Abstract

EsxA-based subunit and DNA vaccines against S. iniae were developed for golden pompano. • The ISA 763A-adjuvanted subunit vaccine achieved the highest protection (RPS of 93.33%). • Both vaccine platforms elicited robust systemic humoral and cellular immune responses. • Subunit and DNA vaccines displayed distinct temporal immune gene expression kinetics. Streptococcus iniae represents an important bacterial agent responsible for streptococcosis in marine fish, leading to substantial economic impacts in aquaculture worldwide. The development of effective vaccines is therefore a critical priority. EsxA, a conserved early-secreted and homolog of antigenic target six (ESAT-6), is involved in bacterial virulence and mediates interactions between the pathogen and its host via the type VII secretion system. In this work, EsxA was examined as a prospective vaccine antigen in golden pompano ( Trachinotus anak ) using both subunit and DNA vaccination strategies. Recombinant EsxA protein was expressed in Escherichia coli BL21(DE3) and administered intraperitoneally as a subunit vaccine, either alone or formulated with the oil-based adjuvant Montanide ISA 763A. Concurrently, a DNA vaccine was developed by cloning the complete e sxA gene into the pVAX1 vector. Vaccinated fish were subjected to challenge with S. iniae at 8 weeks post-immunization to evaluate protective efficacy and assess the host’s innate and adaptive immune responses. A high level of protection against S. iniae challenge was achieved with the EsxA-based subunit vaccine, particularly when formulated with adjuvant ISA 763A, whereas the DNA vaccine elicited moderate yet statistically significant protection. Immunological profiling revealed robust antigen-specific IgM production following subunit vaccination, while DNA vaccination significantly upregulated transcription of key immune-related genes associated with antigen presentation and cellular immunity, including MHC class I and CD8α . Furthermore,