Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein

Abstract

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.

Figure 1

Schematic representation of pftramp, pvtramp and pktramp. The figure shows the localization of the genes encoding PfTRAMP, PkTRAMP (both in gray) and PvTRAMP (in black) in P. falciparum, P. knowlesi and P. vivax chromosomic fragments, respectively, as well as the localization of the adjacent genes evaluated in this study. The arrows above each box indicate the ORF orientation, while the boxes show distribution and organization of the exons along the chromosomal segments. Genes are assigned according to their annotation in PlasmoDB. These chromosomic fragments comprised 21.5 kbp from the P. falciparum chromosome 12 (695,001-716,500 pb), 23.5 kbp from the P. vivax contig CM000455 (1,519,501-1,543,000 pb) and 24 kbp from the P. knowlesi chromosome 14 (1,548,001-1,572,000 pb).

Figure 2

(A) Schematic representation of PvTRAMP indicating the localizations of the predicted signal peptide and transmembrane domain (both in dark gray), as well as the TSR domain (light gray). Localization of the conserved cysteines inside the TSR domain and the synthetic peptides used in this study to obtain anti-PvTRAMP antisera are indicated by arrow heads and white boxes, respectively. (B) PCR amplification of pvtramp from P. vivax genomic DNA and cDNA. Lane 1. Amplification from genomic DNA using primers designed based on the sequence predicted for pvtramp. Lane 2. RT-PCR product amplified from DNAse-treated total P. vivax RNA. (C) Recognition of purified rPvTRAMP by anti-PvTRAMP rabbit sera, as assessed by Western blot. Lane 1: pre-immune sera. Lane 2: hyperimmune sera. Lane H: recognition of purified rPvTRAMP by anti-polyhistidine monoclonal antibody. (D) Western blot analysis of a P. vivax lysate with anti-PvTRAMP rabbit sera. Lane 1: pre-immune sera. Lane 2: hyperimmune sera.

Figure 3

Cellular localization of PvTRAMP as assessed by IFA using hyper-immune anti-PvTRAMP rabbit sera as primary antibody. (A-C) Detection of P. vivax in early schizont stages. (D-I) Parasites in late schizont stage (segmented). The figure shows fluorescence with DAPI and FITC staining, and the merging of both.

Figure 4

ELISA showing reactivity of sera from P. vivax-infected patients against rPvTRAMP. Columns 1-20 correspond to recognition by sera from P. vivax malaria patients. Columns 21-23 showed recognition of rPvTRAMP by healthy individuals that had never had an episode of P. vivax malaria. rPvTRAMP used in this assay was resuspended in urea and thoroughly dialyzed against PBS for its refolding. Each column is shown with its corresponding standard deviation.