SYNTHESIS OF HAEMOPHILUS INFLUENZAE TYPE A OLIGOSACCHARIDES FOR VACCINE DEVELOPMENT

Glycoconjugate vaccines are a very effective way to prevent bacterial and fungal infections. By conjugation of the saccharide antigen to a carrier protein, the T-independent antigen is converted to a T-dependent antigen, resulting in the production of high affinity antibodies as well as memory B-cells. Today, most of the licensed glycoconjugate vaccines extract the required saccharide antigen from bacterial cultures. However, the isolation and purification of the polysaccharides are challenging. Synthetic polysaccharides are an effective alternative with great potential, as they are well-defined and characterized by minimal batch-to-batch variability. The pathogen Haemophilus influenzae (Hi) is a major cause of severe diseases, i.e. meningitis, sepsis and otitis, especially affecting young children. Among 6 identified serotypes, type b (Hib) is the most common and most virulent strain. Additionally, Hib is the first successful example of a vaccine based on synthetic carbohydrate antigens licensed and distributed in Cuba since 2004 with the tradename QuimiHib. In recent years, an increasing rate of infections caused by Hia raised some concern and currently, Hia causes up to 10 % of reported Haemophilus infections. This burden raised some concerns, as no vaccine targeting Hia is currently available or under development. The capsular polysaccharide (CPS) of Hia is a polymer of 4-β-D-Glc-(1→4)-D-ribitol-5-(PO4→) repeating units and is a potential antigen for a future protein-conjugated polysaccharide vaccine. To further explore the CPS of Hia as antigen, we synthesized well-defined oligosaccharides of different chain length of up to five repeating units using state of the art methodology. After synthetic optimization of all required monosaccharide building blocks, they were first assembled by means of fine-tuned glycosylation reactions to obtain disaccharide repeating units and finally combined using the well-known phosphoramidite approach. In particular, a bifunctional disaccharide building block with a phosphoramidite moiety and a temporary protecting group was used for oligomerization. After n coupling(s) using 4,5-Dicyanoimidazole (DCI) as condensating agent and (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO) for the oxidation in a one pot reaction, the temporary protecting group was removed giving the new oligomer (n+1) as new acceptor, which can be coupled again with the bifunctional building block in an iterative cycle. As last step, a C3 linker was introduced on each oligomer followed by a deprotection sequence. Additionally, the resulting oligosaccharides were further conjugated to CRM197 taking advantage of a di-N-hydroxysuccinimidyl adipate linker. Within this work, we successfully prepared five Hia oligomers containing up to five repeating units conjugated to CRM197 carrier protein. The present PhD thesis comprises 10 chapters, including the References list (Chapter 9). Chapter 1 reports a brief overview of the immune response against pathogens, with a particular focus on carbohydrate antigens, as well as the mechanism of action of glycoconjugate vaccines. Chapter 2 describes the development of a historical vaccine and additionally, novel vaccine approaches are explained. In Chapter 3 the bacterium Haemophilus Influenzae (Hi) with its different serotypes is defined and additionally, the successful glycoconjugate vaccine approach targeting type b is specified more precisely. The aim of the thesis is reported in Chapter 4 giving a short overview of the state-of-the-art and explaining further the idea of our approach. The experimental work is discussed in Chapter 5 with a brief summary in Chapter 6 and furthermore, detailed procedures are given in Chapter 7. Besides, all NMRs are published in Chapter 10.