Preclinical Evaluation of the SMNP-Adjuvanted R21 Malaria Vaccine Across Multiple Mouse Strains

Background: Adjuvants can enhance the immunogenicity of virus-like particle (VLP) vaccines. VLP-based malaria vaccines (RTS,S and R21) have demonstrated the potential of this approach and have now been deployed in many countries. However, the efficacy of these vaccines decays over time and could be improved by better vaccine formulations. The development of next-generation saponin-based adjuvants, like SMNP, offers an opportunity to enhance immunogenicity and optimize antigen dose, potentially improving protective immunity. Methods: In this pre-clinical study, we evaluated the immunogenicity and efficacy of the newer, more protective R21 vaccine when formulated with 5 µg of SMNP adjuvant, tested at both a low (0.1 µg) and standard (1 µg) antigen (R21) dose. Three different mouse strains (BALB/c, C57BL/6, and CD1) were immunized using a two-dose regimen with a 3-week interval between doses. Naive mice served as controls. Three weeks post-boost, all animals were challenged with transgenic Plasmodium berghei parasites expressing Plasmodium falciparum circumsporozoite protein. We assessed vaccine-induced humoral responses and compared immunogenicity and protective efficacy across dose groups and strains relative to naive controls. Results: Across all strains, SMNP-adjuvanted R21 induced robust NANP-specific IgG responses, with both the low-dose and standard-dose groups generating high antibody titers. Patterns of immunogenicity varied among mouse strains, but the low-dose R21 + SMNP group consistently elicited responses comparable to the standard-dose group. Although both vaccine doses delayed the onset of blood-stage parasitemia in all mouse strains, the low-dose groups showed a trend toward reduced efficacy compared with the standard dose, but this difference was not statistically significant. Conclusions: Our findings demonstrate that R21 formulated with SMNP is highly immunogenic, and that substantial dose-sparing may be achievable without compromising immune responses. This work supports further exploration of SMNP-adjuvanted VLP vaccines and highlights the potential for dose-optimized strategies to improve the accessibility and scalability of next-generation malaria vaccines.