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Distribution of repetitive and non-repetitive circumsporozoite protein epitopes on Plasmodium falciparum sporozoites and immunochemical characterization of human malaria antisera
Acta Tropica, 51(1992)257-270 © 1992 Elsevier Science Publishers B.V. All rights reserved 0001-706X/92/$05.00
257
ACTROP 00225
Distribution of repetitive and non-repetitive circumsporozoite protein epitopes on Plasmodium falciparum sporozoites and immunochemical characterization of human malaria antisera W. R u d i n , " Ch. P e t i g n a t , a M . T a n n e r a a n d H. M a t i l e b "Swiss Tropical Institute, CH-4002 Basel, Switzerland and bNew Technologies Biology, E Hoffmann-La Roche Ltd., Basel, Switzerland (Received 4 March 1992; accepted 12 May 1992) The presence and distribution of circumsporozoite protein (CSP) epitopes located in the repetitive and non-repetitive regions were studied in three Plasmodiumfaleiparum strains, NF54, IFA5 and IFA6. It was found by immunofluorescence, Western blotting and immunoelectron microscopy that mAbs to epitopes of the repetitive domaine bound similarly to the CSP of all three strains. MAbs to epitopes of the flanking regions yielded either some strain differences (mAbs to the C-terminal end), or reacted only in immunofluorescence tests on whole sporozoites (mAbs to the N-terminal end). Human sera from an area endemic for malaria, two of them positive in ELISA on (NANP)4o and two negative, were tested for their reactivity with epitopes of the flanking regions of the CSP. The presence of antibodies to such epitopes could be demonstrat6d by Western blots and immunocytochemistry independent of the reactivity of the sera to recognize (NANP)40. All tested bound to salivary gland tissues but not to their secretory product in immunocytochemical experiments. Key words: P.falciparum; Sporozoites; CSP epitopes; Human serum
Introduction T h e m a i n surface a n t i g e n o f the s p o r o z o i t e s o f Plasmodium species, is the CSP, which h o m o g e n e o u s l y covers the s p o r o z o i t e surface ( A i k a w a et al., 1981; Y o s h i d a et al., 1981). T h e C S P is expressed 6 d a y s after infection o f the m o s q u i t o on the p l a s m a l e m m a o f oocysts ( P o s t h u m a et al., 1988). O n P . f a l c i p a r u m s p o r o z o i t e s C S P consists o f 37 repeats o f the i m m u n o d o m i n a n t t e t r a p e p t i d e A s n - A l a - A s n - P r o ( N A N P ) a n d 4 repeats o f the sequence A s n - V a l - A s p - P r o ( N V D P ) ( D a m e et al., 1984; E n e a et al., 1984). ( N A N P ) 3 represents the i m m u n o d o m i n a n t d o m a i n ( Z a v a l a et al., 1985) a n d does n o t show v a r i a t i o n a m o n g s t P. falciparum isolates ( A r n o t , 1990). T h e repetitive d o m a i n e is flanked on b o t h sides by c o n s e r v e d regions in all cloned a n d sequenced genes o f the CS p r o t e i n o f v a r i o u s Plasmodium species ( M c C u t c h a n et al., 1988) a n d by variable regions as described for P. falciparum ( L o c k y e r et al., 1989). Correspondence to." W. Rudin, Swiss Tropical Institute, Postfach, CH-4002 Basel, Switzerland; Tel. (061) 284 82 41; Fax, (061) 271 86 54.
258 Antibodies recognizing the synthetic peptide (NANP)3 neutralize sporozoite infectivity in vitro (Hollingdale et al., 1984; Mazier et al., 1986; Zavala et al., 1986). Irradiated sporozoites can protect rodents, monkeys and men from malaria infections, and the repetitive domain of the CSP is thought to be involved in the interaction between sporozoites and hepatocytes (Nussenzweig and Nussenzweig, 1985). However, the efforts to develop a vaccine using (NANP), sequences have not been successful, because only weak T-cell induced immune responses of short duration were achieved (Ballou et al., 1987; Herrington et al., 1987; Etlinger et al., 1988). Synthetic peptides of the non-repetitive sequence are immunogenic in laboratory animals (Ballou et al., 1985; Vergara et al., 1985a,b; Sharma et at., 1986), while antisporozoite antibodies recognizing epitopes of the flanking regions were also found in sera of people living in areas endemic for malaria (Romero et al., 1987; Del Guidice et al., 1988; Stfiber et al., 1990). This is of interest because T helper cells have been shown to be necessary to reach high antibody titers against sporozoites (Spitalny et al., 1977) and that the CSP possesses T cell epitopes in these nonrepetitive regions (Good et al., 1988a,b; Kumar et al., 1988; Sinigaglia et al., 1988). In the present study the binding of mAbs to various CSP epitopes on P.falciparum sporozoites in salivary glands were localized immunocytochemically. These binding sites were compared between the P. falciparum strain NF54 and two field isolates, IFA 5 and IFA 6. In addition, human sera from a malaria endemic area have been tested for antibodies to repetitive and non-repetitive regions of the CSP for the first time.
Material and Methods
Parasites and vector Plasmodiumfalciparum NF54 (Ponnudurai et al., 1981) was used for the immunocytochemical characterization of a series of mAbs to peptides of the CSP. NF54 together with the human isolates P. falciparum IFA 5 and IFA 6 from St. Francis District Hospital Ifakara, Tanzania (Hurt, 1990; Huber, 1991) was used to study the reactivity of various human sera. Anopheles stephensi were obtained from H. Briegel in 1983 and since then bred at Hoffmann La Roche. Mosquitoes were infected 2 to 4 days after emergence with Plasmodium falciparum produced in vitro and fed using membrane feeders (Ponnudurai et al., 1989a). Infected mosquitoes were kept at 80% RH and 27°C until use. Circumsporozoite peptides and fusion proteins The CS peptide (NANP)4 o (Del Giudice et al., 1986) was used for ELISAs to check human sera for antibodies to the repetitive CS protein domain. For immunoblotting, CS fusion proteins expressed in E. coli were used which consisted of repetitive or non-repetitive parts of the CS protein and mouse dihydrofolate reductase (DHFR) linked to six adjacent histidine residues (Stfiber et al., 1990).
Monoclonal antibodies The following mAbs were used in the present study (Table 1): SP3-E9 reactive to NANP epitopes (Boulanger et al., 1988), CS VI-2, CS VI-4, and CS VI-6 reactive
259 to sequences of the non-repetitive region at the C-terminal end of the CS protein (Stiiber et al., 1990), CS FY-IF1, CS FY-4G7 to sequences of the non-repetitive region at the N-terminal end of the CS protein. The mAb Cs VI-3 against D H F R served as a negative control in immunocytochemical experiments. Human sera The human sera tested for their reactivity with CS fusion proteins and sporozoites were collected during the course of a hospital-based study on malaria-resistance and hepatic diseases at St. Francis District Hospital, Ifakara, Tanzania in 1982 (Stahel et al., 1984). The sera originate from an area hyperendemic for malaria with an entomological inoculation rate between 0.7 and 3.0 infectious bites per person per night (Tanner et al., 1986). Indirect immunofluorescence assay ( IFA ) An indirect immunofluorescence assay (Young et al., 1985) was used to estimate the antibody titer of the human sera. About 2000 isolated sporozoites in 5 gl PBS were applied per test area, air dried and stored at - 70°C until use. The following dilutions of the sera were applied in 13 gl aliquots per test area: 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280. Fluorescein isothiocyanate (FITC) conjugated goat anti-human immunoglobulins (Ig; 1:40) (Nordic Immunological Laboratories, The Netherlands) was used as a marker. Enzyme-linked immunosorbent assay ( ELISA ) After disection of the salivary glands, the thorax and abdomen of each mosquito were ground in 200 lal lysis buffer (0.5% bovine serum albumin (BSA), 0.5% Nonidet P-40 (NP-40) in phosphate-buffered saline (PBS, pH 7.2)) with a glass pestel. ELISA plates (Nunc immunoplate maxisorb) coated overnight with 10 tag/ml SP3-E9 in PBS, 100 gl per well at 4°C were rinsed three times with 0.05% Tween 20 in PBS (PBSTween) and pretreated with 100 gl/well 0.5% BSA in PBS (PBS-BSA) at 37°C for 10 min prior to the incubation with 50 gl of the mosquito lysate per well at 37°C for another 10 min. Five PBS-Tween washes were followed with an incubation with 50 lal per well of horseradish peroxidase (HRP)-conjugated SP3-E9 (Wilson and Nakane, 1978) 1:1000 in PBS-BSA, at 37°C for 10 min. After further washings with PBS-Tween, 150 lal substrate solution (50 ml 20 mM tetramethylbenzidine in acetone/ ethanol (1:9) plus 2.5 ml 0.2 M potassium citrate (pH 3.95), and 10 tal 30% H 2 0 2 ) , were added per well. The staining reaction was stopped after 3 min with 75 lal 0.5 M H 2 S O 4 in H20. The absorption was measured in a spectrophotometer at a wavelength of 450 nm. As a control 1600, 800, 400, 200, 100 and 50 NF54 sporozoites per well were used. Western blot Protein probes were electrophoretically separated on a 12.5% SDS-PAGE (Lfiemmli et al., 1970) and then transferred to nitrocellulose (Takacs, 1979) using a Bio-Rad blotting system. The nitrocellulose, cut into 0.5 cm strips, pretreated twice in buffer
260
(0.05 M Tris-HC1, 0.14 M NaC1, 5 mM EDTA, 0.05% NP-40, 0.25% gelatin and 1% BSA (pH 7.3)= Tris-HC1-B-buffer) for at least 1 h before the strips were incubated overnight in mAbs or test sera diluted i :200 in same buffer. The blots, washed again in Tris-HC1-B-buffer, were then reacted with conjugated rabbit anti-mouse Igperoxidase (Nordic) to localize mAb bindings or goat anti-human Ig-peroxidase (Nordic) after treatment with human serum. The substrate 4-chloro-l-naphthol (0.2 mg/ml; Sigma) with 0.005% H 2 0 2 w a s used for the visualisation of the recognized antigens. Four human sera were tested for their reactivity with the following CS polypeptides, which were expressed as fusion proteins with mouse dihydrofolate reductase (DFHR) linked to six adjacent histidine residues (Stfiber et al., 1990) on Western blots: (NANP)I 9, CS 1A, CS F4, and CS VI.
