Harnessing Salmonella as a potent vaccine delivery platform: Targeted HA/NA epitope presentation via alphavirus RdRp-driven expression to boost immune efficacy

In this study, we developed an enhanced Salmonella-based delivery platform for safe and efficient intracellular plasmid delivery, incorporating rationally designed influenza antigen epitopes to boost protective immunity. Two key viral antigens, hemagglutinin (HA) and neuraminidase (NA), were selected for their complementary roles in the viral life cycle and cloned into a eukaryotic expression system driven by RNA-dependent RNA polymerase to ensure robust intracellular expression. HA was engineered to retain conserved epitopes within the stalk region and receptor binding site (RBS), while NA was stabilized in a trimeric conformation via C-terminal fusion with the T4 phage fibritin domain. The two antigens were linked by a furin cleavage site to promote efficient intracellular processing and antigen presentation. Immunization and subsequent challenge studies in mice demonstrated strong antigen-specific humoral and cellular immune responses, with activation of both Th1 and Th2 pathways. Notably, antibody subclass profiling indicated a Th1-skewed response beyond 21 days post-infection. Assessment of long-term immunity confirmed that even 42 days after booster vaccination could successfully respond to vaccinated antigens, elucidating the effectiveness of the Salmonella system. Vaccinated mice were protected from lethal challenge with multiple influenza strains, maintaining body weight and exhibiting significantly reduced viral loads in lung tissues, highlighting the applicability of our epitope-based vaccine model. These findings underscore the potential of the Salmonella-mediated vaccine strategy described here as an effective and scalable approach for influenza immunization.