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Construction of Recombinant Plasmids

#1466


Construction of Recombinant Plasmids Coding Synthesis of Pathogenic Microorganism Protective Antigens and Study of Their Properties

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology

Status
3 Approved without Funding

Registration date
22.02.1999

Leading Institute
State Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk

Collaborators

  • DSTL, UK, Porton Down

Project summary

Development of recombinant vaccine F.tularensis strains synthesising protective antigens of Y.pestis, B.antracis and, B.malei, and testing of protective properties of the strains constructed against experimental plague, antrax and glanders are the major objectives of the project.

The problems on a choice of bacterial vectors for designing live recombinant vaccines are now widely discussed in the literature. Vaccine ВСG and attenuated strains of Salmonellae are the most frequently applied as carriers of heterologous protective antigens [Cardenas L., 1993, Stover C., 1994]. Our proposal to use F.tularensis vaccine strain as a carrier of foreign protective antigens expands the list of possible bacterial vectors, permits to get deeper insight into the mechanisms underlying the formation of protective immunity and to study factors influencing this process.

One of the advantages of tularemia vaccine is the fact, that being moderately reactogenic, it causes an intensive reorganization of humoral and cellular parts of the immune system, providing thus the generation of strong long-term immunity [Tarnvik A., 1989, Sandstrom G., 1994]. F.tularensis strain 15/10, on the basis of which tularemia vaccine has been created, is avirulent for humans and exhibits a reduced virulence for small-sized rods [Olsufiev N.G., 1975]. In USA and countries of western Europe F.tularensis LVS, obtained by the method of selection from vaccine F.tularensis strain 15 is used as a vaccine [Eigelsbach H.T., 1961]. The choice of this microorganism is related to experimental - methodical advantages of it in comparison with other vaccine strains of intracellular pathogens, for example, BCG. Tularemia microbe has rather high growth rate in vivo and in vitro. A simple experimental mouse model of the infection caused has been developed. Cell mediated immune response to F.tularensis LVS is highly specific and consequently is convenient for the study of protective immunity [Conlan J.W., 1994].

Intracellular multiplication of F.tularensis LVS makes it attractive as a model object for the study of intracellular parasitism [Tarnvik A., 1992]. Experiments performed in the 70s have shown that macrophages play the most important role in protection from tularemia infection [Burke D.S., 1977]. The protection of a macroorganism from intracellular parasites depends, mainly, on the ability of T-cells to strengthen the antimicrobe activity of macrophages [Elkins K., 1993]. Thus, it was shown, that peritoneal macrophages of mice are capable of killing the absorbed F.tularensis LVS cells only after addition of gamma-interferone or cytokine, secreted by T-cells [Leiby D., 1992]. It is known, that for effective antibacterial protection of the organism,T-cells should be differentiated into Th1 cell line [Trinchieri G., 1993]. This event occurs in patients infected by tularemia microbe. Thu, CD4 and CD8 cells from patients survived tularemia display Th1 response to various membrane proteins [Sjostedt A., 1990]. Experiments on mice showed also that CD4 and CD8 T-cells play the key role in protection from tularemia. The both cell subspecies thus produce gamma-INF, alpha-TNF, IL-10 and IL-12, playing an important role in protection of the organism from intacellular pathogens. Moreover it was found that these cytokines occur in the liver in two days after subcutaneous infection with LVS strain, and the killed LVS cells do not demonstrate such an activity [Golovliov I., 1995].

Successful experiments on the expression of gene coding the synthesis of L.pneumophilla OmpS protein F.tularensis cells have allowed to develop recombinant F.tularensis strain synthesising L.pneumophilla protective antigen. The obtained variant of vaccine tularemia strain was found to be capable of persisting in the organism of experimental animals preserving the ability to synthesise the foreign protective antigen. This example permits to hope for positive result in case of designing recombinant F.tularensis strains 15/10 with Y.pestis F1-antigen, B.antracis protective antigen and B.malei porine protein.

