Inclusion of an Optimized Merozoite Surface Protein 2-Based Antigen in a Trivalent, Multistage Malaria Vaccine.

merozoite surface protein (MSP)2 is a target of parasite-neutralizing Abs. Inclusion of recombinant MSP2 (rMSP2) as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we used the highly immunogenic MSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. In this study, we exploited the benefits of rMSP8 as a carrier to optimize a rMSP2-based subunit vaccine. rMSP2 and chimeric rMSP2/8 vaccines produced in were evaluated in comparative immunogenicity studies in inbred (CB6F1/J) and outbred (CD1) mice, varying the dose and adjuvant. Immunization of mice with both rMSP2-based vaccines elicited high-titer anti-MSP2 Abs that recognized the major allelic variants of MSP2. Vaccine-induced T cells recognized epitopes present in both MSP2 and the MSP8 carrier. Competition assays revealed differences in Ab specificities induced by the two rMSP2-based vaccines, with evidence of epitope masking by rMSP2-associated fibrils. In contrast to aluminum hydroxide (Alum) as adjuvant, formulation of rMSP2 vaccines with glucopyranosyl lipid adjuvant-stable emulsion, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of Abs to cytophilic IgG subclasses. The rMSP2/8 + glucopyranosyl lipid adjuvant-stable emulsion formulation induced significantly higher Ab titers with superior durability and capacity to opsonize merozoites for phagocytosis. Immunization with a trivalent vaccine including MSP2/8, MSP1/8, and the 25 kDa sexual stage antigen fused to MSP8 (s25/8) induced high levels of Abs specific for epitopes in each targeted domain, with no evidence of antigenic competition. These results are highly encouraging for the addition of rMSP2/8 as a component of an efficacious, multivalent, multistage malaria vaccine.