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

22—29
Copyright ©1986 by The American Society of Tropical Medicine and Hygiene

ANTI-PLASMODIUM FALCIPARUM ANTIBODIES ACQUIRED

BY RESIDENTS IN A HOLOENDEMIC AREA OF LIBERIA
DURING DEVELOPMENT OF CLINICAL IMMUNITY
MATS WAHLGREN,*t ANDERS BJORKMAN,66t HEDVIG PERLMANN,8
KLAVS BERZINS,* ANDPETER PERLMANN@
*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
Parasites
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%.
22
 ANTI-P. FALCIPARUMANTIBODIESAND CLINICALIMMUNITY 23

Study area gamma chains. Specificity of the conjugates was

Blood specimens were collected near Yekepa
Town in northern Liberia in a rural area where
malaria epidemiology has been studied in detail.8
In the 2 villages included in this study, malaria
is holoendemic and perennial. The sporozoite
inoculation rate is 0.2/person-night in the dry
season. P. fakiparum is the main malaria par
asite but P. malariae and P. ovale are also com
monly found.
Sample collection
The study included a survey of 40 children,
aged 2—12years, from Lugbeh Village and 8 ad
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
standing using Hackett's classification. Blood
specimens were collected by finger prick in hep
arinized microhematocrit tubes and centrifuged.
The plasmas were initially kept at —20°C,later
at —70°C.Thick and thin films were made for
microscopic observation.
Parasitological examinations
Blood films were stained with the standard
Giemsa method. Parasite species were identified
and parasite counts were calculated from the
number observed per 100—400 leukocytes, as
suming a constant leukocyte count of 10 x 10@/l.
checked in an ELISA where the plates were coat
ed with purified human myeloma proteins of dif
ferent isotypes.'°
IgG subclasses of the anti-P. fakiparum an
tibodies were determined in an antigen ELISA
with subclass-specific monoclonal mouse anti
bodies as previously described.'0 The mono
clonals were from the same clones as used pre
viously except that the antibodies from clone
JL5 12, earlier used as IgG1 reagent, were sub
stituted by antibodies from clone NL16 (Seward
Labs, London, England). Human sera were ab
sorbed twice with human erythrocyte ghosts and
tested at dilutions 1:200, 1:800 and 1:3,200. Sub
class specificity of the reactions was ascertained
as before.'°
Indirect immunofluorescence assay (IFA)
For the assay of antibodies to parasite antigens
in the membrane of infected erythrocytes, dif
ferent dilutions of test sera were added to mono
layers of glutaraldehyde-fixed and air dried
erythrocytes (5%—l0% parasitemia) as described
by Perlmann et al.5 Slides were stained with bio
tinylated goat antibodies to human immuno
globulin and FITC-conjugated avidin.
Antibodies to intraerythrocytic parasites were
assayed similarly using monolayers of air dried
but unfixed erythrocytes.― Tests were considered
positive when late stage parasites gave a distinct
and bright immunofluorescence.

Immunoprecipitation
ofparasiteantigens
Anti-P.falciparumELISA
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.
 24 WAHLGRENET AL

A low.There
was
asignificant
increase
in1gManti
P.fakiparum activity with increasing age (r=
2.0
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
1.0
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
c&@@
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)
2.0
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 0 O ing(18/38). Elevated activities were seen in some
0
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
YEARS
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
RESULTS
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
@

ANTI-P. FALCIPARUMANTIBODIESAND CLINICALIMMUNITY 25

YEARS

t2: ,@5
12 IC

@
195@

@
‘iì!'-@t3 @;;@6
;4@9 -r
@,,
129,,...
118

V ‘
5

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

>1024C A
p2560 0
5120
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 =
YEARS
100 io2
>10240 0 00
B
FIGURE 4. Relationship between reciprocal IFA ti
00
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