For designing of recombinant F.tularensis strains integrated vector systems on the base of the replicons incapable of stably supporting new genetic information in extrachromosomal state in F.tularensis cells (plasmids ColE1, Sa and others) also deserve attention. Incorporation of certain DNA fragments of F.tularensis chromosome into plasmid pBR322 with ColE1-replicon has allowed for transferring such genetic structure into the chromosome of tularemia microbe by transformation method [Pomerantzev А., 1991]. Phenotypically, it was expressed either in the resistance of the clones obtained to tetracyclin [Pomerantzev А., 1991], or in allelic replacement of the gene of F.tularensis external membrane protein [Antony L.S.D., 1991]. Transferring of the transpozone-like structure Tn10-Km into plasmid pUC19 has also allowed the integration of such recombinant plasmid into F.tularensis chromosome. Thus obtained F.tularensis strains stably inherited the inserted structure, that indicates the usefulness of integrated systems.

The given project will allow to use highly skilled scientific employees and engineers early engaged in BW defense programs as well as laboratory rooms and labware for decision of topical problems of public health in Russian Federation.

The scientific novelty of the planned researches consists in the development of a new bacterial vector, which will have a number of advantages in comparison with the available vectors. Prerequisits for the development of new recombinant of vaccines against infectious diseases caused by intracellular parasites will be established. New data on the mechanism underlying the formation of immunity generated with live bacterial vaccines will be obtained.

The goal of the project is development of recombinant vaccine F.tularensis strains synthesising protective antigens of Y.pestis, B.antracis and, B.malei.

Expected results


— Stable plasmid vectors for construction of recombinant vaccine F.tularensis strains;
— genetic structures determining synthesis of Y.pestis, Bac. antracis, Bur. malei protective antigens, perspective for development of recombinant vaccine strains;
— recombinant vaccine F.tularensis strains with Y.pestis, Bac. antracis, Bur. malei protective antigens and data on their immunobiological and protective properties;
— development of recombinant strains with protective antigens on the base of vaccine F.tularensis strains will allow for the development of new generation vaccines against dangerous diseases.

Scope of activities

The first stage of the project will be devoted to the cloning of selected protective antigens. Simultaneously vector plasmids will be optimized with regard to their stability and expression level of the genes cloned. Then influence of the form of protective antigen representation to the immune system (intracellular and extracellular localization of antigens) on the level of humoral and cellular immunity will be studied. The final stage will involve the researches on protective effect of recombinant F.tularensis strains synthesising protective antigens.

Technical approach and methodology

For identification and determination of the level of Y.pestis F-1, B.antracis and Bur.malei porine protein protective antigens these proteins will be isolated and specific sera for them will be obtained for immunochemical tests.

PCR method will be used for cloning the antigens, the nucleotide sequences of which are already known.

Cloning of Bur.malei porine protein is supposed to carry out on E.coli phage vector. Selection of recombinant phages with porine protein genes will be performed by immunochemical methods. Structure-functional organization Bur.malei porine protein operon will be determined.

Transfer of genes coding the synthesis of protective antigens into the cells of vaccine F.tularensis strain will be carried out on plasmid vectors as well as by using integrative systems.

The kinetics of multiplication of the recombinant strains in the spleen of experimental animals will be eliminated, and the level of antibodies specific for protective antigens will be determined by ELISA method. The level of cellular immunity will be estimated by blast-transformation method. In case of generation of specific immunity to chosen protective antigens in the experimental animals, experiments on the evaluation of protective effect of recombinant vaccine strains against infection of the experimental animals by virulent Y.pestis, Bac. Antracis, Bur. Malei strains will be performed.

Role of foreign collaborators

The form of collaboration:


— regular project discussion by e-mail, fax and phone;
— joint publications;
— cooperative workshops;
— cooperative experiments in the Laboratories of project collaborators and Laboratories of project executors;
— exchange of working materials (plasmids, genetic constructions, sera etc.).


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