Siromolot A. Immunobiological properties of Mycobacterium spp. antigens MPT63 and MPT83 and their role in tuberculosis diagnostic

Knowledge about mechanisms of pathogenesis and development of tuberculosis (TB) is rapidly growing. Interactions of bacterial pathogenic factors with target molecules lead to particular effects in host. Thus, investigation of features and functions of number tuberculosis bacilli antigens is a prerequisite for prevention and treatment of TB. This work is dedicated to investigation of immunobiological properties of MPT63 and MPT83, target cells searching and the possibility of using these proteins for the diagnosis of bovine and human TB. In order to create the molecular instruments necessary for the search of antigens targets with immunochemistry methods and for development of diagnostic test-system fluorescent derivatives of MPT63 and MPT63 and fusion antigenic substance MPT83-MPT63 in one open reading frame were obtained in prokaryotic expression system E.coli. For the first time, putative target cells for the immunodominant antigens Mycobacterium spp. MPT63 and MPT83 were detected. The ability of MPT63 and MPT83 to promote activation and maturation of macrophages by expression of CD11b and F4/80 markers has been established. Also, we demonstrate that the humoral immune response to MPT63, MPT83, MPT83-MPT63 fusion protein and equimolar set of MPT63 and MPT83 is highly distinguished. For each antigen, serum antibody levels were evaluated by using a cutoff value based on optical density index. It has been proven a crucial role of MPT83 for immunogenicity of chimeric protein and/or cocktail of individual antigens under conditions of immunization of laboratory animals. We obtained specific scFv antibodies against MPT63 and MPT83, which could be used for development of the system for quantitative determination of antigens as well as for investigation theirs biological properties. According to the results of the study, it was shown that test-system «IB-Chem Anti-Mycobacterium bovis» is effective in diagnostics in animals infected with M. bovis proper. An industrial sample of a test-system for serological diagnosis of human tuberculosis was developed. The comparative characteristic of the experimental test-system with reference diagnostiс tools is carried out. The specificity of the MPT83-MPT63 based test-system was 69.5% compared to 45.2% for the reference test systems #1 and #2, and the sensitivity was 95.5% compared with 92.5% and 90.2% for test-systems #1 and #2 respectively. Classic BCG vaccine has been used worldwide to prevent TB disease in infants and children, but it has demonstrated limited and variable effectiveness in preventing pulmonary TB in adolescents and adults. The use of alternative antigens and additional approaches for vaccine development urgently needed to protect against TB. Nevertheless, our results establish a novel platform for development of antigenic substances with inherent immunogenic characteristics that are desirable in vaccines.