Construction And Immunogenicity Evaluation Of Epitope-based Vaccines Against S.dysgalactiae And S.aureus

The bovine mastitis is mammary gland inflammation and brings enormous economic losses in dairy industry. Bovine mastitis is caused by a variety of pathogenic microorganisms, among which S. aureus and Streptococcus are the most common. At present, clinical antibiotic therapy can not effectively control S. aureus and Streptococcus infections, which makes vaccine development urgent. Previous studies have confirmed that the cellular immune responses play major roles in the process of preventing S. aureus and Streptococcus infections, In particularly Th1 and Th17 cellular immune responses is crucial in the process of preventing S. aureus infections, Thus, to prime CD4⁺ T cell response is very important for the design a vaccine against bovine mastitis. Epitope is the effective part of an antigen to induce immune responses. Thus, the epitope-based vaccine, which can selectively induce CD4⁺ T cell response and Th1/Th7 cellular immune responses, may be effective in preventing Streptococcus and S. aureus infections. S. dysgalactiae Gap C and S. aureus TRAP protein can induce specific cell immune responses and highly conserved. However, there has been no study to identify the immune-dominant CD4⁺ T cell epitopes on Gap C or TRAP. The epitope-based vaccine covered CD4⁺ T cell and B cell epitope remains to be investigated. In this study, we screened and identified immune-dominant CD4⁺ T cell epitopes of Gap C and TRAP, building a multi-epitope vaccine covering the CD4⁺ T cell and B cell immunodominant epitopes of Gap C and TRAP, seeking to obtain a novel vaccine which effectively prevents Streptococcus and S. aureus infections.In this study, in silico MHCⅡ affinity measurement methods, protein secondary structure and conserved sequence analysis were used to predict potential CD4⁺ T cell epitopes on Gap C and TRAP. Six predictive Gap C（15-mer） and five TRAP（20-mer） peptides were synthesized, then the CD4⁺ T cells of BALB/c（H-2d） and C57BL/6（H-2b） mice, which were immunized by proteins or peptides, were stimulated with potential epitopes. CD4⁺ T cell proliferation and cytokine responses were detected, and the conservative properties of identified epitopes were analyzed. The results showed that Gap C_63-77, TRAP_20-39 and TRAP_94-113 induced specific CD4⁺ T cell proliferation and secreted high levels of IFN-γ and IL-17 A, suggesting that the identified epitopes preferentially elicited polarized Th1/Th17-type responses. CD4⁺ T cell epitopes ofGap C and TRAP were Gap C_63-77, TRAP_20-39, and TRAP_94-113, and the identified epitopes were highly conserved.Based on the above research, the obtained CD4⁺ T cell epitopes Gap C_63-77, TRAP _20-39, TRAP_94-113, and B cell epitopes Gap C_30-36, Gap C_48-57, Gap C_97-103, TRAP_154-163, which were identified in our previous studies, were connected, respectively. The target gene corresponding to the amino acid sequence was synthesized and inserted into p ET32 a vector to construct the recombinant plasmid containing multiple epitopes. Then, the recombinant proteins were expressed and purified to prepare the recombinant epitope-based vaccine PT, PG and PTG. The immunogenicity in the BALB/c and C57BL/6 mice immunized with epitope-based vaccines were analyzed. Cellular immune effects were evaluated with lymphocyte proliferation and cytokine testing, humoral immune effects were evaluated with antibody level detection and phagocytosis test, and immune protection effects were evaluated with challenge assay. The results showed that the epitope-based vaccines PT, PG and PTG induced specific lymphocytes proliferation. At the same time,specific lymphocytes secreted significantly higher levels of IFN-γ, IL-4, IL-10, and IL-17 A, compared to p ET32 a control, suggesting that epitope-based vaccine can induce specific cellular immune responses in mice. The epitope-based vaccines PT, PG and PTG induced significantly higher levels of anti-TRAP and anti-Gap C Ig G, compared to p ET32 a control. Meanwhile, specific antibodies mediated opsonocytophagic killing of S. dysgalactiae and S. aureus, suggesting that epitope-based vaccine can induce specific humoral immune responses in mice. The challenge assay results showed that the epitope-based vaccines PT, PG and PTG had certain immune protection effect. PT could effectively inhibit the infection of S. aureus. However, PG could partially inhibit S. dysgalactiae, and PTG could partially inhibit S. dysgalactiae and S. aureus infection. The results indicated the CD4⁺ T and B cell epitopes of epitope-based vaccine respectively functioned, which can provide a reference for the further study of bovine mastitis vaccines.