Rabbits immunized with NYVAC-Pfl reacted by producing antibodies
to all seven encoded antigens indicating that, at least in this model, all
genes were being expressed in vivo. Subsequent immunization in rhesus
56 C.A. FACER AND M.TANNER

monkeys have confirmed this observation. Phase I trials at the US Walter


Reed Army Institute for Medical Research took place in 1995 and these are to
be followed by phase IIa and IIb trials in October 1997 (Fricker, 1996). The
US FDA foresees no objection to this first DNA malaria vaccine trial in
human volunteers. Nevertheless, before trials proceed in humans, presumably
the riskhenefit ratio will need to be assessed. Additionally, there is no
published evidence that the correct malaria antigen sequence has been gen-
erated in the transfected host cells, that it can be stably maintained or that
plasmids are retained in circular form. The most effective and safe delivery
system and consistency in the delivery are important future considerations.
Concurrent to the production of NYVAC-Pfl is the development of a
multivalent DNA vaccine incorporating P. falciparum genes expressed
predominantly in the liver stages: CS protein, SSP-2 (TRAP), Exp-1,
STARP, LSA-1 and SALSA. Phase 0 trials are now underway (S. Hoff-
man, personal communication).
The DNA vaccines in addition to their superior immunogenicity and
associated protection also provide a further benefit in that they assist in the
identification of protective malaria epitopes which have not yet been
identified by other methods, and allow the fine dissection of the humoral
and cellular responses to a particular antigen.

6. THE FUTURE

There has been a remarkable increase over the past decade in our under-
standing and knowledge of the malaria parasite and the disease that it
causes. Never before has one been in the position to appreciate what a
sophisticated and complex organism we are attempting to defeat through
vaccine development. Nevertheless, we believe that pre-erythrocytic and
asexual blood stage vaccines are now a reality and, given their further
development in order to overcome the remarkable plasticity and diversity
of this parasite, they should assure a high efficacy. However, this will
require the continued funding by the relevant agencies and the determina-
tion and innovation of researchers given this support to produce a vaccine
that will, finally, be universally licensed to effectively control malaria at a
time when it has never been needed more.

ACKNOWLEDGEMENTS

We thank Dr Gerd Pluschke and Dr Tom Smith for reading the manuscript
and for their helpful suggestions.
CLINICAL TRIALS OF MALARIA VACCINES 57

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Phylogeny of the Tissue
Cyst-forming Coccidia

Astrid M. Tenter’ and Alan M. Johnson’

1
Institut fur Parasifologie. Tierarztliche Hochschule Hannover.
Bunteweg 17. 30559 Hannover. Germany and
2Molecular Parasitology Unit. Faculty of Science.
University of Technology. Sydney. GPO Box 123. Broadway.
New South Wales 2007. Australia

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2. Taxonomy. Nomenclature and Currently Used Classifications . . . . . . . . . . . . . . 71
2.1. The family Sarcocystidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.2. The problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3. Traditional Characters Used for the Classification of Tissue
Cyst-forming Coccidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.1. Morphology and ultrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3.2. Life cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3.3. Inference of phylogenetic relationships from phenotypic characters . . . . . 94
3.4. Conflicting hypotheses based on phenotypic characters . . . . . . . . . . . . . . .98
4. The Molecular Answer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.1. Characteristics of SSU rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
4.2. Nucleotide sequence determination . . . . . . . ...................... 102
4.3. Sequence alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.4. Outgroup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.5. Tree-building methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.6. Comparison of different phylogenetic trees inferred from SSU rRNA
sequencedata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5. Phylogenetic Relationships and Genetic Relatedness of Tissue Cyst-forming
Coccidia Inferred from SSU rRNA Sequence Comparisons . . . . . . . . . . . . . . . 112
5.1. Phylogenetic relationships of tissue cyst-forming coccidia
to homoxenous coccidia and other apicomplexan protozoa . . . . . . . . . . . 113
5.2. Phylogenetic relationships of tissue cyst-forming coccidia to each other . 114
5.3. Genetic divergence among tissue cyst-forming coccidia . . . . . . . . . . . . . .117

ADVANCES IN PARASITOLOGY VOL 39 Copyright 0 1997 Academic Press Limited


ISBN &12431739-7 A / / rights of reproduction in any form reserved
70 A.M. TENTER AND A.M. JOHNSON

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

1. INTRODUCTION

The phylum Apicomplexa Levine, 1970 is composed of a group of very


diverse protozoa, all of which are parasitic. Organisms placed into this
phylum are characterized by the presence of a unique apical complex at the
anterior end of the invasive stages in their life cycle (Levine, 1970, 1985;
Levine et al., 1980). The apical complex consists of special organelles that
facilitate the entry of the parasite into its host cell (Sleigh, 1991). However,
to a large extent the phylum Apicomplexa, as well as the taxonomic
groupings within the phylum, have always been regarded as ‘matters of
convenience’ and ‘workable compromise(s)’, erected to contain organisms
that did not obviously belong to more valid taxa (Cox, 1981; Levine, 1988).
Therefore, it is perhaps not surprising that several taxonomic groupings
within this phylum have been the subject of much investigation. One such
group of organisms are the tissue cyst-forming coccidia.
Tissue cyst-forming coccidia are obligately intracellular protozoa with a
typical coccidian life cycle consisting of three alternating phases; that is,
asexual multiplication by merogony (schizogony), sexual reproduction by
gamogony and asexual reproduction by sporogony. Most species of tissue
cyst-forming coccidia are obligately or facultatively heteroxenous; that is,
both an intermediate and a definitive host need to be present for the life
cycle of the parasite to be completed. Usually, the asexual phase of the life
cycle (merogony) leads to the formation of tissue cysts in various tissues of
the intermediate host, while the sexual phase of the life cycle (gamogony)
leads to the formation of oocysts in the intestine of the definitive host.
Comprehensive reviews of the life cycles, biology, morphology, biochem-
istry, immunology, molecular biology, pathogenicity and epidemiology of
several tissue cyst-forming coccidia, in particular those of medical or
veterinary importance, have been published by Dubey and Beattie
(1988), Dubey et al. (1989), Jackson and Hutchison (1989), Luft (1989),
Rommel (1989), Cawthorn and Speer (1990), Current et al. (1990), Dubey
(1990, 1992, 1993), Gothe and Reichler (1990), Johnson (1990), Reming-
ton and Desmonts (1990), Uggla and Buxton (1990), Ho-Yen and Joss
(1992), Dubey and Lindsay (1993), Lindsay et al. (1993), Ruehlmann et al.
(1995) and Tenter (1995).
However, the classification, taxonomy, and nomenclature of tissue cyst-
forming coccidia have been the subject of discussion and controversy for
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 71

many years and are still uncertain today. Although the tissue cyst or oocyst
stages of several tissue cyst-forming coccidia have been known for more
than a century (Miescher, 1843; Kuhn, 1865; Stiles, 1891), it was not until
only two decades ago that the complete heteroxenous life cycles of some of
these parasites were elucidated (Hutchison et al., 1969; Rommel et al.,
1972; Frenkel and Dubey, 1975; Rommel and Krampitz, 1975; Wallace
and Frenkel, 1975). Thus far, the classification of tissue cyst-forming
coccidia has been based on phenotypic characters, such as morphology
and life cycle. However, these characters provide only limited phylogenetic
information, because morphological features are often subject to change
during the developmental cycle of the parasite and the complete life cycles
are not known for all species (reviewed by Rommel, 1989; Dubey, 1993;
Tenter, 1995). Therefore, we still know very little about the phylogeny and
genetic relatedness of different species of tissue cyst-forming coccidia,
both to each other and to members of other taxa in the phylum Apicom-
plexa (see Tadros and Laarman, 1982; Vivier, 1982; Kreier and Baker,
1987; Barta, 1989; Frenkel, 1989; Cox, 1991, 1994; Tenter, 1995).
This review gives an overview of currently used classifications of tissue
cyst-forming coccidia and of recent phylogenetic studies based on mole-
cular data that have provided new insights into the phylogeny and genetic
relatedness of some of these parasites. In particular, the genetic relation-
ships of species placed into the two oldest genera of tissue cyst-forming
coccidia (i.e. Sarcocystis Lankester, 1882 and Toxoplasma Nicolle and
Manceaux, 1909) are discussed.

2. TAXONOMY, NOMENCLATURE AND CURRENTLY USED


CLASSIFICATIONS

The coccidia currently comprise about 2300 named species (reviewed by


Levine, 1988), and thus form the largest group of protozoa in the phylum
Apicomplexa (Table 1). They are obligately intracellular protozoa whose
life cycles characteristically consist of both asexual and sexual phases of
reproduction (Levine, 1985; Cox, 1994). In modern classifications of api-
complexan protozoa (Levine et al., 1980; Hiepe and Jungmann, 1983;
Levine, 1985, 1988; Kreier and Baker, 1987), the coccidia are placed in
this phylum as a subclass, Coccidia Leuckart, 1879, and are further divided
into lower taxa based on traditional characters, such as morphology, the
pattern of their life cycles and the location of their developmental stages
(Table 1).
Several coccidia cause disease and even death in animals and humans
(reviewed by Long, 1990; Kreier, 1993). The taxonomic position of
72 A.M. TENTER AND A.M. JOHNSON

Table 1 Taxonomy of tissue cyst-forming coccidia and some related taxa of


medical or veterinary importance, modified from Levine (1988) and Current et al.
( 1990)

Phylum Apicomplexa Levine, 1970


Apical complex present at some stage, usually consisting of polar ring(s), rhoptries,
micronemes, conoid and subpellicular microtubules; micropore(s) generally present
at some stage; cilia absent; sexuality, when present, by syngamy; all species
parasitic; three classes; about 4600 named species.
Class Sporozoea Leuckart, 1879
Conoid usually present, forming a complete cone; reproduction usually both asexual
and sexual; oocysts with sporozoites, the infective stage resulting from sporogony;
locomotion by gliding, undulation, flexion or flagella; flagella, when present, only on
microgametes; pseudopodia usually absent, when present used for feeding, not
locomotion; homoxenous or heteroxenous; two subclasses; about 4000 named
species.
Subclass Coccidia Leuckart, 1879
Gamonts usually present, normally small and intracellular; conoid not modified into
mucron or epimerite; syzygy usually absent, if present involves gametes; gametes
usually anisogametes; life cycles normally consist of merogony, gamogony and
sporogony; most species parasites of vertebrates; four orders; about 2300 named
species.
Order Eucoccidiida LCger and Duboscq, 1910
Merogony, gamogony and sporogony present; in vertebrates or invertebrates; two
suborders; about 2300 named species.
Suborder Eimeriina LCger, 1911
Macrogamete and microgamete develop independently; syzygy generally absent;
microgamont usually produces numerous microgametes; zygote usually not motile;
sporozoites normally enclosed in a sporocyst; endodyogeny present or absent;
homoxenous or heteroxenous; 14 families; about 1800 named species.
Family Cryptosporidiidae Tyzzer, 1907
Oocysts small, with no sporocyst and four naked sporozoites; development just under
host cell membrane so that the parasite projects into the intestinal lumen;
developmental stages form a feeder organelle or attachment organelle that anchors
the parasite to the base of the parasitophorous vacuole; microgametes without
flagella; developmental stages of some species lacking mitochondria; sporogony
endogenous; homoxenous; in vertebrates; one genus; about five to six species.
Genus Cryptosporidium Tyzzer, 1907
With characteristics of the family; more than 20 named species, only about six of
which are valid; type species Cryptosporidium muris Tyzzer, 1907.
Family Eimeriidae Minchin, 1903
Oocysts with zero, one, two, four or more sporocysts, each with one or more
sporozoites; development in host cell proper; microgametes with two or three
flagella; merogony and gamogony usually within the same host; sporogony typically
exogenous; homoxenous or facultatively heteroxenous; in vertebrates or
invertebrates; 17 genera; about 1550 species.
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 73
Table 1 Continued
Genus Eimeria Schneider, 1875
Oocysts with four sporocysts, each with two sporozoites; merogony intracellular;
sporogony extracellular; a few species have extraintestinal developmental stages;
homoxenous; in vertebrates, rarely invertebrates; about 1200 named species; type
species Eimeria fulciformis (Eimer, 1870) Schneider, 1875.
Genus Zsospora Schneider, 1881
Oocysts with two sporocysts, each with four sporozoites; some species may have
sporozoites andor merozoites that may become dormant intestinally or
extraintestinally and possess the capability of later producing patent infections;
homoxenous, although some species (Cystoisospora Frenkel, 1977) may have an
additional intermediate host in which dormant sporozoites can transmit the infection
to the definitive carnivore host; sporogony endogenous or exogenous; in vertebrates,
rarely in invertebrates; about 250 named species; type species Isospora rura
Schneider, 1881.
Family Sarcocystidae Poche, 1913
Heteroxenous; oocysts containing two sporocysts, each with four sporozoites;
development in host cell proper; sexual stages in intestine of definitive host; asexual
stages in various tissues of intermediate host; two subfamilies; about 150 named
species.
Subfamily Sarcocystinae Poche, 1913
Obligately heteroxenous; asexual multiplication in intermediate host (a prey animal);
first generation meronts in vascular endothelium; last generation meronts (tissue
cysts) in striated muscle or in central nervous system contain metrocytes that form
cystozoites; sexual reproduction and sporogony in intestine of definitive host (a
predator); two or three genera; about 130 named species.
Genus Sarcocystis Lankester, 1882
Last generation meronts typically in striated muscle, some in neural tissue;
merozoites elongate, more than 120 named species; type species Surcocysris
miescheriana (Kuhn, J. 1865) Labbt, 1899.
Genus Frenkelia Biocca, 1968
Last generation meronts typically in central nervous system; merozoites elongate;
definitive hosts raptors; intermediate hosts rodents; two named species; type species
Frenkelia microti (Findlay and Middleton, 1934) Biocca, 1968.
Subfamily Toxoplasmatinae Biocca, 1957
Complete life cycle obligately heteroxenous, but asexual stages usually transmissible
from one intermediate host to another; metrocytes not formed; sporogony
exogenous; four genera; about 20 named species.
Genus Toxoplasma Nicolle and Manceaux, 1909
Meronts in numerous cell types; host cell nucleus outside meront wall; definitive
hosts felids; intermediate hosts consist of numerous species of vertebrates; one
known valid species but about five additional species are named based on tissue cysts
found in intermediate hosts (several may prove to represent non-Toxoplasma
species); type species Toxoplasma gondii (Nicolle and Manceaux, 1908) Nicolle and
Manceaux, 1909.
74 A.M. TENTER AND A.M. JOHNSON

Table I Continued
Genus Besnoitia Henry, 1913
Meronts in fibroblasts and other cells; host cell nucleus within meront wall; tissue
cyst wall thick; definitive hosts mammals; intermediate hosts mammals or reptiles;
about seven named species; type species Besnoitia besnoiti (Marotel, 1913) Henry,
1913.
Genus Hammondia Frenkel and Dubey, 19775
Meronts in numerous cell types; oocysts not infectious to definitive host; definitive
hosts felids or canids; intermediate hosts mammals; about three named species; type
species Hammondia hammondi Frenkel and Dubey, 1975.
Genus Neospora Dubey, Carpenter, Speer, Topper and Uggla, 1988
Oocysts and intestinal stages unknown; merozoites divide by endodyogeny in
numerous tissues, especially brain and spinal cord, usually without formation of
parasitophorous vacuole; tissue cysts thick-walled, found generally in the central
nervous system; in canids; one named species; type species Neospora caninum
Dubey, Carpenter, Speer, Topper and Uggla, 1988.

coccidia of medical and veterinary importance is consistent in several


recent classifications down to the level of the suborder Eimeriina LCger,
1911 (Table 2; EuzCby, 1987; Kreier and Baker, 1987; Levine, 1988;
Current et al., 1990; Eckert et al., 1992). This suborder comprises both
homoxenous and heteroxenous members with a typical coccidian life cycle
consisting of merogony, gamogony and sporogony but, unlike other cocci-
dia, the sexual development of the Eimeriina is characterized by the
independent development of male and female gametes, a zygote that is
not motile and, typically, the enclosure of sporozoites in sporocysts that
form within oocysts (see Table 1; Levine et al., 1980; Hiepe and Jung-
mann, 1983; Levine, 1985, 1988; Kreier and Baker, 1987). However, there
is controversy on the further division of the Eimeriina into families, sub-
families and even genera (see Table 2; Hiepe and Jungmann, 1983; EuzCby,
1987; Kreier and Baker, 1987; Levine, 1988; Current et al., 1990; Eckert et
al., 1992).

2.1. The Family Sarcocystidae

Historically, all eimeriine coccidia that produce sporulated oocysts con-


taining two sporocysts, each with four sporozoites, were placed into the
genus Zsosporu Schneider, 1881 which is classified as a member of the
family Eimeriidae Minchin, 1903 in most recent classifications of coccidia
(see Table 1; Hiepe and Jungmann, 1983; Levine, 1985, 1988; Kreier and
Baker, 1987; Current et al., 1990; Eckert et al., 1992; Mehlhorn and
Ruthmann, 1992; Lindsay and Todd, 1993). After the discovery of hetero-
xenous life cycles of some former members of the genus Zsospora in the
Table 2 Some examples of the classification of tissue cyst-forming coccidia since the elucidation of their life cycles in the 1970s.
Levine Frenkel Frank Tadros and Laarman Dubey
(1973) (1974) (1976) ( 1976) (1977b)
Phylum Protozoa -C
Protozoa Protozoa Protozoa
Subphylum Apicomplexa - * Apicomplexa Apicomplexa
Class Sporozoasida - Apicomplexa Sporozoasida Sporozoasida
Subclass Coccidiasina - Sporozoea * *
Superorder *b - Coccidiomorpha * *
Order Eucoccidiorida - Toxoplasmida Eucoccidiorida Eucoccidiorida
Subordef Eimeriorina (8) Eimeriorina (-) * Eimeriorina (-) Eimeriorina (-)
Family Sarcocystidae Toxoplasmatidae Toxoplasmatidae Eimeriidae Undecided
Subfamily Toxoplasmatinae Toxoplasma Toxoplasma Eimeriinae Toxoplasma
Genus Frenkelia Besnoitia Besnoitia [EimeriaId Hammondia
Toxoplasma Isospora Sarcocystis
Besnoitiinae Sarcocystidae Sarcocystidae Endorimosponnae Frenkelia
Besnoitia Sarcocystis Sarcocystis Endorimospora [Isospora]
Sarcocystinae Hammondia Frenkelia [Levineia]
Sarcocystis Arthrocystis
A rthrocystis Frenkelia

Frenkel Levine Overdulve Frenkel et al. Hiepe and Jungmann


(1977) (1977a) (1978) (1979) (1983)
Phylum - Apicomplexa
Subphylum - *
Class - Sporozoea
Subclass - Coccidia
Superorder - *
Order - Eucoccidiida
Suborder - Eimeriina (-)
Table 2 Continued
Frenkel Levine Overdulve Frenkel et al. Hiepe and Jungmann
(1977) (1977a) (1978) (1979) (1983)
Family Sarcocystidae Eimeriidae Eimeriidae Sarcocystidae or Sarcocystidae
Subfamily Sarcocystinae [Isospora] Isospora Eimeriidae Sarcocystinae
Genus Sarcocystis Toxoplasma Isospora Sarcocystinae Sarcocystis
(Isospora)
Frenkelia Besnoitia Isospora Sarcocystis Frenkelia
(Toxoplasma)
Toxoplasmatinae Sarcocystis Isospora Frenkelia Toxoplasmatinae
(Besnoitia)
Toxoplasma Frenkelia Sarcocystis Toxoplasmatinae Toxoplasma
Besnoitia Toxoplasma Hammondia
Hammondia Besnoitia Besnoitia
[Cystoisospora] Hammondia Cy stoisosporinae
Cy stoisosporinae [Cystoisospora]
[Cystoisospora]
Levine EuzCby Frenkel et al. Kreier and Baker Levine
(1985) (1987) (1987) (1987) (1988)
Phylum Apicomplexa Apicomplexa Apicomplexa Apicomplexa
Subphylum * * * *
Class Sporozoasida Sporozoasida Sporozoea Conoidasida
Subclass Coccidiasina Coccidiasina Coccidia Coccidiasina
Superorder * * * *
Order Eucoccidiorida Eucoccidiorida Eucoccidiida Eucoccidiorida
Suborder Eimeriorina (10) Eimeriorina (7) Eimeriina (-) Eimeriorina (14)
Family Sarcocystidae Isosporidae Sarcocystidae Eimeriidae Sarcocystidae
Subfamily Sarcocystinae Isosporinae Cystoisosporinae Cyclosporinae Sarcocystinae
Genus Sarcocystis [Isospora] [Cystoisospora] [Cyclospora] Sarcocystis
Frenkelia [Cystoisospora] Toxoplasmatinae [Zsospora] Frenkelia
Arthrocystis Toxoplasmatinae Toxoplasma Toxoplasma Arthrocystis
Toxoplasmatinae Toxoplasma Harnrnondia Sarcocystis Toxoplasmatinae
Toxoplasma Harnrnondia Besnoitia Besnoitia Toxoplasrna
Besnoitia Besnoitia Sarcocystinae [Dorisiella] Besnoitia
Sarcocystinae Sarcocystis
Sarcocystis Frenkelia
Frenkelia
Current et al. Eckert et al. Mehlhorn and Ruthmann Dubey Lindsay and Todd
( 1990) (1992) ( 1992) (1993) ( 1993)

Phylum Apicomplexa Apicomplexa Sporozoa Apicomplexa Apicomplexa


Subphylum * * * * *
Class Sporozoasida Sporozoea Sporozoea Sporozoasida Sporozoasida
Subclass Coccidiasina Coccidia Coccidia Coccidiasina Coccidiasina
Superorder * * Eucoccidea * *
Order Eucoccidiorida Eucoccidiida * Eimeriorina Eucoccidiorida
Suborder Eimeriorina ( 13) Eimeriina (-) * * Eimeriorina (-)
Family Sarcocystidae Toxoplasmatidae Eimeriidae Undecided Sarcocystidae
Subfamily Sarcocystinae Toxoplasma [Eirneria] Toxoplasma Sarcocystinae
Genus Frenkelia Neospora [Zsospora] Neospora Sarcocystis
Sarcocystis Harnmondia Toxoplasma Harnrnondia Frenkelia
Toxoplasmatinae Besnoitia Sarcocystis Sarcocystis Toxoplasmatinae
Besnoitia [Cryptosporidiurn] Frenkelia Toxoplasma
Harnrnondia Sarcocystidae [Aggregatal Besnoitia Besnoitia
Neospora Sarcocystis Harnrnondia
Toxoplasma Frenkelia

Numbers in parentheses show the number of families in the suborder Eimeriina.


’Taxonomic level not used in the classification.
No data given by the author(s).
Sister genera not encompassing tissue cyst-forming coccidia are shown in brackets.
78 A.M. TENTER AND A.M. JOHNSON

1970s, the family Sarcocystidae Poche, 1913 was redefined to comprise


two to three subfamilies and five to eight different genera whose life cycles
characteristically alternate between an intermediate host, defined as the
host of the asexual stages, and a definitive host, defined as the host of the
sexual stages (see Section 3.2; Frenkel, 1977; Hiepe and Jungmann, 1983;
Levine, 1985, 1988; Frenkel et al., 1987; Current et al., 1990). In the
intermediate host, which is usually a herbivore or omnivore, one or several
proliferative cycles of merogony (endodyogeny or endopolygeny) occur in
various tissues, with the last cycle leading to the formation of tissue cysts.
In the definitive host, which is usually a carnivore, the sexual phase of
development (gamogony) leads to the formation of oocysts in the intestine.
Gamogony may be preceded by a propagative phase of merogony in the
definitive host. Sporogony occurs inside or outside the host and leads to the
development of isosporan-type oocysts; that is, those containing two sporo-
cysts, each with four sporozoites (see Table 1; Figure 1; Smith, 1981;
Hiepe and Jungmann, 1983; Levine, 1985; Frenkel et al., 1987; Rommel,
1989; Current et al., 1990; Dubey, 1993).

2.2. The Problem

While there is now general agreement in placing all tissue cyst-forming


coccidia into the suborder Eimeriina, their classification into the family
Sarcocystidae has not been generally accepted (see Table 2). By contrast,
the validity of the family Sarcocystidae as well as the validity of genera
and species placed in this family have been subject to controversy and
debate ever since they were introduced as coccidian taxa (Levine, 1973,
1977a, b, 1988; Frenkel, 1974, 1977; Tadros and Laarman, 1976, 1982;
Dubey, 1977a, b; Overdulve, 1978; Rommel, 1978; Frenkel ef al., 1979,
1987; EuzCby, 1980, 1987; Farmer, 1980; Fayer, 1981; Smith, 1981; Baker,
1987; Kreier and Baker, 1987; Levine and Baker, 1987; Current et al.,
1990; Dubey, 1993). In fact, as Cox (1991) stated in a recent review of the
systematics of parasitic protozoa, the ‘systematics of this group has become
one of the most controversial areas of parasitic protozoology’.
Even after the elucidation of their complete life cycles in the 1970s (see
Section 3.2), a total of at least four different families, eight subfamilies,
and 11 genera has been used for the classification of tissue cyst-forming
coccidia (Table 3). Reviews of the history and definitions of these taxa
have been given by Levine (1973, 1977a, 1988), Frenkel (1974, 1977),
Tadros and Laarman (1976, 1982), Dubey (1977a, b), Overdulve (1978),
Rommel (1978, 1989), Frenkel et al. (1979, 1987), EuzCby (1980), Smith
(1981), Hiepe and Jungmann (1983), Greene and Prestwood (1984), Cur-
rent et d. (1990) and Dubey (1993).
I

I Nodevelopment
I on theground
I I
I I

cysts

in the intermediate host


in the intermediate host

FRENKELlA
SARCOCYSTIS
I
I

and gamogony I on the

intermediate hosts

W
Endomites

TOXOPLASMA BESNOlTlA
I

/ Development in
\
and gamogony 1 on the
9 final host and
in the final host 1 ground
I exogenousstages ? \
U I unknown
I I

HAMMONDIA NEOSPORA
Figure I Life cycles of different genera of tissue cyst-forming coccidia. A. The genus Sarcocystis. B. The genus Frenkelia. C. The
genus Toxoplasma. D. The genus Besnoitia. E. The genus Hammondia. F. The genus Neospora. Illustrations are arranged as follows:
development in the intermediate host is shown below the full line, development in the definitive host is shown on the left of the
dashed line, exogenous life cycle stages are shown on the right of the dashed line (modified with permission from Rommel, 1989).
Table 3 Taxa used for the classification of tissue cyst-forming coccidia since the elucidation of their life cycles in the 1970s.
Number of lower
Taxon taxa" References
Family
Eimeriidae Minchin, 1903 5 8 subfamilies Tadros and Laarman (1976), Overdulve (1978), Kreier
5 28 genera and Baker (1987), Mehlhorn and Ruthmann (1992)
Isosporidae Minchin, 1903 3 subfamilies EuzCby (1987)
7 genera
Sarcocystidae Poche, 1913 5 3 subfamilies Levine (1973, 1988), Frenkel (1974), Hiepe and
2-6 genera Jungmann (1983), Frenkel et al. (1987), Current et al.
(1990), Eckert et al. (1992)
Toxoplasmatidae Biocca, 1956 2 4 genera Frenkel (1974), Eckert et al. (1992)
Subfamily
Sarcocystinae Poche, 1913 2-3 genera Levine (1973, 1988), Hiepe and Jungmann (1983),
Euzkby (1987), Frenkel et al. (1987), Current et al.
(1990)
Toxoplasmatinae Biocca, 1957 2-4 genera Levine (1973, 1988), Hiepe and Jungmann (1983),
Euzkby (1987), Frenkel et al. (1987), Current et al.
( 1990)
Isosporinae Wenyon, 1926 2 genera EuzCby (1987)
Cyclosporinae Wenyon, 1926 6 genera Kreier and Baker (1987)
Besnoitiinae Garnham, 1966 1 genus Levine (1973)
Eimeriinae Wenyon, 1926 > 2 genera Tadros and Laannan (1976)
Endorimosporinae Tadros and Laarman, 1976 1 genus Tadros and Laarman (1976)
Cystoisosporinae Frenkel, Heydorn, Mehlhorn and 1 genus Hiepe and Jungmann (1983), Frenkel et al. (1987)
Rommel, 1979
Genusb
Isospora Schneider, 1881' > 240 species Overdulve (1978), Levine (1988), Current et al. (1990),
Dubey (1993)
Sarcocystis Lankester, 1882 > 120 species Levine (1986, 1988), Tenter (1995)
Toxoplasma Nicolle and Manceaux, 1909d 1-9 species Dubey and Beattie (1988), Levine (1988), Current et al.
(1990)
Besnoitia Henry, 1913 6 7 species Levine (1988), Rommel (1989), Dubey (1993)
Frenkelia Biocca, 1968 2 species Levine (1988), Rommel (1989), Dubey (1993)
* Hammondia Frenkel and Dubey, 197Sd 3 4 species Rommel (1989), Dubey (1993)
* Arthrocystis Levine, Beamer and Simon, 1970 1 species Levine and Baker (1987), Levine (1988)
* Endorimospora Tadros and Laarman, 1976' - Tadros and Laarman (1982)
* Levineia Dubey, 1977f - Dubey (1977a, b)
* Cystoisospora Frenkel, 1977' 5 4 species EuzCby (1980), Hiepe and Jungmann (1983), Greene and
Prestwood (1984), Rommel (1989)
Neospora Dubey, Carpenter, Speer, Topper and Uggla, 1 species Dubey (1993), Dubey and Lindsay (1993)
1988

Number of lower taxa depend on the taxonomic scheme used.


Genera that have not been generally accepted are marked with an asterisk (*).
' In some classifications, species of the genus Cystoisospora are included in the genus Isospora.
In some classifications, species of the genus Hammondia are included in the genus Toxoplasma.
A synonym of Sarcocystis and Frenkelia; now obsolete (see Dubey, 1977a; Rommel, 1978).
A synonym of Cystoisospora; now obsolete (see Rommel, 1978; EuzCby, 1980; Dubey, 1993).
84 A.M. TENTER AND A.M. JOHNSON

Some authors place genera of heteroxenous tissue cyst-forming coccidia,


such as Sarcocystis and Toxoplasma, in one family, together with genera
whose members have a homoxenous life cycle and do not form tissue cysts,
such as Eimeria Schneider, 1875 or even Cryptosporidium Tyzzer, 1907
(see Kreier and Baker, 1987; Mehlhorn and Ruthmann, 1992). It has also
been proposed to incorporate tissue cyst-forming coccidia into the genus
Zsospora with further taxonomic differentiation of these parasites at a
subgeneric level (Overdulve, 1978; Kreier and Baker, 1987). Other authors
separate genera of tissue cyst-forming coccidia from homoxenous coccidia
by placing them into one or two families of their own (Farmer, 1980; Hiepe
and Jungmann, 1983; Frenkel et al., 1987; Levine, 1988; Current et al.,
1990; Eckert et al., 1992; Lindsay and Todd, 1993). In some classifications
of coccidia, the genus Toxoplasmu is separated from the genus Sarcocystis
at familial (Frank, 1976; Eckert et al., 1992) or subfamilial level (Tadros
and Laarman, 1976; Farmer, 1980; Hiepe and Jungmann, 1983; EuzCby,
1987; Frenkel et al., 1987; Levine, 1988; Current et al., 1990; Lindsay and
Todd, 1993), whereas other classifications separate tissue cyst-forming
coccidia only at a generic level (Kreier and Baker, 1987; Mehlhorn and
Ruthmann, 1992; Dubey, 1993).
Cox (1994) suggested that the family Sarcocystidae ‘contains at least five
genera and over 100 species, numbers that are very fluid as the group is
being continually reappraised’. For the purpose of this review, the follow-
ing six genera of tissue cyst-forming coccidia in the family Sarcocystidae
are considered valid: Sarcocystis; Toxoplasma; Besnoitia Henry, 1913;
Hammondia Frenkel and Dubey, 1975; Frenkelia Biocca, 1968; and Neo-
sporu Dubey, Carpenter, Speer, Topper, and Uggla, 1988. Their definitions
and life cycles are outlined in Table 1 and Figure 1.
At least four genera of tissue cyst-forming coccidia, Toxoplasma,
Neospora, Besnoitia and Sarcocystis, are of medical andor veterinary
importance. Members of the genus Sarcocystis are characterized by the
development of tissue cysts in striated muscles of the intermediate host, the
absence of asexual multiplication in the definitive host, and an endogenous
phase of sporogony (see Figure 1; Dubey, 1993; Tenter, 1995). By contrast,
members of the genera Toxoplasma, Neospora and Besnoitia form tissue
cysts in various cell types of the intermediate host. Members of the genera
Toxoplasma and Besnoitia have a proliferative phase of merogony preced-
ing gamogony in the definitive host and an exogenous phase of sporogony,
while the complete life cycle of Neospora is still unknown (see Figure 1;
Rommel, 1989; Current et al., 1990; Dubey, 1993).
The genera Toxoplasma and Neospora currently comprise only one valid
species each, whereas the genus Besnoitia consists of at least six species
and the genus Sarcocystis is one of the larger genera of the Apicomplexa,
currently comprising more than 120 species (Dubey, 1993; Tenter, 1995).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 85

However, there is considerable confusion about the taxonomy and nomen-


clature of Sarcocystis species (see Heydorn et al., 1975; Dubey, 1976,
1977b; Tadros and Laarman, 1976,1982; Levine, 1977a, 1986,1988; CCmh
et al., 1978; Markus, 1978; Mehlhorn and Heydorn, 1978; Rommel, 1978;
Frenkel et al., 1979, 1980, 1984, 1987; Levine and Tadros, 1980; Melville,
1980, 1984; Fayer, 1981; Dubey and Fayer, 1983; Mehlhorn et al., 1985;
Baker, 1987; Dubey et al., 1989; Cawthom and Speer, 1990; Cox, 1991;
Mehlhorn and Piekarski, 1995). It is unfortunate that the debate about which
names to use has not ceased, and even today different authors frequently use
different names for the same species. The nomenclature of the Sarcocystis
species included in the studies presented here is given in Table 4. This
nomenclature is consistent with that of Levine (1986) and conforms to the
International Code of Zoological Nomenclature (Melville, 1984).

3. TRADITIONAL CHARACTERS USED FOR THE CLASSIFICATION


OF TISSUE CYST-FORMING COCClDlA

Originally, the erection of protozoan genera now belonging to the group of


tissue cyst-forming coccidia was based on the morphology or location of
asexual stages of the parasites in their intermediate hosts.
The first description of a tissue cyst-forming coccidium was given by
Miescher (1843), who found ‘milky white threads’ in the skeletal muscles
of a house mouse and suspected they had a parasitic origin. In the following
years, similar parasites were found in the muscles of various animals. They
became known as Miescher’s tubules, but their taxonomic position
remained obscure for several decades, and they were variously believed
to be of animal, plant or fungal nature. In 1865, J. Kuhn (1865) described
Miescher’s tubules in striated muscles of pigs and assigned the specific
name Synchytrium miescherianum to them, erroneously assuming their
plant nature. In 1882, Balbiani (1882) and Lankester (1882) were the first
to suggest that these parasites belonged to the protozoan class Sporozoea
Leuckart, 1879. Balbiani (1882) suggested the name ‘sarcosporidies’ for
them, and Lankester (1882) introduced the genus Sarcocystis as a spor-
ozoan taxon. Consequently, the species name Synchytrium miescherianum
was changed to Sarcocystis miescheriana, and the species originally
described by Miescher (1843) was named Sarcocystis muris by LabbC
(1899). However, because S. miescheriana was named before S. muris,
S. miescheriana (Kiihn, 1865) LabbC, 1899 became the type species of the
genus Sarcocystis.
Between 1882 and 1972, numerous species of Sarcocystis were
described on the basis of differences in the host species infected, the
86 A.M. TENTER AND A.M. JOHNSON

Table 4 Nomenclature of Sarcocystis species mentioned in this review, modified


from Levine (1986, 1988).
Surcocystis urieticunis Heydorn, 1985
Surcocystis caprucunis Fischer, 1979
Synonym": Sarcocystis capricanis El-Rafaii, Abdel-Baki and Selim, 1980 lapsus
Calami.
Surcocystis eruzi (Hasselmann, 1923) Wenyon, 1926
Synonyms": Coccidium bigeminum Stiles, 1891 in part; Coccidium bigeminum var.
canis Railliet and Lucet, 1891 in part; Cryptosporidium vulpis Wetzel, 1938;
Endorimospora hirsuta (MoulC, 1888) Tadros and Laarman, 1976; Isospora rivolta
sporocysts of Gassner (1940) and auctores in part; Isospora bigemina large form of
Mehlhom, Heydom and Gestrich (1975) and Heydom, Mehlhom and Gestrich
(1975) in part; Miescheria cruzi Hasselmann, 1923; Lucetina bigemina (Stiles, 1891)
Henry and Leblois, 1926 in part; Sarcocystisfusiformis Railliet, 1897 of Babudien
(1932) and auctores in part; Sarcocystis iturbei Vogelsang, 1938; Sarcocystis
marcovi Vershinin, 1975 in part; Sarcocystis bovicanis Heydom, Gestrich, Mehlhorn
and Rommel, 1975.
Surcocystisfusifonnis (Railliet, 1897) Bernard and Bauche, 1912
Synonyms": Balbiania fusiformis Railliet, 1897; Balbiania siamensis von Linstow,
1903; Balbiania sp. de Jongh, 1885; Sarcocystis blanchardi Doflein, 1901;
Sarcocystis siamensis (von Linstow, 1903); Sarcocystis bubali Willey, Chalmers and
Philip, 1904; Sarcocystis babuli Willey, Chalmers and Phillips, 1904, of Kalyakin
and Zasukhin (1975) lapsus calami.
Surcocystis gigantea (Railliet, 1886) Ashford, 1977
Synonyms": Balbiania gigantea Railliet, 1886; Endorimospora tenella (Railliet,
1886) Tadros and Laarman, 1976; Sarcocystis ovifelis Heydom, Gestrich, Mehlhorn
and Rommel, 1975; Sarcocystis tenella (Railliet, 1886) MoulC, 1886 in part.
Surcocystis mouki Neveu-Lemaire, 1912
Synonym": Sarcocystis orientalis Machul'skii and Miskaryan, 1958 (?).
Surcocystis muris (Railliet, 1886) Labbd, 1899
Synonyms": Coccidium bigeminum var. cati Railliet and Lucet, 1891;
Endorimospora muris (Blanchard, 1885) Tadros and Laarman, 1976; Miescheria
muris Railliet, 1886; Miescheria muris Blanchard, 1885 of auctores; Sarcocystis
muris (Blanchard, 1885) LabW, 1899; Sarcocystis musculi Blanchard, 1885 of
Kalyakin and Zasukhin (1975) lapsus calami.
Surcocystis neuronu Dubey, Davis, Speer, Bowman, de Lahunta,Granstrom,
Topper, Hamir, Cummings and Suter, 1991
Surcocystis teneUa (Railliet, 1886) Mould, 1886
Synonyms": Balbiania gigantea Railliet, 1886 of auctores in part; Coccidium
bigemin" Stiles, 1891 in part; Coccidium bigeminum var. canis Railliet and Lucet,
1891 in part; Cryptosporidium sp. Bearup, 1954; Endorimospora ovicanis (Heydom,
Gestrich, Mehlhom and Rommel, 1975) Tadros and Laarman, 1976;
Hoareosporidiumpellerdyi Pande, Bhatia and Chauhan, 1972 (?); Isospora bigemina
large form of Mehlhom, Heydom and Gestrich (1975) and Heydom, Mehlhom and
Gestrich (1975) in part; Isospora rivolta free sporocysts of Gassner (1940) and
auctores in part; Lucetina bigemina (Stiles, 1891) Henry and Leblois, 1926 in part;
Miescheria tenella Railliet, 1886; Sarcocystis ovicanis Heydom, Gestrich, Mehlhom
and Rommel, 1975.
~~ ~

" For references see Levine and Tadros (1980), Levine (1986, 1988).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 87

dimensions of the tissue cyst, the size or shape of the so-called ‘spores’
within the tissue cyst, or the structure of the tissue cyst wall. By the end of
the century, names had been assigned to nine species of Sarcocystis (see
LabbC, 1899). The list of named Sarcocystis species had increased to 35 by
1926, to 44 by 1932, and to 54 by 1975 (Wenyon, 1926; Babudieri, 1932;
Kalyakin and Zasukhin, 1975). However, only after the elucidation of the
complete heteroxenous life cycles of some of these species in 1972 (see
Section 3.2) did it become clear that what had been known as Miescher’s
tubules for more than a century were in fact last generation meronts (or
tissue cysts) of obligately heteroxenous coccidian parasites in their inter-
mediate hosts, and that the ‘spores’ were last generation merozoites (or
cystozoites); that is, the infectious stage for the definitive host. Since 1972,
the validity of several Sarcocystis species has been evaluated by transmis-
sion studies, and some of the earlier names assigned to species in the genus
Sarcocystis are now obsolete. After an extensive review of the literature,
Levine and Tadros (1980) gave a list of 93 different species of Sarcocystis
with their synonyms and hosts. However, new species of Sarcocystis are
continually being described, even today. Although both the intermediate
and definitive hosts are not known for all species, the last review of the
taxonomy of species in the genus Sarcocystis regarded 122 as valid
(Levine, 1986), and since then at least another 20 species have been named.
Thus, the genus Sarcocystis is now by far the largest genus of tissue cyst-
forming coccidia and is one of only eight genera in the phylum Apicom-
plexa that comprise more than 100 species (see Levine, 1988).
In 1908, asexual stages of another tissue cyst-forming protozoan were
found in the spleen, liver and blood of gondis, a species of North African
rodent, by Nicolle and Manceaux (1908). The authors first thought that this
parasite resembled a species of Leishmania Ross, 1903 and thus assigned to
it the specific name Leishmania gondii. However, a more detailed study of
this parasite showed that it lacked a kinetoplast, and therefore the authors
concluded that the parasite was not related to Leishmania and, in the
following year, proposed the generic name Toxoplasma for it (Nicolle
and Manceaux, 1909). Thus, the second genus of tissue cyst-forming
coccidia was established and Toxoplasma gondii (Nicolle and Manceaux,
1908) Nicolle and Manceaux, 1909 became the type species of the genus
Toxoplasma. However, the tissue cyst stage of T. gondii remained unknown
until the late 1950s (reviewed by Jacobs et al., 1960a, b), and thus no
relationship was recognized between the genus Toxoplasma and the genus
Sarcocystis.
As with Sarcocystis, several species of Toxoplasma were named during
the first half of this century, mainly based on differences in the host
species infected (Wenyon, 1926; GrassC, 1953). However, whereas sev-
eral transmission studies carried out over the last three decades confirmed
88 A.M. TENTER AND A.M. JOHNSON

Surcocystis species to have a relatively high specificity for their intermedi-


ate hosts (reviewed by Levine, 1986; Dubey et al., 1989), T. gondii has
been shown to be probably the most polyxenous protozoan known so far,
being capable of infecting an unusually wide range of intermediate hosts
and many types of host cells (reviewed by Levine, 1961b, 1988; Dubey and
Beattie, 1988). Therefore, most of the Toxoplasmu species described dur-
ing the first half of this century have now been synonymized with T. gondii
(see Levine, 1977b) and, under the assumption that the genus Hummondiu
is valid (see Section 2.2), it may well be that T. gondii is currently the only
valid species in the genus Toxoplusmu (see Table 1; Dubey and Beattie,
1988; Current et ul., 1990; Johnson, 1990).
Taxonomic characters traditionally used for the classification of tissue
cyst-forming protozoa are host-specificity and the morphology or location
of asexual stages in intermediate hosts. However, the advent of electron
microscopy in the second half of this century enabled ultrastructural com-
parisons of these parasites that led to the recognition of the taxonomic
relationship of the genera Surcocystis and Toxoplusmu, to the correct
classification of these parasites as coccidia (see Section 3.1), and finally
to the discovery of their heteroxenous life cycles (see Section 3.2).

3.1. Morphology and Ultrastructure

In early classifications of protozoa which were based primarily on orga-


nelles of locomotion, the genus Surcocystis was either regarded as incertue
sedis or placed into a separate subclass or order within the class Sporozoea
(Figure 2; Butschli, 1882; Blanchard, 1885; Lankester, 1889; LabbC, 1899;
Schaudinn, 1900; Doflein, 1901; Minchin, 1903; LCger and Duboscq, 1910;
Neveu-Lemaire, 1912; Poche, 1913; Wenyon, 1926; Babudieri, 1932; Bar-
retto, 1940; Kudo, 1950; Doflein and Reichenow, 1953). By contrast, the
genus Toxoplusmu was regarded as incertue sedis within the class Sporozoea
or grouped with the homoxenous coccidia in the sporozoan subclass Coc-
cidiomorpha Doflein, 1901 (see Wenyon, 1926; Doflein and Reichenow,
1953; GrassC, 1953), and thus was separated from the genus Surcocystis at
the high level of subclass (Wenyon, 1926; Kudo, 1950; Doflein and Reich-
enow, 1953).
In the late 1950s, the application of electron microscopy provided new
information on the ultrastructure of different asexual stages of Toxoplusmu
and Surcocystis (i.e., endozoites or early generation merozoites of T. gondii
and cystozoites of several Surcocystis species) which confirmed the classi-
fication of these parasites as sporozoa (Ludvik, 1956, 1958a, b, 1960, 1963;
Gavin et ul., 1962). The results obtained by these studies revealed pheno-
typic similarities between the two genera and thus suggested a much closer
CLASSIFICATION OF EVENT CLASSIFICATION OF REFERENCE
SARCOCYSTIS TOXOPLASMA

F
Light microscopy
Discovery of Miescher's tubules 1
(today tissue cyst of Sarcocysris muris)
Description of the tissue cyst of 2
Synchyrrium miescherianum
I882 (today the type species Sarcocysris miescherianu)
Introduction of the genus Surcocysfrs 3
Placement of the Sarcosporidia 3,4
into the class Sporozoea
1882 Discovery of the asexual stages of Toxoplasmu gondii 11
1
-'i
in gondis
Description of the type species Toxoplusma gondir 12
(only asexual stages) 1909

1-
Introduction of the'genus Toxoplasmu 12

Description of several new species of Surcocystis 7. 15, 16


and Toxoplusma
1932 (today partially obsolete) 1926

Subclass or or1 Sarcosporidia Genus incertue sedis in the class 5 , 6. 8-10,


in the class Sporozoea Sporozoea or in the order Coccidiida, 13-17
subclass Coccidiomorpha, class Sporozoea

I
CLASSIFICATION OF EVENT CLASSIFICATION OF REFERENCE
SARCOCYSTIS TOXOPLASM

I
Subclass Sarcosporidia Genus incertae sedis in the order 15, 17

j
in the class Sporozoea Coccidiida, subclass Coccidiomorpha,
1 class Swrozoea

Electron microscopy
1956 1956
Discovery of ultrastructural similarities between 18-2 1
1957 Sarcocystis and Toxoplasma 1957
T
Family Sarcocystidae in the order Family Toxoplasmatidae in the order 22-24
Toxoplasmida, class Toxoplasmea, Toxoplasmida, class Toxoplasmea,

I
subphylum Sporozoa subphylum Sporozoa

-It 1970
Discovery of ultrastructural similarities between
Sarcocystis, Toxoplasma, and Eimeria

Cell culture
Cultivation of the sexual stages of Sarcocystis
25-28

21,37
CLASSIFICATION OF EVENT CLASSIFICATION OF REFERENCE
SARCOCYSTIS TOXOPLASMA

Transmission studies in vivo


Elucidation of the life cycle of Toxoplasma 30-36
Elucidation of the life cycle of Sarcocystis 38-41

1974 Introduction of the genus Hammondia 1974 42,44

Family Sarcocystidae Family Toxoplasmatidae 42,41


in the suborder Eimeriina in the suborder Eimeriina

Elucidation of the life cycle of Frenkelra 43,45,49

1 1
1977 Elucidation of the life cycle of Besnoitiu 1971 46,48

Subfamily Sarcocystinae Subfamily Toxoplasmatinae 48,50-52,


in the family Sarcocystidae in the family Sarcocystidae 54,55, 58

Molecular biology
1988 1988
Inference of the phylogenetic relationship between 53, 56,51,
1996 Sarcocystis and Toxoplasma based on SSU rRNA 1996 59-64

Genus Sarcocystis Genus Toxoplasma


in the family Sarcocystidae in the family Sarcocystidae
92 A.M. TENTER AND A.M. JOHNSON

relationship between Sarcocystis and Toxoplasma than had been believed


previously (Ludvik, 1956, 1958b, 1960, 1963; Goldman et al., 1958).
Consequently, in the first revision of the traditional classification of pro-
tozoa by an international committee of protozoologists, which took into
account ultrastructural and cytochemical data collected until the early
1960s, the two genera Toxoplasma and Sarcocystis were placed together
in the same class Toxoplasmea Biocca, 1957, but were still separated from
the then known eimeriine coccidia at this high taxonomic level (see Figure
2; Biocca, 1956, 1957, 1968; Levine, 1961a, b; Cheissin and Poljansky,
1963; Honigberg et al., 1964). Further electron microscope studies in the
late 1960s revealed ultrastructural similarities between extraintestinal
merozoites of Toxoplasma and Sarcocystis and intestinal merozoites of
several Eimeria species (see Scholtyseck and Piekarski, 1965; SCnaud,
1967; Sheffield and Melton, 1968; Scholtyseck et al., 1970) and thus
indicated coccidian-like life cycles for the former two genera (reviewed
by Scholtyseck and Mehlhorn, 1973).

