Redesign and Evaluation of a Plasmodium falciparum Circumsporozoite Protein-based Malaria Vaccine
Authors:   , Stump William , Burns James M. , Vaidya Akhil B. 
Published: January 2023

Despite success in lowering the global malaria burden over the last decade, progress in further reducing the disease burden has stalled. This stagnation illustrates the need for the development of an effective malaria vaccine. The most studied first-generation malaria vaccine, RTS,S, was deployed in pilot programs in several countries but provides only partial protection in children. RTS,S is a Plasmodium falciparum circumsporozoite protein (PfCSP)-based vaccine that targets the central repeat domain of a major surface antigen of sporozoites fused to a hepatitis B surface antigen carrier to increase immunogenicity. Recent studies indicate that parts of PfCSP not included in RTS,S, the N-terminus and junctional region, contain additional B-cell epitopes that are important for protection. We have expressed and purified four PfCSP-based vaccine antigens using a bacterial expression system. PfCSP constructs include a P. falciparum merozoite surface protein 8 (PfMSP8) carrier. This malaria specific carrier has been shown to enhance immunogenicity, facilitate production and purification of recombinant antigens in correct conformation, expand antibody diversity, and induce superior antibody durability when fused to other recombinant malaria vaccine antigens. We tested the hypothesis that PfMSP8 can be an effective carrier protein for a PfCSP-based vaccine, driving potent antibody responses against a diverse set of protective B cell epitopes. Additionally, we investigated if separation of N- and C-terminal portions of PfCSP can enhance antibody diversity to include non-repeat epitopes in the N-terminus and junctional region of PfCSP. While we did not observe a significant carrier-dependent increase in antibody magnitude, we observed striking differences in PfCSP-specific antibody specificity when comparing PfCSP and chimeric PfCSP-PfMSP8 (PfCSP/8) immunized mice. Fusion of PfMSP8 to PfCSP significantly increased the diversity of B cell epitopes recognized, particularly in the junctional region of PfCSP. Mice immunized with PfCSP primarily recognized conformational PfCSP epitopes while PfCSP/8 immunized mice recognized linear and conformational antibody epitopes in equal proportions. Immunization with a truncated PfCSP C-terminal construct fused to PfMSP8 (PfCSPC/8) induced a higher PfCSP-specific antibody response than immunization with a truncated N-terminal PfCSP construct fused to PfMSP8 (PfCSPN/8), but that increase in magnitude was not improved further by immunization with an admixture of PfCSPN/8 + PfCSPC/8. We also observed that immunization with PfCSPN/8 diversified the antibody response to a greater extent than PfCSPC/8, and that concurrent immunization with both constructs resulted in reduced recognition of N-terminal and junctional region epitopes compared to N-terminal immunization alone. Unexpectedly, immunization with PfCSP/8 or an admixture of PfCSPN/8 + PfCSPC/8 induced more diverse antibody responses in female compared to male mice. Overall, our rPfCSP-based vaccine constructs maintained antibody responses induced by RTS,S while also expanding the antibody diversity to include protective epitopes that are not present in RTS,S. Work to optimize these PfCSP-based malaria vaccines and Plasmodium challenge studies to assess protective efficacy, are ongoing.