Immunoo, tochemistry Salivary glands of P. falciparum-infected A. stephensi were dissected PBS 16 and 20 days after the infective bloodmeal. Fixation with 0.5% glutaraldehyde in 0.2 M Pipes buffer (pH 7.2) for 90 min at RT was followed by a blocking of free aldehyde groups with 0.5 M NH4CI in Pipes for 1 h and washing in buffer overnight. Dehydration in graded ethanol at decreasing temperatures, penetration with the embedding medium, Lowicryl K4M, and polymerisation was performed according to routine procedures (Kellenberger et al., 1980). Colloidal gold particles of 10 nm diameter were produced by reduction of tetrachloroauric acid with sodium citrate and tannic acid (Slot and Geuze, 1985). The gold particles were conjugated with the secondary antibody (De Mey, 1983), rabbi! anti-mouse immunoglobulin antiserum (Dakopatts, Denmark), for the demonstration of mAb bindings and protein A (Pharmacia, Sweden) (Romano and Romano. 1977), for the demonstration of bound human antibodies. Thin sections (50 70 nm) mounted on parlodion-carbon coated copper grids were used for the indirect immunocytochemical localization of antibody binding sites. Sections were preincubated in 5% fetal bovine serum (FBS) in 20 mM PBS for 2 h. then reacted for 2 h with the primary monoclonal antibody or the antiserum diluted in the same buffer. The bound antibodies were visualised by incubation of the sections either in immunogold or protein A-gold solution (A52 s 0.5) for 1 h. Aftei each step the grids were thoroughly washed by floating them on several drops ol buffer and then by rinsing with water. Before observation in the electron microscope~ the sections were stained with uranyl acetate and lead citrate.
Results
Reaction of mAbs with P. falciparum NF 54 sporozoites Immunocytochemical demonstration of the reaction sites of the mAbs on thir sections of NF54 sporozoites revealed different characteristic labelling pattern, (Table 1). MAb SP3-E9 reactive to the NANP domaine bound to the sporozoit~ surface as well as to cytoplasmic structures, predominantly micronemes and rhoptrie,~ (Fig. 1). Cytoplasmic structures of the salivary gland epithelium were labelled onl)
261 TABLE 1 C o m p a r i s o n of the i m m u n o r e a c t i v i t i e s of m A b s to CS peptides a g a i n s t P. falciparum isolates ( I F A 5 and I F A 6) f r o m a h y p e r e n d e m i c area a n d N F 54 in indirect i m m u n o c y t o c h e m i c a l labellings + + , strong binding; + , w e a k binding;
, no binding; n.d., not done.
N a m e s of m A b
Epitope
IFA 5
IFA 6
N F 54
SP3-E9 CS VI-6 CS VI-4 CS VI-2 CS F Y IF1 CS F Y 4 G 7 CS VI-3
(NANP)19 CS VI CS VI CS VI CS F4 CS F4 DHFR
+ + + + + n.d. n.d.
+ + + + + n.d. n.d. n.d.
+ + + + + -
in infected glands (Fig. 1). MAbs CS VI-4 and CS VI-6 to epitopes located at the C-terminal end of CSP bound to the sporozoite surface (Fig. 2), the latter at an obviously higher density. The number of gold particles on intracellular structures was too low to decide whether they represent a real antibody/antigen reaction or unspecific background. No binding to sectioned sporozoites could be found with two mAbs CS FY-1F1 and CS FY-4G7 against epitopes at the N-terminal end of the CSP. The mAb CS VI-3 produced against the carrier protein D H F R for the immunization of mice with CS-protein subunits was used as a control. It never reacted either with parasite structures or mosquito tissues.
Reactivity of mAbs to sporozoites of P. falciparum isolates from a hyperendemic area To select the glands for EM preparation thoraces and abdomens of individual mosquitoes were checked for the presence of CSP by ELISA. About 18% of the mosquitoes with IFA 5 and 30% with IFA 6 gave positive results. The salivary glands of the mosquitoes with the highest absorbance values in the ELISA were processed for immunoelectron microscopy. The binding sites of mAbs to 5 different epitopes of N F 54 CS-protein were studied on thin sections (Table 1). The antibodies recognizing the repetitive (SP3 E9) and the C-terminal end (CS VI-6) of the CSP gave the same results for the two IFA strains of P. falciparum sporozoites as for N F 54 (Table 1). No binding to IFA 5 was found with mAb CS FY-IF1. In contrast to N F 54 (Fig. 4), CS VI-2 reacted with surface antigens of IFA 5 and 6 (Fig. 3), while CS VI-4 did not recognize the sporozoite surface in IFA 5 and 6.
Reaction of human sera with CSP and CSP domains Ten ELISA positive and 10 ELISA negative sera (using (NANP)4 o as antigen, Del Guidice et al., 1986) were selected out of 217 human sera for characterization by indirect immunofluorescence antibody test, Western blot and immunocytochemistry (Table 2). Two sera of each group (1 and 6; 11 and 16) will be described in detail in this study.
