Population genetic structure of the Plasmodium vivax circumsporozoite protein (Pvcsp) in Sri Lanka.

Molecular methods elucidate evolutionary and ecological processes in parasites, where interaction between hosts and parasites enlighten the evolution of parasite lifestyles and host defenses. Population genetics of Plasmodium vivax parasites accurately describe transmission dynamics of the parasites and evaluation of malaria control measures. As a first generation vaccine candidate against malaria, the Circumsporozoite Protein (CSP) has demonstrated significant potential in P. falciparum. Extensive polymorphism hinders the development of a potent malaria vaccine. Hence, the genetic diversity of Pvcsp was investigated for the first time in 60 Sri Lankan clinical isolates by obtaining the nucleotide sequence of the central repeat (CR) domain and examining the polymorphism of the peptide repeat motifs (PRMs), the genetic diversity indices and phylogenetic relationships. PCR amplicons determined size polymorphism of 610, 700 and 710 bp in Pvcsp of Sri Lanka where all amino acid sequences obtained were of the VK210 variant, consisting variable repeats of 4 different PRMs. The two most abundant PRMs of the CR domain, GDRADGQPA and GDRAAGQPA consisted ~2-4 repeats, while GNRAAGQPA was unique to the island. Though, different nucleotide sequences termed repeat allotypes (RATs) were observed for each PRM, these were synonymous contributing to a less polymorphic CR domain. The genetic diversity of Pvcsp in Sri Lanka was due to the number of repetitive peptide repeat motifs, point mutations, and intragenic recombination. The 19 amino acid haplotypes defined were exclusive to Sri Lanka, whereas the 194 Pvcsp sequences of global isolates generated 57 more distinct a.a. haplotypes of the VK210 variant. Strikingly, the CR domain of both VK210 and VK247 variants was under purifying selection interpreting the scarcity of CSP non-synonymous polymorphisms. Insights to the distribution of RATs in the CR region with geographic clustering of the P. vivax VK210 variant were revealed. The cladogram reiterated this unique geographic clustering of local (VK210) and global isolates (VK210 and VK247), which was further validated by the elevated fixation index values of the VK210 variant.

Evaluation of the genetic diversity of domain II of Plasmodium vivax Apical Membrane Antigen 1 (PvAMA-1) and the ensuing strain-specific immune responses in patients from Sri Lanka.

Antigenic polymorphism displayed by malaria parasites is a skewed schema to escape the host immune system. The prevailing genetic diversity at domain II of the Plasmodium vivax Apical Membrane Antigen-1 (Pvama-1DII) was characterized in 64 single clone P. vivax isolates from Sri Lanka, where unstable malaria prevails with low intensity. In Sri Lanka, the Pvama-1DII gene showed meager meiotic recombination with the enclosure of single nucleotide polymorphisms (SNPs). Eleven amino acid (a.a.) variant positions defined 21 a.a. haplotypes with 9 unique to the island, where the predominant haplotype, H1, was identical to the reference Salvador I strain. A further 376 globally dispersed isolates defined 38 a.a. haplotypes (H22-H59), with 4 and 26 haplotypes exclusive to India and Thailand, respectively. The phylogenetic tree revealed no clustering, where most isolates had a very recent common origin. The polymorphism detected in PvAMA-1DII B and T cell epitopes evidenced an immune evasion mechanism exploited by the parasite. Majority of Sri Lankan patients developed antibody responses to both conformational and linear B cell epitopes. The ensuing strain-specific immunity due to extensive antigenic polymorphism was evaluated by aligning a.a. sequences of PvAMA-1DII with the homologous total (IgM+IgG) antibody responses assayed by in-house established indirect ELISAs against 7 PvAMA-1DII overlapping synthetic peptides, P01-P07. While the antibody responses to P01-P03, P06, P07 harbouring P. vivax clinical isolates with polymorphic a.a. haplotype to Sal I was clearly strain-transcending (cross-reactive), individuals with isolates identical to the Sal I strain observed varying antibody prevalence against the seven PvAMA-1DII Sal-I synthetic peptides, with the highest prevalence detected against P04. Synthetic peptide P04, spanning a.a. positions 302-324 of the PvAMA-1DII of the Sal I strain that included the epitope recognized by the invasion inhibitory 4G2 monoclonal antibody of PfAMA-1, was highly conserved in all 440 local and global P. vivax isolates examined. A functional role for this region is reinforced by the highly immunogenic nature of P04, and could point towards a presumably "protective" anti-P04 antibody response that elicited an isotype switch from IgM to IgG, with increasing exposure to malaria exclusively in endemic residents. Thus the conserved and seemingly "protective" nature of the domain II loop of PvAMA-1 makes it a putative contender to be included in a cocktail vaccine against P. vivax asexual erythrocytic stages in Sri Lanka.

Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) in Sri Lanka.

Extensive polymorphism in the genes encoding for surface antigens of Plasmodium falciparum and Plasmodium vivax has been a serious impediment for malaria vaccine development. One such antigen is the merozoite surface protein-1 (MSP-1). The MSP-1 precursor after proteolytic cleavage generates a C-terminal fragment of 42 kDa (MSP-1(42)), which subsequently produces 33 kDa (MSP-1(33)) and 19 kDa (MSP-1(19)) fragments. Since MSP-1(42) is currently being considered as a candidate for vaccine development against blood stage malaria it is important to catalogue the existing diversity in this antigen in natural P. vivax infections. Here we investigated the level of genetic diversity in the PvMSP-1(42) gene fragment in 95 single clone P. vivax infections in Sri Lanka. We observed that the PvMSP-1(19) fragment was highly conserved among these samples, whereas the PvMSP-1(33) fragment exhibited extensive diversity with 39 polymorphic amino acid positions (corresponding to 27 haplotypes, 19 of which were unique to Sri Lanka). Of these 27 PvMSP-1(42) haplotypes, 24 belonged to hypervariable region (HVR) T1-T7 types, while 3 haplotypes were generated by interallelic recombination between T1/T3 (HVRT8-T9) and T2/T3 (HVRT10). In addition, we analysed 107 PvMSP-1(42) sequences (corresponding to 62 haplotypes, H28 to H89) deposited in the NCBI GenBank database from other regions of the world. Seventy-four of these correspond to 9 of the 10 HVR types (HVR-T7 was unique to Sri Lanka). Two novel HVR types, T11 and T12, with a double recombination between HVR-T1/T3 and HVRT6/T2, were derived from South America and Thailand, respectively. T cell epitope polymorphism arising due to non-synonymous substitutions in PvMSP-1(33) may result in differential binding of the polymorphic peptides to class II MHC alleles, inducing different host immune responses. In conclusion, under low transmission and unstable malaria conditions prevalent in Sri Lanka, extensive allelic polymorphism was evident at PvMSP-1(33) due to recombination, mutation, and balancing selection. In contrast, PvMSP-1(19) is highly conserved(,) greatly enhancing its suitability as a malaria vaccine candidate.