20
Ne4
5
FIGURE 3. IFA titers of sera of children
with A. glutaraldehyde (GA)-fixed and air dried in
fected RBC; and B. unfixed and air dried infected RBC.
(Ordinate, reciprocals of endpoint titers; abscissa, age
in years).
DISCUSSION
Blood donors included in this investigation
were 2—15years old, all residents of a malaria
holoendemic area with perennial transmission.8
In this area resistance to the disease develops
with age after repeated infections.'3 A certain
immunity is acquired by the age of 5 years and
only intermittent low grade parasitemia is found
by the age of 15 years. Moreover, a strong neg
ative correlation between age and parasite den
sities as well as between age and spleen size has
been found from the age of 2 and 4 years, re
spectively.'3 Age-related changes in the humoral
immune response were followed in assay systems
recording either total anti-P. fakiparum anti
bodies as well as their isotypes and specificity
patterns or antibodies reacting specifically with
parasite antigens in the membrane of infected
erythrocytesfr@ The latter tests showed a pro
nounced correlation to the development of im
munity, suggesting that these antigens may be
instrumental in the induction of a protective im
mune response.
P. fakiparum antibodieshas a high degree of
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
and adults
age-dependent increase in IgG antibodies was
less pronounced and merely reflected the occur
rence of a few donors with very high antibody
activities in the oldest age groups (12—15years
old). The age-dependent rise in 1gM activity but
not that in IgG activity also showed a weak neg
ative correlation with the parasite densities found
in the serum donors.
Antibody-dependent cell-mediated reactions
involving mononuclear cells or granulocytes are
believed to be important for protection in ma
laria.'4 Such reactions are primarily mediated by
IgG antibodies of IgG, and IgG3 isotype. ‘5There
fore, determination of the IgG subclass of anti
malarial antibodies may be of help in assessing
their possible protective function. We have ear
lier shown that anti-P. fakiparum antibodies in
both acutely ill and immune adults occur in all
4 IgG subclasses.9 This was also seen in the pres
ent investigation. Thus, IgG, and IgG3 antibod
ies were present at all ages and displayed no sta
tistically significant increase with age. Rather, the
findings are in line with the general postnatal
development of these IgG subclasses which reach
50% of the adult level by the age of 1 year.'6 In
contrast, IgG2 antibodies displayed a significant
age-related increase. Whether this was due to the
slower postnatal development of IgG2, attaining
50% of adult levels at the age of 3—4years'6 or
 ANTI-P. FALCIPARUMANTIBODIESAND CLINICALIMMUNITY
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
sera. Although a weak negative correlation was
found between parasite densities and IgG2 an
tibody activities, this was not the case for the
other IgG subclasses. Therefore, the relevance of
this finding, for a possible causal relationship
between elevated antibody levels as detected by
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
what is seen with adult sera from the same area.'8
Antibodies against the major polypeptides in
cluding the major schizont-derived surface gly
coprotein of Mr 195,000 d,'2 were present at all
ages and sometimes at elevated concentrations
even in very young children. The frequency of
negative or weakly reacting sera appeared to be
higher in the younger children than in the older
ones. This was probably due to a generally poor
antibody response in some donors rather than
due to a selective lack of antibodies against in
dividual antigens. It should be emphasized, how
ever, that this assay is of qualitative rather than
quantitative nature. Moreover, as the parasite
extracts had been labeled with 35S-methionine,
antibodies to methionine-poor antigens5'6 were
not detected.
When the sera were analyzed by indirect IFA
of unfixed parasites, no age-dependent changes
in antibody titers were seen and there was no
correlation to parasite densities or spleen size.
This confirms previous results obtained with a
different series of donors from the same area,'3
using the IFA described by Voller and O'Neill.'9
Similar results also have been reported from
Tanzania by Draper Ct al.2°In contrast, others
have shown age-related increases in anti-P. fal
ciparum antibody titers in IFA studies of serum
samples from other West African areas.21'22
However, in those studies, the blood donors
were from areas in which malaria transmission
was seasonally restricted and occurred at lower
rates. In addition, the test groups were divided
differently and included sera from children less
than 2 years of age and from adults older than
27
of
w
>
I
C0
0
a
0 I II
SPLEEN SIZE
FIGURE 5. Percentage of sera positive ( 1/20) in
the IFA with GA-fixed and air dried infected RBC
(ordinate); and spleensizeestimatedaccording to
Hackett: abscissa, 0= normal spleen (n = 6); I = spleen
palpable below costal margin on deep inspiration (n =
7); II = spleen palpable below costal margin, but not
projected beyond a horizontal line half way between
the costa! margin and the umbilicus (n = 18); III =
spleen with lowest palpable point projected more than
half way to the umbilicus but not below a line drawn
horizontally through it (n = 5).
20 years. Other explanations for the differences
in result could be the relatively small number of
sera tested by us and difference in the sensitivity
of the assays used.
In earlier work no correlation has been found
between titers obtained using conventional IFA
or ELISA and those found with the correspond
ing sera in the modified IFA.'8 In contrast to
the ELISA and the IFA of unfixed parasites, that
of glutaraldehyde-fixed and air dried erythro
cytes detects antibodies against only a few par
asite antigens in the membrane of infected cells.5'6
In serum from infants and adult donors, the ma
jority of the antibodies giving positive staining
in this assay are directed against a soluble and
heat stable polypeptide of Mr 155,000 d (Pf 155)
deposited in the erythrocyte membrane by the
bursting schizont or invading merozoites. Pf 155
appears to be important for the invasion process
as it binds to glycophorin A, a putative P. fal
ciparum receptor in the erythrocyte membrane6'23
and human antibodies against Pf 155 very effi
ciently inhibit merozoite reinvasion in vitro.'8
28 WAHLGRENF@AL
This antigen is poor in methionine and therefore
not detected in immunoprecipitation experi
ments performed with 35S-methionine-labeled
parasite extracts.5'6 In this respect it resembles
the heat stable S-antigens described by other in
vestigators.24 However, it appears to lack the ma
jor serological variation characteristic for these
parasite components.6
The relevance of these parasite antigen(s) for
in vivo protection against P. fakiparum malaria
is supported by the present findings showing both
a marked increase in the frequency of antibody
containing sera and antibody titers in parallel
with the acquisition of clinical immunity. Do
nors whose sera contained elevated levels of an
tibodies to these antigens consistently had low
parasite counts and spleens of small or moder
ately enlarged size, suggesting that their immune
system successfully handled the malaria pressure
to which they were exposed.'3 Taken together
with previous findings@―8these results suggest
that P1' 155 and possibly other parasite antigens
in the membrane of infected erythrocytes may
be considered as prime candidates for a vaccine
against the asexual blood stages of P.fakiparum.
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