A novel oral vaccine strategy utilizing a plasmid with CAG promoter-driven alphavirus RdRp for Co-expression of NA epitope and adjuvant in attenuated Salmonella boosts immune responses against H9N2 in mice.

The ongoing evolution of H9N2 avian influenza virus continues to pose a significant threat to the poultry industry and overall biosecurity. Vaccination remains the primary strategy for controlling this pathogen. To develop more effective vaccines, this study employed a novel self-amplified RNA-encoding plasmid system previously constructed based on the Semliki Forest virus (SFV) replicon. This system utilizes the CAG promoter to drive the expression of SFV RNA-dependent RNA polymerase, which initiates the autonomous replication of mRNA that encodes the target gene. This process enables efficient expression of the antigen. First, the self-amplified RNA-encoding plasmid pYL345 (SFV-RFP) was compared with the conventional DNA plasmid pYL347 (RFP) as a control. In vivo mouse imaging and qRT-PCR experiments confirmed that pYL345 mediated significantly higher RFP expression, validating the effectiveness of this self-amplified RNA system. Further comparison was made between the self-amplified RNA vaccine plasmid pYL486 (SFV -neuraminidase (NA)) and the conventional vaccine plasmid pYL423 (NA). To enhance the immune response, the P7 single-chain antibody and the immune adjuvants CXCL15 or CXCL13 were fused and expressed based on pYL486. This resulted in the construction of recombinant plasmids pYL562 (SFV-NA/P7/CXCL15) and pYL564 (SFV- NA/P7/CXCL13). Immunofluorescence and Western blot assays confirmed the successful co-expression of the NA protein and the corresponding adjuvants in all constructed plasmids. Oral delivery using attenuated Salmonella (χYL11218) in mice revealed that self-replicating vaccines (pYL486, pYL562, and pYL564) promoted the maturation of dendritic cells in the mesenteric lymph nodes. This process reshaped splenic T-cell immunity, marked by a reduction in naive T cells and an increase in central memory T cells and effector memory T cells. These vaccines also enhanced the Tfh and germinal center B cell proportions and stimulated robust Th1/Th2-balanced responses from CD4+/CD8+ T cells. The CXCL-15-expressing pYL562 displayed superior efficacy by significantly increasing serum IgG and mucosal sIgA antibody levels, as well as promoting T-cell proliferation. Challenge studies confirmed that these oral self-replicating vaccines effectively protected against G57H9N2 infection, reducing viral loads and weight loss. In conclusion, our SFV-based self-amplified RNA platform delivered orally via Salmonella induces potent systemic and mucosal immunity, representing a promising strategy for H9N2 control.