Advanced Drug Delivery Reviews 43 (2000) 29–43 L

www.elsevier.com / locate / drugdeliv

Application of DNA vaccine technology to aquaculture

¨ Heppell a , Heather L. Davis a,b,c , *
Joel
a
Loeb Health Research Institute at the Ottawa Hospital, 725 Parkdale Avenue, Ottawa, ON, Canada K1 Y 4 E9
b
School of Rehabilitation Sciences, Faculty of Health Sciences and Department of Biochemistry, Microbiology and Immunology,
Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
c
CpG ImmunoPharmaceuticals Inc., Wellesley, MA, USA

Abstract

The aquaculture industry needs to augment its global production and efficiency to meet the increasing consumer needs for
fish and shellfish products. Unfortunately, infectious diseases have been a major impediment to the development and
profitability of fish farms. While vaccines offer the most efficient way to control infectious pathogens, current products have
only been successful against some diseases. These are mostly bacterial, and there are still several important diseases, mainly
of viral and parasitic origin, for which no prophylactic treatment exists. DNA vaccines, compared to traditional antigen
vaccines, have several practical and immunological advantages that make them very attractive for the aquaculture industry.
The early success of DNA vaccines in animal models was very encouraging, but fish are unique in many aspects, and
findings with other classes of vertebrate, namely mammals and birds, do not necessarily apply to aquatic animals. However,
more recent studies with reporter genes showed that fish cells efficiently express foreign proteins encoded by eukaryotic
expression vectors. A piscine-specific backbone vector might eventually improve immune responses to DNA vaccines, but
there is already strong direct evidence for the induction of protective immunity with currently available plasmids. Immune
responses to plasmid DNA injected intramuscularly (IM) into fish are characterized by the production of antibodies, which
have been shown to be neutralizing in two different viral disease models. There is also indirect evidence suggesting the
induction of cell-mediated immunity. Despite this evidence, immune responses to DNA vaccines have only been poorly
characterized in fish because of the limited knowledge of the piscine immune system, and the small number of studies on the
subject. Apart from optimizing the efficiency of DNA vaccines, other important issues, such as safety and production cost
will be determinants for the potential application of this technology in commercial fish farms. Alternative methods of
administration will also have to be developed for small fish and low-valued species, for which IM injection is not practical
and / or cost effective.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Nucleic acid vaccine; Fish; Aquaculture; Disease control; CpG motifs

Contents

1. Introduction ............................................................................................................................................................................ 30
1.1. The need for fish vaccines ................................................................................................................................................ 30
1.2. Advantages of DNA vaccines ........................................................................................................................................... 31

*Corresponding author. Tel.: 1 1-613-798-5555 ext. 7682; fax: 1 1-613-761-5354.

E-mail address: hdavis@lri.ca (H.L. Davis).

0169-409X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0169-409X( 00 )00075-2
30 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43

2. Plasmid constructs................................................................................................................................................................... 31
2.1. Control elements .............................................................................................................................................................. 31
2.2. Antigen encoding gene ..................................................................................................................................................... 31
2.3. DNA as an immunostimulatory molecule for fish ............................................................................................................... 32
3. Methods of administration ....................................................................................................................................................... 32
3.1. Injection .......................................................................................................................................................................... 33
3.1.1. Site of injection...................................................................................................................................................... 33
3.1.2. Dose and volume ................................................................................................................................................... 34
3.1.3. Location of foreign gene expression ........................................................................................................................ 34
3.1.4. Duration of expression and effect on fish tissues....................................................................................................... 34
3.1.5. Alternatives to needle injection ............................................................................................................................... 35
3.2. Oral and immersion vaccination ........................................................................................................................................ 35
4. Immune responses to DNA vaccines ......................................................................................................................................... 36
4.1. Antibody responses .......................................................................................................................................................... 36
4.2. Cellular immune responses ............................................................................................................................................... 37
4.3. Protection against live challenge ....................................................................................................................................... 38
5. Safety of DNA vaccines for fish ............................................................................................................................................... 38
5.1. Fate of injected DNA in fish ............................................................................................................................................. 38
5.2. Safety for the consumer .................................................................................................................................................... 38
5.3. Risks for other fish and the environment ............................................................................................................................ 39
6. Perspectives for DNA vaccines in the aquaculture industry......................................................................................................... 39
7. Conclusions ............................................................................................................................................................................ 40
References .................................................................................................................................................................................. 40

