Production Optimization, Adjuvant Screening and Immunogenicity Evaluation of a Virus-like Vesicle Rabies Vaccine

Background/Objectives: Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), and effective therapeutic treatments are currently lacking. Vaccination remains the primary strategy for rabies control. The Semliki Forest virus-rabies virus glycoprotein (SFV-RVG), a virus-like vesicle rabies vaccine combining Semliki Forest virus replicase and rabies glycoprotein, has shown potential as a promising vaccine candidate. This study aimed to optimize the production of SFV-RVG and evaluate adjuvant formulations to improve its immunogenicity in both mice and dogs. Methods: SFV-RVG production was optimized by determining the optimal multiplicity of infection (MOI) at 0.03 and cell density at 1 × 10⁶-1.3 × 10⁶ cells/mL, followed by scaling up the process in bioreactors. Eleven adjuvant formulations were tested in mice and dogs to assess their effects on immunogenicity. Cytokine analysis and antibody responses were measured, including IFN-γ, IL-4, IgG2a/IgG1 ratios, and neutralizing antibody titers. Results: The optimized SFV-RVG production was successfully scaled up, and M103 adjuvant induced rapid early antibody titers in mice. In dogs, GEL02 led to the highest neutralizing antibody levels, exceeding 40 IU/mL by 28 days post-immunization. Cytokine analysis indicated that both M103 and GEL02 significantly enhanced IFN-γ and IL-4 expression, balancing the Th1/Th2 immune response. SFV-RVG with GEL02 demonstrated stronger immunogenicity than a commercial vaccine, and challenge studies confirmed robust protection against lethal RABV in mice. Conclusions: This study establishes GEL02 as a superior adjuvant for rabies vaccines and provides a scalable SFV-RVG production process. These findings highlight SFV-RVG with GEL02 as a promising rabies vaccine candidate for dogs, offering significant potential for rabies control.