PROTECTIVE EFFECT INDUCED BY THE NEW SUBUNIT TUBERCULOSIS VACCINE WHEN USED AS A BCG BOOST IS ASSOCIATED WITH INHIBITION OF MYCOBACTERIAL DISSEMINATION

Since 1924, BCG vaccine is used to protect children from the most severe forms of tuberculosis. At the same time, the protective effect of BCG in adults is variable. The potential for revaccination with live vaccine is further limited by the rapid spread of HIV infection. The early-secreted Mycobacterium tuberculosis proteins have been used extensively in TB vaccine development, due to their high immunogenicity and have shown protective effect in animal models. The aim of our study was to evaluate the opportunity to increase the anti-TB resistance in experimental animals by re-vaccination with a new subunit vaccine preparation following primary immunization with BCG. To perform such boost vaccination, we used a combination of the Ag85B-TB10.4-FliC chimeric protein, and the plasmid DNA encoding Ag85A antigen. Efficiency of the boost vaccination was evaluated in a model of M. tuberculosis H37Rv aerosol infection of C57BL / 6 laboratory mice, either in the intact animals, or those vaccinated with BCG only, or BCG followed by revaccination with the test vaccine. The data concerning mycobacteria outgrowth from the organs, and life-span of animals after infection were subject to comparative analysis. We have demonstrated that additional boost vaccination with the vaccine under study, as compared with conventional BCG vaccination, leads to further inhibition of mycobacteria dissemination from the site of infection, and significantly prolonged survival of infected animals

Применение нуклеотидного биокорректора из дрожжей рода Saccharomyces cerevisiae в комбинированной терапии туберкулеза

There is information on efficacy of immunostimulating agents as a part of therapy of lung tuberculosis. Nucleotide biocorrector is a mixture of nucleotides and nucleosides derived from baking yeast of the Saccharomyces cerevis genus and presenting wide immunomodulating properties. The aim of this study was to investigate efficacy of this agent along with combined chemotherapy in 52 patients with destructive lung tuberculosis. Of them, 27 patients were treated with nucleotide biocorrector (3 g per day orally during 1 month) and conventional chemotherapeutic medication (the study group) and 25 patients (the control group) received equal chemotherapy alone. All the patients were MBT-positive. Immunological examination (CD3+, CD4+, CD8+, СD16+, and CD20+ lymphocytes and their functional activity, anti-MBT-antibodies) was done before and 1 month after starting the treatment; clinical, radiological and laboratory investigations were performed at the same time and additionally in 3 month. Addition of biocorrector to the standard chemotherapy of lung tuberculosis allowed faster normalization of CD3+, CD4+ and CD8+ lymphocytes, enhanced functional activity of T-cell immunity and increased number of CD20+ B-lymphocytes. Moreover, this therapy has facilitated resolution of lung infiltration, eradication of MBT and recovery of tuberculous cavities by the 3rd month of the treatment. Tolerability of biocorrector was good.Имеются данные о целесообразности включения в комплексную терапию туберкулеза иммуностимулирующих препаратов. Нуклеотидный биокорректор является смесью нуклеотидов и нуклеозидов, выделенных из хлебопекарных дрожжей рода Saccharomyces cerevisiae, и обладает широким спектром иммуномодулирующей активности. Целью исследования явилось изучение его эффективности в комбинированной химиотерапии 52 больных деструктивным туберкулезом легких, из которых 27 больных получали нуклеотидный биокорректор наряду с химиотерапией (основная группа), а 25 больных, получавших только химиотерапию, составили контрольную группу. Все больные выделяли микобактерии туберкулеза с мокротой. Иммунологическое обследование больных (лимфоциты CD3+, CD4+, CD8+, СD16+ и CD20+, их функциональная активность, уровень противотуберкулезных антител) проводилось до и через 1 мес. после начала лечения, клинико-рентгенологические и лабораторные показатели определялись в те же сроки и еще через 3 мес. Все больные получали одинаковую стандартную химиотерапию, кроме того, больные основной группы в течение 1-го месяца получали биокорректор 3 г/сут. перорально. На фоне лечения биокорректором быстрее нормализовалось содержание лимфоцитов CD3+, CD4+ и CD8+, повысились функциональная активность Т-клеточного звена иммунитета и содержание В-лимфоцитов CD20+. Назначение биокорректора способствовало разрешению инфильтрации, прекращению бактериовыделения и закрытию полостей распада к 3-му месяцу лечения. Переносимость биокорректора была хорошей

