Identification of a novel merozoite surface antigen of Plasmodium vivax, PvMSA180

Abstract

Background: Although a number of Plasmodium vivax proteins have been identified, few have been investigated as potential vaccine candidates. This study characterized the Plasmodium vivax merozoite surface antigen 180 (PvMSA180, PVX_094920), a novel P. vivax antigenic protein.

Methods: The target gene was amplified as four overlapping domains (D1, D2, D3 and D4) to enable expression of the recombinant protein using cell-free and bacterial expression systems. The recombinant PvMSA180 proteins were used in protein microarrays to evaluate the humoral immune response of 72 vivax-infected patients and 24 vivax-naïve individuals. Antibodies produced in mice against the PvMSA180-D1 and -D4 domains were used to assess the subcellular localization of schizont-stage parasites with immunofluorescence assays. A total of 51 pvmsa180 sequences from 12 countries (41 sequences from PlasmoDB and 6 generated in this study) were used to determine the genetic diversity and genealogical relationships with DNAsp and NETWORK software packages, respectively.

Results: PvMSA180 consists of 1603 amino acids with a predicted molecular mass of 182 kDa, and has a signal peptide at the amino-terminus. A total of 70.8% of patients (51/72) showed a specific antibody response to at least one of the PvMSA180 domains, and 20.8% (15/72) exhibited a robust antibody response to at least three of the domains. These findings suggest that PvMSA180 is targeted by the humoral immune response during natural infection with P. vivax. Immunofluorescence analysis demonstrated that PvMSA180 is localized on the merozoite surface of schizont-stage parasites, and pvmsa180 sequences originating from various geographic regions worldwide showed low genetic diversity. Twenty-two haplotypes were found, and haplotype 6 (Hap_6, 77%) of pvmsa180 was detected in isolates from six countries.

Conclusions: A novel P. vivax surface protein, PvMSA180, was characterized in this study. Most of P. vivax-infected patients had specific antibodies against particular antigenic domains, indicating that this protein is immunogenic in naturally exposed populations. Genetic analysis of worldwide isolates showed that pvmsa180 is less polymorphic than other well-known candidates and that some haplotypes are common to several countries. However, additional studies with a larger sample size are necessary to evaluate the antibody responses in geographically separated populations, and to identify the function of PvMSA180 during parasite invasion.

Keywords: Haplotype; Humoral immune response; MSA180; Merozoite surface protein; Plasmodium vivax.

Fig. 1

Schematic of recombinant PvMSA180 expression. a Schematic of PvMSA180. The D1 (amino acids [aa] 28–401), D2 (aa 385–800), D3 (aa 785–1180), and D4 (aa 1163–1603) domains were expressed using cell-free and bacterial expression systems. SP signal peptide, P polymorphic region (aa 290–307). b Clustal alignment of the C-terminus sequence of MSA180 of Plasmodium vivax (Pv), Plasmodium falciparum (Pf), Plasmodium knowlesi (Pk), and Plasmodium cynomolgi (Pc). Red bars indicate the conserved aa in the four Plasmodium species, green bars in three species, sky-blue bars in two species, and dark-blue bars in one species. Asterisks denote cysteine residues conserved in the four Plasmodium species

Fig. 2

Expression of recombinant PvMSA180. a Expression of recombinant domains of PvMSA180 in a wheat germ cell-free expression system. Arrowheads indicate the four domains of PvMSA180. D1 domain 1, D2 domain 2, D3 domain 3, and D4 domain 4. b Expression and purification of PvMSA180-D1 (GST-fused) and D4 (His-tagged) in E. coli. c Recombinant PvMSA180-D1 and D4 were probed with anti-GST and anti-His antibodies under reducing conditions. Arrowheads indicate the PvMSA180-D1 (~80 kDa) and D4 (~60 kDa) bands. d Antibody recognition of PvMSA180-D1 and D4 compare to PvDBP-RII recombinant proteins using pooled vivax-patient sera (lane P), malaria naïve healthy sera (lane H), mouse immune serum (lane M), and anti-GST or anti-His antibody (lane R)

Fig. 3

Human IgG response to the four domains of PvMSA180 by protein microarray. a Crude recombinant PvMSA180 was probed with sera from vivax-infected patients (P) and naïve healthy individuals (H). The total prevalence of anti-PvMSA180 IgG significantly differed between patients and naïve healthy individuals. *p < 0.01; ***p < 0.0001 by Student’s t-test. Bars indicate means ± standard deviation. b IgG response to the four domains of PvMSA180 in individual patients. Red and green bars indicate positive and negative reactivity, respectively, in each domain. For total reactivity, red bar indicates 3–4 positive domains; green indicates 1–2 positive domains, and blue indicates the negative domains. The serial numbers of the serum samples are shown on the vertical line

Fig. 4

Localization of PvMSA180 in mature schizont-stage parasites. Schizont-stage parasites were dual-labelled with antisera against PvMSP1-19 (merozoite surface marker) (a and b), PvDBP (microneme marker) (c), or PvRAMA (rhoptry marker) (d). Nuclei are stained with DAPI in the merged images. Scale bar represents 5 µm. DIC differential interference contrast

Fig. 5

Median-joining networks of pvmsa180 haplotypes from isolates worldwide. Genealogical haplotype network showing the relationships among 22 pvmsa180 haplotypes in 51 sequences obtained from P. vivax isolates from 12 countries. H-number, haplotype number. The size of the circles represents the haplotype frequencies and unnumbered circles indicate a single haplotype. Geographical haplotypes are indicated by the colour key. Small red nodes are hypothetical median vectors created by the program to connect sampled haplotypes into a parsimonious network. The distances between nodes are arbitrary