3.2. Life Cycles

The final elucidation of the life cycle of Toxoplasma began with the
discovery that faecal material recovered from a cat previously fed mouse
carcasses and brains infected with Toxoplasma tissue cysts induced Toxo-

Figure 2 Taxonomic milestones in the history of Sarcocystis and Toxoplasma


References: 'Miescher (1843); 2Kiihn, J. (1865); 3Lankester (1882). 4Balbiani
(1882); 'Blanchard (1885); 6Lankester (1875-1889); 7Labbt (1899); *Schaudinn
(1900); 'Doflein (1901); 'winchin (1903); "Nicolle and Manceaux j1908);
2Nicolle and Manceaux (1909); I3Neveu-Lemaire (1912); 14Poche(1913). 'Wen-
yon (1926); I6Babudieri (1932); 17Doflein and Reichenow (1953); "Ludvfk
1956); "Ludvfk (1958b); 'kudvfk (1960); 21Ludvik (1963); 22Biocca (1957);
13Levine (1961b); 24Honi ber et al. (1964); 25Scholtyseck and Piekarski
(1965); 26SCnaud (1967); 'Sh:ffield and Melton (1968); 28Scholtyseck et al.
$1970); 29Fayer (1970); 30Dubey et al. (1970); 31Frenkel et al. (1970);
2Hutchison et al. (1970); 330verdulve (1970); 34Sheffield and Melton $1970);
35Weilandand Kuhn (1970); 36Witte and Piekarski (1970); 37Fayer(1972); 'Hey-
dorn and Rommel (1972a); 39Heydorn and Rommel (1972b); 40Rommel et al.
(1972); 41Rommel and Heydorn (1972); 42Frenkel (1974); 43Peteshev et al.
(1974); 44Frenkel and Dubey (1975); 45Rommel and Krampitz (1975); 46Wallace
and Frenkel (1975); 47Frank (1976); 48Frenkel (1977); 49Krampitz and Rommel
(1977); "Smith (1981); "Levine (1985); "Frenkel et al. (1987); 53Johnson et al.
(1988); 54Levine (1988); "Current et ~1.~61990);56Barta et al. (1991); 57Tenter et
al. (199$ 58Lindsay and Todd (1993. Fenger et al. (1994); 60Holmdahl et al.
bi994); Ellis and Morrison (1995); 'Ellis et al. (1995); 63Jeffries et al. (1997);
A. Jeffries et al. (unpublished information).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 93

p l a s m infection when ingested by other mice (Hutchison, 1965). From


the results of this study, it was concluded that the faeces of cats may
contain an infectious stage of Toxoplasmu, which was eventually identified
as an isosporan-type oocyst of a coccidian by several investigators almost
simultaneously (Hutchison et al., 1969; Kuhn and Weiland, 1969; Siim
et al., 1969; Dubey et al., 1970; Overdulve, 1970; Sheffield and Melton,
1970; Witte and Piekarski, 1970). In 1970, knowledge of the coccidian life
cycle of Toxoplasma was completed by the discovery of sexual stages in
the small intestine of cats (Dubey et al., 1970; Frenkel et al., 1970;
Hutchison et al., 1970, 1971; Weiland and Kuhn, 1970).
At the same time, further evidence for the then hypothetical coccidian
life cycle of Sarcocystis was provided by Fayer (1970, 1972), who inocu-
lated cystozoites of a Sarcocystis species derived from the muscles of
grackles into cell cultures and demonstrated its development up to the
formation of micro- and macrogametes and oocyst-like stages. Finally, a
series of transmission studies in the early 1970s elucidated the sexual phase
of the life cycles of several species of Sarcocystis in their definitive hosts
and provided conclusive evidence for the coccidian nature of these para-
sites (Heydorn and Rommel, 1972a, b; Rommel and Heydorn, 1972; Rom-
me1 et al. 1972).
Thus, knowledge of the life cycles of Toxoplasma and Sarcocystis was
completed more than 150 years after the first discovery of the tissue cyst
stage of this group of coccidia in intermediate hosts. It was finally revealed
that tissue cyst-forming coccidia have a heteroxenous life cycle in which an
asexual phase of development in various tissues of intermediate hosts is
linked to a sexual phase of development in the intestine of carnivorous
definitive hosts. In the succeeding years, numerous experiments involving
transmission of asexual stages of various other tissue cyst-forming proto-
zoa to carnivores showed that long-known members of the genus Zsospora,
such as Zsospora bigemina (Stiles, 1891) Luhe, 1906, Zsospora hominis
(Railliet and Lucet, 1891) Wenyon, 1923 and Zsospora buteonis (Henry,
1932), were in fact developmental stages of a range of different species
belonging to different genera of tissue cyst-forming coccidia (reviewed by
Heydorn et al., 1975; Levine, 1977a; Rommel, 1978; Frenkel et al., 1979).
Thus, it became evident that the oocyst or sporocyst stages of members of
different genera of tissue cyst-forming coccidia had been classified as
members of the genus Zsospora; that is, that different developmental stages
of the same parasite had been associated with two different genera and thus
had been given different generic names.
After the identification of the isosporan-type life cycle stages of Sar-
cocystis and Toxoplasma, it became possible to investigate their complete
life cycles in intermediate and definitive hosts. The results of these
studies provided new and more accurate information on the biology of
94 A.M. TENTER AND A.M. JOHNSON

the parasites as well as the morphology of the developmental stages in their


life cycles, which led to redefinition of the major coccidian families in the
suborder Eimeriina by Frenkel(l974). He suggested that the family Eimer-
iidae should include coccidian genera with monoxenous life cycles, the
family Toxoplasmatidae should include coccidian genera with facultatively
heteroxenous life cycles, and the family Sarcocystidae should include
coccidian genera with obligately heteroxenous life cycles (see Figure 2,
Table 2).
In the following years, studies on other tissue cyst-forming protozoa
(Peteshev et al., 1974;Rommel and Krampitz, 1975;Wallace and Frenkel,
1975;Krampitz et al., 1976;Rommel et al., 1976;Frenkel, 1977;Krampitz
and Rommel, 1977) also revealed heteroxenous coccidian life cycles for
members of the genera Frenkefia and Besnoitia. These studies showed that
the character of facultative homoxeny or heteroxeny is variable at the
generic level among tissue cyst-forming coccidia (Frenkel, 1977) and
hence is not a valid character for familial or subfamilial designations.
Therefore, the family Sarcocystidae was again redefined to include all
heteroxenous (facultatively or obligately) coccidian genera (Frenkel,
1977),but was then further divided into two or three different subfamilies
(see Figure 2; Frenkel, 1977; Smith, 1981;Hiepe and Jungmann, 1983;
Levine, 1985,1988;Frenkel et a f . , 1987;Current et al., 1990;Lindsay and
Todd, 1993).The genus Sarcocystis was placed into the subfamily Sarco-
cystinae Poche, 1913 defined as comprising genera characterized by two
types of reproductive stages (metrocytes and cystozoites) in the tissue cyst,
the absence of asexual reproduction in the definitive host, and an endo-
genous phase of sporogony. By contrast, the genus Toxoplasmu was placed
into the subfamily Toxoplasmatinae Biocca, 1957 defined as comprising
genera that are characterized by only one type of reproductive stage
(cystozoites) in the tissue cyst, an asexual phase of multiplication preced-
ing gamogony in the definitive host, and an exogenous phase of sporogony
(Frenkel, 1977;Frenkel et af., 1979;Hiepe and Jungmann, 1983;Levine,
1985). Genera currently classified in the family Sarcocystinae, such as
Sarcocystis and Frenkelia, have an obligately heteroxenous life cycle,
whereas the life cycle of genera currently classified in the family Toxo-
plasmatinae, such as Toxoplasma, Besnoitia and Hammondia, can be
facultatively or obligately heteroxenous (see Figure 1).

3.3. Inference of Phylogenetic Relationships from Phenotypic


Characters

The underlying theory of biological classifications is that of organic evolu-


tion; that is, all organisms have evolved from a common ancestor. Thus, in
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 95

an ideal situation, biological classifications should be natural; that is, they


should reflect the phylogenetic relationships among the organisms included
in the classification and all taxa used in the classification should be mono-
phyletic, meaning that all members of a taxon have evolved from a com-
mon ancestor of the same or lower evolutionary status.
Thus far, little is known about the phylogenetic relationships of tissue
cyst-forming coccidia to each other or to other genera of coccidia (see
Section 3.4). With higher eukaryotes, information about their phylogenetic
relationships can be derived from the comparison of homologous charac-
ters with fossil records that permit the placement of the organisms under
study into precise evolutionary time frames and the construction of phy-
logenetic trees reflecting highly probable evolutionary histories of these
organisms. However, for soft-bodied protozoa like the Apicomplexa, there
is no fossil record and many intermediate forms that might have been
useful for inferring phylogenetic relationships are lost. Therefore, phylo-
genetic relationships of these parasites need to be inferred from compar-
isons of homologous characters of extant species.
One of the major problems in the reconstruction of organismal phylo-
genies from phenotypic characters is to find those characters that are truly
homologous among the organisms under study and therefore are phylo-
genetically informative (Barta, 1989). In the past, revisions of the tradi-
tional classification of protozoa have been based mainly on new
information obtained on their biology, life cycle or morphology. In
particular, ultrastructural data, which have become available since the
advent of electron microscopy, have greatly influenced current concepts
of taxonomic schemes for protozoa (Honigberg et al., 1964; Levine et al.,
1980; Cox, 1981).
Phenotypic characters used for the classification of tissue cyst-forming
coccidia over the last two decades include host specificity, the pattern of
the life cycle, the degree of heteroxenity, the mode of transmission of
infectious life cycle stages, the type of host cells parasitized, the morphol-
ogy and location of the tissue cyst in the intermediate host, the type of
multiplication in the tissue cyst, the type of development in the definitive
host, the location of sporogony, and the morphology of the oocyst (Table 5 ;
Frenkel, 1974, 1977; Tadros and Laarman, 1976; Levine, 1977b, 1985,
1988; Frenkel ef al., 1979, 1987; Smith, 1981; Hiepe and Jungmann, 1983;
EuzCby, 1987; Dubey et al., 1988; Current et al., 1990).
However, some problems occur with the use of these characters to infer
phylogenetic relationships of tissue cyst-forming coccidia, because many
species descriptions are inadequate, morphological features are often sub-
ject to change during the developmental cycle of the parasite, and the
complete life cycles of many organisms currently classified as coccidia
are still unknown (reviewed by Levine, 1986, 1988; Dubey and Beattie,
Table 5 Some phenotypic characters used for the classification of tissue cyst-forming coccidia.
Character Sarcocystis Frenkelia Toxoplasma Besnoitia Hammondia Neospora
Host range and specifcity
Host range"
definitive host carnivorous birds of prey felids felids felids or canidsd ?
vertebrates
intermediate host herbivorous or rodents or warm-blooded mammals or mammals mammals
omnivorous lagomorphs vertebrates reptiles
vertebrates
Host specificityb
definitive host intermediate high high high high ?
intermediate host usually highd intermediate low low low low
Biology and life cycle pattern
Degree of heteroxenity obligate obligate facultative obligate or obligate 9

facultatived
Type of development
in definitive host gamogony, gamogony, endopolygeny, endopolygeny, endopolygeny, ?
sporogony sporogony gamogony gamogony g~ogonY
in environment none none sporogony sporogony sporogony ?
in intermediate host endopolygeny, endopolygeny, endodyogeny endodyogeny endodyogeny endodyogeny
endodyogeny endodyogeny
Natural route of transmission'
definitive to intermediate host via free via free via sporulated via sporulated via sporulated ?
sporocysts sporocysts oocysts oocysts oocysts
intermediate to definitive host via tissue cysts via tissue cysts via tissue cysts via tissue cysts via tissue cysts ?
definitive to definitive host none none via sporulated none none ?
oocysts or
endozoites
intermediate to intermediate host none or via none or via via tissue cysts via tissue cystsd none via endozoites (or
endozoitesd endozoitesd or endozoites tissue cysts?)
Extraintestinal stages in definitive absent absent present present or absentdpresent or absentd ?
host
Morphology and location of developmental stages
Zygote location in lamina propria in lamina propria in epithelium in epithelium in epithelium ?
Oocyst disporous, disporous, disporous, disporous, disporous, ?
tetrazoic tetrazoic tetrazoic tetrazoic tetrazoic
Tissue cyst
location striated muscles, central nervous many tissues connective tissue striated muscles, central nervous
neural tissue system brain system
shape variabled lobulated or subspherical subspherical subspherical subspherical
subspherical
tissue cyst wall variabled, within thin, within thin, within thick, surrounds thin, within thick, within
host cell host cell host cell host cell host cell host cell
septa present present absent absent absent absent
stages within tissue cyst metrocytes, metrocytes, cy stozoites cystozoites cystozoites cystozoites
cystozoites cystozoites
cystozoite banana-shaped, banana-shaped, crescent-shaped, crescent-shaped, crescent-shaped, crescent-shaped,
broad broad slender slender slender slender
Location of host cell nucleus outside tissue outside tissue outside tissue within tissue outside tissue outside tissue
cyst wall cyst wall cyst wall cyst wall cyst wall cyst wall

(I Refers to all species of the genus.


Refers to individual species within the genus.
Transmission routes may be: (1) orally via ingestion of oocysts, sporocysts or tissue cysts or (2) congenitally via endozoites.
Variable with the species within the genus.
98 A.M. TENTER AND A.M. JOHNSON

1988; Dubey et al., 1989; Rommel, 1989; Tenter, 1995). In addition, it is


often not clear what weight should be assigned to the characters listed
above, and controversy arose about which characters to use for generic
designation and which to use for subfamilial or familial designations
(Tadros and Laarman, 1976, 1982; Frenkel, 1977; Levine, 1977b; Baker,
1987; Frenkel et al., 1987; Levine and Baker, 1987).
As a consequence, the phenotypic characters currently used for the
classification of tissue cyst-forming coccidia are limited in their phylo-
genetic information content, the accuracy of phylogenetic relationships
inferred from them is uncertain and biological classifications based on
these characters are highly subjective. Accordingly, there is great con-
troversy about the division of tissue cyst-forming coccidia into families,
subfamilies, and even genera (see Table 2). Therefore, further studies
using more phylogenetically valid characters are needed to clarify the
relationships of species and genera of tissue cyst-forming coccidia to
each other and to other coccidian genera, and to enable their classification
into valid taxa.

3.4. Conflicting Hypotheses Based on Phenotypic Characters

Based on phenotypic characters, several conflicting hypotheses have been


made with respect to the phylogeny of tissue cyst-forming coccidia.
(i) Landau ( 1974) hypothesized that coccidia of vertebrates evolved
from ancestors that parasitized either the coelomic cavity or tissues of
mesoblastic origin of invertebrates. In the course of their adaptation to
the vertebrate host, the coccidia became localized first in tissues of the
same origin (e.g. reticuloendothelial and blood cells) and later became
adapted to more specialized tissues such as the endodermal tissue of the
intestine. The basis of this adaptation was considered to be carnivorism,
and the acquisition of sporulated oocysts in the life cycle was considered a
secondary feature that enabled the survival of the parasites in the environ-
ment. The author suggested that tissue cyst-forming coccidia, such as the
genera Toxoplasma and Sarcocystis, evolved from an ancestor shared with
other heteroxenous coccidia, such as Lankesterella and Schellackia, and
that homoxenous eimerian coccidia evolved from heteroxenous isosporan
coccidia by secondary simplification of the life cycle.
(ii) By contrast, other authors (Tadros and Laarman, 1982; Kreier and
Baker, 1987) hypothesized that the heteroxenous tissue cyst-forming coc-
cidia evolved from homoxenous ancestors that first inhabited cells of the
alimentary canal of their hosts. Tadros and Laarman (1982) suggested that
an ascending degree of heteroxenity among coccidia with isosporan-type
oocysts can be seen as evidence for evolutionary adaptation from a simpler
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 99

homoxenous life cycle to a more complex heteroxenous life cycle. If true,


this would suggest that obligately heteroxenous coccidia, such as Surco-
cystis, are more highly derived than facultatively heteroxenous coccidia,
such as Toxoplusmu. The authors also suggested that the morphological
similarity between the gamogonic stages of heteroxenous and homoxenous
coccidia, as well as the greater pathogenicity of extraintestinal stages of
Toxoplusmu compared with its intestinal stages, indicate that there is an
older evolutionary link between the host and intestinal stages than between
the host and extraintestinal stages. This hypothesis was also favoured by
Frenkel (1970, 1974, 1989) who postulated that Toxoplusmu originated
phylogenetically from an intestinal coccidium in the ancestors of felids
and later developed the ability to parasitize other hosts and tissues after the
carnivorous habit of the felids’ ancestors became well developed. Thus, the
acquisition of tissue cysts would be a secondary feature that allowed
carnivorism to become an additional means of transmission for the parasite.
Similarly, Surcocystis was postulated to have evolved first in the ancestors
of carnivorous definitive hosts and later to have become adapted to the
utilization of intermediate hosts, with the transfer of the whole phase of
asexual multiplication into the intermediate host being the most highly
derived feature in its life cycle (Tadros and Laarman, 1982; Frenkel, 1989).
The latter hypotheses were supported by a phylogenetic analysis of several
apicomplexan protozoa, based on biological and morphological characters,
by Barta (1989), which suggested that the definitive hosts of these parasites
are their ancestral hosts and that heteroxenous apicomplexan protozoa
evolved independently from different ancestors to adapt to changes in
the feeding behaviours of their definitive hosts.
(iii) From a comparison of the life cycles and ultrastructure of various
apicomplexan protozoa, Krylov (1992) also concluded that the heteroxe-
nous life cycles of some of these parasites developed from homoxenous life
cycles of their ancestors. However, in contrast to the previous authors
Krylov (1992) concluded that only in Toxoplusma is the phylogenetically
older host the definitive host, while in Surcocystis the phylogenetically
older host is the intermediate host.

4. THE MOLECULAR ANSWER?

Over the past decade, the introduction of new molecular biological meth-
ods to the study of phylogenetic relationships has had great impact on
previously held beliefs on the taxonomy, systematics and phylogeny of
almost all life forms (Woese, 1987; Sogin, 1991; Knoll, 1992; Patterson
and Sogin, 1992; Olsen and Woese, 1993; Wainright et ul., 1993; Schlegel,
100 A.M. TENTER AND A.M. JOHNSON

1994). In particular, new techniques for the comparison of macromolecules


that share a common ancestry provided new and independent methods for
testing traditional hypotheses on the phylogenetic relationships of proto-
zoa, and hence a firmer basis for a classification based on evolutionary
theory (Patterson and Sogin, 1992). These techniques use phylogenetically
informative molecules to infer the evolutionary history of the organisms
under study (Felsenstein, 1988).
In particular, comparisons of the sequences of small subunit ribosomal
ribonucleic acid (SSU rRNA) provided new insights into the evolution and
phylogenetic relationships of protozoa (Sogin, 1989, 1991; Johnson et al.,
1990a; Schlegel, 1991). Although there are still many conflicting ideas
about the evolution of protozoa as well as about which molecular char-
acters to use for optimal inference of phylogenetic relationships, there is no
doubt that the information gained from SSU rRNA sequence comparisons
is a valuable complement to ultrastructural data and will have great impact
on future classifications of protozoa (Patterson and Sogin, 1992; Cavalier-
Smith, 1993; Corliss, 1994; Cox, 1994).
Recently, SSU rRNA sequence data have been used to examine the
phylogenetic relationships of tissue cyst-forming coccidia currently classi-
fied in the genera Sarcocystis, Toxoplasma and Neospora to each other, to
other coccidian taxa, and to other apicomplexan protozoa as well as to
examine the extent of genetic divergence among different species of tissue
cyst-forming coccidia (Johnson et al., 1987b, 1988, 1991; Barta et al.,
1991; Gajadhar et al., 1991; Tenter et al., 1992; Gagnon et al., 1993; Ellis
et al., 1994a, b, 1995; Fenger et al., 1994; Holmdahl et al., 1994; Ellis and
Morrison, 1995; Escalante and Ayala, 1995; Luton et al., 1995; Marsh et
al., 1995; Tenter, 1995; Relman et al., 1996; Jeffries et al., 1997).
A wide range of data analysis and tree-building methods has been
developed for the reconstruction of organismal phylogenies from nucleo-
tide sequences (reviewed by Felsenstein, 1988; Swofford and Olsen, 1990;
Beanland and Howe, 1992; Nei, 1992; Hillis et al., 1993; Morrison, 1996).
All of these methods have advantages and disadvantages which may have
great impact on the topology of the phylogenetic tree(s) derived from them.
Therefore, an important aspect of phylogenetic reconstruction is to keep in
mind that none of the different methods are ideal and they are therefore not
guaranteed to produce the ‘true’ phylogenetic tree of the organisms included
in the analysis. However, several of these methods perform well if one
considers that the trees generated by them cannot be expected to represent
the exact evolutionary histories of the organisms under study, but should be
interpreted on the assumption that they are a statistical estimation of the
most likely phylogeny of these organisms. It is beyond the scope of this
chapter to review the different methods currently used in the field of
molecular systematics. However, because various methods have been
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 101

used to generate SSU rRNA sequence data for tissue cyst-forming coccidia
and to infer their phylogenetic relationships, we briefly discuss some of the
factors that may have influenced the topology of the phylogenetic trees
constructed in these studies. More detailed reviews of various aspects of the
reconstruction of organismal phylogenies from nucleotide sequence data
have recently been published by Felsenstein (1988), Hillis and Moritz
(1990), Hillis and Dixon (1991), Beanland and Howe (1992), Miyamoto
and Cracaft (1992), Hillis et al. (1993) and Morrison (1996).

4.1. Characteristics of SSU rRNA

SSU rRNA has been widely used to infer phylogenetic relationships of a


broad range of very diverse organisms, including bacteria, protists and
higher eukaryotes such as animals and plants (Woese, 1987; Sogin,
1989, 1991; Schlegel, 1991; Knoll, 1992; Olsen and Woese, 1993; Wain-
right et al., 1993). The primary and secondary structure of SSU rRNA is
evolutionarily conserved even among very distantly related organisms,
particularly in those regions that determine the core of the secondary
structure of the molecule (Sogin and Gunderson, 1987; Neefs et al.,
1990; Hillis and Dixon, 1991). This enables the alignment of SSU rRNA
sequences derived from very diverse taxa to be used for the construction of
comprehensive phylogenetic trees. However, in other regions of the mole-
cule the sequences vary even between closely related taxa. This double
feature of conservation and variation, a slow rate of evolutionary change in
a clock-like manner, and the universal and abundant nature of SSU rRNA
have made comparisons of SSU rRNA sequences a molecular method of
choice for the inference of phylogenetic relationships in many recent
studies of the phylogeny of very diverse organisms (Sogin, 1989; Baver-
stock and Johnson, 1990; Schlegel, 1991; Olsen and Woese, 1993; Wain-
right et al., 1993).
The different levels of conservation in the SSU rRNA molecule allow
phylogenetic comparisons to be carried out at different taxonomic levels,
such as among distantly related taxa belonging to different phyla or
between more closely related species belonging to only one genus.
Recently, comparisons of SSU rRNA and of SSU rRNA gene sequences
have been used to examine phylogenetic relationships of a range of proto-
zoa placed into the phylum Apicomplexa. These studies have included
members of the genus Perkinsus Levine, 1978, the haemosporidian genus
Plasmodium Marchiafava and Celli, 1885; the piroplasmic genera Theileria
Bettencourt, Franqa and Borges, 1907, Babesia Starcovici, 1893 and
Cytauxzoon Neitz and Thomas, 1948, the homoxenous coccidian genera
Cryptosporidium, Eimeria and Cyclospora Schneider, 1881 and the tissue
102 A.M. TENTER AND A.M. JOHNSON

cyst-forming coccidian genera Toxoplasmu,Sarcocystis and Neospora (see


Gunderson et al., 1986; Johnson et al., 1987b, 1988, 1990b, 1991; Barta el
al., 1991; Gajadhar et al., 1991; Waters et al., 1991, 1993a, b; Ellis et al.,
1992, 1994a, b, 1995; Tenter et al., 1992; Gagnon et al., 1993; Goggin and
Barker, 1993; Allsopp et al., 1994; Escalante and Ayala, 1994, 1995;
Fenger et al., 1994; Holmdahl et al., 1994; Mackenstedt et al., 1994;
Thomford et al., 1994; Waters, 1994; Ellis and Morrison, 1995; Relman
et al., 1996; Jeffries et al., 1997).

4.2. Nucleotide Sequence Determination

SSU rRNA sequence data can be derived either directly from the SSU
rRNA or from its gene. Accordingly, molecular biological methods that
have been employed to generate sequence data for phylogenetic analyses
include gene cloning and sequencing, reverse transcription of RNA and,
more recently, gene amplification by polymerase chain reaction (PCR).
Figure 3 shows phylogenetic trees of species in the genera Sarcocystis,
Toxoplasma and Neospora that have been derived from SSU rRNA
sequence data of these parasites. These trees were obtained using different
methods to generate the nucleotide sequences used for comparison.
The tree shown in Figure 3A was obtained in one of the earlier studies on
tissue cyst-forming coccidia and was constructed from sequence data
generated by reverse transcription of SSU rRNA (Tenter et al., 1992).
This method was developed in the mid 1980s (Qu et al., 1983; Lane et
al., 1985) and has been widely used to generate data derived from phylo-
genetically informative regions of SSU rRNA over the past decade
(reviewed by Johnson and Baverstock, 1989; Barta et al., 1991). Thus, in
the first phylogenetic studies on tissue cyst-forming coccidia this method
was used to obtain SSU rRNA sequences of the parasites (Johnson et al.,
1987b, 1988; Tenter et al., 1992). The advantages of the technique are that
it is quick and inexpensive, and therefore allows the determination of SSU
rRNA sequences of a much larger number of taxa in a shorter period of
time than had been possible with earlier methods. The disadvantages of the
technique are that it generates information on only part of the SSU rRNA
and that the sequence data obtained are only 95-99 % accurate (Lane et al.,
1985; Johnson and Baverstock, 1989; Baverstock and Johnson, 1990). In
addition, the technique is based on RNA as starting material and therefore
requires a reasonable number (usually >lo8) of parasite cells for RNA
extraction.
By the late 1980s, a method became available that employs PCR ampli-
fication and subsequent sequencing of the SSU rRNA gene for the genera-
tion of SSU rRNA sequence data (Medlin et al., 1988). This method has
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 103

A S. tenella
S. capracanis
S. arieticanis
S. cruzi
901 Tgondii

85 E. maxima
-
I
Cryptosporidium sp.

B S. tenella
S.arieticanis
S. gigantea
T. gondii
S.muris
E. tenella
C. parvum
B. bovis
Th. annulata
Perkinsus sp.
Cr cohnii
104 A.M. TENTER AND A.M. JOHNSON

100 7 S. tenella
C
S. arieticanis
S. gigantea
S. muris
I: gondii
97
I
E. tenella
C. pawum
B. bovis
Th. annulata
I ’ Perkinsus sp.
Cr cohnii

S. tenella
- S. arieticanis
-
93 S. firsiformis
S. gigantea
-
100
S. muris
-
98 100 N. caninum
-
71
T. gondii
E. tenella
-63 79 B. bovis
- Th. annulata
C. pawum
Perkinsus sp.
Cr. cohnii

Figure 3 Phylogenetic trees of tissue cyst-forming coccidia inferred from SSU


rRNA sequence comparisons using different methods for phylogenetic analysis. All
four trees were constructed using maximum parsimony analysis of the data, but
different methods were used to generate and align the nucleotide sequences, and
different data sets and outgroups were used in the analyses. Numbers at the nodes
are bootstrap values shown as a percentage. A-C. Trees were constructed from
sequence alignments based on the primary structure of SSU rRNA. A. The data set
used to construct the tree was generated by reverse transcription of SSU rRNA and
consisted of 402 nucleotide positions of semi-conserved SSU rRNA gene regions
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 105

several advantages over the earlier method (Sogin, 1989; Baverstock and
Johnson, 1990). First, it uses DNA, which is more stable than RNA, as
starting material. Only a very small amount (about 50 ng) of DNA is
needed for PCR amplification, and thus the technique is ideal for those
parasites of which only a low number of cells can be obtained. Second, the
fidelity of thermostable DNA polymerases is greater than that of reverse
transcriptase and therefore the sequences obtained are more accurate (prob-
ably 99.7-99.9%) than those obtained by reverse transcription (Medlin et
al., 1988; Saiki et al., 1988; Ellis et al., 1994b). In addition, the entire SSU
rRNA gene sequence can be obtained in both directions. The increased
accuracy of the sequences generated by this method enables the phyloge-
netic comparison of more closely related organisms. Consequently, this
method has now become the method of choice for the generation of SSU
rRNA gene sequences of tissue cyst-forming coccidia (Gajadhar et al.,
1991; Ellis et al., 1994a; Fenger et al., 1994; Holmdahl et al., 1994; Jeffries
et al., 1997). The trees shown in Figure 3B, C, D, which were constructed
in more recent studies on tissue cyst-forming coccidia, were also derived
from sequence data generated by the latter method (Ellis and Morrison,
1995; Ellis et al., 1995).

Figure 3 Continued
(redrawn with permission from Tenter et al., 1992). B-D. Nucleotide sequences
were generated by PCR amplification and sequencing of the SSU rRNA gene. B.
The data set used to construct the tree consisted of the entire SSU rRNA gene
sequences apart from the 5' and 3' ends which were truncated, i.e. 1868 nucleotide
positions (redrawn with permission from Ellis et al., 1995). C. The data set used to
construct the tree consisted of 1567 nucleotide positions of conserved and semi-
conserved SSU rRNA gene regions (redrawn with permission from Ellis et al.,
1995). D. The tree was constructed from a sequence alignment based on the
secondary structure of SSU rRNA. The data set consisted of 1301 nucleotide
positions located in the helices of the SSU rRNA (redrawn with permission from
Ellis and Morrison, 1995).
SSU rRNA sequences of the following taxa were used in the analyses: Protozoa,
phylum Apicomplexa, order Eucoccidiida LBger and Duboscq, 1910, family Sar-
cocystidae: Sarcocystis tenella, Sarcocystis capracanis, Sarcocystis arieticanis,
Sarcocystis cruzi, Sarcocystis fusifonnis, Sarcocystis gigantea, Sarcocystis
muris, ,Toxoplasma gondii, Neospora caninum; family Eimeriidae: Eimeria
tenella, Eimeria stiedai, Eimeria maxima; family Cryptosporidiidae: Cryptospor-
idium parvum, Cryptosporidium sp.; order Piroplasmida Wenyon, 1926: Babesia
bovis, Theileria annulata; order Perkinsida Levine, 1978: Perkinsus sp.; amoebae:
Acanthamoeba castellanii (outgroup in A); dinoflagellates: Crypthecodinium coh-
nii (outgroup in B-D), Prorocentrum micans; Metazoa: Homo sapiens. For sources
of sequences see Tenter et al. (1992), Ellis and Morrison (1995), and Ellis et al.
(1995).
106 A.M. TENTER AND A.M. JOHNSON

4.3. Sequence Alignment

It should be noted that the alignment of the SSU rRNA sequences for
optimal homology is straightforward in regions of relatively conserved
primary and secondary structure of the molecule, but is much more arbi-
trary in the more variable regions. Therefore, for optimal inference of
phylogenetic relationships the latter regions, for which one cannot be
confident of the alignment, should be excluded from the analysis (Hase-
gawa et al., 1985; Baverstock and Johnson, 1990; Beanland and Howe,
1992; Olsen and Woese, 1993; Morrison, 1996). However, there are few
objective criteria from which to determine what regions should be removed
as being phylogenetically uninformative before phylogenetic reconstruc-
tion (Gatesby et al., 1993).
Sequence alignment is probably the most important aspect of the recon-
struction of phylogenetic trees, and is the most problematic (Morrison,
1996). In most of the phylogenetic studies on tissue cyst-forming coccidia
carried out so far, the sequence alignments were based on the primary
structure of SSU rRNA (Johnson et al., 1987b, 1988, 1991; Barta et al.,
1991; Tenter et al., 1992; Gagnon et al., 1993; Ellis er al., 1994a, b, 1995;
Fenger et al., 1994; Holmdahl et al., 1994; Escalante and Ayala, 1995).
While most of these studies used information derived from full-sequence
alignments, with only few sequences of uncertain homology excluded from
the data set used in the analysis (Gajadhar et al., 1991; Gagnon et al., 1993;
Ellis et al., 1994a, 1995; Fenger et al., 1994; Holmdahl et al., 1994;
Escalante and Ayala, 1995; Relman et al., 1996), a few studies concen-
trated on using only information derived from semi-conserved regions of
the molecule; that is, those regions in which the nucleotide sequences were
neither highly variable nor totally conserved among the taxa included in the
analysis and which are believed to contain the phylogenetically informative
nucleotide positions (Johnson et al., 1987b, 1988, 1991; Tenter et al.,
1992). However, the blocks of semi-conserved nucleotide positions were
often chosen by eye, and this limited the number of taxa that could be
aligned.
More recently, several studies have used knowledge of the secondary
structure constraints of SSU rRNA to optimize the alignment (Van de Peer
et al., 1994) and have then used information derived from phylogenetically
informative nucleotide positions located in the helices of the molecule to
infer phylogenetic relationships of tissue cyst-fonning coccidia (Ellis and
Morrison, 1995; Jeffries et al., 1997). This alignment is then edited using
the Dedicated Comparative Sequence Editor of De Rijk and De Wachter
(1993) and it appears to be a significant advance over either full-sequence
alignment based on primary structure or alignment of semi-conserved
nucleotide sequence blocks by eye. Figure 3D shows an example of a
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 107

tree that was constructed from an alignment based on the secondary


structure of SSU rRNA (Ellis and Morrison, 1995), while the trees shown
in Figure 3A, B, C were constructed from alignments based on the primary
structure of the molecule (Tenter et al., 1992; Ellis et al., 1995).

4.4. Outgroup

It is well known that the selection of outgroup for a phylogenetic analysis


can have a very significant effect on the trees obtained (Maddison et al.,
1984; Barta et al., 1991; Escalante and Ayala, 1994). An inappropriate
outgroup that is too distantly related to the ingroup may influence the
alignment because fewer sequences can be unambiguously aligned and
therefore more variable regions may have to be excluded from the data
set used for phylogenetic analysis (see Section 4.3). Such variable regions
may well contain information needed to resolve phylogenetic relationships
at lower taxonomic levels (Barta et al., 1991). However, when the studies
described here were commenced, the optimal outgroup for analyses of
apicomplexan protozoa was still undecided. Therefore, the tree shown in
Figure 3A was rooted using an amoeba as an outgroup (Tenter et al., 1992).
However, based on morphological characters and SSU rRNA sequence
comparisons, it is now believed that the most closely related protozoan
taxa to the Apicomplexa are members of the dinoflagellates (Barta et al.,
1991; Gajadhar et al., 1991; Schlegel, 1991, 1994; Wolters, 1991; Sadler et
al., 1992; Gagnon et al., 1993; Wainright et al., 1993; Escalante and Ayala,
1995). Therefore, a dinoflagellate was used to root the trees shown in
Figure 3B, C, D which were constructed in recent studies on tissue cyst-
forming coccidia (Ellis and Morrison, 1995; Ellis et al., 1995). Depending
on the level of analysis, other studies of the phylogenetic relationships of
tissue cyst-forming coccidia have also used dinoflagellates or have used
taxa that are more closely related to the tissue cyst-forming coccidia, such
as members of the genera Cryptosporidium or Eimeria, as outgroup taxa to
root the trees (Ellis et al., 1994a; Fenger et al., 1994; Holmdahl et al.,
1994; Tenter, 1995; Jeffries et al., 1997).

4.5. Tree-building Methods

Tree-building methods that have been used in phylogenetic studies of


tissue cyst-forming coccidia comprise both distance matrix and charac-
ter-state methods (Johnson et al., 1988, 1991; Barta et al., 1991; Tenter et
al., 1992; Gagnon et al., 1993; Ellis et al., 1994a, 1995; Fenger et al., 1994;
Holmdahl et al., 1994; Ellis and Momson, 1995; Escalante and Ayala,
108 A.M. TENTER AND A.M. JOHNSON

1995; Relman et al., 1996; Jeffries et al., 1997). The more advanced
methods used for phylogenetic reconstruction in recent studies include
neighbour joining, maximum parsimony, and maximum likelihood, with
maximum parsimony being the method that has been used most fre-
quently (Ellis er al., 1994a, 1995; Fenger er al., 1994; Ellis and Morrison,
1995; Escalante and Ayala, 1995; Relman et al., 1996; Jeffries et al.,
1997).
All four trees shown in Figure 3 were derived using maximum parsimony
analysis for tree construction (Tenter et al., 1992; Ellis and Morrison, 1995;
Ellis et al., 1995). This method counts the minimum number of character
state changes (i.e. base substitutions and sometimes also insertions and/or
deletions) that are required for each proposed tree to accommodate the
observed sequence data. That tree or trees requiring the fewest changes (i.e.
the most parsimonious tree(s)), is/are preferred over all other trees (Felsen-
stein, 1988; Swofford and Olsen, 1990; Beanland and Howe, 1992; Hillis et
al., 1993; Morrison, 1996). The robustness of the monophyletic groups
(nodes) of the trees was tested using the bootstrap method. This method
creates a new data set by randomly resampling sites of the original data set
with replacement until the resampled data set is of the same size as the
original one. This can be repeated hundreds of times, with the constructed
tree showing how often the nodes were supported by all of the bootstraps
(Felsenstein, 1985, 1988).

4.6. Comparison of Different Phylogenetic Trees Inferred from SSU


rRNA Sequence Data

As shown in Figure 3, the phylogenetic analyses based on SSU rRNA


sequences or SSU rRNA gene sequences reviewed here gave conflicting
results with respect to the phylogenetic relationships inferred for different
species of tissue cyst-forming coccidia. Phenotypic characteristics of the
species of tissue cyst-forming coccidia and closely related taxa included in
these studies are listed in Table 6.
All four trees shown in Figure 3 are consistent in showing tissue cyst-
forming coccidia (i.e. members of the genera Surcocystis, Toxoplasma and
Neospora), forming a monophyletic group, to the exclusion of homoxenous
coccidia such as Eimeria or Cryptosporidium, and other apicomplexan
parasites such as Babesia, Theileria or Perkinsus. However, the placement
of the different species of the genus Sarcocystis varies among the four
trees. The tree shown in Figure 3A suggests that the genus Sarcocystis is
paraphyletic, with two monophyletic groups. The first group consists of the
pathogenic species with canine definitive hosts; that is, Sarcocystis
tenella (Railliet, 1886) MoulC, 1886, Sarcocystis arieticanis Heydorn,
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 109

1985, Sarcocystis capracanis Fischer, 1979 and Sarcocystis cruzi (Hassel-


mann, 1923) Wenyon, 1926. The second group consists of the non-patho-
genic species transmitted by cats; that is, Sarcocystis gigantea (Railliet,
1886) Ashford, 1977 and Sarcocystis muris (Railliet, 1886) LabbC, 1899.
Both groups are split by Toxoplasma. The tree shown in Figure 3B also
suggests the genus Sarcocystis to be paraphyletic, but this paraphyly is
different from that suggested by the tree shown in Figure 3A. However, it is
similar to the results obtained by Barta et al. (1991) and Ellis et al. (1994b),
who also included SSU rRNA sequences of S. muris, S. gigantea and T.
gondii in phylogenetic analyses of apicomplexan parasites and found that
these two Sarcocystis species were split by Toxoplasma. By contrast, the
trees shown in Figure 3C and D suggest that the genus Sarcocystis is
monophyletic.
As outlined above, the topology of the trees obtained by phylogenetic
analyses of nucleotide sequence data can depend upon the tree-building
method, the method used to generate the nucleotide sequences, the out-
group used for rooting the tree, or the sequence alignment and the data set
used for analysis. As all four trees shown in Figure 3 were constructed
using maximum parsimony analysis it is unlikely that the differences found
among the trees are a result of the tree-building method, because the global
parsimony criterion was used in all cases, even if the three trees obtained in
the more recent studies (Figure 3B, C, D; Ellis and Morrison, 1995; Ellis et
al., 1995) were constructed using a more advanced computer program than
that used for the tree obtained in the earlier study (Figure 3A; Tenter et al.,
1992).
All three trees shown in Figure 3A, B, C were derived from sequence
alignments based on the primary structure of SSU rRNA. The data set used
to construct the tree shown in Figure 3A was obtained by reverse transcrip-
tion of SSU rRNA and consisted of information on 402 nucleotide posi-
tions of semi-conserved regions of the molecule, which included 203
positions that were neither highly variable nor totally conserved among
the taxa included in the analysis (Tenter et al., 1992). Similarly, Barta et al.
(1991), who also observed an apparent paraphyly of the Sarcocystis species,
used 64-188 informative characters in their analyses. The data set used to
construct the tree shown in Figure 3B was obtained by PCR amplification
and sequencing of the SSU rRNA gene and consisted of information on
1868 positions of the SSU rRNA (Ellis et al., 1995). However, this data set
contained positions of uncertain homology. The exclusion of the latter
positions from the data set resulted in information on 1567 positions;
that is, those positions that were obviously homologous in all taxa included
in the analysis. This reduced data set contained 398 positions which were
neither highly variable nor totally conserved among the taxa, and was used
to construct the tree shown in Figure 3C (Ellis et al., 1995). Unlike the two
Table 6 Phenotypic characters of various tissue cyst-forming coccidia and related
Species Definitive Intermediate Degree of Location of tissue cysts Size of Distribution
host host pathogenicity tissue cysts
for the
intermediate host
Genus Sarcocystis
S. arieticanis dog sheep intermediate probably all striated muscles 5 900 pm probably world-wide
S. capracanis canids goat high striated muscles, central nervous 5 1 mm probably world-wide
system, Purkinje fibres
S. cruzi canids, raccoon bovines high striated muscles, central nervous 5 500 pm world-wide
system, Purkinje fibres
S.fusiformis cat water buffalo non-pathogenic oesophageal muscles 5 32 mm probably all countries with
the distribution of the host
S. gigantea cat sheep non-pathogenic predominantly oesophageal, 5 10 mm world-wide
laryngeal and lingual muscles
S. moulei cat goat non-pathogenic oesophageal muscles 5 12 mm Europe, Asia, Africa
S. muris cat, ferret mouse non-pathogenic skeletal muscles 5 6 mm probably world-wide
S. neurona opossumP equines high ? ? North,Central and South
America
S. tenella canids sheep high striated muscles, central nervous 5 700 pm world-wide
system, Purkinje fibres
Genus Toxoplasma
T.gondii felids warm-blooded high' many tissues 5-100 pm world-wide
' vertebrates

Genus Neospora
N. caninum ? mammals high central nervous system 5 100 pm probably world-wide
Genus Isospora
I. felis felids rodents and non-pathogenic mainly lymphoid tissues dormozoited world-wide
some other
mammals

a For further information on these species see Levine (1988), Dubey (1993) and Tenter (1995).
This finding still needs to be confinned by transmission studies, see Fenger et al. (1995).
For risk groups such as pregnant animals or immunocompromised hosts.
Single dormozoites surrounded by a tissue cyst wall.
112 A.M. TENTER AND A.M. JOHNSON

trees shown in Figure 3A and B, the latter tree showed the Sarcocystis
species to be monophyletic, which was supported by high (96100%)
bootstrap values. However, it should be noted that 10 of 37 trees, whose
lengths were within 14 steps of the most parsimonious tree (817 steps long)
found in this analysis, showed paraphyly of the Sarcocystis species, with
the length of the shortest of those trees being 823 steps; that is, only six
steps longer than the most parsimonious tree (Ellis et al., 1995). Thus, there
was no significant number of steps (Felsenstein, 1988) between the most
parsimonious tree, which suggested monophyly of the Sarcocystis species,
and its next best competitor, which suggested paraphyly of the Surcocystis
species.
Therefore, it appears that although all three trees derived from sequence
alignments based on the primary structure of the SSU rRNA were consis-
tent in showing the dog-transmitted pathogenic Sarcocystis species to be
monophyletic, the relationships of the cat-transmitted non-pathogenic Sar-
cocystis species could not be unambiguously resolved by these analyses
(Figure 3A, B, C). By contrast, strong evidence for the monophyly of the
genus Surcocystis was obtained in a recent study on the phylogenetic
relationships of tissue cyst-forming coccidia (Ellis and Morrison, 1995),
in which the sequence alignment was based on the secondary structure of
SSU rRNA (Van der Peer et al., 1994). In this study, monophyly of the
genus Surcocystis was supported by 83% of the bootstrap replicates when
the entire data set containing 2050 positions was used in the analysis and
by 93% of the bootstrap replicates when the data set was restricted to 1301
positions located in the helices of the SSU rRNA molecule (Figure 3D).

5. PHYLOGENETIC RELATIONSHIPS AND GENETIC RELATEDNESS


OF TISSUE CYST-FORMING COCClDlA INFERRED FROM SSU rRNA
SEQUENCE COMPARISONS

Over the past decade, several authors have used SSU rRNA sequence
comparisons to examine the phylogenetic relationships of different species
and genera of tissue cyst-forming coccidia (Johnson et al., 1987b, 1988;
Gajadhar et al., 1991; Tenter et ul., 1992; Gagnon et al., 1993; Ellis et al.,
1994a, b, 1995; Fenger et al., 1994; Holmdahl et al., 1994; Ellis and
Morrison, 1'995; Jeffries et al., 1997). Other authors have included taxa
of tissue cyst-forming coccidia in their studies on different protozoa
currently classified in the phylum Apicomplexa (Johnson et al., 1990b,
1991; Barta et al., 1991; Ellis et ul., 1992; Goggin and Barker, 1993;
Allsopp et al., 1994; Escalante and Ayala, 1995; Relman et ul., 1996). In
addition, comparisons of SSU rRNA sequences have been used to examine
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 113

the extent of genetic divergence among different species and strains in the
genera Sarcocystis, Toxoplasma and Neospora (see Luton et al., 1995;
Marsh et al., 1995; Tenter, 1995). The information gained from these
studies will be an important complement to the information gained from
comparisons of phenotypic characters of tissue cyst-forming coccidia (see
Section 3).
However, when interpreting phylogenetic trees inferred from phenotypic
or molecular data it should be kept in mind that no one method is likely to
generate the ‘true’ phylogenetic tree of the organisms under study. Instead,
the results obtained are a statistical estimation of the probable phylogenies
of the organisms (Felsenstein, 1988; Swofford and Olsen, 1990; Nei, 1992;
Hillis et al., 1993; Morrison, 1996). Thus, rather than aiming at finding the
one tree that shows the ‘most likely’ phylogeny of the organisms, one
should evaluate a range of trees on the assumption that the phylogenetic
relationships that are supported by most or all of those trees are the most
probable phylogenetic relationships of the organisms (see Section 4).

5.1. Phylogenetic Relationships of Tissue Cyst-forming Coccidia to


Homoxenous Coccidia and other Apicomplexan Protozoa

All phylogenetic analyses based on SSU rRNA sequence comparisons


camed out to date are consistent in showing tissue cyst-forming coccidia,
that is, members of the genera Sarcocystis, Toxoplasma and Neospora, to
form a monophyletic group, excluding homoxenous coccidia, such as
members of the genera Eimeria and Cryptosporidium, and other apicom-
plexan protozoa, such as the piroplasm genera Babesia, Theileria and
Cytauxzoon, the haemosporidian genus Plasmodium and the genus Perkin-
sus (see Figure 3; Johnson et al., 1988, 1991; Barta et al., 1991; Tenter et
al., 1992; Gagnon et al., 1993; Fenger et al., 1994; Ellis et al., 1994a, b,
1995; Holmdahl et al., 1994; Ellis and Morrison, 1995; Escalante and
Ayala, 1995; Relman et al., 1996). Thus, the results obtained by all of
these analyses support the hypothesis that tissue cyst-forming coccidia,
such as members of the genera Sarcocystis, Toxoplasma and Neospora, are
monophyletic.
The phylogenetic analyses based on SSU rRNA sequence data of
coccidian taxa also agree in showing the genus Eimeria to be a sister
taxon to the tissue cyst-forming coccidia (see Figure 3; Barta et al., 1991;
Johnson et al., 1991; Tenter et al., 1992; Ellis et al., 1994a, 1995; Ellis
and Morrison, 1995; Relman et al., 1996). Members of the genus Eimeria
are homoxenous coccidia that produce oocysts with four sporocysts, each
containing two sporozoites. However, the position of the genus Cryptos-
poridium, whose members are also homoxenous but produce oocysts
114 A.M. TENTER AND A.M. JOHNSON

containing a single sporocyst or naked sporozoites, is inconsistent in


these analyses. While some studies suggest that Cryptosporidium forms
a monophyletic group with Eimeria and the tissue cyst-forming coccidia
(Johnson et al., 1991; Tenter et al., 1992), others suggest that tissue cyst-
forming coccidia are only distantly related to Cryptosporidium (see
Johnson et al., 1990b, 1991). In fact, some analyses suggest that tissue
cyst-forming coccidia are more closely related to the piroplasms than to
Cryptosporidium (see Figure 3D; Barta er al., 1991; Ellis et al., 1994a,
1995; Holmdahl et al., 1994; Ellis and Morrison, 1995; Relman et al.,
1996).
Therefore, SSU rRNA sequence comparisons, as well as comparisons of
biological and ultrastructural data (Barta, 1989), show members of the
family Sarcocystidae, such as Sarcocystis, Toxoplasma and Neospora, to
share a more recent common ancestor with members of the family Eimer-
iidae, such as Eimeria, than with members of the family Cryptosporidiidae
(LCger, 1911) such as Cryptosporidium. Consistent with this hypothesis, a
recent analysis based on SSU rRNA sequence comparison of a species of
the genus Cyclospora concluded that a monophyletic group of Eimeria and
Cyclospora was the sister taxon of a monophyletic group of Sarcocystis
and Toxoplasma (see Relman et al., 1996). Like the genus Eimeria, the
genus Cyclospora has been classified in the family Eimeriidae on the basis
of phenotypic characteristics, such as a homoxenous life cycle and the
production of oocysts containing two sporocysts each with two sporozoites
(Levine, 1988).

5.2. Phylogenetic Relationships of Tissue Cyst-forming Coccidia to


Each Other

As described above (see Sections 4.6 and 5.1), all four phylogenetic trees
shown in Figure 3 are consistent in showing tissue cyst-forming coccidia
(i.e. members of the genera Sarcocystis, Toxoplasma and Neospora) as a
monophyletic group (Tenter et al., 1992; Ellis and Morrison, 1995; Ellis
et al., 1995). This is in agreement with other phylogenetic analyses of
tissue cyst-forming coccidia based on SSU rRNA sequence comparisons
(Johnson et al., 1988, 1991; Johnson and Baverstock, 1989; Barta et al.,
1991; Gagnon et al., 1993; Ellis et al., 1994a, b; Fenger et al., 1994;
Holmdahl et al., 1994; Escalante and Ayala, 1995; Relman er al., 1996).
In addition, the phylogenetic trees constructed in these studies are con-
sistent in showing the genera Toxoplasmu and Neospora to be monophy-
letic (Ellis et al., 1994a, b; Holmdahl et al., 1994; Ellis and Morrison,
1995; Escalante and Ayala, 1995; Jeffries et al., 1997).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 115

However, as also shown in Figure 3 and described above (see Section


4.6), the analyses of SSU rRNA sequence data of tissue cyst-forming
coccidia gave conflicting results with respect to the phylogenetic relation-
ships among the various Sarcocystis species. While the trees constructed in
these analyses are consistent in showing the dog-transmitted pathogenic
Sarcocystis species, such as S. tenella, S. arieticanis, S. capracanis and S.
cruzi, forming a monophyletic group (Tenter et al., 1992; Ellis and
Morrison, 1995; Ellis et al., 1995; Tenter, 1995; Jeffries et al., 1997), the
position of cat-transmitted non-pathogenic species, such as S. gigantea, S.
muris and S.fusiformis (Railliet, 1897) Bernard and Bauche, 1912, varies
among the different trees (Barta et al., 1991; Tenter et al., 1992; Ellis et al.,
1994b, 1995; Fenger et al., 1994; Ellis and Morrison, 1995; Tenter, 1995;
Jeffries et al., 1997).
As outlined in Section 4, various methods have been used to infer the
phylogenetic relationships of tissue cyst-forming coccidia. However, none
of the analyses in which the alignment of SSU rRNA sequences was based
on the primary structure of the molecule gave a definitive answer with
respect to the phylogenetic relationships of the non-pathogenic Sarcocystis
species to each other, to pathogenic Sarcocystis species, or to other tissue
cyst-forming coccidia (see Figure 3A, B, C; Barta et al., 1991; Tenter et al.,
1992; Fenger et al., 1994; Ellis et al., 1995). By contrast, evidence for
monophyly of the genus Sarcocystis, to the exclusion of Toxoplasma and
Neospora, was obtained when the alignment of SSU rRNA sequences was
based on the secondary structure of the molecule and when either the entire
aligned data set or a subset of the data containing nucleotide positions that
corresponded to the helices was analysed (see Figure 3D; Ellis and Morri-
son, 1995; Jeffries et al., 1997).
Figure 4 shows a consensus tree derived from the phylogenetic analyses
of tissue cyst-forming coccidia based on sequence alignment according to
secondary structure constraints of SSU rRNA (Jeffries et al., 1997). This
analysis included all species of tissue cyst-forming coccidia for which
complete SSU rRNA gene sequences are available to date; that is, eight
species of Sarcocystis, T. gondii and Neospora caninum Dubey, Carpenter,
Speer, Topper and Uggla, 1988. This tree shows monophyly of the genera
Toxoplasma and Neospora as well as of the eight species of Sarcocystis.
Within the monophyletic group of Sarcocystis species, a monophyletic
group of the pathogenic species with canine definitive hosts (i.e. S.
tenella, S. capracanis and S. arieticanis), can also be observed. By con-
trast, the pathogenic species s. neurona Dubey, Davis, Speer, Bowman, de
Lahunta, Granstrom, Topper, Hamir, Cummings and Suter, 1991, whose
definitive host is unknown but has recently been hypothesized to be a
species of opossum (Fenger et al., 1995), appears to be more closely related
to the non-pathogenic Sarcocystis species, which are transmitted by felids,
Degree of Hosts
heteroxenity definitive intermediate
S. tenella obligate canids sheep
S. capracanis obligate canids goat
S. arieticanis obligate dog sheep
S. moulei obligate cat goat
S. gigantea obligate cat sheep
S. fusiformis obligate cat water buffalo
S. muris obligate cat, ferret mouse
S. neurona obligate ? opossum ? horse
N. caninum facultative ? ? mammals
T. gondii facultative felids warm-blooded animals
I. felis facultative cat mammals
E. tenella
Figure 4 Consensus phylogenetic tree of tissue cyst-forming coccidia inferred from SSU rRNA. The analysis included all species
of tissue cyst-forming coccidia for which complete SSU rRNA gene sequences are available to date, i.e. Sarcocystis tenella,
Sarcocystis capracanis, Sarcocystis arieticanis, Sarcocystis moulei, Sarcocystis gigantea, Sarcocystis fusiformis, Sarcocystis muris,
Sarcocystis neurona, Neospora caninum, Toxoplasma gondii and a closely related species of the genus Isospora, Isospora felis.
Eimeria tenella was used as an outgroup. For sources of sequences see Jeffries et al. (1997).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 117

than to the other pathogenic Surcocystis species, which are transmitted by


canids. This is consistent with the results obtained by a phylogenetic
analysis of this species based on primary structure alignment of SSU
rRNA (Fenger et al., 1994). Interestingly, a species currently classified
in the genus Zsospora appears to be monophyletic with Toxoplusmu and
Neosporu. It should be noted that this species, Zsospora felis Wenyon,
1923, belongs to the group of facultatively heteroxenous Zsospora species
(see Table 1; Current et ul., 1990), which some authors believe to belong to
a separate genus Cystoisosporu Frenkel, 1977 that should be classified in
the family Sarcocystidae (see Tables 2 and 3; Smith, 1981; Frenkel et al.,
1987; Rommel, 1989).

