Protective Effects And Mechanism Of Mucosal Immunization With PD-ΔA146Ply Fusion Protein Against Haemophilus Influenzae Infection And Streptococcus Pneumonia In Mice

Obiective: Haemophilus influenzae(H.influenzae)and Streptococcus pneumonia(S.pn),common colonization bacteria on human upper respiratory tract,can cause a variety of invasive and mucosal infection diseases.At present,available approved vaccines against H.influenzae and S.pn exist problems,such as limited serotype coverage,expensive,serotype replacement,therefore it is necessary to develop new vaccine components.The respiratory tract is an important barrier to prevent bacteria to enter the body and the mucosal local immune response plays an important role in resistance to bacterial infection.Our study will fuse two kinds of bacteria vaccine candidate proteins from H.influenzae and S.pn: protein D(PD)and attenuated pneumolysin(Ply)ΔA146Ply and evaluate the protection effect of mucosal immunization with PD-ΔA146Ply fusion protein against H.influenzae and S.pn,investigate the underlying protective mechanisms for providing laboratory basis to develop new vaccines.Methods: Recombinant PD,ΔA146Ply and PD-ΔA146Ply fusion protein were expressed in a prokaryotic expression system and purified by Ni-NTA,then remove the mixed lipopolysaccharide in proteins.C57BL/6 mice were intranasally immunized with the recombinant proteins in different formula.Antibody titers and cytokine levels were measured by ELISA when the vaccination finished.Nasopharynx bacterial clearance and survival rates of vaccinated mice were evaluated.The roles of Th17 immune response in mucosal antibody response and bacterial clearance were assessed in IL-17A-/-mice.Results: We successfully constructed the PD-ΔA146Ply fusion protein expression plasmid and obtained the recombinant proteins with purity above 90%.Western blot results showed that the recombinant proteins could be identified by anti-His antibody.Mucosal immunization with PD-ΔA146Ply fusion protein in C57BL/6 mice can more effectively decrease the nasopharynx colonization of H.influenzae and S.pn compared to PD or ΔA146Ply alone.The survival protective effect elicited by PD-ΔA146Ply in H.influenzae fatal infection model was comparable to PD alone and slightly improved in S.pn fatal infection model compared to ΔA146Ply alone.The higher titers of anti-PD IgG in serum and secretory IgA(sIgA)in saliva could be induced by mucosal immunization with PD-ΔA146Ply,compared to PD alone,and the Ig G isotypes were mainly IgG1,IgG2 a and IgG2 b.The Ig G and sIgA titers of anti-ΔA146Ply induced by PD-ΔA146Ply had no difference compared to ΔA146Ply alone,but the IgG2 a and IgG2 b titers were higher.Mucosal immunization with PD-ΔA146Ply could induce IL-17 A secretion in splenocyte.The sIgA in salvia were significantly lower in IL-17A-/-mice than wild mice and the bacterial clearance ability elicited by PD-ΔA146Ply were also impaired in IL-17A-/-mice.Conclusion: Intranasal immunization with PD-ΔA146Ply fusion protein can effectively defend against colonization and invasive infection of H.influenzae and S.pn infection,providing novel vaccine candidates for simultaneously prevent two kinds of bacterial infection.PD-ΔA146Ply could enhance the immune response strength of PD and modulate the antibody response types of ΔA146Ply.Mucosal immunization with it could affect the sIg A levels of mucosal surface and bacteria clearance through Th17.In general,PD-ΔA146Ply fusion protein was a good vaccine candidate to prevent H.influenzae and S.pn and provide novel vaccine candidates for mucosal vaccination.