An Integrated in silico and in vivo study of nucleic acid vaccine against Nocardia seriolae infection in orange-spotted grouper Epinephelus coioides.

Nocardiosis in aquatic animals caused by Nocardia seriolae is a frequently occurring serious infection that has recently spread to many countries. In this study, DNA vaccines containing potential bacterial antigens predicted using the reverse vaccinology approach were developed and evaluated in orange-spotted groupers. In silico analysis indicated that proteins including cholesterol oxidase, ld-transpeptidase, and glycosyl hydroxylase have high immunogenicity and are potential vaccine candidates. In vitro assays revealed the mature and biological configurations of these proteins. Importantly, when compared to a control PBS injection, N. seriolae DNA-based vaccines showed significantly higher expression of IL1β, IL17, and IFNγ at 1 or 2 days, in line with higher serum antibody production and expression of other cellular immune-related genes, such as MHCI, CD4, and CD8, at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae were recorded in DNA vaccine-cholesterol oxidase (pcD::Cho) injected fish, with a relative survival rate of 73.3%. Our results demonstrate that the reverse vaccinology approach is a valid strategy for screening vaccine candidates and pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses and confer considerable protection against N. seriolae infection.