Study On Immunological Characteristics Of Mycobacterium Tuberculosis EsxV Liposome Nanoparticles Subunit Vaccine

Tuberculosis(TB)is a chronic infectious disease that is widespread worldwide and is caused by Mycobacterium tuberculosis(Mtb)infection.Bacille Calmette-Guérin(BCG),a live attenuated vaccine,did not protect well against adult TB.Compared with live attenuated vaccine,subunit vaccine has better safety,stronger immunogenicity and can provide specific immune protection against bacterial infection.Therefore,the research and development of safe and effective Mtb subunit vaccine is one of the important contents of TB research.In addition,Mtb mainly infect the host through respiratory tract.Currently,subunit vaccines of Mtb that have entered clinical trials are administered through muscle or subcutaneous routes,which cannot effectively stimulate mucosal immune response of the body.Therefore,mucosal immune subunit vaccine against Mtb infection is one of the promising directions in TB vaccine research and development.Mtb antigen Esx V is a protein in the difference region between BCG and Mtb,and it also belongs to the 6k Da early secretion antigen(ESAT-6)family proteins,so it has certain immunogenicity.The Esx V-based subunit vaccine can effectively induce humoral and cellular immune responses,and has certain potential against Mtb infection.Bacterial cyclic dimeric adenosine monophosphate(c-di-AMP)can induce host innate immune response,and as an adjuvant to mucosal vaccines,vaccines can enhance antigen induction and immune response.Liposome nanoparticles(LNP)have been widely used as vaccine carriers.LNP can effectively protect the contents and promote uptake by host cells,thus inducing a stronger immune response in order to prolong the action time of antigens.In this study,antigen Esx V combined with adjuvant c-di-AMP vaccine Esx V:c-di-AMP,Esx V:CP and Esx V:CD encapsulated by long cycle LNP and cationic LNP were constructed.The aim is to explore the immunological characteristics of Esx V,and evaluate the immune effect and anti-MTB infection ability of Esx V:c-di-AMP,Esx V:CP and Esx V:CD after inoculation,so as to provide a theoretical basis for the development of Esx V for TB mucosal vaccine and LNP as a mucosal vaccine carrier.In the study of immunological characteristics of Esx V subunit vaccine,the prokaryotic expression vector p ET28a(+)-esx V was successfully constructed and the target protein was purified recombinant Esx V protein was obtained by affinity chromatography.Esx V can induce significant humoral immune response through mucosal immunization.c-di-AMP as mucosal adjuvant can induce significant Th1/Th2/Th17 cell immune response.Immunological properties of vaccines Esx V:CP and Esx V:CD were studied.Firstly,spherical LNP subunit vaccines Esx V:CP and Esx V:CD were obtained by thin film dispersion method.Compared with Esx V immunity,Esx V:CP and Esx V:CD nasal mucosa inoculation induced increased serum Ig G and lung s Ig A levels,increased secretion levels of IFN-γ,IL-2 and IL-10 cytokines,and increased frequency of IFN-γpositive spleen cells.Esx V:CP and Esx V:CD immunity altered cytokine transcription levels and the proportions of CD4~+and CD8~+T cell subsets in lung tissue,and increased the proportions of central memory T fine and tissue resident T cells,suggesting that Esx V:CP and Esx V:CD may provide long-term protection against pulmonary Mtb infection.Finally,a Mtb H37Ra mouse respiratory tract infection model was established to evaluate the immunoprophylaxis effects of Esx V:CP and Esx V:CD.Detection of antibody levels in each group at 6 w after infection showed that serum antibody levels in Esx V group were decreased,while Ig G1,Ig G2a and Ig G2b levels in Esx V:CP and Esx V:CD groups were still strong after infection.The same phenomenon was observed in lung tissue.The results indicated that the duration of Ig G and Ig A levels induced by Esx V protein alone was less than 10 w,and Esx V:CP and Esx V:CD could prolong the induced lung humoral and mucosal immune responses.Esx V:CP and Esx V:CD can significantly increase the Mtb antigen-specific IFN-γand IL-2 secretion levels of Mtb infected mouse spleen cells,but the frequency of IFN-γpositive spleen cells is similar to that of Esx V group.The detection of the secretion levels of inflammatory cytokines in lung tissue showed that the secretion levels of IL-1βwere enhanced in Esx V:CP and Esx V:CD immune groups,and the secretion levels of TNF-a and IL-6 were similar to those of Esx V.Meanwhile,the transcription levels of IL-2 and IL-4 in Esx V:CD immune group were enhanced in Th1/Th2 lung tissue.Both LNP immunized Mtb mice promoted the accumulation of CD4~+T cells in lung tissue to a certain extent.After immunization,both Esx V:CP and Esx V:CD could alleviate the pathological damage of lung tissue caused by Mtb infection,and the Mtb count of lung showed a downward trend,and the effect of reducing the Mtb count of spleen was more significant in the Esx V:CD immunization group.In conclusion,the immune response induced by Esx V as an antigen is weak,and c-di-AMP can further enhance the immunogenicity of Esx V and promote the humoral and cellular immunity of the host.At the same time,the use of two LNPS can also promote the host immune response.Esx V:CP and Esx V:CD can prolong the presence time of host antibodies and promote the Th1/Th2 immune response.At the same time,it can also reduce the pathological damage caused by Mtb infection to a certain extent,and has a preventive effect on Mtb infected mice.Therefore,Esx V can be used as an antigen for subsequent vaccine studies,the ability of c-di-AMP as a mucosal adjuvant has been further confirmed,and LNP can be used as a carrier of TB vaccine for further studies.