Journal of Immunological Methods, 93 (1986) 55-61
55
Elsevier
JIM 04056
Synthetic peptides as antigens for the detection of humoral
immunity to Plasmodium falciparum sporozoites
Fidel Zavala 1, James P. T a m 2 and Aoi M a s u d a 1
1 Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, N Y 10016, and
2 The Rockefeller University, New York, NY 10021, U.S.A.
(Received10 April 1986, accepted2 May 1986)
The presence of antibodies against P. falciparum sporozoites in humans living in malaria-endemic areas
was measured using as antigen the synthetic peptide (NANP)3 , which represents the immunodominant
region of the circumsporozoite (CS) protein. By using a competitive binding assay it was determined that
antibodies which recognize (NANP)3 do not react with a 22-Mer synthetic peptide representing a
cross-reacting epitope present in an antigen (5.1) from the blood stages of the parasite. Antibodies present
in human sera which react with the 5.1 peptide did not react with (NANP)3. This strongly suggests that
antibodies to (NANP)3 found in sera of individuals living in endemic areas are a reflection of exposure to
P. falciparum sporozoites. These results validate the use of (NANP)3 for epidemiological studies to detect
and measure humoral immunity to P. falciparum sporozoites.
Key words: Syntheticpeptide; Malaria sporozoite; Antibody, human; Immunoradiometricassay
Introduction
The detection of antibodies to sporozoites in
the sera of individuals living in malaria endemic
areas is of epidemiological relevance, since antibody levels should correlate with the rates of
transmission of the disease. In the past, the presence of antibodies to P. falciparum and also to P.
vioax sporozoites has been determined by indirect
immunofluorescence using glutaraldehyde-fixed
sporozoites as antigen (Nardin et al., 1979;
Tapchaisri et al., 1983). In view of the requirement
for sporozoites obtained from the salivary glands
of experimentally infected mosquitoes, this assay
has been seldom used.
Most antibodies to sporozoites are directed
against a single, repetitive epitope of a protein
which covers the whole surface membrane of
sporozoites, the circumsporozoite (CS) protein
(Zavala et al., 1983). Other studies suggested that
synthetic peptides representing this epitope could
be used to measure the levels of antibodies to
sporozoites in human sera. For example, the repetitive epitope of the CS protein of P. falciparum is
contained in a peptide of twelve amino acids
consisting of three tandem repeats of asparaginealanine-asparagine-proline ((NANP)3). In human
sera from endemic areas a highly significant correlation between the titers of antibodies to P.
falciparum sporozoites and the levels of antibodies
to the synthetic peptide (NANP)3 was observed.
Moreover, most or all of the reactivity of these
sera with the parasites was inhibited in the presence of this peptide (Zavala et al., 1985a).
Questions have been raised, however, regarding
the nature of the malaria antigen which stimulates
the production of antibodies to ( N A N P ) 3 . It has
been suggested that some of these antibodies may
be induced not by the CS protein, but by a
cross-reactive antigen named Ag 5.1 found in the
0022-1759/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)
56
blood stages of P. falciparurn (Coppel et al., 1985).
In support of this hypothesis it is known that Ag
5.1 (Coppel et al., 1985; Hope et al., 1985) includes one stretch of amino acids with a high
proportion of proline, asparagine and alanine residues, and also one NANP sequence. Hope et al.
(1984) also identified one monoclonal antibody
raised against blood stages of P. falciparum which
reacted both with the 5.1 antigen and with the CS
protein. Antibodies in pooled human sera from
endemic areas, affinity purified on immobilized
extracts of E. coli expressing the 5.1 antigen,
reacted with P. falciparum sporozoites (Coppel et
al., 1985).
In the present investigation, we studied the
extent and nature of this cross-reaction using individual sera from children and adults living in The
Gambia and in Burkina Faso, areas of high endemicity of P. falciparum malaria.
Materials and methods
Sera
Blood samples from children and adults (not
selected for malaria infection) were collected during the months of November and December of
1982 at the Clinic of the British Medical Research
Council Laboratories, in The Gambia (Fajara).
Other series of sera were collected in Burkina
Faso in September of 1984. The sera were maintained frozen at - 2 0 ° C . Normal serum samples
were obtained from healthy blood donors in New
York City.
Monoclonal antibody
The hybridoma-producing monoclonal antibody 5.1 against a blood stage antigen was identified by McBride et al. in 1982. A sample of the
antibody was kindly provided to us by Dr. J.