5.3. Genetic Divergence Among Tissue Cyst-forming Coccidia

The SSU rRNA molecule is one of the slowest evolving molecules found
throughout the range of living organisms (Hillis and Dixon, 1991). It has
been suggested that SSU rRNA sequence comparison for inference of
phylogenetic relationships is useful only for organisms that diverged
more than 80-100 million years ago (Baverstock and Johnson, 1990).
This technique may reach its limits if organisms are more closely related,
that is, if they diverged less than 80 million years ago. If phylogenetic
relationships are inferred from SSU rRNA sequences of very closely
related species, the extent of microheterogeneity between repeated SSU
rRNA sequences of one species may exceed the extent of nucleotide
differences among the SSU rRNA sequences of different species (Schlegel,
1991; Cai et al., 1992). Usually, phylogenetic relationships inferred from
SSU rRNA sequences become statistically uncertain if the distances among
the compared sequences are less than one nucleotide change per 100
positions (Sogin et ul., 1989; Patterson and Sogin, 1992). Thus, even if
the outgroup, the alignment of homologous nucleotide positions and the
data set used for analysis have all been optimized for inference of phylo-
genetic trees from SSU rRNA sequences, the technique can still be limited
by the organisms themselves. Therefore, the differences found among the
phylogenetic trees of tissue cyst-forming coccidia inferred from SSU rRNA
sequence comparisons (see Sections 4.6 and 5.2) are consistent with the
hypothesis that tissue cyst-forming coccidia are highly derived protozoa. In
other words, the phylogenetic relationships or genetic relatedness of these
parasites are so close that there are few differences among their SSU rRNA
sequences, so that even small changes in the SSU rRNA regions and
methods used for phylogenetic analysis make large differences to the trees
obtained and the probability of obtaining the wrong tree is high (Nei,
1992).
118 A.M. TENTER AND A.M. JOHNSON

In order to test this hypothesis, the mean genetic distances (Swofford,


1991) among the eight Sarcocystis species for which information on the
complete SSU rRNA gene sequences was available (see Figure 4), N.
caninum and two strains of T. gondii were estimated by pairwise nucleotide
comparisons for semi-conserved SSU rRNA gene regions; that is, those
regions that could be unambiguously aligned for all of the taxa included in
the analysis (Figure 5). In addition, pairwise nucleotide comparisons were
carried out between each species of the tissue cyst-forming coccidia, I.
felis, and members of the homoxenous coccidian genera Eimeria and
Cryptosporidium for homologous SSU rRNA positions (Figure 5). These
comparisons indeed revealed very small genetic distances among the dif-
ferent species and genera of the tissue cyst-forming coccidia.
For example, the mean distance among S. moulei Neveu-Lemaire, 1912,
T. gondii and N. caninum (0.0054.022) (i.e. among representatives of
three genera of tissue cyst-forming coccidia) were of the same magnitude
as those observed among the seven species of the genus Eimeria (0.004-
0.022). In addition, the distances between several species of Sarcocystis,
i.e. S. moulei, S. fusifomzis, and S. muris, and T. gondii (0.017-0.022) or
between these Sarcocystis species and N. caninum (0.018) were smaller
than those between dog-transmitted pathogenic Sarcocystis species and
cat-transmitted non-pathogenic Sarcocystis species (0.020-0.030). The dis-
tances among all of the eight species of Sarcocystis varied between < 0.001
and 0.031, while the distances among all species of tissue cyst-forming
coccidia included in this analysis varied between < 0.001 and 0.046. By
contrast, the distances between a member of the tissue cyst-forming cocci-
dia and a member of the homoxenous coccidia included in the present
analysis were much greater (0.0774.1 13), even from their closest rela-
tives, members of the genus Eimeria (0.077-0.105).
It is well known that rates of DNA sequence evolution differ between
different taxonomic groups of eukaryotes (Britten, 1986). However, if we
assume an approximate divergence rate of 2 4 % per 100 million years for
the SSU rRNA of apicomplexan protozoa (see Ochman and Wilson, 1987;
Wilson et al., 1987; Moran et al., 1993; Escalante and Ayala, 1994), the
observed divergences of about 0 . 4 5 4 5 % among the homologous SSU
rRNA gene regions of the tissue cyst-forming coccidia included in the
present analysis equate to an estimated time of divergence of their common
ancestors between about 11 and 225 million years ago. Therefore, the
differences in the placement of the Sarcocystis species in the trees con-
structed in phylogenetic analyses based on SSU rRNA sequence compar-
isons (see Sections 4.6 and 5.2) may indeed result from a relatively close
genetic relationship of the tissue cyst-forming coccidia.
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 119
- -
St I sc’ Sa I Smo’ Sg’ S f 2 Smu’ ~ n ’ Nc TgS48 TgSAl If

s r e n e ~ aI - 0.005 0.010 0.024 0.030 0.024 0.023 0.024 0.031 0.030 0.035 0.032
s capracanrs I 8 - 0.008 0.022 0.029 0.022 0.020 0.022 0.029 0.028 0.030 0.028
s arierrcanis I 15 I3 0.025 0.027 0.025 0.024 0.025 0.033 0.032 0.037 0.033
s mouler ’ 37 33 39 - 0.031 0.000 0.010 0.000 0.018 0.017 0.022 0.018
s gigantea ’ 45 44 42 48 - 0.031 0.031 0.031 0.042 0.041 0.046 0.043
s fuslformrs ‘ 37 33 39 0 48 - 0.010 0.000 0.018 0.017 0.022 0.018
s murrs ’ 35 31 37 15 48 15 - 0.010 0.021 0.020 0.025 0.023
s neurona ’ 37 33 39 0 48 0 15 - 0.018 0.017 0.022 0.0I8
N canrnum 47 44 50 27 64 27 32 27 - 0.000 0.005 0.010
T gondrr S48 46 43 49 26 63 26 31 26 0 - 0.005 0.010
T gondir SAlLlE 53 46 56 33 70 33 38 33 7 7 - 0.012
I Jars 49 43 51 27 66 27 35 27 16 15 18

ic Ea Eb Ema Emi En Ep El Cb Cm cp cw

I.p i s 0.088 0.091 0.090 0.091 0.087 0.088 0.083 0.107 0.099 0.111 0.110
E. acervulina 135 0.011 0.014 0.012 0.014 0.008 0.010 0.134 0.123 0.135 0.134
E brunerri 139 17 0.014 0.013 0.021 0.014 0.017 0.138 0.127 0.136 0.136
E. maxima 138 22 22 0.018 0.022 0.014 0.018 0.138 0.127 0.134 0.135
E. milis 139 18 20 27 0.019 0.012 0.015 0.141 0.128 0.141 0.139
E. necalrix I33 22 32 34 29 - 0.016 0.004 0.134 0.125 0.134 0.132
E. praecox I35 13 21 22 19 25 - 0.012 0.134 0.123 0.133 0.133
E. tenella 127 16 26 28 23 6 19 0.133 0.122 0.132 0.131
C. baileyr I62 205 21 1 210 214 205 205 203 - 0.034 0.024 0.024
C muris I50 I88 193 I94 I95 190 188 I86 52 - 0.045 0.046
c.parvum I69 206 207 204 214 204 203 202 36 69 - 0.003
C. wrairi I68 204 207 206 212 202 203 200 36 70 5 -

St I sc Smo ’ Sg’ Nc TgSAl Ema Emi En Et Cm cw

S. tenella I 0.005 0.024 0.030 0.031 0.035 0.095 0.097 0.089 0.085 0.089 0.100
s capracanis I 8 - 0.022 0.029 0.029 0.030 0.094 0.095 0.087 0.083 0.087 0.100
s moulei’ 37 33 - 0.031 0.018 0.022 0.088 0.088 0.081 0.077 0.095 0.110
S. gigantea ’ 45 44 48 - 0.042 0.046 0.103 0.105 0.099 0.096 0.102 0.113
N canrnum 47 44 27 64 - 0.005 0.087 0.088 0.083 0.079 0.092 0.105
T. gondii SAlLlE 53 46 33 70 7 - 0.092 0.092 0.088 0.084 0.097 0.109
E. maxima 145 144 134 157 134 141 - 0.018 0.022 0.018 0.127 0.135
E. miris I48 145 134 160 134 141 27 0.019 0.015 0.128 0.139
- 0.004
~

E. necarrix 136 133 124 152 127 134 34 29 0.125 0.132


E. lenella I30 127 118 146 121 128 28 23 6 - 0.122 0.131
C. muris I35 133 145 155 140 147 194 195 190 186 - 0.046
C.wrairi I52 152 168 171 160 166 206 212 202 200 70

Figure 5 Genetic distances among tissue cyst-forming coccidia (species of the


genera Sarcocystis, Toxoplasma and Neospora), a heteroxenous species of the
genus Isospora, and homoxenous coccidia (species of the genera Eimeria and
Cryptosporidium) estimated from pairwise nucleotide comparisons for homologous
SSU rRNA gene regions (1561 nucleotide positions). Data shown are absolute
distances (below diagonal) and mean distances adjusted for missing data (above
diagonal) between the following taxa: St, Sarcocystis tenella; Sc, Sarcocystis
capracanis; Sa, Sarcocystis arieticanis; Smo, Sarcocystis moulei; Sg, Sarcocystis
gigantea; Sf, Sarcocystis fusifonnis; Smu, Sarcocystis muris; Sn, Sarcocystis
neurona; Nc, Neospora caninurn; TgS48, Toxoplasma gondii strain S48; TgSAI,
Toxoplasma gondii strain SAILIE; If, Isospora felis; Ea, Eimeria acervulina; Eb,
Eimeria brunetti; Ema, Eimeria maxima; Emi, Eimeria mitis; En, Eimeria necatrix;
Ep, Eimeria praecox; Et, Eimeria tenella; Cb, Cryptosporidium baileyi; Cm,
A.M. TENTER AND A.M. JOHNSON

6. CONCLUSIONS

A 1961 review on ‘Sarcocystis, Toxoplasma and related protozoa’ began


with the words
The members of the class Toxoplasmasida have been and still are a headache
to taxonomists. Their affinities to other protozoa are uncertain, . . . Much of
our difficulty is due to lack of information. As we learn more and more, and
as new facts fall into place, our understanding of the group will improve and
we can expect that some of our present ideas may change. The classification
adopted here is considered reasonable and useful, but it is not necessarily
definitive. (Levine 1961b)
That review was written after the discovery of ultrastructural similarities
between Sarcocystis and Toxoplasma, but before the elucidation of the
heteroxenous life cycles of tissue cyst-forming coccidia. Since then, we
have gained a considerable amount of new knowledge of the life cycles,
biology, morphology, pathogenicity, epidemiology, biochemistry, immu-
nology and, more recently, also molecular biology of this important group
of coccidian parasites. This knowledge has been published in several
thousands of publications, including a large number of books and review
articles. However, the taxonomic relationships of tissue cyst-forming coc-
cidia have remained uncertain and controversial and, except that they have
now been recognized as belonging to the coccidian subclass Eimeriina,
what was said in 1961 about the puzzle of their taxonomy could still be said
even today.
Based on comparisons of phenotypic characters, no consensus has been
reached about the classification of tissue cyst-forming coccidia. Although
their classification into the family Sarcocystidae, as suggested by Frenkel
(1977), has been favoured over other taxonomic schemes by several
authors, it has not been generally accepted. There are still many conflicting
ideas about the classification of tissue cyst-forming coccidia, with respect
to both their position within the subclass Eimeriina and their division into
lower taxa.
As pointed out by Cox (1994), one of the problems in the reconstruction
of apicomplexan phylogeny is that we have knowledge of only about 1% of
all the species that probably exist. Moreover, research has focused on a
small number of species that are of medical or veterinary importance.

Figure 5 Continued
Cryptosporidiurn rnuris; Cp, Cryptosporidiurn parvurn; Cw, Cryptosporidiurn

wrairi. Pathogenic Sarcocystis species using canids as definitive hosts. * Non-
pathogenic Sarcocystis species using felids as definitive hosts. Pathogenic Sar-
cocystis species with uncertain definitive host (probably opossum). For sources of
sequences see Tenter (1995) and Jeffries et al. (1997).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 121

These species are not necessarily representative of the group of parasites


into which they have been classified and thus may not provide valuable
phylogenetic information on that particular group. In the case of tissue
cyst-forming coccidia, names have been assigned to about 150 species and
individual life-cycle stages have been described for probably another 50
species. However, extensive research has been carried out on less than 10%
of these parasites. For example, while more than 16 000 scientific papers
have been published on a single parasite, T. gondii, only about 1000 papers
have been published on all of the more than 120 species of the genus
Sarcocystis put together (see Dubey, 1993).
Until the late 1980s, only a few molecular data were available that could
be used for phylogenetic analyses of tissue cyst-forming coccidia (O’Don-
oghue et al., 1986; Ford et al., 1987; Johnson et al., 1987a). Therefore,
currently used classifications of these parasites have been based exclu-
sively on the comparison of phenotypic characters. However, these char-
acters are limited in their phylogenetic information content and thus little is
known about the phylogenetic relationships of tissue cyst-forming coccidia
to each other and to other genera of coccidia. Accordingly, several con-
flicting hypotheses have been proposed with respect to their phylogeny.
Thus far, two different strategies have been used to infer phylogenetic
relationships or genetic relatedness of tissue cyst-forming coccidia from
molecular data. In the late 1980s, isoenzyme analysis was used to examine
the genetic relatedness of a range of pathogenic and non-pathogenic Sar-
cocystis species of livestock (O’Donoghue et al., 1986). More recently,
several research groups have used SSU rRNA sequence comparisons for
the reconstruction of phylogenetic relationships of species in the genera
Sarcocystis, Toxoplasma and Neospora. Although the results obtained by
these studies have not been consistent in all cases, the following hypotheses
with respect to the phylogeny of tissue cyst-forming coccidia can be made
so far:
(i) Tissue cyst-forming coccidia, such as the genera Sarcocystis, Toxo-
plasma and Neospora, are monophyletic, to the exclusion of homoxenous
coccidia, such as the genera Eimeria, Cyclospora and Cryptosporidium
(see Sections 5.1 and 5.2).
(ii) Within the group of tissue cyst-forming coccidia examined to date,
the genera Toxoplasma and Neospora are monophyletic (see Section 5.2).
(iii) There is evidence to suggest that pathogenic Sarcocystis species
transmitted by canids, such as S. arieticanis, S. capracanis, S. cruzi and S.
tenella, are monophyletic, to the exclusion of S. neurona and non-patho-
genic Sarcocystis species transmitted by felids, such as S. fusiformis, S.
gigantea, S. moulei and S. muris (see Section 5.2). This is consistent with
the results obtained in a study on the genetic relatedness of Sarcocystis
species based on isoenzyme analysis, which also suggested a closer
122 A.M. TENTER AND A.M. JOHNSON

relationship among dog-transmitted pathogenic Surcocystis species, such


as S. cuprucunis, S. cruzi and S. tenellu, than between these species and
cat-transmitted non-pathogenic Surcocystis species, such as S. medusifor-
mis Collins, Atkinson and Charleston (1979), S. gigunteu and S. muris (see
O’Donoghue et ul., 1986).
(iv) Tissue cyst-forming coccidia are very closely related genetically.
For example, the genetic distances among different genera of tissue cyst-
forming coccidia, such as Surcocystis, Toxoplusma and Neosporu, esti-
mated from SSU rRNA sequence comparisons, are of the same order of
magnitude as those observed among different species of the genus Eimeriu
and much lower than those observed between genera of homoxenous
coccidia, such as Eimeriu and Cryptosporidium (see Section 5.3).
(v) The closest relatives of the tissue cyst-forming coccidia appear to be
homoxenous coccidia classified into the family Eimeriidae, such as the
genera Eimeriu and Cyclosporu (see Section 5.1). However, the genetic
distances between genera of tissue cyst-forming coccidia and members of
the family Eimeriidae estimated from SSU rRNA sequence comparisons
are much greater (2-21 times greater) than the genetic distances among the
tissue cyst-forming coccidia (see Section 5.3).
The phylogenetic relationships of tissue cyst-forming coccidia inferred
from SSU rRNA sequence comparisons are consistent with the hypothesis
that these parasites evolved from an ancestor that is shared with other
extant eimeriine coccidia. Thus, these results support the traditional
hypothesis of Tadros and Laarman (1982) that the heteroxenous tissue
cyst-forming coccidia evolved from homoxenous ancestors that parasitized
cells of the alimentary canal of their hosts. The small extent of genetic
divergence observed among SSU rRNA sequences of tissue cyst-forming
coccidia, which is much smaller than that observed among homoxenous
coccidia, also suggests that tissue cyst-forming coccidia are more highly
derived (i.e. phylogenetically younger) than homoxenous coccidia. How-
ever, there is no indication that obligately heteroxenous tissue cyst-forming
coccidia, such as Surcocystis, are more highly derived than facultatively
heteroxenous tissue cyst-forming coccidia, such as Tonoplusmu, as sug-
gested by Tadros and Laarman (1982), nor that the pathogenic species of
the genus Surcocystis are more highly derived than non-pathogenic species
of the genus. However, both SSU rRNA sequence comparisons and iso-
enzyme analysis provided strong evidence for the hypothesis that patho-
genic Surcocystis species transmitted by canids form a monophyletic group
within the genus Surcocystis, to the exclusion of pathogenic Surcocystis
species transmitted by other definitive hosts and non-pathogenic Surcocys-
tis species transmitted by felids. These results are consistent with the
hypothesis that the life-cycle phase of tissue cyst-forming coccidia in their
definitive hosts is phylogenetically older than the life-cycle phase in their
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 123

intermediate hosts, and thus support the results obtained by a phylogenetic


analysis of apicomplexan protozoa based on several biological and mor-
phological characters by Barta (1989), who also postulated that the defi-
nitive hosts of these parasites are their ancestral hosts. Thus, the
development of tissue cysts in an intermediate host of tissue cyst-forming
coccidia indeed appears to be a secondary feature that may have been
acquired to adapt to changes in the feeding behaviour of the definitive
host during the evolution of carnivorism (see Section 3.4; Barta, 1989;
Cox, 1994).
While SSU rRNA sequence comparisons of tissue cyst-forming coccidia
proved to be a very valuable method of inferring their phylogenetic rela-
tionships to homoxenous coccidia, the relationships of some members of
the tissue cyst-forming coccidia, such as non-pathogenic species of the
genus Surcocystis, to each other and to other tissue cyst-forming coccidia
could not be unambiguously resolved by this method. While phylogenetic
trees derived from sequence alignments based on the secondary structure of
the SSU rRNA suggested the Surcocystis species to be monophyletic, trees
derived from sequence alignments based on the primary structure of the
molecule failed to reveal a close relationship among these species. A
possible explanation for the lack of consensus among the different recon-
structed trees with respect to the phylogenetic relationships of some mem-
bers of the tissue cyst-forming coccidia may be the low level of genetic
divergence observed among their SSU rRNA genes described above. This
low level of SSU rRNA gene sequence divergence within the group of
tissue cyst-forming coccidia is unusual for protozoa, which are usually
much more diverse than prokaryotes, animals, plants, or fungi at the
cytological, organizational and molecular level (Sogin, 1989; Schlegel,
1991, 1994; Patterson and Sogin, 1992). It suggests that tissue cyst-forming
coccidia are highly derived protozoa (i.e. that they diverged from each
other only relatively recently) and indeed there is evidence that some
protozoa have continued to evolve so that new groups appeared as late
as some vertebrates (Patterson and Sogin, 1992). Recently, random ampli-
fied polymorphic DNA PCR has been used to compare Toxoplasma, Neo-
sporu and Sarcocystis (see Guo and Johnson, 1995a, b; Joachim et ul.,
1996). The fact that this method, which has been widely used to dis-
criminate between even intraspecific taxa, can be used to compare tissue
cyst-forming coccidia, highlights the short time since divergence of these
parasites. Therefore, further studies using different molecular characters
and more taxa are required to resolve further the phylogenetic relationships
of tissue cyst-forming coccidia to each other.
Thus far, comprehensive phylogenetic studies based on SSU rRNA
sequence comparisons have been carried out only for species of the genera
Surcocystis, Toxoplasma and Neospora. There is currently no published
124 A.M. TENTER AND A.M. JOHNSON

SSU rRNA gene sequence of species of the genera Frenkeliu, Besnoitiu or


Hummondiu. Therefore, we still know very little about the phylogenetic
relationships of the latter three genera (Johnson et ul., 1987a; DardC el ul.,
1992). Only recently, the SSU rRNA gene sequence of I. felis has been
obtained and used for phylogenetic analysis. Interestingly, I. felis did not
take an intermediary position between the tissue cyst-forming coccidia and
the genus Eimeriu, as might have been expected from a comparison of
phenotypic characters. By contrast, I. felis appeared to be monophyletic
with T. gondii and N. cuninum, to the exclusion of all the Surcocystis
species included in the analysis (A. Jeffries et ul., unpublished
information). I. felis is a member of the group of heteroxenous Zsosporu
species that form dormozoites in intermediate (or paratenic) hosts. Some
authors have classified this group of Zsosporu species as a separate genus,
Cystoisosporu, in the family Sarcocystidae (see Frenkel et ul., 1987).
However, this classification has not been generally accepted, because it
is not known whether other Zsosporu species, currently believed to be
homoxenous, possess dormozoites as well. Thus far, no SSU rRNA gene
sequence of the latter group of Zsosporu species is available for phylo-
genetic comparison, and hence further studies are needed to confirm or
refute the validity of the genus Cystoisosporu.
As described in this review, there are distinct differences in the biology,
life cycle and morphology between the heteroxenous tissue cyst-forming
coccidia and homoxenous coccidia. These differences, together with the
monophyly of tissue cyst-forming coccidia consistently inferred from
comparisons of their SSU rRNA gene sequences and the extent of genetic
divergence between tissue cyst-forming coccidia and their closest relatives
(i.e. members of homoxenous genera of the family Eimeriidae) support the
classification of tissue cyst-forming coccidia into the family Sarcocystidae,
to the exclusion of homoxenous eimeriine genera, but the low degree of
genetic divergence among the tissue cyst-forming coccidia revealed by
SSU rRNA sequence comparisons calls into question the relatively high
number of lower taxa that have been assigned to this family. However,
neither phenotypic nor molecular characters should be seen as a panacea
for definitive phylogenetic reconstruction. Rather, the phylogenetic trees
constructed from such data should be viewed as only reasonable estima-
tions of the evolutionary history of the organisms. Clearly, tissue cyst-
forming coccidia show a high degree of biological diversity. Therefore,
until the phylogenetic relationships of this important group of coccidia to
each other have been further resolved by phylogenetic analyses based on
different, but phylogenetically valid, molecular characters that are able to
complement the results obtained so far by SSU rRNA sequence compar-
isons, a reasonable taxonomic compromise appears to be the classification
of tissue cyst-forming coccidia into the family Sarcocystidae based on
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 125

monophyly of this family inferred from SSU rFWA sequence comparisons,


and its further division into genera that are reasonably well accepted, such
as Sarcocystis, Frenkelia, Toxoplasma, Besnoitia, Hammondia and Neo-
spora, based on phenotypic characters.
Over the last decade, phenotypic characters have gradually been replaced
by more objective and more phylogenetically valid molecular characters
for the inference of phylogenetic relationships of a broad range of proto-
zoa. There is no doubt that the information obtained from phylogenetic
analyses based on molecular data, combined with the information obtained
from phenotypic characters, will greatly improve our knowledge of the
phylogeny of tissue cyst-forming coccidia and thus provide a firmer basis
for a classification that reflects the evolutionary histories of these parasites.

ACKNOWLEDGEMENTS

The studies described here were partially funded by grants from the
Australian Research Council and Deutsche Forschungsgemeinschaft. We
thank Kim Luton and Michael Johnson for technical assistance, and Alex
Jeffries, John Ellis, David Morrison, Peter Baverstock and Michel
Rommel for critical discussion, prepublication information and review
of the manuscript.

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Since this review was submitted, some major advances have been made that
have a significant bearing on knowledge of the phylogeny of the tissue cyst-
forming coccidia.
(i) A new method of phylogenetic analysis of 18s rRNA entitled ‘substitution
rate calibration’ finds the genus Sarcocystis to be monophyletic (Van de Peer, Y.,
Van der Auwera, G. and De Wachter, R., 1996. The evaluation of Stramenopiles
and Alveolates as derived by ‘substitution rate calibration’ of small ribosomal
subunit RNA. Journal of Molecular Evolution 42, 201-210).
(ii) Collaborative work by an Australian, a Canadian and a German group has
shown that a homoxenous and a facultatively hetroxenous Isospora species (i.e.
Isospora suis and I. felis, respectively) form a monophyletic clade, which is the
sister group to the Toxoplasma/lveosporaclade to the exclusion of the Sarcocystis
clade (Carreno, R.A., Schnitzler, B.E., Jeffries, A.C., Tenter, A.M., Johnson, A.M.
and Barta, J.R., Phylogenetic analysis of coccidia based on 18s rDNA sequence
comparison indicates that Isospora is most closely related to Toxoplasma and
Neospora; paper submitted for publication). This finding will have major implica-
tions for the classification of the genus Isospora, because it shows that homoxenous
as well and heteroxenous Isospora species should bot be classified in the family
Eimeriidae, but should be classified together with the tissue cyst-forming coccidia.
This would result in a family comprising all coccidia which have oocysts containing
two sporocysts, each with four sporozoites. Depending on the type genus of the
family, the family name would have to be either Sarcocystidae Poche, 1913 or
Isosporidae Minchin, 1903.
Biochemistry of the Coccidia

Graham H. Coombs’. Helen Denton’. Samantha M. A . Brown’


and Kam-Wah Thong2

Infection and Immunity. Institute of Biomedical and


Life Sciences. Joseph Black Building.
University of Glasgow. Glasgow. G12 8QQ. UK and
Discovery Biology I. Pfzer Central Research. Sandwich.
Kent. CT13 9NJ. UK

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
2. Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
2.1. Eimeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
2.2. Toxoplasrna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
2.3. Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
2.4. Sarcocystis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3. Protein and Amino Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.1. Protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.2. Protein catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.3. Amino acid interconversions .................................. 171
4. Polyamine Metabolism ......................................... 171
5. Purine and Pyrimidine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
5.1. Purine metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
5.2. Pyrimidine metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
6. Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
7. Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8. Culturing Coccidia in vitro and Growth Factor Requirements . . . . . . . . . . . . 185
9. Antioxidant Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
10. The Oocyst Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
I1 . Functional Surface Molecules .................................... 188
11.1 Glycosylation ............................................. 189
11.2 GPlanchors .............................................. 189
11.3 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
11.4 Sialidase and other enzymes ................................. 190
11.5 Cell signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

ADVANCES IN PARASITOLOGY VOL 39 Copyrighr 0 1997 Acodernic Press Limited


ISBN 0-12-031739-7 All rights of reproduction in any form resewed
142 G.H. COOMBS H A L .

12. Interaction of Coccidia with the Host Cell ........................... 191


13. Biochemical Action of Anticoccidial Agents. ......................... 194
14. The Current Status and Priorities for the Future ...................... 197
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

1. INTRODUCTION

There are many parasites in the group known as the coccidia, but only
Toxoplasma gondii and Eimeria species have been investigated biochemi-
cally in any detail. Cryptosporidium parvum is now attracting attention,
although at present little is known of its biochemistry, whereas Sarcocystis,
Neospora and Zsospora have been studied in just a few aspects. This review
reflects the emphasis on studying ‘important’ parasites and thus most of the
content concerns them; the extent to which other species are similar
remains to be seen. It is to be expected, however, that closely related
organisms will share many features and one of the aims of this review is
to pin-point biochemical characteristics of the coccidia as a group. Such
common features, especially when they are significantly different from the
host, provide targets that potentially can be exploited with novel anti-
coccidial agents.
The advent of molecular methods for studying evolutionary relationships
has provided many new insights into the phylogeny of protozoa and
enforced a major re-assessment of previously accepted dogma (see Coombs
et af., 1997). Current evidence supports the view that the coccidia are a
related group, although there is increasing evidence that Cryptosporidium
may not be a true coccidian. For the purpose of this review, however, we
have included all organisms that have generally been accepted as coccidia
in recent years. Good knowledge of the phylogenetic relationships between
parasites will be valuable to parasite biochemists in enabling them to make
better predictions on the likely biochemical composition of a parasite and
helping to explain the presence and evolutionary origin of the more pecu-
liar features - such as the plastid-like organelles, mannitol cycle and
pyrophosphate-linked glycolytic enzymes of coccidia. Coccidia have
some components which are similar to their counterparts in plants, such
as dihydrofolate reductasehhymidylate synthase and also their micro-
tubules, but it is important to avoid making general conclusions about
similarities and differences from the very limited data available at present.
Current knowledge of the biochemistry of Toxopfasma and Eimeria is
fragmentary, so in this review we attempt not only to report the known
data but also to use these to hypothesize on possible common adaptations
BIOCHEMISTRY OF THE COCClDlA 143

and highlight areas that deserve to be given priority in future studies.


Coccidia have complex life cycles involving multiple developmental forms
and many environments. Elucidation of how these forms differ at the
molecular level will provide great insight into the key adaptations of
each stage. Unfortunately, this is one of the aspects on which there is
currently very little information. Indeed, discovering how each parasite
stage is adapted to cope with its own particular microenvironment and is
able, in time, to transform to the next developmental stage, which in most
cases lives in a quite different environment, remains one of the foremost
challenges facing the biochemist. The environments encountered by the
various forms of parasites differ quite considerably in many respects, such
as pH and oxygen and nutrient availability, although in many cases the
precise composition is simply not known. The environmental conditions
clearly have a major impact on the parasites’ metabolism. The overall
emphasis of this review is to interpret the reported findings as far as
possible in terms of how the parasites are adapted to their particular
environments, the functional significance of the parasite features, and the
correlation between the ultrastructure of the parasite and its biochemistry.
Coccidia contain a multitude of interesting and unusual organelles, such
as those comprising the apical complex (rhoptries, micronemes, dense
granules), the feeding organelle of the intracellular stage of Cryptospor-
idium, the refractile bodies of Eimeria, the crystalloid body of Cryptospor-
idium, the plastid-like structures of Toxoplasma, Eimeria and Sarcocystis,
and the acidocalcisomes of Toxoplasma. Many of these structures are
shown in Figure 1. Data on these organelles have increased significantly
in recent years (Archbarou et al., 1991; Bonnin et al., 1991, 1995a; Joiner,
1991; Leriche and Dubremetz, 1991; Perkins, 1992; Vermeulen et al.,
1993; Cesbron-Delauw, 1994; Dubremetz, 1995; Metsis et al., 1995;
McFadden et al., 1996; Moreno and Zhong, 1996; Sam-Yellowe, 1996;
Tomley et al, 1996), but analysis of their biochemical structure and function
is at a very early stage and elucidating the roles of these organelles at the
molecular level is a prime objective of current research.
A major obstacle so far to investigations on the biochemistry of coccidia
has been the difficulty of obtaining material for study. One of the most
fascinating and important aspects of coccidia is their ability to invade and
grow within cells of the host but, unfortunately, effective methods for
obtaining the intracellular stages of parasites from infected animals or
for growing the parasites in a host cell line in vitro have been devised
only relatively recently for coccidia other than Toxoplasma, and improve-
ments are still required before some investigations will be possible. This
has been and remains a major limitation on studies of most of the parasites
and their interaction with their host cell. Even more restricting for bio-
chemists is the inability to grow the parasites axenically. Oocysts and
144 G.H. COOMBS EJAL.

Figure I Diagram of a sporozoite of Eimeria to show the principal structures. C,


conoid; DB, dark bodies; ER, endoplasmic reticulum; IM,inner membranous
complex; L, lipid inclusion; MI, mitochondrion; MN, micronemes; MP, micropore;
N, nucleus; NU, nucleolus; OM, outer membrane; P, polar ring; PL, plastid-like
organelle; PP, posterior polar ring; R1,Rz,preconoidal rings; RB,refractile bodies;
RH, rhoptries; The apical complex also characteristically contains dense granules
(not shown). (Modified from Scholtyseck, 1979).

sporozoites, however, can be readily obtained for Eimeria and Cryptospor-


i d i m species, but not Toxoplasmu, and it is these stages that have been
used for most biochemical investigations to date with these parasites. The
tachyzoite stage of Toxoplasma remains the most accessible for study and
is being used extensively at present.
There have been two major reviews on coccidian biochemistry
previously (Ryley, 1973; Wang, 1982) and our aim in compiling this
review was to build upon these seminal articles by concentrating upon
the studies that have been reported since 1980 and emphasizing those since
1990. In recent years there have been a number of useful reviews and
collections of reviews on aspects of the biology of coccidia which have
BIOCHEMISTRY OF THE COCClDlA 145

included information relevant to their biochemistry (Long, 1990; Current


and Garcia, 1991; Petersen, 1993; Smith, 1993; Sterling and Arrowood,
1993; Lindsay and Blagburn, 1994; Marr and Muller, 1995; Martins and
Guerrant, 1995; O’Donoghue, 1995; Clark and Sears, 1996; Gross, 1996;
Steiner and Guerrant, 1996). The emphasis in this review on functionality
means that in general we have not included data concerning molecules of
unknown structure or function (‘major antigens’ and the like). The follow-
ing aspects also are dealt with only when relevant to our understanding of
the biochemistry: cell structure; parasite culture and isolation; taxonomy;
immunity; molecular biology; and pathology. Many of the biochemical
studies are aimed at identifying and characterizing drug targets, thus
chemotherapy is covered briefly in the context of highlighting possible
targets.

2. ENERGY METABOLISM

Only the energy metabolism of Eimeria species has been studied in suffi-
cient detail to produce a reasonably coherent picture and so information on
this parasite dominates this section. The little that is known about other
coccidial parasites is either mentioned as appropriate in Section 2.1 or
detailed separately in Sections 2.2, 2.3 and 2.4.

2.1. Eimeria

2.1.1. Energy Substrates


Current evidence suggests that carbohydrates serve as the major energy
substrates throughout the life cycle of Eimeria, although the possibility that
other compounds may be catabolized for energy generation has been little
explored. Lipid droplets are present to varying extents in all developmental
stages of the parasite and there has been a suggestion (Wilson and Fair-
bairn, 1961), as yet unconfirmed, that these may provide the energy for the
final stages of sporulation and during the dormancy of E. acervulina
oocysts (see Section 2.1.4.(a)). The possibility that Eimeria, like some
other parasitic protozoa, may catabolize amino acids or proteins for energy
production has been investigated only with oocysts and sporozoites, with
apparently negative results (see Section 2.1.3).
(a) Endogenous polysaccharide energy reserves. Granules identifiable
as polysaccharide, by iodine and periodic acid-Schiff (PAS) staining, are
present to varying degrees in almost all developmental forms of E. tenella.
146 G.H. COOMBS ET AL.

The localization of these granules is somewhat variable, although in sporo-


zoites they are predominantly found around the nucleus and refractile
bodies. After the sporozoite has invaded a host cell, the granules become
closely aligned around the refractile bodies. The significance of this is
unknown. Decreases in the number and size of the polysaccharide granules
are associated with sporulation (Patillo and Becker, 1955) and extracellular
survival (Vetterling and Doran, 1969); while resynthesis has been observed
during late merogony (schizogony) and gametogony (Edgar et al., 1944;
Ferguson et al., 1977). Several studies have found a strong relationship
between polysaccharide content and sporozoite or oocyst viability, as
assessed by both dye exclusion and infectivity assays in vivo (Augustine,
1980; Nakai and Ogimoto, 1983a). These observations are consistent with
the use of polysaccharide as an energy reserve.
Originally, the polysaccharide granules were thought to be composed of
glycogen (Wilson and Fairbairn, 1961), but a more thorough chemical and
enzymatic analysis of the isolated granules showed them to be amylopectin
(Ryley et al., 1969), a storage polysaccharide normally found in plants and
fungi. Apart from coccidia, only a few protozoa (including Gregarina
blaberae and the rumen ciliate Entodinium caudatum) have been shown
to use amylopectin as an energy reserve. The average chain lengths of
amylopectin from Eimeria species were found to be between 18 and 23
units, depending on the species. This is slightly shorter than typical plant
amylopectins but similar to the chain profile of amylopectin from G.
blaberae.
Amylopectin phosphorylase has been identified as the major enzyme
involved in amylopectin mobilization during sporogony of Eimeria
(Wang et al., 1975). This enzyme cleaves and phosphorylates glucose
residues from non-reducing ends of amylopectin chains. Presumably, but
this is still to be confirmed, a debranching enzyme is also involved in
mobilization of the polysaccharide. Unsporulated oocysts contain high
levels of amylopectin phosphorylase but the activity decreases almost
linearly during sporulation, so that sporulated oocysts contain less than
8% of the original activity. In most eukaryotes, glycogen phosphorylase is
regulated by phosphorylation and dephosphorylation, the end result of a
CAMP (cyclic adenosine monophosphate) cascade. Wang et al. (1975)
reported, however, that the eimerian enzyme seemed unsusceptible to
activation by phosphorylation and there was no change in CAMP concen-
trations during sporulation. Thus it was concluded that the changes in the
phosphorylase activity must be mediated by a different mechanism.
Karkhanis et al. (1993) identified an amylopectin synthase in extracts of
unsporulated oocysts of E. tenella. This soluble enzyme, optimally active
at pH 7.5, catalysed the transfer of uridine diphosphate (UDP)-glucose to
a primer of either glycogen or, at a higher rate, amylopectin. This speci-
BIOCHEMISTRY OF THE COCClDlA 147

ficity is unusual in that adenosine diphosphate (ADP)-glucose is the


normal substrate for amylopectin synthases from plants, whereas UDP-
glucose is the activated intermediate used by most eukaryotic glycogen
synthases. The enzyme presented typical Michaelis-Menten kinetics for its
substrates and no regulatory feature was noted. If, as seems likely, amylo-
pectin synthesis in Eimeria is analogous to glycogen synthesis in other
eukaryotes, then a self-glucosylating protein and a branching enzyme will
also be involved in the biosynthesis.
Polysaccharide granules have been observed in several other species of
coccidia, being variously identified as either amylopectin or glycogen
(Sheffield et al., 1977; Chaudry et al., 1985; Ferguson and Hutchison,
1987; Current, 1989).
One of the most exciting recent discoveries of coccidian biochemistry is
that mannitol is accumulated in large amounts in some stages of Eimeria
and may serve as an energy reserve during parts of the life cycle. This is
dealt with in detail in Section 2.1.2.(b).
(b) Use of exogenous carbohydrates. The accumulation of carbohydrate
during the intracellular phases of the parasites’ life cycle must be depen-
dent on the uptake of energy substrates from the environment. The bio-
chemical basis of this is poorly characterized, although uptake of some
substrates has been observed with several developmental stages of Eimeria.
Electron microscopical autoradiography was used to assess the incorpora-
tion into growing cells of E. tenella of [3H]glucose administered to infected
chickens. First generation meronts (schizonts) became labelled around the
nucleus, nucleolus, mitochondrion and endoplasmic reticulum, while
macrogametocytes showed less-extensive labelling (Matsuzawa, 1979).
Sporozoites of E. tenella also take up exogenous monosaccharides, and
either catabolize them or convert them to mannitol or amylopectin. Nakai
and Ogimoto (1983a, b, c) incubated sporozoites of E. tenella with [ 14C]-
glucose and showed that radioactivity could be recovered both in amylo-
pectin granules and released C 0 2 . They also investigated the influence of
exogenous carbohydrates on sporozoite viability (assessed by dye exclu-
sion) and amylopectin reserves (assessed by PAS staining). They found
that, in aerobic incubations, glucose, fructose, mannose and maltose were
effective in sparing the utilization of amylopectin, while galactose, glyco-
gen, sucrose, lactose, pyruvate and glycerol were not. The presence of
glucose in the incubation medium also increased the length of time that
sporozoites survived at 41°C and in addition the PAS-positive content of
‘starved’ sporozoites increased when they were placed in medium contain-
ing glucose.
Similarly, Smith and Lee (1986) showed that exogenous glucose greatly
improved the survival in vitro of E. tenella sporozoites over a 24-hour
period. They went on to investigate the mechanism of monosaccharide
148 G.H. COOMBS ETAL.

accumulation using radiolabelled glucose and some of its supposedly non-


metabolizable analogues. The uptake mechanism proved to be saturable
and relatively specific (in that glucose was accumulated faster than some of
its analogues), suggesting that the transport was carrier-mediated.
Glucose uptake did not appear to be influenced by exogenous sodium
levels or by ouabain (an inhibitor of Na+-K+ pumps), but it was inhibited
by phloretin, an inhibitor of facilitated-diffusion systems in many cell
types. These properties are suggestive of a passive carrier-mediated sugar
transport system. Similar transport mechanisms have been described in
other protozoan parasites and are typical of glucose transport systems in
many other cell types. The high capacity but unusually low affinity of the
carrier in sporozoites suggest that it is adapted to function in conditions of
high glucose concentration.

2.1.2. Pathways of Carbohydrate Metabolism


There has not been a comprehensive study of the pathways of carbohydrate
catabolism in any coccidian, including Eimeria. Much of our knowledge of
the enzyme systems operating in the parasites therefore comes from sur-
veys of the isoenzyme activities which have been used to discriminate
between species and strains. Fortunately enzymes involved in carbohydrate
catabolism are generally highly expressed and are particularly suited for
such analyses and so a good number have been reported. Many of the
enzyme activities which have been detected in Eimeria and other coccidian
species are listed in Table 1.
The following sections detail mainly enzymatic evidence for the exis-
tence of key pathways of energy metabolism, and the properties of some of
the enzymes mediating them. Evidence for the functionality of the path-
ways is dealt with in Section 2.1.4. The apparent overall situation is
summarized in Figure 2.
(a) Glycolysis. All the enzymes of the Embden-Meyerhoff glycolytic
pathway have been identified in extracts of sporozoites, merozoites and
oocysts of Eimeria species, and most have also been reported in other
coccidian species (see Table 1). The pathway in E. tenella, C. parvum and
T. gondii is unusual in containing, at least in the stages investigated, a
pyrophosphate-linked phosphofructokinase (PPi-PFK) instead of the con-
ventional ADP-linked enzyme (Peng and Mansour, 1992; Denton et al.,
1994, 1996a). These PPi-PFKs appear to be of the type I variety which are
normally associated with fermentative micro-organisms. Because of their
distribution, and the fact that their use increases the energetic yield of
glycolysis by 50% PPi-dependent PFKs are commonly considered to be
adaptations towards anaerobiosis (see Mertens, 1993). The presence of PPi-
PFK may suggest that these coccidia are adapted for anaerobic modes of
Table I A Enzymes of energy metabolism in coccidiaa
Eimeria
Unsporulated Sporulated Sporozoites Merozoites Meronts Microgame- Macrogame- Toxoplasma Sarcocystis Cryprosporidium
oocysts oocysts tocytes tocytes

Glycolysis
Hexokinase J1, 7 J1, 6 J1 J29,30, 31 J38 E l J42
Glucosephosphate
isomerase Ji’, 8 J6, 8, 25 J1 J21, 30, 31 J32, 33
Pyrophosphate-
phosphohctokinase J1 J1 J1 J1.22 J1
Aldolase J1, 3, 7 J6 (30 J38
Triose phosphate
isomerase Jl X6
Glyceraldehyde 3-phos-
phate dehydrogenase J6
Phosphoglycerate kinase J7
Phosphoglucose mutase JI, 9 J6. 25 J1 J 3 0 , 31 J34 J32, 33
Enolase J7 J6
Pyruvate kinase J1, 7 J1, 6 J1 J1 J38 J1
Lactate dehydrogenase J1, 2, I, 8 J1, 6, 8, 25 J 1 , 19 J30,31 J38 J1, 33, 42
TCA cycle
Citrate synthetase M
Aconitase J30
Iswitrate dehydrogenase $6 J 3 0 , 31 81
a-Ketoglutarate
dehydrogenase $6
Succinate CoA
synthetase E6
Succinate dehydrogenase J 4 . 15 E l 86, 1 J 4 J4 J4 84 84, 15 J1 E1.42
Fumarase 36 J29. 30 J33
Malate dehydrogenase J6 J42
Table 1B Enzymes of carbohydrate metabolism in coccidia"
Eimeria
Unsporulated Sporulated Sporozoites Merozoites Meronts Microgame- Macrogame- Toxoplasma Sarcocystis Cryptosporidium
oocysts oocysts tocytes tocytes

Pentose phosphate pathway


Glucose 6-phosphate
dehydrogenase J5 J6, 8 J 1 , 19 J 2 9 , 20, 31 J38
6-Phosphogluconate
dehydrogenase J8 J8 J30 J34, 38
Gluconeogenesis
Glucose 6-phosphatase J4 J4,25 J4 J4 ?4 J4
Fructose M-bisphos-
phatase Jl
F'yruvate carboxylase J29
Mannitol cycle
Mannitol 1-phosphate
dehydrogenase J20, 21 J 21 J21 J20 J20
Mannitol 1-phosphatase J20, 21 J 21 J21 J21 J20
Mannitol dehydrogenase J20, 21 J 21 J21 J21
(Hexokinase) J20, 21 J 21 J21
Miscellaneous carbohydrate
metabolism
Phosphoglucomutase J7 J6 J30, 31 J34 J32. 33
Amylopectin
phosphorylase J16 J16
Amylopectin synthase J17
a-Glycerophosphate J15
dehydrogenase J15
Phosphoenolpyruvate
carboxykinase J6 J42
Malic enzyme J6
Glycerol 3-phosphate
dehydrogenase J1 J 1 , 30
Amylase J4
Table IC Enzymes of amino acid metabolism and some hydrolases in coccidia"
Eimeria
Unsporulated Sporulated Sporozoites Merozoites Meronts Microgame- Macrogame- Toxoplasma Sarcocystis Ctyptosporidium
oocysts oocysts tocytes tocytes

Glutamate dehydrogenase J14 J14


Aspartate aminotransferase J14 J14 J27. 30 J39
ATPase J4 J4 J4 J 4 , 26 J4 J4
Alkaline phosphatase J4 J4 X19 J4 J4 J4 J4 J28, 30.31 J35, 31
Acid phosphatase J 4 , 10 J 4 , 10, 11 J10, 19 J 4 , 18 J 4 , 18.26 J4,26 J 4 , 18, 26 J27, 28, 41 J35, 31 (42
Non-specific esterase J4 J4 J4 J4 J4 J4 (30, 31
f3-Glucuronidase 84 x4 x4 $4 P4 84 J28
Leucine naphthylamidase x4 ?4 x4 P4 x4 P4
Aconitate hydratase J7
Glyoxalase I Jl
Leucine aminopeptidase Jl, 19 %I9 J19 J19 (19 J30
Adenosylmethionine
decarboxylase J12
Arylsulphatase J13
p-Galactosidase 819 J19 J19 J19 J28 J35 J42
p-G1ucosidase El9 X19 P19 P19
Carboxylic ester
hydrolases P19
S'-Nucleotidase J26 J26 J26 J23

" J, Detected, Ir, apparently absent. Numbers indicate references: 1, Denton et al. (1994,1996aand unpublished observations); 2, Fransden and Cooper
(1972); 3, Mitchell and Daron (1982);4, Michael and Hodges (1973);5, Fransden (1976,1978);6, Smith, N.C. et al. (1994);7, Andrews et al. (1990); 8,
Shirley (1975); Rollinson et al. (1979); 10, Hosek et al. (1988); 11, Farooqui and Hanson (1988); 12, San-Martin Nunez et al. (1987); 13, Farooqui et al.
(1983); 14, Wang et al. (1979); 15, Beyer (1970); 16, Wang et al. (1975); 17, Karkhanis er al. (1993); 18, Heller and Scholtyseck (1970); 19, Fransden
(1970); 20, Schmatz et al. (1989); 21, Michalski et al. (1992); 22, Peng and Mansour (1992); 23, Sibley et al. (1994a); 25, Shirley et al. (1977); 26,
Vetterling and Waldrop (1976); 27, Darde et al. (1992); 28, Manafi et al. (1993); 29, Takeuchi et al. (1980); 30, Darde et al. (1988); 31, Barnert et al.
(1988); 32, Awad-El-Kariem et al. (1993,1995);33, Ogunkolade et al. (1993); 34, Atkinson and Collins (1981);35, Farooqui et al. (1987); 36, Chaudry
et al. (1985); 37, Chaudry et al. (1986b); 38, Gupta er al. (1992); 39, Gupta er al. (1993); 40,Fulton and Spooner (1960); 41, Metsis er al. (1995); 42,
Malek et al. (1996).
152 G.H. COOMBS ET AL.

