Epitopic Specificity of the Human Immune Response to the Invariant ...

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Epitopic Specificity of the Human Immune Response to the Invariant Region of a Polymorphic Plasmodium faciparum Merozoite Surface Antigen Rzepczyk, C. M., Anderson, K. L., Crurshes, P. A., Baxter, E. P., Kere, N., Irving, D., Dyer, S., Jones, G. L. The Journal of Protozoology Research, 2(3): 102111 1992 http://ir.obihiro.ac.jp/dspace/handle/10322/170


帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge

J. Protozool. Res., 2. 102-111 (1992) Copyright © 1992 , Research Center for Protozoan Molecular Immunology

Epitopic Specificity of the Human Immune Response to the Invariant Region of a Polymorphic Plasmodium faciparum Merozoite Surface Antigen C. M. RZEPCZYK1, K. L. ANDERSON1, P. A. CRURSHES1, E. P. BAXTER2, N. KERE3, D. IRVING4, S. DYER4 and G. L. JONES1 1

Queensland Institute of Medical Research, The Bancrroft Centre, Heston, Brisbane, Australia 4029, 2Department of Employment, Vocational Education, Training and Industrial Relations, Brisbane, 3Solomon Islands Malaria Training and Research Insitute, Honiara, Solomon Islands, and 4Biotech Australia, Roseville, Sedney, AUSTRALIA Received 11 June 1992/ Accepted 3 July 1992 Key words: malaria, merozoite surface antigen 2 (MSA2), antibody

ABSTRACT Merozoite surface antigen 2 (MSA2) is a Plasmodium falciparum vaccine candidate. Antibody responses to MSA2 in Melanesians naturally exposed to P. falciparum were investigated by ELISA using the recombinant MSA2 protein known as Ag 1609 and 65 overlapping synthetic peptides spanning predominantly the conserved N and C terminal regions of MSA2. Significant differences were obtained between control and malaria exposed subjects in the ability of sera to bind to Ag 1609. The median absorbance obtained with control sera was 0.36 whereas that obtained with sera from malaria exposed subjects was 1.8. The sera of ninety-five percent of malaria exposed subjects reacted significantly with Ag 1609. The percentage reactivity of the control sera with Ag 1609 was 39%. Overall the serological responses to the synthetic peptides were low and well defined B epitope regions were not delineated. The median absorbances were in general higher for the malaria exposed group than the controls with these differences being significant for five N terminal peptides and 14 C terminal peptides. One peptide (KECTDGNK) at the conserved C terminal end was identified for further investigation as a possible immunodiagnostic epitope. Immunisation of mice with an allelic form of Ag 1609 (namely Ag 1615) produced antibodies to the conserved C terminal of MSA2 but not to the N terminal. INTRODUCTION Considerable information is now available on Plasmodium falciparum proteins which have been identified as being likely vaccine candidate antigens. Among these proteins is merozoite surface antigen 2 (MSA2). MSA2 is the smaller of the two merozoite surface antigens described from P. falciparum (Smythe et al. 1988). Recent studies have shown that MSA2 occurs in a number of allelic forms (Smythe et al. 1990; Thomas et al. 1990). These allelic proteins show very high homology of amino acid sequence at the N and C terminal regions but almost no homology in the central region. The development of malaria vaccines and the search for specific immunodiagnostics for malaria requires a more detailed understanding of the immune responses to malaria antigens (Perlmann et al. 1989). With this in mind, we have examined the recognition of synthetic peptides representing sequences from the N and C terminal 102

ANTIBODY RESPONSE TO MSA2 regions of MSA2 by sera from naturally infected individuals. It is already known from studies with mice that B cell epitopes with specificity for the native protein are found within the conserved regions of MSA2 (Jones et al. 1990; Rzepczyk et al. 1990b) and that mice immunized with such epitopes conjoined to a carrier protein are protected against P. chabaudi challenge (Saul et al. 1992). MATERIALS AND METHODS Subjects and Plasma/Sera Collections: Plasma and sera samples were collected from adult volunteers as follows: 10 Papua New Guineans (Madang province); 46 Solomon Islanders (the majority born on the islands of Guadalcanal and Mailata but many now residing in Honiara on Guadalcanal); and 23 Australian residents (12 Caucasian, 1 Chinese). The Papua New Guinean and the Solomon Islander subjects were all residents of coastal areas where P. falciparum malaria is highly endemic and these individuals can be expected to have had repeated malaria infections. With 3 exceptions, none of the control subjects had visited malaria endemic areas. In the instances where endemic areas were visited, the visits were of short duration, appropriate prophylaxis was taken and none of the controls developed clinical malaria. Plasma (diluted in tissue culture medium) was collected from all subjects and serum samples were obtained from the majority. Recombinant (r) MSA2: Two allelic recombinant r-MSA2 proteins were used in this study. These were Ag 1609 which has the sequence of the FCQ-27/PNG isolate and Ag 1615 with the sequence of the Indochina 1 isolate (Smythe et al. 1990). Details of the derivation of these proteins have previously been described (Rzepczyk et al. 1992). The general structure of the MSA2 protein is depicted schematically in Fig. 1. Ag 1609 was used as a plate antigen in ELISA assays examining the reactivity of human sera. Ag 1615 was used in the murine studies.

