The Construction Of Staphylococcus Aureus Multi-antigen Vaccine And The Evaluation Of Immunoprotective Effect

Staphylococcus aureus?S.aureus?is an important pathogen causing human and animal infections.It harms human and animal health seriously.There is no effective vaccine available.Previous studies have shown that use of multi-antigen and compatibility of novel adjuvants is dominant strategies for the current research of S.aureus vaccines.In this study,ESAT-6-like secretion system protein A,B?EsxA,EsxB?,?-hemolysin?Hla?and Fe-hydroxamic acid uptake protein D2?FhuD2?of S.aureus,were selected as candidate antigens.First,the CDS sequences of EsxA and EsxB were fused.The Hla sequence was mutated to express the non-toxic protein Hla_H35L.The DNA sequences of EsxA-EsxB,Hla_H35Land FhuD2 were ligated to the prokaryotic expression vector,pEasy-Blunt-E1,to form recombinant vectors pEasy-Blunt-E1-EsxA-EsxB,pEasy-Blunt-E1-Hla_H35L and pEasy-Blunt-E1-FhuD2.Three recombinant vectors were transformed into E.coli BL21?DE3?competent cells for proteins expression.After purified by nickel column Ni-NTA and the molecular sieve CL-4B,we finally obtained high-purity EsxA-Esx B,Hla_H35L and FhuD2 proteins.The three proteins,EsxA-EsxB,Hla_H35L and Fhu D2,were together mixed with complete Freund's adjuvant?CFA?or incomplete Freund's adjuvant to form a multiple antigens mixed vaccine(EsxA-EsxB+Hla_H35L+FhuD2+CFA/IFA).At the mean time,EsxA-Esx B,Hla_H35L and FhuD2 were respectively combined with CFA or IFA to make a single antigen vaccine(EsxA-EsxB+CFA/IFA,Hla_H35L+CFA/IFA,and Fhu D2+CFA/IFA).The mice were immunized with the vaccines above mentioned for three times.Then the mice were infected with S.aureus to form pneumonia.Detect and record changes as experimental data,just like body temperature,survival rate,bacterial load of lungs and pathological changes of lungs in each group.By comparing with three monovalent vaccines,we explored the protective immune-response of the mixed antigens vaccine(Esx A-Esx B+Hla _H35L+FhuD2+CFA/IFA).As the result showed,it was found that the mixed antigen immunized group improved the survival rate of the S.aureus infected mice to88.9%and reduced the degree of lungs lesions in the mice and reduced colonizations amount of S.aureus in mouse's lungs compared with the single antigen immunized groups.To evaluate the humoral and cellular immune responses of mice after immunization,the specific antibody against recombinant protein antigens were detected by ELISA method.The cell proliferation of splenic T lymphoid was detected by MTS.Simultaneously,intracellular cytokine staining and flow cytometry were used to detect the proportion of IFN-?⁺CD4⁺cells,IL-4⁺CD4⁺cells,and IL-17A⁺CD4⁺cells in T cells of mice spleen.The results showed that the specific antibody responses against different recombinant protein antigens in the mixed immunized group was significantly increased compared with the single immunized groups,which also greatly improved the proliferative response ability of antigen-specific T cell.The proportion of IFN-?⁺CD4⁺T cells,IL-4⁺CD4⁺T cells,IL-17⁺CD4⁺T cells in the splenic cells of the multi-antigen immunized group was significantly higher than that of other groups.In the second part of the study,a novel multi-antigen vaccine was produced by using the mixed antigens(EsxA-EsxB,Hla _H35L,and FhuD2)compatibility of the polysaccharide adjuvant,EPS029.The immune protective effect was determined by detected the survival rate of the mice,the degree of pathological changes in the lungs,the amount of bacteria loaded in the lungs.The results showed that comparing with the mixed antigens adjuvant-free group(EsxA-EsxB+Hla_H35L+Fhu D2),using EPS029 as an immune adjuvant could increase the survival rate?88.9%?of mice infected by S.aureus,and significantly reduce pathological damage degree of the infected bellows,and reduce colonization amount of S.aureus in lungs.After further study of its immune response characteristics,it showed that when EPS029 was used as an adjuvant to S.aureus,it could simultaneously increase the response level of antigen-specific antibody of IgG1,IgG2a,and IgG2b,and greatly improve proliferative response ability of the antigen-specific T cell as well as the proportion of IFN-?⁺CD4⁺T cells,IL-4⁺CD4⁺T cells,IL-17⁺CD4⁺T cells in the splenic T cells.It indicated that EPS029 was an immune adjuvant could also enhance Th1 type,Th2 type and Th17 type of immune responses.The above results indicated that the vaccine based on the multivalent antigens of S.aureus?Esx,EsxB,Hla and FhuD2?was successfully constructed,which combined with the new adjuvant EPS029 can effectively protect mice against infection of Staphylococcus aureus.This study provided the theoretical basis and experimental data for the development of a new vaccine against the infections of S.aureus.