1. Introduction instances, there are no prophylactic or therapeutic

measures available.
The need for additional or alternate sources of fish Chemicals and antibiotics can be used to control
and shellfish products has made the aquaculture bacterial and parasitic diseases but these products
industry the fastest growing segment of agriculture in often have undesirable side effects such as accumula-
the world [1,2]. The increased demand for aquatic tion in the flesh of animals, development of drug-
animals, which is partly due to greater health aware- resistant strains, and contamination of the aquatic
ness of consumers, and the decline or stagnation of environment [6,7]. For viral infections, where there
natural harvests, have largely contributed to this are no treatments available, the appearance of dis-
rapid growth [1]. However, despite the significant ease in facilities usually requires destruction of
progress toward higher standards of productivity and infected stock before starting anew. Consequently,
quality that fish farming has made in developed most research efforts are focusing on prevention
countries, the industry has still not reached levels rather than treatment with chemicals, which should
comparable to those found with other farmed animals only be considered when vaccines are unavailable for
such as swine and poultry. specific pathogens or when they fail to prevent an
outbreak.
Vaccines have long proven their efficacy in herd
1.1. The need for fish vaccines protection, but in the aquaculture industry, they are
in a relatively early phase of development. Most of
Among problems that the fish and shellfish indus- the commercially available vaccines protect fish
tries have to address, infectious pathogens are the against bacterial diseases, and are simply made of
most important [1,3,4]. It has been estimated that ten inactivated bacteria applied by either immersion or
percent of all cultured aquatic animals are lost injection with an oil adjuvant [3,8,9]. There are
because of infectious diseases alone [5]. Outbreaks currently no vaccines against parasitic pathogens,
can cause severe losses to fish farmers and in many and very few against viruses [9,10]. The high cost of
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
new product development combined with the rela-
tively small size of the industry and low value of
individual animals have largely contributed to this
situation. In fact, many vaccines have been tested
under laboratory conditions, but they were not
commercially viable because of their prohibitive cost
of production, insufficient protection, or their lack of
safety [5,6,8,11,12].
1.2. Advantages of DNA vaccines
The first experiments showing an immune re-
sponse to plasmid-encoded antigens of infectious
pathogens were published several years ago [13–18].
This very promising technology immediately caught
the widespread attention of scientists working in the
field of vaccine development. An early report in
1991 by Hansen et al. [19] showed expression of a
reporter gene in carp muscle after injection of
plasmid DNA, but it was several years before DNA
vaccines were demonstrated in fish [20–22].
For aquatic organisms, as for other farmed ani-
mals, DNA vaccines offer several advantages over
classical antigen vaccines (i.e., live attenuated, whole
killed and subunit vaccines). From a practical point
of view, they are relatively inexpensive and easy to
produce, and all DNA vaccines require identical
production process. Multivalent vaccines can also be
easily prepared by mixing together different plas-
mids, or including more than one antigen-encoding
gene in a single vector for colinear expression, which
will further reduce the cost of production. In addi-
tion, DNA is a very stable molecule, and does not
need to be maintained in a cold environment during
shipment or storage. Finally, the ease of cloning
allows vaccines to be rapidly modified if needed. All
these factors contribute to make DNA vaccines very
attractive to fish vaccine manufacturers.
DNA-based immunization also has immunological
advantages over traditional methods of vaccination.
As shown with mammals, they can induce strong and
long-lasting humoral and cell-dependent immune
responses without boost, similar to that conferred by
live vaccines, but without the risk of inadvertent
infection [23]. In fish, much less is known about the
immune responses following plasmid injection, but
31
there is no indication that it will be very different
from what has been observed with other animals.
2. Plasmid constructs
There are still very few reports on the use of DNA
vaccines in fish, and except for research on trans-
genic animals, which is not the subject of this
review, the question of designing a fish specific
vector has never been addressed. Nevertheless, the
major considerations in vector design for the purpose
of DNA vaccines are presented in this section.
Features not specific to fish, such as bacterial origin
of replication and selection marker, have been re-
viewed elsewhere [23,24] and are not discussed
herein.
2.1. Control elements
Plasmid constructs used for expression of foreign
genes in fish usually contain a mammalian expres-
sion vector backbone. Sequences for transcriptional
control in the standard vectors (promoter, enhancer,
intron, polyadenylation signal, etc.) seem to work
efficiently in aquatic animals. Various promoters have
been assessed for their ability to drive expression of
foreign genes in fish tissues (Table 1). Their efficacy
varies considerably, but overall, the immediate early
promoter of the cytomegalovirus (CMV) gives the
best results. It is also the most widely used in DNA
vaccines reported to date, and its potency has been
demonstrated in fish vaccination trials [20–22,33].
Other transcriptional elements in DNA vaccines have
not been evaluated in fish, but there are indications
from in vitro studies that mammalian introns might
not always be correctly processed in fish cells [34].
Optional features of backbone vectors such as
unrelated Th epitopes to provide additional T help,
CpG immunostimulatory motifs, or genes for
colinear expression of cytokines or costimulatory
molecules, could eventually be added to DNA vac-
cines [23]. Although a better characterization of the
fish immune system is required before similar se-
quences are incorporated into vaccines for aquacul-
ture, a preliminary study suggests that mouse gran-
ulocyte–macrophage colony-stimulating factor
32 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
Table 1
List of promoters tested in fish tissues.
Promoter
Glucocorticoid-responsive mouse
mammary tumor virus (MMTV)
Cytomegalovirus (CMV) immediate early
(alone or with translational promoter)
Carp b -actin
SV40 early
Rabbit b -cardiac myosin heavy chain (MHC)
Human MxA
Artificial (based on human MxA)
Herpes simplex virus thymidine kinase (tk)
with the CMV enhancer
with the SV40 enhancer
(GM–CSF) gene coinjected with an antigen-encod-
ing gene could modulate cell-mediated immune
responses in goldfish [35].
2.2. Antigen encoding gene
In theory, any gene coding for a protein of the
pathogen that induces a protective immune response
in the host species can be used in DNA vaccines, as
long as the codon usage of the gene allows expres-
sion in fish cells. A problem with codon usage could
eventually arise with genes from some bacteria or
lower parasites, but it is unlikely to happen with viral
genes that are normally expressed in host cells using
cellular machinery.
To induce humoral immune responses, B-cells
must first meet circulating antigen to be activated.
Thus, secretion of the foreign protein can influence
the antibody production. However, humoral re-
sponses are possible even when the antigen is not
secreted. For example, antibodies against the cotton-
tail rabbit papilloma virus (CRPV) major capsid
protein (L1) were detected after DNA immunization,
even when L1 had a nuclear localization signal [36].
In the case of non-secreted antigen, B-cells may not
be fully activated unless antigen is released from
transfected cells, such as following lysis by antigen-
specific cytotoxic T-lymphocyte (CTL). It has been
Fish species References
Rainbow trout (Oncorhynchus mykiss) [25]
Rainbow trout (Oncorhynchus mykiss) [21,22,25,26]
Zebra fish (Brachydanio rerio) [21]
Goldfish (Carassius auratus) [27–29]
Xiphophorus sp. [30]
Rainbow trout (Oncorhynchus mykiss) [25]
Tilapia (Oreochromis niloticus) [31]
Common carp (Cyprinus carpio) [19]
Common carp (Cyprinus carpio) [19]
Common carp (Cyprinus carpio) [19]
Common carp (Cyprinus carpio) [19]
Rainbow trout (Oncorhynchus mykiss) [32]
Xiphophorus sp. [30]
Xiphophorus sp. [30]
shown in mice injected with hepatitis B surface
antigen (HBsAg), that appearance of antigen-specific
antibodies is delayed by a few weeks if the protein is
not secreted [37]. Consequently DNA vaccines may
include a signal peptide to direct secretion of the
encoded antigen, but this does not appear to be an
absolute requirement.
2.3. DNA as an immunostimulatory molecule for
fish
Strong immune responses induced by DNA im-
munization are not dependent solely on expression of
antigen. It is now well established that the DNA
molecule itself can act as an adjuvant to enhance the
immune response in mammals [38,39]. In fact, it has
been shown that short DNA sequences containing
unmethylated CpG dinucleotides in specific base
contexts can directly stimulate immunocompetent
cells such as monocytes, B-cells, macrophages and
dendritic cells; these immune stimulatory motifs are
called CpG-S motifs [39]. Adversely, other CpG
motifs that neutralize or block immune activation by
CpG-S motifs are called CpG-N motifs [40,41]. Very
little is known on the effect of CpG motifs in fish,
but preliminary experiments suggest that stimulation
can occur as in mammals, although the optimal
CpG-S motifs for fish are probably different from
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
those previously determined for mice [35,42]. Plas-
mid DNA vectors used to vaccinate fish certainly
contain CpG-S and CpG-N motifs in their sequences.
It is possible that a CpG optimized backbone vector,
containing an optimal number of fish-specific CpG-S
motifs and a reduced number of CpG-N motifs could
improve the immune response in fish, and possibly
allow administration of smaller doses of DNA.
3. Methods of administration
Vaccine administration to aquatic animals poses
obvious technical problems not encountered with
other animals. Nevertheless, in developed countries,
large-scale vaccination is prevalent for high valued
species such as salmon [11,43]. Methods used to
immunize fish can be divided into three major
categories, with several variations in each: injection,
immersion, and oral delivery [7,8,44].
3.1. Injection
3.1.1. Site of injection
Although stressful for the animals and labor
intensive, injection vaccination is usually the most
immunologically effective method to immunize fish
[7,8]. For DNA vaccines, it is thus far the only
method that has been reported in vaccination trials.
Purified plasmid DNA in a small volume of saline or
buffer is usually injected intramuscularly in the
flank, below or close to the dorsal fin, but other sites
have also been tested (Table 2). Injection in the gills
does not result in detectable levels of reporter gene
activity [32], while the outcome with the peritoneal
route was shown to be variable [28,32].
In mice, the site and method of injection of DNA
vaccines have a strong influence on the type of
immune responses elicited [23]. For example, in-
tramuscular (IM) needle injection induces a pre-
dominantly Th1 type response, with a high
IgG2a:IgG1 ratio, interferon (IFN)-g production and
low level of interleukin (IL)-4. On the other hand,
epidermal gene gun inoculation commonly induces a
Th2 response, characterized by the predominance of
IgG1 over IgG2a, less IFN-g and higher IL-4.
Intradermal (ID) injection of DNA with a needle can
induce either Th1 or Th2 profiles [45]. In fish, almost
33
no information is available on the effects of different
routes of administration on immune responses,
owing to the limited studies and the relatively poor
characterization of the piscine immune system.
3.1.2. Dose and volume
The effect of dose and volume of the injected
DNA has been studied more rigorously (Table 2).
Larger volumes are usually associated with a reduc-
tion of the reporter gene activity, but less variation
between individual fish. Some authors have also
noted that an excessively high dose of DNA can
result in a lower expression level of the foreign gene
[30,32]. Typical doses for fish fall in the range of
1–50 mg of DNA, in a volume of 10–50 ml.
However, much lower doses have been found to be
effective [21]. The optimal dose of DNA probably
varies according to the species and size of the
animal, but it does not increase proportionally to the
weight of the fish. Moreover, it has been reported
that the physiological condition of the animal also
influences the expression level. Young and fast
growing carp showed much higher levels of chloram-
phenicol acetyl transferase (CAT) reporter gene
activity than older fish [19].
Interestingly, luciferase reporter gene expression
driven by the CMV promoter is higher in fish than
mouse muscle for the same dose of DNA [21]. It is
not known whether this is due to an increased
stability of the luciferase protein in fish compared to
mammalian cells, to a higher expression level, or to a
better transfection efficiency. However, it suggests
that low doses of DNA, compared to those injected
in mammals, could be used to vaccinate fish. In fact,
doses as low as 0.1 mg of DNA per fish have been
reported to be as effective as 10 mg for inducing
protective immunity [33]. This bodes well for the
potential application of DNA vaccines in fish farms.
3.1.3. Location of foreign gene expression
There are apparent discrepancies in the literature
regarding the site of foreign gene expression after IM
injection of plasmid vectors. In large fish, expression
of reporter genes seems to be mostly restricted to the
site of injection, that is in myocytes as well as in
cells infiltrating the muscle tissue or epithelial cells
lining small capillaries [26]. Transfection of non-
muscle cells, especially professional antigen-present-
34 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43

