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GENE CLONING OF BRUCELLA ANTIGENS WHICH AS VACCINES DO NOT INTERFERE WITH A SPECIFIC DIAGNOSIS

Objective

THIS RESEARCH MIGHT PERMITS TO DESIGN VACCINES AGAINST BRUCELLOSIS WHICH COULD BE DIFFERENTIATED FROM NATURAL INFECTION. IT SHOULD THEN BE POSSIBLE TO VACCINATE CATTLE AGAINST BRUCELLOSIS AND SIMULTANEOUSLY TO STAMP-OUT THE INFECTED CATTLE. PRESENTLY, THIS IS NOT POSSIBLE.
Research was carried out in order to develop a vaccine for brucellosis which includes only the major protective antigens and is devoid of specific antigens useful for the diagnosis. For vaccine development, it was necessary to define more precisely which antigens, lipopolysaccharides (LPS) and/or major outer membrane proteins (OMP), are essential to induce valuable protection. For diagnosis, antigens were looked for that could be used in conjuction with A2. Brucella OMPs are extremely difficult to purify by classical techniques; therefore, genes were cloned using an expression vector. Monoclonal antibodies (Mab) specific to potential antigens were produced and used to passively immunize mice. The immune response of infected animals was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of Brucella antigens, immunoblotting techniques and inhibition enzyme linked immunosorbent assay (ELISA).

Results of passive immunization of mice showed that both A antigens and M antigens of the LPS are essential for protection, whereas OMPs are only weakly protective. The role of these OMPs at the cellular level will be further clarified by using recombinant proteins. The deoxyribonucleic acid (DNA) sequences of both genes were nearly determined and gene expression is under investigation. Mabs specific to 10, 16.5, 19, 25 to 27, 36 to 38, 39, 89, kDa proteins were obtained. Results of Mab binding to Brucella cells indicate that all except the 39 kDa one are OMPs; it is cytoplasmic. After purification by preparative electrophoresis and intradermal injection into infected guinea pigs, the 39 kDa protein induced a strong delayed type hypersensitivity reaction. The 10, 16.5, 19 and 89 kDa proteins were immunogenic in goats and cattle. A2 antigen was purified and Mab directed against this antigen are now being produced. 89 kDa OMPs were also purified by preparative electrophoresis.
DIAGNOSIS OF BRUCELLOSIS IS BASED ON THE SEROLOGICAL DETECTION OF SPECIFIC AGGLUTININS; UNFORTUNATELY, THE VACCINATION AGAINST DISEASE INDUCE ALSO THE SAME AGGLUTININS, AND IT IS NOT POSSIBLE TO KNOW IF AN ANIMAL HAS BEEN VACCINATED OR INFECTED. THIS COULD BE OVERCOME BY USING A VACCINE CONTAINING THE MAJOR ANTIGENS INDUCING PROTECTION, AND DEVOID OF A LEAST ONE SPECIFIC ANTIGEN ALWAYS PRESENT IN THESE BACTERIA.

THE RESEARCH WILL INCLUDE :

- PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST THE MAJOR ANTIGENS,
- PREPARATION OF DNA FROM BRUCELLA,
- CONSTRUCTION OF GENOMIC BRUCELLA LIBRARIES IN E. COLI,
- SELECTION OF RECOMBINED E. COLI CLONES PRODUCING THE DIFFERENT ANTIGENS,
- EVALUATION OF THE PROTECTIVE CAPACITY OF THE CLONES,
- TAXONOMIC STUDY OF THE GENES CODING FOR THE ANTIGENS,
- PURIFICATION, ASSAY, GENE CLONING OF THE ANTIGEN A2.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Institut National de la Recherche Agronomique (INRA)
Address
149 Rue De Grenelle
75341 Paris
France

Participants (1)

INTERNATIONAL INSTITUTE OF CELLULAR AND MOLECULAR PATHOLOGY
Belgium
Address

Bruxelles