Development and immunogenicity of a recombinant PRRSV live vector co-expressing CSFV E2 antigen and porcine IL-18 for multivalent swine vaccination.

Porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) are among the most economically devastating viral pathogens in the global swine industry, causing substantial and often incalculable economic losses. In field vaccination programs, the mutual interference between the immune responses elicited by CSFV and PRRSV vaccines markedly restricts the flexibility of immunization schedules, while the limited cross-protective capacity of current PRRSV vaccines remains an inherent and unavoidable challenge in swine disease management. To address these limitations, the present study aimed to develop a recombinant PRRSV-based live vector vaccine co-expressing two exogenous components: the protective E2 antigen of CSFV and porcine interleukin-18 (IL-18), with the objective of enhancing immunogenicity beyond that achievable with conventional monovalent formulations. Using a reverse genetics system derived from the attenuated PRRSV strain rHuN4-F112, the CSFV E2 gene was inserted between open reading frame 1b (ORF1b) and ORF2a, while a codon-optimized porcine IL-18 gene was integrated between ORF7 and the 3' untranslated region. The resulting recombinant virus, designated rPRRSV-E2-N-IL18, was validated through dual restriction endonuclease digestion, plaque assays, indirect immunofluorescence assay, and Western blotting. Genetic stability was maintained for at least 25 serial passages, with persistent exogenous protein expression confirmed in the F25 generation. For immunogenicity assessment, fifteen PRRSV/CSFV double-negative piglets were randomly allocated into three groups: recombinant vaccine, parental virus, and unvaccinated control. Humoral and cellular immune responses were quantified by ELISA. During the 35-day post-vaccination period, no pyrexia associated with PRRSV infection was observed in the vaccine group, and histopathological examination of the lungs, lymph nodes, and kidneys showed no significant inflammatory lesions. Vaccinated pigs produced specific antibodies against both PRRSV and CSFV E2 within 35 days, and exhibited significantly elevated serum IL-18, IL-4, and IFN-γ levels, indicative of a potentiated Th1/Th2 response. These results confirm that IL-18 may indirectly enhance E2 immunogenicity by promoting Th1/Th2 responses, inducing robust humoral and cell-mediated immunity. This recombinant PRRSV platform represents a promising multivalent live vector vaccination strategy for the simultaneous prevention of PRRSV and CSFV in swine.