Figure 2 Pathways of carbohydrate metabolism operating in Eimeria tenella. 1,


amylopectin phosphorylase (this has not yet been reported); 2, amylopectin
synthase; 3, phosphoglucomutase; 4,hexokinase; 5 , glucosephosphate isomerase;
6, pyrophosphate-dependent phosphofructokinase; 7, pyruvate kinase; 8, lactate
dehydrogenase; 9, mannitol 1-phosphate dehydrogenase; 10, mannitol 1-phospha-
tase; 11, mannitol dehydrogenase. Key to abbreviations: GlP, glucose 1-phosphate;
G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F16P2, fructose 1,6-bisphos-
phate; PEP, phosphoenolpyruvate; MlP, mannitol 1-phosphate; TCA cycle, tricar-
boxylic acid cycle. Energy substrates are in capital letters and underlined, released
end products are boxed, dotted lines indicate regulatory interactions.
BIOCHEMISTRY OF THE COCClDlA 153

energy production, at least for parts of their life cycle (Coombs and Muller,
1995). Like other micro-organisms containing PPi-utilizing glycolytic
enzymes, the coccidia lack cytosolic pyrophosphatase activity. The key
feature of type I PPi-PFKs is their lack of regulatory features. Since PFK is
a key enzyme in regulating glycolytic flux in most eukaryotes, organisms
which possess type I PPi-PFKs must utilize relatively unusual mechanisms
of glycolytic control.
In E. tenella and T. gondii it seems likely that glycolytic control is
exerted, at least partially, through the enzyme pyruvate kinase. Unlike
some other micro-organisms which have a PPi-PFK, the coccidia species
which have been investigated have an ADP-specific pyruvate kinase (PK)
rather than a PPi-specific activity (Denton et al., 1994, 1996a). The PKs of
E. tenella and T. gondii display sigmoidal saturation kinetics with respect
to their substrate phosphoenolpyruvate, but can be activated to hyperbolic
kinetics by certain compounds. The most potent of these activators are
glucose 6-phosphate and fructose 6-phosphate, which are relatively unu-
sual modulators. Fructose 1,6-bisphosphate, the major activator of most
eukaryotic PKs, is largely without effect. Unusually, the PK from C.
parvum shows no evidence of regulatory properties, presenting simple
Michaelis-Menten kinetics with respect to both its substrates (Denton et
al., 1996a). The only other PK so far reported not to be under allosteric
regulation is the type I enzyme from mammalian muscle (Fothergill-Gil-
more and Michels, 1993). Hexokinase, another regulatory enzyme in most
eukaryotes, appears to be unregulated, at least in Eimeria. Interestingly, the
parasite appears to contain only one hexokinase and this is capable of
phosphorylating both glucose (as in glycolysis) and fructose (as in the
mannitol cycle, see Section 2.1.2.(b)) (Schmatz et al., 1989; H. Denton
et al., unpublished observations). The presence of PPi-PFK is a distinct
difference between the coccidia and their hosts. The possibility that this
offers promise for rational chemotherapy is given credence by the report of
Peng et al. (1995) that some phosphonic acid analogues are capable of
specifically inhibiting PPi-PFK purified from T. gondii and also are selec-
tively toxic to parasites within their host cells in vitro.
All coccidia species which have been investigated contain high levels of
lactate dehydrogenase (LDH), the enzyme capable of mediating the oxida-
tion of reduced nicotinamide adenine dinucleotide (NADH) under anaero-
bic conditions. Once again, this might reflect a high dependence on
anaerobic energy generation within the coccidia. The enzyme was purified
from E. stiedai and characterized by Fransden and Cooper (1972). A single
enzyme was identified with an electrophoretic mobility corresponding to
the LDH4 of vertebrates. The enzyme was specific for L-lactate but was
capable of catalysing the reduction of both f3- and a-NAD (although the
rate with the latter was only 3% that with the former). The enzyme subunits
154 G.H. COOMBS EJAL.

proved highly resistant to dissociation and showed no obvious hybridiza-


tion with H subunits from chick heart, implying a fundamentally different
subunit construction to the isoenzymes characterized from vertebrates.
Structural data have now been obtained through analysis of the genes.
Interestingly, Toxoplasma has two genes which appear to be entirely
stage-specific (Yang and Parmley, 1995). In comparison with most
LDHs, the genes encode a five amino acid insert around the active site.
Similar inserts are present in Plasmodium and also Eimeria (A.N. Vermeu-
len, personal communication), indicating that this may be a common
characteristic of the Apicomplexa. There is a large increase in the amount
of the eimerian LDH protein in the intracellular stages (A.N. Vermeulen,
personal communication), suggesting that they depend on fermentative
metabolism to a significant extent. Fructose 1,6-diphosphate aldolase of
E. stiedai has also been purified and identified as a type 1 enzyme typical of
those found in mammalian cells (Mitchell and Daron, 1982; Wang, 1982).
(b) Mannitol metabolism. When Wilson and Fairbairn (1961) measured
the total masses of lipid, protein and anthrone-sensitive carbohydrate in
unsporulated oocysts of E. acervulina, 25% of the oocyst dry mass was left
unaccounted for. This missing component has now been shown to be
predominantly mannitol (Schmatz, 1989; Schmatz et al., 1989), a carbohy-
drate previously known to occur only in fungi. Further investigation
revealed the presence of enzymes associated with a mannitol cycle (Figure
3). With the exception of hexokinase (which, as previously stated, can
accept either glucose or fructose as substrates), the enzymes are all very
specific in their reactions and their Michaelis constant (K,) values suggest
that the pathway acts in only one direction (as shown in Figure 3).
Schmatz and colleagues (1989) reported that mannitol was present at
very high levels (up to 300 m) in unsporulated oocysts of E. tenella but
fell during sporulation to about 10 mM. In contrast, Michalski et al. (1992)
found only small amounts of mannitol (50-80 nmol per lo6 oocysts) in
unsporulated oocysts of the same species. They reported, however, that
mannitol concentrations increased rapidly during the early stages of spor-
ulation, concomitant with a decrease in amylopectin levels, and then
diminished slowly and reached a basal level after 40 h. These changes
correlated with changing activities of the mannitol cycle enzymes. The
apparent discrepancy between the results of these two groups may be due to
the fact that the Michalski et al. (1992) used oocysts obtained directly from
the caecum rather than from faeces (the source used by Schmatz et al.,
1989) and that these may not have been fully mature, so that the initial
changes that occurred in their study may have been due to the final
maturation of the oocysts which normally occurs within the host.
Sporozoites were shown to contain mannitol cycle enzymes and also to
be capable of converting glucose into mannitol (Michalski et al., 1992;
BIOCHEMISTRY OF THE COCClDlA 155

Glucose 6-phosphate

t N
NAD(P)'

A
\
D P Mannitol
~ 1-phosphate wpi
5

ANt:
\

""k
Fructose 6-phosphate

ATP Fructose KADH

Fructose 1,6-bisphosphate

Figure 3 The mannitol cycle in Eimeria tenella. 1, mannitol I-phosphate dehy-


drogenase; 2, mannitol 1-phosphatase; 3, mannitol dehydrogenase; 4, hexokinase;
5, glucosephosphate isomerase; 6, phosphofructokinase.

H. Denton et al., unpublished observations), demonstrating that mannitol


and the mannitol cycle occur in stages other than the oocysts. Recent
evidence suggests, however, that the synthetic part of the cycle is fully
functional only during the sexual phase of the life cycle (Schmatz, 1997)
and that this leads to the large concentration of mannitol in the oocyst.
Most interestingly, this part of the cycle appears to be mainly controlled
through the binding of a protein inhibitor of the first enzyme, mannitol 1-
phosphate dehydrogenase (Schmatz, 1997). It is currently not known how
the degradation of mannitol is regulated. It seems that there is only one
hexokinase isoenzyme (rather than one specific for glucose that participates
in glycolysis and one specific for fructose that functions in mannitol
mobilization) and that it is not tightly controlled. It is clear that there
must be co-ordinated regulation of the fluxes to and from mannitol and
amylopectin and through glycolysis, but the details remain to be elucidated.
The genes encoding the enzymes of the mannitol cycle have now all been
cloned and some have been expressed heterologously to provide active
enzymes (Schmatz, 1997). This is likely to lead to a marked increase in our
knowledge of the enzymes and their potential as drug targets.
The function of the mannitol cycle in coccidia is uncertain, as indeed it is
in fungi. An obvious possibility is that it is acting as an energy reserve
(although why the parasite should require another reserve in addition to
amylopectin is unclear). Several other roles have been proposed (see
156 G.H. COOMBS ETAL.

Schmatz, 1989; 1997), although to date there is little direct evidence for or
against any of them. The following possibilities have been suggested:
1. NADH generated during the breakdown of mannitol may be used
directly for oxidative phosphorylation and so result in energy produc-
tion.
2. The first part of the pathway may act as an electron sink for replenish-
ment of NAD+ under anaerobic conditions.
3. Mannitol may act as an osmoregulator, keeping the oocyst wall rigid
during maturation.
4. Mannitol may have a protective effect against superoxide ions.
5 . Mannitol phosphate may be polymerized to act as a structural compo-
nent in the oocyst or sporocyst wall.
The mannitol cycle also appears to be present in both Toxoplasma and
Cryptosporidium and so it may be a common feature of all coccidia. Both
mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase have
been detected in C. parvum (see Schmatz, 1989) and specific antibodies
have apparently been used to show that mannitol 1-phosphate dehydrogen-
ase, but not its inhibitor, is present in the sexual stages of both Toxoplasma
and Cryptosporidium, whereas both proteins occur in other stages of the
life cycle (Schmatz, 1997).
(c) Tricarboxylic acid. It is currently not certain that Eimeria (or any of
the other coccidia) contains a fully functional tricarboxylic acid (TCA)
cycle at any stage of its life cycle. Of the classical TCA cycle enzymes,
Smith et al. (1994) were able to detect only malate dehydrogenase in
sporulated oocysts of E. tenella. Both phosphoenolpyruvate carboxykinase
(PEPCK) and malic enzyme were present, however, and the authors con-
cluded that Eimeria sporulated oocysts lack a conventional TCA cycle but
contain a PEPCK bypass similar to that in anaerobic protozoa such as
Giardia lamblia (syn. G. duodenalis) and Trichomonas vaginalis (see
Coombs and Muller, 1995). These results, however, conflict with the
assertion that ‘There is ample evidence indicating a functional tri-
carboxylic acid cycle in coccidia in general’ (Wang, 1982). The only
evidence quoted as justification for this statement was the detection of
isocitrate dehydrogenase and malate dehydrogenase in unsporulated
oocysts and the demonstration by cytochemical analysis of succinate
dehydrogenase and isocitrate dehydrogenase in proliferative Toxoplasma.
Succinate dehydrogenase was also apparently detected cytochemically in
several stages of Eimeria (see Beyer, 1970; Michael and Hodges, 1973).
However, we too have been unable to detect this enzyme or NAD+-specific
isocitrate dehydrogenase in oocysts or sporozoites by enzymatic analysis
(see Table 2).
Thus, there is evidence both for and against a functional TCA cycle. It
BIOCHEMISTRY OF THE COCClDlA 157

remains a possibility that the TCA cycle is operative in just some stages of
the parasite. Certainly, sporulation of eimerian oocysts occurs only under
aerobic conditions and can be inhibited by inhibitors of the respiratory
chain (see Section 2.1.2.(d)), suggesting that this form of the parasite may
also have a functional, but perhaps only partial, TCA cycle. Conversely,
however, the end products released by eimerian sporozoites and their lack
of sensitivity to respiratory inhibitors (see Section 2.1.2.(d)) are consistent
with the TCA cycle playing little part in their energy metabolism (see
Section 2.1.4). There are few data on other developmental stages.
(d) Respiratory chain. All developmental stages of Eimeria species
possess distinctive elongate, cristate mitochondria. The processes of spor-
ulation and excystation are associated with vigorous respiratory activity
including the consumption of oxygen and production of carbon dioxide
(see Section 2.1.4 for details). This is reversibly inhibited by cyanide and
other inhibitors of electron transport, implying that it is mediated, at least
partially, by a cytochrome-containing respiratory chain. In contrast, excys-
tation continues and released sporozoites remain viable when exposed to
these inhibitors of electron transport, suggesting that this stage does not
need a functional respiratory chain (Brown et al., 1996).
The detailed composition of the cytochrome chain is yet to be eluci-
dated but there is evidence that it differs from those found in mammalian
mitochondria. Mitochondria isolated from unsporulated oocysts of E.
tenella consumed oxygen in the presence of conventional respiratory
substrates, including NADH and succinate (Wang, 1975; Fry and Wil-
liams, 1984). The isolated mitochondria were uncoupled with respect to
oxidative phosphorylation: oxygen consumption was not dependent on
ADP, and the uncoupler carbonyl rn-chlorophenylhydrazone had no effect
(Fry and Williams, 1984). It remains unclear whether this lack of cou-
pling is an inherent property of the mitochondria or a result of damage
inflicted during the isolation procedure. Spectrophotometric analysis of
the mitochondria revealed absorbance maxima characteristic of type a and
b cytochromes but no clear indication of a type c cytochrome. Interaction
with carbon monoxide suggested that there might be two type a cyto-
chromes present, cytochrome a3 of cytochrome oxidase and an o-type
cytochrome. The mitochondria1 respiration was inhibited by cyanide,
azide, carbon monoxide (inhibitors of cytochrome oxidase) and also by
antimycin A, which blocks co-enzyme Q(Q)-cytochrome reductase.
However, rotenone and amytal were largely without effect, suggesting
that NADH-Q reductase was either absent or presented unusual proper-
ties. Similar findings have been reported for Plasmodium, which is
thought to lack the NADH-Q reductase and to use succinate dehydrogen-
ase as the major feed-in point (Fry, 1991).
The respiratory pathway in Eimeriu has attracted particular interest as
158 G.H. COOMBS ET AL.

the apparent site of action of several anticoccidial drugs. The quinolone


and pyridine coccidiostats and the 2-hydroxynaphthoquinonesappear to act
by blocking different sections of the respiratory chain (Wang, 1975, 1976;
Fry and Williams, 1984; Fry et al., 1984). Resistance to quinolones and
pyridines also appears to be manifested at the level of electron transport, as
much higher concentrations of drug are required to inhibit respiration in
mitochondria from drug-resistant parasites than from those that are drug-
sensitive (Wang, 1975, 1976; Fry and Williams, 1984). The anticoccidial
activities of quinolones (in particular decoquinate) and the pyridone clo-
pidol exhibit peculiar features which provide clues to the structure of the
eimerian respiratory chain. These two drugs have a marked synergistic
effect both in vivo and in vitro; however, acquisition of resistance to one of
the two drugs consistently leads to greater sensitivity to the other (Fry and
Williams, 1984). These observations led to the suggestion that E. tenella
has a branched or parallel electron transport chain, one part of which is
more sensitive to 4-hydroxyquinolones while the other is blocked by
clopidol. If so, resistance to one of the drugs could be mediated by electron
transport being diverted towards the less sensitive of the pathways. This
model would explain both the collateral sensitivities and the apparent
synergism between the two drugs. The pathway more sensitive to clopidol
also appears to be the more readily blocked by cyanide and azide, as
resistance to the drug is associated with decreased sensitivity to these
agents (and the reverse is true for the decoquinate-sensitive route). It is
interesting to view these results in the light of the detection of two type a
cytochromes in the mitochondria: perhaps these represent two terminal
oxidases?
The discovery that plastid-like organelles occur in coccidia and that they
may contain components of a respiratory chain (Hackstein et al., 1995; see
Section 6) suggests that this may contribute to the cell’s respiration. There
are also some data from the use of hydroxamic acids that Toxoplasma
contains a plant-like alternative oxidase (F. Roberts, C. Roberts, L. Mets, J.
Johnson and R. McLeod, personal communication). Re-evaluation of the
conclusions drawn from previous studies, as outlined above, will need to be
undertaken when more details are available on the components of the
plastid and its function.
(e) Other pathways
(i) Pentase phosphate pathway. Glucose 6-phosphate dehydrogenase,
the first enzyme of the pentose phosphate pathway, has been purified and
characterized from unsporulated oocysts of E. stiedai (see Fransden, 1976,
1978). Like mammalian enzymes, the enzyme was specific for NADP and
could accept glucose, at a low rate, as well as glucose 6-phosphate. A large
range of compounds was tested as potential regulators of the enzyme. Of
these the purine triphosphates (adenosine triphosphate, inosine triphos-
BIOCHEMISTRY OF THE COCClDlA 159

phate and guanosine triphosphate: ATP, ITP and GTP) and the pyrimidine
triphosphates (cytidine triphosphate and uridine triphosphate: CTP and
UTP) were effective inhibitors of the enzyme at millimolar concentrations,
while phosphoenolpyruvate (a powerful inhibitor of some bacterial
enzymes) had no effect. There was, however, significant inhibition by oleic
and linoleic fatty acids. 6-Phosphogluconate dehydrogenase activity has
also been detected in starch gels of sporulated and unsporulated oocysts
(Shirley, 1975). From the presence of these enzymes, it seems likely that a
functioning pentose phosphate shunt exists in Eimeria species. It could be
envisaged that this pathway would be particularly important in the rapid
growth situations of merogony and gametogony where NADPH and ribose
requirements would be high. Indeed, James (1980) has presented circum-
stantial evidence that the pathway is very active in isolated meronts (see
Section 2.1.4.(c)).
(ii) Gluconeogenesis. Fructose 1,6-bisphosphatase and glucose 6-phos-
phatase have both been detected in extracts of Eimeria and so it would
seem that the parasites have gluconeogenic capabilities. This may relate to
the importance of amylopectin and possibly mannitol as energy reserves,
although at present there is no evidence that exogenous substrates other
than carbohydrates are used in their synthesis. Perhaps the endogenous
reserves of lipid can be converted to the carbohydrate stores, although this
would probably require a glyoxylate cycle (see below).
(iii) Glyoxylate cycle. No isocitrate lyase or malate synthetase activity
could be detected in crude extracts of E. tenella unsporulated oocysts
(Wang, 1982), implying that a glyoxylate pathway is not present.

2.1.3. Other Catabolic Pathways and Enzymes


The possible use of amino acids as energy substrates has not been investi-
gated in any detail. Anaerobic parasitic protozoa such as Trichomonas
vaginalis and Giardia lamblia, however, do use some amino acids, espe-
cially arginine, in this way (Coombs and Muller, 1995) and so it will be
interesting to see if this is an anaerobic characteristic also shared by
coccidia. Sporozoites of E. tenella did not consume amino acids from
the incubation medium (whether exogenous carbohydrates were available
or not, and under both aerobic and anaerobic conditions), suggesting that at
least this stage of the parasite does not use amino acids as major energy
substrates (H. Denton et al., unpublished observations).
High activities of glutamate dehydrogenase (Wang et al., 1979) and
aspartate aminotransferase (Shirley and Rollinson, 1979) have been
detected in oocyst extracts, but there is no report of any other enzymes
possibly involved in amino acid breakdown. It has been shown that most, if
not all, of the intra-host stages of the parasite are capable of taking up amino
160 G.H. COOMBS ET AL.

acids from the environment, although these were mainly incorporated into
parasite proteins (Krylov and Svanbaev, 1980). There was a marked
decrease in the concentration of most free amino acids during sporulation
of oocysts (H. Denton et al., unpublished observations).
Sarcocystis fusiformis was analysed for aspartate aminotransferase and
alanine aminotransferase (Gupta et al., 1993). Both of these enzymes were
found, indicating that amino acids may be converted to keto acids which
could be catabolized as an energy source.
ATPases are an important group of enzymes that regulate intracellular
ATP and ion levels within cells (Pederson and Carafoli, 1987). ATPase
activity has been detected by histochemical means in most stages of
Eimeria (see Michael and Hodges, 1973; Vetterling and Waldrop, 1976).
The activity in Eimeria sporozoites has been characterized (K.-W. Thong,
unpublished observations), as has that of tachyzoites of T. gondii (see
Takeuchi et al., 1980). Both parasites appear unusual in lacking (or having
very low levels of) Na+/K+-ATPase,the enzyme responsible for maintain-
ing high potassium ion concentrations within most eukaryotic cells. The
apparent absence of this enzyme from Toxoplasma led Takeuchi et al.
(1980) to speculate that the parasite plasma membrane might be freely
permeable to Na+ and K+ and that transmembrane fluxes of these ions
occurring during transition between different environments (in particular
during invasion of host cells) might be important effectors of the parasite’s
metabolism. In apparent support of this conjecture, they demonstrated that
the parasite’s protein synthesis was markedly stimulated by K+ concentra-
tions up to 150 m~ and also showed that there were significantly higher
levels of Na+ than K+ in lysates of tachyzoites.
While lacking a Na+/K+ ATPase, both Eimeria and T. gondii contain
Mg2+-ATPaseactivity (probably the mitochondria1 variety which partici-
pates in oxidative phosphorylation). Ca2+-ATPaseactivity was also detected
in membrane preparations of the parasites. The Mg2+-and Ca2+-ATPasesof
Eimeria presented similar kinetic parameters and pH optima to the equiva-
lent ATPases in chick liver cells, but had significantly different inhibitor
sensitivities (K.-W. Thong, unpublished observations). In particular, the
parasite enzymes were much less affected by azide than their host cell
counterparts, while N-ethyl maleimide proved to be a potent inhibitor of
the eimerian Ca2+-ATPasebut had little effect on the host enzyme. The
activities of a number of ionophores and synthetic anticoccidial drugs were
investigated but they appeared not to inhibit the ATPases in Eimeria.

2.1.4. Variation with Phase of Development


(a) Oocysts and sporulation. The oocyst wall is a complex, highly resistant
structure (Stotish et al., 1978) which seems impermeable to all but the
BIOCHEMISTRY OF THE COCClDlA 161

smallest molecules and respiratory gases (El-Moukdad, 1976). Since it


cannot take in nutrients from the environment, the oocyst is dependent
on its endogenous energy reserves to fuel the processes of sporulation and
to maintain viability during dormancy.
Early studies suggested that the initial phase of sporulation is fuelled by
carbohydrate catabolism (Wilson and Fairbairn, 1961) but the later stages
by lipid oxidation. More recently, consumption of both amylopectin and
mannitol has been reported (see Section 2.1.2.(b)).
The process of sporulation seems to be strictly aerobic and will not
proceed either at low oxygen tension or in the presence of respiratory
inhibitors such as cyanide (see Wang, 1982). Several researchers have
observed vigorous respiratory activity during the process. Wilson and
Fairbairn (1961) found respiratory activity to be maximal at the start of
sporulation; thereafter it fell off steadily with a constant, very low rate
being achieved after 2-3 days. The respiratory profiles reported by Wagen-
bach and Bums (1969) for the sporulation of E. tenella and E. stiedui were
more complex, showing variable rates of respiration throughout the pro-
cess. The rate reached a peak around the binucleate stage (between 10 and
18 h), this maximal activity being followed by a marked depression corre-
lating with the appearance of the early spindle stage. Once sporulation was
complete, respiration fell to a barely detectable level until either the fuel
reserves were exhausted or ingestion by a host caused excystation to occur.
Wagenbach and Bums (1969) noted that sporulated oocysts kept under
anaerobic conditions remained viable for only a quarter of the time of those
kept under aerobic conditions. This was suggested to reflect less efficient
usage of energy substrate under anaerobic conditions, although the poly-
saccharide content of the oocysts was not monitored.
Monitoring of the intra-oocyst concentration of possible fermentation
products during the course of sporulation of E. tenella confirmed that there
was no accumulation (H. Denton et al., unpublished observations), as
would be expected if energy metabolism was fully oxidative. This experi-
ment and conclusion were based upon the assumption that the oocyst wall
is indeed impermeable to metabolites. Support for this idea was provided
by the finding that there were significant concentrations of acetate and
lactate even in unsporulated oocysts, and that the glycerol concentration
was very high (approximately 50 m).
Sporulated oocysts contain considerably lower activities of some glyco-
lytic enzymes than unsporulated oocysts (Table 2), consistent with the
latter, effectively dormant stage having a low glycolytic flux.
(b) Sporozoites and excystation. Excystation occurs efficiently under
both aerobic and anaerobic conditions, and in the presence of respiratory
chain inhibitors such as cyanide (Wagenbach and Bums, 1969; Wang,
1976). The process is associated with the disappearance of amylopectin
Table 2 Activities of enzymes of energy metabolism in Toxoplasma, mouse brain extracts, Cryptosporidium and Eimeria
Enzyme activity (nmol/min/mg protein)"

Sample Hexokinase PPi-PFK PK LDH NADP+ NAD+ SDH


-1CDH -1CDH
T. gondii, 1.92 1.0 (3) 6342203 (3) 46582 1275 (7) 6552 196 (6) 130233 (3) n.d.cO.2 (3) 1326 (3)
tachyzoites
T.gondii, n.a. 265215 (3) 11338+2201 (3) 22592827 (6) 130238 (3) n.d.l.0 (4) n.d.<26 (3)
bradyzoites
Mouse brain n.a. n.d.<1.0 (3) 52192398 (6) n . d ~ O . 3(4) 6425 (3) 2325 (3) 2523 (3)
C. parvum, n.d.<1.0 (3) 331233 (3) 29472775 ( 5 ) 82244 (6) n . d ~ O . 9(3) n.d.<0.9 (3) n.d.c 1.0 (3)
oocysts
E. tenella, 36*4 (3) 191246 (3) 21052572 (3) 26172244 (3) 823 (5) n.d.cO.4 (4) n.d.<0.4 (3)
unsporulated
oocysts
E. tenella, 322 (3) 56224 (4) 124260 (4) 114277 (4) 2028 (5) n.d.<0.6 (4) n.d.<0.6 (3)
sporu1ated
oocysts
E. tenella, 5328 (4) 105215 (5) 13492167 (3) 1254274 (3) 122274 (7) n.d.<0.4 (4) n.d.cO.4 (3)
sporozoites

*SD, n in parentheses; n.d., not detectable, n.a., not assayed. Data from Denton et al., 1994, 1996a.b and unpublished observations. Key to enzymes:
PPi-PFK, pyrophosphate-phosphofructokinase;PK, pyruvate kinase; LDH, lactate dehydrogenase; ICDH, iswitrate dehydrogenase; SDH, succinate
dehydrogenase.
Maximum contribution of host-cell enzymes to activities measured in bradyzoite extracts (nmoVmidmg protein): PFK, 0; PK, 227; LDH, 0; NADP"-
ICDH, 2.8; SDH, 1.1.
BIOCHEMISTRY OF THE COCClDlA 163

granules from the cytoplasm, which suggests that these are the source of
the energy required.
Excystation under aerobic conditions was reported to be accompanied by
vigorous respiratory activity (Vetterling, 1968). This was initiated upon
breakage of the oocyst wall and reached a maximum 2-10 min after
excystation began, before declining to a constant low level which was
maintained until the sporozoites were stimulated to invade host cells. Ryley
(1973) compared the rates of respiration reported by Vetterling (1968) with
the amount of carbohydrate consumed during the equivalent period (Vet-
terling and Doran, 1969) and found that approximately 6 moles of oxygen
were consumed per mole of glucose. This ratio suggested that glucose was
being completely oxidized to carbon dioxide and water and implied that
both a TCA cycle and respiratory chain were operative in this develop-
mental form of the parasite.
The fact that E. tenella sporozoites can excyst and remain motile in the
absence of oxygen, however, suggests that they are facultative anaerobes. It
should be remembered that they exist in the gut lumen, where oxygen
concentrations are low. Ryley (1973) monitored the anaerobic endogenous
metabolism of E. tenella sporozoites and identified lactate as the major end
product (1.49 moYmol glucose); there was also some carbon dioxide and
glycerol production (0.50 and 0.28 moYmol glucose, respectively). These
products accounted for 97% of the amylopectin consumed over the experi-
mental period. Thus it seemed that, under conditions of limited oxygen,
Eimen'a sporozoites obtain energy through glycolytic substrate level phos-
phorylation and depend largely on lactate production for regenerating the
intracellular pool of NAD. The origin of the C 0 2 was unclear, although
some may have been produced via the pentose phosphate pathway or via
pyruvate decarboxylation during the production of acetate.
More recent studies have confirmed these findings with both nuclear
magnetic resonance and enzymatic analyses showing that sporozoites of
E. tenella catabolized glucose to lactate, glycerol and acetate (but not
malate or succinate) under aerobic and anaerobic conditions (H. Denton
et al., unpublished observations). Although somewhat more acetate was
produced when oxygen was present, the total amount of fermentative
products was rather similar under both gaseous conditions - suggesting
that sporozoites have a low capacity for fully oxidative energy metabolism.
These findings are at variance with the conclusions arising from earlier
studies on respiratory rates. The oxygen consumed by sporozoites may
have a role other than energy metabolism.
It has been reported that the invasion rate of E. tenella sporozoites into
host cells in vitro was significantly greater under anaerobic conditions than
when oxygen was present (Wrede et al., 1993). A similar situation has also
been reported for C. parvum (see Upton et al., 1994b). These observations
164 G.H. COOMBS ETAL.

suggest that the sporozoites function better at low oxygen tension, perhaps
because they are specifically adapted towards anaerobiosis.
(c) Zntracellular stages. The metabolic activities of the intracellular
stages of Eimeria are difficult to monitor directly and interpretation of
data is complicated since it is hard to know whether observed changes are
caused by activity of the parasite or alterations in host cell metabolism.
Several groups have attempted to compare the rates of respiration or
anaerobic glycolysis in normal and parasitized caecal tissue, but these
experiments have yielded unreproducible and contradictory results (see
Ryley, 1973). However, a common feature of chicken coccidiosis is a
markedly lowered intestinal pH (van der Horst and Kouwenhoven,
1973). This might well reflect high levels of lactate production by para-
sitized cells, which would imply that there are significant periods of
fermentative metabolism during this phase of the life cycle.
James (1980) showed that isolated meronts of E. tenella were able to
take up and catabolize radiolabelled glucose to carbon dioxide (other
products were not investigated). The rate of production was approximately
three times higher when [l-'4C]glucose was used as opposed to [6-14C]-
glucose, suggestive of high pentose phosphate pathway activity.
Beyer (1970) used cytochemical techniques to investigate the levels of
succinate dehydrogenase and a-glycerophosphate dehydrogenase in the
intracellular stages of E. magma. Growing macrogametes appeared to
have high levels of a-glycerophosphate dehydrogenase but no detectable
succinate dehydrogenase, while the zygote immediately after fertilization
showed high levels of both enzymes. Beyer (1970) interpreted this as
reflecting changes in mitochondria1 activity during the life cycle of the
parasite and considered that, given the ample nutritional means provided
by the host cells, glycolysis can easily cover the energy requirements of the
developing parasites. Later, as the host substrate becomes exhausted and
the merozoites or zygote prepare for an extracellular phase of life, there is a
switch to more efficient aerobic metabolism using the TCA cycle. An
interesting alternative hypothesis is that oxygen becomes limited from
fairly early in the intracellular phase (the parasite grows and multiplies
very rapidly and thus would soon consume the available oxygen) and that
the switch to the potential for aerobic metabolism represents a preadaption
to extracellular existence in an aerobic environment. It seems highly likely
that there are marked changes in energy metabolism during the life cycle of
Eimeria, but more extensive studies are necessary to provide details of the
extent and significance of the variations.
BIOCHEMISTRY OF THE COCClDlA 165

2.2. Toxoplasma

The biochemistry of Toxoplasma attracted rather little attention until rela-


tively recently. The situation changed markedly, however, with the recog-
nition of the parasite as a major cause of mortality in immunosuppressed
individuals. The area of energy metabolism, however, remains ill defined.
The parasite is apparently similar to Eimeria in that it appears to cata-
bolize a substantial proportion of its energy substrates to fermentative end
products even when oxygen is present in abundance. In what remains the
most detailed analysis of T. gondii energy metabolism undertaken to date,
Fulton and Spooner (1960) demonstrated the ability of a range of substrates
to stimulate oxygen consumption by intact tachyzoites of T. gondii. Glu-
cose was the most effective substrate identified and glutamine was almost
as good, while lactate and various intermediates of the TCA cycle were
unable to support respiration. Interestingly, low levels of respiration were
observed in the presence of glucose-free glycogen - a substance likely to
be present at high concentrations in host cells. Toxoplasma itself contains
the polysaccharide amylopectin, presumably as an energy store, which is
present in abundance in bradyzoites but appears to be absent or present at
only low levels in tachyzoites. The explanation for this marked difference
is unknown, but it could reflect the need of the bradyzoite stage (which is
surrounded by a cyst wall that may be largely impervious to exogenous
materials) for an endogenous source of energy to enable it to survive for
prolonged periods. Alternatively, such a source may be required by the
bradyzoite upon release from the tissue cysts. Lactate and acetate were
identified as the two main soluble products of aerobic glucose catabolism
by tachyzoites; traces of propionic, butyric and valeric acids were also
detected. Some 80% of the glucose consumed under aerobic conditions was
accounted for by the products carbon dioxide and lactate (in the approx-
imate ratio 3: l), whereas under anaerobic conditions levels of lactate
production were two to three times greater.
Subsequently, various other compounds were studied for their effects on
respiration. Glucose proved to be the most stimulatory, followed by gluta-
mine, mannose, galactose, ribose, glucose 6-phosphate, fructose, glycogen
and maltose (Aomine, 1981). The aerobic catabolism of glucose to lactate
and acetate by T. gondii tachyzoites was confirmed by further studies using
['HI-NMR (Oshaka et al., 1982). These showed that the overall production
of lactate was much greater than that of acetate, with there being an apparent
delay in the onset of production of the latter. No end product was detected in
the absence of added substrate, in accord with the apparent lack of endo-
genous energy reserves in this stage of the species.
There is only limited information on the enzymes of energy metabolism
in Toxoplasma. Several enzymes of glycolysis have been detected (see
166 G.H. COOMBS ET AL.

Table 1). The glycolytic pathway is unusual (but similar to that of Eimeria)
in that it contains a phosphofructokinase specific for pyrophosphate rather
than ATP and an allosterically-regulated pyruvate kinase (see Section
2.1.2.(a) for details of both).
Ultrastructural studies have revealed mitochondria1 profiles with cristae
in all developmental stages of T. gondii, suggesting that the organelle is
functional. Some TCA cycle enzymes have been reported (see Tables 1 and
2) and there is evidence of a respiratory chain containing cytochrome
oxidase as well as cytochromes b and c. Apparent evidence that the
respiratory chain is indeed functional in energy production comes from
experiments by Werk and Bommer (1980), working on host cell invasion.
Using an in vitro system, they found that preincubating T. gondii tachy-
zoites with cyanide decreased their invasion efficiency by more than 80%.
The invasion rate was, however, restored to normality when glucose was
present during the invasion process. These results suggest that energy
production is far less efficient under the enforced fermentative metabolism
induced by cyanide, and that the cells consequently consume their finite
energy reserves too quickly for cell invasion to occur in most cases.
Recent evidence suggests that bradyzoites may differ from tachyzoites in
relying more heavily on fermentative metabolism and that the respiratory
chain and TCA cycle may be of little significance to bradyzoites (Denton et
al., 1996b; see Table 2). This could partially explain why tachyzoites
differentiate to bradyzoites (Tomavo and Boothroyd, 1995; Gross et al.,
1996). Interestingly, the two forms express different isoenzymes of lactate
dehydrogenase (Yang and Parmley, 1995; see Section 2.1.2.(a)).
An interesting possibility is that tachyzoites utilize host cell ATP in a
way that provides usable energy for the parasite. The evidence is some-
what circumstantial but includes the ‘gathering’ of the host cell mito-
chondria around the parasitophorous vacuole (and the slower growth of
the parasites in host cells lacking mitochondria), the presence of a pore in
the parasitophorous vacuole membrane which allows passage of materials
of molecular mass <1 m a , the formation of a network within the para-
sitophorous vacuole which may act as a conduit for materials from the
host cell, the stimulation of tachyzoite motility by exogenously supplied
ATP, and the release of a highly active nucleoside triphosphate hydrolase
by the parasite from its dense granules into the parasitophorous vacuole
(see Section 5.1.1).

2.3. Cryptosporidium

Very little is known about energy metabolism in Cryptosporidium species.


The apparent lack of mitochondria in C. parvum suggests that oxygen is
BIOCHEMISTRY OF THE COCClDlA 167

unimportant and that glycolysis is the main source of energy generation


(Current, 1989). Such a conclusion is consistent with the finding that
development of the parasite in vitro was enhanced under reduced oxygen
tensions (Upton et al., 1994b) and that the viability of sporozoites was
unaffected by inhibitors of the respiratory chain (Brown et al., 1996).
Glycolytic enzymes have been detected and the presence of lactate dehy-
drogenase suggests that lactate is one end-product (see Tables 1 and 2;
Denton et al., 1996a, b; Malek et al., 1996). Unusual features include the
presence of a pyrophosphate-specific phosphofructokinase and an unregu-
lated pyruvate kinase (see Section 2.1.2.(a)). The apparent lack of hexo-
kinase in oocysts (see Table 2) could be consistent with endogenous
polysaccharide being the sole carbohydrate energy substrate used by this
stage; it has been detected in sporozoites (Malek et al., 1996). The absence
of detectable TCA cycle enzymes (see Tables 1 and 2; Malek et al., 1996)
is consistent with the lack of a mitochondrion.
Mitochondria have been reported to be present in C. muris (see Uni et
al., 1987), suggesting that the metabolism of this species may differ greatly
from that of C. parvum. The viability of sporozoites of C. muris was
affected to some extent by respiratory inhibitors (Brown et al., 1996),
but further studies are required to verify that two supposedly closely related
species do indeed differ so markedly.

2.4. Sarcocystis

Sarcocysts of S. fusifomzis have been shown to consume oxygen (Chaudry


et al., 1986a) and some glycolytic enzymes have been detected (Gupta et
al. 1992), but there have been only a few other investigations (Khulbe et
al., 1989).

3. PROTEIN AND AMINO ACID METABOLISM

3.1. Protein Synthesis

The mechanisms by which coccidia synthesize proteins have been inves-


tigated little in recent years. The few studies reported have mainly been
confined to determinations of the sensitivity of the parasites to some
standard protein synthesis inhibitors (as quoted by Edlind, 1991; see Sec-
tion 13). These inhibitors have activity against coccidia in vitro and in vivo,
suggesting that protein synthesis is a potential target for chemotherapy. It
168 G.H. COOMBS ETAL.

is important to identify and characterize the precise steps that are the key
target sites of these inhibitors so that more effective agents can be
designed.
One of the limitations in studying protein synthesis in the intracellular
stages of the parasites has been the problem of distinguishing between
protein synthesis in the parasite and in the host cell. This has now been
overcome by the use of ricin to inhibit selectively the synthetic process in
the host cell (Gurnett et al., 1995; Carrington et al., 1996). Development of
this procedure should allow studies that previously were not possible and
so, hopefully, will stimulate research on this important topic.
Little detail is known of the mechanisms of protein synthesis. The gene
encoding elongation factor-2, a protein essential for protein synthesis, has
been cloned from C. parvum (Jones et al., 1995). Toxoplasma has been
shown to contain a cyclophilin (High et al., 1994; Page et al., 1995). This is
thought to be involved in protein folding and to be at least one of the
targets of cyclosporins, compounds with anticoccidial activity (see
Section 13).

3.2. Protein Catabolism

Parasite proteinases have attracted considerable attention in recent years


(McKerrow, 1989; North et al., 1990; McKerrow et al., 1993; North and
Lockwood, 1995; Coombs and Mottram, 1997), largely because they are
considered to be good targets for chemotherapeutic attack. Coccidia
undoubtedly possess many proteinases themselves, as is thought to be
the case for all eukaryotic cells, but only a few have been reported to
date. One reason for this is that the coccidial enzymes are not readily
detected using standard techniques, such as substrate sodium dodecylsul-
phate-polyacrylamide gel electrophoresis, which have been used exten-
sively in the analysis of proteinases of other parasitic protozoa. This could
be because the proteinases are present in relatively low abundance in
coccidia, or that the enzymes are more susceptible to denaturation during
electrophoresis, or that they have narrower substrate specificity. An added
complication in studies on Eimeria is that the standard method used for the
experimental excystation of oocysts involves the use of trypsin at high
concentration. Residual trypsin not removed by washing the sporozoites
could interfere with study of the parasite enzymes and there are some
indications that enzymes that have been reported to be of parasite origin
may in fact have been contaminating trypsin.
Wang and Stotish (1975, 1978) looked for proteinases which might be
involved in protein turnover during sporulation or in the release of sporo-
zoites during excystation. Very low proteinase activities were found using
BIOCHEMISTRY OF THE COCClDlA 169

[14C]glycinated haemoglobin as substrate at pH 4 and extracts of sporu-


lated or unsporulated oocysts of E. tenella, whereas none was apparent at
neutral or basic pH. The activity was completely inhibited by phenylmethane-
sulphonyl fluoride (PMSF), implicating a serine proteinase.
Using a different substrate (azocollagen), Farooqui and Hanson (1983)
detected high proteinase activities in sporulated and unsporulated oocysts
and sporozoites of E. tenella. The levels of activity detected were in the
order sporozoites > sporulated oocysts > unsporulated oocysts. Fuller and
McDougald (1990) investigated azocasein digestion by intact sporozoites
and merozoites and detected extremely low levels of activity sensitive to
inhibition by the serine proteinase inhibitor PMSF and to a lesser degree by
tosylamido-2-phenylethyl chloromethyl ketone (TPCK) and tosyl-lysine
chloromethyl ketone (TLCK). The activities associated with the sporo-
zoites and merozoites had different pH optima and inhibitor sensitivities,
implying stage-specific proteinases.
Michalski et al. (1994) succeeded in purifying a 20 kDa proteinase from
E. tenella oocysts, even though it was not readily detected in crude homo-
genates because of some ‘interfering factor’. The purified enzyme had a pH
optimum of 8.0, using either azocasein or gelatin as substrate, and was
sensitive to inhibition by the serine proteinase inhibitors PMSF, aprotonin
and TLCK. It was unaffected by inhibitors specific for other proteinase
types or by divalent cations and so appeared to be a serine proteinase.
Antiserum raised against the enzyme recognized proteins in both oocyst
and sporozoite homogenates on Western blots but did not react with
trypsin, thus confirming that it was indeed of parasite origin. This group
also noted that the proteins in crude oocyst homogenates were hydrolysed
if proteinase inhibitors were not added, thus confirming that proteinases
were present (Michalski et al., 1994). The degradation could be partially
prevented by inclusion of PMSF in the lysis buffer, but a combination of
PMSF and the cysteine proteinase inhibitor E64 was required to achieve
full protection. This provided convincing evidence that oocysts contain
proteinases of both the serine and cysteine types. Incubation of sporozoites
with E64, which does not cross biological membranes, and then detection
of active enzymes using a biotinylated inhibitor suggested that this stage of
E. tenella has a surface-located 46 kDa cysteine proteinase (S.M.A. Brown
et al., unpublished observations).
Adams and Bushel1 (1988) and Fuller and McDougald (1990) used
proteinase inhibitors to investigate the possibility that proteinases might
be involved in host cell invasion by eimerian sporozoites. Using an in vitro
system and preincubation of the sporozoites with various proteinase inhib-
itors, it was found that invasion was significantly inhibited by PMSF and,
in the second study, a range of other inhibitors specific for serine protein-
ases and cysteine proteinases. The use of inhibitors has shown that
170 G.H. COOMBS ETAL.

proteinases of these two classes may also be involved in aiding the para-
site’s penetration through the mucous layer lining the gut epithelium
(S.M.A. Brown et al., unpublished observations).
It has been reported that Eimeria has a gene with high homology with
those encoding aspartic proteinases in other organisms (Laurent et al.,
1993). Immunolocalization studies suggested that the enzyme was asso-
ciated with the refractile bodies of the sporozoite. It is yet to be determined
if the expression of the gene is developmentally regulated and the activity
of the enzyme encoded by the gene has not been described to date. Anti-
bodies raised against the recombinant protein inhibit invasion of a host cell
by sporozoites.
An interesting observation is that there is a ‘collagenase’ associated with
the second generation meronts of Eimeria. It is not certain whether the
enzyme is of parasite or host origin, but it appears that the activity is
important in enabling the meront to migrate through the epithelial layer.
Three leucine aminopeptidase activities were detected when extracts of
unsporulated oocysts of E. tenella were subjected to electrophoretic ana-
lysis (Wang and Stotish, 1978). These activities gradually declined during
sporulation and a different isoenzyme appeared, which was the only activ-
ity that could be detected in sporulated oocysts. It was shown to be located
primarily in the cytoplasm surrounding the sporocysts, but not found in
either sporozoites or merozoites. All four aminopeptidase activities had
similar substrate specificities and were unaffected by inhibitors of serine
proteinases. The enzymes present in unsporulated oocysts had pH optima
in the range 8.0 to 8.5, while that in sporulated oocysts was found to be
more active at pH 8.5-9.0. The latter activity differed from the others in
that it was greatly reduced in the absence of metal ions (Mg2+or Mn2+)and
was inhibited by chelating agents; thus it had characteristics of a metallo-
proteinase. As there is limited protein synthesis occurring during the late
stages of sporulation, it was suggested that the leucine aminopeptidase
activity appearing at that stage may result from the processing of pre-
existing activities. The rather different properties of the isoenzymes, how-
ever, make this unlikely and their presence at different stages during
sporulation suggests that the isoenzymes may perform specific roles in
this process and excystation.
The proteinases of Toxoplasma and Cryptosporidium have been studied
even less extensively than those of Eimeria. Two proteinase activities were
separated from tachyzoites of T. gondii (see Choi et al., 1989). One had
characteristics of a cysteine proteinase and was optimally active at pH 6.0,
the other was an ATP-dependent serine proteinase optimally active at pH
8.5. Several aminopeptidases were detected in T. gondii, using a range of
chromogenic substrates (Manafi et al., 1993). This parasite was also
reported to contain a cystatin-like inhibitor of cysteine proteinases (Irvine
BIOCHEMISTRY OF THE COCClDlA 171

et al., 1992). This is likely to play a role in protecting the parasite from its
own enzymes, although an involvement in aiding survival in the host cell
cannot be ruled out.
Membrane-associated aminopeptidase activity was detected in C. par-
vum sporozoite lysates (Okhuysen er d.,1994) and a role in excystation
was suggested for this enzyme (Okhuysen et al., 1996). A metal-dependent
surface-located cysteine proteinase of C. parvum sporozoites has also
recently been reported (Nesterenko et al., 1995). This finding is consistent
with the observation that E64 inhibits both mucus penetration and host cell
invasion by this parasite (S.M.A. Brown et al., unpublished observations).
The interaction of human natural inhibitors of serine proteinases with C.
parvum has also been studied (Forney et al., 1996a), and it has been
reported that serine proteinase inhibitors affect both oocyst excystation
and host cell invasion (Forney et al., 1996b,c).
The role of proteinases in host cell invasion has been studied in S. muris
by Strobe1 et al. (1992). The results suggested that a cysteine proteinase is
involved and it was postulated that it may enhance parasite invasion by
modifying the parasitophorous vacuole.

3.3. Amino Acid Interconversions

Some enzymes likely to be involved in the interconversions of amino acids


in coccidia have been detected, mainly during isoenzyme studies aimed at
distinguishing species and strains, but there has been no detailed analysis
of the enzymes themselves or of the ability of the parasites to interconvert
amino acids. The enzymes detected to date are detailed in Table 1 and
Section 2.1.3.

4. POLYAMINE METABOLISM

The polyamines spermidine and spermine, and the diamines putrescine and
cadaverine, are widely distributed and have been implicated in a number of
biological functions. The molecules appear to have important roles in cell
division, although their precise mode of action is unclear. In general, the
pathways of polyamine biosynthesis appear relatively similar throughout
nature, with the enzymes ornithine decarboxylase (ODCase) and S-adenosyl-
methionine decarboxylase (SAMDCase) catalysing the rate-limiting steps.
Inhibitors of these enzymes have proved to be effective in inhibiting the
replication of many cell types and have been extensively investigated as
potential antiparasite agents. To date, the effects of such inhibitors provide
172 G.H. COOMBS ETAL.

the most useful information on the significance of polyamine metabolism


in the coccidia species.
a-DL-Difluoromethylornithine (DFMO) is an enzyme-activated irrever-
sible inhibitor of ODCase and an effective antitrypanosomal drug. The
efficacy of DFMO against E. tenella infections in chickens was investi-
gated by Hanson et al. (1981). When adminstered at 0.5% in drinking
water, starting 1 day before infection and continuing for 5 days, the
compound was as effective in preventing symptomatic coccidiosis as
was 0.012% amprolium. Following ‘cure’ with DFMO, the birds were
immune to re-infection by the parasite when challenged 1 week later. If,
however, putrescine was administered along with the DFMO, no cure was
effected. These results are similar to those obtained with other parasites
and imply that DFMO acts specifically by blocking putrescine production,
that its effects are cytostatic rather than directly cytotoxic, and that it is
ineffective if exogenous putrescine is available and can be scavenged by
the parasite.
There have been a few reports on polyamine metabolism in coccidia.
Spennidine was detected in sporulated oocysts of E. acervulina by van der
Horst and Kouwenhoven (1973). SAMDCase has also been investigated
using crude extracts of E. stiedai oocysts (San-Martin Nunez et al., 1987).
The enzyme differed from its counterparts in rat or rabbit liver by having a
considerably lower K, and a slightly higher pH optimum, and by being
relatively insensitive to several potent inhibitors of the mammalian
enzyme. A preliminary investigation of T. gondii has also been reported
(Lipschik et al., 1988). Most interestingly, it has been reported that C.
parvum lacks ODCase but possesses arginine decarboxylase (Yarlett et al.,
1996). This catalyses the conversion of arginine to agmatine, which is the
starting point for polyamine biosynthesis in this coccidian. The parasite also
is able to carry out polyamine back conversion via spermidine:spermine
N’-acetyl transferase (Yarlett et al., 1996).

5. PURINE AND PYRIMIDINE METABOLISM

This is an aspect of parasite biochemistry that has attracted considerable


interest over the years as it has been discovered that many parasite enzymes
and pathways differ greatly from those in the host and so offer attractive
targets for chemotherapeutic attack (Berens et al., 1995); some of these
have been exploited. Coccidia have been studied less extensively in this
respect than some other parasites, but a good amount of data has been
accumulated.
BIOCHEMISTRY OF THE COCClDlA 173

5.1. Purine Metabolism

Coccidia appear to be similar to the majority of parasitic protozoa in being


unable to synthesis purines de novo and therefore being ‘auxotrophs’ for
them (Hassan and Coombs, 1988; Man, 1991; Berens et al., 1995).

5.1.1. Purine Salvage


The pathways operating in Eimeria are summarized in Figure 4. The
parasite is unusual in having a phosphoribosyltransferase (PRTase) that
is active with xanthine, hypoxanthine and guanine (Wang and Simashke-
vich, 1981). It has been suggested that the activity of allopurinol against
this parasite in virro is due to inhibition of this enzyme, although incor-
poration of allopurinol into nucleotides by the enzyme (as occurs in try-
panosomatids) cannot be ruled out. It has also been reported that the
parasite contains adenine PRTase and adenosine kinase, but not kinases
or phosphotransferases active on inosine, guanosine or xanthosine (Wang
and Simashkevich, 1981). Recently, however, a novel activity that phos-
phorylates guanosine to guanosine monophosphate (GMP) has been
described (Maga et al., 1994). Adenosine kinase has been purified from
oocysts of several species and shown to be inhibited by some pyrazolopyr-
imidine ribosides and to metabolize others (Miller et al., 1982), although
these activities did not correlate with the drug’s anti-coccidial effects in
vivo (Krenitsky er al., 1982). Studies of the interconversions of purine
bases and nucleosides by sporozoites and merozoites of E. renella suggest
that this parasite contains adenine deaminase, guanine deaminase, adeno-
sine deaminase, adenosine nucleosidase and guanosine nucleosidase but
lacks nucleoside phosphorylases and AMP deaminase (Lafon and Nelson,
1985). There has been little study of the stage-specificity of activities,
although it appears that adenine deaminase is present in sporozoites and
merozoites but absent from unsporulated oocysts (Wang and Simashke-
vich, 1981; Lafon and Nelson, 1985).
Study of purine salvage by T. gondii was facilitated by growing the
parasite in a host cell lacking hypoxanthine-guanine PRTase and so itself
unable to utilize exogenously supplied hypoxanthine and guanine (Pfef-
ferkorn, 1981). Subsequently, it was reported that the parasite has
PRTases that incorporate all four purine bases into their nucleotides
(Krug et al., 1989). The presence of adenine deaminase and guanine
deaminase suggests that xanthine PRTase and hypoxanthine PRTase
may be the two main anabolic enzymes. These seem to be the prime
route of nucleotide synthesis, as most nucleosides are first hydrolysed to
the base which is then incorporated. This correlates with the lack of most
nucleoside kinase and phosphotransferase activities (Marr, 1991). The
174 G.H. COOMBS ETAL.

