A Novel Designed Vaccine For Staphylococcal α-hemolysin Pneumonia

Staphylococcus aureus is a gram-positive pathogen that causes a myriad of diseases ranging from minor skininfections to life-threatening deep tissue infections and toxinosis. Pneumonia is one of the most commoninvasive diseases caused by this pathogen. S. aureus alpha-hemolysin, a pore-forming cytotoxin, is anessential virulence factor in the pathogenesis of pneumonia. A large number of resistant strains have emergedand lead to ineffectiveness of some antibiotics to control the diseases since antibiotics have been widely usedin clinical treatments. So vaccine should be the key step and the best choice to prevent S. aureus infections.Based on the crystal structure of α-hla heptamer and the α-hla-encoding gene, the mutant form of Hla(hlaH35L), hla1-50, hla5-76, hla158-218and the fused gene hlaD (hla5-76-linker-hla158-218) are clonedinto the prokaryotic expression vector pET28a or pET30a to express the recombinant proteins. The proteinswere detected by Western Blotting using monoclonal antibodies to histidine tag of the expressed proteins asthe first antibodies. The hemolytic activity of hlaH35L is detected.After active immunization with these proteins was performed using the mice pneumonia model,antigen-specific immunoglobulin G responses and protection against staphylococcal pneumonia were elicited.Six groups of mice were respectively immunized with recombinant hla1-50(rHla1-50), rhla5-76, rhla158-218,rhlaD, rhlaH35L and mock (n=12). Proteins were emulsified with Freund’s adjuvant (Freund’s completeadjuvant for the primary immunization, booster immunization was incomplete Freund adjuvant). The boosterimmunity went on the21days after primary immunization. On the35days after primary immunization, themice were challenged with S. aureus Newman strains in LD100, and immune protection in mice immunizedwith rhla1-50, rhla5-76, rhla158-218, rhlaD, rhlaH35L were compared. Passive immunization with theantiserum could antagonize toxin activity. Moreover the immune protection of hlaD was better than the otherones except close to rhlaH35L. Thus, hlaD vaccination or immunotherapy may prevent S.aureus pneumonia.Numbers of cells secreted cytokines IFN-γ and IL-4were detected with ELISPOT. Levels of antibody andcytokines TNF-α, IL-6, IL-17were detected with indirect ELISA. After the spleen lymphocytes inimmunization group were stimulated with the corresponding protein, numbers of cells secreted IFN-γ andIL-4from the mice immunized with rhlaH35L and rhlaD was significantly more than the control. The levelsof IL-17, TNF-αand IL-6from the mice immunized with rhlaH35L and rhlaD were higher than others.In summary, through a series of experiments and analysis, we obtained a nevol fusion protein that has a goodprotective effect against S. aureus α-hla pneumonia, and the size of the fusion protein is appropriate. It willprovide some references for constructing a multi-antigen subunit vaccine against S. aureus α infections.