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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.

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.

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. [ABSTRACT FROM AUTHOR]

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63-specific IFN-γ-secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA-A*0201 restriction of ten predicted MPT63- derived CD8+T-cell epitopes was assessed on the basis of T2 cell line and HLA-A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN-γ enzyme-linked immunospot assay. It was found that five peptides bound to HLA-A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLAA*0201. Five immunogenic peptides (MPT6318-26, MPT6329-37, MPT6320-28, MPT635-14 and MPT6310-19) elicited large numbers of cytotoxic IFN-γ-secreting T cells in HLA-A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA-A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T-SPOT.TB assay (based on ESAT-6 and CFP-10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T-SPOT.TB assay reached 100%. These MPT63-derived HLA-A*0201- restricted CD8+T-cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine. [ABSTRACT FROM AUTHOR]

Background: The clinical course of tuberculosis (TB) infection, bacterial load and the morphology of lesions vary between pulmonary and extrapulmonary TB. Antigens expressed in abundance during infection could represent relevant antigens in the development of diagnostic tools, but little is known about the in vivo expression of various M. tuberculosis antigens in different clinical manifestations. The aim of this study was to study the differences in the presence of major secreted as well as somatic mycobacterial antigens in host tissues during advanced rapidly progressing and fatal pulmonary disease with mainly pneumonic infiltrates and high bacterial load, and to compare this to the presence of the same antigens in TB lymphadenitis cases, which is mainly chronic and self-limiting disease with organised granulomas and lower bacterial load. Methods: Human pulmonary (n = 3) and lymph node (n = 17) TB biopsies, and non-TB controls (n = 12) were studied. Ziehl-Neelsen stain, nested PCR 1S6110 and immunohistochemistry were performed. Major secreted (MPT32, MPT44, MPT46, MPT51, MPT53, MPT59, MPT63, and MPT64) and somatic mycobacterial antigens (Mce1A, Hsp65, and MPT57) were detected by using rabbit polyclonal antibodies. Results: Plenty of bacilli were detectable with Ziehl-Neelsen stain in the lung biopsies while no bacilli were detected in the lymph node biopsies. All the cases were shown to be positive by PCR. Both secretory and somatic antigens were expressed in abundance in pulmonary infiltrates, while primarily somatic antigens were detected in the lymphadenitis cases. Of the secreted antigens, only MPT64 was consistently detected in both cases, indicating a preferential accumulation of this antigen within the inflammatory cells, even if the cells of the granuloma can efficiently restrict bacterial growth and clear away the secreted antigens. Conclusions: This study shows that major secreted mycobacterial antigens were found in high amounts in advanced pulmonary lesions without proper granuloma formation, while their level of staining was very low, or absent, in the lymph node TB lesions with organised granulomas and very low bacillary load, with one exception of MPT64, suggesting its role in the persistence of chronic infection. These findings have implication for development of new diagnostic tools. [ABSTRACT FROM AUTHOR]

Proteins MPT63 and MPT83 which are common for both Mycobacterium tuberculosis and Mycobacterium bovis, due to their high immunogenicity, are thought to play a promising role in the development of immunodiagnostic reagents and vaccines. To enhance the antigenic and immunogenic properties of these proteins, fragments of the mpt83 and mpt63 genes were fused in tandem. In this article we present an effective method for the MPT63-MPT83 fusion product purification by metal-affinity chromatography and in vitro refolding. Our results demonstrate that the antigenic properties of the recombinant proteins obtained are comparable to their native analogues. The anti-rMPT63 and anti-rMPT83 sera were found to be highly reactive against the rMPT63-MPT83 fusion protein, which suggests that the fusion protein retains the antigenic properties of the parent proteins. Our results may potentially contribute to the development of improved diagnostic tools or vaccines against human and/or cattle tuberculosis. [ABSTRACT FROM AUTHOR]

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63-specific IFN-γ-secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA-A*0201 restriction of ten predicted MPT63-derived CD8(+) T-cell epitopes was assessed on the basis of T2 cell line and HLA-A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN-γ enzyme-linked immunospot assay. It was found that five peptides bound to HLA-A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA-A*0201. Five immunogenic peptides (MPT6318-26 , MPT6329-37 , MPT6320-28 , MPT635-14 and MPT6310-19 ) elicited large numbers of cytotoxic IFN-γ-secreting T cells in HLA-A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA-A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T-SPOT.TB assay (based on ESAT-6 and CFP-10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T-SPOT.TB assay reached 100%. These MPT63-derived HLA-A*0201-restricted CD8(+) T-cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)