Table 2
Methods of injection and doses of DNA tested in fish tissues.
Method a Site Gene d Dose (mg) Volume (ml) References
b
IM single Flank CAT 12.5, 25, 50 and 100 100 [19]
injection [31]
CAT 5, 10 and 50 0.1 [20,25]
Luciferase, IHNV-G and -N 10, 25 and 50 100 and 200
lacZ, luciferase 10, 25, 50 and 100 100 [32]
lacZ, luciferase 0.01, 0.1, 1, 10 and 50 5, 10, 25, 50 and 100 [21]
VHSV-G and -N 5, 10 and 50 25 [22]
VHSV-G and -N 0.1, 1 and 10 25 [33]
lacZ 35 50 [27]
lacZ 1, 5, 25, 50 and 125 50 [28,29]
lacZ, luciferase 5, 10, 20 and 40 5 and 6 [30]
IM Flank VHSV-G, 30 100 [26]
multipoint IHNV-G and
injection rabies virus G
IP Peritoneum luciferase 25 100 [32]
injection
lacZ 50 50 [28]
Gill Gills luciferase 25 100 [32]
injection
ID Ventral c lacZ 50 50 [28]
multipoint
injection
Particle Flank Luciferase 0.3, 2, 5, 11 and 25 N.A.e [32]
bombardment
Eye Luciferase 11 N.A. [32]
Flank (midsection) Luciferase 11 N.A. [32]
Flank (close to tail)
Luciferase 11 N.A. [32]
a
Abbreviations: IM 5 intramuscular, IP 5 intraperitoneum, ID 5 intradermal.
b
All injection sites located below or rostroventral to the dorsal fin and above the lateral line are included in this category. The exact
location of the injection varies between experiments.
c
Midline between the pectoral and anal fins.
d
Genes: CAT 5 chloramphenicol acetyltransferase; IHNV-G and -N 5 infectious hematopoietic necrosis virus G and N genes,
respectively; VHSV-G and -N 5 viral haemorrhagic septicaemia virus G and N genes, respectively.
e
Not applicable.

