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Nucleotide sequence polymorphism at the apical membrane antigen-1 locus reveals population history of Plasmodium vivax in Thailand.

ABSTRACT: Apical membrane antigen-1 is a candidate for inclusion in a vaccine for the human malaria parasite Plasmodium vivax. We collected 231 complete sequences of the gene encoding this antigen (pvama-1) from three regions of Thailand, the most extensive collection to date of sequences at this locus. The domain II loop (previously mentioned as a potential vaccine component) was almost completely conserved, with a single amino acid variant (I313R) observed in a single sequence. The 3' portion of the gene (domain II through the stop codon) showed significantly lower nucleotide diversity than the 5' portion (start codon through domain I); and a given domain I sequence might be found in a haplotype with more than one domain II sequence. These results imply a hotspot of recombination between domains I and II. We found significant geographic subdivision among the three regions of Thailand (NW, East, and South) in which collections were made in 2007. Numbers of P. vivax infections have experienced overall declines since 1990 in all three regions; but the decline has been most recent in the NW, and there has been a rebound in numbers of infections in the South since 2000. Consistent with population history, amino acid sequence diversity was greatest in the NW. The South, which had by far the lowest sequence diversity of the three regions, showed signs of a population that has expanded from a small number of founders after a bottleneck.

SUBMITTER: Putaporntip C

PROVIDER: S-EPMC2790030 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Nucleotide sequence polymorphism at the apical membrane antigen-1 locus reveals population history of Plasmodium vivax in Thailand.

Putaporntip Chaturong C Jongwutiwes Somchai S Grynberg Priscila P Cui Liwang L Hughes Austin L AL

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 20090728 6

Apical membrane antigen-1 is a candidate for inclusion in a vaccine for the human malaria parasite Plasmodium vivax. We collected 231 complete sequences of the gene encoding this antigen (pvama-1) from three regions of Thailand, the most extensive collection to date of sequences at this locus. The domain II loop (previously mentioned as a potential vaccine component) was almost completely conserved, with a single amino acid variant (I313R) observed in a single sequence. The 3' portion of the gen ...[more]

PMID: 19643205

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Project description:Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a leading candidate antigen for blood stage malaria vaccine. However, antigenic variation is a major obstacle in the development of an effective vaccine based on this antigen. In this study, the genetic structure and the effect of natural selection of PvAMA-1 among Korean P. vivax isolates were analysed.Blood samples were collected from 66 Korean patients with vivax malaria. The entire PvAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvAMA-1 sequence of each isolate was sequenced and the polymorphic characteristics and effect of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs.Thirty haplotypes of PvAMA-1, which were further classified into seven different clusters, were identified in the 66 Korean P. vivax isolates. Domain II was highly conserved among the sequences, but substantial nucleotide diversity was observed in domains I and III. The difference between the rates of non-synonymous and synonymous mutations suggested that the gene has evolved under natural selection. No strong evidence indicating balancing or positive selection on PvAMA-1 was identified. Recombination may also play a role in the resulting genetic diversity of PvAMA-1.This study is the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Korea. Korean PvAMA-1 had limited genetic diversity compared to PvAMA-1 in global isolates. The overall pattern of genetic polymorphism of Korean PvAMA-1 differed from other global isolates and novel amino acid changes were also identified in Korean PvAMA-1. Evidences for natural selection and recombination event were observed, which is likely to play an important role in generating genetic diversity across the PvAMA-1. These results provide useful information for the understanding the population structure of P. vivax circulating in Korea and have important implications for the design of a vaccine incorporating PvAMA-1.

Project description:BackgroundPlasmodium is an obligate intracellular parasite. Apical membrane antigen 1 (AMA1) is the most prominent and well characterized malarial surface antigen that is essential for parasite-host cell invasion, i.e., for sporozoite to invade and replicate within hepatocytes in the liver stage and merozoite to penetrate and replicate within erythrocytes in the blood stage. AMA1 has long served as a potent antimalarial drug target and is a pivotal vaccine candidate. A good understanding of the structure and molecular function of this Plasmodium protein, particularly its involvement in host-cell adhesion and invasion, is of great interest and hence it offers an attractive target for the development of novel therapeutics. The present study aims to heterologous express recombinant Plasmodium AMA1 ectodomain of P. vivax (rPvAMA1) for the selection of binding peptides.MethodsThe rPvAMA1 protein was heterologous expressed using a tag-free Profinity eXactTM system and codon optimized BL21-Codon Plus (DE3)-RIL Escherichia coli strain and further refolded by dialysis for renaturation. Binding peptides toward refolded rPvAMA1 were panned using a Ph.D.-12 random phage display library.ResultsThe rPvAMA1 was successfully expressed and refolded with three phage-displayed dodecapeptides designated as PdV1 (DLTFTVNPLSKA), PdV2 (WHWSWWNPNQLT), and PdV3 (TSVSYINNRHNL) with affinity towards rPvAMA1 identified. All of them exhibited positive binding signal to rPvAMA1 in both direct phage assays, i.e., phage ELISA binding assay and Western blot binding assay.DiscussionPhage display technology enables the mapping of protein-protein interactions based on a simple principle that a library of phage particles displaying peptides is used and the phage clones that bind to the target protein are selected and identified. The binding sites of each selected peptides toward PvAMA1 (Protein Data Bank, PDB ID: 1W8K) were in silico predicted using CABS-dock web server. In this case, the binding peptides provide a valuable starting point for the development of peptidomimetic as antimalarial antagonists directed at PvAMA1.

