In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

Abstract

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.

A systematic review on malaria sero-epidemiology studies in the Brazilian Amazon: insights into immunological markers for exposure and protection

“…The frequency of responders shows that IgG antibody reactivity to PvMSP-1 19 was 86.7% and the frequency of positive individuals for at least one of the recombinant proteins representing PvMSP-3a and PvMSP-9 sequences was 77 and 76%, respectively. Another paper conducted at those same localities in Rondônia State verified 58% of positive antibody responses to recombinant PvMSP9-RIRII and 32.5% to PvMSP9 E795-A808 protein [34]. In agreement to previous studies, seropositive individuals for PvMSP9-RIRII had a higher median of years of residence in the endemic area.…”

Apical membrane protein 1‐specific antibody profile and temporal changes in peripheral blood B‐cell populations in Plasmodium vivax malaria

“…The concentration of IgG binding to the recombinant protein PvAMA‐1 was measured by enzyme‐linked immunosorbent assay (ELISA) as previously described . The absorbance value (OD) was obtained at 492 nm using an ELISA reader (Spectramax 250, Molecular Devices).…”

Main B‐cell epitopes of PvAMA‐1 and PvMSP‐9 are targeted by naturally acquired antibodies and epitope‐specific memory cells in acute and convalescent phases of vivax malaria

“…This first study, conducted with Brazilian subjects who were exposed to low levels of vivax malaria transmission, provides evidence that two peptides which contain B‐cell epitopes (PvAMA‐1 (S290‐K307) and PvMSP‐9 (E795‐A808) ) are recognized by naturally acquired antibodies and MBCs, which are detectable several months after clinical malaria episodes. The high immunogenicity for both peptides has been previously demonstrated, but there was no inference about the persistence of immune response over time for these antigens.…”

“…For determination of antigen‐specific IgG levels and frequency of antibody‐secreting cells (ASC), we synthesized the peptide sequences SASDQPTQYEEEMTDYQK, corresponding to residues S290‐K307 of PvAMA‐1, and EAAPENAEPVHENA, corresponding to residues E795‐A808 of PvMSP‐9. These peptides were chosen because they are recognized as highly immunogenic B‐cell epitopes . Both were synthesized by fluorenylmethoxycarbonyl (F‐moc) solid‐phase chemistry (purity > 95%).…”

“…Likewise, naturally acquired antibodies to PvMSP‐9 have also been associated with reduction of the risk of clinical malaria among children under 3 years old . Recently, PvMSP‐9 (E795‐A808) was confirmed as the main epitope in C terminal region of PvMSP‐9, and specific antibodies from mice that were immunized with this epitope were also found capable of targeting P vivax merozoites in vitro …”