Scaife, University of Edinburgh. Its properties are
described elsewhere (Hope et al., 1984).
Synthetic peptides
Peptides were synthesized by the stepwise
solid-phase method of R.B. Merrifield (1963) in a
Biosearch peptide synthesizer (Sam II). The
dodecamer, H-(Asn-Ala-Asn-Pro)3-OH or
(NANP)3 was synthesized on a Boc-Pro-OCH 2-
Pam-copolystyrene-l%-divinylbenzene resin (a
0.34 mmol substitution per g of resin) (Mitchell et
al., 1976). The 5.1 peptide H-Asp-Pro-Ala-AspAsn-Ala-Asn-Pro-Asp-Ala-Asp-Ser-Glu-Ser-AsnGly-Glu-Pro-Asn-Ala-Asp-Pro-NH2 was synthesized on a multidetachable benzhydrylamine resin
( p-acyloxybenzhydrylamine-copolystyrene-1%-divinylbenzene resin) (0.4 mmol substitution per
gram of resin) (Tam et al., 1981). A double coupling protocol via dicyclohexylcarbodiimide was
used to give a coupling efficiency of > 99.85%
completion per step. The benzyl-based side chain
protecting groups and tertbutoxycarbonyl (Boc)
for the N-alpha-terminus were used. The cleavage
of H-(NANP)3-OH from the resin support was
effected by HF-anisole (9 : 1, v/v) at 0 °C for 1 h.
However, several other precautions were used for
the synthesis of the 5.1 peptide because of the
sequences in Asp-Asn and Asp-Ser, which were
particularly prone to cyclization reaction to form
aspartimide during the synthesis and acid deprotection of the protected peptide resin, when the
usual beta-benzyl ester protecting group for Asp
was used. The repetitive base neutralization was
shortened to 2 x 1 rain cycles (Tam et al., 1979) to
prevent the base-catalyzed aspartimide formation
during the synthesis. Furthermore, to ensure that
the aspartimide formation is greatly minimized
after the protecting group is removed from the
aspartyl residue, a mild and controlled gradative
deprotection strategy (Tam et al., 1985) was used
in combination with a low deprotection method
(Tam et al., 1983). With these precautions and
improvements, the crude and unpurified 5.1
peptide, when examined by high-pressure liquid
chromatography (HPLC) gave a single symmetrical peak, accounting for 79% of all the peptide
content.
The crude peptides were purified by preparative low-pressure liquid chromatography (100-120
psi) on a reversed-phase C-18 column (2.5 x 30
cm) using an aqueous CV3CO2H (0.05%) and
acetonitrile gradient. The purified material gave a
single symmetrical peak on analytical HPLC ((4 x
300 mm), C-18 reverse phase, Waters Assoc.) and
gave the correct theoretical values of amino acids
by amino acid analysis. The overall yield, after
purification, based on the first amino acid attached to the resin was greater than 68%.
57
Immunoradiometric assay (IRMA)
Antigen conjugates were made by incubating
300/~g of peptide in 1 ml of phosphate-buffered
saline (PBS) containing 0.1% bovine serum albumin (BSA) and 0.25% glutaraldehyde. After
incubation for 5 h at room temperature with constant agitation, 10 #1 of 2 M lysine were added.
After 30 min, the preparation was aliquoted and
stored at - 20 °C.
Flexible polyvinylchloride microtiter plates
(Dynatech, Alexandria, VA) were incubated overnight at room temperature with 20 #1 of antigen
conjugate diluted 100 times in PBS. After washing
three times with PBS, the wells were incubated
overnight with 150/xl of PBS containing 1% BSA
and 0.5 M ethanolamine at pH 8.6 (PBS-BSA-Eth).
The plates were stored frozen at - 2 0 o C.
Serum samples were diluted in PBS-BSA-Eth,
containing also 0.5% Tween 20 and 0.1% NP40.
After centrifugation at 8000 × g for 5 min, 20 /zl
were placed in each well. After incubating for 1 h
at room temperature each well was washed three
times with PBS-BSA-Eth-Tween 20-NP40. Then
30/~1 (7 × 104 cpm) of affinity-purified goat antih u m a n or goat anti-mouse immunoglobulin
(specific acitivty 2 × 107 cpm//~g), diluted in PBSBSA-Eth-Tween 20-NP40 were placed in each well.
After 1 h at room temperature, and washing three
times with PBS-BSA-Eth-Tween 20-NP40, the
wells were dried and counted in a gamma counter.
