Biological Function And Immunological Effect Of Mycobacterium Tuberculosis Recombinant Fusion Protein Subunit Vaccine Dodecin-ESAT-6

Background:Tuberculosis(TB)is a worldwide epidemic infectious disease that seriously endangers human health and brings high public health risk and heavy medical burden to all countries in the world.TB is caused by Mycobacterium tuberculosis(M.tuberculosis).According to the World Health Organization,there are nearly 2 billion people in the world who are potentially infected with M.tuberculosis,of which 5%-10%will develop active tuberculosis in their lifetime.There was an estimated about 10 million TB infected people suffering from active tuberculosis every year,and the number of TB-related deaths is about 1.5 million.According to the latest global TB report of the World Health Organization in 2020,there were about 8.9 to 11million new TB cases in 2019,including 1.51 million TB-related deaths.Although the number of people infected with tuberculosis has declined in recent years,tuberculosis is still the most harmful infectious disease to human.Bacillus Calmette Guerin(BCG)is the only vaccine approved for the prevention of tuberculosis,which can effectively prevent TB in newborns and children.Although BCG has been widely vaccinated,it has not prevented the spread of the global tuberculosis epidemic.BCG can not provide effective protection for adolescents and adults,and can not effectively prevent latent infection of M.tuberculosis.Besides,as a live attenuated vaccine,BCG has its virulence,which has a safety risk for people with low immunity or HIV infection.Therefore,there is an urgent need to develop a new and effective anti-tuberculosis vaccine.Protein subunit TB vaccine has many advantages,such as protein subunit vaccine is not affected by the stimulation of BCG,can induce the long-term and effective activation of CD4~+Th1 cells and CD8~+cytotoxic T cells,clear composition,high yield,short production cycle,easy large-scale production,low cost,no safety risk caused by live bacteria and virus.Studies have found that inactivated Mycobacterium tuberculosis can only cause a transient immune response in animals,but can not induce protective immunity.The proteins secreted in the process of infection and proliferation of Mtb are considered to play an important role in inducing protective immunity.Early secretion of target antigen 6(ESAT-6)is the key virulence protein of Mycobacterium tuberculosis,which is an important Mtb protective antigen deleted by BCG.ESAT-6 encodes multiple T and B cell epitopes,which can effectively induce both T cell and B cell responses.Olsen et al.found that ESAT-6 alone could only induce a weak cellular immune response,and ESAT-6 with DDA and MPL adjuvant could significantly improve the immunogenicity of ESAT-6,and induce the same protective effect as BCG in mice.The low molecular weight of ESAT-6 may be the reason for its low immunogenicity.The fusion protein of ESAT-6 with other tuberculosis antigens is one of the effective methods to improve its immunogenicity.Dodecin protein is a soluble flavoprotein of Mycobacterium tuberculosis,which is encoded by the Rv1498A gene.Dodecin can be detected in the whole-cell lysate,cell membrane extract,cell culture supernatant,and culture medium filtrate of Mycobacterium tuberculosis.It can be used as a new antigen of TB.It was found that dodecin protein is a multimer structure,which forms a compact hollow spherical dodecamer through the salt bridge between subunits,and has thermal stability.Dodecin protein can be used as an oligomeric antigen-carrier to enhance the molecular weight and immunogenicity of ESAT-6.The dodecin-ESAT-6 recombinant fusion protein has the potential to become a new subunit vaccine.In this study,dodecin-ESAT-6 was constructed and analyzed for the first time.It was found that dodecin-ESAT-6 protein was also a multimer with good thermal stability.To evaluate the potential of dodecin-ESAT-6 as a new subunit vaccine,we studied the cellular and humoral immune responses induced by dodecin-ESAT-6 with or without adjuvant and BCG.Our research has important reference value for the development of a new subunit vaccine based on dodecin-ESAT-6.Material and methods:In order to construct and express dodecin-ESAT-6 protein,we first amplified Rv3875 and Rv1498A from Mycobacterium tuberculosis H37Rv genome by polymerase chain reaction(PCR).Then the Rv3875-Rv1498A fusion gene fragment with Nde