An Erythrocyte Membrane-Associated Antigen, PvTRAg-26 of Plasmodium vivax: A Study of Its Antigenicity and Immunogenicity

Abstract

Background:Plasmodium tryptophan-rich (TR) proteins have been proposed as potential vaccine candidate antigens. Among them, P. vivax tryptophan-rich antigens (PvTR-Ags), which have positionally conserved tryptophan residues in a TR domain, are highly antigenic in humans. Several of these antigens, including PvTRAg-26, have exhibited erythrocyte-binding activities. Methods: Subclasses of IgG antibodies against PvTRAg-26 were detected by enzyme-linked immunosorbent assay in 35 P. vivax infected patients and mice immunized with the recombinant antigen to characterize its antigenicity and immunogenicity. Moreover, the antigen-specific immune responses and Th1/Th2-type cytokine patterns of splenocytes from the immunized animals were determined in vitro. The subcellular localization of PvTRAg-26 in ring-stage parasites was also detected by indirect immunofluorescence assay. Results: The IgG1 and IgG3 levels in P. vivax-infected patients were significantly higher than those in uninfected individuals. In the PvTRAg-26-immunized mice, elevated levels of antigen-specific IgG antibodies were observed, dominated by the IgG1 subclass, and Th1-type cytokines were remarkably increased compared with Th2-type cytokines. Additionally, the subcellular location of the PvTRAg-26 protein was closely associated with the caveola-vesicle complex on the infected-erythrocyte membrane in the early ring stage of P. vivax. Conclusions: PvTRAg-26, a P. vivax TR antigen, with high antigenicity and immunogenicity, induces Th1-cytokine response and increases production of IgG1 antibodies. This immune profiling study provided a substantial evidence that PvTRAg-26 may be a potential candidate for P. vivax vaccine development.

Keywords: Plasmodium vivax; immunogenicity; malaria; tryptophan-rich antigens; vaccine candidate.

Figure 1

Schematic diagram showing the expression of PvTRAg-26. (A) Diagram of the gene structure of pvtrag-26; aa, amino acids. (B) Expression and purification of recombinant PvTRAg-26. T: total translation mix, S: supernatant, P: precipitate, Ft: flow through, W: wash; E: elution treated with reducing buffer. (C) Recombinant PvTRAg-26 protein under reducing conditions was probed with an anti-His tag antibody, P. vivax malaria patient serum and immune mouse serum. H: anti-His tag antibody, M: immune mouse serum, P: P. vivax-infected patients' pooled sera.

Figure 2

The levels of IgG subclasses recognizing PvTRAg-26 in sera from P. vivax-infected patients and uninfected individuals. Differences between IgG subclass levels in the negative and positive groups were analyzed using Student's t-tests. ***P < 0.001. The P values for IgG1 and IgG3 were <0.001, and those for IgG2 or IgG4 were >0.05. P < 0.05 was considered to indicate a significant difference.

Figure 3

Levels of serum IgG and IgG subclasses in immunized mice. (A) Antigen-specific IgG levels were detected by ELISA in the sera of mice, as indicated after the final immunizations with PvTRAg-26, ***P < 0.001. (B) Serum IgG subclass pattern in PvTRAg-26-immunized mice, ***P < 0.001.

Figure 4

Proliferation index and cytokine secretion of splenocytes from mice immunized with PvTRAg-26. (A) Splenocytes from mice immunized with or without PvTRAg-26 were stimulated with various concentrations of PvTRAg-26 for 72 h before testing, as indicated. The splenocytes were stimulated with Con A or LPS as positive controls, as indicated, *P < 0.05, **P < 0.01. (B) Cytokine secretion profile of the splenocytes from the antigen-immunized mice.

Figure 5

Localization of PvTRAg-26 in the early ring/trophozoite stage. (A) Ring stage parasites were double-labeled with mouse antisera against PvTRAg-26 (red) and a rabbit anti-Band 3 antibody (green). (B) Ring/trophozoite stage parasites were dual-labeled with mouse antisera against PvTRAg-26 (red) and rabbit antisera against PvCVC81₉₅ (CVC marker, green). Nuclei were visualized with DAPI in merged images. The bar represents 5 μm.