ing cells, could contribute to improved immune
responses. In small fish, expression of the luciferase
reporter gene injected IM was detected in different
organs, including gills, spleen and kidney, but was
highest in muscles [21,25]. Differences between
large and small fish are most likely due to more rapid
spread of injected DNA from the injected muscles in
small fish, but there may also have been variations in
the protocol, sensitivity of the assay and animal
model used. Although promoters can also influence
the location of foreign gene expression, the same
CMV promoter was used in these studies.
3.1.4. Duration of expression and effect on fish
tissues
IM injection induces strong and long-lasting ex-
pression levels of reporter genes, although different
times of peak activity and total duration of expres-
sion have been reported (Table 3). With the lucifer-
ase gene, Anderson and coworkers [25] observed a
diminution of enzyme activity after 7 days, but this
was not confirmed by others [21,32]. In mice,
luciferase transfected cells are not destroyed by CTL
because no (or very low) immune response is raised
against the luciferase protein due to its poor im-
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
Table 3
Duration and peak activity of reporter genes injected into fish muscle.
Reporter gene Fish species
Luciferase Rainbow trout (Oncorhynchus
mykiss)
Zebra fish (Brachydanio rerio)
Xiphophorus sp.
lacZ Goldfish (Carassius auratus)
Chloramphenicol Tilapia (Oreochromis niloticus)
acetyl transferase
(CAT)
munogenicity [46] and, expression has been detected
for nearly 2 years [47]. In any event, it appears that
expression of an antigenic protein will eventually be
curtailed by cellular immune responses against the
antigen-expressing cells. In mice, most muscle cells
expressing immunogenic proteins are destroyed be-
tween day 10 and 20 following gene delivery [46]. In
fish, this elimination process also seems to occur, but
more slowly. The number of b-galactosidase ex-
pressing cells in fish muscle was found to decrease
slowly between day 21 and 70 after injection of the
lacZ gene alone [29], but coinjection with the mouse
GM–CSF gene was shown to have a marked effect
on the rate of disappearance of transfected muscle
fibers [35]. With the G gene of VHSV, there is
indirect evidence that the number of cells expressing
the viral protein at day 84 is reduced by 90%
compared to the maximum number that is found
between 2.5 and 14 days [21].
Histological studies with the lacZ reporter gene
showed that injection of pure DNA solution into
rainbow trout muscle does not cause permanent
tissue damage. The presence of inflammatory cells in
the needle track has been observed early after
injection [32], but there were no signs of necrosis,
and tissue appeared completely regenerated without
scarring by 28 days following injection [21]. In
addition, other negative side effects such as visceral
adhesion and grown retardation, observed with oil-
adjuvanted antigen vaccines [48], have never been
reported with DNA administration.
35
Peak activity Duration References
(days) (days)
7 . 115 [25]
60 . 60 [32]
2.5 . 84 [21]
2.5 . 112 [21]
6 . 10 [30]
21 . 70 [28]
2 ,7 [31]
3.1.5. Alternatives to needle injection
As an alternative to direct injection with a needle,
gene delivery by particle bombardment has been
assessed in fish [32], however, results were not as
good as with direct needle injection, both for expres-
sion level and proportion of animals expressing the
reporter gene. Even if the efficiency of the technique
is improved, it is unlikely that this technology will
be widely employed on fish farms because of its
higher cost and practical limitations (e.g., the DNA
falls off the gold particles in a humid environment).
3.2. Oral and immersion vaccination
For fish farmers, oral vaccines are the ideal
method of immunization because they are easy and
inexpensive to administer, and they are not stressful
for the animals. There are also immunological ad-
vantages associated with oral delivery, such as the
induction of mucosal immunity. Very little infor-
mation is available on mucosal immunity in fish, but
it has been shown to be stimulated by antigens [49].
Oral delivery also has some practical and immuno-
logical drawbacks. First, it is not possible to de-
termine the dose of vaccine ingested by each animal,
although this is not that important as long as good
herd immunity is induced. More importantly, at least
with antigen vaccines, oral delivery appears to be
less effective than injection or immersion for vac-
cinating fish [43].
Immersion vaccination, where animals are placed
36 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
in or sprayed with a concentrated vaccine solution, is
more widely used than oral administration for an-
tigen vaccine delivery, and normally provides better
protection, but still not as good as injection. Com-
pared to injection however, immersion vaccination
requires less manipulation of the fish and can be used
even for very small fish, but it does require more
vaccine solution, especially for large fish.
The greater effectiveness of immersion over oral
delivery is thought to be due to the better absorption
of antigen through the skin and / or gills compared to
the gut, as well as degradation of the vaccine in the
digestive tract [43]. Recent quantitative experiments
have shown that both skin and gills participate in
uptake of vaccine with immersion, with the skin
playing the more dominant role [50,51]. However,
this could depend on the vaccine since other studies
have reported that oral ingestion is the principal
route of antigen entry in bath-immunized fish [52].
At the time of writing this review, there were no
reports of DNA vaccines administered to fish by oral
or immersion routes. Using reporter genes, we have
never observed detectable levels of gene expression
following immersion or oral delivery of purified
plasmid DNA to small fish. However, association of
DNA with transfection agents such as liposomes or
microcarriers can result in transfection of immersed
fish [Heppell et al., unpublished results]. This sug-
gests that immersion and oral vaccination of fish
with formulated DNA vaccines might be feasible and
effective.
4. Immune responses to DNA vaccines
The first line of defense against infection for fish,
as for other animals, is the nonspecific immune
system. This includes physical barriers such as
mucosal surfaces and skin, but also a variety of
leukocytes (e.g., monocytes / macrophages, granulo-
cytes and nonspecific cytotoxic cells) [53] and
substances (e.g., lysozyme, complement, interferon,
C-reactive protein, transferrin and lectin) that non-
specifically inhibit the growth of infectious micro-
organisms [54]. The innate immune system is of
major importance in fish.
The second line of defense is the specific or
acquired immunity. This can be divided in two arms,
cellular and humoral, that rely heavily on T- and
B-lymphocytes respectively. As for mammals, spe-
cific humoral immunity in fish involves recognition
and binding of circulating soluble antigens by B-
cells, which then respond by producing and secreting
antigen-specific antibodies. On the other hand, spe-
cific cell-mediated immunity involves T-cells’ recog-
nition of antigens presented on cell surfaces in
association with molecules of the major histocom-
patibility complex (MHC), which triggers induction
of cytotoxic T lymphocytes (CTL) and T-cell secret-
ing cytokines. In mammals, CD4 and CD8 T-cells
recognize antigen presented by MHC II and MHC I
molecules respectively, but it is not clear if this is
also true in fish as there are no reagents to identify
CD4- and CD8-like molecules [55].
DNA vaccines are known to trigger both non-
specific (via the CpG-S motifs) and specific immune
mechanisms (via the expression of antigen). In the
latter case, they efficiently stimulate both humoral
and cellular immune responses. As such, they behave
