Sequence diversity and evolutionary dynamics of the dimorphic antigen merozoite surface protein-6 and other Msp genes of Plasmodium falciparum

Immune evasion by Plasmodium falciparum is favored by extensive allelic diversity of surface antigens. Some of them, most notably the vaccine-candidate merozoite surface protein (MSP)-1, exhibit a poorly understood pattern of allelic dimorphism, in which all observed alleles group into two highly diverged allelic families with few or no inter-family recombinants. Here we describe contrasting levels and patterns of sequence diversity in genes encoding three MSP-1-associated surface antigens of P. falciparum, ranging from an ancient allelic dimorphism in the Msp-6 gene to a near lack of allelic divergence in Msp-9 to a more classical multi-allele polymorphism in Msp-7 Other members of the Msp-7 gene family exhibit very little polymorphism in non-repetitive regions. A comparison of P. falciparum Msp-6 sequences to an orthologous sequence from P. reichenowi provided evidence for distinct evolutionary histories of the 5` and 3` segments of the dimorphic region in PfMsp-6, consistent with one dimorphic lineage having arisen from recombination between now-extinct ancestral alleles. In addition. we uncovered two surprising patterns of evolution in repetitive sequence. Firsts in Msp-6, large deletions are associated with (nearly) identical sequence motifs at their borders. Second, a comparison of PfMsp-9 with the P. reichenowi ortholog indicated retention of a significant inter-unit diversity within an 18-base pair repeat within the coding region of P. falciparum, but homogenization in P. reichenowi. (C) 2009 Elsevier B.V. All rights reserved.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens

Plasmodium falciparum, the causative agent of human malaria, invades host erythrocytes using several proteins on the surface of the invasive merozoite, which have been proposed as potential vaccine candidates. Members of the multi-gene PfRh family are surface antigens that have been shown to play a central role in directing merozoites to alternative erythrocyte receptors for invasion. Recently, we identified a large structural polymorphism, a 0.58 Kb deletion, in the C-terminal region of the PfRh2b gene, present at a high frequency in parasite populations from Senegal. We hypothesize that this region is a target of humoral immunity. Here, by analyzing 371 P. falciparum isolates we show that this major allele is present at varying frequencies in different populations within Senegal, Africa, and throughout the world. For allelic dimorphisms in the asexual stage antigens, Msp-2 and EBA-175, we find minimal geographic differentiation among parasite populations from Senegal and other African localities, suggesting extensive gene flow among these populations and/or immune-mediated frequency-dependent balancing selection. In contrast, we observe a higher level of inter-population divergence (as measured by F(st)) for the PfRh2b deletion, similar to that observed for SNPs from the sexual stage Pfs45/48 loci, which is postulated to be under directional selection. We confirm that the region containing the PfRh2b polymorphism is a target of humoral immune responses by demonstrating antibody reactivity of endemic sera. Our analysis of inter-population divergence suggests that in contrast to the large allelic dimorphisms in EBA-175 and Msp-2, the presence or absence of the large PfRh2b deletion may not elicit frequency-dependent immune selection, but may be under positive immune selection, having important implications for the development of these proteins as vaccine candidates. (C) 2009 Elsevier B.V. All rights reserved.

Population dynamics of genetically diverse Plasmodium falciparum lineages: community-based prospective study in rural Amazonia

Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum Populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower Virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the Study period. We Suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms.