Preclinical evaluation of a multi-epitope mRNA vaccine platform for broad and durable SARS-CoV-2 protection

Introduction The emergence of immune-evasive SARS-CoV-2 variants has exposed limitations in the breadth and durability of protection conferred by current Spike-based vaccines, highlighting the need for next-generation approaches targeting conserved viral regions. Here, we describe the design and preclinical evaluation of an optimized multi-epitope vaccine, CoV2-BMEPu. Methods CoV2-BMEPu was rationally designed using immunological data from SARS-CoV-2 convalescent cohorts, incorporating conserved and immunodominant regions from the Spike (S), Membrane (M) and Nucleocapsid (N) proteins, together with selected receptor-binding domain (RBD) segments associated with broadly neutralizing antibodies. The construct was engineered as a secreted trimeric antigen and delivered as an mRNA vaccine formulated in lipid nanoparticles (LNPs). In vitro expression, innate immune activation, immunogenicity and protective efficacy were evaluated in cell systems and mouse models. Results mRNA-BMEPu was efficiently expressed in vitro as soluble oligomers and triggered innate immune activation in human macrophages. In C57BL/6 mice, LNP-BMEPu elicited robust binding and neutralizing antibodies against the ancestral virus and antigenically distant Omicron subvariants. Vaccination also induced strong and polyfunctional CD8⁺ T cell and T follicular helper responses that persisted over time. In K18-hACE2 transgenic mice, immunization conferred complete protection against lethal SARS-CoV-2 challenge, with effective control of viral replication and reduced lung inflammation. Discussion These results support CoV2-BMEPu as a next-generation multi-epitope mRNA vaccine candidate capable of inducing broad, durable and protective immunity against current and emerging SARS-CoV-2 variants.