Neutralizing the human Norovirus Histo-Blood Group Antigen Binding Pocket

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There are five genera in the Caliciviridae family, Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus. To date, histo-blood group antigen (HBGA) binding sites are only known for norovirus and lagovirus. Earlier structural studies with norovirus protruding (P) domains showed that genogroup I and II (GI and GII) noroviruses had two equivalent HBGA binding pockets per P dimer. Similarly, lagovirus was found to bind two HBGAs per P dimer. We recently discovered that GII noroviruses had two additional HBGA pockets, making a total of four HBGA binding sites per P dimer. In this proposal, we will perform three main aims in order to better describe calicivirus interactions with HBGAs. In the first aim, we will determine if HBGAs bind to vesivirus, sapovirus, and nebovirus P domains using X-ray crystallography. We have already shown that X-ray crystallography provides a reliable method of determining these binding interactions. Our group has solved over 30 different P domain HBGA complex structures, including several that were previously unknown. We will extend this aim to include norovirus virus-like particles (VLPs) and HBGA complexes in order to gain a better understanding of the binding mechanisms in the context of the intact particles. In the second aim, we will analyze the dose-dependence and manner of HBGA binding using a combination of X-ray crystallography, NMR, and MS. We now know that GII noroviruses have four HBGA pockets per P dimer, but little is known about the multiple binding sites in GI noroviruses and other caliciviruses. In the third aim, we further analyze and develop a monoclonal that binds at the GII norovirus HBGA binding pocket. Currently, this antibody only binds several GII norovirus strains, but with further modifications we expect to produce a broadly-reactive antibody molecule that can bind most GII noroviruses. We will study the ability of the antibody to block HBGA attachment to norovirus and possibly function as a treatment for norovirus infections. Altogether, the data generated from this proposal will provide new information on calicivirus and HBGA interactions and include the development of antibody that can block HBGA from binding to the GII noroviruses - the most clinically important human noroviruses.