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Immunoprobiotics on the basis of recombinant Lactobacillus strains

#1975


Development of Lactobacillus Strains, Synthesizing Tumor Necrosis Factor - ß, to Control Enteropathogens in Farm Animals

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-MIB/Microbiology/Biotechnology
  • AGR-VTH/Vaccines and Theraupetics/Agriculture

Status
8 Project completed

Registration date
22.08.2000

Completion date
21.03.2003

Senior Project Manager
Pobedimskaya D D

Leading Institute
Research Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov

Collaborators

  • US Department of Agriculture / Agricultural Research Center, USA, GA, Athens

Project summary

The objective of the project is to develop a new generation of recombinant, immunologically active, probiotic bacteria for the prevention and treatment of disrupted intestinal microflora in livestock and poultry.
Over the past number of years, the agricultural community has made animal production more efficient through larger scale operations. As a consequence, the intestinal microbial balance in food animals (poultry, swine, bovine, etc) on commercial farms has reflected a herd (flock) characteristic. In many cases, disrupted intestinal flora (disbacterioses) has become a key manifestation resulting in the colonization of the livestock and poultry with human pathological bacteria. This results in reduced production parameters of inpiduals, inhibition of their immune status, and development of infectious diseases. The number of factors involved in the expression of disbacterioses is enormous and the spectrum of these factors is constantly expanding. Development of effective means for the prevention and treatment of disbacterioses (of different etiological agents) is one of the major opportunities of modem veterinary science.
Development and application of bacterial preparations based on representatives of normal intestine microflora, known as probiotics, is one of the most promising approaches to counter and control disbacterioses. It is known that bacteria within the alimentary canal play a significant role in the maintenance of the health of host organism. In particular, the microflora makes a significant contribution by providing protection against infection and also participates in a number of metabolic processes by aiding in the digestive process. Artificial colonization of the intestine with bacteria capable of providing beneficial effect on the intestinal micro-ecology and improving the host animal’s health. This health benefit is accomplished because of the elimination or control of the pathogenic microorganisms, which can be present in enormous numbers, but probiotics also restore the balance and profile of a normal level of endogenous anaerobic bacteria.
A component consequence of the host harboring disrupted intestinal flora is the reduced immunological response together with development of secondary immune deficient states. The proposed probiotic additions would be important in the development of a new generation of ecologically harmless and preventive preparations capable of providing simultaneous correction of the disrupted microflora and stimulating the immune status of the host animal. The approach for accomplishing this task is possible because of developments in modern molecular biology.
Design of recombinant strains of bacteria may be achieved by cloning of genes encoding immunoregulating proteins into cells of normal intestinal microflora (for example, into lactobacillus). This novel approach in the creation of such Lactobacillus spp. strains creates a new potential by using these bacterial carriers for expression of immunomodulating heterologous antigens. The use of lactobacillus as carriers of foreign antigens had been constrained because of the lack of information about the genetics in these bacteria together with the absence of reliable cloning vector systems. However, recent investigations have revealed that Lactobacillus spp. possess plasmids originating from a wide spectrum of host bacteria and thus, can be convenient instruments for vector construction and gene cloning.
In cells of Lactobacillus spp., systems for efficient expression of desirable genes and export into the cytoplasm, subsequent binding to the bacterial surface or secretion of some proteins into the cultural medium is now possible. Such useful properties expressed by Lactobacillus spp. having intestinal colonizing ability and expressing high antagonistic activity against pathogenic bacteria that may be present would be enormous value. Immunomodulating activity and bacteriocin production expressed in such recombinant Lactobacillus spp. make them more desirable from an environmental safety perspective in comparison with attenuated strains of Salmonella, Klebsiella, Listeria and Mycobacteria. Even these attenuated organisms, which have traditionally been used as carriers of heterologous antigens have potential to revert into human virulent pathogens.
The use of natural immuno-mediators is suggested in selected Lactobacillus spp. In particular, tumor necrosis factor -ß (TNF-ß), which posses high immunoregulating activity would provide opportunity to increase such immunopotentiating activity of lactobacterium strains. At present, TNF-ß was found to regulate the formation of the peripheral lymph nodes, the Peyer's patches and spleen. TNF-ß expression causes activation of immune competent cells, directing the antibody producing cells to home into the host immune system. Subsequently, TNF-ß enables stimulation of the expression of 7-integrines and other adhesive molecules on the surface of the intestine and spleen lymphocytes, and induces the synthesis of a number of other endogenous cytokines, as has been experimentally proven.