Individual sera were simultaneously tested in
peptide-coated plates and in control plates coated
with a sham antigen conjugate, prepared as described above, but omitting the peptide. The cpm
in control wells were subtracted from the cpm in
the corresponding peptide-coated plates and the
difference defined as Acpm. The number of cpm
in the control wells varied from 200 to 500 when a
1 / 1 0 dilution of serum was used. The average cpm
of a 10-fold dilution of the normal sera was 259 +
155. This value + 3 standard deviations (742 cpm)
was defined as the normal range.
lmmunofluorescence assay (1FA)
P. falciparum blood forms of isolate FCR-3
were grown in vitro in human erythrocytes according to an established technique (Trager et al.,
1976). 10 #1 of 5% parasitized red blood cells were
then air-dried on immunofluorescence slides and
fixed with acetone. Human serum samples, diluted
10 times, were incubated with the fixed parasites
for 30 min at room temperature. After washing
several times, the slides were incubated for 30 min
with 1 / 4 0 diluted fluorescein isothiocyanate
(FITC)-labeled affinity-purified goat anti-human
immunoglobulin (Kirkegaard and Perry Laboratories). IFA with glutaraldehyde-fixed sporozoites of
P. falciparum was performed according to a technique described elsewhere (Nardin et al., 1979).
Results
Specificity of the 5.1 monoclonal antibody
As documented elsewhere, monoclonal antibody 5.1 raised against blood stages of P.
falciparum cross-reacts with sporozoites (Hope et
al., 1984). We show in Fig. 1 that monoclonal
antibody 5.1 also reacts with the immobilized synthetic peptide (NANP)3 , which represents the
immunodominant epitope of the P. falciparum
circumsporozoite protein.
The complete amino acid sequence of the 5.1
)3
/
E 2~
c)
//
I-i ~~~'/
o
o.6
5.1
coated plate
(NANP)
3
coated plate
unrelatecl
peptide
1.2 2.,5 5.0
IO 2o
concentration of anti-5.1Mab (iug/ml)
Fig. 1. Reactivityof monoclonalanti-P, falciparum blood form
antibody (5.1) with synthetic peptides. Increasing concentrations of 5.1 monoclonal antibody were incubated in plastic
wells containingimmobilized(NANP)3or 5.1 syntheticpeptide.
After 1 h, 30 ~1 (7 × 10 4 epm) of 125I-labeled goat anti-mouse
immunoglobulin were placed in each well; the wells were
incubated 1 h, washed thoroughly, dried and counted in a
gamma counter.
58
antigen has been determined. The sequence which
most resembles (NANP)3 is contained between
amino acids 116-137 (Coppel et al., 1985; Hope et
al., 1985). This stretch of 22 amino acids (for the
entire sequence, see the materials and methods
section), which we have designated as the 5.1
peptide, is rich in alanine, praline and asparagine
and includes one N A N P sequence.
To demonstrate that indeed the 5.1 peptide
contained the epitope recognized by the 5.1 antibody, the 22-Mer was synthesized and used as
antigen in an IRMA. In Fig. 1 we show that the
5.1 antibody binds to the immobilized 5.1 synthetic peptide, but not to an unrelated immobilized peptide ( G Q P Q A Q G D G A N A ) . Moreover,
2.0
the 5.1 peptide in the fluid phase inhibited the
reactivity of the 5.1 antibody both with immobilized (NANP)3 and 5.1 peptides (Fig. 2). In this
experiment the 5.1 antibody (6 /~g/ml) was
incubated for 1 h at room temperature with increasing amounts of 5.1 peptide and then added to
wells of microtiter plates containing either peptide
5.1 or (NANP)3. The amount of bound monoclonal antibody was measured with a second radiolabeled antibody, namely, affinity-purified goat
anti-mouse Ig. As shown in Fig. 2 the 5.1 antibody
bound to a similar extent to the immobilized
(NANP)3 or 5.1 peptide. In addition, the 5.1
peptide in the fluid phase inhibited the homologous and heterologous reactions at similar molar
concentrations.
The 5.1 peptide at a concentration of 10 -4 M
also totally inhibited the immunofluorescence observed upon interaction of monoclonal antibody
5.1 with P. falciparum sporozoites and blood
stages. Taken together, these results demonstrate
that the 5.1 peptide contains the epitope recognized by monoclonal antibody 5.1, in both blood
stages and sporozoites of P. falciparum.
