| Tuberculosis(TB)is still one of the diseases that seriously threaten people's life and health all over the world.The increasing incidence of multi-drug resistant tuberculosis(MDR-TB)coinfection with HIV and the enormous number of people with latent tuberculosis infection(LTBI)make it harder to prevent and control TB epidemic.Protective vaccination and rapid and accurate diagnosis are the key for controlling TB.BCG is the only TB vaccine approved for clinical use now,however,it shows poor protection against Mycobacterium tuberculosis(M.tuberculosis)infection.New vaccines under development including both therapeutic vaccines and preventive vaccines.Therapeutic vaccines are designed to inhibit the relapse of LTBI and to shorten the course of treatment for active TB patients.For example,a therapeutic vaccine developed with the whole bacterial protein antisens extracted from Mycobacterium vaccae(M.vacccae)has entered phase ? clinical trials.However,the immune mechanism of M.vaccae in TB immunotherapy is still unknown,while the rapid developments in genomics and immunoproteomics technologies laid a good foundation for further study.So part one of this study was designed to investigate the cross immune response of M.vaccae to M.tuberculosis and BCG to explore the immunization mechanism of whole bacterial protein antigen extract against M.tuberculosis infection.The complete genome sequence of M.vaccae(ATCC 95051)was determined by using IIlumina HiSeq 2000 sequencing platform and a total of 5941 coding sequences were annotated.Comparative analysis between M.vaccae and 3 M.tuberculosis genomes and 16 M.bovis was conducted by BLAST.The results showed a total of 2164 M.vaccae genes have homology with M.tuberculosis which are distributed in different functional categories.For the cross immunological research,M.vaccae(ATCC 95051),M.tuberculosis(H37Rv)and BCG(China strain)were cultured with L-J solid media,from which the bacteria were harvested.The whole bacterial protein antigens prepared by ultrasonic disruption were used to immunize BALB/c mice respectively.Then the cross-immune response were detected The humoral immunity(sera antibodies)was tested with enzyme-linked immunosorbent assay(ELISA).The cellular immunity was evaluated by the detection of various cytokines with cytokine release assay(CRA).The mice immunized with the three mycobacterial antigens could produce high levels of antibodies titer(IgG)and cytokines(IFN-? and IL-2),but slightly high levels of cytokine IL-4 and IL-10.The results of the cross immune reactivity tests showed that the antigens of ATCC 95051,H37Rv and BCG were able to cross-react in the immunized mice,all of which generated a significantly high level of IFN-?,IL-2 and antibody IgG titer,to each other respectively.The positive protein antigens of M.tuberculosis reacting with IgG or IgM in M.vaccae immunized mice was determined by using M.tuberculosis protein microarray and the protein-protein interaction analysis was done by STRING 11.0 online.The results showed that 424 shared protein antigens reacted to IgG.212 to IgM,including 72 proteins recognized by both IgG and IgM.From the protein-protein interactions results,we can infer that antigens can induce humoral immune response independently or interact with other antigens.Preventive vaccines are aimed at the healthy people who are not infected with M.tuberculosis.A key breakthrough in the development of preventive vaccines is to find more protective antigens.Several dominant antigens were identified in our previous study by means of reverse vaccinology.These antigens can be used not only in vaccine construction,but also in the establishment of immunological diagnostic methods.Serological detection technology is rapid,easy and economical immunological diagnostic method.Compared to diagnosis methods aimed to detect pathogens,such as sputum smear,sputum culture and Gene-Xpert,which can only identify TB patients with bacteria positive,serological examination can also detect bacteria negative TB patients.Part two of this study was aimed to identify novel diagnostic antigens or multiple-antigen combination sets for serological diagnosis and screen antigens for vaccine development.20 M.tuberculosis proteins were expressed and purified,of which the performance in serological diagnosis was determined by using 258 human sera and the 4 kinds of animal sera with ELISA.Perl software was used for the selection of multiple antigen combination set with optimal sensitivity and specificity.Rv0432 and Rv0934 yielded good serological performance with sensitivity and specificity of 87.90%/66.34%and 68.79%/84.16%?respectively.And two combinations?Rv1886c-Rv0934 and Rv1886c-Rv2318-Rv0934.provided the best performance,the sensitivity and specificity was 80.25%/73.27%and 81.53%/70.30%,respectively.The reactions with animal sera showed that 12 proteins were with good specificity to M.tuberculosis immunized mice,which showed no cross immune reaction with M.vaccae or BCG immunized mice.Evaluation of an antigen includes both antigenicity and immunogenicity.Next in part three,the immunogenicity of Rv0674 was evaluated by means of immunizing BALB/c mice Lusing Ag85B protein as a positive control.Antibodies were tested using ELISA and cytokines were determined by CRA.Rv0674 could induce a high level of IFN-? and interleukin-2 as well as a high IgG titer,indicating that Rv0674 is essential in humoral and cellular immunity in mice.Moreover,the analysis of the cytokine profile and IgG isotype showed that Rv0674 was characterized as a T-helper 1/2(Th1/Th2)-mixed-type protective immunity with the predominance of Th1 cytokines.In conclusion,there are cross immune responses of M.vaccae to M.tuberculosis and BCG,which provide a certain immunological foundation for using M.vaccae in developing new antituberculous vacccnes.RvI886c-Rv0934 and Rv1886c-Rv2318-Rv0934 can serve as novel serological diagnostic markers.Rv0674 may be a good potential candidate for the development of TB serological diagnosis and a new TB vaccine. |
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