Genetic Diversity and Antigenic Polymorphism in Plasmodium falciparum : Extensive Serological Cross-Reactivity between Allelic Variants of Merozoite Surface Protein 2

Abstract

Diversity in the surface antigens of malaria parasites is generally assumed to be a mechanism for immune evasion, but there is little direct evidence that this leads to evasion of protective immunity. Here we show that alleles of the highly polymorphic merozoite surface protein 2 (MSP-2) can be grouped (within the known dimorphic families) into distinct serogroups; variants within a serogroup show extensive serological crossreactivity. Cross-reactive epitopes are immunodominant, and responses to them may be boosted at the expense of responses to novel epitopes (original antigenic sin). The data imply that immune selection explains only some of the diversity in the msp-2 gene and that MSP-2 vaccines may need to include only a subset of the known variants in order to induce pan-reactive antibodies.Allelic diversity in malaria parasites is widely assumed to be a mechanism for immune evasion (7,24). Among surface proteins, diversity arises from multiple point mutations or variations in the numbers, lengths, and sequences of amino acid repeats. Repeat diversity may arise from intragenic recombination, misalignment of repeated DNA sequences (6), or complementary-strand slippage (12, 13) during DNA replication. The AT-rich nature of the Plasmodium falciparum genome may facilitate the generation of new variants (28). The maintenance of variants in the population implies that they have a selective advantage (e.g., evasion of preexisting immune responses) or that the mutations are selectively neutral and that population diversification occurs randomly.In some genes (e.g., the circumsporozoite surface protein [csp] gene), point mutations cluster in areas immunodominant for T cells (16) and can lead to loss of epitope recognition (38) or antagonism of T-cell responses (26), although it is not clear that either of these leads to the avoidance of protective immunity. Similarly, monospecific antibodies differentiate between allelic variants of merozoite surface proteins (13, 23), but the human antibody response is commonly directed at conserved or semiconserved epitopes (8, 34) and direct evidence for the evasion of antibody-dependent immunity is lacking.Comparison of the frequencies of nonsynonymous-to-synonymous mutations (20) for P. falciparum genes provides evidence of diversifying selection in msp-1 (11), which encodes merozoite surface protein-1, but the data are less clear for msp-2, which encodes merozoite surface protein-2 (9, 11, 21), and the significance of the findings is uncertain, given the limitations of the method (37). Comparison of intra-and interpopulation variances in allele frequencies provides evidence for selection on a polymorphic region of msp-1 (5) and dimorphic regions of msp-2 (4), but there are no data for polymorphic regions of msp-2. Comparison of inter-and intraspecific levels of variation suggests balancing selection on the erythrocyte-binding antigen EBA-175 (25) and the apical membrane antigen AMA-1 (22). However, except for msp-1, for which selection may be mediated by strain-specific antibo...

Epitope-Specific Regulation of Immunoglobulin Class Switching in Mice Immunized with Malarial Merozoite Surface Proteins

“…We also hypothesized that the crucial interactions for class switching were more likely to take place between MSP2 and T cells than between MSP2 and B cells, as following natural infection, human antibodies to MSP2 recognize predominantly dimorphic and polymorphic sequences, with little or no antibody to the conserved sequences being detectable (52), suggesting that there are no dominant B-cell epitopes within the highly conserved regions of the molecule. Furthermore, although conserved or cross-reactive epitopes for antibodies are present throughout the MSP2 molecule (24), dominant epitopes for human (41,53) and murine (40) T cells have been identified within the conserved sequences of MSP2. The data presented here essentially support these predictions in that (i) mice immunized with full-length MSP2 made little or no antibody to the conserved C terminus, (ii) we have identified a dominant T-cell epitope within the conserved C terminus of the molecule which is able to drive class switching to IgG2b, and (iii) this peptide is not, in itself, a major target for anti-MSP2 antibodies.…”

“…Discrete domains of MSP2 were produced in E. coli as fusion proteins with the C-terminal region of GST (44) using the pGEX expression system (Amersham Pharmacia Bioscience, Little Chalfont, United Kingdom). The production and validation of proteins representing the dimorphic sequences of serogroup A (Di-A) and serogroup B (Di-B) and polymorphic sequences from each serogroup (Poly-A and Poly-B) have been described previously (24). Conserved 5Ј and 3Ј sequences of the MSP2 gene (Con-N], ϳ320 bp, and Con-C, ϳ325 bp) were amplified using specific primers (CN5Ј [CCAGTACCAGTAGGAGGC] and CN3Ј [GAAGAG AATTATATGAATATGGC]), ligated into pGEX-3, validated by DNA sequencing (44), and expressed in E. coli.…”

“…1). MSP2 variants can be grouped into two major serogroups, type A (typified by cloned isolate 3D7) and type B (e.g., isolates FCR3 and HB3) (21,45); certain B-cell epitopes appear to be conserved, giving rise to antigenic crossreactivity within each family (20,24). Thus, cross-reactive epitopes within dimorphic or polymorphic sequences, or conserved sequences within the N and C termini of the protein (Con-N and Con-C, respectively), may explain the apparent ability of all MSP2 serotypes to drive IgG3 class switching.…”

In Immunization with Plasmodium falciparum Apical Membrane Antigen 1, the Specificity of Antibodies Depends on the Species Immunized

“…Also, it is clear that some adults in areas of endemicity produce a larger proportion of allele-specific antibodies (7,40), and these antibodies may provide selective advantages for some parasites. Previous studies with other malaria proteins (CSP and MSP2) have suggested that T-cell responses could be the source of immune selection pressure (15,16). While there is no clear evidence for AMA1, Polley and colleagues (40) proposed the possibility of selection pressures on AMA1 domain III by T-cell-mediated immunity.…”

Defining malaria burden from morbidity and mortality records, self treatment practices and serological data in Magugu, Babati District, northern Tanzania

“…Serum was eluted from filter papers as described by Corran et al (2008). Immunoglobulin G antibodies against blood stage antigens were determined by indirect enzyme-linked immunosorbent assay (indirect ELISA), as previously described (Drakeley et al, 2005) using recombinant MSP-119 (Wellcome Genotype), produced as previously described by Franks et al (2003) or recombinant AMA-1 (3D7). Briefly, flat bottom 96-well microtitre plates (Nunc, Roskilde, Denmark) were coated overnight with 50 μl of 0.5 mg/ml dilution of the specific antigen.…”