Eimeria

Guanine - I0
Xanthine Hypoxanthine a Adenine
If

Toxoplasma

I Guanosine
4 I
I Xanthosine I lnosine Adenosine I

Figure 4 Pathways of purine salvage apparently operating in coccidia. 1, GMP


synthetase; 2, IMP dehydrogenase; 3, AMP dearninase; 4, hypoxanthine-guanine-
xanthine PRTase; 5 , adenine PRTase; 6, adenylosuccinate synthetase; 7, adenylo-
succinate lyase; 8, adenosine kinase; 9, nucleoside phosphorylase; 10, guanine
dearninase; 11, adenine dearninase; 12, purine nucleosidase; 13, adenosine deami-
nase; 14, guanosine phosphotransferase; GMP, guanosine 5’-rnonophosphate;
XMP, xanthosine 5’-monophosphate; IMP, inosine 5’-monophosphate; AMP, ade-
nosine 5’-rnonophosphate.

exception is adenosine kinase, which is present at high activity (Krug et


al., 1989) and has been studied in some detail (Iltzsch et al., 1995). A
gene encoding hypoxanthine-guanine PRTase in T. gondii has now been
cloned and sequenced (Cote et al., 1992; Vasanthakumar et al., 1994) and
the binding specificity of xanthine and guanine PRTase has been studied
(Naguib et al., 1995). A subsequent report, however, suggests that Toxo-
plasma is similar to Eimeria in possessing just one PRTase that is active
towards xanthine, hypoxanthine and guanine (Donald et al., 1996). There
are two genes encoding hypoxanthine-guanine-xanthine PRTase, although
BIOCHEMISTRY OF THE COCClDlA 175

the activities of the isoenzymes seem similar. Recombinant enzymes have


now been produced. The pathways apparently present in T. gondii are
summarized in Figure 4. Toxoplasma stages expressing hypoxanthine-
guanine-xanthine PRTase are sensitive to 6-thioxanthine, apparently
because it is converted by the enzyme to a rogue mononucleotide which
is toxic. This suggests that this coccidian enzyme could be targeted by
pro-drugs.
Adenine nucleotides appear to be the preferred purine source of the intra-
cellular parasite and it seems that the parasite may consume host cell ATP
for this purpose (Pfefferkorn and Pfefferkorn, 1977; Schwartzman and
Pfefferkorn, 1982). Tachyzoites, but apparently not sporozoites, contain
large amounts of a highly active nucleoside triphosphate hydrolase (Asai et
al., 1983b, 1986). This enzyme appears to be unique to Toxoplasma, no
equivalent being present in Eimeria or Plasmodium. It is a 63 kDa enzyme,
constituting some 3% of the total cell protein. The activity comprises two
isoenzymes with differing substrate specificity (Asai et al., 1995); not all
strains have both isoenzymes. The enzyme is activated by reductive
dithiols such as diothiothreitol (DTT) and also by reduced thioredoxin,
although they do not participate in the catalytic mechanism itself (Asai and
Kim, 1987). There is evidence that the enzyme is a component of the dense
granules and is released into the parasitophorous vacuole after invasion
(Sibley et al., 1994a, c), and so it is probably involved in the hydrolysis of
exogenous adenine nucleotides. It is capable of hydrolysing a broad spec-
trum of nucleoside triphosphates and diphosphates. The enzyme could also
be important in providing energy for the parasite (see Section 2.2). It has
been suggested that the enzyme is a potential target for diagnostic reagents
and chemotherapy and a gene encoding the enzyme has now been cloned
and analysed (Johnson et al., 1989; Morency et al., 1992; Bermudes et al.,
1994). Recombinant enzymes have been produced and are being used for
drug screening. The mononucleotides resulting from the action of nucleo-
side triphosphate hydrolase are dephosphorylated before being taken up
(Schwartzman and Pfefferkorn, 1982). It has been reported that Toxo-
plasma may lack a surface 5’-nucleotidase activity (De Carvalho and De
Souza, 1989), although more recently such an enzyme has been described
(see Sibley et al., 1994a). The acid phosphatase activities that have also
been shown to be located in dense granules and rhoptries (Metsis et al.,
1995) could be involved in some way.
Little has been reported on the mechanisms of purine uptake by Eimeria
or Toxoplasma. The uptake of adenosine, inosine, hypoxanthine and ade-
nine by Toxoplasma was shown to be rapid but non-concentrative. There
appeared to be a single adenosine transporter located in the plasma mem-
brane of tachyzoites (Schwab et al., 1995), although the same transporter
may also act in the uptake of inosine and the two bases. It has been
176 G.H. COOMBS EJAL.

suggested that the anticoccidial agent arprinocid may act through inhibiting
hypoxanthine transport (Wang, 1982); if so this unusual transporter could
be the target.
There has been no report on purine metabolism in Cryptosporidium.

5.1.2. Purine Nucleotide Interconversions


The results of studies in which the fates of radiolabelled purines supplied to
the parasites were determined suggest that both T. gondii and E. tenella are
unable to convert GMP to AMP, though the reverse is possible (Pfefferkorn
and Pfefferkorn, 1977; Lafon and Nelson, 1985; Krug et al., 1989). This
appears to be due to the absence of GMP reductase (see Hassan and
Coombs, 1988). The lack of this enzyme means that guanine and xanthine
are incorporated only into guanine nucleotides and the parasites cannot
satisfy all their purine requirements by salvage of these bases alone.
Adenine, hypoxanthine and the respective nucleosides (adenosine and
inosine) can, however, meet all the purine needs of the parasites. The
only enzyme involved in these interconversions that has been studied is
inosine monophosphate (IMP) dehydrogenase (Hupe et al., 1986). The
enzyme in Eimeria appears to represent a useful chemotherapeutic target.
It is inhibited by mycophenolic acid and this compound also inhibits the
growth of the intracellular parasite in vitro.

5.2. Pyrimidine Metabolism

Many parasitic protozoa both synthesize pyrimidines de novo and salvage


them preformed. Both of these routes are significant in T. gondii and
probably Eimeria species, although the data for Eimeria are very limited.
Only salvage has been studied for C. parvum.

5.2.1. De Novo Synthesis of UMP


The presence of de novo biosynthesis of pyrimidines in T. gondii was
suggested from incorporation studies with bicarbonate and aspartate
(Schwartzman and Pfefferkorn, 1981), although earlier studies had shown
that orotate itself is incorporated only at very low levels (see Hassan and
Coombs, 1988). All six enzymes of the pathway have also been detected
and characterized to some extent (Hill et al., 1981; O’Sullivan et al., 1981;
Asai et al., 1983a; Pfefferkorn, 1988). The first three, carbamoylphosphate
synthetase (CPS), aspartate transcarbamylase (ATCase) and dihydro-
orotase (DHOase), exist as separate entities. Interestingly, ATCase is
relatively insensitive to feedback inhibition by UTP and CTP (an important
BIOCHEMISTRY OF THE COCClDlA 177

regulatory mechanism in many micro-organisms) and so is unlikely to be


the control point of the pathway in T. gondii. The next enzyme, dihydro-
orotate dehydrogenase, is particulate and probably linked to the respiratory
chain. The final two activities, orotate phosphoribosyltransferase (OPRT-
ase) and orotate monophosphate (OMP) decarboxylase (ODCase), appear
to exist as a bifunctional enzyme.
Only the oocyst stage of Eimeria has been studied in detail (Hill et al.,
1981). ATCase, OPRTase and ODCase were detected, albeit at low levels,
but CPS and DHOase were not found. It is to be expected that pyrimidines
will not be required in large amounts during this stage of the life cycle and
so the presence of some of the enzymes suggests that the biosynthetic
pathway is likely to be significant in the multiplicative forms. Unfortu-
nately, no information is yet available on these forms. The presence of de
novo synthesis of pyrimidines in sporozoites was suggested by the finding
that aspartate and orotate were incorporated into pyrimidine nucleotides
(Krylov, 1982)

5.2.2. Pyrimidine Salvage and Nucleotide Catabolism


There has been no report on pyrimidine salvage enzymes of Eimeria, but
several studies on the incorporation of pyrimidines have been carried out.
Early autoradiographic studies indicated that uridine and cytidine, but not
thymidine, can be incorporated into the nucleic acids of the intracellular
parasite and an important role for uridine was also suggested by the finding
that uridine analogues are growth inhibitory (see Hassan and Coombs,
1988). Intracellular stages of E. tenella also incorporate uracil and, indeed,
the amount of incorporation can be used as a measure of parasite devel-
opment (Schmatz et al., 1986). Subsequently, it was shown that purified
merozoites and meronts both take up exogenous uracil and incorporate it
into their nucleic acids (Geysen et al., 1991).
Toxoplasma gondii has been studied more extensively. The two key
enzymes in the tachyzoites appear to be uridine phosphorylase, which
catalyses the reversible phosphorolysis of uridine, deoxyuridine and thy-
midine, and uracil PRTase (Iltzsch, 1993; Iltzsch and Tankersley, 1994;
Donald and Roos, 1995). These enzymes account for the ability of the
parasite to incorporate uridine, deoxyuridine and uracil. Cytidine/deoxy-
cytidine deaminase, which can deaminate both cytidine and deoxycytidine,
is also present but the low activity of this enzyme and its relatively low
affinity for its substrates means that neither cytidine or deoxycytidine are
incorporated into the parasite in significant amounts. Other salvage
enzymes are absent, except for nucleoside 5’-monophosphate phospho-
hydrolase activity toward monophosphates of uridine, deoxyuridine,
thymidine, cytidine and deoxycytidine (UMP, dUMP, TMP, CMP,
178 G.H. COOMBS ET AL.

dCMP), thus precluding the salvage of other pyrimidine metabolites


(Iltzsch, 1993). The results suggest that UMP is central to pyrimidine
salvage as well as resulting from the de novo biosynthetic pathway. The
central role of uridine phosphorylase in pyrimidine salvage and its differ-
ence from mammalian enzymes, which normally are specific for either
nucleosides or deoxynucleosides and do not metabolize both, led to a
search for nucleobase analogues which are specific inhibitors of the para-
site enzyme and so have potential as antiparasite agents (Iltzsch and
Klenk, 1993; El Kouni et al., 1996). The results confirmed that there are
both similarities and differences between the catalytic sites of the parasite
and mammalian enzymes. The effects of salvage inhibitors on T. gondii
have also been studied (Youn et al., 1990).
Upton et al. (1991) studied C. parvum in Madin-Darby bovine kidney
(MDBK) cells and found that this coccidian is similar to T. gondii and
Eimeria species in that it incorporates exogenous uracil.

5.2.3. Nucleotide Interconversions and Folate Metabolism


UMP resulting from de novo synthesis or salvage pathways has to be
converted not only to UTP and CTP but also to deoxyribonucleotides.
There is scant information available on this aspect of coccidian metabo-
lism, but the sensitivity of Toxoplasma growth and DNA synthesis to
hydroxyurea (Kasper and Pfefferkorn, 1982) suggests that reduction occurs
at the diphosphate level and involves nucleoside diphosphate reductase.
Thymidylate synthase, the enzyme catalysing the conversion of dUMP
to TMP, is unusual in many parasitic protozoa in being a bifunctional
enzyme that also has dihydrofolate reductase activity. This is also true
for coccidia (Garrett et al., 1984; Ivanetich and Santi, 1990). Interestingly,
however, the eimerian enzyme is considerably larger (240 kDa) than the
Toxoplasma enzyme (120 kDa; Kovacs et al., 1990) and also the bifunc-
tional enzymes present in trypanosomatids and Plasmodium species (Gar-
rett et al., 1984). A gene encoding the bifunctional enzyme in C. parvum
has now been characterized (Gooze et al., 1992) and sequence analysis has
provided a possible explanation for the parasite’s innate resistance to anti-
folates (Vasquez et al., 1996). The bifunctional protein of T. gondii has
been heterologously expressed and characterized (Trujillo et al., 1996).
The observation that 5-fluorouracil, a specific inhibitor of thymidylate
synthase from other sources, also inhibits replication of T. gondii (see
Pfefferkorn and Pfefferkorn, 1977) demonstrated that this coccidian
enzyme may be a good drug target. The peculiarity of the coccidian
dihydrofolate reductases in comparison with those of the respective hosts
has already been exploited by the development of drugs such as pyrimetha-
mine and aminopyrimidine (see Section 13) and attempts are being made to
BIOCHEMISTRY OF THE COCClDlA 179

obtain better inhibitors (Gangjee et al., 1993, 1995, 1996; Martinez et al.,
1996; Piper et al., 1996).
Enzymes of de novo folate synthesis were searched for in T. gondii
(Kovacs et al., 1989). The parasite was shown to use para-aminobenzoic
acid and convert it into reduced folates, of which 5-formyltetrahydrofolate
was the most abundant. This confirmed the importance of folate biosynth-
esis in the parasite. This had been deduced previously from the parasite’s
sensitivity to sulphonamides (see Section 13), which are thought to target
dihydropteroate synthetase. Better inhibitors are being pursued (Allegra et
al., 1990; Chi0 et al., 1996). The presence of a folate biosynthetic pathway
in Eimeria has also been demonstrated by the parasite’s inability to use
exogenous folate and sensitivity to sulphonamides (see Section 13),
although none of the enzymes has been investigated.

6. NUCLEIC ACIDS

Modem techniques of molecular biology have started to have a large


impact on studies on coccidia, as with many parasites. Information on a
number of genes has been gained and comparison with known structures
has revealed the likely identity of the corresponding protein in a number of
cases. These include, in addition to those mentioned elsewhere in the
review, heat shock proteins (HSPs) (Laurent et al., 1994; Dunn et al.,
1995; Khramtsov et al., 1995; Lyons and Johnson, 1995; Clark et al.,
1996), tubulin (Edlind et al., 1994; Zhu and Keithly, 1996; Edlind,
1997), a BiP homologue (Dunn et al., 1996a), NAD transhydrogenase
(Kramer et al., 1993) which encodes a protein apparently associated with
the refractile body (Vermeulen et al., 1993) and which, from its pattern of
expression, is functional during sporulation and in the sporozoite rather
than the intracellular forms (A.N. Vermeulen, personal communication),
acetyl co-enzyme A synthetase in C. parvum (see Khramtsov et al., 1996),
and haemolysin in the same parasite (Steele et al., 1995). In other cases,
however, the genes show no significant homology to any already charac-
terized and so the role of the gene product remains to be elucidated.
Undoubtedly, a great amount of additional information will be forthcoming
when the various genome sequencing projects currently in progress, or
planned, are completed (Wan et al., 1996). Analysis of ribonucleic acid
(RNA) levels themselves and the expression of different genes has shown
that, not surprisingly, there is variation during the life cycle (Ellis and
Thurlby, 1991; Herbert et al., 1992; Abrahamsen et al., 1994, 1995).
Identification and analysis of the varying genes should provide useful
new insights into the specific adaptations of individual developmental
180 G.H. COOMBS ETAL.

stages. Progress has been made in some cases. For instance, it was shown that
different lactate dehydrogenase genes are expressed in tachyzoites and
bradyzoites of Toxoplasma (see Parmley et al., 1995; Yang and Parmley,
1995). The application of molecular techniques, including the development
of successful methods of reverse genetics, has progressed most rapidly with
Toxoplasma amongst the coccidia and they are now very amenable for such
studies (Sibley et al., 1993, 1994b; Donald and ROOS,1994; Messina et al.,
1995; Soldati et al., 1995; Seeber and Boothroyd, 1996). There has recently
also been some progress with the development of transfection procedures for
Eimeria. Full coverage of these advances in molecular biology of coccidia
would be inappropriate in this article; other recent reviews have summarized
the situation (Bonnin et al., 1995b; Boothroyd et al., 1995; Savira, 1995).
A most exciting advance has been the finding in coccidia of extranuclear
circular deoxyribonucleic acid (DNA) closely related to that of plastids
(Egea and Lang-Unnasch, 1995; McFadden et al., 1996). Similar DNA
appears to be a characteristic of the Apicomplexa and that in Plasmodium
has been studied most extensively (Feagin, 1994; Williamson et al., 1994;
Wilson et al., 1996; Wilson, 1997). It appears, however, that there is high
conservation between the different groups. Toxoplasma contains eight
copies of the circular DNA and it has now been demonstrated that the
DNA is contained within a distinct organelle located close to the nucleus
(McFadden et al., 1996). The name ‘apicoplast’ has been suggested for the
organelle. The DNA includes genes encoding RNA polymerase, transfer
RNA and various ribosomal proteins. Evidence has also been presented for
the presence of a chlorophyll a-Dl complex in Toxoplasma by Hackstein et
al. (1995). As this is a key component of the respiratory chain of plastids,
this is highly suggestive of electron transport occurring in the organelle. The
importance of this is uncertain (see Section 2.1.2.(d)) and the functional
significance of the plastid is still a matter of debate (Hackstein et al., 1997;
Wilson 1997). One possibility, in addition to energy metabolism, is the
provision of essential proteins. The findings that some herbicides thought to
act by inhibiting the components of the respiratory chain encoded by the
plastid have anticoccidial activity (Hackstein et al., 1995) and that malaria
parasites are sensitive to inhibitors of plastid metabolism (see McFadden et
al., 1996) have led to an explosion of interest in this aspect of the parasites’
biochemistry and attempts to exploit it as a drug target. Recent findings (F.
Roberts, C. Roberts, L. Mets, J. Johnson and R. McLeod, personal commu-
nication) from studies using specific inhibitors have suggested that the
plastid may be involved in haem biosynthesis and also that it may contain
the shikimate pathway, which in plants is central to the biosynthesis of
aromatic amino acids, para-aminobenzoic acid and ubiquinone. The plastid
DNA does not have genes for any of the proteins of these pathways and so
they would have to be nuclear-encoded, as is the case in plants.
BIOCHEMISTRY OF THE COCClDlA 181

It has been reported that some Eimeria species may contain viral nucleic
acids. Sporulated oocysts of E. nieschulzi were found to have an RNA-
dependent RNA polymerase activity which was absent from both E. tenella
and E. acervulina. This activity correlated with an unknown nucleic acid
species which may represent the genomic RNA of a possible virus (Sepp et
al., 1991; Roditi et al., 1994). Another study performed on E. necatrix and
E. maxima isolated RNA molecules, possibly of viral origin and thought to
be located in the cytoplasm of the parasite (Ellis and Revets, 1990).
The DNA content of S. muris was analysed using a DNA-specific Feul-
gen stain to measure the overall DNA content of the various developmental
stages. All the stages studied had haploid DNA except for early zygotes
which were diploid. These zygotes later underwent nuclear meiosis to
produce two haploid daughter cells (Mackenstedt et al., 1990).
The ways in which nucleic acids are synthesized, processed and catabo-
lized in coccidia have been little studied. Ribonuclease P of T. gondii has
been partially characterized (Mack et al., 1994), a type I1 topoisomerase
gene has been identified in C. pawum by Christopher and Dykstra (1994),
and it has been reported that DNA polymerase activity of T. gondii corre-
lated with virulence (Makioka and Ohtomo, 1995).
Studies on the mechanisms controlling gene expression in coccidia are in
their infancy. A most exciting recent finding, however, is that an inhibitor
of histone deacetylase has potent anticoccidial activity (Darkin-Rattray et
al., 1996; Singh et al., 1996). The compound, a natural product isolated
from fungi growing in a rain forest in Costa Rica, is a tetrapeptide called
‘apicidin’ - the name chosen to reflect the finding that the compound is
active against all members of the Apicomplexa tested. Histone deacetylase,
which is active towards acetylated lysine residues, is responsible for con-
trolling the binding of histones to DNA and so modulating gene expression.
This discovery suggests that this and other transcription factors may be
good targets for chemotherapeutic attack.

7. LIPID METABOLISM

Lipid distribution has been investigated using cytochemical techniques in


several coccidia. Large numbers of intensely staining lipid droplets are
present in the macrogametocyte stages of E. tenella, T. gondii (see Ryley,
1973) and I. belli (see Lindsay and Blagburn, 1994). At least in Eimeria,
these droplets appeared to persist through the process of zygote develop-
ment and into the sporulated oocysts, where they accumulated within the
sporocyst structures. Small lipid droplets have also been reported in the
microgametocytes of the above-mentioned species, as well as in the
182 G.H. COOMBS ETAL.

sporozoites of Eimeria. In the remaining stages of Eimeria, as well as the


tachyzoites of Toxoplasma, lipid staining is very diffuse and suggestive of
the presence of only structural rather than storage lipids (Fransden, 1970;
Ryley, 1973).
Although the presence of these lipid droplets suggests a role as an energy
store, the part that they play is far from clear. Using quantitative mass
spectroscopy, Wilson and Fairbairn (1961) found that the lipid content of
E. acervulina oocysts decreased by about 50% during the later stages of
sporulation and they concluded that the lipids were being used as an energy
substrate. However, a contradictory conclusion was reached by Weppel-
man et al. (1976), working with E. tenella oocysts. Using gravimetric
analysis they found that, although the fatty acid content of the oocysts
fell by about 50% during sporulation, this decrease was almost compen-
sated for by a 30% increase in the weight of non-saponifiable lipids (in
particular C24 and C26 fatty alcohols). From this they concluded that there
was no net oxidation of lipids during sporulation, but rather a general
incorporation of fatty acids into fatty alcohols. The newly synthesized fatty
alcohols appeared to remain in the cytoplasm, although whether in the
oocyst fluid, the sporocysts or the sporozoites was not ascertained.
Compositional studies have been carried out on tachyzoites of T. gondii
by Foussard et al. (1991a,b), sporulated and unsporulated oocysts of E.
tenella by Weppelman et al. (1976) and Stotish et al. (1978), and oocysts of
C. parvum by Mitschler et ul. (1994). In the last study, the host MDBK
cells, which are often used for growth in vitro of coccidial parasites, were
also analysed. The results obtained for phospholipid compositions are
summarized in Table 3A. Phosphatidylcholine (PC) represents the major
phospholipid in both T. gondii and C. parvum and was also detected in the
oocyst walls of E. tenella, although quantitative data were not obtained for
this parasite. The proportion of PC in both the former two parasites was
found to be high in comparison to MDBK cells and some other protozoa,
for example Plasmodium falciparum (45%) and Trypanosoma cruzi (44%).
Recent work indicates that, at least in Toxoplasma tachyzoites, part of
the high PC content may be accounted for by the rhoptry contents. These
organelles are of special interest because of their occurrence uniquely in
members of the Apicomplexa and their apparent involvement in the for-
mation of the parasitophorous vacuole. In search of evidence for such a
role, Foussard et al. (1991b) investigated the lipid composition of a rhoptry
fraction of T. gondii tachyzoites. The composition proved highly unusual in
two ways. First, it contained a high proportion of PC (75%) but lacked
several other phospholipids found in whole cell extracts. Second, the
cholestero1:phospholipid ratio was extremely high (3:2). A membrane
structure containing high proportions of PC and cholesterol would be an
extremely stable bilayer and very resistant to fusion with other membranes.
BIOCHEMISTRY OF THE COCClDlA 183

Table 3A Phospholipid composition of some coccidia extracts


Phospholipid Eimeria tenella Toxoplasma Cryptosporidium MDBK cells
(oocyst wall) gondii parvum
(tachyzoites) (oocysts)
PC J 62.024.2 65.5 24.1 40.62 1.8
PE J 11.224.2 7.321.8 27.2 2 3.6
PYPS J 15.0i5.6 1.820.9 12.6i2.3
SM ? 8.024.3 24.4 2 5.2 14.02 1.5
CL ? ? 0.920.8 5.820.5
LPC J ? ? ?

Results are expressed as % of total phosphate; means * SD.


Key: PC, phosphatidylcholine; PE, phosphatidylethanolamine;PI, phosphatidylinositol; PS,
phosphatidylserine; SM, sphingomyelin; CL, cardiolipin; LPC, lysophosphatidyl choline;
MDBK, Madin-Darby bovine kidney; J , detected but not quantified; ?, not investigated.

Table 3B Fatty acid composition of some coccidia extracts


Fatty acid Eimeria tenella T. gondii C. parvum (oocysts)
Unsporulated Sporulated (tachyzoites) Phospholipids Neutral lipids
oocysts oocysts
~ ~ ~~

C 14:O 1 2 9 0 5
C 16:O 12 8 17 31 27
C 16:l 1 3 n.d. trace 3
c 18:O 10 17 11 16 20
c 18:l 75 68 31 22 27
C 18:2 trace trace 20 29 15
c 20:o n.d. n.d. 6 trace 1

Results are expressed as % of total phospholipid in the fraction.


Key: n.d., not detected.
Data from Weppelman er af., 1976; Stotish et al., 1978; Foussard ef a[., 1991a,b; Mitschler er
al., 1994.

This correlates well with the known properties of the parasitophorous


vacuole of T. gondii (see Joiner, 1991; Sibley et al., 1994a).
In contrast with the rhoptries, the pellicle of T. gondii has a low choles-
tero1:phospholipid ratio of 0.4:1 (Foussard et al., 1991a). This correlates
well with the high membrane fluidity observed by fluorescence polarization
(Gallois et al., 1988). Hexacosanol is the predominant lipid present (-60%)
in the oocyst walls of E. tenella, with phosphatidylcholine, phosphatidyl-
ethanolamine, phosphatidylserine and lysophosphatidylcholine being the
four predominant phospholipids (Stotish et al., 1978).
The phospholipid composition of C. parvum cysts is unusual in that the
amount of cardiolipin (CL) is extremely low compared, for example, with
the levels in MDBK cells. CL in eukaryotic cells is almost exclusively a
184 G.H. COOMBS ET AL.

component of the inner mitochondria1 membrane, so this observation is


consistent with the suggestion that C. parvum either lacks a mitochondrion
altogether or has only a rudimentary structure (Current, 1989). It should be
remembered, however, that CL was not detected at all in T. gondii and this
parasite contains structures that resemble mitochondria on morphological
criteria; there is also evidence for a functional TCA cycle and respiratory
chain (see Section 2.1.2).
The fatty acid compositions reported for coccidia are summarized in
Table 3B. It is important to remember that the various sets of data are not
directly comparable as they refer to different lipid fractions. However,
Toxopfasma and Cryptosporidum are similar in containing 16:0, 18: 1 and
18:2 as their major acyl chains, while Eimeria contains predominantly 18:l
and has only trace amounts of 18:2. Mitschler et al. (1994) noted that the
fatty acid profile of C. parvum was significantly different from that of
MDBK cells and concluded that C. parvum must have the ability either to
synthesize or selectively to sequester specific acyl chains.
Cholesterol is the only sterol that has been detected in either Toxoplasma
or Cryptosporidium, and it has also been identified in Sarcocystis and
Eimeria species. In Eimeria, several other components are also found in
the non-saponifiable lipid fraction. From their retention times in gas-liquid
chromatographic analysis, van der Horst and Kouwenhoven ( 1973) claimed
that two of these components in E. acewulina were squalene and proges-
terone. Weppelman et al. (1976), however, using E. tenella extracts, found
no evidence for these compounds but identified the non-saponifiable lipids
as cholesterol and a range of fatty alcohols with 22-32 carbon atoms
(hexacosinol was the most abundant). In the unsporulated oocysts, the
majority of these alcohols were located in the oocyst wall. It seems likely
that they contribute to the impermeability and general resistance of this
structure.
To date, there have been very few reports on the mechanisms of lipid
uptake or metabolism in coccidia, although a phospholipase A2 secreted by
T. gondzz is known (Marin et a f . , 1996). This lack of study is reflective of
the situation in parasitology in general where, until recently, most lipid
research had been confined to compositional analyses with little reference
to the function or metabolism of the molecules. The situation with other
parasitic protozoa is beginning to change, with recent research showing
that some, parasites possess unusual mechanisms of lipid metabolism
(including, in particular, the synthesis of glycosylphosphatidylinositol
(GP1)-anchors - see Section 11.2) and uptake systems which might
provide targets for drug attack (in, for example, malaria parasites; Vial,
1996). This appears to be an area of coccidian biochemistry worthy of
increased research effort.
BIOCHEMISTRY OF THE COCClDlA 185

8. CULTURING COCClDlA IN VlTRO AND GROWTH FACTOR


REQUIREMENTS

Propagation in vitro and axenic cultivation of coccidia are two of the


biggest problems yet to be solved and the need to develop a system to
study and monitor these parasites has prompted many studies. Axenic
cultivation is still not possible with any coccidian. Growth intracellularly,
however, is relatively straightforward for Toxoplasma and is used routinely
with tachyzoites; importantly, there are now procedures reported for pro-
ducing bradyzoites (Weiss et al., 1994). Methods are also available for
Eimeria, with first and second generation meronts being produced (Doran
and Augustine, 1978). Cryptosporidium has proved less easy, although
recent reports that infection rates as high as 50% have been achieved
with both MDBK cells and fallopian tube epithelial cells (Yang et al.,
1996) are encouraging. Most emphasis to date has been placed on disco-
vering an appropriate host cell line (Upton et al., 1994a, c) and developing
methods for monitoring growth (You et al., 1996), although there has been
some interest in determining the best culture conditions with respect to
nutrient and gaseous requirements (Augustine and Jenkins, 1996). Sodium
pyruvate seemed to increase development of E. tenella in MDBK cells,
whereas changing glucose availability appeared to have no effect. The
removal of the amino acids arginine, glutamine, isoleucine and tryptophan
adversely affected the growth of E. tenella (see Strout and Schmatz, 1990).
Parasite stage, source and inoculum size were all also shown to be impor-
tant factors, as were environmental conditions including temperature,
humidity and oxygen levels (Upton et al., 1994b). For example, twice as
many E. tenella sporozoites invaded host cells in a 4% oxygen environment
than did so at atmospheric levels (Strout and Schmatz, 1990).
Studies on the growth factor and nutrient requirements of Eimeria have
been performed by eliminating individual components from the culture
medium and observing the resulting effect (Strout and Schmatz, 1990;
Upton and Tilley, 1992). It was observed that, whilst all of the growth
factors present in Medium 199 were needed for merogony, gametogony
did not require the presence of calciferol or folic acid (Strout and
Schmatz, 1990). Similarly biotin, folic acid, inositol, thiamine, vitamin
E, menodione, para-aminobenzoic acid and vitamin B12 were shown to
be important for first generation merogony and oocyst formation (Strout
and Schmatz, 1990). Following parasite growth in vitro over a 7-day
period showed that development of E. tenella was enhanced by doubling
the standard concentrations of insulin, ascorbic acid, choline, thiamine,
riboflavin, nicotinamide and glutathione (Strout and Schmatz, 1990).
Tryptophan is thought to be essential for Toxoplasma as the parasite died
186 G.H. COOMBS ETAL.

in human macrophages treated with interferon-y, which results in trypto-


phan starvation.

9. ANTIOXIDANT MECHANISMS

The ‘respiratory burst’ which leads to the production of oxygen metabo-


lites, such as hydroxyl free radical, hydrogen peroxide and superoxide,
represents a powerful defence mechanism of many cells against parasite
invasion. Many parasites, however, have evolved strategies that enable
them to overcome or avoid such attack. Oxygen radicals can also be by-
products of the use of oxygen by the parasites themselves. The utilization
of antioxidants and/or antioxidant enzymes by some parasitic helminths
and protozoa to scavenge oxygen radicals has been well documented
(Docampo, 1995). Such antioxidant mechanisms may play an important
role in intracellular survival and are potential targets for novel antiparasitic
drugs which would complement the host’s immune mechanisms. Research
into the antioxidant mechanisms of coccidian parasites, however, has so far
been limited to Toxoplasma (see Murray and Cohn, 1980; Murray et al.,
1980, 1985; Hughes et al., 1989) and Eimeria (see Hughes et al., 1989;
Michalski and Prowse, 1991; Ovington and Smith, 1992; Prowse et al.,
1992).
The ability of these parasites to withstand toxic oxygen metabolites and
the part such molecules play in parasite killing have been investigated to
some extent. Toxoplasma gondii tachyzoites were found to be susceptible
to the direct effects of hydrogen peroxide and the range of oxygen meta-
bolites (hydroxyl radical, superoxide, singlet oxygen) generated by
xanthine-xanthine oxidase in an in vitro model system (Hughes et al.,
1989). By adding scavengers to the system, it was confirmed that the major
toxic metabolites were hydroxyl free radical and singlet oxygen. The same
conclusions came from studies using monocytes and activated macro-
phages (Murray er a f . ,1985). Furthermore, there is circumstantial evidence
suggesting a close correlation between macrophage oxidative capacity and
the ability to inhibit intracellular parasite development in vivo (Murray and
Cohn, 1980). It is to be remembered, however, that there is also evidence of
oxygen-independent killing mechanisms effective against T. gondii which
can be enhanced by cytokines (Murray et al., 1985; Catterall et al., 1986;
McCabe and Remington, 1986). Current data also suggest an important role
for nitric oxide (Hayashi et al., 1996).
There is recent evidence to suggest that oxygen radicals may play a role
in resistance to Eimeria infection (Ovington and Smith, 1992; Prowse et
al., 1992). Sporozoites of E. tenella and E. bovis were also shown to be
BIOCHEMISTRY OF THE COCClDlA 187

susceptible in vitro to a number of oxygen radicals including hydrogen


peroxide, superoxide and hydroxyl free radical (Hughes et al., 1989;
Michalski and Prowse, 1991). Interestingly, E. bovis sporozoites were
about five-fold more susceptible than T. gondii tachyzoites to hydrogen
peroxide (Hughes et al., 1989). This correlated to some extent with the
different levels of two antioxidant enzymes, superoxide dismutase (SOD)
and catalase (CAT), which are both twice as active in T. gondii as in
Eimeria species. In contrast, the two parasites have similar glutathione
peroxidase activity (Hughes et al., 1989).
SOD, CAT and glutathione peroxidase are the only antioxidant enzymes
that have been characterized in T. gondii (see Sibley et al., 1986; Hughes et
al., 1989) and E. tenella (see Hughes et al., 1989; Michalski and Prowse,
1991). The parasite enzymes are electrophoretically quite distinct from
those of the host cells (Sibley et al., 1986; Hughes et al., 1989). T. gondii
tachyzoites possess an Fe-SOD (but no other form) and CAT, both with a
pH optimum of about 8. The activities of these enzymes were very similar
in parasite lines differing greatly in virulence to mice, suggesting that they
are not major factors determining virulence.
Unsporulated oocysts of E. tenella contain high levels of SOD, including
two forms of each of CdZn-SOD, Fe-SOD and Mn-SOD, and low CAT
activity (Michalski and Prowse, 1991). Sporulated oocysts, however, have
only low levels of SOD and contain only one form of the enzyme (Mn-
SOD). Oxygen is used during sporulation and so it appears that the multiple
enzymes must play an important part in protecting the parasite from the
superoxide generated as a by-product during this process. E. tenella sporo-
zoites also possess only low levels of Mn-SOD and CAT (Michalski and
Prowse, 1991). This also correlates well with the largely anaerobic nature
of these forms (see Section 2.1). It was reported from another study that E.
bovis sporozoites have low levels of SOD and CAT, whereas the mero-
zoites lack CAT but contain much higher levels of SOD (Hughes et al.,
1989). The significance of these findings awaits elucidation. Isoelectric
focusing revealed just two isoenzymes of SOD in the sporozoites but at
least 10 isoenzymes in the merozoites of E. bovis.
Glutathione plays an important part in countering oxygen radicals in
many cells. Trypanosomatids also contain the spermidine-glutathione
complex known as trypanothione. To date, however, there has been no
report on the presence or absence of glutathione and related compounds in
coccidia. The high concentrations of mannitol detected in Eimeria (see
Section 2.1) may represent an important mechanism for dealing with host
oxygen radicals since mannitol is an effective radical scavenger.
Thus the current data are fragmentary but suggest that coccidia have a
number of antioxidant mechanisms, at least some of which are develop-
mentally regulated. The complexity of the situation may be a reflection of
188 G.H. COOMBS ETAL.

the intricate systems involved in the host-parasite interactions and the


variety of environments encountered during the parasites’ life cycles.

10. THE OOCYST WALL

The oocyst wall of E. tenella has two layers, an outer layer 10 nm thick and
an inner 90 nm layer (Stotish et al., 1978). Studies using gas-liquid
chromatography have revealed that glucose is the predominant neutral
sugar, with small amounts of mannose and galactose (Stotish et al.,
1978). Unsporulated oocyst walls were found to be 67% protein, 14% lipid
and 19% carbohydrate. They contain an unusual polysaccharide, initially
thought to be chitin but which, on further analysis, was found to differ from
it in detail (Ryley, 1973). One of the protein components has been char-
acterized (Eschenbacher et al., 1996).
There appear to be two types of C. pawurn oocyst, designated ‘thin
walled’ (comprising 20% of the total number) and ‘thick walled’ (Cur-
rent, 1989). The thin-walled oocysts are thought to be auto-infective,
hence the initial infection is recycled (a unique feature of Cryptospor-
idium among coccidia), whereas the thick-walled oocysts are passed out
in the faeces to infect other hosts. It has been reported that these thick-
walled oocysts have three membranes and an outer wall of two chitinous
layers, while the thin-walled oocysts have a single unit membrane (Ster-
ling and Arrowood, 1993). More recently, Cryptosporidium oocysts were
reported to be similar to those of Eimeria in having two layers - an
outer 10 nm layer and a thicker inner one (Petersen, 1993). The oocyst
wall composition of three species of Cryptosporidiurn has been examined
using surface labelling and antibodies (Tilley et al., 1990; Nina et al.,
1992). The oocysts of C. baileyi, C. muris and C. pawum could all be
distinguished, but C. parvum and C. baileyi were shown to be more
similar to each other than either was to C. muris.
The structure of the oocyst wall of T. gondii has not been studied in
detail.

11. FUNCTIONAL SURFACE MOLECULES

Parasite surfaces have received particular attention from scientists because


of their crucial role in the interaction of a parasite with its host. Many of
the studies, however, including those on coccidia, have focused on more
general properties (e.g. that by Drozd and Schwartzbrod, 1996) or on
BIOCHEMISTRY OF THE COCClDlA 189

identifying surface molecules by various labelling and antibody techniques.


Most of the molecules detected in this way have been characterized only
with respect to their mobility on gels and the presence or absence of
carbohydrates or lipids, although the application of molecular techniques
has begun to provide structural data as well (Cesbron-Delauw et a f. , 1994;
Odberg-Ferragut et al., 1996). Thus in most cases their function remains a
mystery. Surface molecules with a defined activity have been identified for
a few parasites and some of these have been studied in detail and char-
acterized very well (see Smith, J.E., 1994). Unfortunately, this is not the
case for any coccidia and this is an area that deserves, and is starting to
receive, much greater attention.

11.l.Glycosylation

Many molecules on cell surfaces are glycosylated both for protection and
as one mechanism of implementing and regulating interactions with the
environment and other cells. Glycosylation is not limited to surface mole-
cules and such processing also occurs with, for example, lysosomal pro-
teins. There have been several reports of the presence of glycoproteins in
Eimena and Toxoplasma (Schwarz et al., 1993), but in most cases the
subcellular location of the molecules has not been established and very few
structural or mechanistic details are known. Dieckmann-Schuppert el al.
( 1992) reported that exogenous dolichol pyrophosphoryl oligosaccharides
were utilized by T. gondii for glycosylation of its proteins. It was not until
1993 that biochemical evidence was obtained showing that T. gondii
tachyzoites synthesized N-linked glycoproteins and the lipid-linked glycan
precursor for N-linked oligosaccharides (Odenthal-Schnittler et al., 1993).

11.2. GPI Anchors

Many surface molecules are attached to the plasma membrane via GPI
anchors. Such anchors occur widely in eukaryotic cells but seem to be
particularly important in parasitic protozoa (McConville and Ferguson,
1993). However, this is an area in which coccidia have been investigated
in much less detail than some other protozoan parasites, notably kineto-
plastid flagellates. The major surface proteins on the tachyzoites of T.
gondii do have GPI anchors (Nagel and Boothroyd, 1989; Tomavo el al.,
1992a) and the parasite is capable of synthesizing GPI and also N- and
0-glycans (Tomavo et al., 1992b; Schwarz and Tomavo, 1993). Some
undefined antigens on the surface of Eimeria sporozoites are also attached
via GPI anchors (Gurnett et al., 1990).
190 G.H. COOMBS ETAL.

11.3. Lectins

Lectins are the class of carbohydrate-binding proteins present on most cell


surfaces. They are thought to be involved in cell-cell interactions, includ-
ing invasion of the host cell by parasites.
N-acetylglucosamine-specificlectins are the main lectins of C. parvum
oocysts (Llovo et al., 1993). The lectins of C. parvum sporozoites have also
been studied, with the aim of elucidating their role in sporozoite attachment
to host cells (Thea et al., 1992). Of the glycoproteins tested, bovine
submaxillary mucin (BSM) and the blood group antigen-related PI glyco-
protein proved to be the most inhibitory of sporozoite haemagglutination
activity. Galactose and N-acetylgalactosamine were the most effective
monosaccharide inhibitors (Joe et al., 1994). Using invasion studies with
MDCK cells, C. parvum sporozoites preincubated with BSM or fetuin
showed a reduction in attachment by 77% and 63%, respectively (Joe et
al., 1994). Invasion was also reduced by 28% and 27%, respectively, thus
supporting the theory that lectins play a major role in C. parvum host cell
invasion.
Three Eimeria species and various developmental stages of E. tenella
have been examined for lectin activity using haemagglutination. It was
found that Eimeria has lectins that are developmentally regulated (Strout et
al., 1994), suggesting stage-specific functions. The lectin specificity of
each species was also found to be different. L( -)-fucose and D(+)-arabinose
inhibited the lectins of E. tenella and E. acervulina, respectively, whilst
none of the 30 monosaccharides tested inhibited E. maxima (see Strout et
al., 1994). Additionally, the pH optima of the lectins from the three species
were found to differ and, to an extent, to correlate with the conditions
prevailing in the part of the gut that each infects (Strout et al., 1994). These
results suggest that lectins may play a role in site selection by these
coccidial parasites. Lectins seem to play a role in host-cell invasion,
although the study of Baba et al. (1996) suggested that a lectin-like
receptor on the host cell recognizes galactose on the sporozoite surface.

11.4. Sialidase and Other Enzymes

Sialidase is present on the surface of invasive stages of E. tenella, the


activity being some 20-fold higher on merozoites than sporozoites (Pelle-
grin et al., 1993). It has ‘been suggested that the enzyme plays a role in
desialylating intestinal mucins and so reducing the viscosity of the envir-
onment and aiding migration of the parasite. The enzyme could also be
involved in modifying the surface of the host cell before and during
BIOCHEMISTRY OF THE COCClDlA 191

invasion. It remains to be seen whether the presence of a surface sialidase


is a common feature of coccidia.
There have been reports of other surface molecules on coccidia, but as
yet few have been confirmed as enzymes. An aminopeptidase (Okhuysen
et al., 1994) and cysteine proteinase (Nesterenko et al., 1995) in C. parvum
both appear to be surface located. These could be involved in aiding the
parasite’s penetration of the mucous layer andfor invasion of a host cell.
Sporozoites of Eimeria also appear to have a surface-located cysteine
proteinase which may function in a similar way (S.M.A. Brown et al.,
unpublished observations), although the activity is yet to be characterized.
Toxoplasma has an adenosine transporter in its surface membrane (Schwab
et al., 1995).

11.5. Cell Signalling

Cell signalling is an aspect of biochemistry that has attracted enormous


attention in recent years and some progress has now been made in under-
standing its role in parasite biology. This remains an area of coccidian
biology, however, that is largely unexplored. Genes encoding protein
kinases and a phosphatase in T. gondii have been reported (Russell and
Dwyer, 1993; Ng et al., 1995), and evidence for proteins binding guanosine
triphosphate (GTP) has been presented (Halonen et al., 1996). A Ca2+-
dependent protein kinase, with some plant-like features, has been cloned
from E. tenella (Dunn et al., 1996b). Interestingly, it appears to be
cytosolic in sporozoites but to move to the apical tip during host cell
penetration.

12. INTERACTION OF COCClDlA WITH THE HOST CELL

The ways in which the parasite interacts with its host cell is one of the most
fascinating and important aspects of coccidian biology, but unfortunately at
present it is also one of the least well understood. Information is available
on the events that occur during the invasive process itself (Dubremetz and
Entzeroth, 1993; Dubremetz and Schwartzman, 1993; Kasper and Mineo,
1994; Sibley et al., 1994a; Dubremetz and McKerrow, 1995; Morisaki et
al., 1995; Sibley, 1995; Sam-Yellowe, 1996), but we have only a rudimen-
tary understanding of how the parasite interacts with the cell in which it is
growing. Because little is known of the mechanisms at the biochemical
level, these aspects do not fall within the remit of this review and it will
192 G.H. COOMBS ETAL.

refer only to some of the more recent studies and include a few comments
on major areas that warrant more detailed biochemical study.
All coccidia inhabit a parasitophorus vacuole when intracellular. Cryp-
tosporidiurn differs from most other coccidia, however, in that the para-
sitophorous vacuole remains at the surface of the host cell rather than lying
deep within the cytoplasm (Lumb et al., 1988; Sterling and Arrowood,
1993). The parasite is therefore said to be extracytoplasmic. The signifi-
cance of this difference in intracellular location is totally unknown. Much
remains to be discovered concerning the environments within the parasi-
tophorous vacuoles, but current evidence suggests that they may differ
quite considerably and recently it was reported that the vacuole resulting
from the invasion of Toxopfasma sporozoites differs distinctly from that
which contains tachyzoites. It will be interesting to discover if there is
similar stage specificity with other coccidia.
In all coccidia studied, three types of organelles have been reported to be
involved in host cell invasion, namely rhoptries, micronemes and dense
granules. These are distinct types of organelles, although we are only
beginning to obtain an insight into the part that each plays in the invasion
process. It is thought that the contents of the organelles are secreted during
host cell invasion and that this results in changes to the host cell membrane
which aid entry of the parasite. Rhoptries are thought to contain mainly
enzymes, although they also contain unusually large amounts of lipid.
Some of the molecules present in micronemes and dense granules have
also been identified, but the precise way in which they function is yet to be
determined.
Laminin on the surface of T. gondii tachyzoites has been suggested to
enhance host cell entry (Hall and Joiner, 1991; McLeod et al., 1991;
Furtado et af., 1992). Tachyzoite binding to host cells was inhibited by
antibodies to both laminin and a-6-p-1 integrins. It has been suggested that
laminin may facilitate host cell invasion by creating a bridge between the
tachyzoite and host cell. A fibronectin-like molecule has been identified on
E. tenefla (see Lopez-Bernad et uf., 1996).
Invasion of host cells was inhibited when E. tenefla sporozoites were
preincubated with phopholipase A2, possibly due to the sporozoite surface
membrane being modified (Crane and McGaley, 1991). Yet, when T.
gondii tachyzoites were treated in the same way, the infection rate of
host cells increased. It appears that T. gondii possesses a calcium-requiring
phospholipase activity which is involved in some way with host-cell
penetration (Saffer and Schwartzman, 1991; Marin et af., 1996). The
surface-located sialidase activity present on Eimeria (see Section 11.4) is
likely to play a part in host cell invasion (Pellegrin et af., 1993) and
parasite proteinases also seem to play a role in the invasion process (see
Section 3.2).
BIOCHEMISTRY OF THE COCClDlA 193

The parasite’s motility is important for invasion (King, 1988). Actin is


localized in the anterior region of the tachyzoites of T. gondii and appears
to play a role in this parasite’s movement and also invasion of its host cell
(Endo et al., 1988; Yasuda et al., 1988; Dobrowolski and Sibley, 1996).
Actin has also been detected in the sporozoites of Eimeria (see Baines and
King, 1989). Myosin was also shown to occur at the anterior pole of
Toxoplasma (see Schwartzman and Pfefferkorn, 1983) and actomyosin was
detected in sporozoites of Eimeria (see Preston and King, 1992). Two
classes of myosin genes have been described in Toxoplasma, encoding
proteins of 93 and 114-125 kDa respectively. Both classes are quite diver-
gent from those of other organisms. Other genes, as yet uncharacterized,
are also known to exist in the parasite. Motility of, and host cell invasion
by, Cryptosporidium and Eimeria were blocked by cytocholasins, indicat-
ing that actin is involved in these processes (Russell and Sinden, 1981;
Russell, 1983). An actin gene of C. parvum has been analysed ( I m et al.,
1992). Microtubules were apparently detected in Toxoplasma (see
Schwartzman and Krug, 1985). Microtubule inhibitors blocked C. parvum
infection (Wiest et al., 1993) and colchicine also decreased invasion by T.
gondii (see Aguirre-Cruz et al., 1996). Taxol, an inhibitor of microtubule
biosynthesis, also inhibited intracellular replication of Toxoplasma (R.
Estes, D. Mack, N. Vogel and R. McLeod, personal communication).
Tubulin gene sequences have been reported and the potential of coccidial
tubulins as drug targets has been discussed (Edlind et al., 1994).
An important factor in cell invasion by some coccidia is the type of host
cell involved. Toxoplasma and Eimeria, however, are relatively promiscu-
ous and invade almost any cell type (Melendez, 1996), although the
efficiency of invasion differs. For example, T. gondii tachyzoites infect
anchorage-dependent cells more easily than anchorage-independent cells
(Nam and Choi, 1992). This may be due to the differences in membrane
rigidity. Other coccidia appear to show much greater specificity for their
host cells, although the mechanisms determining this are unclear.
It seems likely that modification of the host cell by the parasite is a
common phenomenon of intracellular parasitism and provides a means
whereby the parasite ensures the availability of the necessary nutrients.
The ways in which malaria parasites affect the host erythrocyte has been
the subject of much study, but the molecular adaptations of coccidia for
living within their host cells remain largely unknown. Toxoplasma releases
material into, and modifies, the parasitophorous vacuole and this is thought
to play a crucial role in aiding survival (Dubremetz and Entzeroth, 1993;
Beckers et al., 1994; Ossorio et al., 1994; Sibley et al., 1994c, 1995;
Lecordier et al., 1995; Speer et al., 1995). The ‘feeder’ organelles that
are apparent with intracellular coccidia are, as the name suggests, thought
to be involved in the parasite’s obtaining nutrients, but this process is yet to
194 G.H.COOMBS ETAL.

be investigated at the molecular level. There is evidence from the use of


antibodies specific for coccidian antigens that the parasite products are
inserted into the host-cell’s surface membrane, but the significance of this
has not been elucidated.

