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Plasmodium falciparum-specific antibodies tend to be short-lived, but their cognate memory B cells (MBCs) circulate in the peripheral blood of exposed subjects for several months or years after the last infection. However, the time course of antigen-specific antibodies and B-cell responses to the relatively neglected parasite Plasmodium vivax remains largely unexplored. Here, we showed that uncomplicated vivax malaria elicits short-lived antibodies but long-lived MBC responses to a major blood-stage P vivax antigen, apical membrane protein 1 (PvAMA-1), in subjects exposed to declining malaria transmission in the Amazon Basin of Brazil. We found that atypical (CD19 + CD10 - CD21 - CD27 - ) MBCs, which appear to share a common precursor with classical MBCs but are unable to differentiate into antibody-secreting cells, significantly outnumbered classical MBCs by 5:1 in the peripheral blood of adult subjects currently or recently infected with P vivax and by 3:1 in healthy residents in the same endemic communities. We concluded that malaria can drive classical MBCs to differentiate into functionally impaired MBCs not only in subjects repeatedly exposed to P falciparum, but also in subjects living in areas with low levels of P vivax transmission in the Amazon, leading to an impaired B-cell memory that may affect both naturally acquired and vaccine-induced immunity., (© 2019 John Wiley & Sons Ltd.)

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9 E795-A808 ) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9 E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9 E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9 E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.