Project description:ObjectiveThe apical membrane antigen-1 (AMA-1) is considered as a promising candidate for development of a malaria vaccine against Plasmodium parasites. The correct conformation of this protein appears to be necessary for the stimulation of parasite-inhibitory responses, and these responses, in turn, seem to be antibody-mediated. Therefore, in the present investigation, we expressed the Plasmodium vivax AMA-1 (PvAMA-1) ectodomain in Escherichia coli (E. coli), purified it using standard procedures and characterized it to determine its biological activities for it to be used as a potential target for developing a protective and safe vivax malaria vaccine.Materials and methodsIn this experimental investigation, the ectodomain of PvAMA-1 antigen (GenBank accession no. JX624741) was expressed in the E. coli M15pQE30 expression system and purified with immobilized-metal affinity chromatography. The correct conformation of the recombinant protein was evaluated by Western blotting and indirect immunofluorescence antibody (IFA) test. In addition, the immunogenic properties of PvAMA-1 were evaluated in BALB/c mice with the purified protein emulsified in Freund's adjuvant.ResultsIn the present study, the PvAMA-1 ectodomain was expressed at a high-level (65 mg/L) using a bacterial system. Reduced and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as Western blot analysis confirmed the appropriate conformation and folding of PvAMA-1. The evaluation of immunogenic properties of PvAMA-1 showed that both T helper-1 and 2 cells (Th1 and Th2) responses were present in mice after three immunizations and persisted up to one year after the first immunization. Moreover, the antibodies raised against the recombinant PvAMA-1 in injected mice could recognize the native protein localized on P. vivax parasites.ConclusionWe demonstrate that our recombinant protein had proper conformation and folding. Also, there were common epitopes in the recombinant forms corresponding to native proteins. These results; therefore, indicate that the expressed PvAMA-1 has the potential to be used as a vivax malaria vaccine.

Project description:Mexico advanced to the pre-elimination phase in 2009 due to a significant reduction in malaria cases, and since 2000, Plasmodium vivax is the only species transmitted. During the last two decades, malaria transmission has been mostly local and isolated to a few regions. It is important to gain further insights into the impact of control measures on the parasite population structure. Hence, the aim of the current study was to determine detailed changes in P. vivax genetic diversity and population structure based on analysing the gene that encodes the apical membrane antigen 1 (pvama1). This analysis covered from control to pre-elimination (1993-2011) in a hypo-endemic region in southern Mexico.The 213 pvama1 I-II sequences presently analysed were grouped into six periods of three years each. They showed low genetic diversity, with 15 haplotypes resolved. Among the DNA sequences, there was a gradual decrease in genetic diversity, the number of mixed genotype infections and the intensity of positive selection, in agreement with the parallel decline in malaria cases. At the same time, linkage disequilibrium (R2) increased. The three-dimensional haplotype network revealed that pvama1 I-II haplotypes were separated by 1-11 mutational steps, and between one another by 0-3 unsampled haplotypes. In the temporal network, seven haplotypes were detected in at least two of the six-time layers, and only four distinct haplotypes were evidenced in the pre-elimination phase. Structure analysis indicated that three subpopulations fluctuated over time. Only 8.5% of the samples had mixed ancestry. In the pre-elimination phase, subpopulation P1 was drastically reduced, and the admixture was absent.The results suggest that P. vivax in southern Mexico evolved based on local adaptation into three "pseudoclonal" subpopulations that diversified at the regional level and persisted over time, although with varying frequency. Control measures and climate events influenced the number of malaria cases and the genetic structure. The sharp decrease in parasite diversity and other related genetic parameters during the pre-elimination phase suggests that malaria elimination is possible in the near future. These results are useful for epidemiological surveillance.

Dataset Information

Nucleotide sequence polymorphism at the apical membrane antigen-1 locus reveals population history of Plasmodium vivax in Thailand.

Publications

Nucleotide sequence polymorphism at the apical membrane antigen-1 locus reveals population history of Plasmodium vivax in Thailand.

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