Construction And Immunogenicity Evaluation Of Novel Vaccines Against Mycobacterium Tuberculosis

Tuberculosis is an infectious disease that seriously threatens human health worldwide.According to the statistics of the World Health Organization(WHO),there were an estimated 1.7 million TB deaths and 10.4 million people fell ill with TB in 2016.BCG is the only licensed vaccine against TB.Although it provides effective protection against M.tb in children,BCG has highly variable efficacy(0-80%)against adult pulmonary TB.With the emergence and increasing prevalence of drug-resistant and multi-drug resistant tuberculosis,there is an urgent need to develop more desirable novel vaccines against TB.Mycobacterium tuberculosis,the main pathogen causing tuberculosis,is an intracellular parasite.Therefore,the cellular immune response is critical to the resistance to infection.The induction of a strong cellular immune response is a major research direction in this field.At present,new tuberculosis vaccines in clinical trials are mainly attenuated live vaccines,protein subunit vaccines,and viral vector vaccines.Selected antigens of these vaccines are mainly secreted proteins that have been demonstrated in animal studies or human trials to induce strong immune responses,such as TB10.4,ESAT6,MPT64,Ag85 B,and Ag85 A.The above-described vaccine attempts to use the virus or BCG for antigen expression,or use a recombinant protein containing a specific adjuvant as an immunogen to induce the body to produce a cellular immune response against Mycobacterium tuberculosis.Although many achievements have been made in the development of vaccines against TB,there are still no new vaccine products coming into the market.Therefore,this paper is intended to construct a MVA-based(MVAH4)and adenovirus-based(Ad H4)vaccine expressing Ag85B-TB10.4 fusion protein,a MVA-based(MVATH4)vaccine with highly expressing Ag85B-TB10.4,and Ag85B/TB10.4-BLP protein subunit vaccine for investigating the immune effects of homologous and heterologous viral vector prime-boost immunization strategies,viral vectors with different antigen expression capabilities,and BLP as an adjuvant.The data will provide experimental data and theoretical basis for the research and development of novel vaccine against tuberculosis.Viral vector vaccines can induce a strong cellular immune response.MVA and adenovirus are two commonly used viral vectors in the vaccine research and development,which have an important potential in the development of vaccines against tuberculosis.However,recombinant viral-based vaccines currently in clinical trials employ vaccination strategies that administrate multiple doses of the same recombinant vaccine.However,this vaccination strategy easily leads to the host anti-carrier immune response,which greatly reduces the effectiveness of the vaccine.Studies have shown that the prime-boost immunization strategy using different vaccines can effectively avoid the above problems.Therefore,in this study,MVAH4 and Ad H4 recombinant viral-based vaccines expressing the Ag85B-TB10.4 fusion protein were constructed,and the differences in immune responses generated by homologous and heterologous prime-boost immunization strategies were studied.Both Ad H4 and MVAH4 can induce immune responses in mice.The immunization strategy containing Ad H4(Ad H4-Ad H4,Ad H4-MVAH4 and MVAH4-Ad H4)induces higher levels of antigen-specific antibodies,produces higher levels of IFN-?,and a higher proportion of activated CD8+ T cells(CD69+CD8+ T cells).All immune strategies induce a balanced Th1 and Th2 immune response.The number of IFN-?-producing splenocytes sensitive to CD8+ T-cell restricted peptides of Ag85B(9-1p and 9-2p)and Th1-related cytokines(IFN-? and TNF-?)in the Ad H4-MVAH4 heterologous prime-boost regimen immunization group were significantly higher than that in the other viral vector-based vaccines and the BCG immunized groups,respectively.These results indicate that an immunization regimen involving Ad H4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that of regimens containing BCG or MVAH4 alone,and the Ad H4-MVAH4 prime-boost regimen may generate an ideal protective effect.The homologous prime-boost immunization results of Ad H4-Ad H4 or MVAH4-MVAH4 showed that the MVAH4-induced immune response was significantly weaker than that of Ad H4 at the same immunization dose.In order to solve this problem,this study tried to add the t PA signal peptide sequence to the N-terminal gene of the antigen protein to construct the MVATH4 recombinant vaccine.The expression of antigen protein increased significantly in MVATH4 infected cells comparing to MVAH4.After mice were immunized with MVATH4,the immune response was detected.Compared with the MVAH4 group,the levels of antibodies,cytokine secretion,and the proportion of activated T cells were significantly elevated in MVATH4 group,and the response to Th1 and Th2 responses was not changed.In addition to immunogens and immunization strategies,adjuvants are also key factors that influence efficacy of vaccine.At present,several of the protein subunit vaccines against tuberculosis in clinical trials adopt the strategy of mixing the antigen protein with an adjuvant that activates the TLR4 or TLR9 pathway,for inducing an antigen-specific cellular immune response.However,studies have shown that M.tb expresses a series of TLR2 ligand components after infecting cells.These TLR2 activators play an important role in inducing an immune response against tuberculosis in the immune system.Bacterial-like particles(BLP)are peptidoglycan shells prepared by the acid treatment of Lactococcus lactis,which commonly used in the food industry.Clinical experiments have shown that BLP has good safety.Previous studies have confirmed that BLP produced by Lactococcus lactis is a TLR2 activator,which does not interact with TLR3,4,5,7,8 and 9.In this study,BLP particles were used as adjuvants,and Ag85 B and TB10.4 proteins were linked to BLP through PA.Evaluation of the immune response after immunization of mice revealed that the level of specific antibody and cytokine secretion induced by the Ag-BLP immunization group was higher than that of the protein immunization group,but that was still mainly a humoral immune response.Besides,the response pattern of Th1/Th2/Th17 in Ag-BLP immunization group was not the same as that of the BCG immunization group.In summary,this study constructed MVAH4 and Ad H4 viral vector vaccines that express the Ag85B-TB10.4 fusion protein,and evaluated the immune responses induced by two vaccines under different immunization regime.Next,MVATH4 that added with the t PA signal peptide sequence was constructed,and the enhancement of the vaccine-induced immune response was verified.In addition,we used BLP as an adjuvant in protein subunit vaccine.But that did not effectively induce cellular immune responses after immunization of mice.In this study,different vaccines were investigated in terms of immunogenicity.In-depth response mechanisms and vaccine protection require further research.