more like a live vaccine, except without the risk of
infection, and perform better than inactivated vac-
cines or purified macromolecules, which induce
mainly humoral responses.
4.1. Antibody responses
DNA vaccines allow expression of antigens in
situ. Therefore, foreign proteins, especially those of
viral origin, can be presented to the immune system
in their proper conformation with post-translational
modifications and intracellular trafficking identical to
those of antigens produced during natural infection.
Since many B-cell epitopes are conformational,
presentation of antigenically relevant epitopes to the
immune system may be more readily attainable with
DNA vaccines than with other type of vaccines, such
as inactivated or subunit vaccines, where conforma-
tional epitopes might be lost [24].
In mammals, the strength and longevity of anti-
body responses induced by DNA vaccination depend
on the animal and antigen model. Humoral responses
are usually long-lived in mice [56], but appear to be
shorter in non-human primates [24]. A single dose of
antigen-encoding DNA is sufficient to induce a
protective immune response in some cases, but
depending on the antigen, the route of administration
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
and the animal species, boosts might be required
[23]. The antibody isotype induced is generally IgG,
although serum IgM and IgA have also been reported
[57,58].
Few studies have examined the antibody response
in fish following DNA administration. Comparison
between published experiments is difficult because
species and conditions used were different, but they
all showed that DNA vaccines induce antigen-spe-
cific immunoglobulins in fish. Antibodies against
b-galactosidase have been detected in goldfish as
early as 7 days after injection of lacZ encoding DNA
[29]. In rainbow trout, antibodies to the G protein of
viral haemorrhagic septicaemia virus (VHSV) were
detected 23 days after injection with a plasmid
encoding the G gene [26]. The concentration of
serum antibodies peaked at 3 to 8 weeks and
remained high for several weeks after [20,29],
although the number of antibody-forming cells ap-
peared to decline rapidly [29]. In the first report of a
fish DNA vaccine, seroconversion was detected in
only a small percentage of immunized rainbow trout
(2 / 15) [20], but later studies demonstrated that it is
possible for all vaccinated fish to have significant
levels of antibody [26–28]. In fact, Russell et al. [27]
showed that after IM injection of a DNA vector
coding for b-galactosidase, goldfish seroconvert even
more readily than mice. This may relate to the less
developed connective tissue compartmentalization of
muscles in fish compared to higher vertebrates,
which could make piscine muscle fibers more access-
ible to direct DNA transfer and increasing the
possibility for DNA to spread to other tissues
[21,27]. As in mammals, the antibody response in
fish is dose-dependent, and seroconversion is incom-
plete when low doses of DNA are used [28].
Boosting on the third week following priming,
does not seem to produce a stronger or earlier
humoral immune response [20]. As well, Lorenzen
and coworkers [22] have shown that the strength of
the neutralizing activity in sera collected from rain-
bow trout immunized with plasmid DNA encoding
the G gene of VHSV was not significantly different
before and after challenge with live virus. These
results suggest that near maximal humoral responses
are induced by a single dose of DNA vaccine.
However, the effect of boost on the duration of
protection has not been evaluated.
37
Only tetrameric forms of antibodies (IgM-like)
have been originally isolated from fish serum, but it
is now well established that fish have different
antibody isotypes, although the functional relevance
of this structural diversity is not quite clear, and
considerable variations exist between piscine classes
[59]. The type of immunoglobulin associated with
IM injection of DNA in fish has not been de-
termined. However, the avidity of the antibodies
appears to be similar with DNA- and protein-im-
munization [28].
4.2. Cellular immune responses
The in vivo synthesis of antigen with DNA-based
vaccination is a desirable feature because it mimics a
natural infection by intracellular pathogens with
respect to MHC class I antigen presentation. This is
associated with strong cell-mediated responses in-
cluding CTL. Such Th1-biased responses may be
reinforced by the presence of CpG-S motifs in the
plasmid backbone [60]. MHC class I-restricted,
CD8 1 CTL have been demonstrated in lymph nodes
or spleen of mice injected IM with plasmid DNA
[24]. Generation of memory T lymphocyte responses
has also been demonstrated with a variety of plasmid
constructs [24]
Cellular immune responses in fish are more dif-
ficult to assess owing to the relatively poor under-
standing of the piscine immune system and the
general unavailability of inbred strains of animals
and appropriate reagents. Nevertheless, there is
indirect evidence for activation of this arm of the
immune system in fish. A lymphocyte proliferation
assay performed on isolated kidney leukocytes re-
covered from fish at different time points following
DNA immunization, showed that antigen-specific
restimulation had a kinetic very similar to that of the
antibody production [29]. Indirect evidence of cel-
lular immune responses has also been reported. For
example, results showing activation of Mx gene
expression in muscle after IM administration of
antigen-coding but not control plasmid [26], suggest
the production of interferon in association with an
antigen-specific response. Mx genes encode inter-
feron-inducible proteins that confer nonspecific
protection against viral infection in mammals [61].
38 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
They have also been described in fish, although their
antiviral function has not yet been established [62].
Furthermore, upregulation of MHC class II ex-
pression by DNA vaccines has also been shown in
fish, at the site of injection [26]. Expression of these
genes is indicative of the recruitment of activated
immunocompetent cells, such as B-cells, macro-
phages and activated T-cells, that are associated with
MHC II expression [55]. Such cells could display
antigenic peptides and provide help to both B and T
lymphocytes.
Detection of specific CTL in vaccinated fish has
not yet been possible, but there is some indirect
evidence indicating that DNA immunization also
induces this type of immune response. Fish muscle
co-injected with plasmids encoding luciferase (a non-
antigenic reporter gene) and VHSV-G protein had
greatly decreased luciferase activity by day 28 after
DNA injection, compared to control muscles co-
injected with luciferase-encoding DNA and a non-
expressing control plasmid [21]. This apparent loss
of antigen-expressing muscle cells is immune me-
diated since it occurs in normal but not immuno-
incompetent (SCID) mice [46]. In fish, these results
suggest the induction of a VHSV-G specific CTL
response [21]. Moreover, a DNA vaccine encoding
the nucleocapsid N gene of VHSV was also shown to
provide protection against live virus in the absence
of neutralizing activity in sera from vaccinated fish
[22], suggesting that cellular immune responses
might be responsible, at least in part, for the protec-
tion.
4.3. Protection against live challenge
Although immune responses to DNA vaccines in
fish are yet poorly characterized, it is known that
they can offer very good protection in small scale
challenge trials. Rainbow trout vaccinated with DNA
expressing the G protein of infectious hematopoietic
necrosis virus (IHNV) had a relative percent survival
(RPS) of 75 with live challenge [20]. Similarly, trout
DNA-vaccinated against VHSV had RPS values up
to 97 and 78, depending on the vaccine dose, after
homologous and heterologous challenge respectively
[21,22,33].
While it is likely that both humoral and cell-
mediated immune responses are induced, the impor-
