Expression and immunogenicity of a rabies virus glycoprotein–HSV-2 ICP35 fusion antigen delivered by an mRNA–LNP formulation

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Rabies virus (RABV) is a zoonotic pathogen with an almost 100% case-fatality rate and limited post-exposure prophylaxis options. mRNA-based antigen delivery represents a promising approach but requires further optimization. We prepared a cationic lipid nanoparticle (LNP) delivery system using the thin-film hydration method and characterized its physicochemical properties and delivery efficiency. An mRNA encoding a fusion antigen comprising rabies virus glycoprotein (G) and herpes simplex virus type 2 infected cell polypeptide 35 (ICP35), lacking canonical 5' and 3' untranslated region sequences, was constructed. In vitro protein expression of the G-ICP35 mRNA was confirmed. Immunogenicity was evaluated in BALB/c mice following three intramuscular immunizations with G-ICP35 mRNA, RABV G/N proteins, or their combination. Tail vein blood samples were collected on days 7, 21, and 39 after the final immunization, and rabies virus-specific antibody titers and T-cell responses were assessed. The cationic LNP formulation efficiently mediated intracellular delivery and expression of the G-ICP35 mRNA. In vivo, the mRNA-LNP formulation induced detectable humoral and cellular immune responses, including rabies virus-specific IgG production and antigen-specific T-cell activation. Notably, co-immunization with mRNA and subunit vaccines further enhanced immune responses compared with either vaccine alone, achieving peak rabies virus-specific IgG titers of 694.5 IU/mL. These findings demonstrate the feasibility of a DCChol-based mRNA-LNP formulation for fusion antigen delivery and support further investigation of combined mRNA and subunit vaccine strategies.