262 Sera 11 and 16, although negative in ELISA, showed a positive fluorescence in the IFAT on air-dried sporozoites up to a dilution of 1 in 320. Serum 1 bound extensively to (NANP)19, CS VI, and CS 1A. Serum 6 reacted with the fusion protein CS F4 rather than CS 1A as compared to serum 1. The two
263 TABLE 2 Reaction of human sera from a hyperendemicarea with different CS-protein domains - A characterization by indirect immunofluorescence antibody test (IFAT), Western blot (WB) and immunocytochemistry (ICC) E + = ELISA OD > 0.3 (Del Giudice et al., 1986); a: highest dilution showing a positive fluorescence on air dried sporozoites; + + +, strong labelling; + +, medium labelling; +, weak labelling; + / - , uncertain weak labelling. ELISA
Human serum
WB CS 1A
WB CS VI
WB CS F4
WB (NANP)
IFAT"
ICC
E+
1 6
++ +
+++ +++
+ +++
+++ +++
1/1280 1/1280
+++ ++
E-
I1 16
++
+++ ++
++ ++
_+ +
1/320 1/320
+++ +
E L I S A negative sera 11 a n d 16 showed a very weak b i n d i n g to ( N A N P ) I 9 o n Western blots as c o m p a r e d to the E L I S A positive sera or were negative. They b o u n d to the fusion proteins CS F4 a n d CS VI (serum 11), a n d in addition, CS 1A, for serum 16 (Figs. 5 a n d 6). All sera tested showed a weak reaction with D H F R alone. As a c o n t r o l the m A b s specific for the blotted CSP a n d m A b CS VI-3 to D H F R were applied. The m A b s to the CS polypeptides b o u n d specifically to the respective peptides, whereas CS VI-3 recognized D H F R in all fusion protein. The selected h u m a n sera also reacted with thin sections o f salivary glands infected with P. falciparum N F 54. Serum 1 recognized epitopes on the sporozoite surface as well as cytoplasmic structures (Fig. 7). This reaction could be drastically reduced either by p r e - i n c u b a t i o n of the sections with the m A b SP3-E9 ( N A N P ) or by adding the synthetic peptide ( N A N P ) 5 o to the serum prior to its application to the sections (Fig. 8). The same d i s t r i b u t i o n of the m a r k e r b u t in a lower density, was achieved with serum 6 (Fig. 11). Serum 11 b o u n d p r e d o m i n a n t l y to cytoplasmic structures, m i c r o n e m e s a n d rhoptries, of sporozoite sections. Only a few gold particles were located on the parasite surface (Fig. 9). W i t h this serum, the labelling density could n o t be reduced by i n c u b a t i o n o f the serum with ( N A N P ) 5 o or by blocking the antigen with the m A b SP3-E9 (Fig. 10). Serum 16 which reacted with epitopes located in the flanking regions of the CSP on Western blots, gave only a very weak labelling o n sporozoites, n o t showing any clear association with particular structures (Fig. 12). All four sera b o u n d to the salivary gland epithelium of infected mosquitoes, however i n d e p e n d e n t from their reactivity for N A N P . In a final step, the reaction o f the h u m a n sera with thin sections of the P.falciparum
Figs. 1-4. Plasmodiumfalciparumsporozoites in salivary glands of Anophelesstephensi. Binding sites of mAbs visualized with rabbit anti-mouse Ig-gold. E = salivary gland epithelium, S = sporozoite, SV = secretory vacuole, arrowhead = microneme/rhoptry system, all bars indicate 0.5 gm. Fig. I. Strain NF 54, mAb SP3 E9 produced against (NANP)50. Sporozoites, surface and cytoplasmic structures, and gland epithelium are labelled. Fig. 2. Strain NF 54, mAb CS V1-6 produced against non-repetitive epitope at the C-terminal end of the CS-protein. Specific labelling of the sporozoite surface. Fig. 3. Strain IFA 6, mAb CS VI-2 produced against non-repetitive epitope at the C-terminal end of the CS-protein. Surface labelling of sporozoites. Fig. 4. Same antibody as Fig. 3, but reacted with strain NF 54. Sporozoite surface not labelled.
264
i ¸i
il
CS F4
im
CS VI CS 1A
il ~,ii! i
~iiii~i~i:~: i~ ~i ~ ~
ili ii ~'~i~
NANP
iiiii~iil~ ~ .
u
DHFR
DHFR
i zi!
1 6 11 16 K1 K2 K3 K4 sera mAbs
1 6 11 16 K 1 K 2 K 3 K 4 sera mAbs
Figs. 5 and 6. Reactivity on Western blots of h u m a n sera (1, 6, 11, 16) from an area endemic for malaria (Ifakara, Tanzania) against various D H F R - f u s i o n proteins (Stfiber et al., 1990) representing epitopes of the non-repetitive (Fig. 5) and repetitive (Fig. 6) region of the PlasmodiumJalciparum CSP. K 1 m A b CS VI-3 (anti D H F R ) , K z m A b CS V|-6, K 3 m A b CS FY-I FI, K 4 m A b SP3 E9, CS 1A = C-terminal epitope of CSP.
isolates IFA 5 and IFA 6 in A. stephensi salivary glands was compared to their reaction with N F 54 sporozoites. These labelling experiments demonstrated similar binding sites for the human sera on both isolates as for N F 54: surface and cytoplasm for sera 1 and 6, predominantly cytoplasm for sera 11 and 16. The marker densities, which were high for the sera 1 and 11 and low for the sera 6 and 16, also corresponded to the results for N F 54.
Discussion For P. berghei (Aikawa et al., 1981) and for P. falciparum (Posthuma et al., 1987) the sporozoites within the mosquito salivary glands are completely covered by CS protein. This was confirmed with mAb SP3-E9 (to the repetitive domain) and the mAbs to epitopes of the flanking region at the C-terminal end of the CS protein. The difference between the antibodies to the repeats and those to the flanking region was in the labelling of the cytoplasm. The SP3-E9 obviously recognized epitopes on cytoplasmic structures, probably micronemes and rhoptries, as has been shown for P. knowlesi (Fine et al., 1984). It is assumed that the molecules which reacted are intracellular precursors of the CS protein (Yoshida et al., 1981; Cochrane et al., 1982; Nardin et al., 1982). Why the antibodies to three different epitopes of the C-terminal region only recognized intracellular antigens poorly or not at all cannot be answered with the present study. The N A N P region is immunodominant (Dame et al., 1984; Zavala et al., 1985), and consequently a fewer epitopes of the flanking regions will be exposed at the parasite surface. An intracellular masking of the binding region by amino acids could be another explanation for the lack of reactivity (Posthuma et al., 1987). Finally, the steric configuration of the intracellular domains
265
Figs. 7-12. Reactivity of human sera from an area endemic for malaria against Plasmodiumfalciparum (NF 54) sporozoites in salivary glands of Anopheles stephensi. S = sporozoite, SV= secretory vacuole, arrowhead=microneme/rhoptry system, all bars indicate 0.5 jam. Fig. 7. Human serum 1, positive in ELISA against (NANP)4o, 1:500. Fig. 8. Human serum 1 inhibited with synthetic peptide (NANP)5 o. Fig. 9. Human serum 11, negative in ELISA against (NANP)4o, 1:500. Fig. 10. Human serum I 1 inhibited with synthetic peptide (NANP)s o. Fig. 11. Human serum 6, positive in ELISA against (NANP)4o, 1:500. Fig. 12. Human serum 16, negative in ELISA against (NANPLo , 1:500.