I and Hind III restriction sites was amplified by overlapping extension PCR(SOEing PCR)and cloned into p ET28a expression vector.We successfully constructed expression vectors p ET28a-dodecin and p ET28a-dodecin-ESAT-6.They were then transformed into Rosetta(DE3)plys S strain and induced by IPTG to express the target protein with 6×His Tag.The target protein was purified by Ni ion affinity chromatography and identified by SDS-PAGE and Western blot.To study the multimer stability of dodecin and dodecin-ESAT-6,we used the metal bath method to heat treat dodecin and dodecin-ESAT-6 proteins and analyzed their molecular weight changes by SDS-PAGE.In order to study the multimers of ESAT-6,we used ESAT-6 monoclonal antibody to identify and analyze it by SDS-PAGE and Western blot.Our results confirmed the existence of ESAT-6dimer and multimer.To study the immunomodulatory effect of dodecin-ESAT-6 on macrophages,we used phorbol ester(TPA)to induce THP-1 monocytes to differentiate into macrophages and detected the expression of IL-1β,IL-6,IL-12p40,TNF-α,IFN-γand IL-10 m RNA by RT-q PCR,the commercial ELISA kits were used to detect and analyze the above cytokines in the cell culture supernatant to study its regulatory effect.In order to further study the signaling pathway of dodecin-ESAT-6-induced pro-inflammatory factor secretion,THP-1 macrophages were pretreated with TLR2 or TLR4 inhibitor,then the secretion levels of TNF-α,IL-1β,and IL-6 were detected by ELISA to study their excellular receptors;THP-1 macrophages were pretreated with MEK1/2inhibitor,p38 inhibitor or NF-κB inhibitor and then the secretion levels of TNF-α,IL-1β,and IL-6 were detected by ELISA to study their intracellular signaling pathways.To study the effect of recombinant protein on macrophage surface molecules,we used dodecin,ESAT-6,and dodecin-ESAT-6 co-incubated with RAW264.7 macrophages for 24 hours,and then CD80-FITC.CD86-PE and MHC II-APC monoclonal antibodies were used to label the positive cells,and the expression levels of macrophage surface molecules were detected by flow cytometry.To study the effect of recombinant protein on macrophage apoptosis,THP-1 cells were incubated with dodecin,ESAT-6,or dodecin-ESAT-6 for 24hours separately,and the apoptosis level was detected by flow cytometry using Annexin V-FITC and PI apoptosis detection kit.In order to study whether the recombinant protein has a toxic effect on cells,we used the lactate dehydrogenase(LDH)detection method to detect the LDH released by cells incubated with dodecin,ESAT-6,or dodecin-ESAT-6 for 6,24,and 48 hours separately.To study the proteins’effects on the intracellular survival ability of M.smegmatis,macrophages were infected with M.smegmatis at MOI of 10,and the CFU of survival M.smegmatis was detected at 6,24,48,and 72 h.We then evaluated the immune response induced by dodecin-ESAT-6 in mice.To evaluate the immunological effect induced by dodecin-ESAT-6 protein or combined with DDA/MPL protein adjuvant on mice,we established dodecin-ESAT-6 protein alone group,DDA/MPL adjuvant alone group,and dodecin-ESAT-6+DDA/MPL combined group.After the mice were immunized three times,the serum of each group was collected,the titers of Ig G,Ig G2a,and Ig G1antibodies produced by the mice were detected by indirect ELISA,and the ratio of Ig G2a/Ig G1 was calculated.The spleen lymphocytes of each group were separated by spleen lymphocyte isolation kit,after 72 hours of antigen stimulation,the spleen lymphocytes were labeled with CD3-PE-Cy5,CD4-FITC,and CD8-PE flow cytometry antibodies,and CD4~+and CD8~+T lymphocyte subtypes were analyzed by flow cytometry.The antigen-specific proliferation of splenic lymphocytes in immunized mice was detected by the CCK-8 method.The levels of IFN-γ,IL-2,IL-4,and NO were detected by ELISA and Griess nitric oxide detection method respectively.In order to evaluate the potential of dodecin-ESAT-6 as a subunit vaccine,we set dodecin,ESAT-6,dodecin-ESAT-6,and BCG prime dodecin-ESAT-6 boost immunization groups,and used the above immunological methods to detect and analyze the humoral and cellular immune responses of each group.Results:We successfully constructed pET28a-dodecin and p ET28a-dodecin-ESAT-6 recombinant protein expression plasmids,and identified them by PCR,double enzyme digestion,and DNA sequencing to ensure that the constructed recombinant plasmids had no mutation or deletion.We transformed the recombinant plasmids p ET28a-dodecin