Brucella Vaccine Strains Genotyping
Comparative Molecular-Genetic and Immunochemical Characterization of Brucella Vaccine Strains
Tech Area / Field
- AGR-VTH/Vaccines and Theraupetics/Agriculture
- AGR-DIS/Disease Surveillance/Agriculture
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Federal Centre of Toxicological and Radiation Safety of Animals, Russia, Tatarstan, Kazan
Project summaryBrucellosis, which is recorded in many countries of the world, affects various species of wild and agricultural animals, noticeably cripples wild fauna and animal breeding, as well as constitutes a considerable menace for human health. For example, the employees of the yellowstone national Park in the USA come to bat of brucellosis in bisons. yellowstone bisons are the last remaining reservoirs of bovine brucellosis in the United States. Paired cultural and serological tests show about 50% of all seropositive bison, and almost 70% of higher titer bison cows, have detectable infection. The risk of brucellosis transmission to domestic animal is great. Thereby the goal of this project is development of the strategy of prevention and prophylaxis of brucellosis in bisons of Yellowstone National Park.
The absence of reliable and secure means for vaccine prophylaxis of the disease in this animal species predetermines the necessity of a comprehensive comparative estimation of the efficiency of the actual brucellosis vaccines on purpose to determine their ability to prevent brucellosis in bisons. The compulsory preliminary characterization of the Brucella abortus (see p. 24) vaccine strains under study aimed in inpidualizing their phenotypic and genotypic differences should be the first stage of such comparative research. on the chance of establishing of the identity of some vaccine strains at this stage of our investigations it will be reasonable to raise a question about the expediency of exception of a part of the vaccines from further examination. The minority of vaccine strains will allow shrinking the number of animal, which is planed to be used at the phases of vaccines' potency testing.
The genus Brucella is pided into six species B. melitensis, B. abortus, B. suis, B. canis, B. ovis, B. neotomae. Traditionally distinction between species and biovars is performed by differential tests based on phenotypic characterization of lipopolysaccharide antigens, phage typing, dye susceptibility, СО2 requirement, H2S production and other metabolic properties. By an analysis of approximately 25 phenotypic characteristics the different Brucella strains can be further pided into 19 biovars. Differentiation of inpidual strains below the biovar level has so far been impossible.
Presently various techniques, namely DNA-DNA hybridization, definition of a restriction fragment-length polymorphism by pulsed-field gel electrophoresis, various modification of PCR, other methods for amplification of target genes and, at last, complete genome sequencing, are used for examination of the features of the Brucella genetic organization [Allardet-Servent A. et al., 1988; Grimont F. et al., 1992; Cloeckaert A. et al., 1995, 1996; Ficht T.A. et al., 1990, 1996; Halling S.M. et al., 1990; Verger J.M. et al., 1998; Fekete A. et al., 1992; Bricker B.J. et al., 1994; Ouahranibettashe S. et al., 1996; Tcherneva A. et al., 1996, 2000]. The majority of the used genetic techniques allows to determine species and intraspecies polymorphism of Brucella, and some of them can fetch out even strain dissimilarities.
The authors of the project had an experience in indication, identification and genotyping of Brucella by definition of a restriction fragment-length polymorphism, DNA-DNA hybridization and PCR techniques [Faizov T.Kh. et al., 1989; Ryskov A.P. et al., 1990; Faizov T.Kh. et al., 1998; Dentovskaya S.V., 2000; Dentovskaya S.V., Kulichenko A.N., 1999, 2000; Sharova I.N. et al., 2000].
For the first described in 1995 [Schena M et al., 1995], DNA microarray methods have already made a marked influence on many fields, including cellular physiology [Spellman P.T. et al., 1998], cancer biology [Khan J. et al., 1998], and pharmacology [Marton M.J. et al., 1998]. The first results of gene expression profiling of the host-pathogen interaction have just begun to emerge.
Development of a whole-genome DNA microarray for a fully sequenced microorganism is conceptually simple. Effective microbial gene-finding programs can quickly and accurately predict most ORFs [Delcher A.L. et al., 1999]. DNA fragments representing each of the genes can be obtained by PCR amplification that uses ORF-specific oligonucleotides. Homology-searching algorithms may be used to choose regions of genes that will not cross-hybridize with other regions of the genome. After a simple purification step, PCR fragments will be arrayed by a robotic arrayer. This powerfull approach was used to construct a 4,290-ORF E. coli microarray [Tao H. et al., 1999] and a 3,834-ORF Mycobacterium tuberculosis microarray [Wilson M. et al., 1999] and others.
