Novel approaches to the development of L-vaccines against TB
Project Status: 3 Approved without Funding
Duration in months: 36 months
Objective
Project objective. Development of a new preparation for prophylaxis of latent TB infection in cattle. Current situation in the given field of research. Laboratory specialists have been studying the problem of animal tuberculosis since 1976 year. During this time it had been developed various diagnostic, preventive preparations for large livestock farms. It is necessary to develop a vaccine from L-form mycobacteria and use it for the prevention of tuberculosis in successful and unsuccessful with TB economic entities, where it is impossible to treat the cattle through systematic allergic researches.
Preparation of a vaccine from L-form mycobacteria is relevant. Application of this vaccine will reveal one of possible pathogenetic mechanisms of endogenous infection and allow to identify specific ways of further struggle with the disease. Development of a technology for producing L-form vaccine is important for differentiation of postvaccinal reactions. The given vaccine would not pose a risk of development of infection in an animal that was observed when using a live vaccine. Studies on the development of L- vaccine against tuberculosis will be conducted for the first time. Impact of the proposed project on the progress in this field.
A new anti-tuberculosis preparation will make it possible to use it as a highly effective prophylactic mean. Competence of the project participants in this field.
Specialists of the Microbiology Laboratory are engaged in the development, introduction and production of diagnostic, preventive medications to protect animals from tuberculosis, as well as develop express-methods for laboratory diagnosis of tuberculosis. Expected results and their future application. At successful completion of the project it will be developed a new preparation for prophylaxis of latent TB infection of cattle in successful and unsuccessful with TB economic entities, where systematic allergic studies are inefficient. Project relevance to the ISTC goals. Despite of project participants with the knowledge in the field of weapons of mass destruction the given Project has only peaceful purposes thereby relevant to the ISTC goals. Also it is planned to involve scientists from the participant organizations in the international scientific community by providing information on the project at international conferences and seminars. Data on the volume of work. It is planned to perform the following works under the Project:
- it will be selected L-cultures of Mycobacterium tuberculosis and studied culture-morphological, biochemical, biological and molecular genetic properties.
- it will be worked out methods of accumulation of bacterial mass for the vaccine development;
- it will be worked out technological parameters of L- vaccine production;
- it will be constructed an experimental preparation
- it will be worked out doses and studied protective properties of an experimental vaccine in laboratory animals and compared with the analogue in in vivo experiments on laboratory animals for further application as a preventive anti-TB preparation. The role of foreign collaborators. Scientific advice, scientific exchange, coordination of joint actions, joint seminars (including working seminars), meetings, analysis of technical reports, joint use of rare materials and samples, joint or parallel studies, discussion of information received, verification of results using independent methods or technical means, joint publications and execution of intellectual property rights. To implement the above, and in case of conferences on similar topics it is planned trips to the CIS and abroad countries. Technical approaches and methodology. The project will include the following stages:
For the researches will be used mycobacteria culture - M. bovis-8, isolated from pathological material in previous years.
At the beginning of the experiment it will be conducted full biological control of this culture in order to confirm its initial typical properties (culture and morphological property, biochemical property, PCR method and biological sample).
After refreshment, the culture will be inoculated on Lowenstein-Jensen’s egg medium, and then will be adapted to Pavlov’s nutrient medium and only three passages an obtained film of bacterial mass will be reinoculated on synthetic medium of Soton.
Translation of vegetative tuberculosis culture M. bovis-8 into L-form with the purpose of accumulation of bacterial mass in vitro will be carried out on a nutrient medium to produce Mycobacterium tuberculosis spheroplasts.
Main properties of mycobacterial L-forms will be studied by determination of nucleotide sequences of certain regions in L-form M. bovis genome, encoding protective antigens. The given experiment will be performed using polymerase chain reaction (PCR) using specific primers that allow to detect the presence of genetic markers specific to mycobacteria. The primer will be constructed using different computer programs as Oligo 6 and Vector NTI Suite 9. Specific DNA regions will be accumulated in a thermal cycler GeneAmpPCR 9700, AppliedBiosystems and TC-512 of the company Techne.
DNA will be subjected to dideoxy sequencing using terminating dideoxy nucleotides (according to Sanger’s method) on an automatic 16-capillary sequencer GeneticAnalyser 3130 xl, AppliedBiosystems. POP-7 will be used as a polymer for capillary.
Terminating DNA products will be worked out by cycle sequencing. Purification of DNA from unbound dyes will be carried out by gel filtration on Centri-Sep column or CleanSeqReagent according to the instructions supplied with the kit.
After study of properties of M. bovis L-forms it will be constructed an experimental preparation for further application as anti-TB preparation.
Efficacy of the preparation will be assessed in the in vivo experiments on laboratory animals infected with a suspension of virulent culture of Mycobacterium tuberculosis. Pathological and bacteriological examinations of internal organs of infected animals will show the preparation’s efficacy.