266 at the C-terminal end of the CS protein could be different from that being exposed after the integration of the protein into the membrane. MAb CSVI-2 did not react with NF54 sporozoites but with those of the strai~ Ro 59 (Stfiber et al., 1990), while mAbs to epitopes at the N-terminal end (CS FYIF1 and CS FY-4G7) also did not bind in immunocytochemical experiments. In the case of the epitope CSVI-2 a strain specificity of the respective domain is describec (Stiiber et al., 1990), whereas the same arguments as mentioned above for the mAb., to C-terminal epitopes could be valid for the lack of intracellular binding. Thougl~ the N-terminal region is exposed on the parasite surface our results suggest eitheJ masking of the epitopes by overlapping of the molecules or an insufficient preservation of the protein during preparation for electron microscopy is responsible for the negative results. The same antibodies also did not react with the parasite-derivec CS protein in Western blots but with the native protein at the parasite surface a,~ measured by IFAT and with recombinant proteins (Stfiber et al., 1990). All mAbs which bound to CS protein on sporozoites also gave a labelling in th( cytoplasm of infected salivary gland epithelium. This probably results from th( shedding of CS protein during penetration of the epithelium as has been repeatedl5 demonstrated in vivo and in vitro (Posthuma et al., 1987b; Hamilton et al., 1988 Boulanger et al., 1988; Stewart and Vanderberg, 1988; Golenda et al., 1990). Area~ between the parasites binding antibodies to repetitive domains as well as non repetitive C-terminal domains indicate that the shedding, most recently shown tc begin with the secretion of the protein at the anterior end of the sporozoite (Stewar and Vanderberg, 1991), continues within the secretory vacuoles of the salivary glanc cells. The demonstration by ELISA of CSP released in the haemolymph durin~ migration can be used to identify infected mosquitoes, e.g., to select infected salivar~ glands as in the present study. The antibodies applied to P. falciparum NF54 sporozoites were also reacted witt thin sections of salivary gland sporozoites of the two field isolates IFA 5 and IF~ 6. Similar results between strains were observed with the mAbs SP3-E9 (NANP and CSVI-6. This fits with earlier findings describing the absence of antigenic variation for the repetitive domain of P. falciparum CS protein (Arnot, 1990) and conservative character of the CS VI-6 epitope (Stiiber et al., 1990). Just the opposit~ was found with the other two mAbs, CS CI-2 and CS VI-4, to carboxy-termina epitopes. CS VI-2 bound to the sporozoite surface of the isolates IFA 5 and IFA ( but not to N F 54 sporozoites and vice versa for CS VI-4. The immunocytochemica labellings therefore support the assumed strain specificity of part of the epitopes ii the flanking region studies (Stiiber et al., 1990). The possibility of specific recognition by human sera of the epitopes recognizec by the mAbs was examined by immunocytochemical techniques. Most of antibodie produced after immunization of humans with sporozoites are directed against th~ repetitive (NANP)3 domain of the CSP (Zavala et al., 1983; Zavala et al., 1985) However, there is also immune reactivity of human sera with non-repetitive sequence of the CSP ofP.falciparum (Del Giudice et al. 1988; Stiiber et al., 1990) and P. viva: (Romero et al., 1987). Out of a big series of human sera from Ifakara, Tanzania four have been compared in this study. Two of them were highly positive in ELIS/ on (NANP)4o (Del Giudice et al., 1986) the other two were negative but all fou sera reacted with air-dried sporozoites in IFAT. Thus, the lack of antibodies to th, repetitive sequence is not a reliable criterion for the absence o f antibodies t~
267
P. falciparum sporozoites in human sera. The reaction of the same sera on Western blots with DHFR-fusion peptides representing sequences of the CS protein confirmed the ELISA-results for the recognition of the NANP-epitopes. From the four sera selected for a characterization with different techniques, those two, which were negative in ELISA but positive in IFAT, did not recognize the repetitive domain, but at least one epitope of the non-repetitive region. The four sera tested yielded four different combinations of reactivity against fusion peptides of the flanking regions. All of them bound to CS VI, representing a sequence at the C-terminal end. This resulted in a more or less dense surface labelling of sporozoites in immunocytochemical experiments, which could neither be inhibited by blocking the antigens with the mAb SP3-E9 to the repetitive domain nor by adding (NANP)5o to the serum. In addition, there was always a reaction with at least one of the fusion peptides CS IA, another C-terminal epitope, and/or CS F4, representing the N-terminal region. The results achieved for sera 1 and 6 by immunogold labellings on salivary gland sporozoites correspond to the reactions on Western blots and to the binding sites found for the mAbs produced against the CS sequences which have been used for the blots. However, it is not possible to sort out how far antibodies to the flanking regions have contributed to the labelling. The inhibition experiments to eliminate the binding to (NANP), showed that the antibodies to this epitope are clearly the dominant part of the antibody-content in the serum. A demonstrated crossreactivity of mAbs to N A N P with the CS VI region (Stfiber et al., 1990) may be responsible for a reduction of the surface labelling to a lower density than has been shown for mAb CS VI-6. The binding of serum 11 to N F 54 sporozoites could not be inhibited by preincubation of the serum with (NANP)5 o. Thus, the presence of antibodies to the repetitive domain is excluded. On the other hand, serum 11 cannot contain antibodies reactive against the epitope CS F4, the mAb to this epitope did not bind in immunocytochemical labellings on N F 54, and against the epitope CS VI, this epitope has been shown to be predominantly located on the sporozoite surface. Therefore, it can be concluded that serum 11 recognizes at least one additional epitope, which is responsible for a conspicuous labelling of cytoplasmic structures. Serum 16 contained antibodies to the epitopes CS VI, CS 1A, and CS F4, and gave a very weak labelling of the parasite surface and intracellular organelles. Compared to results with the other sera, labelling of the CS VI epitopes on the parasite surface could be expected, whereas intracellular binding occurred possibly due to a recognition of the CS 1A region or to binding of antibodies to other epitopes than those dealt with in the present paper. Finally, the application of the four human sera to thin sections of salivary gland sporozoites from the P.falciparum isolates IFA 5 and IFA 6 did not result in labelling patterns qualitatively different from those on N F 54 sporozoites. Whether this is due to the presence of identical epitopes in all three strains or due to the presence of antibodies to different antigen types in the human sera cannot be concluded. Positive results in IFAT for the reactivity of human sera against air dried sporozoites do not imply that antibodies to the repetitive domain of the CS protein are present. It has been shown by immunogold labellings, that there are responses to epitopes in the flanking regions of the CSP in residents of an area hyperendemic for malaria. Given the immunodominance of the repetitive domains (Zavala et al., 1985), and the existence of sera containing antibodies exclusively to non-repetitive epitopes
268 it can be assumed, that these antibodies persist for a longer period t h a n those to th( repeats. Epitopes at the c a r b o x y - t e r m i n a l end are exposed at the sporozoite surface whereas the epitopes at the a m i n o - t e r m i n a l end seem n o t to be accessible.
Acknowledgments We wish to t h a n k Mrs. E. Fluri, Mrs. C. Kn611 a n d Mr. X. Petitjean for their skillfu technical assistence a n d Dr. P.F. Billingsley, Imperial College for Science a n d Technology, L o n d o n , for critical discussion of the manuscript. We are grateful tc Prof. W.L. K i l a m a , director general of the T a n z a n i a n N a t i o n a l Institute for Medical Research, for his s u p p o r t of the field studies within the K i l o m b e r o Health ResearclP r o g r a m m e that is supported by grants from the Swiss D e v e l o p m e n t C o - o p e r a t i o n Research clearance was granted by the T a n z a n i a n C o m m i s s i o n for Science anc Technology (reference R A 47/307).
