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Identification and characterization of merozoite surface protein 1 epitope.

ABSTRACT: Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum-derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmodium falciparum and did not find any sequence similarity to human and mouse reference proteins. MSP1 is a known target of the immune response and a single CTL epitope binding to the HLA-A*0201 is available for merozoite form. Here, we report the results from the computational characterization of MSP1, precursor (1720 residue) and screening of highest scoring potential CTL epitopes for 1712 overlapping peptides binding to thirty four HLA class-I alleles and twelve HLA class-I supertypes (5 HLA-A and 7 HLA-B) using bioinformatics tools. Supertypes are the clustered groups of HLA class-I molecules, representing a sets of molecules that share largely overlapping peptide binding specificity. The prediction results for MSP1 as adhesin and adhesin-like in terms of probability is 1.0. Results also show that MSP1 has orthologs to other related species as well as having non allergenicity and single transmembrane properties demonstrating its suitability as a vaccine candidate. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.

SUBMITTER: Singh SP

PROVIDER: S-EPMC2770364 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Identification and characterization of merozoite surface protein 1 epitope.

Singh Satarudra Prakash SP Mishra Bhartendu Nath BN

Bioinformation 20090817 1

Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum-derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmod ...[more]

PMID: 20011145

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Project description:Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines.

Project description:BACKGROUND: Malaria caused by Plasmodium vivax is a major public health problem worldwide that affects 70-80 million people in the Middle East, Asia, Western Pacific, South America and the Caribbean. Despite its epidemiological importance, few antigens from this parasite species have been characterized to date compared to Plasmodium falciparum, due in part to the difficulties of maintaining an in vitro culture of P. vivax. This study describes the identification of the P. falciparum thrombospondin-related apical merozoite protein homologue in P. vivax (PvTRAMP) and examines its potential to be further evaluated as vaccine candidate. METHODS: The gene encoding PvTRAMP was identified through an extensive search of the databases hosting the genome sequence of P. vivax. Genes adjacent to pvtramp were identified in silico to determine the degree of similarity between the protein sequences encoded by equivalent chromosomic fragments in P. falciparum and Plasmodium knowlesi. The pvtramp gene was amplified from cDNA of P. vivax schizont stages, cloned and expressed in Escherichia coli. Anti-PvTRAMP antisera was obtained by inoculating rabbits with PvTRAMP B cell epitopes produced as synthetic peptides in order to assess its recognition in parasite lysates by Western blot and in intact parasites by indirect immunofluorescence. The recognition of recombinant PvTRAMP by sera from P. vivax-infected individuals living in endemic areas was also assessed by ELISA. RESULTS: The PfTRAMP homologue in P. vivax, here denoted as PvTRAMP, is a 340-amino-acid long antigen encoded by a single exon that could have a potential role in cytoadherence, as indicated by the presence of a thrombospondin structural homology repeat (TSR) domain. According to its transcription and expression profile, PvTRAMP is initially located at the parasite's apical end and later on the parasite surface. Recombinant PvTRAMP is recognized by sera from infected patients, therefore, indicating that it is targeted by the immune system during a natural infection with P. vivax. CONCLUSIONS: The results of this work support conducting further studies with PvTRAMP to evaluate its immunogenicity and protection-inducing ability in the Aotus animal model.