STUDIES ON PROTECTIVE EFFECTS OF A VACCINE, BASED ON RECOMBINANT Ag85, TB10 AND FliC PROTEINS

At present time, there is an obvious need for a new generation of vaccines as the most effective preventive approach, in order to stop spreading of tuberculosis infection. So far, the most popular strategy is aimed at heterological vaccination. The idea is to use BCG, or improved BCG, or attenuated M. tuberculosis for primary vaccination. For the further booster vaccination one may apply thw s.c. subunit or vector vaccines, containing protective mycobacterial proteins. The aim of our investigation was to evaluate protective effects of a new vaccine based on recombinant bacterial proteins Ag85, ТВ10 and FliC. We used a model with aerosol M. tuberculosis H37Rv infection, and compared lung and spleen CFU counts and life-span of vaccinated versus non-vaccinated С57BL/6 mice. As a result, we revealed three vaccine variants with comparable protective capacity against BCG using our experimental model. The most promising variant is suggested for testing in preclinical trials

Monoclonal antibodies against Mycobacterium tuberculosis, detecting the antigen in urine of the pulmonary tuberculosis patients

Protease-resistant protein and lipid mycobacterial antigens are present in biological fluids of pulmonary tuberculosis patients. Anti-tuberculosis monoclonal antibodies (MAb) reacting to proteins participating in the molybdopterin synthesis have been obtained. With the help of two MAb recognizing different epitopes on one antigen with molecular mass of 32, 50 and 75 kilodalton. With the help of which the two-site sandwich of IFA type capable of detecting the mycobacterial antigen in antigenic preparations of х M. tuberculosis and in urine samples of tuberculosis patients. However the specificity of this test is fairly low since when testing urine samples this antigen was found in the half of healthy donors. It is due to the fact that in human urogenital tract some opportunistic pathogens are present which belong to normal bacterial population with which the tested MAb have the cross-reaction

Diagnosis of Bactec samples by immunoglobulins of mouse hyperimmune sera obtained against modified antigens of the cell wall of Mycobacterium tuberculosis

The objective: using double-site enzyme immunoassay (ELISA) to evaluate the specificity of the antigens of digestive and chemically modified cell walls (CW) of M. tuberculosis.Subjects and methods. Hyperimmune sera of mice were obtained against modified CW antigens; immunoglobulins of different subclasses were isolated from them. With their help, 152 Bactec cultures with mycobacterial and non-mycobacterial growth from patients with lung diseases were tested by ELISA.Results. When CW was treated with proteinase K (prK), the protein content decreased by 10 times, and upon hydrolysis of NaOH, by more than 30 times. In immunoblotting, there was a narrowing of the spectrum of recognized antigens by the sera of hyperimmune mice (compared with whole CW), which indicated a decrease in their immunogenicity. Modification of WC of M. tuberculosis disavows 54 kDa antigen, causing a strong IgG1 subclass response.Diagnostic efficacy in ELISA with Bactec cultures increases with the use of immunoglobulins obtained against antigens treated with proteinase K – 79.14% (Pr.A) and 86.68% (Pr.G), when compared with immunoglobulins against the original drug – 70.69% (Pr.A) and 69.11% (Pr.G). Specificity increases significantly when using IgG1 antibodies after immunization with CW treated with prK (71.92% versus 25.93% in the initial preparation). Thus, new antigens of M. tuberculosis were identified, new antibody preparations for diagnosis in microbiological cultures were created against them

