Evaluation of Recombinant SIP, Enolase, and GAPDH as Subunit Vaccine Candidates Against Streptococcus iniae in Four-finger Threadfin (Eleutheronema tetradactylum): Immunogenicity, Protective Efficacy, and Multivalent Potential.

Streptococcus iniae is a highly virulent pathogen causing severe streptococcosis outbreaks and high mortality in cultured fish, including four-finger threadfin fish in Taiwan. This study evaluated the immunogenicity and protective efficacy of recombinant enolase, surface immunogenic protein (SIP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), administered individually and in combination, as subunit vaccines against S. iniae infection. Recombinant proteins were expressed in Escherichia coli, purified, and emulsified in an oil-based adjuvant before intraperitoneal vaccination of threadfin fish. Immune responses were assessed through serum IgM levels, lysozyme activity, immune gene expression, serum bactericidal activity, and relative percent survival (RPS) after challenge with virulent S. iniae. The results demonstrated successful expression of target proteins and significant elevation of antigen-specific IgM antibody level, particularly in the combination vaccine group. Enhanced lysozyme activity and upregulation of complement component C2, T cell marker CD8, and cytokines (TNF-α, IL-12, IL-10) indicated robust innate and adaptive immune activation. Serum bactericidal assays revealed the highest bacterial killing in fish vaccinated with the combination formulation. Post-challenge survival was greatest in fish receiving formalin-killed cell vaccine (RPS 86.2%), followed by moderate protection conferred by recombinant SIP (rSIP) (72.4%) and the combination vaccine (52.7%). Recombinant enolase and GAPDH provided lower protection levels. These findings highlight the potential of rSIP and multivalent subunit vaccines as promising strategies for controlling S. iniae infections in aquaculture.