Project description:BackgroundThe development of an effective malarial vaccine is an urgent need. Most glycosylphosphatidylinositol (GPI)-anchored proteins of Plasmodium parasites are exposed to neutralizing antibodies, and several are advanced vaccine candidates. In the present study, Plasmodium vivax Pv32 (PVX_084815) as a hypothetical, predicted GPI-anchored and cysteine-rich motif was identified from our previous findings with a focus on its antigenic profiling. The orthologue gene pv32, a predicted GPI anchor of P. falciparum PF3D7_1434400, has still not been well studied.MethodsThe gene information of pv32 was obtained from PlasmoDB. Recombinant Pv32 protein was expressed and purified using a wheat germ cell-free expression system and a glutathione-Sepharose column. Naturally acquired immune response to recombinant Pv32 protein was evaluated using a protein microarray with 96 parasite-infected patients and 96 healthy individuals. Antibodies against recombinant Pv32 proteins from immune animals were produced, used and analyzed for the subcellular localization of native Pv32 protein by an immunofluorescence assay. A total of 48 pv32 sequences from 11 countries retrieved from PlasmoDB were used to determine the genetic diversity, polymorphisms and genealogical relationships with DNAsp and NETWORK software packages.ResultsPv32 is encoded by a conserved gene with two introns that are located on chromosome 13 and expressed as a 32 kDa protein in mature asexual stage parasites. Immunofluorescence data showed that Pv32 localized on the merozoite surface in schizont-stage parasites. The recombinant Pv32 was recognized by 39.6% of antibodies from P. vivax-infected individuals compared with healthy individuals. Low levels of nucleotide diversity (π = 0.0028) and polymorphisms of pv32 were detected within worldwide isolates.ConclusionsThis study shows the identification and characterization of the hypothetical protein, Pv32. Pv32 provides important characteristics, including a merozoite surface protein, a predicted GPI motif and Cysteine-rich motif among Plasmodium species. These results suggested that Pv32 is immunogenic with a merozoite surface pattern to antibodies during natural infection in humans.

Project description:BackgroundPlasmodium falciparum merozoite surface protein-1 (PfMSP-1) and -2 (PfMSP-2) are major blood-stage vaccine candidate antigens. Understanding the genetic diversity of the genes, pfmsp-1 and pfmsp-2, is important for recognizing the genetic structure of P. falciparum, and the development of an effective vaccine based on the antigens. In this study, the genetic diversities of pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum were analysed.MethodsThe pfmsp-1 block 2 and pfmsp-2 block 3 regions were amplified by polymerase chain reaction from blood samples collected from Myanmar patients who were infected with P. falciparum in 2013-2015. The amplified gene fragments were cloned into a T&A vector, and sequenced. Sequence analysis of Myanmar pfmsp-1 block 2 and pfmsp-2 block 3 was performed to identify the genetic diversity of the regions. The temporal genetic changes of both pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum population, as well as the polymorphic diversity in the publicly available global pfmsp-1 and pfmsp-2, were also comparatively analysed.ResultsHigh levels of genetic diversity of pfmsp-1 and pfmsp-2 were observed in the Myanmar P. falciparum isolates. Twenty-eight different alleles of pfmsp-1 (8 for K1 type, 14 for MAD20 type, and 6 for RO33 type) and 59 distinct alleles of pfmsp-2 (18 for FC27, and 41 for 3D7 type) were identified in the Myanmar P. falciparum population in amino acid level. Comparative analyses of the genetic diversity of the Myanmar pfmsp-1 and pfmsp-2 alleles in the recent (2013-2015) and past (2004-2006) Myanmar P. falciparum populations indicated the dynamic genetic expansion of the pfmsp-1 and pfmsp-2 in recent years, suggesting that a high level of genetic differentiation and recombination of the two genes may be maintained. Population genetic structure analysis of the global pfmsp-1 and pfmsp-2 also suggested that a high level of genetic diversity of the two genes was found in the global P. falciparum population.ConclusionDespite the recent remarkable decline of malaria cases, the Myanmar P. falciparum population still remains of sufficient size to allow the generation and maintenance of genetic diversity. The high level of genetic diversity of pfmsp-1 and pfmsp-2 in the global P. falciparum population emphasizes the necessity for continuous monitoring of the genetic diversity of the genes for better understanding of the genetic make-up and evolutionary aspect of the genes in the global P. falciparum population.

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Identification and characterization of merozoite surface protein 1 epitope.

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Identification and characterization of merozoite surface protein 1 epitope.

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