Live vaccines inducing high protection have lag number negative consequences connected with infection of macrophages and replication of bacterial cells within host that in any case is undesirable for host (Kunkle R.A. et al., 1995; Noskov A.N. et al., 1995; Wergifosse P. et al., 1995). New methods of priming the specific cytotoxic lymphocytes can be of great importance for the development of vaccines against various intracellular pathogens.
The matter of fact is that the structural basis of T cell immune recognition is now well understood. CD4+ and CD8+ T cells possess clonally distributed receptors (abTCR) which are specific for complex of peptides derived from protein antigens and specifically bound to class I or II MHC molecules (Rock K. et al., 1996). The reason for the existence of two distinct classes of MHC molecules, both of which possess the peptide binding activity and are recognized by abTCR, appears to relate to different function of CD4+ and CD8+ T cells. CD4+ T cells interact productively with B cells, macrophages, and dendritic cells that primarily acquire a multiplicity of antigenic proteins from the external milieu. These proteins do not necessarily constitute markers for active infection of the antigen presentation cells (APCs), and the results T cell effector function is not constrained to the APC. In contrast, CD8+ cells or cytotoxic T lymphocytes (CTLs) are specialized for destruction of cells that have been infected or undergone malignant transformation, where the presence of a particular protein marks the cell for death before mature virus, intracellular bacteria or further tissue invasion occurs. For activation CTLs it is necessary that the presented protein antigen has translocated into cytosol of АРС and was presentation to class I MHC molecules. Macrophages infected, e.g. with Brucella, might be a good target for cytotoxic lymphocytes (CTL), if CTL were induced, and abTCR complexes recognizing molecules MHC-I along with Brucella CTL-peptides were generated at their surfaces. In order to stimulate CTL, we have used a vehicle involving nontoxic components of Anthrax Toxin: Protective Antigen (PA) and associative domen of Lethal Factor (LFn).
The main tasks of proposed project are:
1. Comparative molecular-genetic and immunochemical characterization of Brucella abortus vaccine strains.
– to define phenotypic characteristics of the Brucella vaccine strains under study;
– to define restriction fragment-length polymorphism of the vaccine strains of Brucella by means of DNA-DNA hybridization and pulsed-field gel electrophoresis;
– to conduct genotyping of the Brucella vaccine strains by means of various modifications of PCR, permitting to fetch out differences between single strains (REP-PCR, ERIC-PCR, AP-PCR, IS-an-PCR);
– to determine molecular-genetic characteristics of the Brucella vaccine strains by the use of AFLP markers;
– to test a possibility of usage of the persity in variable-number tandem repeats of genome DNA for genotyping of the Brucella vaccine strains;
– to estimate a degree of genetic persity of the Brucella vaccine strains;
– to estimate a degree of possible persity of expression of the representative range of genes of the Brucella vaccine strains.
2. Development Brucella molecular vaccine (BMV) preparations for prophylaxis and treatment of brucellosis in cattle.
– encapsulation of BMV preparation in microspheres/gelatin capsule which not only protects the protein from degradation but which also allows sustained release of the protein in the vaccine, thus reducing the need for big boosting. As BMV preparations will be used fused proteins consisting from BCSP31/Omp25 and associative domen Lethal Factor of Anthrax toxin;
– testing of prepared preparations in murine and guinea models at peroral administration;
– construction of new fused proteins consisting from L7/L12 Brucella ribosomal protein and associative domen LF for delivery to MHC I;
– determination of CTL peptides for Omp25 by means peptide-panel (synthesis of 22 peptides and testing of CTL lysis of target-cells obtained by synthetic peptides) as peptide component for BMV;
– development cell model (macrophages/macrophage-like cell lines J774.A or RAW264.7) of experimental brucellosis with using B. abortus strains S19, RB51 and 82;
– studying the protective properties of BMV on the guinea pigs model.
The project realization will be an essential contribution into development of applied biology and veterinary medicine as well as into solution of significant social problem, control for brucellosis, being of serious danger for bisons in the yellowstone national Park in the USA. Moreover, performance of the project will allow the scientists and engineers of the Stare Research Center for Applied Microbiology and All-Russian Research Veterinarian Institute, engaged previously in studies in the area of biodefense, to reorientate the circle of their scientific interests and to use the gained experience to carry out fundamental and applied studies, connected with solution of international scientific and technical problems in the fields of biology and veterinary medicine, to create long-term prospects for fruitful activity within the framework of International Scientific Association. These issues correspond well to the task of ISTC.