| Tuberculosis (TB) is a major health threat caused by Mycobacterium tuberculosis (M. tuberculosis). With the increasing drug-resistant strains and HIV infection, TB remains one of the major diseases with high morbidity and mortality worldwide. Central to the success of M. tuberculosis as a pathogen is its ability to persist for long periods of time in latent state in human cells and may develop into active disease. The current vaccine, Bacilli Calmette-Guerin (BCG), is widely used in many areas of the world. It protects children from severe TB, but can not prevent reactivation of latent TB and is unreliable against the pulmonary TB in adults. Therefore, novel vaccines and vaccination strategy which target adult TB, especially latent infection are urgently needed.Objective: To search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG) and to control or even eradicate M. tuberculosis in all stages of infection including the persister bacteria, antigens of Mtb10.4(Rv0288) expressed in replicating bacilli and HspX (also called Acr, Hsp16.3, Rv2031c) highly expressed in dormant bacilli were fused together to construct a multistage fusion protein Mtb10.4-HspX (MH for short) and its immunogenicity and protective efficiency were analyzed.Methods:The DNA sequences coding mature protein of Mtb10.4and HspX were generated by PCR amplification. The plasmid encoding Mtb10.4-HspX was generated by inserting the genes Mtb10.4and HspX into the multiple cloning sites of the expression vector pET30a (+) respectively and was transformed into E. coli strain BL21for production of the fusion protein MH. Three successive chromatographic purification steps were involved in the purification of MH.The human T-cell responses to MH were evaluated using ELISPOT. MH was emulsified in an adjuvant composed of N, N’-dimethyl-N,N’-dioctadecylammonium bromide (DDA) and mycobacterial cord factor trehalose-6,6-dimycolate (TDM) to construct subunit vaccine. Mice received inoculations three times at Ost,3rd, and6th week, mice received PBS or BCG as the control, and the immunogenicity of MH was analyzed at6weeks after the last immunization. The mice primed with BCG and boosted by MH subunit vaccine two times at the12th and14th week were used to analyze its immunity boosting BCG at6weeks after the last immunization. All groups were challenged with5×105CFU of virulent M.tuberculosis H37Rv via caudal vein at24th week,10weeks after the last boosting. Protection was assayed by counting colony forming units (CFUs) of bacteria in infected lungs and spleens at the42days after the challenge.Results:The fusion protein MH was stably produced in E. coli in supernatant, and was purified by hydrophobic chromatography, ion-exchange chromatography and gel filtration chromatography successively. MH induced higher numbers of IFN-y-producing T cells in groups of TB patients and persons latently infected with M. tuberculosis. The mice immunized with MH three times produced high levels of cytokines(IFN-γ、IL-17) and antibodies(IgG1、IgG2b、IgG2c). The frequencies of IFN-y and IL-17secreting lymphocytes from the mice primed by BCG and boosted by MH were higher than that immunized with BCG without boosting (p<0.05) or PBS (p<0.05). BCG-prime and MH-boost induced a significant reduction in the number of bacteria in the lungs compared to PBS controls (P<0.05), a reduction compared to BCG group (P=0.069), and presented the smallest lesions among the three groups (p<0.05).Conclusion:MH in adjuvant DDA-TDM generated strong antigen-specific humoral and cell-mediated immunity, and had the capability to enhance BCG-primed immunity and the protective efficacy against M. tuberculosis in mice. These findings suggest that MH in DDA-TDM has the potential to be a good multistage tuberculosis vaccine candidate. |
PDF Full Text Request