Immunogenicity Of S.aureus MntC Enhanced By T Cell Epitope And Identification Of CD4~+T Cell Epitope On GapC

Staphylococcus aureus(S.aureus)is an important opportunistic pathogen causing a variety of diseases such as skin and soft tissue(SSTIs),pneumonia,infective endocarditis and sepsis.It is also one of the most important infectious pathogens causing bovine mastitis,which seriously impacts the animal husbandry and dairy industry.Due to the increasing reports of drug-resistant strains of S.aureus,the effect of antibiotics on the treatment of S.aureus infection is diminishing.Therefore,it is particularly important to optimize the use of prophylactic or therapeutic vaccines to control S.aureus infections.Manganese transport protein C(Mnt C)is a key protein for S.aureus to obtain manganese,and it can elicit protective immunity against S.aureus.Agglutinin-like sequence 3(Als3),an adhensin of Candida albicans(C.albicans),can induce robust cellular immune responses and cross-protection against S.aureus.And target of RNAIII-activating protein(Tra P)is a membrane-related protein of S.aureus,which can induce specific cellular immune response and good immune protection against S.aureus infection.In order to further improve the immunogenicity of Mnt C,the two CD4~+ T cell epitopes of Als3 and Tra P were respectively fused with Mnt C,and the specific immune response and immunoprotection induced by the fused proteins were tested.A flexible linker was used to connect the Als3 T cell epitope or Tra P T cell epitope and the N-terminal of Mnt C to form fused proteins,which was named ATM or TTM.The BALB/c mice were intramuscularly inoculated with the fusion proteins emulsified with equal-volume of complete Freund's adjuvant,and the booster vaccination with the fusion proteins emulsified with incomplete Freund's adjuvant was given on 21 th post the first inoculation.While Mnt C,Als3 T cell epitope(Als3 T),Tra P T cell epitope(Tra P T),Mnt C and Als3 T cell epitope mix(Mnt C +Als3 T),Mnt C and Tra P T cell epitope mix(Mnt C + Tra P T)and PBS,emulsified with the above adjuvant,were used as the controls.After booster immunization,the concentration of cytokine secreted by CD4~+ or CD8~+ T cells,the level of antibody in serum and the subclasses of Ig G were analyzed,and the mice were challenged with S.aureus Newman strain or C.albicans SC5314 strain.The results showed that ATM and TTM could induce CD4~+ T cells to secrete higher levels of IFN-?,IL-17 A and IL-4,as well as antibodies than Mnt C,and ATM could also induce CD8~+ T cells to produce higher levels of IFN-? and certain levels of IL-17 A and IL-4.After challengewith S.aureus,ATM and TTM could significantly reduce the bacterial load in the liver,spleen,lung and kidney of the mice.At the same time,ATM elicited robust cross-immunoprotection against C.albicans challenge,which significantly reduced the fungal load in the liver,spleen and kidney of the mice.These results indicate that ATM can induce stronger immune protection against S.aureus infection and certain immunity against C.albicans infection.It also demonstrates that both the Als3 T cell epitope and the Tra P T cell epitope can significantly enhance the immunogenicity of Mnt C protein,and the Als3 T cell epitope is better than the Tra P T cell epitope.Since the above CD4~+ T cell epitopes could enhance the immunogenicity of the antigen,therefore,we analyzed and identified the CD4~+ T cells of S.aureus Gap C by bioinformatics prediction,protein property analysis and cytokine detection,which are 104 DKAQAHIEAGA KKVLISAPA123 and 314 YDNEMSYTAQ LVRTLAYLAE333.They both could induce remarkable immune protection against S.aureus infection.The above research provides a reference for developing the novel and high-efficiency vaccines against S.aureus infection.