Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys.

TLDR: Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model, and the soluble and refolded forms of PcMSP-142 and PvM SP-142 proteins also appeared to have a similar partially protective effect.

Abstract: Progress towards a vaccine against Plasmodium falciparum malaria is advancing rapidly, with several candidate antigens being tested for safety and efficacy in humans (4); comparatively, however, the development of a vaccine against P. vivax malaria has lagged behind. Unlike P. falciparum, where sporozoite challenge studies using the chloroquine-sensitive 3D7 strain are available, the relapsing nature of P. vivax hepatic stages and the lack of an in vitro culture system precludes any sporozoite challenge studies of P. vivax vaccine candidates in humans. Therefore, as more recombinant vaccine products become available, there will be an increasing need to compare and down-select vaccine candidate antigens in preclinical studies using animal models of P. vivax infection. P. cynomolgi, which infects rhesus macaques in southeast Asia, is a closely related species to P. vivax; human transmission of P. cynomolgi has also been reported (25). The two parasites share a similar clinical course of infection (26), a reticulocyte-specific invasion (17), the presence of Schuffner's dots on infected erythrocytes (2), and a dormant liver hypnozoite stage that is responsible for a relapsing blood stage infection (23). P. cynomolgi and P. vivax have similar genomic GC content, and rRNA analysis confirms their close taxonomic relatedness (43). High homology of prime vaccine candidates such as the apical membrane antigen 1, 97% (13); circumsporozoite protein, 90% (16); erythrocyte binding protein, 76% (32); and the 42-kDa fragment of the merozoite surface protein 1 (MSP-1), 75% (31); have been reported. Although it is believed that the P. cynomolgi-rhesus model can serve as a model for testing P. vivax antigens, there is only one previous report where this model was used to test the efficacy of a recombinant P. vivax vaccine (22). The merozoite surface protein 1 (MSP-1), found on the surface of Plasmodium merozoites, has been a prime vaccine candidate for many years (10). Following its synthesis as a 200-kDa precursor, the MSP-1 molecule undergoes step-wise proteolytic processing resulting in a glycosyl-phosphatidylinositol-anchored 42-kDa protein (MSP-142) on the surface of free merozoites (7). This 42-kDa intermediate undergoes secondary processing at the time of invasion, releasing a 33-kDa soluble polypeptide (MSP-133) and leaving behind on the invading merozoite a glycosyl-phosphatidylinositol-anchored 19-kDa form (MSP-119) (7). Depending on the species, MSP-119 contains 10 or 11 cysteine residues that form five disulfide bonds. Immunization with recombinant MSP-142 and MSP-119 raises antibodies that inhibit invasion of merozoites in vitro (9) and protects the immunized animals against live parasite challenge (29). We have previously reported a process for the production of correctly folded human vaccine-grade P. vivax MSP-142 protein in the bacterial expression host Escherichia coli Origami(DE3) (14). This bacterial strain has mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhances the disulfide bond formation of recombinant proteins expressed in its cytoplasm, hence resulting in a soluble product (6). However, the transposon-mediated genetic modifications of E. coli carried both the tetracycline and the kanamycin resistance genes as selectable markers and therefore made the cells incompatible with the production of human use vaccines that relied on plasmids carrying the Tetr or the Kanr genes. The use of a compatible expression host E. coli BL21(DE3) resulted in the production of an MSP-142 protein that was insoluble and located in the inclusion body fraction of the cells. In this report we have shown that soluble MSP-142 protein can be obtained from the BL21(DE3) cells by in vitro refolding of the inclusion body-derived protein under controlled redox conditions. Both the soluble and refolded P. vivax MSP-142 products appeared to be structurally similar based on biophysical analysis and reactivity with conformational epitope-specific monoclonal antibodies. It was our goal, therefore, to seek evidence of the immunological equivalence of these proteins in a parasite challenge model. Towards that end we report here the results of a vaccination study comparing the immunogenicity and efficacy of the soluble and refolded P. vivax MSP-142 products in the P. cynomolgi-rhesus model. We have also produced equivalent homologous constructs of P. cynomolgi MSP-142 protein both in their soluble and refolded forms, and these two proteins were also used as positive control immunogens in this study.