and p ET28a-dodecin-ESAT-6 into Rosetta(DE3)plys S.The expression clones of dodecin-ESAT-6 and dodecin recombinant protein were obtained.After induced by IPTG,the target protein with 6×his protein tag was successfully expressed and purified by Ni ion affinity chromatography column,the purified proteins were identified by SDS-PAGE and Western blot.SDS-PAGE and Western blot analysis of ESAT-6,dodecin,and dodecin-ESAT-6 showed that ESAT-6 protein was a 6 k Da monomer and it can form 24k Da multimer and 12 k Da dimer.Our results showed that dodecin-ESAT-6 and dodecin-ESAT-6 could withstand 95℃heat treatment and SDS-PAGE denaturation without changing its multimer form,indicating dodecin-ESAT-6and dodecin-ESAT-6 were stable multimer proteins.We found that dodecin-ESAT-6 could induce macrophages to produce high levels of IL-1β,IL-6,IL-12p40,and TNF-αm RNA expression and cytokine secretion,and low levels of IFN-γsecretion,but had no effect on anti-inflammatory factor IL-10;Our further study found that dodecin-ESAT-6mediated the pro-inflammatory cytokine secretion through TLR2 and TLR4receptors and ERK,p38 MAPK and NF-κB intracellular signaling pathways.Flow cytometry analysis showed that dodecin-ESAT-6 could up-regulate the expression levels of CD80,CD86,and MHC-II on the surface of RAW264.7macrophages,while dodecin and ESAT-6 had a low-level inhibitory effect on the expression of macrophage surface molecules.In addition,we also found that dodecin,ESAT-6,and dodecin-ESAT-6 could induce low-levels of apoptosis in THP-1 macrophages.In vitro cytotoxicity test showed that none of them had obvious cytotoxicity.ESAT-6 could increase the intracellular survival ability of M.smegmatis,but dodecin and dodecin-ESAT-6 did not affect the intracellular survival of M.smegmatis.Our animal experiments showed that dodecin-ESAT-6 with DDA/MPL improved higher levels of cellular and humoral immune responses than dodecin-ESAT-6 protein alone.The antibody titer,Ig G2a/Ig G1 ratio,CD4~+T lymphocyte ratio,antigen-specific splenic lymphocyte proliferation,IFN-γ,IL-2,and NO levels of adjuvanted dodecin-ESAT-6 group were significantly higher than those of protein alone group.Dodecin,ESAT-6 and dodecin-ESAT-6proteins induced different degrees of immune responses in mice.The antibody titer,Ig G2a/Ig G1 ratio,percentages of CD4~+CD8~+T cells,antigen-specific splenic lymphocyte proliferation,levels of IFN-γ,IL-2,and NO of dodecin-ESAT-6 immunized group were significantly higher than those of dodecin group and ESAT-6 group.The immune response induced by ESAT-6 and dodecin-ESAT-6 tended to be Th1 predominant;The BCG prime dodecin-ESAT-6 boost strategy induced a stronger Th1 type immune response than dodecin-ESAT-6,the antibody titer levels of Ig G and Ig G2a,percentages of CD4~+CD8~+T cells,antigen-specific splenic lymphocyte proliferation,levels of IFN-γ,IL-2,and NO were significantly higher than those of dodecin-ESAT-6 group.Conclusion:In this study,The recombinant fusion protein of dodecin-ESAT-6 was constructed using dodecameric protein dodecin from Mycobacterium tuberculosis.The biological function and immune regulation mechanism of dodecin-ESAT-6 were studied in vitro,and the immune response induced by dodecin-ESAT-6 was evaluated in a mouse model.Our results showed that dodecin-ESAT-6 was a stable multimer protein.Dodecin-ESAT-6has good immunogenicity in vitro,it can induce macrophages to produce high levels of pro-inflammatory cytokines.The secretion of TNF-α,IL-1β,and IL-6induced by dodecin-ESAT-6 is mediated by TLR2 and TLR4 receptors and ERK,MAPK,and NF-κB intracellular signaling pathways.This study found that dodecin-ESAT-6 can significantly up-regulate CD80,CD86,and MHC-II on the surface of macrophages.Our study found that dodecin-ESAT-6 has good safety in vitro and in vivo:it has no cytotoxicity and pro-apoptotic effect on human THP-1 macrophages,does not increase the intracellular survival ability of M.smegmatis in macrophages,does not influence the weight of immunized mice,and has no vaccine-related toxicity reactions.Compared with dodecin and ESAT-6,dodecin-ESAT-6 can induce stronger Th1 type cellular and humoral immune responses in mice.The enhanced immune strategy of dodecin-ESAT-6with BCG can induce stronger Th1 type cellular and humoral immune responses than those of dodecin-ESAT-6 in mice.Our study provides a valuable reference for the development of a novel subunit vaccine based on dodecin-ESAT-6.