The Immune Effect And Mechanism Of A Novel Adjuvant MTOM Combined With Mycobacterium Tuberculosis Subunit Vaccine

[Objective]Although Bacillus Calmette-Guerin(BCG)is the most widely used in the world,can most effectively prevent infant severe tuberculosis(TB),the ability of protecting against adolescent and adult with pulmonary tuberculosis is obviously insufficient.With the advent of Mycobacterium tuberculosis(M.tb)and human immunodeficiency virus(HIV)co-pandemic,and multi-drug resistant tuberculosis(MDR-TB)are emerging,the situation of TB is still very serious.It is an urgent need to develop more effective TB vaccine.Subunit vaccine is an important type of a new generation of TB vaccine,having the advantages of safety and stability.However,the lack of suitable and approved adjuvant significantly hampered the developments of the TB subunit vaccine.MTO—consisting of monophosphoryl lipid A(MPL),trehalose-6,6’-dibehenate(TDB)and MF59—is developed as an adjuvant for the novel TB vaccine to evoke the Thl-type T cell responses,while it is insufficient to induce multifunctional IL-2~+ T cells,which has a limited ability to confer protection against M.tb infection.Heat-killed Mycobacterum vaccae(Mv),which can evoke cytotoxic CD8+ and CD4+ T cells responses and has strong adjuvanticity,was here combined with MTO to produce a new adjuvant.called MTOM.The TB fusion protein Rv3407-PhoY2-Ag85A-Rv2626c-RpfB(WH121)was mixed with MTO,Mv and MTOM respectively to construct three subunit vaccines,and the protective efficacy and immune responses were compared in a TB mouse model.[Methods]1.Fusion protein WH121 was prepared and quantified.2.The preparation of adjuvants and vaccines:adjuvant Mv,MTO and MTOM were prepared.and combined with WH121 protein to construct subunit vaccines,respectively.3.C57BL/6 mice were divided and immunized:mice were divided into WH121 group,Mv group,MTO group,MTOM group,WH121/Mv group,WH121/MTO group and WH121/MTOM group.200 μL of WH121 protein or adjuvant,or WH121 vaccine preparations were given to mice subcutaneously(s.c.)twice at 3 week intervals.The BCG China vaccine was used as a positive control,and PBS served as a negative control.200 μL of BCG(1 × 106 CFU)and PBS were administered once s.c.at the time of the first vaccination.4.The evaluation of protective effects against M.tb infection:six weeks after the last immunization,C57BL/6 mice were challenged i.n.with virulent M.tb H37Rv strain(100 CFU).The animals were sacrificed after four weeks,spleens and lungs were obtained and the CFU numbers per organ were enumerated.Lung tissue was sectioned and stained with HE and acid-fast staining.The bacterial loads of per organ and the pathological changes of lung were observed.5.Six weeks after the last immunization,the immunogenicity of each vaccined group was compared:1)The serums were Separated,and the levels of IgG and its subclasses were analyzed by ELISA assay.2)Spleen cells from mice were prepared.PPD-and WH121-specific IFN-γ,TNF-αand IL-2 were detected by ELISA assay.3)PPD-and WH121-specific IFN-y,TNF-a and IL-2 multifunctional CD4+ and CD8+ T cells in per mouse were detected by intracellular cytokine staining and multicolor flow cytometry.[Results]1.Compared with PBS group,bacterial loads of lung and spleen,and lung pathological damage in each vaccined group were improved in different degrees.Relative to WH121/MTO or WH121/Mv groups.the bacterial load,the pathological damage of lung,and the acid fast bacilli in WH121/MTOM group were significantly reduced(p<0.05,p<0.001),and its protective efficacy matched that of BCG.2.Compared with BCG,mice vaccinated with WH121/MTOM produced higher levels of WH121-specific IgG,IgG1,and IgG2a(P<0.001).the ratio of IgG2a/IgGl substantially increased,elicited a shift of the IgG subclass towards a Thl-type response.WH121/MTOM induced higher levels of these antibodies than WH121/MTO or WH121/Mv,and the conversion trend of the IgG subclass towards Thl-type responses was more obvious.3.Compared with BCG,the WH121/MTOM vaccine induced higher levels of WH121-specific TNF-α and IL-2(P<0.001).The levels of WH121-specific IFN-γ,TNF-α and IL-2 induced by WH121/MTOM were higher than those induced by WH121/MTO or WH121/Mv(P<0.05,P<0.001).4.Compared with MTO or Mv alone,the single.double,and triple IL-2~+,CD4+ and CD8+T cells elicited by MTOM significantly increased(p<0.05).5.Compared with MTOM group,all the combinations of CD4+T cell cytokine responses to WH121 were elevated to a greater degree in the WH121/MTOM group(P<0.05).Relative to BCG,WH121/MTOM showed higher levels of both PPD-and WH121-specific IFN-γ+IL-2~+ CD4+ T cells than BCG(P<0.05).6.Compared with MTOM group,the WH121/MTOM vaccine increased six combinations of CD8+T cell responses to WH121 in addition to IFN-γ+TNF-α+CD8+ T cells(P<0.05).Relative to BCG,the frequency with which WH121-specific CD8+ T cells expressed single,double,or triple IL-2~+ significantly increased in the WH121/MTOM group(P<0.05).[Conclusions]1.Novel adjuvant MTOM consisting of Mv and MTO,has the ability to augment Th1-type responses and IL-2~+ T cells,thus could be used for the development of vaccine candidates against other intracellular pathogens.2.This work can lay the foundation for further preclinical evaluation of WH121/MTOM in other animal models,and help to accelerate the research and development of TB vaccines.