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The goal of this study was to characterize serum immunoglobulin G (IgG) antibody responses during experimental immunization of laboratory mice by purified recombinant proteins MPT63, MPT83 of M. tuberculosis and artificial fusion protein MPT83-MPT63 and obtain the recombinant single chain variable fragments of antibodies (scFv) against these antigens. This study demonstrates that the humoral immune response to MPT63, MPT83, MPT83-MPT63 fusion protein and equimolar set of MPT63 and MPT83 was highly different. For each antigen, serum antibody levels were evaluated by a cutoff value based on optical density index. 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 also specific scFv antibodies against MPT63 and MPT83. These antibodies can be used for the development of the system for quantitative determination of antigens as well as for their biological properties investigation. Thereby, based on the results of the immune response and mycobacterial proteins antigenicity we showed highly immunogenicity properties of N-terminal part of MPT83 antigen for enhencement of ELISA sensitivity. We suggest MPT83-MPT63 fusion protein as a potential candidate on the role of antigenic substance for the serological diagnosis of tuberculosis.

The aim of the work was to evaluate in vitro the cytostatic effect of recombinant fragments of the non-toxic point mutant of diphtheria toxin — CRM197, which was suggested as a potent medication for treatment of triple negative breast cancer. For this purpose, non-toxic recombinant derivatives of diphtheria toxin — CRM197, subunit B SbB and receptor domain Rd had been isolated by Ni-NTA agarose affinity chromatography and their effect on the growth of individual colonies of triple negative breast cancer MDA–MB–231 cells were characterized by such parameters as average colony area, perimeter and circularity index. According to the obtained results, CRM197 and SbB, whose molecules contain the translocation domain Td, exhibited the same cytostatic effect against MDA–MB–231 cells, reducing the area and perimeter of individual colonies. Rd protein did not affect the last two parameters that characterize the size of the colonies, but changed the form of the margin of colonies, as evidenced by an increase in the circularity index. It is supposed that Td may be involved in the implementation of cytostatic action due to its inherent pore-forming activity in relation to lipid membranes. It is concluded that Rd and Td, unlike the catalytic domain of diphtheria toxin, play important roles in the implementation of the cytotoxic properties of CRM197, while SbB consisting of Rd and Td is the structural DT fragment of smallest molecular weight that can be used as the analog of CRM197.

The aim of the research was to compare the characteristics of nanoparticles with different chemical structure and size (colloidal gold Gold 1 and Gold 2, calcium phosphate CaP and poly(lactideco-glykolid) PLGA 1 and 2) to find the most efficient carriers of antigen — recombinant diphtheria toxoid for per os immunization. According to the MTT test, all studied particles show no significant cytotoxic impact on the studied cells in vitro, with the exception of CaP nanoparticles, which in high concentrations have cytotoxic effect on the U937 cells, and Gold nanoparticles 1 and 2, that are able to inhibit growth of the L929 cells. The most effective phagocytosis by macrophage-like cells J774 is observed for PLGA nanoparticles 1 and 2 with the immobilized antigen, while Gold nanoparticles 1 and 2 with antigen can interact with the surface of these cells without being phagocytated by them. In BALB/c mice immunized per os with antigen immobilized on PLGA 1 and 2 as well as Gold 2 carriers, the concentration of specific IgA antibodies in blood significantly increases after the second immunization, compared with controls. In the group of mice treated with PLGA 2 conjugated antigen, the concentration of specific IgG in blood after the third immunization also increases. These results show the promise of nanoparticles PLGA 1 and 2 as adjuvant for immunization per os.

Diphtheria is a highly contagious life-threatening disease caused by the toxi genic strains of Corynebacterium diphtheria, which are transformed by a bacteriophage carrying the toxin gene. Diphtheria causative agent and its major virulence factor diphtheria toxin are well studied, but outbreaks of disease still occur worldwide. Rapid development of new methods in immunology and molecular biology is currently leading to improvement of prophylaxis, diagnosis and treatment of diphtheria. This review highlights the microbiological, epidemiological and immunological aspects of diphtheria infection, role of diphtheria toxin and others virulence factors in diphtheria pathogenesis and role of humoral anti-toxic immunity in the protection against disease. Perspectives in development of new diagnostic tests, anti-diphtheria vaccines, immunobiological preparations and antidotes for prevention of diphtheria infection, and other anti-diphteria means was also discussed.