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Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1).

ABSTRACT: Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P?=?0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

SUBMITTER: Bueno LL

PROVIDER: S-EPMC3119695 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1).

Bueno Lilian Lacerda LL Lobo Francisco Pereira FP Morais Cristiane Guimarães CG Mourão Luíza Carvalho LC de Ávila Ricardo Andrez Machado RA Soares Irene Silva IS Fontes Cor Jesus CJ Lacerda Marcus Vinícius MV Chavez Olórtegui Carlos C Bartholomeu Daniella Castanheira DC Fujiwara Ricardo Toshio RT Braga Erika Martins EM

PloS one 20110621 6

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association betwe ...[more]

PMID: 21713006

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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.

Dataset Information

Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1).

Publications

Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1).

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