Construction Of A Staphylococcus Aureus Subunit Vaccine And Preliminary Study On Its Immunoprotective Effect

The Staphylococcus aureus(S.aureus)is one of the most important pathogens for human and animals which caused many diseases,such as bacteremia,toxic shock syndrome,endocarditis,osteomyelitis,pneumonia and so on.Since the 1960s,with the extensive use of antibiotics,many drug-resistant strains have appeared that brought serious challenges for the treatment of S.aureus-infections.In the face of the growing drug-resistance of S.aureus,development of effective vaccines against this pathogen is particularly important.In the present study,we selected S.aureus surface proteins TraP,Efb,Csa1,Rap,MntC,GapC and FhuD2 as candidate antigens,and we constructed the antigens recombinant expression vectors.We expressed and purified the recombinant proteins.Then,mice were immunized with the purified recombinant proteins.We measured the antibody level by enzyme-linked immunosorbent assay(ELISA).Two weeks after the last immunization,mice were challenged with S.aureus USA 300 by nasal drops.After the challenge,in order to clarify the immunoprotective effect of the protein,we examined changes in body temperature,lung pathological changes,lung bacterial load and survival rate.The results showed that the TraP,Efb,Csa1,Rap,MntC,GapC and FhuD2antigen recombinant expression vectors constructed successfully and purified by induced expression to obtain pure protein.Compared with the PBS control group,TraP,Efb,Csa1,Rap,MntC,GapC and FhuD2 antigens can significantly enhance the specific IgG antibody titer in mice serum.In the challenge experiment,TraP,Csa1,Rap,MntC,and GapC antigens can improve the resistance to S.aureus-infection and the survival rate of mice in each group is as follows:TraP:80%,Efb:20%,Csa1:80%,Rap:60%,MntC:80%,GapC:60%,FhuD2:30%.Next,we selected TraP?Csa1?MntC as candidate antigens because the survival rate of these three antigens is up to 80%and higher than other antigens.These three antigens were mixed in equal proportions and then combined with Lactobacillus polysaccharide adjuvant EPS29 and EPS30 to prepare a multi-antigen mixed vaccine,and the mice were respectively immunized.The antibody level measured by enzyme-linked immunosorbent assay(ELISA).Two weeks after the last immunization,mice were challenged with S.aureus USA 300 by nasal drops.After the challenge,in order to clarify the immunoprotective effect of the mixed immunization,we examined changes in body temperature,lung pathological changes,lung bacterial load and survival rate.The results showed that TraP+Csa1+MntC+CFA/IFA,TraP+Csa1+MntC+EPS29,TraP+Csa1+MntC+EPS30immunized groups were resistant to S.aureus infection compared with PBS group,EPS29 group,EPS30 group and TraP+Csa1+MntC group.And these three groups can reduce the colonization of Staphylococcus aureus in the lungs;Compared with PBS,EPS29,EPS30,TraP+Csa1+Mntc,theTraP+Csa1+MntC+CFA/IFA,TraP+Csa1+MntC+EPS29,TraP+Csa1+MntC+EPS030 immunization groups can significantly increase the specific IgG antibody in mice serum and the antibody titers of IgG antibody subclass IgG1,IgG2a and IgG2b.TraP+Csa1+Mntc+CFA/IFA,TraP+Csa1+Mntc+EPS29,TraP+Csa1+Mntc+EPS30 also significantly enhanced the proliferation and cytokines secretion in T lymphocytes.Compared with PBS,EPS29,EPS30andTraP+Csa1+MntC,TraP+Csa1+MntC+CFA/IFA,TraP+Csa1+MntC+EPS29,TraP+Csa1+MntC+EPS30 significantly enhanced the ratio of CD4~+IFN-?~+T cells,CD4~+IL-4~+T cells,CD4~+IL-17~+T cells,CD8~+IFN-?~+T cells and CD8~+IL-17~+T cells.