Fig. 1.

Schematic representation of MSA2 showing position of first N-terminal (E65) and C-terminal (G1) peptides.

Malarial Peptides: 40 octapeptides from the N-terminal region (named E65 to El04) as well as 27 octapeptides from the C-terminal region (named G1 to G27) of the mature FCQ-27/PNG form of MSA2 were synthesised and purified as previously described (Jones et al. 1990). The N terminal peptides (E65 to E104) were staggered by one residue at a time and extended into the variable region of the FCQ-27/PNG sequence. The sequences of these peptides are shown in Table 1. C terminal peptides (G1 to G27) were staggered by two residues at a time and extended into the presumptive cleaved acylation region which is common between different parasite isolates (Smythe et al. 1990). The sequences of these peptides is shown in Table 2. Peptides E100 and G18 were not available for study. Another three synthetic peptides were included in the study: peptide N (KNESKYSNTFINNAYNMSIRRSM) which 103

ANTIBODY RESPONSE TO MSA2 covers all the conserved N terminal sequences of the mature protein and peptides C1 (APENKGTGQHGHMHGSRNMHPQNTSDSQKEC) and C2 (TDGNKENCGAATSLLNNSSNIASINK) which contain the conserved C terminal sequences of the mature protein (Smythe et al. 1988; Rzepczyk et al. 1992). Coupling of Peptides to BSA: The procedures were described previously (Jones et al. 1989a, 1990). Briefly, E and G series peptides, and peptides N (KNESKYSNTFINNAYNMSIRRSM) and C2 (TDGNKENCGAATSLLNNSSNIASINK) were synthesized with an added N terminal cysteine to facilitate coupling to bovine serum albumin (BSA) using the heterobifunctional reagent maleimidocaproyloxysuccinimide (MCS) (Lee et al. 1980). In the case of peptides C1 (APENKGTGQHGHMHGSRNMHPQNTSDSQKEC) , the C terminal cysteine which is part of the natural sequence of MSA2 was used for coupling to BSA. The extent of coupling was monitored. Control plates were coated with sham coupled BSA (BSA-. Table 1 Synthetic Peptides Spanning N terminal region of MSA2 (E series)

*E80 is the last peptide to contain totally conserved amino acids. Table 2 Synthetic Peptides Spanning C Terminal Region of MSA2 (G series)

* Variant amino acid residue.