13. BIOCHEMICAL ACTION OF ANTlCOCClDlAL AGENTS

In general, agents that are effective against the genus Eimeria are also
effective for Toxoplasma, Sarcocystis and Isospora (see Stuart and Lindsay,
1986; Kirkpatrick and Dubey, 1987; Cawthorn and Speer, 1990; Johnson,
1990; McDougald, 1990), but until recently the vast majority of research
effort had been devoted to the economically important avian parasites. The
realization that toxoplasmosis and cryptosporidiosis are important infec-
tions related to the acquired immune deficiency syndrome (AIDS) resulted
in increased efforts to obtain better chemotherapy against these infections;
with promising results concerning the former (see below), but little success
so far with the latter. Although many different compounds, including those
active against Eimeria, have been tested against Cryptosporidium (see, for
example, Woods et al., 1996), only halofuginone (Current and Blagburn,
1990; Naciri et al., 1993; Khaw and Panosian, 1995) was found to be
effective and it has yet to be shown to be useful clinically. More recently,
paromomycin has been reported to have some activity against Cryptospor-
idium (see Wallace et al., 1993; Scaglia et al., 1994; Tzipori et al., 1995;
Verdon et al., 1995; Flanigan et al., 1996), as have the ionophore lasalocid
(Rehg, 1993) and benzimidazoles (Fayer and Fetterer, 1995). There is little
information on chemotherapy for Caryospora or Hammondia (see Kirkpa-
trick and Dubey, 1987; Upton and Sundermann, 1990; Dubey, 1993) and
only one report on Besnoitia (see Shkap et al., 1987). Infections with
Neospora are difficult to treat; clindamycin and sulphadiazine can be
effective if used early in infection, and trimethoprim and pyrimethamine
can also produce cures (Dubey and Lindsay, 1993). Therefore, the main
focus of this section is on Eimeria and Toxoplasma.
The drugs of choice for avian Eimeria are polyether ionophores (Dutton
et al., 1995; see Table 4),which have captured approximately 80% of the
current market (worth in total about US$350X lo6 per year). It is expected
that these compounds will dominate the market for many years to come,
because there has been no commercial launch of any novel class of syn-
thetic or semisynthetic avian anticoccidial drug since the early 1980s. This
is a major concern as resistance problems have arisen with current agents
(Chapman, 1993; Edgar, 1993; Vertommen and Peek, 1993). Some
vaccines are now available and perhaps they will set the trend for control
BIOCHEMISTRY OF THE COCClDlA 195

of avian coccidiosis in the future (Buxton, 1993; Wallach, 1993; Barriga,


1994; Buxton and Innes, 1995). However, more impetus in coccidial
biochemistrykhemotherapy research probably remains the most likely
way of providing an answer to the current problem. Furthermore, elucida-
tion of the mechanisms of action of, and resistance to, current drugs may
lead to novel chemical strategies for obtaining the new anticoccidial drugs
that are urgently required.
There are two good reviews covering the biochemical action of avian
anticoccidial drugs (Wang, 1982; Looker et al., 1986). McDougald (1982,
1990) and Haberkorn (1996) also commented on the mechanism of action
of these drugs in their reviews of the anticoccidial chemotherapy literature.
There have been few advances in this area in recent years and so the current
understanding is simply summarized in Table 4. It needs to be stressed that,
in most cases, the precise mechanism of action is not known. When
compared to the detailed information available on, for instance, many
antimalarial drugs, current knowledge of the biochemical action of avian
anticoccidial drugs is superficial. There have been only a few recent papers
on the effects of drugs on parasite morphology and the site of action of
anticoccidial drugs (Smith, C.K. and Stout, 1980; Maes et al., 1988;
Verheyen et al., 1988; Guyonnet et al., 1990; Daszak et al., 1991; Rather
et al., 1991; Zhu and McDougald, 1992; Conway et al., 1993; Ferguson et
al., 1994). Studies on the mechanism of resistance to ionophores in Eimeria
have revealed differences in drug uptake (Augustine et al., 1986) and
protein content (Zhu et al., 1994). However, despite the major impact
that drug resistance has had on anticoccidial chemotherapy (Chapman,
1993; Edgar, 1993; Vertommen and Peek, 1993), and studies on the basis
of resistance to some compounds (Pfefferkorn et al., 1989; Pfefferkorn and
Borotz, 1994), the mechanisms of drug resistance have not been elucidated
in detail in any case. More basic but focused research on coccidia is needed
to provide better understanding of drug-parasite interactions.
The standard treatment for acquired (non-congenital) toxoplasmosis is
the combined use of pyrimethamine and sulphonamide (Chang, 1993;
Georgiev, 1994; Khaw and Panosian, 1995), which inhibit dihydrofolate
reductase (DHFR) and dihydropteroate synthetase, respectively, of folate
metabolism and exhibit synergism. Other DHFR inhibitors, including tri-
methoprim, piritrexim and trimetrexate, have been used and also found to
be efficacious (Araujo and Remington, 1992; Georgiev, 1994). Recent
efforts to design more selective inhibitors of T. gondii DHFR have met
with some success (Gangjee et al., 1995, 1996; Rosowsky et al., 1995a, b,
c; Jackson et al., 1996; Piper et al., 1996) and determination of the activity
in vivo of these inhibitors is awaited.
Prenatal therapy of congenital toxoplasmosis is based primarily on spir-
amycin, whereas clindamycin is the standard agent for chemoprophylaxis
Table 4 Mechanism of action of commercially available avian anticoccidial drugs
Compound class Examples Mechanism of action
Polyether ionophore Salinomycin Perturb ion gradients
Lasalocid
Maduramicin
Semduramicin
Carbanilide/pyrimidine Nicarbazin Oxidative phosphorylation uncoupler?
Febrifugine Halofuginone Not known
Triazine Diclazuril Pyrimidine metabolism?
Toltrazuril Mitochondrial respiration? (see Harder and Haberkom, 1989)
Chlorophyll a-D1 complex? (see Hackstein et al., 1995)
Quinolone Decoquinate Mitochondrial respiratiodelectron transport
Buquinolate Topisomerase?
Pyridinol Clopidol Mitochondrial respiratiodelectron transport
Thiamine analogue Amprolium Thiamine uptake and utilization
Nitrobenzamide Zoalene Nicotinamide antagonist?
Nitromide
Guanidine Robenidine Oxidative phosphorylation uncoupler?
Benzylpurine Arprinocid Purine salvage?
Ion chelation (N-oxide active metabolite)?
Organic arsenical Roxarsone Binds protein sulphydryl groups?
Polyketide Oxytetracycline Protein synthesis?
Chlortetracycline
Sulphonamide Sulphadimethoxine Dihydropteroate synthetase
Sulfaquinoxiline
Aminopyrimidine and Ormethoprim and Dihydrofolate reductase and
sulphonamide sulphadimethoxine dihydropteroate synthetase
BIOCHEMISTRY OF THE COCClDlA 197

of newborn infants (Georgiev, 1994). These two agents, and other macro-
lides and azalides, are known to inhibit parasite protein synthesis (Blais et
al., 1993a, b). Interestingly, recent evidence suggests that the target site is
the plastid ribosomes rather than cytoplasmic or mitochondrial ribosomes
(Pfefferkorn and Borotz, 1994; Beckers et al., 1995). Eimeria also contains
similar plastid-like DNA, and so it is possible that the anti-Eimeria activity
of macrolides and azalides also operates through targeting plastid
ribosomes.
Another promising drug for the treatment of toxoplasmosis is the hydroxy-
naphthoquinone atovaquone (Georgiev, 1994). This is thought to act by
inhibiting the ubiquinol-cytochrome c reductase component (complex 111)
of the mitochondrial respiratory chain (Hudson, 1993). The role of this in
the parasite is not entirely clear as current evidence suggests that both
tachyzoites and bradyzoites are largely fermentative (see Section 2). It is
possible that the electron transport chain is primarily involved in pyrimi-
dine biosynthesis, as appears to be the case for the asexual bloodstream
forms of P. falciparum.
There have been several recent reports of novel experimental compounds
with anticoccidial activities. Some of these interesting anticoccidial
'leads' and their putative biochemical action are listed in Table 5. It is
apparent that macrolides/azalides are well represented, suggesting that
protein synthesis provides an important coccidial target. On the other
hand, nothing is known about the identity of the molecular targets of
many of the compounds. The recent discovery that a histone deacetylase
inhibitor has good anticoccidial activity (see Section 6) suggests that
transcription factors may be exploitable drug targets. It remains to be
seen whether any of these compounds or their analogues will become
viable commercial products.

14. THE CURRENT STATUS AND PRIORITIES FOR THE FUTURE

The information gained so far permits some tentative conclusions to be


drawn on the biochemical features that are characteristics of coccidia.
Future studies will reveal whether or not these speculations are correct.
Very many questions remain unanswered. Some are of fundamental
importance and are key issues. For example, what are the microenviron-
ments in which the parasites live? Detailed information would greatly aid
our attempts to understand how the parasites are adapted to their infection
sites and may well help in the development of satisfactory culture methods.
What determines host cell specificity of the parasites? This is particularly
intriguing for the several Eimeria species which infect the same host
Table 5 Putative mechanisms of action of potential anti-coccidial agents
Compound Organism Activity Putative mechanism Reference
Artemesinin Toxoplasma in vitro Free radical? Holfels et al., 1994
Azithromycin Cryptosporidium in vitrolin vivo Protein synthesis Araujo et al., 1991
Toxoplasma Rehg, 1991
Araujo and Remington, 1992
Blais et al., 1993a
Vargas et al., 1993
Benzimidazole Eimeria in vivo Not known Bayer AG, 1994
Carboxyemimycin Eimeria in vivo Orotic acid analogue Matsuno et al., 1984
Pyrimidine metabolism?
Clarithromy cin Toxoplasma in vivo Protein synthesis Edlind, 1991
Georgiev, 1994
Cyclosporin A Eimeria in vitrolin vivo Cyclophilin? McCabe et al., 1986
Toxoplasma P-glycoprotein? Rose and Hesketh, 1989
Harrison and Stein, 1990
Page et al., 1995
Cytosaminomycin Eimeria in vitro Not known Haneda et al., 1994
Difluoromethy lomithine Eimeria in vivo Ornithine decarboxylase McCann et al., 1981
Diolmycin Eimeria in vitro Not known Tabata et al., 1993c
Epiroprim Toxoplasma in vitrolin vivo Dihydrofolate reductase Chang et al., 1994
Martinez et al., 1996
Frenolicin B Eimeria in vivo Not known Omura et al., 1985
Hy&ox ycoumarin Eimeria in vivo Not known Upjohn Company, 1992
Hydroxynaphthoquinone Toxoplasma in vitrolin vivo Mitochondria1 electron Fry and Williams, 1984
(Atovaquone) Eimeria transport (complex III) Fry et al., 1984
Fry and Pudney, 1992
Romand et al., 1993
Hynapene Eimeria in vitro Not known Tabata et al., 3993b
Paromomycin Cryptosporidium in vivo Protein synthesis Edlind, 1991
Fayer and Ellis, 1993
F’rimaquine Eimeria in vivo Via free radical? Baker et al., 1986
Sarcocystis Oxidative phosphorylation? Bisby, 1990
Matsuno et al., 1991
Rifabutin Toxoplasma in vivo DNA-dependent RNA Araujo ct al., 1994, 1996a,b
polymerase Olliaro et al., 1994
Roxithromycin Toxoplasma in vivo Protein synthesis Araujo et al., 1991
Sinefungin Cryptosporidium in vivo Methylation reactions? Brasseur et al., 1994
ws-5995 Eimeria in vitrolin vivo Not known Devi et al., 1994
Xanthoquinodin Eimeria in vitrolin vivo Not known Tabata et al., 1993a
200 G.H. COOMBS ET AL.

animal but live within cells in different parts of the gut. Why? How similar/
different are the species that invade different hosts (e.g. E. bovis in cattle
compared with E. tenella in chickens and C. pamum in humandcattle and
C. muris in mice) and what determines the host specificity? Comparative
studies on different species are one way of pin-pointing the key adaptations
of the parasites for their particular host. Similarly, comparative studies of
the different developmental stages during the life cycle of a single parasite
species can help to elucidate the special adaptations of the stages. One area
requiring urgent attention with Toxoplusma is to determine how the brady-
zoites differ from the tachyzoites, and so explain the differing sensitivity to
drugs of the two forms. How do the sporozoites find an appropriate host
cell and penetrate the mucus lining the gut? It seems likely that the parasite
excretes some materials to disrupt the mucous layer, but this requires
further study. The oocyst of C. muris excysts in the stomach, how does
it avoid the lethal effects of the acidity? Perhaps only those oocysts that
excyst in the mucous layer can produce viable sporozoites or maybe the
parasite has a mechanism for neutralizing the acid in its microenvironment,
for instance by secreting urease in a similar fashion to Helicobacter pylori.
There are also several areas of biochemistry that have attracted, and are
attracting, considerable interest in other parasites but which are yet to be
seriously addressed in coccidia. These include cell signalling, the cell
cycle, cell differentiation and the subcellular organization of metabolism.
To be able to exploit rationally the biochemical peculiarities of a para-
site, an extensive amount of data on the putative target is usually required
(see Coombs and Croft, 1997). Such data have been obtained for coccidia
in very few instances to date, although the recent interest in protein
synthesis, mannitol metabolism and pyrophosphate-linked enzymes is
encouraging. Obtaining new drugs by empirical means requires adequate
methods of screening for activity. Screens which simulate conditions in
vivo are crucial for progress; unfortunately the in vitro methods currently
available are far from ideal.
What should be the priorities for the future?
The use of anticoccidial drugs is shadowed by resistance development.
Understanding the mechanisms of resistance is always useful, but it
becomes essential when there is no alternative drug that can be used.
Knowledge of the resistance mechanisms may enable the design of resis-
tance reversers that can overcome the immediate problem (see Coombs and
Croft, 1997). This is the strategy that has been pursued with some success
in malaria and cancer chemotherapy, but as yet little is known about the
mechanisms underlying resistance to anticoccidial compounds.
Advances in parasitological procedures are required, whether one wishes
to pursue the rational or the empirical route to drug discovery. One of the
most urgent needs for future biochemical work on many stages of several
BIOCHEMISTRY OF THE COCClDlA 20 1

coccidia is the development of satisfactory culture systems in vitro. With-


out these, many biochemical approaches are simply not feasible. Similarly,
the methods currently used for gently lysing and fractionating the parasites
are, in the main, not adequate. It has been possible to purify some of the
organelles of the apical complex and studies on them are in progress. The
procedures used at present, however, do not allow study of other organ-
elles, such as the refractile bodies of eimerian sporozoites, and yet isolation
of the organelles is one important way of determining their structure and
elucidating the role they play in the cell. The plastid-like organelles are
already attracting immense interest, particularly as drug targets with
renewed interest in various herbicides as potential anticoccidial agents
(see for example Arrowood et al., 1996; Stokkermans et al., 1996), and
elucidating their functional significance to the parasite is a priority in the
rational approach to chemotherapy (for instance, are they important in
energy generation, perhaps as a source of essential proteins? -see Section 6).
Perhaps other approaches can be used to find some of the answers. It is
undoubtedly true that some of the big advances in the near future will arise
from the application of molecular techniques to identify and characterize
coccidian genes of interest and to express them heterologously to provide
protein for study. Such technology will allow the investigation of proteins
that are insufficiently abundant to make purification a practical proposition.
There are also exciting possibilities of using reverse genetics to delete
parasite genes and so discover the roles that the encoded proteins play.
This approach is already providing fascinating insights with other parasitic
protozoa. The application of this technology to Toxoplasma has begun,
using the tachyzoite stage. Similar techniques are being developed for
Eimeria and may soon follow for Cryptosporidium, although the lack of
a good culture system in vitro means that growing the mutant lines may
prove very difficult.
The past 15 years have been a period when there have been great
advances in our understanding of the biochemistry of many parasitic pro-
tozoa, but this is not really the case for the coccidia. There have been some
exciting discoveries concerning Eimeria and Toxoplasma, but fewer than
could have been expected. We hope, however, that, by bringing together in
this review the new information that has been discovered and highlighting
areas and approaches that hold promise for further investigation, the next
decade may prove more fruitful for coccidian biochemistry.
202 G.H. COOMBS ET AL.

ACKOWLEDGEMENT

HD and SMAB acknowledge receipt of SERCBBSRC CASE studentships


with Pfizer.

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Genetic Transformation of Parasitic Protozoa

John M. Kelly

Department of Medical Parasitology. London School of Hygiene and


Tropical Medicine. Keppel Street. London. WCIE 7HT. UK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2. Transfection of Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.2. Stable transformation of Leishmania by episomal vectors . . . . . . . . . . . . 229
2.3. Gene targeting in Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
2.4. Functional analysis of Leishmania genes . . . . . . . . . . . . . . . . . . . . . . . . . 233
2.5. Analysis of gene expression in Leishmania ....................... 237
3. Transfection of Trypanosoma brucei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
3.1. Development of stable transformation vectors ..................... 239
.
3.2. Analysis of gene expression in T brucei . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.3. Transfection as a tool for functional analysis in T. brucei . . . . . . . . . . . . .246
4. Transfection of Trypanosoma cruzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.1. Development of vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.2. Transfection as a tool for functional analysis of T. cruzi genes . . . . . . . . . 250
4.3. Analysis of gene expression in T. cruzi . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
5. Transfection of the Apicomplexa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
5.1. Development of transfection techniques for Toxoplasma gondii . . . . . . . 252
5.2. Development of transfection techniques for Plasmodium . . . . . . . . . . . . . 256
6. Transfection of Intestinal Protozoan Parasites ......................... 257
6.1. Transfection of Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
6.2. Transfection of Giardia lamblia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
7 . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

ADVANCES IN PARASITOLOGY VOL 39 Copyright 0 1997 Academic Press Limited


ISBN 0-12-031739-7 All rights of reproduction in any form reserved
228 J.M. KELLY

1. INTRODUCTION

Since the development of recombinant DNA technology in the early 197Os,


transfection procedures have been applied to a wide range of organisms,
both prokaryotic and eukaryotic. These genetic manipulation experiments
have been essential for the unravelling of the mechanisms of gene expres-
sion and are the main approach used to define the function of gene products
(Oliver, 1996). In this chapter recent progress in the development and
application of these techniques to the parasitic protozoa is outlined. A
review is timely since transfection procedures have now been established
for most protozoan species that are major causes of human disease. Further-
more, laboratory scientists working on parasitic protozoa are increasingly
adopting a ‘twin-track’ strategy, with genome sequencing as the principal
approach to identifying new genes and transfection as the principal approach
to defining their function. The major practical benefit from this work will be
the rapid identification and assessment of many novel chemotherapeutic
targets and the development of new strategies for vaccination.
The terms ‘transfection’ and ‘transformation’ are often used interchange-
ably in the context of gene transfer experiments. For the purpose of this
chapter ‘transfection’ is defined as the introduction of foreign DNA into a
cell, and ‘transformation’ as the stable acquisition by a cell of new gene(s)
by uptake of foreign DNA. Transfection techniques are central to molecu-
lar biology and can be traced back to the discovery by Frederick Griffith
that non-virulent strains of pneumococci can be transformed into virulent
forms with an inheritable phenotype (Griffith, 1928). It was not until 1944,
however, that the nature of the transforming agent was discovered when in
a series of rigorous experiments Avery and colleagues demonstrated that
acquisition of the transformed phenotype was mediated by DNA (Avery et
al., 1944). Although not universally accepted at the time, this was one of
the landmark discoveries of 20th century science, in that it demonstrated
unequivocally that DNA was the source of genetic information.

2. TRANSFECTION OF LEISHMANIA

2.1. Background

Stable transformation systems applicable to parasitic protozoa were first


developed for the trypanosomatids Leishmania, Trypanosoma brucei and
Trypanosoma cruzi. In seeking to establish these gene transfer systems two
main problems were perceived; first, what sequences would be required in
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 229

the vector DNA to facilitate expression of the drug-selectable marker gene


and, second, how would this recombinant DNA be introduced into the
parasite? Preliminary approaches to addressing these questions were
made using transient transfection of the trypanosomatids Leptomonus sey-
mouri (Bellofatto and Cross, 1989) and Leishmania enriettii (Laban and
Wirth, 1989). In these experiments, no attempt was made to select the
transfected cells; rather, transfection efficiency was assessed by measuring
the level of activity which resulted from transient expression of the bacter-
ial reporter gene chloramphenicol acetyltransferase (cut) which was
included in the vector. At first it was thought that the choice of sequences
used to flank the cut gene in these types of experiment might present
problems because the mechanisms which regulate trypanosomatid gene
expression are unusual or unique in several aspects (Graham, 1995). For
example, in trypanosomatids many genes are arranged in tandem arrays,
polycistronic transcription of genes transcribed by RNA polymerase I1
appears to be the rule, and transcription is discontinuous with every
mRNA having the same 39 nucleotide 5 ’-spliced leader sequence derived
from the mini-exon genes.
In the absence of information on the nature of promoters or polyadenyla-
tion signals, sequences from the 5 ’-upstream region of the mini-exon genes
and the 3’-downstream region of the a-tubulin genes were used to flank cut
in the construct used in the L. seymouri experiments (Bellofatto and Cross,
1989). For the L. enriettii experiments, sequences from the 5 ’ - and 3’-
flanking regions of the a-tubulin genes were chosen (Laban and Wirth,
1989). Transfection was carried out by electroporation in each case. Elec-
troporation is the procedure whereby cell membranes are permeablised by
a short electric pulse allowing uptake of exogenous DNA; this technique is
commonly used in transfection experiments with other organisms (for
review, see Nickoloff, 1995). Transient expression of CAT activity was
detected in both L. seymouri and L. enriettii. These experiments therefore
helped to identify the genetic components necessary for expression in
trypanosomatids and demonstrated that electroporation techniques could
be applied to these organisms. Electroporation is now the predominant
method used to transfect parasitic protozoa, although the biolistic process
whereby cells are bombarded with tungsten microparticles coated in vector
DNA has also been used successfully in the case of the insect trypanoso-
matid Crithidiu fusciculutu (Vainstein et al., 1994).

2.2. Stable Transformation of Leishmania by Episomal Vectors

The first successful stable transformation of a parasitic protozoan was


achieved with Leishmania. This followed the construction of plasmid
230 J.M. KELLY

vectors using flanking sequences derived from the genes encoding a-tubu-
lin (Laban et al., 1990) and the bifunctional enzyme dihydrofolate reduc-
tase-thymidylate synthetase (dhfr-ts) (Kapler er al., 1990). These
transfection vectors contained a bacterial origin of replication, an ampi-
cillin-resistance (amp) gene and had the respective Leishmania sequences
placed on either side of the neomycin phosphotransferase (neo) gene. This
gene, which is encoded by the bacterial transposon Tn5, confers resistance
to the aminoglycoside G418 (Southern and Berg, 1982). After electropora-
tion, transformed Leishmania cells selected on the basis of G418 resistance
were obtained. These transformants were found to contain multiple extra-
chromosomal copies of the plasmids, which in some cases were organized
in large circular episomes composed of head-to-tail repeats of the vector.
Integration into the parasite genome was not demonstrated. The neo RNA
transcribed from these vectors was found to be correctly processed in terms
of polyadenylation and spliced leader addition. Although the mechanisms
by which transcription of these transfected genes are regulated remain to be
defined, it is clear that promoter elements, as recognized in other eukar-
yotic systems, are not involved. The spliced leader acceptor site and the
associated upstream polypyrimidine stretch both seem to be important for
efficient expression (Curotto de Lafaille et al., 1992).
These first-generation constructs have since been modified to function as
Leishmania expression vectors (LeBowitz et al., 1990; Tobin et al., 1993).
In the case of the dhfr-ts derived vectors (the pX family) it was found that
they could also be used to confer transfection-mediated G418 resistance on
several Leishmania species, and on the genera Endotrypanum and Crithi-
dia, but not on T. cruzi or T. brucei (Coburn et al., 1991). More recently,
several other Leishmania expression vectors have been developed, includ-
ing one which also functions in T. cruzi (Kelly et al., 1992) (Figure l), and
one which is completely deficient in Leishmania-derived sequences (Papa-
dopoulou et al., 1994a). This latter finding suggests that in Leishmania
specific signals may not be required for episomal replication or for the
initiation of transcription. The range of drug-selectable markers available
for Leishmania transfection experiments has also been extended to include
the hygromycin phosphotransferase (hyg) gene (Cruz et al., 1991), the N-
acetylglucosamine-1-phosphate transferase (nagt) gene (Lui and Chang,
1992a), the streptothricin acetyltransferase (sat) gene (Joshi et al., 1995)
and the phleomycin (ble)and puromycin (pac) resistance genes (Freedman
and Beverley, 1993). LeBowitz et al. (1992) have also reported the con-
struction of a Leishmania expression vector in which the herpes simplex
virus thymidine kinase gene (HSVtk) functions as a negative selectable
marker. Expression of HSVtk renders transformants susceptible to nucleo-
side analogues such as ganciclovir, enabling cells which have lost the
construct to be rapidly selected from a parasite population.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 231

500 bp
H

Figure I The pTEX vector which facilitates the expression of transfected genes
in Trypanosoma cruzi and Leishmania (Kelly et al., 1992). Both the multiple
cloning site (MCS) and the neor gene are flanked by the 5'-upstream and 3'-
downstream regions of the T. cruzi gapdh genes (thin line). The thick line,
including the amp' gene, represents sequences derived from the bacterial plasmid
pBluescript. Genes cloned into the MCS can be expressed at high levels in
transfected parasites. Other protozoan episomal expression vectors conform to
this general design.

In addition to plasmid vectors two types of cosmid shuttle vector are


now available for work with Leishmania (Ryan et al., 1993a; Kelly et al.,
1994) (Figure 2). Cosmids are modified plasmids. They contain an anti-
biotic resistance gene (often amp) as a selectable marker in bacteria, and
are engineered to carry the cos sequences that are essential for packaging
of bacteriophage h (see legend to Figure 2). Using cosmids it is possible
to selectively clone recombinant molecules containing DNA inserts of
between 30 and 45 kb. The Leishmunia cosmid shuttle vectors contain
additional drug-selectable marker genes (hyg or neo) flanked by
sequences that facilitate their expression in the parasite. These cosmids
can be introduced into Leishmania by electroporation where they repli-
cate episomally in multiple copies. The ability to introduce large DNA
fragments into Leishmania is a powerful additional approach to genome
analysis especially in organisms in which many genes are arranged in
large tandem arrays.
232 J.M. KELLY

Eco RI

Bam HI

pcosTL Sma I
8.5kb
Sac II cos Eco RI
Bst XI
Sac I

ADa I
Kpn i

Figure 2 pcosTL, a Trypanosoma cruzi and Leishmania cosmid shuttle vector


(Kelly et al., 1994). This vector is typical of many cosmids in that it has two cos
sites (derived from the cohesive ends of h phage) which are located on either side
of a ‘blunt-end’ restriction site (Sma I). Library construction involves ligation of
size fractionated parasite DNA (30-50 kb fragments obtained by partial Sau 3a
digestion) to vector arms (obtained by Barn HUSma I digestion) in the presence of
5 mM ATP. Because these conditions are unfavourable for ‘blunt-end’ ligations the
majority of products are linear molecules; either vector arms joined at the Barn HI
site or recombinant molecules in which the vector arms are joined to genomic DNA
fragments via ligation at the compatible Barn HUSau 3a sites. It is then possible to
selectively clone recombinant molecules containing large DNA inserts because A.
in vitro packaging systems will only package DNA molecules that carry cos
sequences separated by 37-52 kb of DNA. The packaged recombinant molecules
can then be introduced into Escherichia coli by infection where they subsequently
replicate as plasmids under ampicillin selection. Cosmid shuttle vectors such as
pcosTL contain an additional drug selectable marker (in this case neo‘) which
allows the recombinant cosmids to be propagated in the appropriate eukaryotic
cells. In pcosTL, the T. cruzi gapdh sequences located on either side of the neo
gene ensure expression of the functional RNA that permits the vector to confer
G418 resistance on transfected T. cruzi and Leishmania.

2.3. Gene Targeting in Leishmania

In the preliminary reports of Leishmania transfection, drug resistance was


observed to be episomally mediated. However, it soon became apparent
that integration of exogenous DNA into the parasite genome could also be
achieved and that the mechanism involved homologous recombination.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 233

Integration was favoured when low amounts of linearized vector DNA was
used for electroporation. In the first successful targeted integration, the
dhfr-ts genes of the avirulent L. major strain CC-1 were replaced with the
neo drug-selectable marker after simultaneous integration at both alleles
(Cruz and Beverley, 1990). In the construct used in this experiment, the
dhfr-ts flanking regions were placed on either side of the neo gene. As
expected from the role of DHFR-TS in folate biosynthesis, homozygous
‘knockouts’ could be isolated when electroporated cells were selected in
the presence of a thymidine supplement. Similar results were obtained
when the dhfr-ts genes were replaced consecutively with neo then hyg
(Cruz et al., 1991). In contrast, when this experiment was repeated with
a virulent L. major strain (LV39), auxotrophic mutants could not be iso-
lated (Cruz et al., 1993). Replacement of a single dhfr-ts gene with neo was
achieved, but it was then not possible to obtain dhfr-ts mutants using a
second replacement vector containing the hyg gene, even when selection
was carried out in the presence of thymidine. Instead, the transformed lines
were found to have undergone alterations in chromosome number, either
aneuploidy or tetraploidy, to accommodate the double replacement of dhfr-
ts with neo and hyg. This experiment, while demonstrating the plasticity of
the Leishmunia genome, also suggests that in this virulent L. major strain,
DHFR-TS performs an additional essential function which is independent
of its role in thymidine synthesis.
The a-tubulin genes of L. enriettii are organized in two allelic clusters
containing a total of 30 genes (Landfear et al., 1983), a common type of
organization for trypanosomatid genes. The applicability of gene targeting
techniques to study such multicopy arrays was demonstrated when the L.
enriettii a-tubulin genes were replaced with drug-selectable markers (Cur-
Otto de Lafaille and Wirth, 1992). The vectors were constructed such that
the neo and hyg genes were positioned between sequences derived from the
flanking regions of the tubulin clusters. In cases where replacement of both
clusters was observed, a-tubulin genes could be found within extrachro-
mosomal elements which were presumably generated as part of the deletion
process.

2.4. Functional Analysis of Leishmania Genes

The development of plasmid and cosmid shuttle vectors and of procedures


to facilitate gene ‘knockout’ represent a major technical advance that will
allow the functional analysis of Leishmania genes to proceed in parallel
with genome sequencing. The molecular basis of virulence, the mechan-
isms of drug resistance and of gene expression, and the assessment of
parasite-specific enzymes as novel chemotherapeutic targets are amongst
234 J.M. KELLY

questions now being addressed using these techniques. Many of the initial
functional studies have been aimed at elucidating the mechanisms of drug
resistance.
In Leishmania, drug resistance has often been associated with gene
amplification (Beverley, 1991; Ouellette and Papadopoulou, 1993). For
example, exposure of cells to a number of apparently unrelated drugs has
been correlated with amplification of the H locus (Beverley et al., 1984) in
the form of large extrachromosomal circles. Using plasmid shuttle vectors
it has been possible to dissect the 40 kb H locus and to identify the genes
which confer drug resistance when overexpressed. Thus the gene that
confers arsenite resistance was identified after fragments of the L. major
H region were ligated into a Leishmania expression vector and introduced
by transfection back into the parasite at high copy number (Callahan and
Beverley, 1991). Constructs which produced resistance to both arsenite and
trivalent antimonials were shown to contain a P-glycoprotein-related gene,
ImpgpA. The mechanism of antimonial resistance conferred by overexpres-
sion of lmpgpA has been shown to result from decreased influx, possibly
caused by the interaction of PGPA with other Leishmania proteins (Call-
ahan er al., 1994). In an independent study with L. tarentolae, a similar
resistance phenotype was observed (Papadopoulou et al., 1994b). In addi-
tion, these authors also found an association between overexpression of the
P-glycoprotein-related gene and resistance to pentavalent antimonials.
The pattern of drug resistance observed in Leishmania cells that over-
express 1mpgpA does not conform to the multi-drug-resistant (MDR) phe-
notype that is found in mammalian cells as a result of overexpression of P-
glycoproteins. However, an MDR phenotype has been identified in L.
donovani. It results from overexpression of a Leishmania mdr homologue
(ldmdr I ) , either by gene amplification or by transfection (Henderson et al.,
1992). This gene, which is not localized in the H region, is associated with
cross-resistance to vinblastine, puromycin and the anthracyclines. In L.
enriettii, stepwise selection with vinblastine has been shown to result in
the generation of V-circles, 40 kb extrachromosomal elements which con-
tain an mdr homologue (lemdr I ) (Chow et al., 1993). Using a homologous
recombination approach to disrupt specific sites within the V-circle, it was
demonstrated that vinblastine resistance results only from overexpression
of the lemdr I gene (Wong et al., 1994).
Transfection procedures have been used in the functional analysis of
another novel gene associated with the H region, ltdh, a short chain
dehydrogenase (Papadopoulou et al., 1994~).Plasmid-mediated overex-
pression of this gene was shown to confer resistance to methotrexate, an
inhibitor of DHFR. The mechanism of resistance is thought to involve
increased production of reduced folates by a pathway that involves pterin
and which can compensate for the inhibition of DHFR by methotrexate.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 235

Experiments with homozygous ltdh mutants, which were obtained by


targeted gene deletion, suggest that the product of ltdh is a key enzyme
that converts a pterin derivative into an essential co-factor. It is this co-
factor which is converted into reduced folates and so compensates for
DHFR inhibition. The uniqueness of LTDH suggests that it may have
potential as a target for specifically designed anti-parasitic drugs. Other
potential chemotherapeutic targets in Leishmania which have been studied
using transfection include lmcpa, a single copy gene that encodes a
cysteine proteinase. Double-knockout of this gene had no obvious pheno-
typic effect suggesting that the enzyme is non-essential for parasite survi-
val (Souza et al., 1994). The role of other Leishmania cysteine proteinases
is currently being investigated using similar approaches (see also Figure 3).

Figure 3 Targeted gene deletion in Trypanosoma cruzi. In the T. cruzi X10.6


strain the genes encoding the major cysteine protease are organized in two allelic
clusters of 14 and 23 tandemly repeated copies (Tomas and Kelly, 1996). To
address the function of this enzyme we have designed vectors to delete the clusters
after integration by homologous recombination (M.C.Taylor and J.M. Kelly,
unpublished). (A) Organization of the cysteine protease gene clusters showing
the 1.4 kti coding sequence (open box) and the 400 bp intergenic regions (thin
line). The 3'-proximal gene contains a divergent sequence towards the 3'-end
(shaded). (B) Linearized vector used to delete the entire cluster. The neo gene
(filled box) is arranged between the 5 ' - and 3'-flanking regions of T. cruzi gapdh.
The thick lines represent the sequences derived from the flanking regions of the
cysteine protease gene cluster which are arranged to target integration. (C) Follow-
ing transfection and G418 selection, drug-resistant organisms were isolated in
which one of the gene clusters had been replaced by the vector DNA.
236 J.M. KELLY

In Leishmania, tunicamycin resistance has also been associated with the


amplification of large extrachromosomal elements, although these are dis-
tinct from the H and V circles. Transfection experiments have confirmed
that resistance results from amplification of the nagt gene and the con-
comitant increased expression of the protein product (Lui and Chang,
1992a). By exploiting nagt as a selectable marker in a plasmid vector, it
has been possible to overexpress the major surface metalloprotease, Gp63,
in a L. amazonensis cell line deficient in the enzyme (Lui and Chang,
1992b). The transformed parasites had a slightly enhanced ability to bind to
macrophages.
Experiments involving transfection have also been used in other studies
aimed at investigating the interaction of Leishmania parasites with the
immune system. For example, recombinant L. major which express and
secrete active murine interferon-y (IFN-y) have been inoculated into nude
mice and a genetically susceptible BALB/c strain (Tobin et al., 1993).
Disease progression was reduced in the T-cell deficient nude mice, but not
in the BALB/c strain. This observation is consistent with earlier studies
(Sadick et al., 1990) which showed that inoculation of genetically suscep-
tible mice with exogenous IFN-y alone fails to impede the inappropriate
Th2 cell response that L. major elicits. The role of other cytokines in
modifying T-cell responses should be amenable to investigation using
this approach. In another study (Kaye et al., 1993) the ability of Leishma-
nia to target macrophages, and the phagolysosome in particular, has been
exploited to investigate antigen processing using transfected Leishmaniu
modified to express ovalbumin and p-galactosidase. These reporter proteins
were successfully targeted to the phagolysosome where they were correctly
processed for CD4+ T-cell recognition.
This ability of transfected Leishmania to present foreign antigens to the
immune system has stimulated interest in the potential of this technology to
produce live vaccines. In an initial study, partial protection against malaria
was achieved after mice were inoculated with recombinant L. enrietrii
which expressed Plasmodium yoelii circumsporozoite protein (PyCSP)
(Wang et al., 1995). PyCSP was shown to be present at high levels on
the Leishmania surface and the protected mice made a cytotoxic T-lym-
phocyte response against the protein. Using transfection techniques it has
also been possible to produce attenuated Leishmania for use in vaccination
studies. Auxotrophic L. major, in which the dhfr-ts genes had been deleted
by targeted integration, were able to protect mice against a challenge with a
virulent L. major strain (Titus et al., 1995). Although the thymidine-
dependent attenuated parasites persisted in the mice for up to 2 months,
they were incapable of causing disease in either susceptible or immunode-
ficient strains. The main advantage of using this direct approach to attenua-
tion is that reversion to a virulent phenotype should not be a problem,
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 237

although it cannot be completely excluded (Papadopoulou et al., 1994~).


The production of dhfr-rs mutants from other Leishmania species may also
prove to be a safe and effective approach to vaccination against the other
forms of leishmaniasis. In addition, it may be possible to use the dhfr-ts
Leishmania as a multi-vaccine vehicle by manipulating the recombinant
organisms to express protective antigens from other pathogens.
The development of cosmid shuttle vectors (Ryan et al., 1993a; Kelly et
al., 1994) has made it possible to apply ‘functional cloning’ techniques to
Leishmania. These vectors can contain large DNA inserts (> 40 kb) allow-
ing the entire parasite genome to be accommodated by as few as 1000
cosmids. It is therefore feasible to use ‘shotgun’ transfection with cosmid
genomic DNA libraries to isolate genes by functional complementation or
on the basis of a conferred phenotype such as drug resistance. In the first
applications of this approach (Ryan et al., 1993b; Descoteaux et al., 1995)
functional complementation has been used to isolate genes required for
synthesis of lipophosphoglycan (LPG), the complex glycoconjugate which
is the most abundant molecule on the surface of the Leishmania promas-
tigote and which is essential for infection. The stategy has involved trans-
fection of LPG-deficient L. donovani mutants with a cosmid library derived
from the wild-type parasite; the transfected parasites were then ‘panned’
using antibodies to select transformants that expressed the normal LPG
phenotype. This resulted in the identification of two novel parasite genes;
one encodes a putative glycosyltranferase thought to be responsible for
addition of galactofuranose residues during LPG biosynthesis (Ryan et al.,
1993b), and the second encodes a protein that may mediate the transport of
LPG precursors or biosynthetic enzymes into the secretory network (Des-
coteaux et al., 1995). Other independently derived LPG mutants are cur-
rently under investigation by these workers in an attempt to genetically and
biochemically dissect the pathway of LPG biosynthesis. These type of
experiments illustrate that functional complementation using cosmid shut-
tle vectors has enormous potential as an approach to identifying and
characterizing protozoan genes. Identification and characterization of viru-
lence factors, novel drug-resistance genes and the triggers of differentiation
are a few of the questions which can be addressed given suitable screening
systems.

2.5. Analysis of Gene Expression in Leishmania

As with other organisms, transfection has proved to be a crucial experi-


mental tool for analysis of Leishmania gene expression. Various studies
have suggested that regulation of gene expression occurs predominantly at
the post-transcriptional level. For example, attempts to identify RNA
238 J.M. KELLY

polymerase 11-dependent promoters have not been successful in Leishma-


nia and the essential requirement for expression has been shown to be the
presence of an upstream trans-splice acceptor site and an adjacent pyrimi-
dine-rich tract (Curotto de Lafaille et al., 1992). Indeed, transfection
experiments vectors have shown that even when the upstream region of a
gene is entirely replaced by a synthetic pyrimidine tract and AG dinucleo-
tide splice site, expression can still occur (Curotto de Lafaille et al., 1992;
Papodopoulou et al., 1994a). Another unusual aspect of trypanosomatid
gene expression is the lack of a recognizable motif identifying the site of
polyadenylation. Following an elegant series of experiments involving both
stable and transient transfection of L. major (LeBowitz et al., 1993), it was
proposed that the site of polyadenylation is specified by the location of the
trans-splice site of the immediate downstream gene. This coupling
mechanism could provide a means of co-ordinating expression of genes
linked within polycistronic transcripts and could act as an additional point
of control at the post-transcriptional level.
When L. amazonensis promastigotes are exposed to mammalian tem-
peratures there is an increase in the level of several transcripts including
the heat shock protein 83 (hsp83) mRNA. In contrast to the situation in
other organisms, this increase does not appear to be associated with upre-
gulation of transcription (Argaman et al., 1994). Rather, increased mRNA
stability seems to be the major mechanism involved since studies have
shown that at lower temperatures degradation of hsp83 mRNA is more
rapid. To gain further insights into how the level of expression is con-
trolled, the cat gene was flanked by the hsp83 intergenic sequences and
inserted into the Leishmania expression vector pX (Argaman et al., 1994).
When L. amazonensis promastigotes were transformed with this construct,
cat mRNA was found to accumulate in response to increased temperature.
Since upregulation of transcription is not involved, this suggests that
sequences in the hsp83 intergenic region could mediate the temperature-
dependent stability of the transcript.
In an analogous approach Ramamoorthy et al. (1995) used stable trans-
formation of L. chagasi to investigate the role of gp63 intergenic regions in
stage-specific expression. Using P-galactosidase as a reporter gene in the
pX vector, these authors found that incorporation of gp63 3 '-untranslated
regions (-UTRs) alone had no significant effect on expression. However,
when the 3'-UTR of mspS (a gp63 gene normally expressed only in sta-
tionary phase promastigotes) was inserted together with its downstream
intergenic region, there was a 20-fold increase in P-galactosidase activity at
the appropriate stage. When a similar experiment was performed using the
equivalent sequences obtained from gp63 genes that are expressed consti-
tutively in culture or are expressed in logarithmic growth phase, no altera-
tions in the level of activity was observed. These experiments imply that
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 239

different gp63 genes are differentially regulated at the post-transcriptional


level and that sequences in the intergenic regions have a role in the process.
The sequence elements that mediate transcriptional control of protein
coding genes in Leishmunia have not so far been identified. However,
some progress has been made in delineating sequences involved in con-
trolling expression of the mini-exon genes that encode the spliced leader
sequence. As in other trypanosomatids, the Leishmania mini-exon genes
are organized in a series of tandem arrays. In both L. tarentolae (Saito et
al., 1994) and L. amazonensis (Agami et al., 1994) it has been demon-
strated in experiments using transfection studies and linker scanning
analysis that transcription of each mini-exon gene is controlled indepen-
dently by conserved upstream elements. Nucleotides between positions
-30 and -70 seem to be important in both cases, although sequences at
other positions also contribute. The polymerase that mediates transcrip-
tion of the mini-exon genes has some properties similar to RNA poly-
merase I1 (Saito et al., 1994).
Transcription factors which function in Leishmania have not so far been
characterized. However, a L. major 28 kDa DNA-binding protein desig-
nated hexamer binding protein (HEXBP), which contains zinc finger motifs
and which binds to oligonucleotides derived from the 5’-untranslated
region of the gene encoding Gp63 has been identified (Webb and McMas-
ter, 1993). The precise biological function of this single-stranded DNA-
binding protein remains to be elucidated since deletion of both copies of
the gene has no apparent phenotypic effect and does not alter the abun-
dance of gp63 mRNA (Webb and McMaster, 1994). It is clear that much
remains to be discovered about the nature of proteins that regulate gene
expression in Leishmania. The availability of genetic manipulation systems
such as those described here, should facilitate rapid progress in this area.

3. TRANSFECTION OF TRYPANOSOMA BRUCE/

3.1. Development of Stable Transformation Vectors

The first successful T. brucei transfection experiments were reported in


1990 when several groups described transient transfection of procyclic
I

forms using electroporation (Clayton et al., 1990; Rudenko et al., 1990;


Zomerdijk et al., 1990). In contrast to the situation in Leishmania, tran-
scription in these systems was found to be promoter dependent. Following
on from these experiments, stable transformation was soon achieved with
the neo gene being used as a selectable marker (ten Asbroek et al., 1990;
240 J.M. KELLY

Lee and Van der Ploeg, 1990; Eid and Sollner-Webb, 1991). In each of
these cases, transformation was mediated by integration which was found
to occur exclusively by homologous recombination, with the formation of
episomes being rare (ten Asbroek et al., 1993). Transfection with linear
DNA is considerably more efficient than with circular DNA and transfor-
mation efficiencies levels as high as 1 X have been reported (ten
Asbroek et al., 1993). Less than 1 kb of homologous flanking sequence was
found to be necessary for targeted insertion into the tubulin locus. Other
selectable markers including the hyg (Lee and Van der Ploeg, 1991) and ble
(Jefferies et al., 1993) resistance genes have now also been used success-
fully in T. brucei transformation experiments.
In these initial reports, transformation of bloodstream form T. brucei was
not described. However this has now been achieved (Carruthers et al.,
1993), largely as a result of improvements in culturing methods, particu-
larly the development of a technique for obtaining high plating efficiency
of colonies on agarose plates (Carruthers and Cross, 1992). In these blood-
ation experiments, constructs were targeted to the
For reasons that are still unclear, transformation in
these and other experibents with bloodstream parasites (Kelly et al., 1995;
Rudenko et al., 1995) *as 1000-fold less efficient than with the procyclic
forms. Despite this, th$ development of bloodstream form transfection
represents a significant step forward which has greatly extended the poten-
tial applications of these techniques to the African trypanosome.
Unlike Leishmania and other trypanosomatids, stable transformation of
T. brucei with episomal vectors has proved difficult to achieve routinely.
Integration is the favoured mechanism of transformation and the require-
ments for autonomous replication appear to be more stringent than in
other trypanosomatids. For example, after transfection with a circular
construct containing the neo gene flanked by the tubulin intergenic
regions, ten Asbroek et al. (1993) obtained only a single transformant
containing extrachromosomal plasmid. This was in the form of a circular
pentamer and was lost soon after drug pressure was removed. In contrast,
however, Patnaik et al. (1993) have reported an episomal vector which
exhibits greater stability in T. brucei procyclics in the absence of drug
pressure, and which replicates as a single copy element. This vector
(pT13-11) contains the neo gene flanked by the 5’- and 3’-regions of
the procyclic acidic repetitive protein (parp) gene, including the promo-
ter. Stable episomes were isolated after random fragments of T. brucei
DNA had been introduced into the construct prior to ‘shotgun’ transfec-
tion and selection of drug-resistant transformants. Since the ability of
pT13-11 to replicate and segregate in T. brucei resulted from the inser-
tion of a single DNA fragment, one possibility is that both these proper-
ties were conferred by closely linked genomic elements. Further studies
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 241

have also shown a close linkage between elements responsible for repli-
cation and transcription (Patnaik er al., 1994a); a region encompassed by
the parp promoter seems to play a crucial role in plasmid replication.
Transfection experiments with pT 13-1 1 and derivatives thereof have
demonstrated that these episomes can exist in a broad range of trypano-
somatids. However, there is variation in the requirements for stable
replication (Patnaik et al., 1994b) between trypanosomatids with the
requirements of T. brucei being the most stringent.
An analogous approach was used by Metzenberg and Agabian (1994) to
study episome-mediated transformation in T. brucei. Xba I fragments of T.
@
eucbrF&+w
.a e cloned randomly into a plasmid which contained the parp
N m o t e r placed in'front of the hyg gene. After electroporation of procyclics
with the resultant libraw of molecules, drug-resistant cells in which trans-
formation was episomally mediated were isolated. The vector was found to
contain a 1 kb insert thar was derived from a kinetoplast DNA (kDNA)
minicircle. Vector molechles were confined to the nucleus and were not
detected in the mitochondrion. If these kDNA sequences function by pro-
viding an origin of replication, it will imply a common mechanism of
episome maintenance in the nucleus and mitochondrion of T.brucei. Simi-
lar to observations described in a previous report (ten Asbroek et al., 1993),
the vector molecules were present in several copies per cell and were
maintained as supercoiled extrachromosomal elements composed of head-
to-tail repeats (7-9 copies). The formation of large episomes composed of
tandemly linked vector molecules has also been reported by Lee (1995). The
construct used in this transfection experiment contained the parp promoter
linked to the neo gene and was found to exhibit mitotic stability in procyclic
trypanosomes, although the mechanism involved is unclear. At the moment,
therefore, there is no consensus on the absolute requirements for episomal
stability, replication and segregation in T. brucei.
More recently, the construction of trypanosome artificial chromosomes
(TACs) has been reported (Lee et al., 1995). These vectors contain theparp
promoter, use the hyg gene as a selectable marker, and have telomere
repeats and subtelomeric sequences at the termini of the linear molecule.
In transfected cells, the molecules were mitotically stable and remained
linear, but were found to have increased in size due to the addition of new
sequences at internal sites. These additional sequences were derived from
the mini-chromosomes and from a subset of larger chromosomes. Although
the development of TACs is still at a early stage, and further work will be
required, they could have applications in the study of gene expression,
particularly the switching events involved in antigenic variation (see
below).
To extend the range of tools available for functional analysis, Wirtz and
Clayton (1995) have recently reported the development of an inducible
2 42 J.M. KELLY

expression system for T. brucei. Because inducible T. brucei promoters


have not been identified, the authors made use of the Escherichia coli
tetracycline(Tc)-responsive Tet repressor (tetR) as the central component
of their system. First they engineered trypanosomes to constitutively
express the Tet repressor by introducing a modified copy of the TnlO-
encoded tetR gene into the * under hygromycin selection. An
expression construct wzdhe-ich the ble selectable marker
and the luc reporter genes were placed under the control of the parp
promoter, and the Tet operator sequences werd placed immediately adja-
cent to the site of transcription initiation. The best results were achieved
when this construct was targeted in the reverse /orientation into the rDNA
non-transcribed spacer. Addition of as little as 50 n g / d Tc could produce a
10 000-fold increase in luciferase activity; when the Tc was withdrawn, the
luciferase activity fell to background levels. Further experiments have
shown that the level of inducible expression can be regulated by Tc in a
dose-dependent manner. Inducible expression systems should have many
potential uses. For example, they will greatly aid the study of genes which
express products that are potentially toxic or which function in cell-cycle
control or differentiation.

