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Am. J. Trop. Med. Hyg.. 35(1), 1986, pp.

Copyright ©1986 by The American Society of Tropical Medicine and Hygiene


*Department of Immunology, University of Stockholm, S-106 91 Stockholm, Sweden,
Yekepa Clinical Research and Training Unit of the Liberian Institute
for Biomedical Research, Liberia, and tDepartment of Infectious Diseases,
Karolinska Institute, Roslagtulls Hospital,
S-11489 Stockholm, Sweden

Abstract. Sera from 48 children and adolescents (2—15years of age), residing in a malaria
holoendemic area of Liberia were investigated for specificities and isotypes of anti-P.
falciparum antibodies. No clear-cut relationship to the development of clinical immunity
was found when the overall antibody activities to total parasite antigens were determined
by enzyme-linkedimmunosorbent assay(ELISA).Although therewas a certainriseof
1gM, total IgG- and IgG2 antibody activities, this was most pronounced at ages when a
clinical but nonsterile immunity is already present. When the sera were investigated by
immunoprecipitation of 35S-methionine labeled parasite polypeptides, the total number of
parasite antigens precipitated was similar at all ages. Analysis by indirect immunofluores
cence (IFA), registering antibodies to intracellular parasite antigens, revealed no age
dependent changes in antibody titers. In contrast, when the sera were assayed by a novel
IFA, specific for a restricted number of parasite antigens in the membrane of infected
erythrocytes, the frequency of positive sera as well as the anti-P. fakiparum titers rose in
parallel with the development of clinical immunity. Thus, theseantigensappearedto be
important inducers of protective immune responses and may be suitable candidates for a
vaccineagainsttheasexualblood stagesof P.fakiparum.

In highly endemic malarious areas, a relative ical immunity (age, spleen size, parasite densities)
clinical immunity is normally acquired by the with the occurrence of antibodies of different iso
age of 5 years and severe malaria becomes less types or P. fakiparum specificities. Sera from 48
frequent. A fully developed but nonsterile im children or adolescents, aged 2—15 years and
munity is generally present by 10 years of age living in a holoendemic area of Liberia, were
and Plasmodium fakiparum malariaisusually assayed for anti -P.fakiparum antibodies by sev
kept at subpatent levels in adults. Serum anti eral different methods. Using a novel immuno
bodies isolated from immune donors living in fluorescence assay,@'7 we found a positive cor
The Gambia have a protective capacity when relation between the development of clinical
injected into children acutely infected with P. immunity and the appearance of antibodies di
fakiparum.―2 Similarly, passive transfer of an rected against P. fakiparum antigens in the
tibody via the placenta has been suggested to membrane of infected erythrocytes.
protect newborns against the disease.3
Dissection by recently developed techniques
of the humoral response in clinically immune MATERIALS AND METHODS
individuals ascompared tothatinnonimmunes
may be expected to provide further insights in
the protective capacities of different anti-P. fal The Tanzanian strain F32 ofPlasmodiumfal
ciparum antibodies. In this study we have at ciparum, isolated in 1978, was used for all the
tempted to correlate different parameters of din tests. The parasites were cultivated in red blood
cells (RBC), blood group 0, at a hematocrit of
Accepted 3 July 1985. 5%.

Study area gamma chains. Specificity of the conjugates was

checked in an ELISA where the plates were coat
Blood specimens were collected near Yekepa ed with purified human myeloma proteins of dif
Town in northern Liberia in a rural area where ferent isotypes.'°
malaria epidemiology has been studied in detail.8 IgG subclasses of the anti-P. fakiparum an
In the 2 villages included in this study, malaria tibodies were determined in an antigen ELISA
is holoendemic and perennial. The sporozoite with subclass-specific monoclonal mouse anti
inoculation rate is 0.2/person-night in the dry bodies as previously described.'0 The mono
season. P. fakiparum is the main malaria par clonals were from the same clones as used pre
asite but P. malariae and P. ovale are also com viously except that the antibodies from clone
monly found. JL5 12, earlier used as IgG1 reagent, were sub
stituted by antibodies from clone NL16 (Seward
Labs, London, England). Human sera were ab
Sample collection sorbed twice with human erythrocyte ghosts and
tested at dilutions 1:200, 1:800 and 1:3,200. Sub
The study included a survey of 40 children,
class specificity of the reactions was ascertained
aged 2—12years, from Lugbeh Village and 8 ad
as before.'°
olescent boys (aged 15) from Bonah Village. No
subject had taken any antimalarial drug for the
last three months. Age and sex were recorded
and spleen size was estimated with the child Indirect immunofluorescence assay (IFA)
standing using Hackett's classification. Blood
For the assay of antibodies to parasite antigens
specimens were collected by finger prick in hep
in the membrane of infected erythrocytes, dif
arinized microhematocrit tubes and centrifuged.
ferent dilutions of test sera were added to mono
The plasmas were initially kept at —20°C,later
layers of glutaraldehyde-fixed and air dried
at —70°C.Thick and thin films were made for
erythrocytes (5%—l0% parasitemia) as described
microscopic observation.
by Perlmann et al.5 Slides were stained with bio
tinylated goat antibodies to human immuno
globulin and FITC-conjugated avidin.
Parasitological examinations
Antibodies to intraerythrocytic parasites were
Blood films were stained with the standard assayed similarly using monolayers of air dried
Giemsa method. Parasite species were identified but unfixed erythrocytes.― Tests were considered
and parasite counts were calculated from the positive when late stage parasites gave a distinct
number observed per 100—400 leukocytes, as and bright immunofluorescence.
suming a constant leukocyte count of 10 x 10@/l.