tance of antibodies alone for the IHNV and VHSV
models is evident. In fact, passive immunization of
small rainbow trout (700 degree-day old) with serum
from adult fish previously immunized with DNA
encoding the G gene of IHNV or VHSV provided
100% protection against challenge of the host fish
with the respective virus [26].
5. Safety of DNA vaccines for fish
Concerns about the safety of DNA vaccines are
very different for food source animals and humans
[63] and are also somewhat different for fish and
other farmed animals. For example, in food source
animals there is less concern about integration of
DNA into the genome (except in germ cells) and the
possibility to induce autoimmune diseases. In mam-
mals, DNA vaccines can induce production of anti-
DNA antibodies, but these are mostly against single-
stranded DNA and are non-pathogenic. Indeed,
autoimmune reactions against the host’s DNA have
never been observed, even in lupus-prone strains of
animals [64–66].
Integration of plasmid DNA into the host genome
is very unlikely, and the risk of an insertional
mutagenic event, which is even less, has been
estimated to be 1000 times lower than the rate of
spontaneous mutation [67]. Although the potential
for integration remains a concern for humans [68], it
is less worrisome for fish and other farmed animals
with short life spans. Exceptions would be stable
integration into germ cells, especially in fish that will
be used for reproduction or wild-stock enhancement.
Integration of plasmid DNA in the host genome has
not been demonstrated in fish yet [25,29], and the
presence of the transfected gene product or DNA
itself in gonads or eggs from injected fish has not
been assessed.
5.1. Fate of injected DNA in fish
The persistence of DNA after IM injection in fish
differs somewhat from that in mammals. Unlike
findings with mice, where degradation of the plasmid
DNA starts only a few hours after injection [69],
DNA administered to fish is more stable, with
relatively little degradation being detected by South-
ern blotting. Plasmid could be detected in injected
muscle even at the latest time point tested, that is 63
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
days after injection [25]. With the polymerase chain
reaction (PCR), plasmid DNA was also detected in
injected muscle at all time point tested (the latest
being 70 days after injection), but not in other tissue
extracts [26,29]. The persistence of plasmid DNA is
associated with long term expression of the encoded
antigen [29].
5.2. Safety for the consumer
The only potential risk to the consumer is that
associated with ingestion of plasmid DNA remaining
in the injected flesh. In mice, it has been estimated
that 1 to 2% of orally ingested plasmid DNA survive
the gastrointestinal tract and persist as short frag-
ments in the nuclei of intestinal epithelia and Peyer’s
patches cells, in the peripheral white blood cells and
in the cells of the spleen and liver [70,71]. In spleen
cells, plasmid fragments have been found in rare
instances to be covalently linked to DNA with a high
degree of homology to mouse genes [71]. Moreover,
DNA orally administered to pregnant mice (50 mg of
plasmid daily for 1–2 weeks) was found to cross the
placenta and was subsequently detected in various
organs of fetal and newborn animals, but the associa-
tion with abnormalities has not been investigated
[72]. It must be realized, however, that the amounts
of DNA given to these mice greatly exceed what
might be ingested by eating a vaccinated fish. Most
(if not all) of the plasmid DNA injected into the fish
would have been degraded by the time the flesh is
eaten, some months or years later. Even if the fish
were eaten immediately after injection, the amount
of plasmid DNA consumed would be extremely
minute compared to the total amount of nucleic acid
from all sources, including bacteria and viruses,
normally ingested daily. Moreover, licensed veteri-
nary vaccines made of inactivated or attenuated
pathogens already contain large amount of nucleic
acid, mostly from the genome of the pathogen, as a
normal component. From this point of view, DNA
vaccines should be no more dangerous than currently
used vaccines.
5.3. Risks for other fish and the environment
Most of the injected plasmids that fail to enter the
nucleus of cells are rapidly degraded by nucleases in
the extracellular space and in the cytoplasm. Further-
39
more, DNA that survives in transfected cells will be
degraded when these cells are destroyed by antigen-
specific CTLs. Nevertheless, a small proportion of
DNA can survive for prolonged periods in injected
muscle tissue of fish. The prolonged expression of
foreign genes was initially thought to be a risk for
tolerization to the gene product. This would affect
not only the immunized fish, but also other fish,
since tolerant animals could become carriers and
continuously shed infectious agents into the environ-
ment. However, tolerization has never been demon-
strated in DNA-immunized animals for any of the
many animal models of DNA vaccines developed to
date [23]. Moreover, any amount of antigen secreted
into an aqueous environment by putative tolerized
animals would be extremely small and immediately
diluted.
The potential risks for the environment, when
vaccinated fish are released or escape from farms,
are extremely minute. DNA injected fish are not
transgenic animals, and it would be very unlikely
that they could pass on the plasmid, or part of it, to
other organisms or to the next generation of fish.
Therefore, the risk of integration in other organisms
is extremely small and even if did occur, prob-
abilities for a mutagenic event to happen would be
even lower, with the risk of an integrative event in a
stem cell being infinitesimally small, as discussed
previously. Again, risks are unlikely to be greater
than with current widely used vaccines.
6. Perspectives for DNA vaccines in the
aquaculture industry
Results obtained with the first experiments on
DNA vaccines for fish are very encouraging, but
several issues still have to be addressed before it will
be feasible to have large-scale application of this
technology on fish farms. Administration by injec-
tion has been shown to be very efficient, and will
likely be the first method developed for commercial
DNA immunization of fish. However, alternative
routes to deliver nucleic acid vaccines are required
for very small fish. Oral or immersion vaccination
would be more appropriate in this case. These
methods are also desirable for fish of lower value,
such as channel catfish, for which the cost of
40 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
carrying out the injection procedure would be
prohibitive.
Vaccines are normally not administered before the
fish are immunocompetent, although DNA vaccines
have performed well in neonates of other species
[73–76]. This is possibly due to the fact that antigen
expression can continue until the immune system is
mature enough to respond to it [23]. Protection in
fish only needs to last long enough for animals to
reach market size or for some diseases, a size where
the pathogen is no longer a risk. Long-term protec-
tion by DNA vaccines still has to be demonstrated in
fish, and this will be most important for diseases
affecting adult animals.
There are also certain requirements to be met prior
to the commercial application of DNA vaccines. The
most important is the need to meet specific regula-
tions from government agencies. It is also desirable
to gain public acceptation of the technology, espe-
cially by fish farmers, who will be the actual
customers for the vaccine, and by the consumers
who will eat the fish.
While DNA vaccines are generally inexpensive to
manufacture, it may be possible to further reduce the
cost of vaccine delivery to fish. For example, plas-
mids could be added to existing multivalent protein
vaccines. It has already been shown that DNA
administered IP with an oil adjuvant, similarly to
