Development of an oral Lactobacillus casei-based recombinant vaccine expressing GCRV-II multi-epitope fusion protein and its protective efficacy against grass carp hemorrhagic disease.

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) results in severe economic losses in aquaculture. Vaccination is an effective strategy for controlling viral infections. The subunit vaccines are considered promising because of their safety, strong immunogenicity, and ease of production. In this study, antigenic epitopes derived from the outer capsid proteins VP4, VP5, and NS38 of GCRV genotype II (GCRV-II) were identified, and oral Lactobacillus casei recombinant vaccines were developed to evaluate their protective efficacy in grass carp. Based on sequence homology, epitope prediction and structural analyses, three dominant epitopes, VP4274-367, VP5105-336 and NS38143-338, were selected and fused into a recombinant protein. The immunogenicity of fusion protein was confirmed by enzyme-linked immunosorbent assay (ELISA). Injection of the fusion protein significantly enhanced the expression of immune-related genes, increased serum neutralizing antibody levels and reduced the expression of inflammatory cytokines. After GCRV-II challenge, the vaccinated fish showed a survival rate of 66.7%, compared with 6.7% in the injectable control group, and exhibited the lowest tissue viral loads among all groups. To develop an oral vaccine suitable for aquaculture, the fused epitope sequence was inserted into the secretory expression vector pVE5523 and expressed in L. casei. The recombinant strain showed high tolerance to heat and acidic conditions and stably colonized the intestine for up to 14 d. Oral administration of feed containing freeze-dried recombinant L. casei significantly upregulated immune-related genes (ifn1, cd4-2, mhc-Ⅱα,igm and igt) as well as lysozyme and complement C3 levels. After GCRV-II challenge, orally vaccinated fish reached a survival rate of 70%, much higher than the 23.33% survival observed in the oral control group, with significantly decreased tissue viral loads. These results demonstrate that the recombinant L. casei vaccine LC-VP4274-367-VP5105-336-NS38143-338 is a promising oral vaccine candidate for protecting grass carp against GCRV-II infection.