Genetic modification of cholera toxin B subunit for mucosal anti-infectious or anti-pathologic vaccination

Sammanfattning: The development of mucosal vaccines requires antigen delivery systems that can efficiently help to present vaccine antigens to the mucosal immune system. En efficient and well known antigen carrier/delivery system is the B subunit of cholera toxin (CTB). The present works describes the construction, expression, purification and structural and functional characterization of three novel genetic fusion proteins based on CTB, prepared for mucosal administration purposes. The first protein, between CTB and the B-chain of human insulin, was constructed for the purpose of testing its ability to induce oral tolerance in an antigen-specific fashion and thus be a potential candidate for immunotherapy of autoimmune (type I) diabetes. Previous work showed that chemical conjugation of whole human insulin to CTB can increase the mucosal tolerogenic potential of insulin and suppress autoimmune diabetes in animal models. In the present work, we demonstrate that a genetic fusion protein linking the B-chain of insulin to the carboxy end of CTB can be efficiently produced and purified. The fusion protein could bind to GM1 receptors, induced strong insulin-specific stimulation of specific T-cells in vitro and was at least as efficient as chemical CTB-insulin conjugate in inducing immunological suppression in vivo. The second fusion protein was constructed with the aim of providing a potential vaccine candidate against schistosomiasis capable of suppressing both the infection and the extent of immunopathology. In many chronic infections caused by bacteria, viruses, and parasites, immunopathology is a significant part of the disease complications. Schistosomiasis a helminth infection that affects several hundred million people in tropical areas is a classical example of such a chronic disease. Our results show that mucosal administration of a genetically engineered fusion protein between CTB and a peptide representing amino acid residues 24-43 and 191-212 of Schistosoma mansoni GST (Sm28GST) suppressed liver granuloma formation in mice infected with S. mansoni and also decreased the worm burden and egg production. These effects were associated with peripheral suppression of specific DTH in treated animals, and with Sm28 GST-specific antibody production, respectively. The third fusion protein, in which two CTB monomers were tandem-linked with the help of an immunologically active linker peptide (c-myc), was made to investigate whether it would be possible to generate other oligomeric forms of CTB than the "normal" CTB homopentamers and whether such novel structures could also bind to cells and present antigens to the immune system. The results show that this novel fusion protein formed large structures composed of polymers made up of a basic unit of ca. 150 kDa in molecular size, which could correspond to a basic unit containing 12 CTB monomers and 6 c-myc linkers. The assembled structures could bind to GM1 and be recognized by CTB pentamer-specific monoclonal antibodies. Mucosal immunization studies in mice indicated that the fusion protein was powerfully immunogenic giving rise to antibodies that recognized both native pentameric CTB and novel structures within the molecule including the linker-peptide, c-myc. Work in progress with other peptide linkers including the insulin B chain suggests that the length and confirmation of the peptide will determine the structure of the assembled molecules. However, a common finding is that the individual CTB monomers of the constructed dimers are able to efficiently oligomerize with corresponding monomers of adjacent dimer molecules thereby forming complex structures with GM1-binding activity and ability to present the linking-peptide or protein to the mucosal immune system. In conclusion, the results show that genetic fusion proteins between CTB and defined peptide antigens can be made and exhibit properties that make them promising candidates as vaccines or immunotherapeutic agents for the induction of either or both of immune responses and so-called oral tolerance after mucosal administration.