Research is being done to understand the mechanism of cellular immunity in swine protection against different viral diseases and to develop new vectors for vaccine development. This work is based on the use of a cloned bacterial outer membrane lipoprotein (from Pseudomonas aeruginosa) as a vector to make fusion proteins containing protective epitopes, in view of the development of subunit and/or live vaccines against important swine diseases such as Affican swine fever (ASF). The lipoprotein presents several advantages, such as small molecular weight, localization in the membrane, high expression and binding to peptidoglycan (a natural adjuvant).
4 vectors have beendeveloped based on the lipoprotein gene of Pseudomonas aeruginosa containing different cloning sites. Monoclonal antibodies against the lipoprotein have been produced and characterized. ASF virus particles have been produced from strain Lisbon 60 in pig macrophages. ASF virus deoxyribonucleic acid (DNA) has been purified and purity controlled in restriction mapping. Haelll fragments of ASF virus DNA have been shotgun cloned in the engineered Hpal site of one Lpp vector. Positive clones have been detected by colony hybridization using digoxigenin labelled ASF virus DNA, in the presence of excess of pig DNA. The clones have been confirmed using an antibody which recognizes the lipoprotein but not fusion proteins. Clones have been detected which produce membrane bound fusion proteins by sodium dodecyl dulphat-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using another monoclonal antibody whose epitope is not disrupted by the introduction of new amino acids in the Lpp sequence. 2 large fusion proteins have been obtained so far, one of 29 kDa and one of 0 kDa (size of the Lpp was 7 kDa).
The project is based on the use of a cloned bacterial outer membrane lipoprotein (from Pseudomonas aeruginosa) as a vector to make fusion proteins containing protective epitopes in view of the development of subunit and/or live vaccines against important swine diseases like African Swine Fever (ASF). The lipoprotein presents several advantages, i.e. a small molecular weight, localisation in the membrane, high expression, binding to peptidoglycan (a natural adjuvant).
More specifically, the vector will be manipulated by inserting polylinkers to facilitate the cloning of viral DNA and cloned in a vector for controlled expression under the dependence of the lac promoter. DNA fragments from the strain Lisbon 60 of ASF will be cloned and expression of epitopes detected with an anti ASF serum. The important ASF viral epitopes inducing non-specific and specific cellular immune defense mechanisms will be tested by natural killer (N/K) cell activity, lymphoproliferative responses and CTL activity.