1.5
pl~~
ted plate
x
E
1.0
(NANP) 3 coated
t).
o
\\
05-
0
I
0
10-8
I0 -7
10-6
10-5
10-4
concentration of 5.1 peptide (M)
Fig. 2. Binding of the anti-blood form monoclonalantibody to
5.1- and (NANP)3-coated wells in the presence of soluble 5.1
peptide. The competitivebinding assay was done by incubating
the monoclonal antibody 5.1 at a concentration of 6 pg/ml
with increasing molar concentrations of the 5.1 synthetic
peptide. After 1 h at room temperature an aliquot of the
mixture was placed in wells coated with 5.1 or (NANP)3
peptide; the wellswereincubatedfor I h, washed and incubated
for an additional hour with radiolabeled goat anti-mouse immunoglobulin.
Specificity of human serum antibodies reacting with
the 5.1 and (NANP)3 peptides
As shown previously, the proportion of sera of
individuals from an endemic area which reacted
with (NANP)3 increased with age, ranging from
22% in children 1-14 years old, to 84% in adults
over 34 years of age (Zavala et al., 1985a). Among
randomly selected sera from individuals over 20
years of age several displayed titers which were
higher than 1/160. The antibody class was predominantly IgG (data not shown).
In the present study we assayed 80 human sera
from adults living in Burkina Faso and The
Gambia for the presence of antibodies against the
synthetic peptides (NANP)3 and 5.1 by means of
a solid-phase IRMA. By indirect immunofluorescence, 98% of these sera contained antibodies to
blood-forms of P. falciparum. While 80% of these
sera also contained antibodies against (NANP)3,
only 5% reacted specifically with the 5.1 peptide.
The specificity of the reaction with (NANP)3
was examined by preincubation of twelve randomly selected positive sera from Burkina Faso
with a 1 0 - 4 M solution of (NANP)3 or of 5.1
59
SenJ'n
Inhibitor Peptide antigens
(Peptide)
5.1
(NANP) 3
+
f
940
?
o
I
],
(NANP) 3
t
+
5.1 U
g
5.1 U
0z
,,
i
0
Sera alone
Sera + 5.1 peptide
Sera + (NANP) 3 peptide
[,
i
i
I
i
i
2
I
0
]-5-
I+
I
I
I
2
I
3
I
4
j
I
6
Acpm X 10-3
Fig. 4. Binding of antibodies of human sera to immobilized 5.1
and (NANP)3 peptides in the presence of soluble synthetic
peptides. The competitive binding assay was performed as
described in the footnote of Fig. 2.
o
x
3
o
Sera alone
Sera + 5.1 peptide
Sera + (NANP) 3 peptide
Fig. 3. Binding of serum antibodies from humans living in
Burkina Faso (A) and The Gambia (B) to immobilized
(NANP)3 in the presence of soluble synthetic peptides. Ten
times diluted serum samples were incubated with PBS or 10-4
M concentration of soluble peptides 5.1 or (NANP)3. After 1 h
at room temperature, an aliquot of the mixture was placed in
an (NANP)3-coated plate to determine the binding of antipeptide antibodies as described in the footnote of Fig. 2. All
tests were performed in duplicate and the vertical bars span the
values obtained.
peptide, prior to adding the sera to the (NANP)3coated wells. The binding of antibodies to the
wells was totally inhibited by (NANP)3, but not
by the 5.1 peptide (Fig. 3A). Identical results were
obtained using 14 (NANP)3 positive sera from
The Gambia (Fig. 3B).
As mentioned, only four sera recognized the 5.1
peptide in the IRMA. In contrast to the results
described above, the reaction of these sera was
totally inhibited by preincubation with the 5.1
peptide (10 -4 M) but was not affected by
(NANP)3 at the same molar concentration (Fig.
4). It should be pointed out that three out of these
four sera also contained antibodies to (NANP)3.
This reactivity, however, was only inhibited by
(NANP)3 and not affected by the 5.1 peptide in
solution. Another relevant finding was that antibodies of a human volunteer (G.Z.), vaccinated
with sporozoites (Clyde et al., 1973), reacted
strongly with (NANP)3 but not with the 5.1
peptide (Fig. 4).
Discussion
The present findings complement our earlier
studies on the antigenic properties of the synthetic
peptide (NANP)3- They strongly suggest that antibodies to (NANP)3, found in sera of individuals
living in malaria endemic areas, are a reflection of
exposure to P. falciparum sporozoites.
