Recombinant Brucellosis Vaccine
Recombinant Brucellosis Vaccine Development Using Bacterial Salmonella Vector Expressing Brucella Protective Antigens
Tech Area / Field
- AGR-VTH/Vaccines and Theraupetics/Agriculture
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- Chiron S.r.l., Italy, Siena\nTexas A&M University / College of Veterinary Medicine, USA, TX, College Station\nUnited States Department of Agriculture / Agricultural Research Service / National Animal Disease Center, USA, IA, Ames
Project summaryLive vaccines on the basis of attenuated Brucella abortus RB51 and 19 (in USA), B.abortus 82, 82pn and 75/79 (in Russia) are currently used to control brucellosis in animals. Live bacterial vaccines are an effective vehicle to generate protective immunity against intracellular parasites. However, the vaccines fail to provide 100% protection against brucellosis. Moreover, the vaccines are not safe enough for humans and can be infectious after vaccination. These vaccines have been documented not infrequently to induce mild brucellosis with allergic complications.
Thus, development of an effective and safe vaccine against brucellosis remains a priority. Studies on the construction of new vaccines and improvement of available vaccines to control brucellosis in humans and animals, including rare species such as American bisons, are in progress. There are several strategies to improve the available brucellosis vaccines, or to develop new vaccines, such as development of live vaccines modified with protective antigens, DNA-vaccines and other approaches. It was shown that strengthening of the efficiency of the vaccines can be achieved by increasing expression level of protective antigens of Brucella vaccine strains, e.g. B.abortus RB51.
Cu/Zn superoxide dismutase, L7/L12 ribosomal protein and a number of other antigens, including major proteins of the outer membranes (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1 and others) are very attractive as protective antigens. However, attempts to develop a sufficiently effective vaccine on the base of these antigens have been failed despite the fact that these antigens possess expressed variable protective properties. Brucella escape from the immune response of the host and have their own strategy to survive in macrophages. This strategy implies that coordinated expression of various pathogenicity factors allows the bacteria to successfully overcome the macrophage barrier. Systematic study of these factors localized on the cell membrane as well as the study of the secretory proteins may solve some of the problems related to the evaluation of vaccine strains and to the development of new vaccines.
Since the host immune system responds to the products of expression and translation of various genes of the microorganism, it seems reasonable to use methods of analysis of prokaryotic cell proteome for the assay of these complicated protein patterns. Identification of proteins on 2D-electrophoregrams by using N-terminal microsequencing and mass-spectrometry will undoubtedly make the investigations more complex and expensive. However, for comparative analysis of dimeric maps of Brucella vaccine strains, it is not necessary to perform sequencing of all identified proteins. It is more informative to study their immunologic properties. Therefore, in early stages of the project, it seems reasonable to identify and isolate a large group of membrane proteins of each Brucella vaccine strain, to study their immunological properties and to determine which antigens are the most significant for designing of new subunit and recombinant vaccines.
For the construction of these vaccines, it is important to not only select optimum protective antigens but also to identify an efficient delivery system which will provide antigen presentation. This delivery system could be either a molecular vector for target delivery of protective factors to the cytosol, or an attenuated bacterial strain having the capacity to survive in phagocytes is similar to Brucella strains. One of the attenuated Salmonella strains could be used. Salmonella, as bacterial vectors, have a number of advantages in comparison with other intracellular parasites. They can be used both as peroral and parenteral vaccines; they induce both local and systemic immunity, including production of serum antibodies and secretory immunoglobulins; they can also generate cellular immunity and antibody-dependent cytotoxicity. There is a large body of experimental and clinical data to confirm the safety of Salmonella strains. Methods for genetic manipulation with Salmonella are well-known: plasmid vectors constructed for E.coli are usually appropriate for Salmonellas; there are methods for stabilization of plasmids and methods for transferring of plasmid DNA into the chromosome. There are plenty of experimental data on immunization of lab animals with attenuated Salmonella expressing foreign antigens. These data provide evidence that it is possible to develop vaccines to protect vaccinated animals from infection. Presently many heterologous antigens have been expressed and characterized in Salmonella vaccine strains.
Construction of attenuated Salmonella strains expressing Brucella immunogenic proteins for the development of vaccines against brucellosis in animals is the objective of the proposed project.
Attenuated Salmonella strains used in Russia as vaccines against salmonellosis in domestic farm animals will be used as bacterial vectors. Safety of these bacterial strains has been proven by vaccination of millions of farm animals. In addition other attenuated strains, including those identified by the authors of the project will be used.
The duration of the project is three years. The experiments will be performed with three major aims:
– construction of recombinant Salmonella strains expressing Brucella protein antigens (L7/L12, Omp25, Omp31); studies on the immune response to the Brucella protein antigens delivered into the gut associated lymphoid tissue by a bacterial vector;
– studies on membrane and secretory sub-proteomes of vaccine strains of Brucella (new Brucella antigens with promise for the use in recombinant vectors will be selected);
– determination of significant immunological criteria for the prediction and evaluation of protective properties of Brucella vaccines.
Investigations in these three approaches will brucellosis vaccines delivering one or several protective Brucella antigens by Salmonella vectors. Safety and accessibility of the Salmonella vector will allow for an integrated immunological investigation of recombinant vaccines and for selection of immunological criteria to predict protection. Oral immunization is also more similar to that of natural infection.
The following results are expected:
– development of plasmid vectors for transfer and expression of Brucella protective antigens in Salmonella;
– construction of genetic structures and Salmonella recombinant strains bearing the genes of Brucella protein antigens (L7/L12, Omp25, Omp31 and others isolated and characterized within the project);
– production of anti-brucellosis sera and diagnostic reagents for detection of Brucella protective antigens expressed in Salmonella;
– identification of native membrane and secretory proteins of Brucella vaccine strains (with post-translation modifications) as standards for proteomic analysis and immunological tests;
– development of 2D-reference maps of membrane and secretory proteins of Brucella vaccines strains; comparative evaluation of representative and quantitative characteristics of biosynthesis in various vaccine strains;
– definition of immunological criteria for evaluation of protectivity of Brucella antigens (protein preparations and recombinant strains);
– generation of experimental data on the studies of immune response to Brucella protein antigens delivered into gut associated lymphoid tissue by attenuated Salmonella strains;
– development of laboratory examples of recombinant brucellosis vaccines on the basis of attenuated Salmonella strain expression of Brucella protein antigens.
Specialists with long experience in the field of genetic engineering and molecular-biological research and skills in designing attenuated strains and recombinant vaccines will be involved in the project.
The present research meets ISTC objectives by providing opportunities to specialists engaged previously in the development of biological protection to redirect their skills to solving basic and applied tasks in the fields of veterinary medicine and microbiology. The project will promote integration of Russian scientists into the world scientific community. Results obtained by the proposed project will promote the solution of an important problem of national and international veterinary and public health significance, namely control of brucellosis.