Ring stage dominated P. fakiparum cultures
A trophozoite-schizont-enriched antigen frac were labeled until late schizont stage with 355W
tion, obtained by centrifugation on a Percoll gra methionine, solubilized and precipitated with 10
dient, was used as antigen as described previ @dundiluted test serum and a polyvalent rabbit
ously.9 For post-coating, plates were incubated anti-human immunoglobulin serum. The precip
5 hr at room temperaturewith 0.25% gelatin itated antigen antibody complexes were analyzed
instead of 2% bovine serum albumin as this fur by sodium dodecyl sulphate-polyacrylamide gel
ther decreased the background. All sera were electrophoresis (SDS-PAGE) under reducing
tested at dilutions 1:1,000, 1:5,000 and 1:25,000. conditions and fluorographed.'2
1gM antibodies were assayed with a goat anti
human 1gM preparation conjugated with alkaline
phosphatase (ALP) (Sigma Chemical Co., St. Statistical analysis
Louis, Missouri). IgG antibodies were assayed
with ALP-conjugated rabbit antibodies specific Spearman's rank correlation test was used. P
for the Fc fragment of human immunoglobulin values >0.05 were considered insignificant.

A low.There
P.fakiparum activity with increasing age (r=
0.66, P = 8.9 x l06). IgG antibodies to P. fal
ciparum were present in all 48 sera. There was
1.5 also an age-dependent increase in antibody ac
o tivity but the statistical significance of this cor
relation was less pronounced (r = 0.38, P = 1.1 x
10_2; Fig. lB).
0 8 C@@O IgG1and IgG3antibodies'°
werepresentin al
0.5 00 o most all sera (46/48 and 47/48, respectively) and
o showed a similar rise with age as the total IgG
antibodies. However, this age correlation was not
5 lo 15 statisticallysignificant(r = 0.27,P = 7.5 x 10-2
YEARS for IgG, and r = 0.28, P = 6.0 x 10-2 for IgG3,
respectively). In contrast, IgG2 antibodies which
>2.0 were detectable in 75% of the samples (29/38;
10 samples excluded because of lack of serum)
rose with age similarly to what was seen for 1gM
(r= 0.69,P = 3.6 x l0@).IgG4 antibodies were
1.5 present in 50% of the samples available for test
0 0 O ing(18/38). Elevated activities were seen in some
1.0 0 0 O children younger than 4 years as well as in some
08 o 0 O 8 adolescents, and there was no increase with age
0 O 0
@ 00 o O (r=0.l3,P=4.6 x 10').
O 0 ° @o8
0.5 In individual donors, there was no correlation
00 o8
0 0 between parasite densities and the 0D405 for total
IgG, IgG,, IgG3 and IgG4 antibodies, respective
5 10 15 ly. However, a weak negative correlation was
found for 1gM and IgG2 (r = 0.243; r = 0.246,
FIGURE 1. ELISA values of sera (diluted 1/1,000; respectively).
ordinate 0D@5) of children and adolescents (abscissa,
age in years) in the P.fakiparum ELISA. A. 1gM reac
tivity; B. IgG reactivity. Immunoprecipitation of355-methionine-labeled
P. falciparum polypeptides
To establish whether or not there was a major
ELISA difference in the antigens recognized by the anti
P. fakiparum antibodiesoccurringat different
Allvaluesareexpressedasthetestvalueminus ages, serum samples were reacted with 35S-me
background (i.e., incubation buffer). The 0D@5 thion.ine-labeled parasite extracts, precipitated
for sera of healthy Swedish blood donors at the with rabbit anti-human immunoglobulin and
dilutions presented varied between 0—0.05(1gM, subjected to SDS-PAGE and autofluorography.
IgG), 0—0.075 (IgG,, IgG3, IgG4) and 0—0.1for The results of a representative experiment are
IgG2. shown in Figure 2. In general, all sera precipi
tated some 20 parasite derived polypeptides of
Antibodyisotypes different apparent molecular weights. The major
antigens seen were of M@195,000 d, 150,000 d,
1gM antibodies9 were present in 42/48 sera 140,000 d, 129,000 d, 118,000 d, 90,000 d,
tested (0D405 > 0.1). Figure 1 shows the age 80,000 d and 50,000 d, respectively. Antibodies
dependent changes of these antibodies at serum to these antigens were present at all ages
dilutions of 1:1,000. Similar results were ob between 2—12years and there was no signif
tained when the sera were tested at dilutions of icant age-dependent difference in the intensity of
1:5,000 or 1:25,000 although the readings ob the signals seen on the fluorographs. Identical
mined at these dilutions were frequently rather banding patterns but slightly stronger signals were



