A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

Abstract

BackgroundA vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.Methodology/Principal FindingsWe selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance.Conclusions/SignificanceWe produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity.Trial RegistrationClinicalTrials.gov NCT00663546

Evolution of the merozoite surface protein 7 ( msp7 ) family in Plasmodium vivax and P . falciparum : A comparative approach

“…Then such paralogs that underwent an adaptive divergence in the P. vivax lineage, have been under purifying selection in the extant P. vivax populations indicating functional constrains. Interestingly, there is evidence that two of those genes (PVX_082675 and PVX_82680) are immunogenic and the latter is recognized by semi-immune individuals without fever after being challenged with sporozoites indicating that those antibodies could be associated with protection (Arévalo-Herrera et al, 2016; Hostetler et al, 2015). …”

Evidence of functional divergence in MSP7 paralogous proteins: a molecular-evolutionary and phylogenetic analysis

“…Recent protein-protein interaction assays have shown that MSP7 proteins do not appear to bind to the same host receptor [26]; moreover, these proteins seem to be forming different protein complexes in the parasite [7, 27–30], maybe to perform different parasite-host interactions. Such results flout the functional redundancy hypothesis [15, 18]; functional divergence in MSP7 paralogs thus appears to be probable.…”

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies

“…! have improved success rates in the production of soluble protein for use in immunoepidemiology [43] and receptor-ligand interaction studies [22,44]. We chose to use transiently transfected, non-adherent HEK293E cells for protein expression; 15 of 22 proteins (68%) were expressed with detection by anti-pentahistidine antibody in our study.…”