Recently, novel plasmid vectors were developed at the RCT & HRB for transfer and expression of heterologous information into Lactobacillus spp. cells and other grampositive and gramnegative bacteria. A collection of Lactobacillus spp. isolated from animals, silage, and milk products has been established. The isolated strains were identified by polymerase chain reactions with genus specific primers. Lactobacillus plantarum strain BS916, and Lactobacillus sp. strain BS928 were isolated from com silage and from canned vegetables, respectively. Two plasmids (PBS196 and pBS197) from L. plantarum strain 195 were cloned and mapped. Distribution of these plasmids among L. plantarum strains, isolated from plant substrates (including L. plantarum strain BS916) was observed using DNA-DNA-hybridization. It was observed that plasmid pBS196 can be mobilized by conjugative plasmid pAM. Plasmid pBS196 is a good candidate for construction of vector systems.
Additionally, highly purified preparations of TNF-ß were produced at the RCT & HRB. Immunobiological properties of these preparations have been comprehensively studied. It was shown that the TNF-ß preparations are very effective in combination with probiotics used for decreasing disrupted intestinal microflora syndrome in farm animals. TNF-ß also was demonstrated to elevate the resistance of farm animals to bacterial and viral infections. Cloning of the TNF-ß gene in Lactobacillus cells allows for construction of novel recombinant strains. These strains will provide not only normalization of intestinal microflora, but also will prolong the cytokine effect for several days, which will ensure high stimulation of both local and general immunity. Novel immunoprobiotics are to be developed based on the constructed strains.
The proposed project involves the selection of Lactobacillus spp. strains, which should serve as carriers of desired heterologous antigens. Vector plasmids capable of being transferred and expressed in Lactobacillus spp will be constructed. Recombinant plasmids bearing the TNF-ß gene and new recombinant Lactobacillus spp. strains secreting TNF-ß will be obtained. In order to stabilize the expression of TNF-ß gene in Lactobacillus spp. cells, the gene will be inserted into the Lactobacillus spp. chromosome. The expression of TNF-ß gene will be determined following this genetic manipulation. Cultural, biochemical and physiological properties of the constructed recombinant strains will be evaluated. Pharmacodynamics and immunobiological properties of the new strains will be evaluated in vivo employing chicken challenge studies. The antagonistic activity to pathogenic bacteria will be studied in these chicken challenge studies. Toxicological studies of recombinant Lactobacillus spp. strains will be carried out on lab animals. The most promising strains will be used to develop immunoprobiotics. Methods of producing these recombinant probiotics will be optimized (including cultural conditions, lyophilization and storage). The development of the recombinant immunoprobiotics will be tested under appropriate biosecure facilities both at the ARS cooperator’s laboratory and in Russia.
After successful results are obtained, patent rights will be sought, and licensing of the intellectual property will be obtained with an appropriate business interest. Efficiency of the immunoprobiotic application will be tested after obtaining FDA approval for commercialization. Following these above steps, extension of the novel treatment will be studied by cooperating with ARS to identify pertinent antigens expressed by human pathogens involved in livestock and poultry colonization. The results obtained can be used for developing industrial technology in the production of novel immunoprobiotics. These will find application in practical veterinary medicine for controlling human enteropathogens in livestock and poultry.
This proposal provides a new approach in the construction of immunoprobiotics, and involves the cloning of selected cytokine controlling genes in Lactobacillus spp. The use of Lactobacillus spp. as carriers of heterologous antigens expands the list of candidates of bacterial vectors, permits substantial insight into the mechanisms underlying genetic control and expression of the antigens in the given bacteria. The factors will be studied, which influence these processes. The results obtained shall open a wide avenue for the use of live Lactobacillus spp. as vectors for cloning many protective antigens, and development on their basis new preventive and therapeutic formulations.
The proposed project meets the objectives of ISTC to engage highly skilled weapons scientists and engineers as well as to use the available in RCT&HRB laboratory facilities and lab ware for developing studies about regulating the immune response of livestock and poultry to human pathogens. The proposal will result in creating a new generation of probiotic vaccines having wide applications in veterinary medicine.
The USDA-ARS collaborators will make contributions to the project by discussing various technical aspects of the project and participate in the planning for its success. The ARS would be involved in overseeing the progress of the project and would be involved in live animal challenge studies. Further, as identification of pertinent antigens is determined, the ARS would work cooperatively with the Principal Investigator to express these in the selected Lactobacillus spp. Information exchange would be ongoing during the course of the project. The USDA-ARS would participate in symposia and workshop meetings, would assist in publication of resulting papers, would provide patent and information support, and would assist and support project participants to participate in international meetings.
The objectives of the project are:
1- to construct a plasmid vector bearing genes encoding for TNF-ß. This vector must be capable of being transferred and expressed in the Lactobacillus spp. cells; to develop techniques for genetic transfer of the recombinant plasmid into Lactobacillus spp. and construct recombinant strains of Lactobacillus spp. synthesizing TNF-ß;
2- to study pharmacodynamics, antagonistic activity and immunobiological properties of recombinant strains of Lactobacillus spp. synthesizing TNF-ß; to carry out the toxicological studies of recombinant strains on lab animals; to develop novel immunoprobiotics on the base of TNF-ß synthesizing Lactobacillus spp. strains and to elaborate optimal methods for their application.


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