Site-Directly Attenuated F. Tularensis Strains
Immunobiological Study of Attenuated Francisella tularensis Strains Producted by Site-Directed Mutagenesis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- BIO-SFS/Biosafety and BioSecurity/Biotechnology
3 Approved without Funding
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Centre de Recherches du Service de Sante des Armees, France, La Tronche\nLudwig-Maximilians-Universität / Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Germany, Munich
Project summaryTularemia is the zoonotic feral nidal specially dangerous infection caused by the gram-negative coccobacillus Francisella tularensis. The bacterium is widely distributed in nature and has been isolated from about 250 wildlife species, many of which can transmit disease to humans in a myriad of ways: direct contact with infected animals, ingestion of contaminated water, inhalation of aerosolized organisms or via vectors that include mosquitoes, ticks and flies (Morner, 1992; Abd et al., 2003). The high morbidity and mortality, its potential for aerosolization, its low infectious dose for a man and the ease of propagating the organism in vitro make this disease a potential agent of a biological weapon (El Sahly, 2009), and causes necessity of development of an effective vaccine.
In spite of the fact that existing vaccine strain F. tularensis subsp. holarctica 15 (Olsuf’ev et al., 1960), obtained in the 40th of the last century in Russia and used till now in terrain of the countries of the former USSR, and its derivative LVS as the result numerous passages through artificial nutrient mediums, used in the countries of Europe and Boreal America, has more than a semi-centennial history, there are still some problems which hamper its licensing. First, genetic analysis of LVS has revealed that it contains multiple genetic defects in comparison with clinical isolates, including both point mutations and large deletions (Rohmer et al., 2006). However the relative contributions of these inpidual defects to the attenuation of the strain, and the capacity for reversion remain unknown, that is strain it is not genetically determined. Second, the safety of the LVS vaccine in immunocompromised inpiduals has not been evaluated. Furthermore, the protection induced by LVS, or how it mitigates patho-physiological processes, are not well understood (Conlan et al., 2007). Third, the strain has circumscribed protectivity against high dose (more than 100 CFU) of respiratory challenge with strain F. tularensis subsp. tularensis (type А) and has high residual virulence for the person and animals due to respiratory method of vaccination (McCrumb, 1961, Hornik. & Eigelsbach,, 1966). Fourth, passage of this vaccine strain on peptone cysteine agar gave rise to two phenotypic variants distinguishable by their colony colour, blue or grey. Blue are capable to induce protective immunodefence at mice while grey do not possess such ability (Eigelsbach et al., 1961). During batch production of the vaccine (at cultivation in liquid nutrient medium) the blue variant could still give rise to the grey variant, which could represent as much as 20% of some preparations (Sandstrom, 1994). Finally, there are no easily measurable correlates of protection for LVS that could be used to demonstrate that vaccination elicited an immune response guaranteed to protect against a subsequent encounter F. tularensis.
Thus, the basic criteria for rational selection of perspective tularemia strains - base for development live tularemia vaccine - are precisely certain genetical characteristics, stability of inheritance of beneficial properties, ability to induce optimum protective immunodefence against F. tularensis.
The main goal of this project is study of immunobiological properties of the site-directly attenuated F. tularensis strains.
The tasks of the project include: search of genetic determinants, inactivation of which results in supression of pathogenic properties at conservation of immunogenic potential of F. tularensis; creation of a collection of F. tularensis avirulent immunogenic strains, obtained by site-directly inactivation of genes influencing pathogenicity; сonfirmation of the role of target genes by complementation method; studying of influence of recombination system on stability of obtained strains; evaluation of growth properties for obtained strains on solid and liquid nutrient mediums, stability to bactericidal action of normal rabbit serum, phagocytosis and persistence in organs of laboratory animals at different routes of introduction and in different terms after challenge; definition of cytotoxity of chosen strains (histological researches of organs of laboratory animals) at different routes of introduction of bacterial culture; investigation of immunogenic properties of obtained strains by a level humoral (antibody formation) and cell-mediated (subpopulation proliferation of T lymphocytes and synthesis of cytokines) immune responses; study of protective properties of perspective strains; selection of optimum variants of an immunization with avirulent immunogenic F. tularensis strains using various models of laboratory animals.