The opposing view originated from the observation that one out of numerous monoclonal antibodies obtained by immunization with blood
stages of P. faiciparum also reacted with
sporozoites. This antibody recognized an antigen
of M r 23 000 in all asexual stages of the parasite
and was designated Ag 5.1. In addition, antibodies
60
in the sera of individuals living in malaria endemic
areas reacted both with the 5.1 antigen and with
the surface of glutaraldehyde-fixed sporozoites
(Hope et al., 1984; Coppel et al., 1985). The idea
that the production of antisporozoite antibodies
was stimulated by blood stages was further
strengthened by the finding that the Ag 5.1 had a
region with a certain similarity to the repeats of
the CS protein of P. falciparum.
We re-examined this question because of its
obvious relevance for the interpretation of any
seroepidemiological studies measuring levels of
antibodies to sporozoites in endemic areas.
A 22-Mer peptide encompassing the region of
Ag 5.1 which resembles (NANP) 3 was synthesized
and shown to react directly with the monoclonal
antibody 5.1 and to inhibit its binding to
sporozoites or blood stages of the parasite. Next
we studied the reactivity of individual sera from
The Gambia and Burkina Faso with the synthetic
peptides 5.1 and (NANP)3. As summarized in Fig.
4, we found no evidence for the presence of antibodies which interacted with both peptides. The
preincubation of sera with peptide (NANP)3 only
removed their reactivity with (NANP)3 but not
with peptide 5.1. The reverse was also true, i.e.,
pre-incubation with peptide 5.1 only removed the
reactivity with the Ag 5.1. Moreover the 5.1 epitope does not seem to be very immunogenic. Although 98% of the sera had antibodies to blood
stages of P. falciparum, only 5% reacted with the
synthetic peptide 5.1. In contrast 80% of the serum
samples reacted with (NANP)3, the immunodominant epitope of the CS protein. We conclude that
the occurrence of cross-reactivity between antibodies to Ag 5.1 and CS protein in the sera of
individuals from these two malaria endemic areas
is a rare event, if in fact it occurs at all.
This conclusion is in agreement with extensive
experimental evidence in rodent and monkey
malarias showing that the humoral immunity to
sporozoites and to blood stages is strictly stagespecific, and with the observations of Nardin et al.
(1979) showing no correlation between levels of
antibodies to sporozoites and blood stages in The
Gambia. Furthermore, children from endemic
areas who have had several malaria attacks are
frequently found to have high titers of antiblood-form antibodies, but not anti-sporozoite
antibodies. Therefore, the present results validate
the use of the synthetic peptide (NANP)3 for the
detection and measurement of humoral immunity
to P. falciparum sporozoites.
Some interesting applications of an IRMA using
(NANP)3 as antigen can be envisaged. For example, sero-epidemiological studies could determine
whether the presence of anti-sporozoite antibodies
and resistance to malaria are causally related.
Such studies were never attempted in the past
because of the difficulty in obtaining sufficient
numbers of sporozoites to perform IFA reactions
and because of the uncertainties as to the nature
of the parasite antigens recognized by the serum
antibodies. An additional advantage of using the
synthetic peptide as antigen in epidemiological
surveys is that the (NANP)3 epitope is represented
in CS proteins of P. falciparum strains from different geographical areas of the world (Weber et
al., 1985; Zavala et al., 1985b). The use of
enzyme-labeled antibodies for an ELISA adaptation of this assay gave almost identical results (not
shown).
Also, P. falciparum malaria vaccines, produced
either by recombinant DNA techniques or containing synthetic peptides, are likely to be tested
in the near future. The precise measurement of
levels of anti-sporozoite antibodies in endemic
areas, immediately before and following the
immunization, will be necessary to evaluate and
compare different vaccine preparations, to determine the effects of dosage, adjuvants, etc. The
immunoradiometric assay here described seems
ideally suited for this purpose provided that the
candidate vaccines contain the P. falciparum CS
protein or the repetitive epitope.
Acknowledgements
The authors would like to thank Drs. Ruth and
Victor Nussenzweig for continued support and
critical comments. We also thank Dr. Fulvio
Esposito for providing samples of human sera,
Ms. Monica Samper for technical assistance, and
Roger Rose for typing the manuscript. This work
was supported by grants from the Agency for
International Development, the National Institutes
of Health, and the U N D P / W o d d Bank/WHO
61
S p e c i a l P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in
Tropical Diseases.
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