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270 Posthuma, G., Slot, J.W. and Geuze, H.J. (1987) Usefulness of the immunogold technique in quantitatic of a soluble protein in ultra-thin sections. J. Histochem. Cytochem. 35 (No. 4), 405 410. Posthuma, G., Meis, J.F.G.M., Verhave, J.P., Hollingdale, M.R., Ponnudurai, T., Meuwissen, J.H.E. ~ and Geuze, H.J. (1988) Immunogold localization of circumsporozoite protein of the malaria parasi Plasmodiumfalciparum during sporogony in Anopheles stephensi midguts. Eur. J. Cell Biol. 46, 18-2 Romano, E.L. and Romano, M. (1977) Staphylococcal Protein A bound to colloidal gold: A usef~ reagent to label antigen-antibody sites in electron microscopy. Immunochemistry 14, 711. Romero, P., Heimer, E.P., Herrera, S., Felix, A.M., Nussenzweig~ R.S. and Zavala, F. (1987) Antigen analysis of the repeat domain of the circumsporozoite protein of Plasmodium vivax. J. Immunol. 13' 1679-1682. Sharma, S., Gwadz, R.W., Schlesinger, D.H. and Godson, G.N. (1986) lmmunogenicity of the repetiti~ and the nonrepetitive peptide regions of the divergent CS protein of Plasmodium kno~lesi. J. Immune 137, 357 361. Sinigaglia, F., Guttinger, M., Gillessen, D., Doran, D.M., Takacs, B., Matile, H., Trzeciak, A. and Pinl J.R. (1988) Epitopes recognized by human T lymphocytes on malaria circumsporozoite-protein. Eu J. lmmunol. 18, 633-636. Slot, J.W. and Geuze, H.J. (1985) A new method of preparing gold probes for multiple-labeling cytochen istry. Eur. J. Cell Biol. 38, 87 93. Spitalny, G., Verhave, J.P., Meuwissen, J.H.E.Th. and Nussenzweig, R.S. (1977) Plasmodium berghe T cell dependence of sporozoite-induced immunity in rodents. Exp. Parasitol. 42, 73-81. Stahel, E., Tanner, M., Gyr, N.~ Weiss, N., Hess, Ch. and Betschart, B. (1984) Seroepidemiology ( hepatitis B virus in rural Tanzania: Relation of hepatitis B markers and liver function tests in patien~ with clinical liver disease and in healthy controls. East Afr. Med. J. 61,806 811. Stewart, M.J. and Vanderberg, J.P. (1988) Malaria sporozoites leave behind trails of circumsporozoil protein during gliding motility. J. Parasitol. 35, 389. Stewart, M.J. and Vanderberg, J.P. (1991) Malaria sporozoites release circumsporozoite protein froI their apical end and translocate it along their surface. J. Protozool. 38, 411 421. Stfiber, D., Bannwarth, W., Pink, J.R.L., Meloen, R.H. and Matile, H. (1990) New B cell epitopes i the Plasmodiumfalciparum malaria circumsporozoite protein. Eur. J. Immunol. 20, 819 824. Takacs, B. (1979) Electrophoresis of proteins in polyacrylamide slab gels. In: I. Lefkovits and B. Pern: (eds.), Immunological Methods, Vol. 1, Academic Press, New York, pp. 81--105. Tanner, M., Del Giudice, G., Betschart, B., Biro, S., Burnier, E., Degr6mont, A., Engers, H.D., Freyvoge T.A., Lambert, P.H., Pessi, A., Speiser, F., Verdini, A.S., and Weiss, N. (1986) Malaria transmissio and development of anti-sporozoite antibodies in a rural african community. Mere. Inst. Oswaldo Cru 81, (Suppl. II), 199 205. Vergara, U., Gwadz, R., Schlesinger, D., Nussenzweig, V. and Ferreira, A. (1985b) Multiple non-repeate epitopes on the circumsporozoite protein of Plasmodium knowlesi. Molec. Biochem. Parasitol. 1~ 283 292. Vergara, U., Ruiz, A., Ferreira, A., Nussenzweig, R.S. and Nussenzweig, V. (1985a) Conserved grout" specific epitopes of the circumsporozoite proteins revealed by antibodies to synthetic peptide~ J. Immunol. 134, 3345 3348. WHO (1988) Vaccination Certificate Requirements and Health Advice for International Travel. WHC Geneva. Wilson, M.B. and Nakane, P.K. (1976) In: W. Knapp, K. Holubar and G. Wick (Eds.), Immune fluorescence and related staining techniques, Elsevier/North-Holland, Amsterdam, pp. 215 224. Young, J.F., Hockmeyer, W.T., Gross, M., Ballou, W.R., Wirtz, R.A., Trosper, J.H., Beaudoin, R.L Hollingdale, M.R., Miller, L.H., Diggs, C.L. and Rosenberg, M. (1985) Expression of Plasmodiut circumsporozoite proteins in Escherichia coli for potential use in humane malaria vaccine. Science 22~ 958 962. Yoshida, N., Potocnjak, P., Nussenzweig, V. and Nussenzweig~ R.S. (1981) Biosynthesis of Pb44~ th protective antigen of sporozoites of Plasmodium berghei. J. Exp. Med. 154, 1225 1236. Zavala, F., Cochrane, A.H., Nardin, E.H., Nussenzweig, R.S. and Nussenzweig, V. (1983) Circumspore zoite proteins of malaria parasites contain a single immunodominant region with two or more identic~ epitopes. J. Exp. Med. 157, 1947 1957. Zavala, F., Tam, J.P., Cochrane, A.H., QuakyL I., Nussenzweig, R.S. and Nussenzweig, V. (1985 Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science 228 1436 1440. Zavala~ F., Tam, J.P. and Masuda, A. (1986) Synthetic peptides as antigens for the detection of humora immunity to Plasmodiumfalciparum sporozoites. J. Immunol. Methods 93, 55 61.
257
ACTROP 00225
Distribution of repetitive and non-repetitive circumsporozoite protein epitopes on Plasmodium falciparum sporozoites and immunochemical characterization of human malaria antisera W. R u d i n , " Ch. P e t i g n a t , a M . T a n n e r a a n d H. M a t i l e b "Swiss Tropical Institute, CH-4002 Basel, Switzerland and bNew Technologies Biology, E Hoffmann-La Roche Ltd., Basel, Switzerland (Received 4 March 1992; accepted 12 May 1992) The presence and distribution of circumsporozoite protein (CSP) epitopes located in the repetitive and non-repetitive regions were studied in three Plasmodiumfaleiparum strains, NF54, IFA5 and IFA6. It was found by immunofluorescence, Western blotting and immunoelectron microscopy that mAbs to epitopes of the repetitive domaine bound similarly to the CSP of all three strains. MAbs to epitopes of the flanking regions yielded either some strain differences (mAbs to the C-terminal end), or reacted only in immunofluorescence tests on whole sporozoites (mAbs to the N-terminal end). Human sera from an area endemic for malaria, two of them positive in ELISA on (NANP)4o and two negative, were tested for their reactivity with epitopes of the flanking regions of the CSP. The presence of antibodies to such epitopes could be demonstrat6d by Western blots and immunocytochemistry independent of the reactivity of the sera to recognize (NANP)40. All tested bound to salivary gland tissues but not to their secretory product in immunocytochemical experiments. Key words: P.falciparum; Sporozoites; CSP epitopes; Human serum
Introduction T h e m a i n surface a n t i g e n o f the s p o r o z o i t e s o f Plasmodium species, is the CSP, which h o m o g e n e o u s l y covers the s p o r o z o i t e surface ( A i k a w a et al., 1981; Y o s h i d a et al., 1981). T h e C S P is expressed 6 d a y s after infection o f the m o s q u i t o on the p l a s m a l e m m a o f oocysts ( P o s t h u m a et al., 1988). O n P . f a l c i p a r u m s p o r o z o i t e s C S P consists o f 37 repeats o f the i m m u n o d o m i n a n t t e t r a p e p t i d e A s n - A l a - A s n - P r o ( N A N P ) a n d 4 repeats o f the sequence A s n - V a l - A s p - P r o ( N V D P ) ( D a m e et al., 1984; E n e a et al., 1984). ( N A N P ) 3 represents the i m m u n o d o m i n a n t d o m a i n ( Z a v a l a et al., 1985) a n d does n o t show v a r i a t i o n a m o n g s t P. falciparum isolates ( A r n o t , 1990). T h e repetitive d o m a i n e is flanked on b o t h sides by c o n s e r v e d regions in all cloned a n d sequenced genes o f the CS p r o t e i n o f v a r i o u s Plasmodium species ( M c C u t c h a n et al., 1988) a n d by variable regions as described for P. falciparum ( L o c k y e r et al., 1989). Correspondence to." W. Rudin, Swiss Tropical Institute, Postfach, CH-4002 Basel, Switzerland; Tel. (061) 284 82 41; Fax, (061) 271 86 54.
258 Antibodies recognizing the synthetic peptide (NANP)3 neutralize sporozoite infectivity in vitro (Hollingdale et al., 1984; Mazier et al., 1986; Zavala et al., 1986). Irradiated sporozoites can protect rodents, monkeys and men from malaria infections, and the repetitive domain of the CSP is thought to be involved in the interaction between sporozoites and hepatocytes (Nussenzweig and Nussenzweig, 1985). However, the efforts to develop a vaccine using (NANP), sequences have not been successful, because only weak T-cell induced immune responses of short duration were achieved (Ballou et al., 1987; Herrington et al., 1987; Etlinger et al., 1988). Synthetic peptides of the non-repetitive sequence are immunogenic in laboratory animals (Ballou et al., 1985; Vergara et al., 1985a,b; Sharma et at., 1986), while antisporozoite antibodies recognizing epitopes of the flanking regions were also found in sera of people living in areas endemic for malaria (Romero et al., 1987; Del Guidice et al., 1988; Stfiber et al., 1990). This is of interest because T helper cells have been shown to be necessary to reach high antibody titers against sporozoites (Spitalny et al., 1977) and that the CSP possesses T cell epitopes in these nonrepetitive regions (Good et al., 1988a,b; Kumar et al., 1988; Sinigaglia et al., 1988). In the present study the binding of mAbs to various CSP epitopes on P.falciparum sporozoites in salivary glands were localized immunocytochemically. These binding sites were compared between the P. falciparum strain NF54 and two field isolates, IFA 5 and IFA 6. In addition, human sera from a malaria endemic area have been tested for antibodies to repetitive and non-repetitive regions of the CSP for the first time.
