Identification and characterization of Pv50, a novel Plasmodium vivax merozoite surface protein

Abstract

Background: Plasmodium vivax contains approximately 5400 coding genes, more than 40% of which code for hypothetical proteins that have not been functionally characterized. In a previous preliminary screening using pooled serum samples, numerous hypothetical proteins were selected from among those that were highly transcribed in the schizont-stage of parasites, and highly antigenic P. vivax candidates including hypothetical proteins were identified. However, their immunological and functional activities in P. vivax remain unclear. From these candidates, we investigated a P. vivax 50-kDa protein (Pv50, PVX_087140) containing a highly conserved signal peptide that shows high transcription levels in blood-stage parasites.

Results: Recombinant Pv50 was expressed in a cell-free expression system and used for IgG prevalence analysis of patients with vivax malaria and healthy individuals. Immune responses were analyzed in immunized mice and mouse antibodies were used to detect the subcellular localization of the protein in blood-stage parasites by immunofluorescence assay. A protein array method was used to evaluate protein-protein interactions to predict protein functional activities during the invasion of parasites into erythrocytes. Recombinant Pv50 showed IgG prevalence in patient samples with a sensitivity of 42.9% and specificity of 93.8% compared to that in healthy individuals. The non-cytophilic antibodies IgG1 and IgG3 were the major components involved in the antibody response in Pv50-immunized mice. Pv50 localized on the surface of merozoites and a specific interaction between Pv50 and PvMSP1 was detected, suggesting that Pv50-PvMSP1 forms a heterodimeric complex in P. vivax.

Conclusions: Increased immune responses caused by native P. vivax parasites were detected, confirming its immunogenic effects. This study provides a method for detecting new malaria antigens, and Pv50 may be a vivax malaria vaccine candidate with PvMSP1.

Keywords: Antigenicity; Immunogenicity; Merozoite surface protein; Plasmodium vivax; Protein interaction; Pv50.

Fig. 1

Primary structure and sequence characterization of Pv50. a Schematic diagram of Pv50. The Pv50 protein contains 460 amino acids, with a calculated molecular mass of 50.4 kDa. Indicated are the signal peptide (amino acid (aa) positions 1–19) and lipid attachment site (aa, 1–25). Truncated Pv50 (20–460 aa) was constructed for expression. b Phylogenetic tree showing the relationships among protein sequences of P. cynomolgi, P. inui, P. fragile, P. coatneyi, P. vivax, P. knowlesi and P. malariae 50 genes. The position of P. vivax pv50 is indicated with a red arrow. c Sliding window plot showing the nucleotide diversity of the pv50 in 64 worldwide isolate sequences with window size of 25 and a step size of 5

Fig. 2

Recombinant Pv50 protein for expression. a The purification progress of Pv50 (46 kDa) was evaluated by 12.5% SDS-PAGE. b Western blot analysis of recombinant Pv50 with penta anti-His antibody (His), mouse immune sera (M), mixed vivax patient sera (P), rabbit immune sera (R), and schizont-stage parasite lysates (PL) under reducing conditions probed with IgG antibody from Pv50 rabbit immune sera. Arrowheads indicate specific bands for each recombinant protein. Abbreviations: M, protein marker; T, total translation mix; S: supernatant; P, purification; Ft: flow through; N, elution under non-reducing; R, elution under reducing

Fig. 3

IgG antibody responses to Pv50 using protein microarrays. Recombinant Pv50 from the sera of malaria patients (positive) and healthy individual samples (negative) from the ROK was probed. Significant differences in total IgG prevalence were observed with high specificity between patients with vivax and healthy individuals (P < 0.0001). The P-values were calculated using a Mann-Whitney test. The bar indicates the mean plus ± SD

Fig. 4

Localization of Pv50 mature schizont stage. Schizont-stage parasites were dual-labeled with antisera against Pv50 (red color), PvMSP1-19 (merozoite surface marker, green color), and nuclei were stained with DAPI (blue). Scale-bar: 2.5 μm

Fig. 5

Immunogenicity analysis of Pv50. OD₄₅₀ value of culture medium with recombinant Pv50 (2.5, 5 and 10 µg/ml), ConA, LPS, or only culture medium as control. a Proliferation of splenocytes is shown after stimulation with Con A or LPS as a positive control. The results are expressed as the mean value of OD₄₅₀ ± SD. b Cytokine levels from 72 h cultured supernatants of splenocytes from Pv50-immunized BALB/c mice stimulated in vitro with Pv50. The black horizontal bars represent the cytokine geometric means ± 3SD. c Specific IgG1 (black), IgG2b (dark gray), IgG3 (light gray) and IgG2a (white) against the immunogen itself were determined by ELISA. The results are expressed as the mean titers ± SD

Fig. 6

The interaction between Pv50 and PvMSP1. The interaction were visualized by probing mouse and rabbit immune sera, and staining parasites with probes termed anti-Mouse MINUS and anti-rabbit PLUS, the hybridization probes were labeled with Texas Red (Red), and nuclei were stained with DAPI (blue). a PvMSP1 interacted with Pv50 in the schizont stage. b PvMSP10 did not interact with Pv50 in the schizont stage. c PLA assay developed without first antibody as blank control. Scale-bar: 2.5 μm

Fig. 7

Kinetics for interaction between Pv50 and P. vivax MSP1. Reaction mixtures (1 μl) containing 10 μg/ml Cy5-conjugated Pv50 were applied to protein arrays for 30 min. Interactions of Pv50 with PvMSP1 were determined as described in methods. The results are expressed as the means of fluorescence intensities ± SD from three separate experiments