A recombinant pseudorabies virus co-expressing G2b and G2c S1 broadens neutralizing antibody responses against PEDV variants and enhances pre-existing vaccine-induced antibody responses

Porcine epidemic diarrhea virus (PEDV) G2c variants harbor substantial antigenic alterations that reduce the cross-neutralizing activity of antibodies induced by current vaccines and may contribute to vaccine failure in the field. To explore a vaccine candidate capable of broadening antibody responses against genetically and antigenically diverse PEDV strains, we generated three gene-deleted pseudorabies virus (PRV) recombinant vaccines expressing S1 proteins of G2b, G2c, or both G2b and G2c PEDV strains. The biological safety, genetic stability and immune efficacy of the three candidates were evaluated. All recombinant viruses exhibited genetic stability and preliminary safety in mice. Immunization induced robust PEDV-specific antibody responses, including IgG, IgG1, IgG2a and neutralizing antibodies against different PEDV strains. Notably, the recombinant PRV co-expressing G2b and G2c S1 elicited higher neutralizing antibody titers and broader neutralizing antibody responses against classical, epidemic, and emerging PEDV variants than either single-S1 vaccine. Enhanced antibody responses were accompanied by markedly increased germinal center B and follicular helper T cell populations in draining lymph nodes and spleen, indicating improved germinal center reactions. In a G2b commercial vaccine-primed mouse model, the dual-S1 recombinant virus further increased antibody responses against CV777 and the G2c variant JXCY while maintaining responses against AJ1102. Collectively, these findings demonstrate that a recombinant PRV co-expressing G2b and G2c S1 broadened serum neutralizing antibody responses against representative PEDV variants in mice and effectively expands immunity elicited by current vaccines. This study provides a promising strategy for addressing antigenic mismatch and improving vaccine-induced immunogenicity against representative PEDV variants.