Material and Methods
Parasites and vector Plasmodiumfalciparum NF54 (Ponnudurai et al., 1981) was used for the immunocytochemical characterization of a series of mAbs to peptides of the CSP. NF54 together with the human isolates P. falciparum IFA 5 and IFA 6 from St. Francis District Hospital Ifakara, Tanzania (Hurt, 1990; Huber, 1991) was used to study the reactivity of various human sera. Anopheles stephensi were obtained from H. Briegel in 1983 and since then bred at Hoffmann La Roche. Mosquitoes were infected 2 to 4 days after emergence with Plasmodium falciparum produced in vitro and fed using membrane feeders (Ponnudurai et al., 1989a). Infected mosquitoes were kept at 80% RH and 27°C until use. Circumsporozoite peptides and fusion proteins The CS peptide (NANP)4 o (Del Giudice et al., 1986) was used for ELISAs to check human sera for antibodies to the repetitive CS protein domain. For immunoblotting, CS fusion proteins expressed in E. coli were used which consisted of repetitive or non-repetitive parts of the CS protein and mouse dihydrofolate reductase (DHFR) linked to six adjacent histidine residues (Stfiber et al., 1990).
Monoclonal antibodies The following mAbs were used in the present study (Table 1): SP3-E9 reactive to NANP epitopes (Boulanger et al., 1988), CS VI-2, CS VI-4, and CS VI-6 reactive
259 to sequences of the non-repetitive region at the C-terminal end of the CS protein (Stiiber et al., 1990), CS FY-IF1, CS FY-4G7 to sequences of the non-repetitive region at the N-terminal end of the CS protein. The mAb Cs VI-3 against D H F R served as a negative control in immunocytochemical experiments. Human sera The human sera tested for their reactivity with CS fusion proteins and sporozoites were collected during the course of a hospital-based study on malaria-resistance and hepatic diseases at St. Francis District Hospital, Ifakara, Tanzania in 1982 (Stahel et al., 1984). The sera originate from an area hyperendemic for malaria with an entomological inoculation rate between 0.7 and 3.0 infectious bites per person per night (Tanner et al., 1986). Indirect immunofluorescence assay ( IFA ) An indirect immunofluorescence assay (Young et al., 1985) was used to estimate the antibody titer of the human sera. About 2000 isolated sporozoites in 5 gl PBS were applied per test area, air dried and stored at - 70°C until use. The following dilutions of the sera were applied in 13 gl aliquots per test area: 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280. Fluorescein isothiocyanate (FITC) conjugated goat anti-human immunoglobulins (Ig; 1:40) (Nordic Immunological Laboratories, The Netherlands) was used as a marker. Enzyme-linked immunosorbent assay ( ELISA ) After disection of the salivary glands, the thorax and abdomen of each mosquito were ground in 200 lal lysis buffer (0.5% bovine serum albumin (BSA), 0.5% Nonidet P-40 (NP-40) in phosphate-buffered saline (PBS, pH 7.2)) with a glass pestel. ELISA plates (Nunc immunoplate maxisorb) coated overnight with 10 tag/ml SP3-E9 in PBS, 100 gl per well at 4°C were rinsed three times with 0.05% Tween 20 in PBS (PBSTween) and pretreated with 100 gl/well 0.5% BSA in PBS (PBS-BSA) at 37°C for 10 min prior to the incubation with 50 gl of the mosquito lysate per well at 37°C for another 10 min. Five PBS-Tween washes were followed with an incubation with 50 lal per well of horseradish peroxidase (HRP)-conjugated SP3-E9 (Wilson and Nakane, 1978) 1:1000 in PBS-BSA, at 37°C for 10 min. After further washings with PBS-Tween, 150 lal substrate solution (50 ml 20 mM tetramethylbenzidine in acetone/ ethanol (1:9) plus 2.5 ml 0.2 M potassium citrate (pH 3.95), and 10 tal 30% H 2 0 2 ) , were added per well. The staining reaction was stopped after 3 min with 75 lal 0.5 M H 2 S O 4 in H20. The absorption was measured in a spectrophotometer at a wavelength of 450 nm. As a control 1600, 800, 400, 200, 100 and 50 NF54 sporozoites per well were used. Western blot Protein probes were electrophoretically separated on a 12.5% SDS-PAGE (Lfiemmli et al., 1970) and then transferred to nitrocellulose (Takacs, 1979) using a Bio-Rad blotting system. The nitrocellulose, cut into 0.5 cm strips, pretreated twice in buffer
260
(0.05 M Tris-HC1, 0.14 M NaC1, 5 mM EDTA, 0.05% NP-40, 0.25% gelatin and 1% BSA (pH 7.3)= Tris-HC1-B-buffer) for at least 1 h before the strips were incubated overnight in mAbs or test sera diluted i :200 in same buffer. The blots, washed again in Tris-HC1-B-buffer, were then reacted with conjugated rabbit anti-mouse Igperoxidase (Nordic) to localize mAb bindings or goat anti-human Ig-peroxidase (Nordic) after treatment with human serum. The substrate 4-chloro-l-naphthol (0.2 mg/ml; Sigma) with 0.005% H 2 0 2 w a s used for the visualisation of the recognized antigens. Four human sera were tested for their reactivity with the following CS polypeptides, which were expressed as fusion proteins with mouse dihydrofolate reductase (DFHR) linked to six adjacent histidine residues (Stfiber et al., 1990) on Western blots: (NANP)I 9, CS 1A, CS F4, and CS VI.
Immunoo, tochemistry Salivary glands of P. falciparum-infected A. stephensi were dissected PBS 16 and 20 days after the infective bloodmeal. Fixation with 0.5% glutaraldehyde in 0.2 M Pipes buffer (pH 7.2) for 90 min at RT was followed by a blocking of free aldehyde groups with 0.5 M NH4CI in Pipes for 1 h and washing in buffer overnight. Dehydration in graded ethanol at decreasing temperatures, penetration with the embedding medium, Lowicryl K4M, and polymerisation was performed according to routine procedures (Kellenberger et al., 1980). Colloidal gold particles of 10 nm diameter were produced by reduction of tetrachloroauric acid with sodium citrate and tannic acid (Slot and Geuze, 1985). The gold particles were conjugated with the secondary antibody (De Mey, 1983), rabbi! anti-mouse immunoglobulin antiserum (Dakopatts, Denmark), for the demonstration of mAb bindings and protein A (Pharmacia, Sweden) (Romano and Romano. 1977), for the demonstration of bound human antibodies. Thin sections (50 70 nm) mounted on parlodion-carbon coated copper grids were used for the indirect immunocytochemical localization of antibody binding sites. Sections were preincubated in 5% fetal bovine serum (FBS) in 20 mM PBS for 2 h. then reacted for 2 h with the primary monoclonal antibody or the antiserum diluted in the same buffer. The bound antibodies were visualised by incubation of the sections either in immunogold or protein A-gold solution (A52 s 0.5) for 1 h. Aftei each step the grids were thoroughly washed by floating them on several drops ol buffer and then by rinsing with water. Before observation in the electron microscope~ the sections were stained with uranyl acetate and lead citrate.
Results
Reaction of mAbs with P. falciparum NF 54 sporozoites Immunocytochemical demonstration of the reaction sites of the mAbs on thir sections of NF54 sporozoites revealed different characteristic labelling pattern, (Table 1). MAb SP3-E9 reactive to the NANP domaine bound to the sporozoit~ surface as well as to cytoplasmic structures, predominantly micronemes and rhoptrie,~ (Fig. 1). Cytoplasmic structures of the salivary gland epithelium were labelled onl)
261 TABLE 1 C o m p a r i s o n of the i m m u n o r e a c t i v i t i e s of m A b s to CS peptides a g a i n s t P. falciparum isolates ( I F A 5 and I F A 6) f r o m a h y p e r e n d e m i c area a n d N F 54 in indirect i m m u n o c y t o c h e m i c a l labellings + + , strong binding; + , w e a k binding;
, no binding; n.d., not done.
N a m e s of m A b
Epitope
IFA 5
IFA 6
N F 54
SP3-E9 CS VI-6 CS VI-4 CS VI-2 CS F Y IF1 CS F Y 4 G 7 CS VI-3
(NANP)19 CS VI CS VI CS VI CS F4 CS F4 DHFR
+ + + + + n.d. n.d.
+ + + + + n.d. n.d. n.d.
+ + + + + -
in infected glands (Fig. 1). MAbs CS VI-4 and CS VI-6 to epitopes located at the C-terminal end of CSP bound to the sporozoite surface (Fig. 2), the latter at an obviously higher density. The number of gold particles on intracellular structures was too low to decide whether they represent a real antibody/antigen reaction or unspecific background. No binding to sectioned sporozoites could be found with two mAbs CS FY-1F1 and CS FY-4G7 against epitopes at the N-terminal end of the CSP. The mAb CS VI-3 produced against the carrier protein D H F R for the immunization of mice with CS-protein subunits was used as a control. It never reacted either with parasite structures or mosquito tissues.
