Final Report Summary - CRYPTOVACC (Development of fungal conjugate vaccines based on synthetic oligosaccharide structures)
Cryptococcus neoformans is an opportunistic encapsulated yeast that causes cryptococcal meningoencephalitis (cryptococcosis) in immunocompromised individuals, thus, the people directly benefitting from a vaccine against C. neoformans would be patients with an impaired immune system, either produced (e.g. organ transplant patients) or acquired (e.g. AIDS patients), which are most susceptible to fungal infections.
The cell wall of C. neoformans is surrounded by a major polysaccharide capsule (GXM), which is a main virulence factor and thus a potential target for the development of a capsular polysaccharide (CPS) based vaccine. The development of commercial glycoconjugate vaccines as safe and most efficient vaccines has had huge impact on vaccine schemes and the vaccine market and many of the new vaccines licensed are of this kind, however, there are at the moment no commercial glycoconjugate vaccines developed against fungal infections. C. neoformans candidate vaccines based on native part structures of the capsular polysaccharide did not only elicit protective antibodies, but also non-protective and even deleterious (disease-enhancing) antibodies. This controversial result is related to the microheterogeneity of the native CPS which doesn’t permit extrapolation of any information on the structure of the immunogenic epitopes or use of native material in vaccines.
The present project address the challenge of preparation of a glycoconjugate vaccine against C. neoformans by the use of synthetic part structures of the C. neoformans CPS; these structures are well-defined both in size and composition, and suitable for structure-activity relationship studies to identify a immunological protective epitope that can be used in a vaccine.
The main objectives of the project are listed below:
* access to thioglycoside building block donors to be used in the construction of well defined part structures of the CPS of Cryptococcus neoformans;
* preparation of spacer equipped part structures of the CPS of Cryptococcus neoformans;
* printing of the above structures on glass plates and screening with fluorescent labelled lectins;
* screening of the Cryptococcus neoformans microarray plates with monoclonal antibodies;
* preparation of candidate vaccine protein conjugates;
* training of the researcher in (a) planning a multistep synthesis, (b) preparing oligosaccharides [oligosaccharide synthetic strategies], (c) printing of microarray, (d) screening of lectins, (e) preparing and analysing glycoconjugates.
The first goal of the project was the synthesis of large thioglycoside building blocks to be used in glycosylation reactions. The planned synthetic strategy involved insertion of an orthogonal protective group on the 3 position of the mannose residues. In this way, these compounds will allow of the mannose backbone in either direction. They can act both as glycosyl donors and acceptors (upon removal of the orthogonal protecting group). Selective, high yielding and reproducible procedures were implemented for the synthesis of the set of thioglycoside donors required [8 derivatives ranging in size from mono- to hexasaccharide (serotype A and serotype D)]. These were then used in the construction of large part structures of the CPS of C. neoformans through iterative glycosylation reaction on spacer equipped acceptors. Thus, a unique library of synthetic well-defined C. neoformans GXM structures was obtained ranging in size from mono- to octadecasaccharides.
The permanent protecting groups (benzyl groups) were all removed simultaneously by means of hydrogenolysis (H2-Pd/C). Also, the azido group on the linker was reduced to an amine. In this way, compounds suitable for microarray printing or protein conjugation reactions were prepared.
Part of the above library was printed onto activated glass slides. The quality of the printing was verified using fluorescent labelled lectins. Thus, a unique microarray of C. neoformans CPS structures has been produced for the identifying of protective epitopes.
The microarray has been sent to our biological collaborator for interaction studies with a library of C. neoformans CPS-recognizing monoclonal antibodies, both protective and non-protective. A work that is presently on-going. Using this approach, but with only three hexasaccharide structures, we have earlier been able to identify an immunogenic but not protective epitope. We now hope that with this substantially enlarged (both concerning numbers and size of compounds) library also protective epitopes will be identified. These epitopes will then be conjugated to a carrier protein to form a candidate vaccine that will be used in mice immunisation experiments.
Through collaboration with a world leading vaccine company we have access to a carrier protein which has proven its effectivity as part of a number of commercial glycoconjugate vaccines. During a stay at this company the Marie Curie post-doc fellow, Dr Guazzelli, was trained in glycoconjugate synthesis, purification, and analysis, why this skillset is now available in our lab.
Dr. Guazzelli prepared through multi step (for larger structure 50+ steps) total synthesis a library of part structures of the capsular polysaccharide (CPS) of C. neoformans. The multidisciplinary nature of the project as well as the exposure to the industry sector gave him a diverse skill–set and professional maturity.