t2: ,@5
12 IC


‘iì!'-@t3 @;;@6
;4@9 -r

V ‘

FIGURE 2. Fluorograph of SDS-PAGE after separation of―-S methionine-labeled P.fakiparum polypeptides

immunoprecipitated with undiluted sera (10 1d)of children of different ages or of 3 healthy Swedish blood donors
(C). Molecular weights of some of the major polypeptides are indicated x l0@.

obtained with the sera from the 8 adolescent boys, the adolescent sera these titers were very high
aged 15 years (not shown). However, as seen (Fig. 3). Furthermore, for the 43 donors inves

from Figure 2, the frequency of sera having a tigated, there was a significant negative correla
generally reduced activity in this test was higher tion between parasitemia and antibody titers in
in the younger age groups than in the 8—12-year this assay (r = 0.43, P = 6.9 x l0@). As seen
old children. from Figure 4, parasite densities > 100/mm3
blood were rarely found in donors whose anti
Antibodies to P. falciparum antigens in the body titers were >1:80 and none or very few
membrane of inftcted erythrocytes parasites were found in donors of high titered
sera. In addition, children with elevated anti
With sera diluted 1:20, this test detects an body titers to the parasite antigens in the eryth
tibodies against parasite antigens in the mem rocyte surface rarely had enlarged spleens. Rath
brane of infected erythrocytes, primarily those er, spleen size appeared to be negatively correlated
containing ring stages and early trophozoites. The to antibody titer. The proportion of positive sera
predominant antibody recognizes an antigen of ( 1:20) as a function of spleen size according to
Mr 155,000 d. Intraerythrocytic parasites or non Hackett is shown in Figure 5.
infected erythrocytes are not stained.4'5
The frequency of sera giving positive immu Antibodies to intracellular parasites
nofluorescence in this test rose significantly with
age. Thus, for the 43 sera available, the propor These were assayed in an IFA using parasitized
tion of positive sera (at dilutions 1:20) was erythrocytes as above but without fixation. All
29%, 50% and 88% in the age groups 2—5,6—9 43 sera tested contained parasite staining anti
and 10-15 years, respectively. The frequency of bodies with titers ranging from 1:320 to >1:
positive sera from Liberian adults from the same 10,240. However, there was no age-dependent
areas is also approximately 90%.@ The endpoint change in antibody levels (Figure 3). Moreover,
titers of the test sera also showed a significant there was no age-dependent correlation between
age-dependent increase (43 sera available for parasitemia and antibody titers, or between the
testing, r = 0.56, P = 2.2 x l0@). In some of latter and spleen size (data not shown).

