Oral immunization of Nile tilapia with Chlorella-bioencapsulated multi-epitope fusion antigen effectively protects against Streptococcus agalactiae infection.

Streptococcus agalactiae is an important bacterial pathogen of farmed tilapia; however, practical oral vaccines with stable protective efficacy remain limited because of poor antigen stability and low intestinal delivery efficiency. In this study, three immunodominant epitopes derived from the S. agalactiae surface immunogenic protein Sip, C5a peptidase ScpB, and leucine-rich repeat protein LrrG, as well as a multi-epitope fusion antigen (MEFA) constructed from these epitopes, were individually expressed in Chlorella sorokiniana, thereby establishing a microalgae-based oral vaccine system. Absolute quantitative qPCR and Western blot analyses confirmed that Sip, ScpB, LrrG, and MEFA were all stably expressed in the recombinant microalgae. A 28-day feeding trial in GIFT Nile tilapia showed that the recombinant algal biomass did not affect fish growth performance, while oral immunization significantly increased serum peroxidase, catalase, acid phosphatase, and alkaline phosphatase activities and induced antigen-specific IgM responses. Immune gene analysis further showed that different antigens induced distinct tissue-specific and time-dependent response patterns: Sip tended to induce an early pro-inflammatory and humoral immune activation; ScpB and LrrG were more closely associated with cellular immunity and mucosal antigen presentation; and MEFA induced a more durable and balanced immune response during the middle and late stages of immunization. Following S. agalactiae challenge, the MEFA group showed the highest relative percent survival (RPS) of 87.5%, with alleviated clinical symptoms, reduced gill and intestinal tissue damage, and decreased transcription levels of multiple bacterial virulence-related genes in host tissues. These results indicate that recombinant Chlorella can serve as both an antigen expression vehicle and a natural delivery carrier, and that bioencapsulated MEFA is a promising oral vaccine candidate for the control of streptococcosis in tilapia.