Potential role of adjuvants is moving on with success stories in controlling bacterial diseases in fishes. Montanide™ ISA 763A VG and Montanide™ ISA 763B VG- developed by SEPPIC, commercial adjuvants used for i.p. injection in fish, are proved to be excellent in animal vaccines. The efficiency of these adjuvants in protection and in improving immune response (Wangkahart et al. 2023) including aluminum hydroxide, flagellin, chitosan oligosaccharide, CpG oligonucleotides, liposome, poly I:C, MARCOL 52, Freund’s complete and incomplete adjuvants, are a range of effective adjuvants tried in vaccine studies (Jiao et al. 2010, Zheng et al. 2012, Dalmo et al. 2016).
Nile tilapia aquaculture is affected by Streptococcosis caused by S. agalactiae (Dangwetngam et al.2016, Barato et al. 2015). In this study, we have analysed the effects of flagellin and IFN-γ mixed with SAIV vaccine in providing protection and enhanced immune response in Nile tilapia. Recent studies have shown up with the role of flagellin as promising vaccine adjuvant and immunostimulant for fish aquaculture. It has been observed that flagellin from Y. ruckeri, potently activate the expression of inflammatory cytokines, antimicrobial peptides and acute phase proteins in vitro and in vivo (Wangkahart et al. 2019). In vivo studies showed that there is a remarkable induction in the expression of IL-12 family members and cytokines in Th2 and Th17 pathways but have little effects in Th1 pathway following flagellin stimulation (Wangkahart et al. 2019 ).
This study emphasizes the role of flagellin and IFN-γ to boost the immune response and protective efficacy in Nile tilapia infected with S. agalactiae. The immune parameters are analysed post vaccination. We administered vaccine through injection mode and fishes were injected with a booster at 21 dpv. Fish serum samples were collected at two time points (15 dpv and 36 dpv), before booster and after booster respectively. It was observed that the SOD and CAT activity in serum was remarkably enhanced in the fish administered with SAIV adjuvanted with flagellin and IFN-γ. The same effect was observed in the activities of LZM and MPO. The enhancement in the innate immune parameters was observed after booster vaccination at 36 dpv. LZM activity is essential in non-specific immune response in the fish and enhancement in immune response and is crucial to fight the invading bacteria (Ashfaq et al.2019). We also tested serum bactericidal activity post vaccination and found that higher levels of its activity in fish treated with SAIV adjuvanted with flagellin and IFN-γ. This finding further supports the related outcome from the previous studies where adjuvanted vaccines enhancing non-specific immune response in the fishes with their potential bactericidal activity accompanied by elevated LZM activity, complement factors and other associated molecules (Ellis 1999, Hoare et al. 2019).
Specific antibody production is a measure of preventing bacterial infection and we examined the specific IgM antibody levels since it is the main antibody in the teleost fish (Ye et al. 2013). ELISA is a widely used sensitive method for the detecting and quantifying specific humoral antibody response to fish pathogens (Ristow et al. 1993). Vaccinated fishes are examined for humoral immune response by detecting and quantifying specific IgM antibodies in the sera post vaccination on 15 dpv and 36 dpv. We observed that specific IgM antibody level were enhanced in fish sera vaccinated with SAIV adjuvanted with flagellin, IFN-γ and its combination (G3, G4 and G5 respectively) when compared to negative control (G1) and positive control (G2) groups. Enhanced IgM antibody levels were significantly elevated in the fish sera at 36 dpv. The findings clearly indicate the potential role of molecular adjuvants in stimulating the humoral immunity in fish which again promisingly support the previous data where adjuvants enhance the immune response in tilapia infected with S. agalactiae (Wangkahart et al. 2023, Wangkahart et al. 2021). It was observed that specific IgM antibodies level were increased in SAIV vaccine adjuvanted with Montanide 763A VG and 763B VG. The IgM antibody level in these groups were also increased at an early stage (Wangkahart et al. 2023 ).
The role flagellin and IFN-γ in enhancing the efficacy of SAIV vaccine in conferring protection was measured. Many studies have proved a consistent correlation between the high expression of specific antibodies and protective efficacy of vaccine (Wangkahart et al. 2021). The RPS value of 59.37%, 71.87% and 81.25% was observed for SAIV vaccine adjuvanted with flagellin, IFN-γ and its combination, respectively, while the RPS of the positive control group was 15.62%. The RPS of fish injected with vaccine adjuvanted with both flagellin and IFN-γ showed enhanced protection. The protective efficacy of flagellin was already studied (Wangkahart et al. 2019) and the present finding is found to be in accordance with the earlier observation. An enhanced RPS was observed in fish injected with SAIV adjuvanted with Montanide 763A VG (77.42%) and Montanide 763B VG (74.19%) when compared to the unadjuvanted vaccine control group (61.29%) (Wangkahart et al. 2023). In another study, survival rate of fish administered with S. agalactiae ghost vaccine (SAGV) adjuvanted with Montanide 763B VG (80.0%), SAGV with Montanide GEL02 (84.4%) was found to be improved when compared to control group which was only 33.3% (Wangkahart et al. 2021). Our findings are supporting the previous studies with the analogous observations.
The histopathological examinations of head kidney, spleen, liver, kidney, gills and brain were performed in vaccinated and non-vaccinated fish. No histological changes were observed significantly in the head kidney, kidney, gills, liver and brain tissues of the fish vaccinated with SAIV adjuvanted with both flagellin and IFN-γ and challenged with S. agalactiae whereas decreased melanomacrophage cells was observed in the spleen tissues.
Immune related gene expression analysis remains well linked with the fish vaccination studies giving insights to the disease resistance offered by the respective vaccine administered. In this study, the expression levels of eight adaptive immune-related genes in spleen and head kidney of fish following immunization, such as CD4, IgM, MHC-I, MHC-II, TCRb, and innate immune response, such as IL-1β, IL-6 and IL-8 were analyzed to see whether the molecular adjuvants flagellin and IFN-γ affected their expression in vaccinated fish. There were no upregulation of the expression of major proinflammatory cytokines at 15 dpv and 36 dpv., suggesting that the fish at these time points retained to resting state without heightened inflammation or due to trafficking of inflammatory cells from tissues to the vaccination site. Interestingly, the expression of both MHC-II and IgM was coordinately increased at 36 dpv in the spleen of adjuvanted vaccines fish and in the head kidney in IFN-γ containing vaccine groups. The heightened transcript expression of IgM is in agreement of the increased specific IgM antibody titer in molecular adjuvanted vaccine groups found in this study. MHC-II molecules are constitutively expressed on the surface of professional antigen-presenting cells (APCs), including dendritic cells, B cells and macrophages, and can be upregulated by IFN-γ leading to antigen presentation to CD4+ T cells and immune activation (Wijdeven et al. 2018). It is noteworthy that MHC-II expression in head kidney was only heighten in IFN-γ containing vaccine groups and these groups also showed higher antibody titer and protection against live bacterial infection, implying different mechanisms initiated by flagellin and IFN-γ. Our study suggests that MHC-II upregulated by IFN-γ present processed peptide to CD4+ T cells leading to activation and differentiation into T helper cell subsets and specific antibody response and protection.