3.2. Analysis of gene expression in T. brucei

One of the ways in which T. brucei adapts to changes in its environment is


by altering expression of its surface antigens. In the insect procyclic form
of the parasite, P A W (also known as procyclin) is the predominant surface
molecule, whereas in the bloodstream form it is the variant surface glyco-
protein (VSG). There is a repetoire of more than 1000 antigenically distinct
vsg genes, but only one of these is expressed at a time. The parasite evades
the host immune system by changing its VSG coat. This switching often
involves in situ activation of transcription or DNA re-arrangements in
which the active vsg gene is replaced at an expression site by a different,
previously silent vsg gene (for review see Pays et al., 1994; Borst and
Rudenko, 1994; Graham, 1995). Transfection techniques are now central to
investigations into the mechanisms which control these switching events
and which regulate stage-specific expression. Currently, three T. brucei
promoters have been characterized: the rRNA promoter (Zomerdijk el al.,
1991), the vsg promoter (Zomerdijk et al., 1990; Jefferies et al., 1991) and
the parp promoter (Rudenko et al., 1990; Sherman et al., 1991; Brown et
al., 1992). Transcription in each of these cases is a-amanitin resistant, a
property characteristic of RNA polymerase I involvement (Chung et al.,
1992). In other eukaryotes, the sole function of RNA polymerase I is to
transcribe rRNA genes (Nogi et al., 1991).
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 2 43

In T. brucei, the parp genes are arranged in arrays of two or three copies
and are transcribed polycistronically under the control of a single upstream
prorp6(P=., 1994). Transient transfection assays coupled with
M e r scanning analysh have been used to delineate the upstream regula-
tory sequences and to evhluate their contribution to transcriptional control
(Sherman et al., 1991; Bro\wn et al., 1992). These experiments revealed the
presence of a bipartite core control element situated just upstream of the
transcription initiation site and other features characteristic of a RNA
polymerase I promoter. In the bloodstream form of the parasite, parp
mRNA is completely absent even though transcription of the parp genes
still occurs at about 10% of the level measured in procyclics (Pays et al.,
1990). This suggests that other mechanisms in addition to control of
promoter activity must play a significant role in the stage-specific expres-
sion of PARP. To investigate this further, Vanhamme et al. (1995) targeted
a reporter construct into the parp promoter region. They confirmed the
presence of transcriptional activity in the bloodstream form and obtained
evidence for regulation at the level of RNA elongation. Using a variety of
transfection based techniques, Berberof et al. (1995) have demonstrated
that sequences in the 3'-UTR of parp mRNA also play an important part in
regulation, presumably acting at the level of RNA maturation andor
stability. Hehl et al. (1994) have identified an additional regulatory element
in the 3'-UTR of parp mRNA. This 16-nucleotide sequence has the poten-
tial to form a stable hairpin loop structure, does not affect mRNA stability,
but is required for efficient translation.
The major surface antigen of procyclics in the related parasite Trypano-
soma congolense is the protein GARP (glutamic acidalanine rich protein).
Although it shares no sequence similarity, this molecule appears to be a
functional analogue of PARP. Indeed, when GARP is expressed in T.
brucei procyclics following stable transformation, it can be localised on
the cell surface (Hehl et al., 1995). Like the parp genes, those expressing
GARP are also arranged in tandem repeats. In a series of experiments,
which utilized transient transfection assays and nuclear run-ons (Graham et
al., 1996), the garp promoter has been localized to a region about 500 bp
upstream of the start codon of the 5'-proximal gene; this compares to less
than 100 bp in the case of parp. In addition, it was found that transcription
of the garp genes is a-amanitin sensitive, suggesting that the process may
be mediated by RNA polymerase 11. This could be related to the observa-
tion that the garp genes are constitutively active like other protein coding
genes transcribed by RNA polymerase 11. In T. congolense bloodstream
forms, the garp mRNA is present but the protein is not detectable implying
that GARP expression is controlled predominantly at the translational a n d
or post-translational level.
In bloodstream form T. brucei, the actively expressed vsg gene is
244 J.M. KELLY

situated in a telomeric site which also contains a number of other expres-


sion site associated genes (ESAGs). Although there may be up to 20 such
expression sites, only one is active at a time. The vsg gene is co-transcribed
with the ESAGs as part of a polycistronic transcript under the control of a
single upstream promoter (Borst and Rudenko, 1994). Transfection tech-
niques have been central to addressing two of the main questions relating to
vsg gene regulation; that is, what are the mechanisms underlying stage-
specific expression, and how does the bloodstream form trypanosome
suppress expression of all vsg genes apart from the active one?
Using approaches such as nuclear run-on assays and ultraviolet inactiva-
tion of transcription, the vsg promoter had been localized to a region 45-
60 kb upstream of the vsg gene (Johnson et al., 1987; Pays et al., 1989). It
has now been possible with transient transfection, using the cat gene as a
reporter, to fine map the promoter and to demonstrate that it is active in
both the bloodstream and procyclic forms of the parasite (Zomerdijk et al.,
1990; Jefferies et al., 1991). In procyclics, however, transcription termi-
nates within several hundred nucleotides. This was demonstrated by
nuclear run-on assays and confirmed by experiments in which marker
genes were targeted into the vsg promoter region (Rudenko et al., 1994).
These experiments also showed that the vsg expression sites are constitu-
tively active in procyclics, although at a reduced level. In addition, most if
not all expression sites appear to be simultaneously active. The down-
regulation of promoter activity in procyclics was found to be dependent
on the genomic context, but independent of orientation. Thus evidence
suggests that the stage-specific transcription of the vsg genes results from
controls operating at the levels of both initiation and elongation.
Recent studies have also shown that silencing of non-active vsg gene
expression sites in bloodstream parasites is a positional effect that is
independent of the promoter that is being repressed (Horn and Cross,
1995; Rudenko et al., 1995). When the vsg promoter was replaced with
the parp or rDNA promoters using targeted integration, these promoters
could be inactivated and reactivated in the context of the vsg expression
site. These experiments strongly suggest that in the vsg expression site,
promoter activity does not depend on sequences specific to the vsg pro-
moter, and therefore other mechanisms such as telomeric silencing must be
involved in regulating expression.
T. brucei also contains a distinct subset of vsg genes (20-30 copies) that
are expressed only in the metacyclic stage of development. These genes are
randomly activated in the salivary glands of the tsetse fly during differ-
entiation from the procyclic form. The metacyclic vsg (m-vsg) genes are
located adjacent to the telomeres on larger chromosomes and generally
only one gene is expressed per cell. Expression of the m-vsg genes has
recently been investigated using nuclear run-on assays and transient trans-
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 245

fection (Graham and Barry, 1995). The genes encoding the M-VSG seem to
be distinct from other trypanosome genes in that they are transcribed as
monocistronic transcripts and their stage-specific expression is regulated
exclusively at the level of transcription. This distinct mechanism of control
is thought to facilitate the random activation of m-vsg genes which could
enhance transmission by presenting the host immune system with an anti-
genically diverse parasite population.
Several studies have shown that post-transcriptional regulation is an
important feature of gene expression in the trypanosomatids (Graham,
1995). This is a consequence of polycistronic transcription; linked genes
are probably co-transcribed at equivalent rates and any differences in the
level of steady state mRNA must therefore result from control exerted at
the post-transcriptional level. In bloodstream form T. brucei, such mechan-
isms account for the greater than 100-fold difference in the abundance of
the mRNAs encoding vsg and some of the upstream ESAGs. Sequences in
the 3’-UTR of vsg mRNA have been shown to mediate stage-specific
expression (Berberof et al., 1995). A 97-nucleotide region, immediately
upstream of the vsg mRNA polyadenylation site, was found to confer a
three-fold stimulation of reporter gene expression in bloodstream parasites
and to downregulate by the same extent in procyclics. This nine-fold
difference between the two stages suggests that elements within this 3’-
UTR sequence make a significant contribution to the stage-regulated
expression of vsg.
Downstream of trypanosomatid genes consensus signals for polyadeny-
lation such as the AAUAAA sequence have not been identified. In Leish-
mania, for example, it has been shown that the site of polyadenylation is
specified not by a consensus signal sequence but by the position of the
splice acceptor site of the downstream gene (above) (LeBowitz et al.,
1993). Consistent with this, analysis of transcription using permeabilized
cells has shown that in T. brucei, trans-splicing of the downstream gene
must precede polyadenylation during the synthesis of transcripts derived
from the tubulin array (Ullu et al., 1993). To identify signals required for
polyadenylation of T. bmcei tubulin mRNA, Matthews et al. (1994) pro-
duced an expression construct in which the intergenic region of the p- and
a-tubulin genes was placed between an upstream cat and downstream luc
gene. The intergenic region was then mutagenized with a series of block
substitutions. After introduction of the resulting constructs into T. brucei
by electroporation, the RNA expressed by the reporter genes was analysed.
It was inferred from the results that pyrimidine-rich elements located
upstream of the a-tubulin splice acceptor site were required for accurate
polyadenylation of the upstream p- and for trans-splicing of the downstream
a-tubulin genes. Similar conclusions were drawn from an analogous series
of experiments aimed at investigating polyadenylation of the parp mRNA
246 J.M. KELLY

(Schurch et al., 1994; Vassella et al., 1994). This coupled reaction of trans-
splicing and polyadenylation of co-transcribed genes may be an important
site of gene regulation in trypanosomes.
RNA polymerase I1 promoters have not been characterized in T. brucei
and several studies have implicated the 3’-UTR as the major mediator of
stage-specific regulation of pol I1 transcribed genes. For example, aldolase
mRNA is present at increased levels in bloodstream parasites, although
there is no change in transcription rate (Hug et al., 1993). To test whether
or not the aldolase 3’-UTR could influence the level of expression it was
placed downstream of the luc gene in a transfection construct. After
electroporation of bloodstream and procyclic forms, the stage specificity
of luc expression was found to be similar to that shown by the aldolase
genes. In other experiments, it was also demonstrated that 3’-UTRs derived
from the hexose transporter genes could confer stage specificity on reporter
genes after stable transformation (Hotz et al., 1995). Although the mechan-
isms remain to be defined, it appears that control operates at several levels,
including alterations in RNA stability.

3.3. Transfection as a Tool for Functional Analysis in T. brucei

One of the first uses of T. brucei transfection techniques was as a tool to


characterize glycosome targeting signals. Glycosomes are microbody-like
organelles unique to the kinetoplastids that are thought to be related to the
peroxisome microbodies found in many other eukaryotes (for review, see
Sommer and Wang, 1994). Several housekeeping enzymes are sequestered
in glycosomes, including the first seven enzymes of glycolysis. The gly-
cosomal enzymes are encoded by nuclear DNA and are synthesized on free
ribosomes in the cytosol. Import into the glycosome is a rapid post-transla-
tional event and does not involve protein modification or peptide cleavage.
The topogenic signal is thought to reside in the sequence or structure of the
mature enzyme. In the case of peroxisomes a carboxyl-terminal tripeptide,
SKL and variants thereof, has been shown to function as a minimal signal
for targeting proteins (Gould et al., 1989). For example, removal of the
carboxyl-terminal SKL sequence from firefly luciferase blocks its import
into peroxisomes.
To investigate glycosome import signals in T. brucei, the luc gene was
inserted into the tubulin locus by homologous recombination (Sommer et
al., 1992). When the location of the expressed protein was analysed, it was
found to be sequestered in glycosomes. The importance of the carboxyl-
terminal tripeptide was demonstrated in a second experiment when deletion
of the SKL sequence was shown to block import into the glycosome. After
a number of transfection experiments involving expression of a series of
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 247

modified luc genes, it was demonstrated that the glycosome tripeptide


targeting signal was degenerate and could consist of (A/C/G/H/N/P/S/T)
(WK/M/N/Q/R/S) (I/L/M/Y). These results indicated that the requirements
for glycosome import are less stringent than those for peroxisome import.
Using transfection, a second reporter protein (CAT) has also been targeted
to the T. brucei glycosome after attachment of ‘SKL-like’ tripeptides to the
carboxyl-terminus (Blattner et al., 1992). In these transient transfection
experiments, the tripeptides CKL and SRL proved to be the most efficient
targeting signals.
Transfection experiments have also been used to define the glycosome
targeting signal of the T. brucei enzyme phosphoglycerate kinase (PGK)
(Fung and Clayton, 1991; Blattner et al., 1992; Sommer et al., 1993). In
this enzyme the carboxyl-terminal tripeptide sequence is SSL. The experi-
ments demonstrated that this tripeptide will function as a minimal glyco-
some targeting signal, but will do so more efficiently in the context of the
PGK carboxyl-terminal hexapeptide NRWSSL (Blattner et al., 1992). This
implies that sequences in addition to the tripeptide could also have a role in
the sequestration of glycosomal enzymes. Indeed, some glycosomal
enzymes lack the ‘SKL-like’ tripeptide and are thought to have amino-
terminal (e.g. aldolase) or internal (e.g. the PGK isoenzyme, 56PGK)
targeting signals. Attempts to define the precise nature of these signals
are underway.
Other T. brucei genes subjected to functional analysis using transforma-
tion techniques include those that encode components of the transferrin-
binding complex. Bloodstream T. brucei obtains iron by a receptor-
mediated process that involves uptake of host transferrin. Subunits of the
T. brucei transferrin-binding complex are encoded by ESAG 6 (Schell et
al., 1991) and ESAG 7. The ESAG 7 encodes a product that resembles a
truncated version of the ESAG 6 protein, but lacks the glycosylphosphatidyl-
inositol-(GP1)-anchor addition site. To study the function of both these
proteins, the ESAG 6 and 7 genes were co-expressed in procyclics, a form
of the parasite that does not bind transferrin and normally does not express
products of the ESAGs. Both genes were cloned into a polycistronic
transcription cassette under the control of the rRNA promoter, with the
neo or hyg gene as a selectable marker (Ligtenberg et al., 1994). The
constructs were then consecutively targeted to the rDNA locus after trans-
fection of procyclics. Transformed parasites were found to express protein
products similar to the expected molecular weights. Parasites transfected
with constructs that expressed ESAG 6 or ESAG 7 alone did not bind
transferrin. Reconstitution of the surface transferrin-binding complex was
only achieved in double transfectants; thus the products of both genes are
required for transferrin binding. It was noticed, however, that in these
transformants the transferrin-binding complex was not confined to the
2 48 J.M. KELLY

flagellar pocket as in bloodstream parasites, but was spread over the entire
surface. In addition, the reconstituted complex did not mediate transferrin
uptake. These results suggest that other bloodstream form-specific proteins
could be required for correct localization of the complex and to facilitate
transferrin uptake.
The ability to confer transfection-mediated drug resistance on T. brucei
has been exploited to investigate the mechanisms of genetic exchange. T.
brucei genetic hybrids can be isolated after co-transmission of mixed
parasite populations through tsetse flies (Jenni et al., 1986). However,
genetic exchange is a rare event and is a non-obligatory part of the life
cycle. To improve the efficiency of hybrid selection, transformation has
been used to confer either neo or hyg resistance on parasites prior to
passage through the fly. Hybrids were then successfully selected on the
basis of double drug resistance (Gibson and Whittington, 1993; Gibson and
Bailey, 1994). This technique has great potential and should be a useful
additional approach for those seeking to understand the mechanisms of
genetic exchange in T. brucei.

4. TRANSFECTION OF TRYPANOSOMA CRUZl

4.1. Development of Vectors

In the first T. cruzi transfection experiments, electroporation was used in


combination with a transient expression system (Lu and Buck, 1991). The
plasmid vector developed for these experiments was constructed by insert-
ing a fragment of DNA containing an intact mini-exon gene repeat in front
of cat. Transient expression of CAT was detected after transfection of T.
cruzi epimastigotes with this construct. This demonstrated that electropora-
tion procedures were applicable to T. cruzi and defined the minimal
requirements for expression.
Stable transformation of T. cruzi was first achieved using the pTEX
shuttle vector (Kelly et al., 1992) (see Figure 1). This vector was con-
structed using flanking sequences derived from the two tandemly repeated
glycosomal glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes
(Kendall et al., 1990). pTEX contains a multiple cloning site (MCS) and
has the neo gene as a selectable marker. Genes inserted into the MCS can
be expressed at high levels in transfected cells. The vector replicates
episomally, predominantly in the form of large circular elements composed
of multiple head-to-tail repeats. Similar extrachromosomal elements have
been detected after transfection of Leishmania (Kapler et al., 1990), T.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 249

brucei (Metzenberg and Agabian, 1994) and L. seymouri (Bellofatto et al.,


1991). The vector copy number and the level of expression of transfected
genes can both be enhanced by growing the transformed cells at progres-
sively higher G418 concentrations. This phenomenon, which has also been
observed in Leishmania, probably results from random segregation of
vector DNA conferring a selective advantage on daughter cells that contain
a higher vector copy number.
pTEX has also been used to confer stable transformation on Leishmania,
but attempts to transform C. fasiculata and T. brucei have not been suc-
cessful. In both T.cruzi and Leishmania, RNAs derived from the transfected
genes are trans-spliced at the site used during processing of the gapdh
transcript. Other developments in T.cruzi shuttle vectors include the con-
struction of plasmids based on the flanking sequences of the gene encoding
the surface antigen Gp72, and vectors that are capable of conferring
phleomycin and tunicamycin resistance (Nozaki and Cross, 1994). In
addition, the construction of a cosmid shuttle vector has also been reported
(Kelly et al., 1994) (see Figure 2). This vector is maintained at high copy
number within T. cruzi as circular extrachromosomal elements. Genes
contained within the cosmid insert can be expressed at high levels in
transfected cells.
It was found in T. cruzi that transformation could be mediated by
integration, which occurred exclusively by homologous recombination
(e.g. see Figure 3). Hariharan et al. (1993) described the inactivation of
the PUB1 2.5 polyubiquitin locus by targeted gene disruption. The trans-
fections were carried out with circular DNA, integration occurred with as
little as 228 bp of sequence identity between vector and target, and
tranformants were selected on the basis of G418 resistance. In these
experiments episomes were not detected. Disruption of the TCR2 7
gene, which encodes an antigen containing 14 amino-acid repeats, has
also been achieved by targeted integration (Otsu et al., 1993). In this
case, however, transformants were only detected when linearized vector
was used. It can be inferred from transfection experiments in which both
of the TCR27 alleles were disrupted that the product of this gene is not
essential for cell viability. However, it was observed that the null mutants
grow more slowly than wild-type parasites in cultured mammalian cells
(Otsu et al., 1995).
The factors that determine the route by which transformation is mediated
in T. cruzi remain to be precisely determined. However, it is clear from
these early experiments that T. cruzi has more in common with Leishmania
than with T. brucei.
250 J.M. KELLY

4.2. Transfection as a Tool for Functional Analysis of T. cruzi Genes

Amongst the first T. cruzi genes to be subjected to functional analysis using


transfection procedures were those encoding the surface glycoprotein
Gp72. Null mutants were created when the single pair of gp72 genes
were replaced following targeted insertion of the neo and hyg resistance
genes (Cooper et al., 1993). At the morphological level, the null mutants
were characterized by an abnormal flagellar phenotype in which normal
attachment to the parasite cell membrane was lost. Although they were still
able to infect mammalian cells, the mutants had a greatly impaired ability
to survive within the triatomine insect vector (Ribeiro de Jesus et al.,
1993). To confirm that the observed phenotype was due entirely to disrup-
tion of the gp72 genes, the gene was re-introduced using a plasmid expres-
sion vector. As expected, this resulted in complementation of the mutant
phenotype by restoration of the normal flagellar morphology (Nozaki and
Cross, 1994).
Transfection techniques have also been utilized to investigate the trypa-
nothione system, a potential target for broad-spectrum anti-trypanosomatid
chemotherapy. Trypanosomatids are thought to be devoid of catalase and
glutathione reductase, and it has been proposed that the trypanothione
system is a major component of anti-oxidant defence (Fairlamb and Cer-
ami, 1992). For example, protection against hydrogen peroxide is thought
to involve a trypanothione-dependent peroxidase activity, although this has
not been detected in T. cruzi. To investigate the role of this system, the L.
donovani trypanothione reductase (TR) gene (Taylor et al., 1994) was
introduced into T. cruzi and L. donovani at high copy number using the
pTEX shuttle vector (Kelly et al., 1993). TR is an NADPH-dependent
oxidoreductase which is central to the control of thiol metabolism and
functions by maintaining trypanothione (the low molecular thiol unique
to the trypanosomatids) in its reduced form. Transformed cells were char-
acterized by a greatly increased level of TR (10-14-fold) and by an
enhanced ability to regenerate dihydrotrypanothione, the reduced form of
the thiol. However, recombinant cells did not have increased resistance to
agents such as nifurtimox, which are thought to induce oxidative stress, and
they metabolized hydrogen peroxide at the same rate as non-transformed
cells. These experiments suggest that trypanothione-dependent reactions
are not rate limiting in the utilization of hydrogen peroxide and that
resistance to nifurtimox, the main drug used to treat acute Chagas’ disease,
will not arise due to amplification of the TR gene. The role of TR is
currently being investigated using gene-knockout and other approaches
to downregulate enzyme activity.
Using a plasmid vector based on the calmodulin-ubiquitin 2.65 locus, it
has been possible to express functional mammalian cytokines in T. cruzi.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 251

When La Flamme et al. (1995) introduced the genes encoding murine


interleukin-2 (IL-2)and IFN-y into T. cruzi, IL-2 was found to be secreted
from the cells, whereas IFN-y was not. However, the replacement of the
IFN-y signal sequence with the corresponding region from IL-2 resulted in
efficient secretion of IFN-y. Differences were noticed though, in the site of
cleavage of the signal sequence in the T. cruzi system compared with the
murine cells. This could imply that there are differences in signal sequence
processing between T. cruzi and mammalian cells.
A rapid method for subcellular localization of proteins in T. cruzi, which
involves a combination of epitope tagging and transfection, has recently
been reported (Tibbetts et al., 1995). This approach involved modification
of the MCS of the pTEX vector (see Figure 1) so that in transfected T.
cruzi, any protein expressed from the construct has an additional 10 amino
acid carboxyl-terminal tag derived from an epitope of human c-myc. To
test the system, the T. cruzi mitochondria1 70-kDa heat shock protein gene
(mt-hsp70) was ligated into the MCS and the resulting construct then
transfected into T. cruzi epimastigotes. Using a monoclonal antibody spe-
cific to the c-myc sequence, the tagged mt-hsp7O was localized to the
kinetoplast as previously reported for the wild-type protein. This method
should be a simple and rapid approach for determining the location of
parasite proteins, provided that the tag does not interfere with the localiza-
tion process.

4.3. Analysis of Gene Expression in T. cruzi

The mechanisms of gene expression in T. cruzi have been less well studied
than those in Leishmania or T. brucei. However, transfection techniques
are now being applied to this area and steady progress can be expected. As
yet, no RNA polymerase 11-dependent promoters have been characterized
and, similar to the situation in Leishmania, it is thought that they are not
required for episomal expression. Transient transfection assays have, how-
ever, been used to identify and characterize the rRNA promoter in T. cruzi
(Tyler-Cross et al., 1995). In the presence of a suitable 3’-splice site, the
rRNA promoter was shown to mediate expression of the cat gene, at a level
100-fold greater than previously obtained with the mini-exon gene promo-
ter (Lu and Buck, 1991). Unexpectedly, the promoter did not function in all
T. cruzi isolates, indicating. a restricted host range. This may reflect the
extensive diversity of the T. cruzi species and can be correlated with the
observation that pol I promoters have been less well conserved during
evolution than pol I1 or pol I11 promoters. It will be interesting to determine
whether there is a relationship between promoter activity in various strains
and other taxonomic parameters such as isoenzyme profiles.
252 J.M. KELLY

As has been reported for Leishmania and T. brucei (see above), recent
studies with T. cruzi suggest that the 3’-UTRs of genes play an important
role in the control of gene expression (Nozaki and Cross, 1995). In a series
of stable transformation experiments using episomal vectors containing the
luc gene as a reporter, it was shown that the 3’-UTRs of the stage-specific
genes gp72, gp82 and amastin (ama) could mediate a considerable down-
regulation in the level of luc mRNA in transformed epimastigotes, com-
pared with the control situation in which the 3’-UTR was derived from the
constitutively expressed gapdh gene. In amastigotes, the ama 3 ’-UTR was
shown to confer upregulation, such that the overall stage-specific effect
compared with epimastigotes was greater than 100-fold. Since previous
studies have shown that the ama gene is transcribed at the same rate in
epimastigotes and amastigotes (Teixeira et al., 1994), these findings imply
that regulation at the level of mRNA stability is a major point of control for
stage-specific expression in T. cruzi.

5. TRANSFECTION OF THE APICOMPLEXA

5.1. Development of Transfection Techniques for Toxoplasma


gondii

The obligate intracellular parasite Toxoplasma gondii is a widespread


human pathogen. Although often asymptomatic, infections can result in
congenital disease and can be life threatening in immunocompromised
patients. Attempts to develop stable transformation techniques for T. gondii
were initially hampered by the lack of an in vitro culturing system and by
problems thought to be associated with the transfection of intracellular
parasites. Another complication was that most drugs that have been used to
select transformants in other systems (e.g. aminoglycosides such as G418)
are also toxic to the mammalian cells in which T. gondii replicate. In
addition, because the parasite is haploid at the tachyzoite stage, it was
judged that in the initial transfection experiments vectors should be
designed to avoid the targeted replacement of important or essential genes.
As with trypanosomatids, the parameters necessary to achieve transfec-
tion were first addressed using transient systems. In the first successful
report (Soldati and Boothroyd, 1993), transfection was carried out with a
plasmid vector which had been constructed by placing the 5’- and 3’-
flanking regions of the T. gondii surface antigen 1 ( s a g l ) gene either
side of cat. Freshly purified T. gondii tachyzoites were electroporated
with this construct in a potassium phosphate buffer (cytomix) similar in
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 253

ionic composition to the cytosol (Van den Hoff et al., 1992) and CAT
activity was detected after 8 h. When the sagl 5’-flanking fragment was
replaced with the corresponding regions from the genes encoding a-tubulin
and rhoptry protein 1 (ropl), the amount of CAT expressed was eight- and
four-fold higher respectively. Remarkably, in these transient experiments
up to 15% of electroporated cells expressed the transfected gene product.
To characterize the upstream sequences that govern expression of sagl,
transient transfection experiments coupled with deletion analysis were
performed (Soldati and Boothroyd, 1995). These experiments identified
six repeated sequence elements situated in the region between 35 and
190 bp upstream of the first of two transcription start sites that were
essential for transcription initiation.
The sagl-based vector used in the transient experiments was modified
prior to being utilized in experiments aimed at conferring stable trans-
formation using the cat gene as a selectable marker (T. gondii are
susceptible to chloramphenicol under conditions in which host cells are
unaffected). The modified vector (SBCATl) contained the 5‘- and 3’-
flanking sequences of sagl in addition to a copy of the B l gene, which is
highly re-iterated in T. gondii (Kim et al., 1993). Both linearized and
circular forms of SBCATl were found to mediate stable transformation
after electroporation, with linear DNA being more efficient. In all cases,
SBCATl became integrated into the B l locus. Attempts were then made
to delete the single copy sagl and ropl genes using vectors designed for
gene replacement. Knockout of the ropl gene was achieved, although in
the majority of transformants the gene was not deleted and the vector
DNA had integrated by non-homologous recombination. Transformants
in which the sagl gene was deleted were not isolated, suggesting that
loss of this gene could confer a selective disadvantage. However, the
precise function of SAGl remains unclear since mutant T. gondii that do
not express the antigen can still invade and proliferate in mammalian
cells.
To study the role of SAGl, Kim and Boothroyd (1995) used transfection
to complement these mutants and a novel approach to isolate the transfor-
mants. The sag1 gene was introduced into the parasite by electroporation
and transformants that expressed SAGl on the cell surface were selected
directly using a fluorescence-activated cell sorter (FACS). The success of
this rapid selection method suggests that it can be readily adapted to allow
direct isolation of recombinant parasites that express other cell surface
proteins encoded by transfected genes. Restriction enzyme-mediated inte-
gration is another technique that has been used to improve transformation
procedures in T. gondii (Black et al., 1995). This technique, which simply
involves performing the electroporation step in the presence of a restriction
enzyme, is thought to enhance random integration by inducing breaks in
254 J.M. KELLY

the genomic DNA. In experiments with T. gondii it was found that Not1
increased the efficiency of stable transformation by almost 50-fold; indeed
the enhancement of integration occurred even if the vector DNA was not
linearized prior to electroporation. This technique has been used to facil-
itate the co-transfection of T. gondii with four different constructs, only one
of which expressed a drug-selectable marker (CAT).
As an alternative approach, stably transformed T. gondii have also been
generated on the basis of pyrimethamine resistance conferred by expression
of a modified dhfr-ts gene (Donald and Roos, 1993). The gene was mod-
ified by introduction of point changes to the sequence by analogy with the
mutant gene associated with pyrimethamine resistance in malaria. This
modified gene was cloned into a plasmid containing the endogenous
dhfr-ts flanking sequences and was found to confer pyrimethamine resis-
tance after transfection. Integration was mediated by non-homologous
recombination in these experiments and gene replacement was not
observed. However, when other constructs containing larger fragments of
contiguous genomic DNA sequence (up to 16 kb) were used, targeted
integration occurred in half the cases (Donald and Roos, 1994). In these
experiments circular plasmids were found to be more efficient than linear
DNA at undergoing integration by homologous recombination. Reciprocal
cross-over resulting in duplication of the dhfr-ts locus appeared to be the
most common mechanism involved, although complete allelic replacement
also occurred at low frequency. One drawback with using pyrimethamine
resistance for selection of recombinant T. gondii is the possibility of
accidentally acquired infection, since the drugs of choice used to treat
toxoplasmosis are normally anti-folates.
Another T. gondii transformation system that has been developed is
based on the naturally occurring tryptophan auxotrophy of the parasite
(Sibley et al., 1994). The transformation vector was constructed such
that the E. coli trpB gene (which encodes the fJ-subunit of tryptophan
synthase, an enzyme that catalyses the synthesis of tryptophan from indole
and serine) was placed between the 5’- and 3’-flanking sequences of the
sag1 gene. After electroporation, transformants were selected by growth in
tryptophan-deficient medium supplemented with indole. In these experi-
ments, transformants were obtained using both circular and linear con-
structs, and integration occurred at distinct non-homologous sites normally
resulting in tandemly repeated copies of the vector.
Two recent reports (Messina et al., 1995; Soldati et al., 1995) have
described the use of phleomycin to select transformed T. gondii. Messina
et al. constructed vectors in which the ble gene was placed between
sequences derived from the flanking regions of the sagl or the gral or
gra2 genes. After transfection, the tachyzoites were allowed to invade host
cells without drug selection. Following lysis of the cell monolayers, the
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 255

parasites were incubated with phleomycin prior to re-invasion of host cells.


When these cells lysed, the process was repeated. Transformation frequen-
cies in the range 10-4-10-5/pg DNA were obtained by this approach. With
a similar strategy, Soldati et al. (1995) were able to complement a T. gondii
ropl knockout using co-transfection and phleomycin selection. Although
deletion of the ropl gene did not effect growth or invasion properties of the
parasite, it led to alterations in the ultrastructure of the secretory organelles
known as rhoptries. These changes could be complemented by a plasmid
containing the ropl gene which integrated at random sites in the T. gondii
genome.
In T. gondii, when transfection is carried out with vectors in which
only short regions of flanking sequence are placed on either side of the
drug-selectable gene, integration occurs predominantly by non-homolo-
gous recombination. By exploiting this, and the fact that T. gondii is
haploid at the tachyzoite stage, it has been possible to use insertional
mutagenesis and marker rescue to clone the uracil phosphoribosyltransfer-
ase (urpt) gene (Donald and Roos, 1995). The strategy involved using
transfection to confer pyrimethamine resistance on a population of para-
sites (mediated by random integration) followed by selection with 5-
fluorodeoxyuridine (FUDR). This compound is metabolized to 5-fluorour-
acil which leads to fatal inhibition of thymidine monophosphate-(-TMP)
synthesis. Since UPRT is a non-essential enzyme in T. gondii, those
parasites which survive FUDR selection should contain a disrupted urpt
gene. Four independent mutants were isolated and gene disruption was
confirmed after plasmid rescue. This experiment demonstrates the feasi-
bility of insertional mutagenesis as an approach to facilitate the cloning of
non-essential T. gondii genes provided that a suitable screening system is
available. Furthermore, it suggests that a single transfection experiment
may result in saturation mutagenesis of the entire genome. It should also be
possible to use urpt as a negative selectable marker so that after gene
knockout has been achieved, parasites which subsequently lose the inte-
grated vector DNA can be selected. This may be of particular use when the
vector confers pyrimethamine resistance through expression of a mutant
dhfr-ts gene.
In summary, there are now several genetic approaches available to
address important questions regarding T. gondii biology. Transformation
occws at high frequency and vector DNA can be integrated by homologous
or non-homologous routes. Thus, gene knock out, complementation and
insertional mutagenesis are all feasible. Although episomal vectors are not
yet available for work on T. gondii, their development remains possible
(Roos er al., 1995).
256 J.M. KELLY

5.2. Development of Transfection Techniques for Plasmodium

For several years the development of a genetic transformation system


applicable to Plasmodium species has been widely perceived as the single
most important technical advance required by laboratory workers involved
in malaria research. The lack of such a system has greatly inhibited the
ability to investigate important aspects of drug resistance, antigenic varia-
tion, cytoadherence and parasite differentiation. The problems encountered
in developing a transfection system for Plasmodium have been similar to
those experienced with T. gondii. There is no readily usable axenic culture
system for Plasmodium, transfected DNA must cross four membranes to
reach the nucleus, and the parasite is haploid for most of its life cycle, a
potential problem if transformation is mediated by homologous recombi-
nation. Other factors that had to be considered in vector design were that
most Plasmodium promoters appear to be stage specific and that the AT-
richness of some Plasmodium genomes could result in inefficient transla-
tion of drug-selectable markers derived from other organisms. Many frus-
trating research years were spent attempting to develop Plasmodium
transfection procedures.
In 1993, the first successful transfection of a malaria parasite was
reported with the transient expression of firefly luciferase in the avian
pathogen Plasmodium gallinaceum (Goonewardene et al., 1993). The plas-
mid vector used in this experiment was constructed by inserting the luc
gene in-frame into the coding region of the P. gallinaceum sexual stage
surface antigen gene pgs28. The 5'- and 3'-flanking regions of pgs28 were
also included in the vector. After electroporation of extracellular female
gametes and fertilized zygotes it was possible to detect luciferase activity
within 24 h. The next advance reported was the transient transfection of the
human malaria parasite Plasmodium falciparum (Wu et al., 1995). The
vectors used in these experiments contained the cat gene as a reporter and
had flanking sequences derived from the P. falciparum genes encoding heat
shock protein 86 (hsp86) and histidine rich proteins 2 and 3 (hrp2 and 3).
Surprisingly, using electroporation it was possible to transfect parasites
within red blood cells; CAT activity could be detected after transfection in
both ring stages and schizonts. Transfections were also performed with a
construct containing a synthetic cat gene which had been modified to
conform to the P. falciparum codon bias. However, the level of expression
in parasites transfected with this construct was not significantly different
from that observed in experiments in which the wild-type cat gene was
used, suggesting that inefficient translation of non-malarial transcripts may
be less of a problem than expected.
Stable transformation was finally achieved using the rodent parasite
Plasmodium berghei (van Dijk et al., 1995). The approach taken involved
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 257

selectable marker. This gene, which contains the Ser"' -


using the dhfr-ts gene from a pyrimethamine-resistant P. berghi strain as a
Asn mutation
associated with drug resistance in P. berghei, was cloned into a plasmid
together with its association flanking regions and used to transfect extra-
cellular merozoites by electroporation. These cells were then inoculated
into rats, and transformants selected on the basis of pyrimethamine resis-
tance. Drug-resistant parasites, which contained several copies of the
vector in the form of episomal monomers, were isolated. Upon removal
of drug pressure the plasmids were gradually lost, suggesting that they
undergo random segregation during cell division. In these experiments
integration into genomic DNA was not observed. However, it has since
been reported that linear constructs can be targeted to the subtelomeric
repeat region of chromosomes where they are stably maintained (van Dijk
et al., 1996). This was achieved after a 2.2 kb fragment of DNA, derived
from a tandemly repeated element located in these subtelomeric regions,
was incorporated into the vector. The integration events were site specific
and appeared to be mediated by reciprocal exchange.
Pyrimethamine resistance has also been used as the basis for a recently
described stable transformation system for P. falciparum. Asexual erythro-
cyte-stage parasites were transfected with plasmids containing mutant dhfr-
ts genes associated with drug resistance (Wu et al., 1996). After selection
over several weeks, transformed P. falciparum were isolated in which the
vector DNA was found to be present as unrearranged episomes. When
these cells were maintained in culture for a further 2-3 months, integration
into chromosomal DNA was detected. This occurred by homologous
recombination, with integration confined to the dhfr-ts locus, or to the
hrp2 or hrp3 loci which provided the 5 ' - and 3'-flanking sequences used
in the constructs.
Although development of Plasmodium transformation systems is still at
an early stage, and the precise mechanisms involved remain to be defined,
it is clear that major technical advances have been made. Over the next few
years these systems will have a significant impact on attempts to unravel
the biology of the malaria parasite.

6. TRANSFECTION OF INTESTINAL PROTOZOAN PARASITES

6.1. Transfection of Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis, a major public


health problem in many countries. One of the main goals of researchers
258 J.M. KELLY

working on this parasite is to identify and characterize the molecules


responsible for the invasive phenotype. The recent development of a stable
transformation system for E. histolytica is a significant step forward in
addressing this question. Initially, three groups described transient trans-
fection of the trophozoite stage of the parasite using the cat (Nickel and
Tannich, 1994) and luc (Purdy et al., 1994; Gilchrist et al., 1995) genes as
reporters. In these experiments the flanking sequences from the actin, lectin
(hgll and hg13) and ferredoxin genes were used in the construction of the
transfection vectors. Both the 5'- and 3'-regions of these genes were found
to be required for efficient expression of the reporter genes, suggesting that
each plays a role in regulation of expression. These transfection experi-
ments allowed the electroporation conditions for E. histolytica to be opti-
mized and reports of stable transformation soon followed (Hamann et al.,
1995; Vines et al., 1995).
One group was successful with a construct in which the actin-flanking
sequences were positioned to facilitate expression of the neo selectable
marker (Hamann et al., 1995). After electroporation of trophozoites, G418-
resistant cells were isolated which were characterized by the presence of
unaltered multiple episomal copies of the vector DNA. These episomes
could be maintained in the continued presence of G418, but were lost soon
after removal of drug pressure. Integration into the E. histolytica genome
was not detected. The construct was then modified by incorporation of cat
together with the 5'-flanking region of the E. histolytica lectin gene and the
3'-flanking region of the actin gene. This second construct, which could
confer G418 resistance and facilitate high level expression of CAT, will
have obvious uses as an E. histolytica expression vector. In a second report
of stable transformation, Vines et al. (1995) used the lectin-flanking
sequences in a vector also designed to confer drug resistance by expression
of neo. Transformation was mediated by episomal DNA and integration
was not detected.

6.2. Transfection of Giardia larnblia

Giardia lamblia is a flagellated intestinal protozoan parasite which has


worldwide distribution. In humans, infections can result in severe diarrhoea
although they can also be asymptomatic. Giardia has attracted general
interest because it represents one of the earliest eukaryotic lineages. Two
different approaches to infection of G. lamblia have now been reported.
Using a conventional transient transfection strategy, Yee and Nash (1995)
detected luciferase activity in trophozoites after electroporation with a
plasmid in which luc had been placed between the flanking regions of
the G. lamblia glutamate dehydrogenase (gdh) gene. In a series of experi-
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 259

ments, the highest level of expression was obtained with a construct con-
taining a gdhnuc fusion gene in which the first 18 codons of gdh preceded
the Zuc sequence. Luciferase activity was considerably reduced in parasites
transfected with constructs in which either the 5’- or 3’-flanking regions of
gdh had been removed.
In a novel approach, Yu ef aZ. (1995) used a viral vector to mediate
transient expression of luciferase in transfected G. Zamblia. This followed
on from an earlier study (Furfine and Wang, 1990) which demonstrated that
the 7 kb double-stranded RNA G. ZambZia virus (GLV) (Wang and Wang,
1986) can be introduced into the parasite by electroporation in its single-
stranded form. A cDNA clone was constructed in which the 5’- and 3’-
UTRs of GLV cDNA were placed either side of the Zuc gene. After
electroporation of G. Zamblia trophozoites with an in vitro transcript
derived from this cDNA, it was shown that the RNA could be replicated
and transcribed to produce anti-sense RNA. In addition, luciferase activity
could be detected up to 120 h after transfection. Luciferase expression was
dependent on the presence of the UTRs and of a putative packaging site.
This system has obvious scope for development, and the authors suggest
that it may be possible to use G. ZambZia infected with recombinant GLV
that express a toxic product as a therapeutic approach to treatment of
giardiasis.

7. CONCLUDING REMARKS

Genetic transformation techniques have now been established for most of


the medically important protozoan parasites. These technical break-
throughs have created much excitement in the molecular parasitology
community and progress continues at a rapid rate. At the time of writing,
major advances are being reported in the application of genetic transforma-
tion procedures to malaria parasites. The future impact of these techniques
on malaria research can be judged by recent progress in the trypanosomatid
field where transfection has become a routine research tool. Significant
progress has been made in our understanding of the mechanisms of gene
expression and drug resistance, new approaches to vaccine development
are .being considered, and functional analysis can now be addressed
directly. The mechanism of chloroquine-resistance in Plasmodium falci-
parum will certainly be one of the first questions tackled when this new
technology is applied to malaria. We are also entering a new era in
molecular parasitology with the advent of the parasite genome projects.
Already there has been a considerable increase in the rate at which
sequence data are becoming available; at the moment this is mainly in
260 J.M. KELLY

the form of expressed sequence tags (ESTs). It is clear that the traditional
approaches to the cloning and sequencing of parasite genes are becoming
redundant and that, in future, the major research effort will be aimed at
functional analysis of protein sequences derived from the genome projects.
Transformation techniques will allow us to investigate these questions in
the context of the parasite itself.

ACKNOWLEDGEMENTS

I thank David Baker, Anita Skinner and Martin Taylor for critical reading
of this manuscript. I also thank Martin Taylor for producing Figure 3. The
financial assistance of the Leverhulme and Wellcome Trusts, the Medical
Research Council and the UNDPNorld B a n W H O Special Programme
for Research and Training in Tropical Diseases (TDR) is gratefully
acknowledged.

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The Radiation-attenuated Vaccine against
Schistosomes in Animal Models:
Paradigm for a Human Vaccine?

Patricia S . Coulson

Department of Biology. The University of York.


PO Box No. 373. York. YO1 SYW. UK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
1.1. Thedisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
1.2. The need for a vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
1.3. Laboratory animals as hosts for schistosomes ..................... 274
2. Parasite Migration and Development in the Naive Animal . . . . . . . . . . . . . . . . 275
2.1. Migration in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2.2. Elimination of parasites during a primary infection . . . . . . . . . . . . . . . . . 278
2.3. Migration and development in other laboratory animals . . . . . . . . . . . . .280
3. The Radiation-attenuated Vaccine in Mice ............................ 282
3.1. Protection involves a specific immune response . . . . . . . . . . . . . . . . . . .282
3.2. Parameters of parasite attenuation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
4. lnductionoflmmunio/ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
4.1. The effect of radiation on the parasite ........................... 284
4.2. Persistence of attenuated schistosomula ......................... 286
4.3. Immunogenic potential of attenuated schistosomula . . . . . . . . . . . . . . . .289
5. Immune Responses During Priming ................................ 290
5.1. Lymph nodes draining sites on the migration route . . . . . . . . . . . . . . . . . 290
5.2. The lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
.
6 The Immune Effector Mechanism .............. .................... 295
6.1. The site of challenge parasite elimination ......................... 295
6.2. The nature of the immune response ............................. 296
6.3. Events in the immune lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
6.4. Potential mechanisms of parasite elimination . . . . . . . . . . . . . . . . . . . . . . 303
6.5. The central role of IFNy in the effector mechanism . . . . . . . . . . . . . . . . . .305
7 . The Irradiated Vaccine in Other Laboratory Hosts ...................... 307
7.1. The rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

ADVANCES IN PARASITOLOGY VOL 39 Copyright 0 1997 Academic Press Limited


ISBN 0-12431739-7 All rights of reproduction in any form reserved
272 P.S.COULSON

7.2. Primates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310


7.3. Domestic livestock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
8. Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

1. INTRODUCTION

1.I. The Disease

Human schistosomiasis is caused by trematodes of the genus Schistosoma


which have a complex life cycle involving an intermediate snail host.
Water provides a suitable environment for the snails to thrive, and the
medium through which the free-living stages of the schistosome life cycle
locate and infect their respective hosts. Thus, the development of irrigation
schemes and the construction of dams to conserve water, together with the
inevitable concentration of human populations in these areas, have
enhanced the transmission of schistosomiasis (Hagan and Gryseels,
1994). The disease is estimated to afflict more than 200 million people,
with a further 600 million at risk of infection, and is now endemic in 74 of
the world’s tropical and subtropical countries (Bergquist, 1995). It is hardly
surprising that it is recognized as one of the most widespread parasitic
diseases of man, and an important public health problem.
In the principal species infecting humans, parasites take up residence in
the blood vessels of the hepatic portal system (S. mansoni, S. japonicum),
or the vesical plexus (S. haematobium), and can persist within their host for
many years. Schistosome eggs are produced continually by the adult
female and are the principal stimuli for immunopathology and disease.
The developed egg contains a live embryo, the miracidium, which has to
hatch in fresh water and locate its intermediate host in order for the life
cycle to be perpetuated. To this effect, approximately half of the eggs
deposited pass into the lumen of the gastrointestinal or urinary tract and
are liberated from the host. The remainder are either trapped in local tissue
sites or break free within blood vessels (von Lichtenberg, 1987) and, in the
case of S. mansoni, become lodged in the presinusoidal portal venules of
the liver (Cheever, 1969).
The host’s response to soluble secretions from the mature egg is an
intense delayed-type hypersensitivity reaction (DTH), culminating in the
formation of a granuloma (Boros and Warren, 1970). Relentless emboliza-
tion of S. mansoni or S. japonicum eggs eventually causes diffuse portal
inflammation and fibrosis, with permanent venous obstruction (von Lich-
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 273

tenberg, 1987). This, in turn, can lead to portal hypertension, hepatome-


galy, splenomegaly and the formation of oesophageal varices. Similarly,
sustained deposition of S. haemafobium egg clusters into a urothelial patch
results in fibro-inflammatory swellings capable of obstructing the urinary
tract. Generally, the disease manifestations are chronic and can lead to a
debilitating condition. In some individuals there is a slow insidious transi-
tion from reversible to severe pathology, and problems arise in distinguish-
ing potentially severe cases from mild ones during subclinical stages of
infection (von Lichtenberg, 1987).
Control of the disease is based upon many factors, such as health
education, diagnostic techniques, snail control and, in particular, chemo-
therapy (Mott, 1987). All these strategies have limitations, the foremost
being the expense involved. However, the mass treatment of infected
human populations with chemotherapeutic drugs has been effective in
reducing the prevalence of disease (Mahmoud ef al., 1983). Unfortunately,
such treatment cannot completely reverse the pathological damage caused
by the long-term deposition of eggs in host tissues, nor does it influence re-
infection. Moreover, the potential for the eventual emergence of drug-
resistant strains of parasite remains a persistent threat.

1.2. The Need for a Vaccine

Whilst chemotherapy is a valid tool as a short-term remedy for morbidity,


there is clearly a need for a vaccine to help accomplish control more
definitively. Ideally, the putative vaccine would be administered as a single
dose before natural exposure to the parasite. It is worth noting that schisto-
somes do not replicate in the mammalian host so that although sterile
immunity would normally be the ultimate goal of a vaccine, in this case
it is not an absolute requirement. Partial protection, resulting in a reduction
in an individual’s worm burden or in worm fecundity, would prevent
infection intensity rising above the threshold at which severe clinical
disease occurs, and also influence the level of transmission (Chen and
Mott, 1988). Sher and colleagues (1989) have contended that ‘the argu-
ments in favour of the development and deployment of antischistosomal
vaccines seem indisputable, despite complacent comments from current
advocates of chemotherapy’.
One of the questions posed when contemplating the feasibility of
achieving a vaccine against schistosomes is whether acquired immunity
occurs in humans. It has frequently been reported that, in areas that are
endemic for human schistosomiasis, children have higher intensities and
prevalences of infection than adults (e.g. Kloetzel and Da Silva, 1967).
Likewise, after chemotherapeutic cure of existing schistosome infections,
274 P.S. COULSON

younger individuals in an endemic population become re-infected at much


higher intensities than adults. For many years it was not clear whether these
differences in re-infection rates were the result of age-related changes in
the amount of exposure to parasites (Warren, 1973) or the development of a
protective immunity. However, extensive field studies have measured indi-
vidual intensities of re-infection after treatment of S. mansoni (Butterworth
et al., 1985, 1988; Dessein et al., 1988) and S. haematobium (Wilkins et
al., 1987), whilst also monitoring the amount of contact the same indivi-
duals had with infective water. These studies showed that the disparities in
intensity of infection between children and older age-groups are too great
to be accounted for simply by differences in exposure. It would therefore
seem likely that a decrease in susceptibility to re-infection is due, at least in
part, to the slow development of a protective immunity rather than to non-
specific, age-related changes in human behaviour or physiology (Butter-
worth, 1994). Nevertheless, the subject remains open to speculation and the
responsible mechanisms may turn out to be multi-factorial (Gryseels,
1994). Research on the cellular and humoral components of responses in
infected humans has highlighted a number of potential effector responses
(Butterworth, 1994); whether any of them could provide the basis for a
vaccine remains to be seen.

1.3. Laboratory Animals as Hosts for Schistosomes

Owing to the difficulties inherent in identifying and defining immune


mechanisms in the human host it has proved necessary to use animal
models of schistosomiasis. Progress in this area has been greatly
enhanced by the fact that several laboratory animals are susceptible to
the parasite, although to varying degrees depending on the host and
schistosome species. For instance, in mice and primates the three major
schistosome species infecting man develop to sexually mature adults
capable of producing viable eggs. At the other end of the spectrum is
the rat in which S. mansoni survives for only 4-6 weeks before the
majority of worms are eliminated. Hamsters, guinea pigs, gerbils, rabbits
and a variety of ungulates are also susceptible to one or more schisto-
some species. Much of the research has focused on S. mansoni, simply
because it is the easiest of the three species to maintain in the laboratory.
Likewise, the ability of the mouse to manifest many pathological features
of human schistosomiasis, and to develop immunity, coupled with its
relatively low cost and ease of production, makes it a suitable model
for study. Thus, the bulk of information outlined below deals with this
host-parasite combination; reference made to other schistosome species
will be clearly indicated.
RADIATION-ATTENUATEDVACCINE AGAINST SCHISTOSOMES 275

The resistance to schistosomes that develops in chronically infected


animals appears to be directed against migrating challenge larvae, leaving
the adult worms of the initial exposure unharmed. Smithers and Terry
(1969) adopted the term ‘concomitant immunity’ to describe this phenom-
enon, and it was taken as evidence for the development of protective
immunity against schistosomiasis. However, it has been demonstrated
that a major component of concomitant immunity in mice is artefactual
(Wilson et al., 1983). Thus, pathological changes occur in the hepatic
portal vasculature, due to egg deposition in the liver by worms of the
primary infection, that influence the development of larvae from a subse-
quent exposure. As a consequence, it is impossible to evaluate the con-
tribution of any immunologically specific mechanism of parasite
elimination in this model.
Fortunately, a considerable level of immunity is induced in a variety of
experimental animals following their exposure to radiation-attenuated
schistosomes. Since the attenuated parasites do not develop to maturity
there is no egg deposition and hence no associated pathology. Given the
proviso about the influence of pathology on resistance (Wilson, 1990), it is
not surprising that studies on immunity to schistosomes have increasingly
concentrated on this seemingly more straightforward models. The goal of
the research is to recreate similar conditions using a vaccine composed of
purified or recombinant antigens ultimately for use in humans.

2. PARASITE MIGRATION AND DEVELOPMENT IN THE NAIVE


ANIMAL

In order for schistosomes to establish in the mammalian host, cercariae


must penetrate the skin, transform into schistosomula, locate their favoured
site in the vasculature and mature to adulthood. This complex part of their
life cycle provides several opportunities for interactions between the host’s
immune system and parasites at different stages of development, at various
sites in the body. Therefore, before addressing the question about how
immunity is generated in laboratory animals vaccinated with irradiated
cercariae, it is essential to appreciate the way in which schistosomes reach
their site of parasitization, and what happens to those that fail to do so.