most current fish vaccines, induced nearly as high
antibody production as pure plasmid injected IM
[28]. Mixing protein and DNA vaccines has been
found to be effective in mammals [H.J. Baker and
B.F. Smith, personal communication]. Different plas-
mids may also be coinjected without losing immuno-
genicity of either components [26], although colinear
expression with more than one gene on a single
plasmid is more cost effective from a manufacturing
point of view. Finally, improvement of the vector
backbone to express greater amounts of the foreign
protein or to have stronger immune responses against
the antigen may also allow lower doses of DNA to
be used and thus further reduce the cost.
7. Conclusions
The development of DNA vaccines for fish is still
in an early stage. Nevertheless, experiments with
model antigens give very promising results, with
induction of both humoral and cellular immune
responses. Moreover, their efficacy in challenge with
live pathogens has been demonstrated with two
important viral diseases for which no commercial
vaccines are available.
The increasing need for effective fish vaccines in
the rapidly growing aquaculture industry is a driving
force for the development of new products. DNA
vaccines could circumvent many of the limitations
associated with traditional methods of vaccination,
but in the end, their usefulness will depend upon a
favorable balance of efficacy, safety and cost [77].
The first two factors should not be a problem for
DNA vaccines, but the cost efficiency still has to be
demonstrated for low-valued species of fish.
References
[1] F.P. Meyer, Aquaculture disease and health management, J.
Anim. Sci. 69 (1991) 4201–4208.
[2] D.T. Hanfman, The Status and Potential of Aquaculture in
the United States: an Overview and Bibliography, U.S.
Department of Agriculture, National Agricultural Library,
1993.
[3] P.M. Aasjord, E. Slinde, Fish vaccine: development, pro-
duction and use of bacterial vaccines, with special reference
to salmon, in: A.M. Martin (Ed.), Fisheries Processing:
Biotechnological Applications, Chapman & Hall, London,
1994, pp. 432–465.
[4] E. Mialhe, E. Bachere, V. Boulo, J.P. Cadoret, C. Rousseau, V.
CedeZo, E. Saraiva, L. Carrera, J. Calderon, R.R. Colwell,
Future of biotechnology-based control of disease in marine
invertebrates, Mol. Mar. Biol. Biotechnol. 4 (1995) 275–
283.
[5] J.C. Leong, J.L. Fryer, Viral vaccines for aquaculture, Annual
Rev. Fish Dis. 3 (1993) 225–240.
[6] C.B. Munn, The use of recombinant DNA technology in the
development of fish vaccines, Fish Shellfish Immunol. 4
(1994) 459–473.
[7] C.McL. Press, A. Lillehaug, Vaccination in European sal-
monid aquaculture: a review of practices and prospects, Br.
Vet. J. 151 (1995) 45–69.
[8] S.G. Newman, Bacterial vaccines for fish, Ann. Rev. Fish
Dis. 3 (1993) 145–185.
[9] R.A. Schnick, D.J. Alderman, R. Armstrong, R. Le Gouvello,
S. Ishihara, E.C. Lacierda, S. Percival, M. Roth, World wide
aquaculture drug and vaccine registration progress, Bull. Eur.
Ass. Fish Pathol. 17 (1997) 251–260.
[10] P.J. Midtlyng, Novel vaccines & new vaccination strategies
for fish, Bull. Eur. Fish Pathol. 17 (1997) 239–244.
[11] J.C. Leong, E. Anderson, L.M. Bootland, P.-W. Chiou, M.
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
Johnson, C. Kim, D. Mourich, G. Trobridge, Fish vaccine
antigens produced or delivered by recombinant DNA tech-
nologies, Dev. Biol. Stand. 90 (1997) 267–277.
[12] J.R. Winton, Molecular approaches to fish vaccines, J. Appl.
Ichthyol. 14 (1998) 153–158.
[13] G.J. Cox, T.J. Zamb, L.A. Babiuk, Bovine herpesvirus 1:
immune responses in mice and cattle injected with plasmid
DNA, J. Virol. 67 (1993) 5664–5667.
[14] H.L. Davis, M.-L. Michel, R.G. Whalen, DNA-based im-
munization induces continuous secretion of hepatitis B
surface antigen and high levels of circulating antibody, Hum.
Mol. Genet. 2 (1993) 1847–1851.
[15] J.B. Ulmer, J.J. Donnelly, S.E. Parker, G.H. Rhodes, P.L.
Felgner, V.J. Dwarki, S.H. Gromkowski, R.R. Deck, C.M.
DeWitt, A. Friedman, L.A. Hawe, K.R. Leander, D. Mar-
tinez, H.C. Perry, J.W. Shiver, D.L. Montgomery, M. Liu,
Heterologous protection against influenza by injection of
DNA encoding a viral protein, Science 256 (1993) 1745–
1749.
[16] B. Wang, K.E. Ugen, V. Srikantin, M.G. Agadjanyan, K.
Dang, Y. Fefaeli, A.I. Sato, J. Boyer, W.V. Williams, D.B.
Weiner, Gene inoculation generates immune responses
against human immunodeficiency virus type I, Proc. Natl.
Acad. Sci. U.S.A. 90 (1993) 4156–4160.
[17] M. Sedegah, R. Hedstrom, P. Hobart, S.L. Hoffman, Protec-
tion against malaria by immunization with plasmid DNA
encoding circumsporozoite protein, Proc. Natl. Acad. Sci.
U.S.A. 91 (1994) 9866–9870.
[18] Z.Q. Xiang, S. Spitalnik, M. Tran, W.H. Wunner, J. Cheng,
H.C. Ertl, Vaccination with a plasmid vector carrying the
rabies virus glycoprotein gene induces protective immunity
against rabies virus, Virology 199 (1994) 132–140.
[19] E. Hansen, K. Fernandes, G. Goldspink, P. Butterworth, P.K.
Umeda, K.-C. Chang, Strong expression of foreign genes
following direct injection into fish muscle, FEBS Lett. 290
(1991) 73–76.
[20] E.D. Anderson, D.V. Mourich, S.C. Fahrenkrug, S. LaPatra, J.
Shepherd, J.C. Leong, Genetic immunization of rainbow
trout (Oncorhynchus mykiss) against infectious hematopoietic
necrosis virus, Mol. Mar. Biol. Biotechnol. 5 (1996) 114–
122.
[21] J. Heppell, N. Lorenzen, N.K. Armstrong, T. Wu, E. Loren-
zen, K. Einer-Jensen, J. Schorr, H.L. Davis, Development of
DNA vaccines for fish: vector design, intramuscular injection
and antigen expression using viral haemorrhagic septicaemia
virus genes as model, Fish Shellfish Immunol. 8 (1998)
271–286.
[22] N. Lorenzen, E. Lorenzen, K. Einer-Jensen, J. Heppell, T.
Wu, H.L. Davis, Protective immunity to VHS in rainbow
trout (Oncorhynchus mykiss, Walbaum) following DNA
vaccination. Fish Shellfish Immunol. 8 (1998) 261–270.
[23] H.L. Davis, M.J. McCluskie, DNA vaccines for viral dis-
eases, Microbes and Infections 1 (1999) 7–21.
[24] J.J. Donnelly, J.B. Ulmer, J.W. Shiver, M.A. Liu, DNA
vaccines, Ann. Rev. Immunol. 15 (1997) 617–648.
[25] E.D. Anderson, D.V. Mourich, J.C. Leong, Gene expression
in rainbow trout (Oncorhynchus mykiss) following in-
41
tramuscular injection of DNA, Mol. Mar. Biol. Biotechnol. 5
(1996) 105–113.
[26] P. Boudinot, M. Blanco, P. de Kinkelin, A. Benmansour,
Combined DNA immunization with the glycoprotein gene of
viral hemorrhagic septicemia virus and infectious hemato-
poietic necrosis virus induces double-specific protective
immunity and nonspecific response in rainbow trout, Virolo-
gy 249 (1998) 297–306.
[27] P.H. Russell, T. Kanellos, I.D. Sylvester, K.C. Chang, C.R.
Howard, Nucleic acid immunisation with a reporter gene
results in antibody production in goldfish (Carassius auratus
L.), Fish Shellfish Immunol. 8 (1988) 121–128.
[28] T. Kanellos, I.D. Sylvester, C.R. Howard, P.H. Russell, DNA
is as effective as protein at inducing antibody in fish, Vaccine
17 (1999) 965–972.
[29] T. Kanellos, I.D. Sylvester, A.G. Ambali, C.R. Howard, P.H.
Russell, The safety and longevity of DNA vaccines for fish,
Immunology 96 (1999) 307–313.
[30] P.M. Schulte, D.A. Powers, M. Schartl, Efficient gene
transfer into Xiphophorus muscle and melanoma by injection
of supercoiled plasmid DNA, Mol. Mar. Biol. Biotechnol. 7
(1998) 241–247.
[31] A. Rahman, N. Maclean, Fish transgene expression by direct
injection into fish muscle, Mol. Mar. Biol. Biotechnol. 1
(1992) 286–289.
[32] ´
M. Gomez-Chiarri, S.K. Livingston, C. Muro-Cacho, S.
Sanders, R.P. Levine, Introduction of foreign genes into the
tissue of live fish by direct injection and particle bombard-
ment, Dis. Aquat. Org. 27 (1996) 5–12.
[33] N. Lorenzen, E. Lorenzen, K. Einer-Jensen, J. Heppell, H.L.
Davis, Genetic vaccination of rainbow trout against VHS
virus: Small amounts of DNA protect against a heterologous
serotype, Virus Res. (1999) in press.
[34] B.M. Bearzotti, E. Perrot, C. Michard-Vanhee, G. Jolivet, J.
