Immunological Evaluation of mRNA‐LNP Vaccines Targeting Conserved S2 and NSP3 Domains of SARS‐CoV‐2

ABSTRACT

The evolution of SARS‐CoV‐2 necessitates next‐generation vaccine strategies targeting conserved viral elements to ensure broad and durable protection. In this study, we designed and evaluated novel mRNA‐based vaccines encoding conserved domains of SARS‐CoV‐2, including the spike S2 subunit, NSP3 Macrodomain I (Mac I), and papain‐like protease (PLpro). These mRNAs were synthesized, cloned, and encapsulated into lipid nanoparticles (LNPs) with 70%–80% efficiency and favorable physicochemical properties. In vitro characterization showed efficient antigen expression with 60%–80% cell survival. Immunization of BALB/c mice induced strong humoral responses, particularly against S2 and Mac I, marked by IgG titers, class switching, and durable antibody presence up to 84 days post‐booster. PLpro elicited minimal antibody responses, likely due to limited stability or intrinsic low immunogenicity. Combinatorial LNPs encoding multiple antigens maintained robust expression and elicited comparable immune responses despite dose splitting. Anti‐S2 antibodies effectively inhibited spike protein uptake in vitro. Importantly, the selected antigenic regions exhibited high sequence conservation across major SARS‐CoV‐2 variants, underscoring their broad‐spectrum vaccine potential. These findings support further development of domain‐targeted, multi‐antigen mRNA vaccines to enhance cross‐variant protection and T‐cell‐mediated immunity.