The molecular adjuvant effect of the C-terminal peptide of complement C5a in a teleost fish

The activation of the complement system can generate the component C5a, which has potent immunoregulatory activities, such as promoting phagocytosis, respiratory burst, and chemotaxis of leukocytes. Our previous study has shown that teleost C5a exhibits excellent molecular adjuvant function, however, the functional region and the underlying mechanism are still waiting for elucidation. Here, the sequence of grass carp C5a was determined through multiple sequence alignment. The homologous modeling showed that grass carp C5a mainly consists of α-helices. We synthesized the C-terminal peptide (37 amino acid residues) of C5a (C5a-CP) and found that the leukocytes of grass carp could be significantly chemoattracted by C5a-CP, and the phagocytic activity of grass carp IgM⁺ B cells, an important antigen-presenting cell in teleost fish, could be significantly enhanced by C5a-CP. Further study revealed that leukocyte populations, including IgM⁺ B cells, IgM^- lymphocytes, and myeloid cells, all expressed the receptor of C5a (C5aR), and the pro-phagocytic activity of C5a-CP on IgM⁺ B cells could be blocked by anti-C5aR antibodies, suggesting that C5a-CP exerted the immunomodulatory effect through C5aR. To test the molecular adjuvant effect of C5a-CP, we immunized grass carp with a mixture of inactivated Aeromonas hydrophila or genotype II grass carp reovirus (GCRV-Ⅱ) and C5a-CP. The results showed that C5a-CP significantly increased the serum specific IgM level and total IgM concentration and reduced the mortality of immunized grass carp after A. hydrophila challenge, indicating that C5a-CP has excellent vaccine adjuvant functions. In summary, this study revealed that the C-terminus is the functional region of the immunomodulatory activity of teleost C5a and the C5a-CP is an excellent candidate adjuvant for vaccines.