Chemical Approaches to Study Flu Infectivity

Influenza epidemics caused by influenza viruses occur every year worldwide. Even though many cases are mild, the disease also leads to severe cases and even results in deaths. As influenza pandemics emerge infrequently and are hard to predict, this illness remains a threat and major challenge for clinicians worldwide. Continuous evolution of influenza viruses makes vaccine preparations elaborate and not always effective. Current available drugs have adverse side effects and more and more problems of strain resistances arise. To reduce the danger of this disease a better understanding of influenza virus transmission and pathogenesis is crucial. Our cells are covered by a dense layer of glycans (polysaccharides) that play an important role in many biological processes. Based on our current scientific knowledge flu viruses bind to cells of the human respiratory tract by recognizing sialic acids on glycans. The type of the sialic acid-linkage to the premature sugar unit determines the receptor specificity of flu viruses (avian versus human viruses) and presents a barrier for transmission. Recent studies indicate that due to the antigenic drift not only the terminal sialic acids and the linkage to the premature sugar but also the underlying glycan structure plays an important role in the recognition process of the virus to the host cells. The aim of this project is to investigate which structural features of glycans on cells in our respiratory tract contribute to the infectivity of flu viruses. We expect to identify a unique glycan structure that is pivotal for the recognition of human host cells by various flu viruses. With this new information in hand, novel promising anti-influenza drugs that are not based on the inhibition of the virus propagation but on the prevention of the virus to enter host cells will be developed. Multivalent inhibitors will be prepared that bind to the virus in competition with human cells and thus prevent the infection at a very early stage. In the course of this project, we will prepare chemoenzymatically around 30 glycans that have been identified by mass spectrometry to be in our respiratory tract. They will be used in binding studies with hemagglutinin proteins of various flu viruses to uncover not yet known ligands biologically relevant for virus binding to host cells. Further studies with the uncovered glycans will elucidate which glycans contribute to the infectivity of various flu viruses and finally lead to the identification of a unique glycan structure that is crucial for the attachment process to our cells. By applying this unique glycan structure with biocompatible carrier systems novel multivalent hemagglutinin inhibitors will be developed.

Glycans are sugar-based chains that are found on the outer surface of all cells. They play an essential role in the communication between cells and the outside world and are involved in every major disease. However, there is still little known how glycans actually perform their functions. To unravel the roles of glycans play in diseases, infections, immune disorders and cancer, well-defined glycans are needed. The goal of this project was to develop a method for the preparation of O-glycans to be able to investigate their role in viral infections. The project was performed at Utrecht University in the international group of Prof. Geert-Jan Boons. Prof. Boons is a well-known expert in the field of glycoscience. His group could establish an efficient chemoenzymatic method for the preparation of complex N-glycans. With their existing knowledge, in the course of the project a method for the facile chemoenzymatic preparation of various mucin-type O-glycans was developed. Mucins are a class of glycoproteins that carries the greatest number of O-glycans. These O-glycans are attached via an O-glycosidic linkage to the amino acid threonine or serine of the peptide backbone of mucins. O-glycans on mucins have been found to be structurally divers. They contain various terminal epitopes. The developed method where the cores were synthesized chemically, and the extension was done enzymatically could be applied to synthesize a library of heterogeneous O-glycans. Mucins contribute to many biological processes eg. they are involved in immune signaling, barrier formation and host-pathogen interactions. They are present in mucous secretion and are expressed at epithelial surfaces of gastrointestinal and respiratory tract. In contrast to N-glycans mucin O-glycans are underrepresented in current microarray platforms. To facilitate O-glycan interactomics studies, there is a need for a collection of structurally defined complex O-glycans. To address this deficiency, the prepared O-glycan library was printed as a microarray. The microarray was used for the investigation how the different O-glycan cores influence binding preferences of various host and microbial glycan binding proteins.