Mycobacterium tuberculosis antigens MPT63 and MPT83 increase phagocytic activity of murine peritoneal macrophages.

Macrophages (MΦ) are the most described and characterized target and host of mycobacteria. Like other cells of innate immunity MΦ have a wide range of receptor molecules which interact with different pathogen associated molecular patterns (PAMPs). Immunodominant proteins MPT63 and MPT83 that are synthesized in abundance by Mycobacterium bovis or Mycobacterium tuberculosis strains could be involved in development of tuberculosis infection. The aim of this study was to search for effects of these mycobacterial antigens on target cells. For this aim full-sized sequences of MPT83 (rMPT83full) and MPT63 antigens were cloned into plasmid pET24a(+). The increase of phagocytic activity of murine peritoneal macrophages was demonstrated, but not of macrophage-like cells from J774 cell line, which were treated by rMPT63 and rMPT83full proteins for 24 h. This effect of such antigens can be considered as a way to facilitate the consumption of mycobacterial cells by macrophages to avoid other effector mechanisms of innate and adaptive immunity.

RECOMBINANT SINGLE CHAIN VARIABLE FRAGMENT ANTIBODIES (scFv) AGAINST Pro144-Leu155 FRAGMENT OF HUMAN PROTEIN C.

The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10(-9) M. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.

[Fluorescent derivatives of diphtheria toxin subunit B and their interaction with Vero cells].

Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.

[Toxin-neutralizing properties of antibodies to diphtheria toxin recombinant subunits A and B and a new method of their estimation].

Development of complications during diphtheria depends to a large extent on toxin-neutralizing antibodies level in the patient's blood. Active immunization of people with diphtheria anatoxin is widely used for diphtheria prevention and passive immunization with hyperimmune antitoxic horse serum is used for diphtheria treatment. A traditional component of anti-diphtheria vaccines--diphtheria anatoxin has a number of serious disadvantages, which are mainly associated with complicated procedure of its production. Thus, the search for new antigen substances, which can effectively stimulate protective humoral response to diphtheria toxin, is an urgent task in anti-diphtheria vaccine development. Furthermore, one of the most important objects is the development of new in vitro methods for estimation of diphtheria toxin-neutralizing polyclonal and monoclonal antibodies, which allow to avoid using active diphtheria toxin and toxin-sensitive laboratory animals. Comparative studies of toxin-neutralizing antibodies induction after immunization of laboratory animals with recombinant subunits A and B of diphtheria toxin were carried out. The new method for detection of protective antibodies in serum was proposed. This method is based on the ToBI test (Toxin Binding Inhibition test); namely on the property of anti-diphtheria antibodies to inhibit the biding of toxin subunit B fused with enhanced green fluorescent protein (EGFP) to the sensitive to diphtheria toxin Vero cells. The ability of subunit B to induce toxin-neutralizing antibodies in laboratory animals (rabbits and guinea pigs) was confirmed by the intradermal test, which is traditionally used to detect protective antitoxic antibodies in the serum, and by flow cytometry method, developed for this purpose. The results suggest that diphtheria toxin recombinant subunit B may be used for the induction of the protective immune response. The new developed approach for estimation diphtheria toxin-neutralizing antibodies is more ethical and safe and can substitute successfully the traditional methods.

[Construction of immune library of murine immunoglobulin genes and screening of single-chain Fv-antibodies specific to diphtheria toxin B subunit].

The aim of this work was to obtain the panel of recombinant single-chain Fv-antibodies against diphtheria toxin B subunit, the main diagnostic and pathogenic antigen of Corynebacterium diphtheriae. For this purpose we have constructed the immune library of murine immunoglobulin genes. A number of scFv specific to diphtheria toxin B subunit were acquired from the obtained library after one round of selection by phage-display. ScFv encoding DNA-fragments of eight clones were subcloned into plasmid pET-22b(+). It was shown that selected scFv were highly specific to diphtheria toxin B subunit, with affinity constant for different clones ranged from 10(7) to 10(9) M(-1).

[Cytotoxicity of the B subunit of diphtheria toxin to human histocytic lymphoma U937].

The B subunit of diphtheria toxin (DT) is responsible for interaction with receptor on the cell surface and translocation of the catalytically active A subunit across endosomal membrane into the cell cytosole. Receptor for DT and its B subunit is membrane-anchored precursor of heparin-binding epidermal growth factor-like growth factor (pro-HB-EGF), which under the action of metalloproteases turns into soluble form (sHB-EGF), which acts as a potent mitogen for different cell types. Since free B subunit of DT has no catalytic activity it is considered to be nontoxic. However its influence on the cells in vitro remains to be investigated. The aim of this study was to examine the influence of diphtheria toxin B subunit on viability of diphtheria-sensitive cells using B subunit recombinant analogues. It was shown that diphtheria toxin B subunit recombinant analogue at a concentration of 12.8 x 10(-7) M had a cytotoxic effect on the human histocytic lymphoma cell line U937, which expresses a large amount of sHB-EGF. Besides, the similar cytotoxic effect had a fusion protein which consisted of a B subunit and an enhanced green fluorescent protein (EGFP). However recombinant EGFP alone didnot influence the cell viability. Annexin-V-FITC/PI staining demonstrated that maximal cytotoxic effect had been elicited after 48 hours of cultivation. Cytotoxic test with trypan blue and propidium iodide staining excluded the direct influence of investigated proteins on the integrity of plasma membrane because of the ability of B subunit to pore formation. So, we offer a hypothesis that realization of cytotoxic effect of diphtheria toxin B subunit and its derivative on the U937 cell culture occurs via inhibition of mitogenic activity of sHB-EGF resulted in induction of apoptosis.