Chemical Tularemia Vaccine
Development of a Chemical Tularemia Vaccine and Studying F. Tularensis Outer Membrane Components Responsible for Protective Immunity Formation
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Gamalei Institute of Epidemiology and Microbiology, Russia, Moscow\nState Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- FOA National Defence Research Establishment / Department of NBC Defence, Sweden, Umeä
Project summaryTularemia is a zoonotic bacterial disease, which occurs epidemically in many countries in the Northern hemisphere. It is caused by very small Gram-negative coccobacteria with diameter of 0,2-0,5 mm. Francisella tularensis occurs in two main biovars. Highly virulent in humans and rabbits F.tularensis boivar tularensis (type A) is a dominant biovar in North America. F. tularensis biovar palaearctica (type B) occurs in Asia, Europe and is less spread in North America. F. tularensis has no serologic subspecies. The pathogenic S-form is a natural form of F. tularensis occurence in natural conditions. S-cells of the virulent strains are covered with capsules. Lipopolysaccharide (LPS) is one of the main components of the capsules.
F. tularensis belongs to facultative intracellular pathogens, which live in mononuclear phagocytes and other cells and cause granulomatous diseases. Survival rate in phagocytes is one of the key characteristic features of intracellular bacteria. At present F. tularensis is regarded as a model micro-organism when studying mechanisms of immunity formation and determining strategy for development of subcellular vaccines against intracellular pathogens.
One of the main achievements in tularemia protection is, certainly, development of live tularemia vaccines (LTV), application of which allows one to decrease tularemia rate to sporadic or group outbreaks. At the same time, LTV, as well as any other live vaccine, has some shortcomings and the principle one is introduction of foreign genetic information to humans and long persistence of the vaccine strain. One of the directions with good prospects in the field of improvement and development of different ways of efficient protection from tularemia agent is development of a new generation of immunoprotective preparations on the basis of protective antigenic components of tularemia bacteria. But, the problems we face in developing subcellular tularemia vaccines as well as efficient molecular vaccines against other intracellular bacteria (tuberculosis, plague, lepra agents) remain to be unsolved and are of great interest for infectious immunology. Studying F. tularensis bacteria for many years has not resulted in revealing pathogenicity factors and elucidating the processes of intracellular parasitism in detail. Besides, until now, the question on the protective component of bacteria and the nature of the protective immunity in tularemia still remains in dispute.
The leading role in forming resistance to tularemia belongs to cell-mediated immune reactions. The main antigens, recognised by specific T-lymphocytes, are F. tularensis outer membrane (OM) proteins. However, application of only T-cell-reactive OM proteins for vaccination of animals did not lead to the expected results. A small protective effect was also manifested when adjuvants (ISCOMs) were combined with F. tularensis OM proteins.
Possibly, regulation of early cytokine expression is the course of low induction of the protective effect, when only OM proteins are used. This idea is supported by the studies, which show that in mice early expression of IL-12, TNF-a and INF-g (the most important stimulators of T-cell differentiation along Th1-pathway) is induced by alive tularemia bacteria, but not isolated agents. Unlike OM proteins, LPC is not capable of stimulating T-lymphocytes, but at the same time it is one of the main carbohydrate polymers of the cell wall. LPS efficiently interacts with antibodies, which are present in sera of patients infected or vaccinated with live tularemia vaccine. Despite the fact that F. tularensis PLS has no toxic properties, typical for endotoxins of Gram-negative bacteria, and does not protect laboratory animals, challenged with virulent strains, from death, it is one of the key antigens participating in forming immunity against tularemia. The significance of LPS for successful vaccination against tularemia was supported by experiments using OM preparations and lipopolysaccharide protein (LPS-protein) complexes. A single subcutaneous immunisation of guinea pigs with OM preparations protected 60-100% of the animals for a month after their challenge with virulent F. tularensis strains (type B). The level of specific protection achieved using different series of OM preparations was comparable to that induced by LTV.
Ideas about mechanisms of forming immunity against intracellular bacteria and data obtained on protective properties of OM preparations and LPS-protein complexes of F. tularensis using experiments form the basis for this Project.
In the course of Project execution we expect to obtain laboratory samples of a chemical tularemia vaccine, immunisation with which would protect laboratory animals from subcutaneous challenge with virulent F. tularensis strains (type B) in a dose of 100 lethal doses. We shall try to modify the immune response in order to channel it along Th1-pathway and, at the same time, to stimulate the active proliferation of long-term memory cells. To stimulate T-cell response along Th1-pathway we are going to use cytokines. IL-12, the presence of which is necessary at the early stages of the process leading to differentiation of T-cells along Th1-pathway, can be one of them. We plan to study the role of INF-a and other cytokine analogs in the stimulation of long-term memory cells when forming tularemia immunity.