DURATION OF IMMUNE RESPONSE INDUCED BY THE VACCINE BASED ON RECOMBINANT Ag85, TB10 AND FliC PROTEINS

Already about one hundred years BCG remains the only widely used tuberculosis (TB) vaccine. It is known that intensity of the BCG-induced Th1 immune response decreases over time and comes to naught within 10-15 years. It significantly distinguishes BCG from the vaccines providing a lifelong protection such as vaccines against poliomyelitis or measles, and can be bound to natural restriction of duration of a persistence of the BCG-induced CD4+ T-cells. Data on insufficient ability of BCG to stimulate life-long immunological memory is accumulating. Earlier in our lab on the model of rather resistant to TB C57BL/6 mice infection with the virulent laboratory strain of Mycobacterium tuberculosis (Mtb) H37Rv protective activity (comparable to that of BCG Russia) of 3 subunit vaccine variants was demonstrated as assessed by lung and spleen CFU counts and life span of animals after infection. The aim of this study was to study the characteristics and duration of the immune response induced by the most effective variant of these vaccines. Groups of C57BL/6 mice were vaccinated intramuscularly twice with two-week intervals with 10 μg protein conjugated to 200 μl of an aluminum hydroxide emulsion. Immune response (production of specific antibodies, vaccine proteinstimulated production of interferon gamma and proliferation in vitro) was monitored during 10 months after vaccination. We have shown that the test vaccine induces in mice the formation of long-term immunological memory to a bacterial antigen. Moreover, in the presence of glutoxim the immunological memory spectrum shifts to a "protective" type, i.e. the predominance of the cellular component of the immune response over the antibody response is stimulated. The next step will be the investigation of vaccine effectiveness for revaccination after primary BCG immunization

A Mechanism of Gold Nanoparticle Aggregation by Immunoglobulin G Preparation

Conjugates of gold nanoparticles (GNPs) and antibodies are widely used in various fields of biochemistry and microbiology. However, the procedure for obtaining such conjugates remains precarious, and the properties of conjugates differ significantly for different antibody clones. One of the most common problems is the aggregation of GNPs in the course of their conjugation with antibodies. This article considers an example of the conjugation of monoclonal antibodies with non-stable aggregating product. The composition of the antibody preparation was studied using electrophoresis, asymmetrical flow field-flow fractionation, and ultracentrifugation. It was shown that the component that causes the aggregation of the GNPs is the light chains of immunoglobulins that appear due to the spontaneous decay of the antibodies. After separation of the fraction with a molecular weight of less than 30 kDa, stable conjugates of antibodies with GNPs were obtained. The high functional activity of the obtained conjugates was confirmed by immunochromatography

Моноклональные антитела против Mycobacterium tuberculosis, выявляющие антиген в моче больных туберкулезом легких

Protease-resistant protein and lipid mycobacterial antigens are present in biological fluids of pulmonary tuberculosis patients. Anti-tuberculosis monoclonal antibodies (MAb) reacting to proteins participating in the molybdopterin synthesis have been obtained. With the help of two MAb recognizing different epitopes on one antigen with molecular mass of 32, 50 and 75 kilodalton. With the help of which the two-site sandwich of IFA type capable of detecting the mycobacterial antigen in antigenic preparations of х M. tuberculosis and in urine samples of tuberculosis patients. However the specificity of this test is fairly low since when testing urine samples this antigen was found in the half of healthy donors. It is due to the fact that in human urogenital tract some opportunistic pathogens are present which belong to normal bacterial population with which the tested MAb have the cross-reaction.В биологических жидкостях больных туберкулезом легких присутствуют протеазоустойчивые белковые и липидные микобактериальные антигены. Получены противотуберкулезные моноклональные антитела (МАТ), реагирующие с белками, участвующими в синтезе молибдоптерина. С помощью двух МАТ, распознающих разные эпитопы на одном антигене с молекулярной массой 32, 50 и 75 кДа, создан двухсайтовый ИФА типа«сэндвич», способный выявлять микобактериальный антиген в антигенных препаратах M. tuberculosis и в образцах мочи больных туберкулезом. Однако специфичность этого теста невысока, так при исследовании образцов мочи был выявлен антиген у половины здоровых доноров. Это связано с тем, что в урогенитальном тракте человека присутствуют некоторые условно патогенные микроорганизмы, относящиеся к нормальной микрофлоре, с которыми перекрестно реагируют исследуемые МАТ