Dataset Information

Analysis of Plasmodium vivax Apical Membrane Antigen-1 (PvAMA-1) Haplotypes among Iranian Isolates.

ABSTRACT: Plasmodium vivax apical membrane antigen-1(PvAMA-1) is a surface protein with polymorphic sites. This study was aimed to analyze the polymorphic amino acid residues at PvAMA-1 in different infected age groups. 92 blood samples were collected from the south and southeast of Iran. The DNA coding for the domain I (DI), DII, and partial DIII of this antigen was amplified by Nested-PCR, and sequenced. Nucleotide mutations were found in 49 sites and based on the amino acid sequence, 30 variable sites were detected. Age distribution of malaria cases showed that the majority of the patients were between 10 to 30 years old. The scattering plot haplotypes by age showed an increasing incidence rate with age during childhood, whereas, incidence was the lowest in patients under five years old. Comparison of the polymorphic sites of PvAMA-1 in Iranian isolates with those found in other geographic regions of the world indicated nine common variable positions. In addition, a significant dependence was found between some particular substitutions and age categories. Dependence between particular substitutions and age groups suggests that certain residues in AMA-1 are responsible for clinical attacks in different ages, likely as a result of host immune pressure. The crystal structure of the PvAMA-1 showed that the amino acid substitutions that changed the protein charge were exclusively located in loops and turns where, the interactions with antibodies could occur. These data provide the necessary information for an AMA-1 based malaria vaccine design to be effective across all ages.

SUBMITTER: Nedaei F

PROVIDER: S-EPMC6004292 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Analysis of <i>Plasmodium vivax</i> Apical Membrane Antigen-1 (PvAMA-1) Haplotypes among Iranian Isolates.

Nedaei Fatemeh F Noormohammadi Zahra Z Naddaf Saied Reza SR Mohammadi Somayeh S Esmaeili Rastaghi Ahmad Reza AR

International journal of molecular and cellular medicine 20171116 4

<i>Plasmodium vivax</i> apical membrane antigen-1(PvAMA-1) is a surface protein with polymorphic sites. This study was aimed to analyze the polymorphic amino acid residues at PvAMA-1 in different infected age groups. 92 blood samples were collected from the south and southeast of Iran. The DNA coding for the domain I (DI), DII, and partial DIII of this antigen was amplified by Nested-PCR, and sequenced. Nucleotide mutations were found in 49 sites and based on the amino acid sequence, 30 variable ...[more]

PMID: 29988191

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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.

Dataset Information

Analysis of Plasmodium vivax Apical Membrane Antigen-1 (PvAMA-1) Haplotypes among Iranian Isolates.

Publications

Analysis of <i>Plasmodium vivax</i> Apical Membrane Antigen-1 (PvAMA-1) Haplotypes among Iranian Isolates.

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