ANTIBODY RESPONSE TO MSA2 ELISA Assays: With human sera/plasma. Wells of flat bottom ELISA plates (Titertek activated immunoassay plate, The Netherlands) were precoated with 5 µg/ml of the peptide BSA-conjugates, Ag 1609 or with BSA-MCS. One well was used for each antigen and a separate plate was used for each donor. Coated plates were stored at 4OC in a humid environment until further processed. The plates were then blocked with a solution containing 5% skim milk powder in PBS and rinsed in PBS. The next day plates were incubated with the test sera diluted in PBS containing 5% skim milk powder, 0.05% Tween 20 with 0.1 % BSA (plasma was used where serum was unavailable). After rinsing in PBS/Tween, anti-human globulins conjugated to horse-radish peroxidase and the substrate 2,2’ azino-di (3-ethylbenzthiazoline sulphonate were added successively according to the manufacturer's instructions (Amersham U. K.). The absorbance was measured directly at 30 minutes using an automated microplate reader. Sera and plasma were used at a final dilution of 1 in 50. With murine sera. The ELISA protocol was as previously described (Rzepczyk et al. 1990b) and is comparable to the method described for the human sera/plasma. Mouse sera were tested at a final dilution of 1/100 against four plate antigens. The antigens were Ag 1615, peptides N (KNESKYSNTFINNAYNMSIRRSM) , C1 (APENKGTGQHGHMHGSRNMHPQNTSDSQKEC) and peptide C2 (TDGNKENCGAATSLLNNSSNIASINK). Immunisation of Mice with r-MSA2: The procedure has been described previously (Rzepczyk et al. 1990b). Briefly, four mice per group were pre-bled to provide day 0 sera and were then immunised intraperitoneally with Ag 1615 at 10 µg per injection in a 1:1 emulsion of complete Freund's adjuvant (CSL, Melbourne, Australia). One month later, animals received a second injection of Ag 1615 emulsified in incomplete Freund's adjuvant (CSL). Animals were bled two weeks after the second injection (day 42). Two strains of mice were used. They were C57BL/10ScSn (H-2b)(abbreviated to BL/10) and B10.BR (H-2k). Only female mice up to 12 weeks old were used. Statistical Analysis: The Mann-Whitney test was used throughout to determine differences between the control and malaria exposed groups because the absorbance data for some of the malaria peptides were found to be non-normally distributed. A total of 66 comparisons were made and to adjust for these multiple comparisons, a significance level of p < 0.0007 (0.05/66) was considered statistically significant. All data were analysed using the SPSS/PC+ statistical package. RESULTS Background Absorbances: The median absorbance to the control antigen (BSA-MCS) was 0.17 in both the malaria exposed and unexposed subjects and there was no significant differences between the two groups in the measured response to this antigen (p = 0.6272). The absorbances from all 79 subjects were used to calculate the level above which sera were deemed to show specificity for a particular antigen. The absorbance above which only 1 % of the distribution occurred was taken as the cut off value. This value was 0.424 and all sera giving an absorbance reading > 0.424 were regarded as showing a specific response. Recombinant (r) MSA2: Ninety-five percent of the sera from malaria-exposed individuals showed significant responses to Ag 1609 whereas only 39% of the sera from non-exposed individuals recorded positive antibody responses by the same criteria. The median absorbance obtained in the ELISA for all the sera from malaria-exposed subjects was 1.8 (range 1.68) whereas that for the non-exposed subjects was 0.36 (range 1.51). These differences were statistically significant (p = 0.0000).


ANTIBODY RESPONSE TO MSA2 N-terminal Peptides (E series): The median absorbances obtained in the ELISA for each of the E series peptides, within the exposed and non-exposed groups is shown in Fig. 2. The results show that the reactivity of these peptides with the sera from both malaria exposed and control subjects was very low. The median absorbances for all the N terminal peptides, with the exception of E91, did either not exceed the cutoff value of 0.424 or were just marginally above this value. Significant differences between the two subject groups were observed with respect to five peptides, namely E73 (p = 0.0004), E88 (p = 0.0006), E94 (p = 0.000), E99 (p = 0.0004), and E101 (p = 0.000) with the median absorbances of the malaria exposed subjects being higher. The percentage of subjects from each of the groups responding to these peptides is shown in Fig 3. C-terminal Peptides: The median absorbance obtained for each of the G series peptides is shown in Fig. 4. The reactivity of the sera from both subject groups with the G series peptides was overall higher than that observed with the E series peptides. For fourteen peptides the absorbances obtained differed significantly between the malaria exposed and non-exposed groups, with responses in the exposed group having a higher median value. These peptides were G2-G7, G9-G10, G15-G17, G20, G23-G24. P = 0.000 for all of these except for G16 and G23 where p was 0.0001 and for G24 where p = 0.0002. Fig. 5 shows that while many of the G series peptides were recognised by over 80% of the sera from malaria subjects they were also recognized by high proportion of the control sera. Peptide G15 was an exception in this regard as it was recognised by fewer than 15% of the control sera. The median absorbance recorded for G15 in the malaria exposed group was also relatively high whereas the control median absorbance did not exceed the background value (Fig. 4). Although peptides G10 and G20 were recognised by < 10% of the control sera, they were recognised by only approximately 60% of the exposed subjects (Fig. 5). Median absorbances obtained to these peptides were also lower than those obtained with G15. Marine Response to r-MSA2: Antibodies to the N terminal of MSA2 were not detected in mice immunized with Ag 1615 although titres of 103 were obtained to both the C1 and C2 peptides which together encompass the region covered by G1-G27. The results are summarised in Table 3. DISCUSSION The ability of an individual to mount an immune response to a particular antigen or to defined epitopes depends on a number of interrelating factors. The antigen must be in a particular milieu which promotes or at least does not not impede its recognition by the immune system. Our results indicate that the MSA2 protein is recognised by most individuals in endemic areas and this confirms our previous finding with this antigen (Rzepczyk et al. 1989). The ability to mount an effective humoral response also depends on the recognition of appropriate T cell epitopes in the antigen by T helper cells. We have already shown that MSA2 contains several T cell epitopes recognised by Melanesians (Rzepczyk et al. 1990a). A complicating factor in studies on antibody response to malarial antigens is the high level of cross reactivity between these antigens (Anders 1986). Thus some of the positive responses recorded to MSA2 may well be due to antibody elicited to other malarial antigens cross reacting with epitopes in MSA2. In addition, as some non-exposed donors also gave positive ELISA readings to MSA2, a malaria non-specific component in the humoral responses is suggested. While the use of sera at higher dilution may have given increased discrimination between the sera from the control and malaria specific groups, this apparent non-specificity could be due to some sharing of sequence between MSA2 and the antigens



Fig.2 Recognition of N-terminal peptides by human sera in ELISA. Clear bars, malaria exposed subjects; filled bars, malaria unexposed subjects.