>1024C A
p2560 0
1280 0 0 0 128( 0
0 00 8 0
320 320
00 00 00
000 00 0
80 0 0
0 80 0 0
20 0 000 0 0
Neg CO 000 0 000000 00 20 0 000 0
5 10 15 Neg 0000 @CO0 0 =
100 io2
>10240 0 00
FIGURE 4. Relationship between reciprocal IFA ti
5210 00000 00000 0 ters (ordinate) to GA-fixed and air dried infected RBC
000 00 00 0 00 00 and parasite densities (abscissa, number of parasites!
1280 0 0 0 0 0 000 0 mm3 of blood).
00 0 0
320 0 0

80 The ELISA used for determination of total anti

P. fakiparum antibodieshas a high degree of
Ne4 parasite specificity.9 Overall levels of 1gM anti
bodies increased with the age of the serum do
YEARS nors, starting at the age of 6 years and reaching
essentially adult levels at ages >10 years. The
FIGURE 3. IFA titers of sera of children and adults
age-dependent increase in IgG antibodies was
with A. glutaraldehyde (GA)-fixed and air dried in
fected RBC; and B. unfixed and air dried infected RBC. less pronounced and merely reflected the occur
(Ordinate, reciprocals of endpoint titers; abscissa, age rence of a few donors with very high antibody
in years). activities in the oldest age groups (12—15years
old). The age-dependent rise in 1gM activity but
DISCUSSION not that in IgG activity also showed a weak neg
ative correlation with the parasite densities found
Blood donors included in this investigation in the serum donors.
were 2—15years old, all residents of a malaria Antibody-dependent cell-mediated reactions
holoendemic area with perennial transmission.8 involving mononuclear cells or granulocytes are
In this area resistance to the disease develops believed to be important for protection in ma
with age after repeated infections.'3 A certain laria.'4 Such reactions are primarily mediated by
immunity is acquired by the age of 5 years and IgG antibodies of IgG, and IgG3 isotype. ‘5There
only intermittent low grade parasitemia is found fore, determination of the IgG subclass of anti
by the age of 15 years. Moreover, a strong neg malarial antibodies may be of help in assessing
ative correlation between age and parasite den their possible protective function. We have ear
sities as well as between age and spleen size has lier shown that anti-P. fakiparum antibodies in
been found from the age of 2 and 4 years, re both acutely ill and immune adults occur in all
spectively.'3 Age-related changes in the humoral 4 IgG subclasses.9 This was also seen in the pres
immune response were followed in assay systems ent investigation. Thus, IgG, and IgG3 antibod
recording either total anti-P. fakiparum anti ies were present at all ages and displayed no sta
bodies as well as their isotypes and specificity tistically significant increase with age. Rather, the
patterns or antibodies reacting specifically with findings are in line with the general postnatal
parasite antigens in the membrane of infected development of these IgG subclasses which reach
erythrocytesfr@ The latter tests showed a pro 50% of the adult level by the age of 1 year.'6 In
nounced correlation to the development of im contrast, IgG2 antibodies displayed a significant
munity, suggesting that these antigens may be age-related increase. Whether this was due to the
instrumental in the induction of a protective im slower postnatal development of IgG2, attaining
mune response. 50% of adult levels at the age of 3—4years'6 or

reflects an antigen-dependent restriction of the

IgG2 antibOdies presently is not known. No dis
tinct age pattern of this type was found for IgG4
antibodies present in a more limited number of
sera. Although a weak negative correlation was
found between parasite densities and IgG2 an w
tibody activities, this was not the case for the >
other IgG subclasses. Therefore, the relevance of I
this finding, for a possible causal relationship C0
between elevated antibody levels as detected by a
ELISA and parasite densities, is uncertain.
No age-dependent differences in the formation
of antibodies to individual parasite antigens were
revealed by analyzing the sera by immunopre
cipitation after SDS-PAGE of biosynthetically
labeled P.fakiparum extracts. The total number
of parasite polypeptides precipitated was similar
at all ages and not significantly different from 0 I II
what is seen with adult sera from the same area.'8 SPLEEN SIZE
Antibodies against the major polypeptides in
cluding the major schizont-derived surface gly FIGURE 5. Percentage of sera positive ( 1/20) in
the IFA with GA-fixed and air dried infected RBC
coprotein of Mr 195,000 d,'2 were present at all (ordinate); and spleensizeestimatedaccording to
ages and sometimes at elevated concentrations Hackett: abscissa, 0= normal spleen (n = 6); I = spleen
even in very young children. The frequency of palpable below costal margin on deep inspiration (n =
negative or weakly reacting sera appeared to be 7); II = spleen palpable below costal margin, but not
higher in the younger children than in the older projected beyond a horizontal line half way between
the costa! margin and the umbilicus (n = 18); III =
ones. This was probably due to a generally poor spleen with lowest palpable point projected more than
antibody response in some donors rather than half way to the umbilicus but not below a line drawn
due to a selective lack of antibodies against in horizontally through it (n = 5).
dividual antigens. It should be emphasized, how
ever, that this assay is of qualitative rather than 20 years. Other explanations for the differences
quantitative nature. Moreover, as the parasite in result could be the relatively small number of
extracts had been labeled with 35S-methionine, sera tested by us and difference in the sensitivity
antibodies to methionine-poor antigens5'6 were of the assays used.
not detected. In earlier work no correlation has been found
When the sera were analyzed by indirect IFA between titers obtained using conventional IFA
of unfixed parasites, no age-dependent changes or ELISA and those found with the correspond
in antibody titers were seen and there was no ing sera in the modified IFA.'8 In contrast to
correlation to parasite densities or spleen size. the ELISA and the IFA of unfixed parasites, that
This confirms previous results obtained with a of glutaraldehyde-fixed and air dried erythro
different series of donors from the same area,'3 cytes detects antibodies against only a few par
using the IFA described by Voller and O'Neill.'9 asite antigens in the membrane of infected cells.5'6
Similar results also have been reported from In serum from infants and adult donors, the ma
Tanzania by Draper Ct al.2°In contrast, others jority of the antibodies giving positive staining
have shown age-related increases in anti-P. fal in this assay are directed against a soluble and
ciparum antibody titers in IFA studies of serum heat stable polypeptide of Mr 155,000 d (Pf 155)
samples from other West African areas.21'22 deposited in the erythrocyte membrane by the
However, in those studies, the blood donors bursting schizont or invading merozoites. Pf 155
were from areas in which malaria transmission appears to be important for the invasion process
was seasonally restricted and occurred at lower as it binds to glycophorin A, a putative P. fal
rates. In addition, the test groups were divided ciparum receptor in the erythrocyte membrane6'23
differently and included sera from children less and human antibodies against Pf 155 very effi
than 2 years of age and from adults older than ciently inhibit merozoite reinvasion in vitro.'8

