Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant<i>Pf</i>s25, a Leading Transmission-Blocking Vaccine Candidate

Abstract

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a <i>Plasmodium falciparum</i> multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP1₁₉) to <i>P. falciparum</i> MSP8 (<i>Pf</i>MSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP1₁₉ Here, using the <i>Pf</i>MSP1/8 construct, we further optimized the recombinant <i>Pf</i>MSP8 (r<i>Pf</i>MSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate <i>Pf</i>s25. The production of r<i>Pf</i>s25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of <i>Pf</i>s25 block sexual-stage development in mosquitoes. The sequence encoding mature <i>Pf</i>s25 was codon harmonized for expression in <i>Escherichia coli</i> We produced a r<i>Pf</i>s25-<i>Pf</i>MSP8 fusion protein [r<i>Pf</i>s25/8(CΔS)] as well as unfused, mature r<i>Pf</i>s25. r<i>Pf</i>s25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric r<i>Pf</i>s25/8(CΔS) was expressed and easily purified, with the <i>Pf</i>s25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native <i>Pf</i>s25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of <i>Pf</i>MSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.