Project Objective: To study F. tularensis OM components, responsible for forming protective immunity and to develop approaches to obtaining a chemical tularemia vaccine based on OM preparations and a molecular vaccine on the basis of LPS-protein complexes.
Main tasks to solve the above problem:
1. To obtain laboratory samples of the chemical tularemia vaccine on the basis of F. tularensis outer membranes and evaluate the protective activity of different vaccine samples.
2. To determine the possible application of the LPS-protein complex (LPS-recombinant 17 kDa protein) for development of a molecular tularemia vaccine.
3. To characterise the protective immune host response to inoculation with preparations of the chemical vaccine.
a) To study the contribution of T-cell subpopulations to the acquired immunity.
b) To study cytokine expression in response to the administration of the chemical tularemia vaccine.
c) To determine phenotypic and functional characteristics of the cells formed once more in response to the administration of the chemical tularemia vaccine.
d) To determine the expression of T-cell receptors in the T-cells formed once more in response to inoculation with the chemical tularemia vaccine.
4. To modify the immune response using natural immunomodulators (IL-12, INF-g, INF-a) - inductors of Th1-response and formation of memory T-cells.
Results of theoretical value
New data, obtained in the course of the Project execution, about the nature and efficiency of the tularemia protective immunity, obtained using subcellular vaccines, will be of great interest for infectious immunology and improvement of means for specific protection against intracellular bacteria.
Results of practical value
We plan to obtain laboratory samples of the chemical tularemia vaccine, immunisation with which would protect laboratory animals from subcutaneous challenge with virulent strains (type B) of F. tularensis in doses of 100 lethal doses.
Results of commercial value
· production technology and composition of tularemia vaccines;
· recombinant strain producing 17 kDa protein of F. tularensis.
Technical Approach and Methodology
To solve the problems set in the Project, it is planned to use a complex of modern methods of microbiology, immunology, molecular biology, gene engineering, hybridoma technology.
In this work we plan to use virulent and vaccine F. tularensis strains. Bacteria will be cultivated on solid and liquid media. Outer membranes, LPS, proteins, LPS-protein complexes will be isolated using centrifugation, gel filtration, ion-exchange chromatography, ultrafiltration. F. tularensis antigens will be characterised by immunochemistry, immunoblotting, polyacrilamide gel electrophoresis, ultraviolet spectroscopy, circular dichroism, infrared spectroscopy. When obtaining the strain producing recombinant 17 kDa protein we shall use methods of gene engineering and oligonucleotide synthesis. The effect of subcellular tularemia vaccines on the immune system will be studied using cellular immunology methods. PCR method will be used for determination of cytokine mRNA. To determine the cell phenotype in response to the inoculation with the vaccine we plan to apply flow cytometry. The immune response will be modified using natural immunomodulators (IL-12, INF-g, INF-a, prothymosin-a) in increasing doses.
International co-operation under the project
In order to establish new contacts and for further integration into the international scientific collaboration network, the following activities will be scheduled within the framework of this project:
· elaboration of common research strategy and collaborative programmes;
· exchange of information and materials required for successful research;
· training of specialists in specific areas of partner competence, scientific missions for exchange of experience;
· arrangement for meetings and seminars with participation of collaborators;
· elaboration of an assistance programme for Russian partners (consultations, advice, preparation of publications, provision with computer programmes for modelling of molecular structures etc.);
· definition of new research projects and search for potential foreign partners through collaborators.
We have established partnership with Prof. G. Sandström (Department of Infectious Diseases, University of Umeä, S-901 85 Umeä, Sweden), who has been dealing with tularemia problems for a long time and will have an interest in carrying out joint studies under the Project. Besides, we have got scientific contacts with Prof. Ioshiro Ohara (Department of Microbiology, Kanazawa Medical University Uchinado-machi, Kahoku-gun Ishikawa 920-02 Japan), Dr. C. Tram (Institute Pasterus, Unit de bactriology molculaire et mdicale, France), Dr. D.M.Waag (U.S. Army Medical Research Institute of Infectious Diseases Fort Detrick, Federick, Maryland 21702-5011 USA), who are able to get interested in the execution of this Project and to become its collaborators.
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