2.1. Migration in Mice

The subject of schistosome migration and development in the murine host


has been studied intensively over the past 15 years (reviewed by Wilson,
276 P.S. COULSON

1987). Following primary exposure of the mouse to S. mansoni, between 30


and 50% of the cercariae applied will eventually mature. The route of
migration taken by larval schistosomes from the skin infection site to the
hepatic portal system has been the focus of much debate, although evidence
has accumulated in favour of an intravascular passage in the direction of
blood flow. Studies have benefited from the advent of compressed organ
autoradiography which has enabled isotopically labelled parasites to be
detected in mouse tissues (Georgi, 1982). In terms of estimating accurately
the number of schistosomula in an organ, this method surpasses both
histological techniques and the recovery of parasites from tissues that
have been minced finely and incubated in culture medium.
The route of migration of S. mansoni in the mouse is summarized in
Figure 1. Parasites successfully penetrating the skin spend, on average,

Infection
++I

rLungs

Figure 1 Migration of S. mansoni in the mouse. Schistosomula undertake an


intravascular route of migration from the skin to the hepatic portal system. The
majority of penetrants leave the skin via the bloodstream with a small fraction
exiting via the lymphatics and successfully negotiating the lymph nodes (LN). The
lungs represent the first capillary bed encountered by the larvae, and they are
obliged to remain in this location undergoing morphological adaptations before
continuing their migration. Schistosomula reaching the venous compartment of the
lungs pass to the left side of the heart and are dispersed to systemic organs in
proportion to the fractional distribution of cardiac output. Those delivered to
splanchnic organs gain access to the hepatic portal system (see shaded area) via
the mesenteric capillaries, and most are retained in the liver; any which negotiate
the hepatic sinusoids are returned to the lungs via the venous circulation. Parasites
distributed to other systemic organs traverse the capillaries and also return to the
lungs. Two to three circuits of schistosomula around the pulmonary-systemic
vasculature are adequate to produce the adult worm population.
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 277

between 2 and 5 days negotiating the epidermis, basement membrane and


dermis before effecting entry into blood vessels (Miller and Wilson, 1978);
once within the lumen they are rapidly transported by blood flow to the
lungs. Whilst the majority of schistosomula leave by this route, a fraction
of penetrants (10-20%) exit via the lymphatics (Wheater and Wilson,
1979; Mountford et al., 1988) and enter the circulation from the thoracic
duct. Arrival in the pulmonary vasculature marks the beginning of a 3-4-
day period of development for the schistosomulum, including elongation
(Wilson et al., 1978) and loss of mid-body spines (Crabtree and Wilson,
1980), which is thought to adapt it for onward migration.
Schistosomula passing to the venous compartment of the lungs are
carried to the left side of the heart and dispersed to systemic organs in
proportion to the fractional distribution of cardiac output. Parasites gaining
access to splanchnic organs negotiate the capillary beds to enter the hepatic
portal system, and the majority are retained in the liver. Those distributed
to other systemic organs traverse the capillaries and reach the venous
compartment to be carried back to the lungs (Miller and Wilson, 1980;
Wilson and Coulson, 1986). The minor proportion of parasites that suc-
cessfully negotiates the liver sinusoids also returns to the lungs. Injection
of radiolabelled schistosomula into the hepatic portal system and their
subsequent detection in the lungs have provided estimates of the trapping
efficiency of the liver (72-86%, Wilson and Coulson, 1986; 70-75%,
Mangold et al., 1986); the latter study also showed that schistosomula
which exit from the liver have a reasonable chance of returning there.
The high rate of trapping, together with the probability of being distributed
to the hepatic portal system, means that only two to three circuits of
parasites around the pulmonary-systemic vasculature are required to gen-
erate the observed population of adult worms (Wilson, 1987).
Accumulation of schistosomula in the liver commences around day 8
after infection and is virtually complete by day 21 (Miller and Wilson,
1980; Wilson et al., 1986). Within days of arriving in the liver, the
elongate, migrating parasites shorten to the dimensions of skin worms;
motility diminishes and they begin to feed on blood and increase in mass
(Wilson et al., 1978). It is not known why these morphological changes
occur only when a schistosomulum reaches the liver, rather than in other
organs. It may be in response to raised nutrient levels or chemical compo-
nents unique to the hepatic portal blood (Wilson et al., 1978). Alterna-
tively, schistosomula may experience difficulty in negotiating the narrow
hepatic sinusoids (see Miller and Wilson, 1980). When they arrive in the
liver, parasites lodge in the smallest hepatic portal distributaries (Wheater
and Wilson, 1979); as they grow they move upstream into progressively
larger vessels. By 6 weeks after infection virtually all worms have paired in
278 P.S. COULSON

the liver and migrated, via the hepatic portal vessels, to the mesenteric
veins where egg laying commences.

2.2. Elimination of Parasites during a Primary Infection

The fate of the 50-70% of penetrants that fail to mature in the naive mouse
has received considerable attention. It was originally thought that most
schistosomula died in the skin shortly after penetration, as only a small
number could be recovered when this tissue was minced and subsequently
incubated in culture medium for several hours (Clegg and Smithers, 1968).
However, fundamental to interpretation of these data is the assumption,
now shown to be erroneous, that all viable parasites are recovered by the
assay (Miller and Wilson, 1980; Coulson and Wilson, 1988). Whilst noting
an inflammatory response in the skin, numerous investigators carrying out
histological studies failed to find dead or dying parasites in this tissue (e.g.
Stirewalt, 1959; von Lichtenberg et al., 1976; Mastin et al., 1983).
Autoradiographic tracking showed that the vast majority of labelled para-
sites penetrating the skin of naive mice eventually left this tissue and
arrived in the lungs (Mangold and Dean, 1983; Dean et al., 1984; Wilson
et al., 1986). In addition, if the skin was bypassed by intravenous injection
of lung schistosomula directly to the lungs, the level of maturation did not
differ significantly from that which followed percutaneous infection
(McLaren et al., 1985; Mangold et al., 1986). There would thus appear
to be no major elimination of schistosomula in the skin of naive mice.
Since the eventual number of parasites maturing in the hepatic portal
system was much lower than the peak number detected in the lungs by
autoradiography, Georgi et al. (1983) concluded that elimination occurred
either in the latter organ, en route to the liver, or in the liver itself. The last
possibility was shown to be unlikely by the injection of lung schistosomula
directly to the hepatic portal system (Mangold et al., 1986; Coulson and
Wilson, 1988). The outcome was a high level of maturation (70-85%)
which, given the efficiency of trapping in the liver, would suggest that this
organ provides a safe haven for migrating parasites.
The exact site and timing of elimination can only be defined by establish-
ing whether a decline in parasite numbers in a tissue represents death and
clearance or exit to another organ on the migration route. In order to
achieve this all mouse tissues must be sampled to permit the construction
of an accurate balance sheet of parasite numbers. In the study where this
was carried out (Wilson et al., 1986), it transpired that no decline in the
total population occurred until later than 14 days after infection. Recruit-
ment of parasites to the hepatic portal system was complete by day 21, at
which time schistosomula in other locations, and hence destined to die,
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 279

were distributed in the ratio of 3.9: 1.9: 1 in the lungs, systemic organs and
skin infection area. The lungs were therefore considered to be the prevalent
site of parasite elimination in the naive mouse.
Autoradiographic tracking of isotopically labelled schistosomula,
extracted from the lungs of donor animals and injected as a pulse at various
points on the migration route, has provided estimates of organ transit times
(Wilson and Coulson, 1986). A mean transit time of 30-35 h was required
for a schistosomulum to traverse the pulmonary capillary bed, whilst
passage through non-splanchnic systemic organs was more rapid taking
only 16 h. Therefore, despite the morphological changes which are thought
to facilitate their migration through capillaries, schistosome larvae appear
to find the lungs a difficult organ to traverse.
Histological and ultrastructural examination of schistosomula in the
lungs of naive mice revealed that newly arrived parasites were intravas-
cular and did not attract an inflammatory response (Mastin et al., 1983; von
Lichtenberg et al., 1985; Crabtree and Wilson, 1986). However, inflam-
matory foci developed in the vicinity of schistosomula with time postinfec-
tion (von Lichtenberg et al., 1985), particularly at later sampling points
when a substantial proportion of the parasites was located partly or com-
pletely in alveoli (Crabtree and Wilson, 1986). Passage through the lungs is
thought to be an active process involving muscular exertion by the schis-
tosomulum; this could lead to rupture of the thin blood-air barrier causing
the larva to enter an alveolus. It was concluded that the intra-alveolar
parasites observed in the above study accounted for a large proportion of
the non-maturing population of a primary infection. Corroborating evi-
dence for this hypothesis was provided by transfer experiments in which
schistosomula were extracted from the lungs of donor mice and delivered,
via the trachea, to the alveoli of naive recipients (Coulson and Wilson,
1988). Such parasites had a limited capacity to recover from alveolar sites
and complete their migration, unlike their cohorts introduced directly into
the vasculature. The alveolus would thus appear to represent a cul-de-sac
on the migration route; the fate of a parasite in this inhospitable location is
most likely death due to starvation. It has, however, been suggested that
schistosomula in alveoli could be expelled after passing via the trachea into
the gastrointestinal tract (Crabtree and Wilson, 1986; Dean and Mangold,
1992). In this context, radiolabelled larvae have been detected by auto-
radiography in the trachea, and lumina of the eosophagus, stomach and
intestines (Georgi et al., 1986).
Less systematic application has been made of parasite recovery and
autoradiographic tracking techniques to investigate the migration and site
of elimination of S. haematobium and S. juponicum. Whereas the limited
data point to similarities in the pattern of migration for the three schisto-
some species, differences are highlighted in the kinetics of the process.
280 P.S. COULSON

Thus, S. haematobium larvae are slower than S. mansoni in their exit from
the skin, as judged by autoradiography (Georgi et al., 1986); their peak
accumulation in the lungs is also later, with an apparent retention of
parasites in this organ. Conversely, S. japonicum undergo a rapid migra-
tion, with maximal yields of parasites being obtained from the lungs on
days 3 4 , in comparison to day 6 for S. mansoni larvae (Gui et al., 1995).
Petechial haemorrhages, visible to the naked eye, also appear on the lungs
during the first week of infection (Mitchell et al., 1991; Gui et al., 1995),
but are seldom seen in S. mansoni-infected mice, suggesting a more
aggressive migration on the part of the former parasite. The rapidity of
S. japonicum migration is also borne out by the recovery of a small number
of worms from the liver as early as day 3, with the peak occurring around
days 7-10, approximately 2 weeks sooner than that for S. mansoni (Gui et
al., 1995). Autoradiographic tracking has confirmed that virtually all S.
japonicum larvae leave the skin infection site by day 4, the time when peak
numbers are detected in the lungs (Laxer and Tuazon, 1992; P.S. Coulson,
N.A. Moloney and R.A. Wilson, unpublished data). Both studies confirmed
that parasite numbers reach a maximum in the liver between days 6 and 10,
but Laxer and Tuazon (1992) noted a reduction thereafter, leading them to
suggest that this organ represents a major site of attrition. However, no
evidence for this was found by Coulson et al. (unpublished data) nor by
Mitchell et al. (1991) who recovered parasites from the hepatic portal
system at frequent intervals after infection.
Although the final level of maturation in the mouse depends to some
extent on the geographical strain of S. japonicum, it is usually in the region
of 45-75% (Mitchell et al., 1991), and hence considerably higher than for
S. mansoni. On the other hand, maturation of S. haematobium ranges from
only 7% (Dean et al., 1996) to 15% (Agnew et al., 1989). There would thus
appear to be a relationship between the number of worms establishing in
the mouse and the speed of migration through the lungs. Whether this is
due to differences in the migratory capacity of the schistosome species or
to the host’s response to their presence in the lungs remains to be dis-
covered.

2.3 Migration and Development in Other Laboratory Animals

2.3.1. The Rat


Although most schistosomes do not survive beyond 6 weeks after infection
in the rat, and any that do are stunted, the course of migration and devel-
opment before this time is very similar to that observed in more permissive
hosts. The process was originally investigated by recovery of S. mansoni
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 28 1

from the lungs and portal tract of rats at various times after a primary
exposure (Perez et al., 1974; Miller and Wilson, 1978, 1980; Knopf et al.,
1983; Ford et al., 1984a). These studies established that schistosomula
spent 2-3 days in the skin before migrating to the lungs. Parasites were
detected in the hepatic portal system from day 9, with numbers rising to a
maximum between 3 and 4 weeks before declining as they were eliminated
from the liver.
Autoradiographic tracking (Knopf et al., 1986; Ward and McLaren,
1989; P.S. Coulson and R.A. Wilson, unpublished data) revealed that there
was a rapid and highly efficient migration out of the skin, so that less than
10% of penetrants remained in this location by day 6 whilst > 85% were
detected in the lungs. In the study where all tissues were sampled (P.S.
Coulson and R.A. Wilson, unpublished data), significant numbers of schis-
tosomula were located in systemic organs on days 9 and 12 postinfection.
All three studies confirmed that the first parasites reached the liver between
days 6 and 9, with numbers remaining virtually steady from days 10/12 to
perfusion at day 21. These data indicate that migration of S. mansoni in the
rat proceeds at a faster rate than in the mouse, with arrival in the lungs and
liver occurring approximately 2 days earlier. One other notable discrepancy
was the lack of a substantial number of larvae in the lymph nodes draining
the skin exposure site (P.S. Coulson and R.A. Wilson, unpublished data). A
maximum of only 2.5% of penetrants was detected on day 6, whereas a
comparable study in mice revealed a peak of 16% on day 5 (Mountford et
al., 1988).
Despite the greater speed of migration in the rat compared to the mouse,
maturation is much lower, reaching at best 25-30%. The situation is
complicated in the rat because it is difficult to ascertain whether the plateau
in parasite numbers in the liver is the result of a cessation in migration to
this organ. Although this is the most likely explanation, a turnover in the
worm population, in which gain is matched by loss as the parasites suc-
cumb to a destructive mechanism in the liver, cannot be ruled out. How-
ever, there is reason to believe that, as in the mouse, the lungs present a
significant obstacle to the migrating larvae. A histological investigation
revealed that the rat develops significant cellular reactions around larvae in
the lungs 5-7 days after exposure to a primary infection (von Lichtenberg
and Byram, 1980). Furthermore, when a balance sheet of parasite numbers
in all rat tissues was constructed (Coulson and Wilson, unpublished data),
it was found that four times as many larvae were detected in the lungs
compared to other systemic organs at a time when migration to the liver
appeared to be complete. It would therefore be reasonable to suggest that
the majority of non-maturing parasites are eliminated in the naive rat lung.
282 P.S. COULSON

2.3.2. Primates
Compared to the wealth of information on schistosome migration in the
mouse, little is known about the subject in man or in related primates. The
limited data available were obtained from an autoradiographic tracking
study in the olive baboon Papio anubis (Wilson et al., 1990). Exit of
schistosomula from the skin exposure site was rapid, with 72% of applied
S. mansoni parasites being detected in the lungs on day 5 . Schistosomula
made an equally rapid transit to the liver, since 60% of the adult worm
burden had arrived by day 9. No parasites could be detected in the brain or
kidneys at any sampling time, making it impossible to comment on the
systemic phase of migration; nor were they detected in lymph nodes
draining the exposure site. The final adult population, measured by portal
perfusion, indicated a maturation of 78%, a level similar to that recorded
by Sturrock et al. (1984a). The data suggest that, in comparison to rodents,
schistosome migration in baboons is faster and more successful, possibly
because the lungs do not represent a significant obstacle to migrating
larvae. One might postulate that a similar situation pertains in humans
but it remains an open question that is impossible to resolve.

3. THE RADIATION-AlTENUATED VACCINE IN MICE

Whilst irradiated cercariae were first considered as a potential vaccine


against schistosomiasis more than 30 years ago (e.g., Villella et al.,
1961; Perlowagora-Szumlewicz and Olivier, 1963), detailed analyses of
their effects in mice did not emerge until the late 1970s.

3.1. Protection Involves a Specific Immune Response

There is abundant evidence to indicate that the resistance displayed by


mice vaccinated with irradiated cercariae is mediated by specific immune
mechanisms, unlike that observed in chronically infected animals. For
example, it is schistosome species-specific (Cheever et al., 1983; Bickle
et al., 1985; Aitken et al., 1988), and can be transferred across a parabiotic
union between vaccinated and naive animals (Dean et al., 1981a; Coulson
and Wilson, 1997). It is dependent on functional populations of T and B
lymphocytes, although it has no requirement for complement (Sher et al.,
1982a), IgM antibodies (Correa-Oliveira and Sher, 1985), or components
of an immediate hypersensitivity response (IgE and mast cells; Sher et al.,
1983). Extensive research over the last decade has established the promi-
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 283

nence of T-cell responses in the acquisition of resistance (see Sections 5


and 6).

3.2. Parameters of Parasite Attenuation

Attenuation can be achieved by exposure of parasites to radiation from an


ultraviolet (UV; Dean et al., 1983), X-ray (Hsu et al., 1981) or gamma
source (Minard et al., 1978a). The last has become the most widely used
and, unless otherwise stated, studies referred to hereafter have used this
method of attenuation. Following a single vaccination of mice with irra-
diated larvae, the resistance develops as early as 1.5 weeks postexposure
(James et al., 1981; Ratcliffe and Wilson, 1992) and persists for at least 15
weeks (Minard et al., 1978a). Maximal protection has been achieved with
as few as 50 to as many as 30 000 irradiated larvae (Dean, 1983). In
general, additional vaccinations do not boost the level of resistance
(Dean, 1983), although Hsu et al. (1981), using X-irradiated larvae,
reported a progressive increase in resistance to levels greater than 90%
when the number of immunizing exposures was raised from one to five.
The dose of radiation applied to parasites is an important factor influen-
cing the eventual level of protection (Minard et al. 1978a; Bickle et al.,
1979b), although opinions have differed regarding the optimum amount.
Studies in the United States initially used 56/50 krad (e.g. Minard et al.,
1978a; Mangold and Dean, 1984) but more recently irradiation doses of
15-20 krad have resulted in higher levels of protection than 50 krad (Rey-
nolds and Ham, 1992; Richter et al., 1995). In the United Kingdom,
20 h a d has consistently proved the most effective (e.g. Bickle et al.,
1979b; P.S. Coulson, unpublished data). The reasons for these discrepan-
cies are unresolved, but may involve differences in the method of calibra-
tion of the radioactive source or conditions of snail maintenance (James
and Dobinson, 1985).
Mouse strains differ widely in the extent of immunity developing after
exposure to attenuated cercariae (Murrell et al., 1979). Those of the
C57BL/6 strain are consistently high responders for S. mansoni (e.g. James
et al., 1981; Richter et al., 1993b) and a single exposure to 500 optimally
irradiated cercariae routinely induces 60-75% protection (assessed by
reduced adult worm burdens) against a challenge with normal cercariae
administered 5 weeks after vaccination. CBA strain mice have been used in
most vaccination studies involving S. japonicum (Moloney et al., 1985)
and S. haematobium (Agnew et al., 1989), although in the case of the latter
parasite, Dean et al. (1996) have reported much higher levels of protection
with C57BL/6 and BALB/c mice.
To date, the level of immunity stimulated by irradiated larvae surpasses
284 P.S. COULSON

any generated in mice by vaccines comprising purified or recombinant


molecules. Regrettably, in recent independent trials of six designated
schistosome vaccine candidates, the stated goal of consistent induction of
40% protection or better was not achieved with any of the antigen for-
mulations (TDR news, June 1996). However, administration of living
(albeit attenuated) parasites to humans would be inappropriate for both
practical and ethical reasons (von Lichtenberg, 1985). The most important
ethical implication concerns the fact that in mice a fraction of larvae
migrate through systemic organs on their journey to the hepatic portal
system; there is no way of predicting the dose of radiation needed to
attenuate parasites so that they would not expire in, for example, the human
brain, with potentially serious pathological complications. Effort must
therefore be sustained to establish defined antigen vaccines for use against
human schistosomiasis. As irradiated parasites presently offer the most
efficient and reliable means of protecting laboratory animals against schis-
tosomes, it is anticipated that a thorough understanding of the mechanisms
of immunity in this model could serve as a paradigm for the development
of a suitable antigen vaccine for use in man.

4. INDUCTION OF IMMUNITY

Why should radiation-attenuated parasites induce a protective response


when an equal number of normal parasites does not?

4.1. The Effect of Radiation on the Parasite

One hypothesis, proposed by Wales and Kusel (1992), is that radiation in


some way alters antigen expression so that vaccinating parasites are more
immunogenic than their normal counterparts. These authors suggest that
disruption of antigen conformation could occur as a direct consequence of
radiation damage, or result from inhibition of protein synthesis. Irradiation-
induced abnormalities in protein structure would then stimulate the
immune system to recognize molecules that are usually of minimal
antigenicity. However, data from other studies do not lend support to
this theory. For example, analyses performed by Simpson et al. (1985)
have demonstrated that irradiation of cercariae did not qualitatively affect
antigen expression, nor they find any evidence for differences in antibody
specificity between vaccinated and normally infected mice. Moreover, both
antibody and T cells from vaccinated mice recognized viable (i.e. unatte-
nuated) schistosomula (Correa-Oliveira et al., 1984), a finding which can
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 285

be taken to imply that antigens are shared between immunizing and normal
larvae. This is hardly surprising when one considers that the host’s immune
system primed by antigens from irradiated larvae has to be capable of
responding to those from normal parasites, in order to initiate the effector
mechanism.
Once it had been established that optimally irradiated parasites did not
reach maturity (e.g. Minard et al., 1978a), their survival pattern was an
obvious target for investigation. In early studies, only a small proportion
(5%) of optimally irradiated larvae could be recovered from the lungs of
mice, yet some appeared to persist in this organ from 6 to 21 days after
infection (Minard et al., 1978b; Bickle et al., 1979a). It was therefore
concluded that irradiated schistosomula died at the site of administration,
en route to, or in the lungs. However, both the above studies used recovery
of parasites from minced tissues as their assay. Apart from known ineffi-
ciency of this technique to retrieve all schistosomula present, it might be
anticipated that radiation-damaged parasites would be even less readily
extractable than normal ones.
Indeed, the extent of migration proved greater than these original studies
indicated. Using a quantitative histological technique (Mastin et al. (1983)
demonstrated that a large proportion of attenuated schistosomula were
located in the lungs (57% on day 7) and persisted there until at least 21
days after infection, although by this time most were dead. There was a
slight retardation of migration out of the skin but death at this site appeared
negligible. Attenuated schistosomula, and the residual inflammatory foci
resulting from their destruction, were also detected in the lungs of vacci-
nated mice in a study by von Lichtenberg et al. (1985); whilst most
parasites had died prior to day 14, resolution of residual foci was pro-
tracted. Using autoradiography tracking, Mangold and Dean (1984) con-
firmed that migration of attenuated larvae between skin and lungs
paralleled that of normal schistosomula except for the slight delay men-
tioned above. These authors also concluded that the majority of irradiated
larvae died in the lungs during the first 3 weeks of infection. An assessment
of the ultrastructure of attenuated schistosomula fixed in situ in the lungs
has revealed that, as far as could be seen, they had undergone the normal
developmental changes associated with this stage of migration (Mastin et
al., 1985). The only apparent difference between normal and irradiated
schistosomula seemed to be one of location since many of the latter were
detected in alveoli, rather than blood vessels, by day 13 postexposure.
However, it was subsequently demonstrated that a large number of normal
parasites also enter alveoli at later times during infection (Crabtree and
Wilson, 1986). It is difficult to quantify the numbers of parasites in alveolar
locations, but one might postulate that a larger proportion of irradiated than
normal schistosomula suffer this fate.
286 P.S. COULSON'

Whilst the above investigations established that migration of attenuated


parasites terminated prematurely, there was little to suggest how this was
brought about by irradiation. However, the findings of a recent comparison
of normal and irradiated schistosomula have provided a potential explana-
tion (Harrop and Wilson, 1993a). The study differed from the one carried
out earlier, where irradiated parasites were examined in situ (Mastin et d.,
1985), in that the larvae were derived by artificially transforming cercariae
and culturing them in vitro for several days. Scanning electron microscopy
revealed marked distinctions in surface morphology between normal and
irradiated larvae at the lung stage of development (Figure 2). The latter
parasites exhibited random constrictions at intervals along the length of the
body, probably resulting from overcontraction of circular muscle fibres;
they were also less motile than normal larvae. The authors postulated that
the disruption in neuromuscular co-ordination accounts for the limited
migratory capacity of attenuated schistosomula in vivo.

4.2. Persistence of Attenuated Schistosomula

An apparent conclusion to be drawn from the above studies is that an


optimum dose of radiation does not cause rapid death of schistosomula,
since many are able to persist in various sites for up to 3 weeks.
Investigations were made to discover whether this protracted period of
contact between host and parasite was an important component in the
development of a protective response. A requirement for persistence of
irradiated schistosomula in order to induce immunity was unquestionably
demonstrated by terminating the infection with drugs, or by excising the
skin exposure site at intervals after vaccination. Bickle (1982) found that
immunizing larvae had to be in contact with the mice for between 1 and 2
weeks in order to stimulate optimum protection. In a skin excision study
carried out by Mangold and Dean (1984), a minimum period of 8-11
days was established.
The persistence of attenuated schistosomula was also the subject of a
subsequent autoradiographic study (Mountford et aZ., 1988), which
extended the findings of Mangold and Dean (1984) by paying particular
attention to the lymph nodes draining organs on the parasite migration
route. It was discovered that normal (i.e. non-attenuated) schistosomula
migrated rapidly through lymph nodes draining the skin exposure site,
since numbers peaked at 16% of applied parasites on day 5 and declined
to less than 3% on day 7. This confirmed the observations of an earlier
histological study which noted the absence of dead larvae in draining
lymph nodes and the presence of parasites in the efferent lymphatics
(Wheater and Wilson, 1979). In contrast, fewer attenuated parasites were
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 287

Figure 2 Differences between normal and irradiated larvae at the lung stage of
development. Scanning electron micrographs of schistosomula cultured for 8 days
in vitro. Scale bar = 10 pn. A. Normal schistosomulum showing the smooth
outline of the body and characteristic loss of midbody spines but presence of
anterior (a) and posterior (p) spines. B. Schistosomulum derived from an irradiated
cercaria showing constrictions of the body at random points along its length
(arrows), and presence of anterior (a) and posterior (p) spines (reproduced from
Harrop and Wilson, 1993a, with permission).
288 P.S. COULSON

detected in draining lymph nodes (peak of 11.5% on day 3,but they


persisted throughout the second week after exposure (Mountford et al.,
1988). Furthermore, proportionally greater amounts of parasite-released
material were detected in the nodes of vaccinated mice compared to those
infected with normal cercariae, most probably as a result of an increased
turnover of parasite proteins caused by irradiation. These findings were
taken as proof that attenuated schistosomula are sequestered in this
location.
In order to establish whether persistence of attenuated larvae in the
draining lymph nodes was obligatory for the induction of immunity, the
fate of parasites was investigated using a ‘vaccination’ regime that fails to
elicit significant protection. When mice were dosed orally, 24 h after
vaccination, with the compound Roll-3125, there was an early death
and rapid clearance of larvae whilst they were still in the skin (Mountford
et al., 1989). High levels of parasite-released material were detected in this
site, but negligible amounts were present in draining nodes. In another
study, using 80 h a d irradiated parasites (which induce little or no protec-
tion), it was established that few migrated from the skin exposure site to the
draining nodes and none reached the lungs (Constant et al., 1990). Addi-
tionally, Mountford and Wilson (1990) have shown that surgical excision
of skin-draining lymph nodes, 5 days before vaccination and up to 10 days
after, caused a significant reduction in the level of immunity compared to
that in sham-operated controls. These data lend strong support to the
hypothesis that persistence of radiation-attenuated parasites in the skin
and its draining lymph nodes, together with the prolonged release of
antigen in the latter site, are major factors in the induction of resistance.
Whilst there is little doubt that the skin-draining lymph nodes play a
central role in the induction of immunity, the incomplete ablation of
resistance when the nodes are absent (Mountford and Wilson, 1990)
implies that a component of the response must be elicited elsewhere. It
was postulated by Mastin er al. (1983) that the persistence and subsequent
death of irradiated schistosomula in the lungs could provide the appropriate
antigenic stimulus for the induction of an immune response against chal-
lenge parasites. When optimally attenuated cercariae are applied percuta-
neously (Mountford et al., 1988) or attenuated schistosomula are
administered intradermally (Coulson and Mountford, 1989), a sizable pro-
portion migrate as far as the lungs; in both instances a high level of
protection results. However, if attenuated schistosomula are introduced
directly to the lungs (i.e. bypassing the lymph nodes) they induce only
moderate levels (intratracheal route) or virtually no protection (intravenous
route), despite the larvae remaining in this location for at least nine days
(Coulson and Mountford, 1989). It would therefore appear that irradiated
larvae must persist in both lymphatic tissue and lungs in order to generate
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 289

an optimum immune response. The precise nature of the lung involvement


in priming is presently unclear. There may be a requirement for presenta-
tion of antigens in the pulmonary tissues and/or lung-draining lymph
nodes, or the larvae may simply act as a stimulus for the recruitment of
reactive cells from the circulation.

4.3. Immunogenic Potential of Attenuated Schistosomula

An extended period of contact between host and irradiated parasite may be


necessary to allow production and processing of a sufficient amount of the
relevant antigen(s) to stimulate the immune system. Alternatively, the
requirement for schistosomular persistence might signify that the relevant
antigens are not confined to early developmental stages or that they exist
only on later stage parasites. Attempts have therefore been made to protect
mice by vaccinating them with parasites of different ages. Attenuated skin-
stage schistosomula, administered intramuscularly, were capable of indu-
cing immunity, although the level was not as high as when mice were
vaccinated percutaneously with attenuated cercariae (e.g. Bickle et af.,
1979b; Sher and Benno, 1982). Studies on the immunogenicity of older
parasites have provided conflicting results. In one, attenuated lung schis-
tosomula induced significant immunity when administered by a variety of
routes (Dean et af., 1981b), in another they were poorly immunogenic
(Sher and Benno, 1982). Regardless of the route of injection, little protec-
tion was induced with day 21 or 28 irradiated liver worms (Dean et af.,
198 lb). Overall, these studies suggested that as parasites age their ability to
induce resistance diminishes.
In an effort to shed more light on this subject a comparison was made
between the immunogenicity of newly transformed and day 8 lung schis-
tosomula (Coulson and Mountford, 1989). However, unlike previous stu-
dies where parasites were attenuated after recovery from normally infected
donor mice (Dean et al., 1981b; Sher and Benno, 1982), the schistosomula
were derived from donors exposed to irradiated cercariae. (The migratory
potential of the larvae may differ in these two circumstances and hence
explain the disparate results.) A striking finding was that lung-stage larvae,
injected intradermally, induced at least as much immunity as attenuated
cercariae administered percutaneously, revealing that antigens specific to
the skin stage were not required for the development of immunity. Indeed,
the protective antigens could be specific to the lung schistosomulum since
there is no way of assessing whether the protection elicited by newly
transformed larvae depends upon them developing to the lung stage.
290 P.S. COULSON

5. IMMUNE RESPONSES DURING PRIMING

Since local antigen presentation in the skin-draining lymph nodes, and


possibly the lungs, is considered to be crucial for the induction of immu-
nity, investigations have focused on responses in these locations.

5.1. Lymph Nodes Draining Sites on the Migration Route

Constant et al. (1990) discovered that following vaccination, there was a


large fold-increase in cell numbers in the skin-draining (axillary) and lung-
draining (mediastinal) lymph nodes. However, changes in an irrelevant
node (draining the forelimbs) and the spleen were undetectable or slight
by comparison, thereby indicating marked localized rather than systemic
sensitization. Events in the axillary nodes preceded those in the mediastinal
lymph node by 7 or more days and it was presumed that the delay in
reactivity in the latter site reflected the time taken for the attenuated larvae
to migrate from the skin to the lungs. Proliferative responses in these
lymphatic tissues have been characterized in vivo by measuring the incor-
poration of the thymidine analogue bromo-deoxyuridine into dividing cells
(Constant and Wilson, 1992). Both the axillary and mediastinal lymph
nodes of vaccinated mice showed a significant increase in the proportion
and absolute number of proliferating cells. When the surface phenotype of
these cells was determined, the proliferative response was found to occur
predominantly in the T-cell compartment of the nodes. Whilst the total
number of B lymphocytes in the lymph nodes increased dramatically,
further investigation revealed that this was most likely due to their pre-
ferential retention rather than proliferation.
In vitro studies of lymphocyte blastogenesis have shown that cells
extracted from lymph nodes draining either the skin (James et al., 1981;
Lewis and Wilson, 1982; Pemberton et al., 1991; Richter et al., 1993b) or
lungs (Lewis and Wilson, 1982; Pemberton et al., 1991) proliferate in
response to a variety of schistosome antigens. The last two groups of
researchers also noted that the levels of antigen-specific proliferation by
splenocytes were low in comparison to those of lymph nodes. In the most
detailed of the studies, Pemberton et al. (1991) found that proliferation of
cells from the axillary node peaked on day 5, declining sharply thereafter,
whilst cells from the mediastinal lymph node responded later with peak
blastogenesis occurring 18 days postvaccination. The majority of this
activity was ablated by pretreatment of the cultures with anti-CD4 mono-
clonal antibody (mAb), implicating a role for T helper cells in the early
events during priming.
RADIATION-ATTENUATEDVACCINE AGAINST SCHISTOSOMES 29 1

At this point it would be pertinent to consider recent advances in our


understanding of the role of T-helper lymphocytes in immune responses.
The CD4+ T-helper subset has been divided into Thl and Th2 cells on the
basis of the profile of cytokines they secrete in culture (Mosmann and
Coffman, 1987). Originally defined in a panel of long-term murine T-
helper-cell clones, these two phenotypes have since been confirmed in
mouse immune responses (Mosmann and Coffman, 1989) and amongst
human T-cell clones (Romagnani, 1992). The Thl subset is characterized
by interleukin (1L)-2 and interferon gamma (IFNy) production, whilst Th2
cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13. Cytokines such as IL-3
and granulocyte-macrophage colony stimulating factor (GM-CSF) are
common to both T-helper subsets (Mosmann and Moore, 1991). The
two major patterns of cytokine synthesis initially recognized appear to
correlate with the induction of cell-mediated (Th 1) or humoral (Th2)
immunity, thus providing a possible explanation for the separate and
often reciprocal regulation of these responses. Since the original defini-
tion, it has become clear that a number of other cytokine secretion
phenotypes exist. For instance, a non-polarized clone, termed Tho,
expresses most or all of the cytokines made by Thl and Th2 cells and
may represent a subset that is at an intermediate stage of differentiation
(Firestein et al., 1989). More recently, it has been established that the
dominant factors determining subset variation are lymphokines and other
cytokines (Seder and Paul, 1994). Thus, if IL-4 is present during the
priming period the resultant CD4+ T cells produce IL-4 upon restimula-
tion, and the development of IFNy-secreting cells is inhibited. In the
absence of IL-4, priming for IFNy production occurs but this is markedly
enhanced by IL-12. Presently, there are no authenticated cell markers
available to distinguish the Thl and Th2 subsets; their status can only be
estimated indirectly through determination of their specific secretory
activity.
In order to establish whether a particular T-helper-cell subset is induced
by vaccination with irradiated parasites, Pemberton et al. (1991) examined
the profile of cytokines produced by cultures of lymph nodes cells. During
the early stages (around day 5 postvaccination) they detected antigen-
stimulated production of IL-2, IL-3, IL-4 and IFNy by cells from skin-
draining lymph nodes, indicative of a Tho or a mixed Thl/Th2 response.
However, at later times (between days 18 and 23), IL-3 and IFNy were
predominant with virtually no IL-4 secretion. IFNy was also the most
abundant cytokine produced by mediastinal lymph node cell cultures.
Whilst this cytokine profile does not conform precisely to the classical
definition for Thl cells, it would nevertheless appear that a polarized
response is eventually generated in the lymph nodes draining tissues on
the parasite migration route.
292 P.S. COULSON

Analysis of skin-draining lymph nodes from mice exposed to non-immu-


nizing (normal; Pemberton et al., 1991 or 80 krad-irradiated; Mountford et
al., 1992) cercariae revealed an initial pattern of cytokine secretion similar
to that in protected animals, but production was not sustained. Moreover,
increases in total cell content were less pronounced and of shorter duration
(Constant et al., 1990; Pemberton et al., 1991; Mountford et al., 1992).
Indeed, when the cellularity of the axillary nodes was taken into account,
optimally attenuated larvae generated a T-cell population at day 22 that had
a 24-fold greater potential for IFNy production than that from mice exposed
to normal parasites (Pemberton et al., 1991). Attenuated larvae thus appear
to act like a natural adjuvant, delivering themselves to a site where antigen
processing and presentation is intense. By releasing antigenic material over
an extended period, they provide a continual stimulus for expansion of the
lymphocyte pool.
During the natural course of lymphocyte traffic, the schistosome-reactive
T-helper cells generated in the lymph nodes will leave this site and ulti-
mately enter the bloodstream. From 14 days after vaccination of mice, a
large and sustained expansion of the circulating leucocyte pool could be
detected, and it was established that this was mainly due to changes in
lymphocyte numbers (Menson et al., 1989). Additionally, splenocytes
(Pemberton et al., 1991; Pearce et al., 1991), and in particular splenic
CD4+ T cells (Caulada-Benedetti et al., 1991), recovered from mice up to 4
weeks after vaccination produced substantial amounts of IFNy in response
to schistosome antigen. Since the spleen plays a fundamental role in
filtering the blood, this finding can be taken as evidence for the presence
of circulating schistosome-reactive lymphocytes in vaccinated mice. More
conclusive proof is afforded by the results of a study using an assay of
classical DTH (Ratcliffe and Wilson, 1991). The initiation of a DTH
response relies upon the recruitment of specifically sensitized T-helper
cells from the bloodstream to a site of secondary challenge (e.g. the pinna
or footpad). The size of the swelling measured at this peripheral site
provides a crude estimate of the number of reactive cells in the circulation.
Footpad DTH to adult schistosome antigen was first observed on day 10
and reached its maximum 17 days after vaccination before declining almost
to baseline levels by day 35. The response was completely abrogated by
treatment of mice prior to footpad challenge with a cytotoxic rat mAb to
murine CD4.

5.2. The Lungs

The technique of bronchoalveolar lavage (BAL), in which fluid is instilled


into the distal airways and retrieved, yields reproducible recoveries of
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 293

viable cells for functional and phenotypic analyses. As a means of exam-


ining pulmonary immunological events, this approach provides an alter-
native to histopathological evaluation of leucocyte responses, and has the
additional benefit of being quantitative. Typically, BAL recoveries from
the naive mouse lung comprise greater than 90% macrophages with very
few lymphocytes.
When BAL was performed on mice vaccinated with irradiated cercariae
an influx of leucocytes into the pulmonary airways was detected from 14
days onwards. This infiltrate, characterized by flow cytometry (Aitken et
al., 1988) or differential staining (Menson et af., 1989), contained lym-
phocytes, macrophages and eosinophils. The inflammatory response in the
airways peaked at day 21 but elevated numbers of T lymphocytes persisted
at least until 10 weeks after vaccination (Aitken et af., 1988). At all
sampling times after vaccination the major fraction of the infiltrating
lymphocytes comprised T cells of the CD4 subset (Coulson and Wilson,
1993). An intense eosinophilia also peaked at day 21, accounting for 30%
of the total leucocytes at this time, but it was extremely transient by
comparison (Menson et af., 1989). Exposure of mice to an equivalent
number of normal cercariae resulted in an earlier circulating lymphocyto-
sis, of shorter duration, and far fewer lymphocytes and macrophages
infiltrated the lungs than after vaccination.
The period of rapid influx of cells corresponded to histological observa-
tions of cell accumulation around vaccinating larvae in the lungs between
days 13 and 21 (Mastin et al., 1983). The build-up of cellular foci occurs
over the interval during which footpad DTH is diminishing (Ratcliffe and
Wilson, 1991), leading these authors to suggest that the decrease in footpad
reactivity (i.e. circulating sensitized T cells) is a consequence of DTH-
mediating T lymphocytes being recruited from the bloodstream to the
lungs. A follow-up study, using 5 1-chromium-labelled splenocytes,
demonstrated that the capacity for cell recruitment to the lung from the
circulation was at its most intense between days 13 and 19 postvaccination
(Ratcliffe and Wilson, 1992). It would thus appear that the fate of many of
the sensitized, circulating T-helper lymphocytes is to enter the pulmonary
tissues.
In terms of their functional properties, it was found that by day 14
postvaccination, BAL cultures contained infiltrating lymphocytes capable
of producing abundant quantities of IFNy and IL-3 upon stimulation with
larval antigen; ablation experiments revealed that CD4+ T cells were the
source of the secreted IFNy (Smythies et al., 1992b). However, after a peak
at day 21, production of both cytokines diminished, despite the fact that
numerous lymphocytes were present in the cultures. Since antigen-stimu-
lated production of IL-2 and IL-4 was virtually undetectable in BAL
cultures at any sampling time, the cytokine phenotype of the lymphocyte
294 P.S. COULSON

population recruited to the lungs was essentially identical to that of lymph


node cells at later times after vaccination (see Section 5.1). In comparison
to the situation in vaccinated mice, only small amounts of IFNy were
produced by BAL cultures from mice exposed to normal, unirradiated
cercariae.
The cytokine profile in the lungs has also been examined at the mRNA
level by Wynn et al. (1993, using semi-quantitative, reverse transcription-
polymerase chain reaction (RT-PCR). These researchers observed elevated
expression of mRNA for several of the Th2-associated cytokines (IL-4, IL-
5 and IL-6) in whole lung tissue between days 6 and 28 postvaccination
but, somewhat surprisingly, exposure of mice to normal cercariae resulted
in similar increases in cytokine message. However, the major difference
between the two groups of mice was in the kinetics of IFNy expression.
Thus, whilst an elevated level of IFNy mRNA was induced in the lungs by
normal larvae, this was not apparent until 12 days after the increase seen
with irradiated larvae.
One role for the inflammatory cytokine IFNy is to activate pulmonary
macrophages. With this in mind, Menson and Wilson (1989) characterized
the activation state of individual macrophages by their capacity to mount
an oxidative burst. They were able to show that highly activated macro-
phages could be recovered from the lungs between 14 and 28 days post-
vaccination, coincident with the interval when airway T cells produced
abundant IFNy if cultured in vitro (Smythies et al., 1992b). Furthermore,
during the same period the proportion of Ia+ alveolar macrophages
increased up to five times normal; upregulation of Ia is also consistent
with an activation phenotype (Menson and Wilson, 1990). These observa-
tions provide strong circumstantial evidence for IFNy activity in vivo, and
it is conceivable that macrophage products play a part in the recruitment of
cells to the lung after vaccination.
Intriguingly, airway lymphocytes could not be stimulated to proliferate
in vitro in response to mitogen or antigen (Smythies er al., 1992b). It was
considered that a lack of IL-2 may have been the cause of non-proliferation
but addition of recombinant IL-2 to the BAL cell cultures (at doses that
augmented splenocyte responses) did not stimulate blastogenesis (L.E.
Smythies, unpublished data). It would therefore appear that the T-helper
lymphocytes present in the lungs after vaccination represent a differen-
tiated cell population whose function is to generate rapid release of cyto-
kine upon stimulation with recall antigen. As such, it is typical of a murine
effector/memory cell population described by Budd et al. (1987). Indeed,
this status was confirmed when phenotypic analysis and flow cytometry
were performed (Coulson and Wilson, 1993); virtually all the CD4+ T
lymphocytes from the airways were CD44hi and CD45RB - , unlike those
from the lymph nodes and circulation. What is more, a large proportion of
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 295

this population was schistosome specific, as judged by the ability to


upregulate the inducible chain of the IL-2 receptor in response to recall
antigen. The relevance of the CD4+ T-cell population resident in the lungs
at the time of challenge, to protection, is addressed in Section 6.3.

6. THE IMMUNE EFFECTOR MECHANISM

The most reliable indicator of challenge elimination is the reduction in


recovery of mature worms from the hepatic portal system of immune
animals compared to previously uninfected controls (Dean, 1983). Unfor-
tunately, this yields no information on the site(s) at which elimination takes
place. Before progress could be made in understanding the mechanism
causing immune elimination of challenge parasites in vaccinated mice, it
was first necessary to establish where it occurred.

6.1. The Site of Challenge Parasite Elimination

There has been a great deal of controversy regarding the major site of
challenge parasite elimination, the main issues of which were addressed in
a pair of debate articles (Wilson and Coulson, 1989; McLaren, 1989).
Studies to date have provided conflicting results, with some implicating
the skin and others the lungs. Evidence in support of elimination in the skin
comes from several lines of investigation. Mincing and incubation of skin
and lungs of vaccinated mice yielded lower numbers of challenge schisto-
somula compared to normal animals (Miller and Smithers, 1980). Histo-
pathological studies of the skin of vaccinated mice showed damaged and
dead challenge parasites within subcutaneous inflammatory foci (Hsu et al.,
1983; Ward and McLaren, 1988). The level of resistance was reduced if the
skin was bypassed by direct injection of challenge larvae intravenously to
the lungs (McLaren et al., 1985). MAbs, which selectively deplete neu-
trophils (McLaren et al., 1987) or CD4+ T cells (Piper and McLaren, 1993),
diminished resistance most effectively if they were administered at the
time of challenge. Likewise, serum from multiply vaccinated mice con-
ferred partial protection only if transferred at early times after infection,
before schistosomula reached the lungs (McLaren and Smithers, 1988).
Finally, results from autoradiographic tracking have been interpreted to
mean that challenge parasites do not reach the lungs in similar numbers to
those in normal mice (Kamiya et al., 1987).
Running counter to this is the wealth of evidence in favour of elimina-
tion of parasites at a later stage in their migration. For example, similar
296 P.S. COULSON

numbers of challenge larvae were recovered from the lungs of normal and
vaccinated mice, by mincing and incubation (Minard et al., 1978b; Stek et
al., 198la). Histopathological studies revealed that, despite intense inflam-
matory responses in the skin, there was no evidence of challenge parasite
damage or elimination in this site (Mastin et al., 1983; von Lichtenberg et
al., 1985). Little or no reduction in the level of resistance was observed
when the skin was bypassed (Dean et al., 1981b; Mangold et al., 1986).
The most consistent level of protection was obtained when immune serum
was administered several days postinfection, coincident with parasite
migration through the lungs (Mangold and Dean, 1986). Furthermore,
autoradiographic tracking demonstrated that challenge parasites eventually
reached the lungs of vaccinated mice in similar numbers to those in normal
mice (Dean et al., 1984). However, these data do not establish the precise
site of challenge elimination, only that it occurs at some point after arrival
of schistosomula in the lungs.
In order to determine the site more accurately, an autoradiographic
tracking study was performed, in which all organs were sampled to provide
an estimate of the total number of challenge parasites in the mouse as well
as their location (Wilson et al., 1986). It was discovered that identical
proportions reached the lungs of normal and vaccinated mice but in the
latter there was a greatly reduced systemic phase of migration and hepatic
portal accumulation, indicating that fewer larvae travelled beyond this site.
When migration was complete in vaccinated mice a large fraction of
schistosomula was still detectable in the lungs compared to other tissues.
It was therefore concluded that the majority of parasite elimination took
place in this organ, although the process was extended, starting around 7-
12 days after challenge and continuing at least to day 35.
The disparity between observations of skin and lung elimination may
reside, for instance, in the different mouse strains and/or parasite isolate
used in the various laboratories, although there is little evidence to support
this notion (Dean et al., 1995). However, whilst it is difficult to reconcile
the data on the site of elimination, all workers agree that the target of the
immune response is the lung schistosomulum. Even when elimination was
considered to occur in the skin, the larvae trapped in inflammatory reac-
tions in the subcutaneous tissues have succeeded in developing from skin-
stage to lung-stage worms (Ward and McLaren, 1988).

6.2. The Nature of the Immune Response

6.2.1. Cellular Immunity


A hallmark of classical cell-mediated immunity is that it can be adoptively
transferred to a naive recipient with sensitized T cells from an immune
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 297

donor. Hence, in the absence of additional evidence, the failure of adoptive


transfer of immunity with lymphoid cells from vaccinated mice (Bickle et
al., 1985; R. Aitken and P.S. Coulson, unpublished data) would make it
difficult to propose a role for a cell-mediated response. However, compre-
hensive studies by James, Sher and co-workers have firmly established that
a single exposure to attenuated cercariae stimulates a state of cell-mediated
immunity in mice, analogous to that induced by bacille Calmette-GuCrin
(BCG) or Corynebacterium parvum. A major breakthrough in their
research was the discovery of an inbred mouse strain (P) which fails to
develop vaccine-induced immunity (James and Sher, 1983).
Whilst not relating events to a particular organ, several lines of inquiry
have implicated the macrophage as an essential component of the immune
response in once-vaccinated mice (reviewed by James, 1986). For example,
upon stimulation with schistosome antigen, T cells from immunized mice
produce cytokines such as IFNy which activate normal peritoneal macro-
phages for non-specific as well as larvicidal activity (James et al., 1983,
1984). Activated macrophages can be elicited which are capable of killing
skin-stage schistosomula in an in vitro cytotoxicity assay (e.g. Mahmoud et
al., 1979; James et al., 1984). Furthermore, vaccination of inbred mouse
strains, with known ‘macrophage defects’, does not result in protection
against a challenge infection (James and Sher, 1983; James et al., 1983). It
has been demonstrated conclusively that P-strain mice have a selective
defect in T-cell function, failing to produce cytokine( s) to activate macro-
phages at the site of specific antigen challenge (James et al., 1986).
Additional support for a cell-mediated mechanism of immunity operat-
ing in the once-vaccinated mouse is provided by ablation studies. In vivo
administration of a cytotoxic rat mAb to the murine CD4 marker selec-
tively depletes T-helper lymphocytes, and results in abrogation of protec-
tive immunity (Kelly and Colley, 1988; Vignali et al., 1989a; Phillips et
al., 1991). Whereas this treatment is highly successful in once-vaccinated
mice, it has less effect after multiple vaccinations. Mice exposed twice to
irradiated cercariae showed a 70% reduction in protection compared to a
100% reduction in once-vaccinated animals (Vignali et al., 1989a). The
resistance of mice hyperimmunized by five exposures to irradiated cercar-
iae was unaffected by the treatment, regardless of when it was applied
(Kelly and Colley, 1988). These findings could be construed as evidence
for the participation of antibody in the immune mechanism operating in
multiply vaccinated mice (see below).

6.2.2. Humoral Immunity


Although classical humoral responses can be demonstrated after chal-
lenge (e.g. Correa-Oliveira et al., 1984), it has so far proved difficult
298 P.S. COULSON

to characterize their function in immune elimination. The experiments of


Dean et al. (1981a) indicate that vaccine-induced resistance can be trans-
ferred to naive mice via a parabiotic union allowing passage of both
humoral and cellular factors. Apart from this, evidence for the role of
antibody in once-vaccinated mice is sparse. There was an absence of
striking differences in schistosome-specific antibody titres between mice
developing high or low levels of protection, suggesting that additional or
alternative mechanisms are involved (James et al., 1981). Moreover,
attempts to transfer immunity with serum from once-vaccinated highly
resistant donors to naive recipients have generally failed (Bickle et al.,
1985; Mangold and Dean, 1986; P.S. Coulson and R.A. Wilson, unpub-
lished data). On the other hand, two or more vaccinations generate anti-
body titres capable of mediating passive transfer of resistance (e.g.
Mangold and Dean, 1986; McLaren and Smithers, 1988; Richter et al.,
1993a), with recipients displaying between 20 and 60% reduction in worm
burdens depending on the protocol.
The protective factors in immune serum appear to involve IgG antibodies
(Mangold and Dean, 1986), particularly those of the IgGl isotype (Delgado
and McLaren, 1990), although IgM may play a synergistic role (Jwo and
LoVerde, 1989). The serum is effective if administered 1 h before (McLa-
ren and Smithers, 1988) or up to several days after exposure (Mangold and
Dean, 1986; Richter et al., 1993a). Transfer of sera from mice given
multiple exposures to UV-irradiated S. japonicum cercariae also consis-
tently protects naive recipients against homologous challenge (Moloney et
al., 1987). The major protective effect is similarly mediated by IgG anti-
bodies which act at or about the lung-stage of S. japonicum migration
(Moloney and Webbe, 1990; Dunne et al., 1994). However, the mechanism
of immune elimination of larvae of either schistosome species in recipients
of such sera has not been explored.
Analysis of cellular responses in multiply vaccinated mice indicates an
increased level of Th2-associated reactivity, measured as antigen-specific
IL-4 and IL-5 production, especially in lymph nodes draining the vaccina-
tion site (Caulada-Benedetti et al., 1991). This is consistent with the
observation of increased serum levels of IgGl antibodies against schisto-
some antigens in these mice (Delgado and McLaren, 1990; Caulada-Ben-
edetti et al., 1991), since IgGl is known to be a Th2-dependent
immunoglobulin isotype (Finkelman et al., 1990). In comparison, sera
from once-vaccinated mice contain a balance of IgGl and IgG2a (Thl)
isotypes (Mountford et al., 1994). Paradoxically, splenocytes from multiply
vaccinated mice continue to secrete substantial amounts of IFWy (Caulada-
Benedetti et al., 1991; Reynolds and Harn, 1992), and these animals retain
the ability to produce activated macrophages upon specific antigen chal-
lenge (Caulada-Benedetti et al., 1991).
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 299

Taken together, the data suggest that whilst Thl -dependent immune
mechanisms predominate in mice vaccinated once with irradiated cercar-
iae, protection after multiple exposures results from a mixture of Thl and
Th2