The technical approach and methodology will involve: the mutagenesis of F. tularensis genes will be carried out by method of allelic replacement with use of specially designed for this purpose vector pPV (I.Golovliov, A. Sjostedt, A.N. Mokrievich, V.M. Pavlov, 2003), transmitted into F. tularensis cells by conjugation method, and the vector pHV33′SacBΔB (the unpublished data), introduced there by transformation method; the complementation of inactivated genes will be carried out with use of integration vectors or low copied plasmid pHV33, that is stable hereditable in an autonomous state in F. tularensis cells; estimation of ability of obtained mutants to be associated and absorbed by macrophages and to survive in them on macrophage-like cells of line J774.1A and marrowy macrophages, determination of cell infection will be done by means of agar plate inoculation; estimation of persistence of bacterial cells in vivo on contamination of organs of laboratory animals, euthanized in different terms after challenge; determination of cytotoxity of strains at different routes of introduction by histological researches of organs of laboratory animals; evaluation of intensity of humoral immunity by immunoenzyme method; evaluation of intensity of cell-mediated immunity by quantity of CD4+ and CD8+ T-cells (using flow cytofluometry method), in RBTL test and on level of synthesis of interferon-γ, interleukin-2, interleukin-12 by T lymphocytes (using immunoenzyme method); estimation of ability of investigated strains to activate macrophages on level of synthesis of the tumor necrosis factor- by immunoenzyme method; study of protective properties of perspective strains and selection of optimum variants of immunization on murine model and model of Guinea pigs will be carried out using the vaccine strain no. 15 (Gaisky strain).
The expected results. The new data on genetic determinants which can serve as targets for genetic-engineering manipulations with the purpose of change of virulence at conservation of immunogenic properties will be obtained. The collection of avirulent immunogenic F. tularensis strains will be created and the functional role of inactivated genes will be confirmed. Use of genes of recombination system will allow to stabilize immunogenic properties of perspective strains and to exclude their possible reversion to wild type strain. The evaluation of growth properties on artificial nutrient mediums, a survival rate in biological fluids, multiplications in vitro and in vivo will be carried out. Study of cytotoxity of strains at different routes of introduction will be carried out. Immunological properties of strains (humoral and cell-mediated immunity, a level of synthesis of cytokines) will be tested. Protective properties of perspective strains will be investigated and variants of an immunization on various models of laboratory animals will be optimized. Strains obtained as a result of performance of the Project can form a base for creation of the live tularemia vaccine. Uniqueness and novelty of expected results consist in the fact that developed strains of F. tularensis will is stably avirulent and immunogenic. The results of the project including fundamental scientific data and applied methods will be published in peer-reviewed Russian and international scientific journals.
Implementation of this project will accord with ISTC goals and objectives: the outcome will be an essential contribution to development of basic biology and applied medical microbiology. Creation of potential vaccine F. tularensis strains having high protective properties will be the essential contribution to struggle against possible exhibitings bioterrorism, being of serious danger for the world community. Additionally, the project implementation will allow the scientists, engineers, and technicians of the Institute of Immunological Engineering previously engaged in studies in the area of biodefense to reorient the field of their scientific interests, to use their experience for solution of international basic and applied research problems, to create long-term prospects for fruitful collaboration in international commonwealth of learning.
Authors of the project have a good experience of research in the field of development of genetic methods of F. tularensis study (see section 2.1).
The role of the foreign collaborators/partners will include: taking part in elaboration of common research strategy and collaborative programs; regular project discussion by means of e-mail, fax, and phone; performance of joint investigations; training of specialists in specific areas according to collaborator competence, scientific missions for exchange of experience; making arrangements for meetings and seminars; participation in preparation of manuscripts of articles and scientific presentations.