Reactivity of mAbs to sporozoites of P. falciparum isolates from a hyperendemic area To select the glands for EM preparation thoraces and abdomens of individual mosquitoes were checked for the presence of CSP by ELISA. About 18% of the mosquitoes with IFA 5 and 30% with IFA 6 gave positive results. The salivary glands of the mosquitoes with the highest absorbance values in the ELISA were processed for immunoelectron microscopy. The binding sites of mAbs to 5 different epitopes of N F 54 CS-protein were studied on thin sections (Table 1). The antibodies recognizing the repetitive (SP3 E9) and the C-terminal end (CS VI-6) of the CSP gave the same results for the two IFA strains of P. falciparum sporozoites as for N F 54 (Table 1). No binding to IFA 5 was found with mAb CS FY-IF1. In contrast to N F 54 (Fig. 4), CS VI-2 reacted with surface antigens of IFA 5 and 6 (Fig. 3), while CS VI-4 did not recognize the sporozoite surface in IFA 5 and 6.
Reaction of human sera with CSP and CSP domains Ten ELISA positive and 10 ELISA negative sera (using (NANP)4 o as antigen, Del Guidice et al., 1986) were selected out of 217 human sera for characterization by indirect immunofluorescence antibody test, Western blot and immunocytochemistry (Table 2). Two sera of each group (1 and 6; 11 and 16) will be described in detail in this study.
262 Sera 11 and 16, although negative in ELISA, showed a positive fluorescence in the IFAT on air-dried sporozoites up to a dilution of 1 in 320. Serum 1 bound extensively to (NANP)19, CS VI, and CS 1A. Serum 6 reacted with the fusion protein CS F4 rather than CS 1A as compared to serum 1. The two
263 TABLE 2 Reaction of human sera from a hyperendemicarea with different CS-protein domains - A characterization by indirect immunofluorescence antibody test (IFAT), Western blot (WB) and immunocytochemistry (ICC) E + = ELISA OD > 0.3 (Del Giudice et al., 1986); a: highest dilution showing a positive fluorescence on air dried sporozoites; + + +, strong labelling; + +, medium labelling; +, weak labelling; + / - , uncertain weak labelling. ELISA
Human serum
WB CS 1A
WB CS VI
WB CS F4
WB (NANP)
IFAT"
ICC
E+
1 6
++ +
+++ +++
+ +++
+++ +++
1/1280 1/1280
+++ ++
E-
I1 16
++
+++ ++
++ ++
_+ +
1/320 1/320
+++ +
E L I S A negative sera 11 a n d 16 showed a very weak b i n d i n g to ( N A N P ) I 9 o n Western blots as c o m p a r e d to the E L I S A positive sera or were negative. They b o u n d to the fusion proteins CS F4 a n d CS VI (serum 11), a n d in addition, CS 1A, for serum 16 (Figs. 5 a n d 6). All sera tested showed a weak reaction with D H F R alone. As a c o n t r o l the m A b s specific for the blotted CSP a n d m A b CS VI-3 to D H F R were applied. The m A b s to the CS polypeptides b o u n d specifically to the respective peptides, whereas CS VI-3 recognized D H F R in all fusion protein. The selected h u m a n sera also reacted with thin sections o f salivary glands infected with P. falciparum N F 54. Serum 1 recognized epitopes on the sporozoite surface as well as cytoplasmic structures (Fig. 7). This reaction could be drastically reduced either by p r e - i n c u b a t i o n of the sections with the m A b SP3-E9 ( N A N P ) or by adding the synthetic peptide ( N A N P ) 5 o to the serum prior to its application to the sections (Fig. 8). The same d i s t r i b u t i o n of the m a r k e r b u t in a lower density, was achieved with serum 6 (Fig. 11). Serum 11 b o u n d p r e d o m i n a n t l y to cytoplasmic structures, m i c r o n e m e s a n d rhoptries, of sporozoite sections. Only a few gold particles were located on the parasite surface (Fig. 9). W i t h this serum, the labelling density could n o t be reduced by i n c u b a t i o n o f the serum with ( N A N P ) 5 o or by blocking the antigen with the m A b SP3-E9 (Fig. 10). Serum 16 which reacted with epitopes located in the flanking regions of the CSP on Western blots, gave only a very weak labelling o n sporozoites, n o t showing any clear association with particular structures (Fig. 12). All four sera b o u n d to the salivary gland epithelium of infected mosquitoes, however i n d e p e n d e n t from their reactivity for N A N P . In a final step, the reaction o f the h u m a n sera with thin sections of the P.falciparum
Figs. 1-4. Plasmodiumfalciparumsporozoites in salivary glands of Anophelesstephensi. Binding sites of mAbs visualized with rabbit anti-mouse Ig-gold. E = salivary gland epithelium, S = sporozoite, SV = secretory vacuole, arrowhead = microneme/rhoptry system, all bars indicate 0.5 gm. Fig. I. Strain NF 54, mAb SP3 E9 produced against (NANP)50. Sporozoites, surface and cytoplasmic structures, and gland epithelium are labelled. Fig. 2. Strain NF 54, mAb CS V1-6 produced against non-repetitive epitope at the C-terminal end of the CS-protein. Specific labelling of the sporozoite surface. Fig. 3. Strain IFA 6, mAb CS VI-2 produced against non-repetitive epitope at the C-terminal end of the CS-protein. Surface labelling of sporozoites. Fig. 4. Same antibody as Fig. 3, but reacted with strain NF 54. Sporozoite surface not labelled.
264
i ¸i
il
CS F4
im
CS VI CS 1A
il ~,ii! i
~iiii~i~i:~: i~ ~i ~ ~
ili ii ~'~i~
NANP
iiiii~iil~ ~ .
u
DHFR
DHFR
i zi!
1 6 11 16 K1 K2 K3 K4 sera mAbs
1 6 11 16 K 1 K 2 K 3 K 4 sera mAbs
Figs. 5 and 6. Reactivity on Western blots of h u m a n sera (1, 6, 11, 16) from an area endemic for malaria (Ifakara, Tanzania) against various D H F R - f u s i o n proteins (Stfiber et al., 1990) representing epitopes of the non-repetitive (Fig. 5) and repetitive (Fig. 6) region of the PlasmodiumJalciparum CSP. K 1 m A b CS VI-3 (anti D H F R ) , K z m A b CS V|-6, K 3 m A b CS FY-I FI, K 4 m A b SP3 E9, CS 1A = C-terminal epitope of CSP.
isolates IFA 5 and IFA 6 in A. stephensi salivary glands was compared to their reaction with N F 54 sporozoites. These labelling experiments demonstrated similar binding sites for the human sera on both isolates as for N F 54: surface and cytoplasm for sera 1 and 6, predominantly cytoplasm for sera 11 and 16. The marker densities, which were high for the sera 1 and 11 and low for the sera 6 and 16, also corresponded to the results for N F 54.
Discussion For P. berghei (Aikawa et al., 1981) and for P. falciparum (Posthuma et al., 1987) the sporozoites within the mosquito salivary glands are completely covered by CS protein. This was confirmed with mAb SP3-E9 (to the repetitive domain) and the mAbs to epitopes of the flanking region at the C-terminal end of the CS protein. The difference between the antibodies to the repeats and those to the flanking region was in the labelling of the cytoplasm. The SP3-E9 obviously recognized epitopes on cytoplasmic structures, probably micronemes and rhoptries, as has been shown for P. knowlesi (Fine et al., 1984). It is assumed that the molecules which reacted are intracellular precursors of the CS protein (Yoshida et al., 1981; Cochrane et al., 1982; Nardin et al., 1982). Why the antibodies to three different epitopes of the C-terminal region only recognized intracellular antigens poorly or not at all cannot be answered with the present study. The N A N P region is immunodominant (Dame et al., 1984; Zavala et al., 1985), and consequently a fewer epitopes of the flanking regions will be exposed at the parasite surface. An intracellular masking of the binding region by amino acids could be another explanation for the lack of reactivity (Posthuma et al., 1987). Finally, the steric configuration of the intracellular domains
265
Figs. 7-12. Reactivity of human sera from an area endemic for malaria against Plasmodiumfalciparum (NF 54) sporozoites in salivary glands of Anopheles stephensi. S = sporozoite, SV= secretory vacuole, arrowhead=microneme/rhoptry system, all bars indicate 0.5 jam. Fig. 7. Human serum 1, positive in ELISA against (NANP)4o, 1:500. Fig. 8. Human serum 1 inhibited with synthetic peptide (NANP)5 o. Fig. 9. Human serum 11, negative in ELISA against (NANP)4o, 1:500. Fig. 10. Human serum I 1 inhibited with synthetic peptide (NANP)s o. Fig. 11. Human serum 6, positive in ELISA against (NANP)4o, 1:500. Fig. 12. Human serum 16, negative in ELISA against (NANPLo , 1:500.