Fig.3. Percentage of subjects with antibodies recognising N-terminal peptides. Clear bars, malaria exposed subjects; filled bars, malariaunexposed subjects. 107


Fig.4. Recognition of C-terminal peptides by human sera in ELISA. Clear bars, malaria exposed subjects; filled bars, malaria unexposed subjects.

Fig.5. Percentage of subjects with antibodies recognising C-terminal peptides. Clear bars, malaria exposed subjects; filled bars, malaria unexposed subjects. 108

ANTIBODY RESPONSE TO MSA2 Table 3. Relative recognition of synthetic peptides N and C terminal conserved regions of MSA2 following immunisation with r-MSA2*

* Ag 1615 was the r-MSA2 protein used as immunogen. ** r-MSA2 = Ag 1615; Peptide N = KNESKYSNTFINNAYNMSIRRSM Peptide C1 = APENKGTGQHGHMHGSRNMHPQNTSDSQKEC Peptide C2 = TDGNKENGAATSLLNSSNIASINK Peptides N, C1, C2 were coupled to BSA before use as plate antigens. # Mean absorbance ± standard deviation obtained in ELISA. Data based on four mice per group. ## Represents highest titre obtained in ELISA for each group of four mice. An O. D. < 0.3 was regarded as the endpoint. All control sera had titres < 102

of commonly occurring micro-organisms (Rzepczyk et al. 1989) or to antibodies in these individuals recognising epitopes within MSA2 as mimotopes (Geysen et al. 1987). In respect of epitopic specificity of antibody responses, a number of authors have indicated that domains of a protein with greater flexibility and with greater surface availability are more likely to contain epitopes against which an immune response would be mounted (Jameson and Wolf 1988). Antibodies thus very often react with N-terminal segments of a protein antigen since these fulfil the criteria of availability and flexibility. We have previously demonstrated (Jones et al. 1991) that the C-terminal part of MSA2 has, overall, a much higher antigenic index than the N-terminal part of MSA2. This feature is supported by our present results from mice immunised with r-MSA2 (Table 3) in which antibodies were detected to the C terminal of the MSA2 protein but not to the N terminal. Our studies on the epitopic specificity of the human antibody response also showed poor recognition of the N terminal region. While the response to the C terminal peptides was higher, the interpretation of the data with the C terminal peptides is difficult in view of the substantial malaria non-specific binding seen with some of these peptides. Poor recognition of both N and C terminal sequences has been reported in monkeys which had been repeatedly infected with the CAMP strain of MSA2 and this was also found to be the case with a human serum sample tested (Thomas et al. 1990). There is also some data that the immunodominant B


ANTIBODY RESPONSE TO MSA2 epitopes recognised by humans may be located in the central regions rather than at the N and C terminal of MSA2 (Smythe et al. 1990). Evidence has recently been obtained that peptides E71, G5 and G12 when conjoined to diphtheria toxoid can protect mice infected with P. chabaudi (Saul et al. 1992) via a humoral mechanism. If these results translate to the human situation, it will be necessary that the vaccine immunogen induces an antibody response to epitopes located in conserved regions of MSA2. The studies reported here suggest that the immunisation of humans with MSA2 proteins either as entire recombinant proteins in vaccines or with the native protein as a result of natural infection will not produce antibody to E71 and may well only produce low level antibody to the other two peptides, G5 and G12. Better targeting of required responses in vaccination may necessitate the use of defined synthetic peptides or suitably truncated recombinant proteins as immunogens rather than entire proteins (Jones et al. 1989b). Peptide G15 identified in this study as being relatively well recognized by sera from individuals from endemic areas while being minimally recognised by the sera of control subjects may warrant further investigation as an indicator of malarial immune status in man. Peptides from other malarial antigen, particularly the circumsporozoite protein and Pf155/RESA are already being employed in this context (Petersen et al. 1990; Del Guidice et al. 1987).

ACKNOWLEDGMENTS We wish to thank Diana Battistutta for her statistical advice. This study was supported by the Australian National Health and Medical Research Council and by the Generic Technology Component of the Australian Industry and Development Act, 1968.

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