Attal, M.-C. Theron, C. Puissant, M. Dreano, J.J. Kopchick,
R. Powell, F. Gannon, L.-M. Houdebine, D. Chourrout, Gene
expression following transfection of fish cells, J. Biotechnol.
26 (1992) 315–325.
[35] T.S. Kanellos, I.D. Sylvester, V.L. Butler, A.G. Ambali, C.D.
Partidos, A.S. Hamblin, P.H. Russell, Mammalian granulo-
cyte–macrophage colony-stimulating factor and some CpG
motifs have an effect on the immunogenicity of DNA and
subunit vaccines in fish, Immunology 96 (1999) 507–510.
[36] J.J. Donnelly, D. Martinez, K.U. Jansen, R.W. Ellis, D.L.
Montgomery, M.A. Liu, Protection against papillomavirus
with a polynucleotide vaccine, J. Infect. Dis. 173 (1996)
314–320.
[37] M.-L. Michel, H.L. Davis, M. Schleef, M. Mancini, P.
Tiollais, R.G. Whalen, DNA-mediated immunization to the
hepatitis B surface antigen in mice: aspects of the humoral
response mimic hepatitis B viral infection in humans, Proc.
Natl. Acad. Sci. U.S.A. 92 (1995) 5307–5311.
[38] D.M. Klinman, G. Yamshchikov, Y. Ishigatsubo, Contribution
of CpG motifs to the immunogenicity of DNA vaccines, J.
Immunol. 158 (1997) 3635–3639.
[39] A.M. Krieg, A.-K. Yi, S.J. Chorr, H.L. Davis, The role of
CpG dinucleotides in DNA vaccines, Trends Microbiol. 6
(1998) 23–26.
42 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43
[40] A.M. Krieg, T. Wu, R. Weeratna, S.M. Efler, L. Love-
Homan, L. Yang, A.-E. Yi, D. Short, H.L. Davis, Sequence
motifs in adenoviral DNA block immune activation by
stimulatory CpG motifs, Proc. Natl. Acad. Sci. U.S.A. 95
(1998) 12631–12636.
[41] A.M. Krieg, CpG DNA: a novel immunomodulator, Trends
Micobiol. 7 (1999) 64–65.
[42] J. Heppell, J. Sanchez-Dardon, A.M. Krieg, H.L. Davis,
Stimulation of rainbow trout immune cells with oligodeoxy-
nucleotides bearing CpG motifs, in: Proc. 3rd Int. Symp.
Aquatic Ani. Health, 1998, p. 101.
[43] A.E. Ellis, Recent development in oral vaccine delivery
systems, Fish Pathol. 30 (1995) 293–300.
[44] A.E. Ellis, in: A.E. Ellis (Ed.), Fish Vaccination, Academic
Press, New York, 1988, p. 255.
[45] A.D. Cohen, J.D. Boyer, D.B. Weiner, Modulating the
immune response to genetic immunization, Faseb J. 12
(1998) 1611–1626.
[46] H.L. Davis, C.L. Brazolot Millan, S.C. Watkins, Immune-
mediated destruction of transfected muscle fibers after direct
gene transfer with antigen-expressing plasmid DNA, Gene
Ther. 4 (1997) 181–188.
[47] J.A. Wolff, J.J. Ludtke, G. Acsadi, P. Williams, A. Jani,
Long-term persistence of plasmid DNA and foreign gene
expression in mouse muscle, Hum. Mol. Genet. 1 (1992)
363–369.
[48] T.T. Poppe, O. Breck, Pathology of Atlantic salmon Salmo
salar intraperitoneally immunized with oil-adjuvanted vac-
cine, A case report. Dis. Aquat. Org. 29 (1997) 219–226.
[49] A.G. Zapata, A. Chiba, ´ A. Varas, Cells and tissues of the
immune system of fish, in: G. Iwama, T. Nakanishi (Eds.),
The Fish Immune System: Organism, Pathogen, and En-
vironment, Academic Press, San Diego, 1996, pp. 1–62.
[50] M. Ototake, G.K. Iwama, T. Nakanishi, The uptake of
bovine serum albumin by the skin of bath-immunised
rainbow trout Oncorhynchus mykiss, Fish Shellfish Immunol.
6 (1996) 321–333.
[51] J.D. Moore, M. Ototake, T. Nakanishi, Particulate antigen
uptake during immersion immunisation of fish: the effective-
ness of prolonged exposure and the roles of skin and gill,
Fish Shellfish Immunol. 8 (1998) 393–407.
[52] R.A. Robohm, R.A. Koch, Evidence for oral ingestion as the
principal route of antigen entry in bath-immunized fish, Fish
Shellfish Immunol. 5 (1995) 137–150.
[53] C.J. Secombes, The nonspecific immune system: cellular
defenses, in: G. Iwama, T. Nakanishi (Eds.), The Fish
Immune System: Organism, Pathogen, and Environment,
Academic Press, San Diego, 1996, pp. 63–104.
[54] T. Yano, The nonspecific immune system: humoral defense,
in: G. Iwama, T. Nakanishi (Eds.), The Fish Immune
System: Organism, Pathogen, and Environment, Academic
Press, San Diego, 1996, pp. 105–158.
[55] M.J. Manning, T. Nakanishi, The specific immune system:
cellular defenses, in: G. Iwama, T. Nakanishi (Eds.), The
Fish Immune System: Organism, Pathogen, and Environ-
ment, Academic Press, San Diego, 1996, pp. 159–205.
[56] H.L. Davis, M. Mancini, M.-L. Michel, R.G. Whalens, DNA-
mediated immunization to hepatitis B surface antigen:
longevity of primary response and effect of boost, Vaccine 14
(1996) 910–915.
[57] D.M. Justewicz, M.J. Morin, H.L. Robinson, R.G. Webster,
Antibody-forming cell response to virus challenge in mice
immunized with DNA encoding the influenza virus hemag-
glutinin, J. Virol. 69 (1995) 7712–7717.
[58] R.R. Deck, C.M. DeWitt, J.J. Donnelly, M.A. Liu, J.B.
Ulmer, Characterization of humoral immune responses in-
duced by an influenza hemagglutinin DNA vaccine, Vaccine
15 (1997) 71–78.
[59] S.L. Kaattari, J.D. Piganelli, The specific immune system:
humoral defense, in: G. Iwama, T. Nakanishi (Eds.), The
Fish Immune System: Organism, Pathogen, and Environ-
ment, Academic Press, San Diego, 1996, pp. 207–254.
[60] D.J. Lee, M. Corr, D.A. Carson, Control of immune re-
sponses by gene immunization, Ann. Med. 30 (1998) 460–
468.
[61] J. Pavlovic, P. Staeheli, The antiviral potentials of Mx
proteins, J. Interferon Res. 11 (1991) 215–219.
[62] J.C. Leong, G.D. Trobridge, C.H.Y. Kim, M. Johnson, B.
Simon, Interferon-inducible Mx proteins in fish, Immunol.
Rev. 166 (1998) 349–363.
[63] K. Yamanouchi, T. Barrett, C. Kai, New approaches to the
development of virus vaccines for veterinary use, Rev. Sci.
Tech. Off. Int. Epiz. 17 (1998) 641–653.
[64] D.S. Pisetsky, Immunologic consequences of nucleic acid
therapy, Antisens Res. Develop. 5 (1995) 219–225.
[65] G. Mor, M. Singla, A.D. Steinberg, S.L. Hoffman, K. Okuda,
D.M. Klinman, Do DNA vaccines induce autoimmune
Disease?, Hum. Gene Ther. 8 (1997) 293–300.
[66] D.S. Pisetsky, DNA and the immune system, Ann. Internal
Med. 126 (1997) 169–171.
[67] W.W. Nichols, B.J. Ledwith, S.V. Manam, P.J. Troilo, Po-
tential DNA vaccine integration into host cell genome, Ann.
N.Y. Acad. Sci. 772 (1995) 30–39.
[68] F.R. Vogel, N. Sarver, Nucleic acid vaccines, Clin. Mi-
crobiol. Rev. 8 (1995) 406–410.
[69] J.A. Wolff, P. Williams, G. Ascadi, S. Jiao, A. Jani, C.
Chong, Conditions affecting direct gene transfer into rodent
muscle in vivo, BioTechniques 4 (1991) 474–485.
[70] R. Schubbert, C. Lettmann, W. Doerfler, Ingested foreign
(phage M13) DNA survives transiently in the gastrointestinal
tract and enters the bloodstream of mice, Mol. Gen. Genet.
242 (1994) 495–504.
[71] R. Schubbert, D. Renz, B. Schmitz, W. Doerfler, Foreign
(M13) DNA ingested by mice reaches peripheral leukocytes,
spleen and liver via the intestinal wall mucosa and can be
covalently linked to mouse DNA, Proc. Natl. Acad. Sci.
U.S.A. 94 (1997) 961–966.
[72] R. Schubbert, U. Hohlweg, D. Renz, W. Doerfler, On the fate
of orally ingested foreign DNA in mice: chromosomal
association and placental transmission to the fetus, Mol. Gen.
Genet. 259 (1998) 569–576.
[73] A. Bot, S. Bot, A. Garcia-Sastre, C. Bona, DNA immuniza-
tion of newborn mice with a plasmid-expressing nucleopro-
tein of influenza virus, Viral Immunol. 9 (1996) 207–210.
 J. Heppell, H.L. Davis / Advanced Drug Delivery Reviews 43 (2000) 29 – 43 43

[74] A. Bot, S. Antohi, S. Bot, A. Garcia-Sastre, C. Bona,
Induction of humoral and cellular immunity against influenza
virus by immunization of newborn mice with a plasmid
bearing a hemagglutinin gene, Intl. Immunol. 9 (1997)
1641–1650.
[75] D.E. Hassett, J. Zhang, J.L. Whitton, Neonatal DNA immuni-
zation with a plasmid encoding an internal viral protein is
effective in the presence of maternal antibodies and protects
against subsequent viral challenge, J. Virol. 71 (1997) 7881–
7888.
[76] A.M. Prince, R. Whalen, B. Brotman, Successful nucleic acid
based immunization of newborn chimpanzees against hepati-
tis B virus, Vaccine 15 (1997) 916–919.
[77] C.W. Beard, P.W. Mason, Out on the farm with DNA
vaccines, Nature Biotechnol. 16 (1998) 1325–1328.