266 at the C-terminal end of the CS protein could be different from that being exposed after the integration of the protein into the membrane. MAb CSVI-2 did not react with NF54 sporozoites but with those of the strai~ Ro 59 (Stfiber et al., 1990), while mAbs to epitopes at the N-terminal end (CS FYIF1 and CS FY-4G7) also did not bind in immunocytochemical experiments. In the case of the epitope CSVI-2 a strain specificity of the respective domain is describec (Stiiber et al., 1990), whereas the same arguments as mentioned above for the mAb., to C-terminal epitopes could be valid for the lack of intracellular binding. Thougl~ the N-terminal region is exposed on the parasite surface our results suggest eitheJ masking of the epitopes by overlapping of the molecules or an insufficient preservation of the protein during preparation for electron microscopy is responsible for the negative results. The same antibodies also did not react with the parasite-derivec CS protein in Western blots but with the native protein at the parasite surface a,~ measured by IFAT and with recombinant proteins (Stfiber et al., 1990). All mAbs which bound to CS protein on sporozoites also gave a labelling in th( cytoplasm of infected salivary gland epithelium. This probably results from th( shedding of CS protein during penetration of the epithelium as has been repeatedl5 demonstrated in vivo and in vitro (Posthuma et al., 1987b; Hamilton et al., 1988 Boulanger et al., 1988; Stewart and Vanderberg, 1988; Golenda et al., 1990). Area~ between the parasites binding antibodies to repetitive domains as well as non repetitive C-terminal domains indicate that the shedding, most recently shown tc begin with the secretion of the protein at the anterior end of the sporozoite (Stewar and Vanderberg, 1991), continues within the secretory vacuoles of the salivary glanc cells. The demonstration by ELISA of CSP released in the haemolymph durin~ migration can be used to identify infected mosquitoes, e.g., to select infected salivar~ glands as in the present study. The antibodies applied to P. falciparum NF54 sporozoites were also reacted witt thin sections of salivary gland sporozoites of the two field isolates IFA 5 and IF~ 6. Similar results between strains were observed with the mAbs SP3-E9 (NANP and CSVI-6. This fits with earlier findings describing the absence of antigenic variation for the repetitive domain of P. falciparum CS protein (Arnot, 1990) and conservative character of the CS VI-6 epitope (Stiiber et al., 1990). Just the opposit~ was found with the other two mAbs, CS CI-2 and CS VI-4, to carboxy-termina epitopes. CS VI-2 bound to the sporozoite surface of the isolates IFA 5 and IFA ( but not to N F 54 sporozoites and vice versa for CS VI-4. The immunocytochemica labellings therefore support the assumed strain specificity of part of the epitopes ii the flanking region studies (Stiiber et al., 1990). The possibility of specific recognition by human sera of the epitopes recognizec by the mAbs was examined by immunocytochemical techniques. Most of antibodie produced after immunization of humans with sporozoites are directed against th~ repetitive (NANP)3 domain of the CSP (Zavala et al., 1983; Zavala et al., 1985) However, there is also immune reactivity of human sera with non-repetitive sequence of the CSP ofP.falciparum (Del Giudice et al. 1988; Stiiber et al., 1990) and P. viva: (Romero et al., 1987). Out of a big series of human sera from Ifakara, Tanzania four have been compared in this study. Two of them were highly positive in ELIS/ on (NANP)4o (Del Giudice et al., 1986) the other two were negative but all fou sera reacted with air-dried sporozoites in IFAT. Thus, the lack of antibodies to th, repetitive sequence is not a reliable criterion for the absence o f antibodies t~
267
P. falciparum sporozoites in human sera. The reaction of the same sera on Western blots with DHFR-fusion peptides representing sequences of the CS protein confirmed the ELISA-results for the recognition of the NANP-epitopes. From the four sera selected for a characterization with different techniques, those two, which were negative in ELISA but positive in IFAT, did not recognize the repetitive domain, but at least one epitope of the non-repetitive region. The four sera tested yielded four different combinations of reactivity against fusion peptides of the flanking regions. All of them bound to CS VI, representing a sequence at the C-terminal end. This resulted in a more or less dense surface labelling of sporozoites in immunocytochemical experiments, which could neither be inhibited by blocking the antigens with the mAb SP3-E9 to the repetitive domain nor by adding (NANP)5o to the serum. In addition, there was always a reaction with at least one of the fusion peptides CS IA, another C-terminal epitope, and/or CS F4, representing the N-terminal region. The results achieved for sera 1 and 6 by immunogold labellings on salivary gland sporozoites correspond to the reactions on Western blots and to the binding sites found for the mAbs produced against the CS sequences which have been used for the blots. However, it is not possible to sort out how far antibodies to the flanking regions have contributed to the labelling. The inhibition experiments to eliminate the binding to (NANP), showed that the antibodies to this epitope are clearly the dominant part of the antibody-content in the serum. A demonstrated crossreactivity of mAbs to N A N P with the CS VI region (Stfiber et al., 1990) may be responsible for a reduction of the surface labelling to a lower density than has been shown for mAb CS VI-6. The binding of serum 11 to N F 54 sporozoites could not be inhibited by preincubation of the serum with (NANP)5 o. Thus, the presence of antibodies to the repetitive domain is excluded. On the other hand, serum 11 cannot contain antibodies reactive against the epitope CS F4, the mAb to this epitope did not bind in immunocytochemical labellings on N F 54, and against the epitope CS VI, this epitope has been shown to be predominantly located on the sporozoite surface. Therefore, it can be concluded that serum 11 recognizes at least one additional epitope, which is responsible for a conspicuous labelling of cytoplasmic structures. Serum 16 contained antibodies to the epitopes CS VI, CS 1A, and CS F4, and gave a very weak labelling of the parasite surface and intracellular organelles. Compared to results with the other sera, labelling of the CS VI epitopes on the parasite surface could be expected, whereas intracellular binding occurred possibly due to a recognition of the CS 1A region or to binding of antibodies to other epitopes than those dealt with in the present paper. Finally, the application of the four human sera to thin sections of salivary gland sporozoites from the P.falciparum isolates IFA 5 and IFA 6 did not result in labelling patterns qualitatively different from those on N F 54 sporozoites. Whether this is due to the presence of identical epitopes in all three strains or due to the presence of antibodies to different antigen types in the human sera cannot be concluded. Positive results in IFAT for the reactivity of human sera against air dried sporozoites do not imply that antibodies to the repetitive domain of the CS protein are present. It has been shown by immunogold labellings, that there are responses to epitopes in the flanking regions of the CSP in residents of an area hyperendemic for malaria. Given the immunodominance of the repetitive domains (Zavala et al., 1985), and the existence of sera containing antibodies exclusively to non-repetitive epitopes
268 it can be assumed, that these antibodies persist for a longer period t h a n those to th( repeats. Epitopes at the c a r b o x y - t e r m i n a l end are exposed at the sporozoite surface whereas the epitopes at the a m i n o - t e r m i n a l end seem n o t to be accessible.
Acknowledgments We wish to t h a n k Mrs. E. Fluri, Mrs. C. Kn611 a n d Mr. X. Petitjean for their skillfu technical assistence a n d Dr. P.F. Billingsley, Imperial College for Science a n d Technology, L o n d o n , for critical discussion of the manuscript. We are grateful tc Prof. W.L. K i l a m a , director general of the T a n z a n i a n N a t i o n a l Institute for Medical Research, for his s u p p o r t of the field studies within the K i l o m b e r o Health ResearclP r o g r a m m e that is supported by grants from the Swiss D e v e l o p m e n t C o - o p e r a t i o n Research clearance was granted by the T a n z a n i a n C o m m i s s i o n for Science anc Technology (reference R A 47/307).
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