The overall results of the project represent a fundamental advance in the development of a synthetic glycoconjugate vaccine against Cryptococcus neoformans.
The cell wall of C. neoformans is surrounded by a major polysaccharide capsule (GXM), which is a main virulence factor and thus a potential target for the development of a capsular polysaccharide (CPS) based vaccine. The development of commercial glycoconjugate vaccines as safe and most efficient vaccines has had huge impact on vaccine schemes and the vaccine market and many of the new vaccines licensed are of this kind, however, there are at the moment no commercial glycoconjugate vaccines developed against fungal infections. C. neoformans candidate vaccines based on native part structures of the capsular polysaccharide did not only elicit protective antibodies, but also non-protective and even deleterious (disease-enhancing) antibodies. This controversial result is related to the microheterogeneity of the native CPS which doesn’t permit extrapolation of any information on the structure of the immunogenic epitopes or use of native material in vaccines.
The present project address the challenge of preparation of a glycoconjugate vaccine against C. neoformans by the use of synthetic part structures of the C. neoformans CPS; these structures are well-defined both in size and composition, and suitable for structure-activity relationship studies to identify a immunological protective epitope that can be used in a vaccine.
The main objectives of the project are listed below:
* access to thioglycoside building block donors to be used in the construction of well defined part structures of the CPS of Cryptococcus neoformans;
* preparation of spacer equipped part structures of the CPS of Cryptococcus neoformans;
* printing of the above structures on glass plates and screening with fluorescent labelled lectins;
* screening of the Cryptococcus neoformans microarray plates with monoclonal antibodies;
* preparation of candidate vaccine protein conjugates;
* training of the researcher in (a) planning a multistep synthesis, (b) preparing oligosaccharides [oligosaccharide synthetic strategies], (c) printing of microarray, (d) screening of lectins, (e) preparing and analysing glycoconjugates.
The first goal of the project was the synthesis of large thioglycoside building blocks to be used in glycosylation reactions. The planned synthetic strategy involved insertion of an orthogonal protective group on the 3 position of the mannose residues. In this way, these compounds will allow of the mannose backbone in either direction. They can act both as glycosyl donors and acceptors (upon removal of the orthogonal protecting group). Selective, high yielding and reproducible procedures were implemented for the synthesis of the set of thioglycoside donors required [8 derivatives ranging in size from mono- to hexasaccharide (serotype A and serotype D)]. These were then used in the construction of large part structures of the CPS of C. neoformans through iterative glycosylation reaction on spacer equipped acceptors. Thus, a unique library of synthetic well-defined C. neoformans GXM structures was obtained ranging in size from mono- to octadecasaccharides.
The permanent protecting groups (benzyl groups) were all removed simultaneously by means of hydrogenolysis (H2-Pd/C). Also, the azido group on the linker was reduced to an amine. In this way, compounds suitable for microarray printing or protein conjugation reactions were prepared.
Part of the above library was printed onto activated glass slides. The quality of the printing was verified using fluorescent labelled lectins. Thus, a unique microarray of C. neoformans CPS structures has been produced for the identifying of protective epitopes.
The microarray has been sent to our biological collaborator for interaction studies with a library of C. neoformans CPS-recognizing monoclonal antibodies, both protective and non-protective. A work that is presently on-going. Using this approach, but with only three hexasaccharide structures, we have earlier been able to identify an immunogenic but not protective epitope. We now hope that with this substantially enlarged (both concerning numbers and size of compounds) library also protective epitopes will be identified. These epitopes will then be conjugated to a carrier protein to form a candidate vaccine that will be used in mice immunisation experiments.
Through collaboration with a world leading vaccine company we have access to a carrier protein which has proven its effectivity as part of a number of commercial glycoconjugate vaccines. During a stay at this company the Marie Curie post-doc fellow, Dr Guazzelli, was trained in glycoconjugate synthesis, purification, and analysis, why this skillset is now available in our lab.
Dr. Guazzelli prepared through multi step (for larger structure 50+ steps) total synthesis a library of part structures of the capsular polysaccharide (CPS) of C. neoformans. The multidisciplinary nature of the project as well as the exposure to the industry sector gave him a diverse skill–set and professional maturity.
The overall results of the project represent a fundamental advance in the development of a synthetic glycoconjugate vaccine against Cryptococcus neoformans.