A novel self-amplified RNA vaccine co-expressing NA and HA1 delivered by Salmonella confers potent protection against H9N2 influenza in chickens.

The ongoing evolution of the H9N2 avian influenza virus presents a growing threat to both the poultry industry and public health. In this context, vaccination is not only a vital preventive measure but also the primary strategy for controlling viral transmission and preventing outbreaks. To enhance vaccine efficacy, this study successfully established a novel self-amplified RNA vaccine platform utilizing the Semliki Forest virus (SFV) replicon. The core mechanism involves utilizing the CAG promoter to drive the expression of SFV RNA-dependent RNA polymerase (RdRp), which initiates the autonomous replication of mRNA encoding target antigens, thereby creating a self-propagating cascade amplification reaction that achieves sustained and elevated antigen expression in vivo. Initially, we constructed the self-amplified RNA plasmid pYL345 (SFV-EF1α-RFP) and conventional DNA plasmid pYL347 (EF1α-RFP) as a control. In vivo chick imaging and quantitative reverse transcription polymerase chain reaction (qRT-PCR) experiments confirmed that pYL345 (SFV-EF1α-RFP) significantly enhances RFP expression, validating the effectiveness of self-replicating RNA system. Afterward, we compared the self-amplified RNA plasmid pYL486 (SFV-EF1α-NA) with the conventional vaccine plasmid pYL423 (EF1α-NA). Quantitative analysis of NA protein and RdRp via qRT-PCR validated the successful construction of the novel self-amplified RNA system. To further boost the immune response, we integrated the HA1 antigen into pYL486 (SFV-EF1α-NA) to create the recombinant plasmid pYL615 (SFV-EF1α-NA-HA1). Immunofluorescence and Western blot assays confirmed the successful co-expression of NA and HA1 proteins across all constructed plasmids. Utilizing an oral Salmonella delivery system, we conducted protection studies against avian influenza virus infection. Compared with other groups, oral immunization with the novel self-amplified RNA vaccine S615 (pYL615) delivered by Salmonella significantly increased humoral immunoglobulin G and mucosal secretory immunoglobulin. A levels, enhanced cellular immune responses, promoted T lymphocyte proliferation and differentiation, and upregulated cytokine expression (IL-4 and IFN-gamma). Challenge experiments demonstrated that dual-antigen co-expressing pYL615, conferred enhanced protection to chickens against the G57 subtype H9N2 viral challenge, alleviated infection-induced weight loss, reduced viral shedding in oropharyngeal and cloacal samples, decreased lung and tracheal virus titers, decreased pro-inflammatory cytokine production (IL-6 and IL-1β), and mitigated tracheal and pulmonary damage. Overall, this study successfully developed a novel self-amplified mRNA vaccine platform based on the SFV replicon. Through oral Salmonella delivery, we demonstrated its capacity to elicit robust systemic and mucosal immune responses, offering a promising innovative strategy for controlling H9N2 avian influenza.