This antigen is poor in methionine and therefore Vaccines. Cold Spring Harbor Laboratory, Cold
not detected in immunoprecipitation experi Spring Harbor.
7. Franzén,L., and Wahlgren, M., 1985. Malaria
ments performed with 35S-methionine-labeled diagnosis: Parasitological and serological tests.
parasite extracts.5'6 In this respect it resembles PostgraduateDoctor, 8: 386—392.
the heat stable S-antigens described by other in 8. Bjorkman, A., Hedman, P., Brohult, J., Willcox,
vestigators.24 However, it appears to lack the ma M., Diamant, I., Pehrsson, P. 0., Rombo, L.,
jor serological variation characteristic for these and Bengtsson, E., 1985. Different malaria con
trol activities in an area of Liberia. Effects on
parasite components.6 malariometric parameters. Ann. Trop. Med.
The relevance of these parasite antigen(s) for Parasit., 79: 239—246.
in vivo protection against P. fakiparum malaria 9. Wahlgren, M., Berzins, K., Perlmann, P., and
is supported by the present findings showing both Björkman, A., 1983. Characterization of the
a marked increase in the frequency of antibody humoral immune response in Plasmodium fa/
ciparum malaria. I. Estimation of antibodies to
containing sera and antibody titers in parallel P. fakiparum or human erythrocytes by means
with the acquisition of clinical immunity. Do of micro ELISA. C/in.Exp. ImmunoL, 53: 127-
nors whose sera contained elevated levels of an 134.
tibodies to these antigens consistently had low 10.Wahlgren,M., Berzins, K., Perlmann,P.,and
Persson, M., 1983. Characterization of the hu
parasite counts and spleens of small or moder moral immune response in P/osmodiumfakip
ately enlarged size, suggesting that their immune arum malaria. II. IgG subclass levels of anti P.
system successfully handled the malaria pressure fa/ciparum antibodies in different sera. C/in.
to which they were exposed.'3 Taken together Exp. ImmunoL, 54: 135—142.
11. Lundgren, K., Wahlgren, M., Berzins, K., Troye
with previous findings@―8these results suggest Blomberg,M., Perlmann, H., and Perlmann, P.,
that P1' 155 and possibly other parasite antigens 1983. Monoclonal, parasite and anti RBC an
in the membrane of infected erythrocytes may tibodies produced by stable EBV-transformed B
be considered as prime candidates for a vaccine cell lines from malaria patients. /. Immunol,,
131: 2000—2003.
against the asexual blood stages of P.fakiparum.
12. Holder, A. A., and Freeman, R. R., 1982. Bio
synthesis and processing of a P/asmodium fa/
ciparum schizont antigen recognized by im
mune serum and a monoclonalantibody. J. Exp.
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