High-Resolution Mapping of Human Norovirus Antigens via Genomic Phage Display Library Selections and Deep Sequencing.

Norovirus (NoV) infections are a leading cause of gastroenteritis. The humoral immune response plays an important role in the control of NoV, and recent studies have identified neutralizing antibodies that bind the capsid protein VP1 to block viral infection. Here, we utilize a NoV GI.1 Jun-Fos-assisted phage display library constructed from randomly fragmented genomic DNA coupled with affinity selection for antibody binding and subsequent deep sequencing to map epitopes. The epitopes were identified by quantitating the phage clones before and after affinity selection and aligning the sequences of the most enriched peptides. The HJT-R3-A9 single-chain variable fragment (scFv) antibody epitope was mapped to a 12-amino-acid region of VP1 that is also the binding site for several previously identified monoclonal antibodies. We synthesized the 12-mer peptide and found that it binds the scFv antibody with a KD (equilibrium dissociation constant) of 46 nM. Further, alignment of enriched peptides after affinity selection on rabbit anti-NoV polyclonal antisera revealed five families of overlapping sequences that define distinct epitopes in VP1. One of these is identical to the HJT-R3-A9 scFv epitope, further suggesting that it is immunodominant. Similarly, other epitopes identified using the polyclonal antisera overlap binding sites for previously reported monoclonal antibodies, suggesting that they are also dominant epitopes. The results demonstrate that affinity selection and deep sequencing of the phage library provide sufficient resolution to map multiple epitopes simultaneously from complex samples such as polyclonal antisera. This approach can be extended to examine the antigenic landscape in patient sera to facilitate investigation of the immune response to NoV.IMPORTANCE NoV infections are a leading cause of gastroenteritis in the United States. Human NoVs exhibit extensive genetic and antigenic diversity, which makes it challenging to design a vaccine that provides broad protection against infection. Antibodies developed during the immune response play an important role in the control of NoV infections. Neutralizing antibodies that act by sterically blocking the site on the virus used to bind human cells have been identified. Identification of other antibody binding sites associated with virus neutralization is therefore of interest. Here, we use a high-resolution method to map multiple antibody binding sites simultaneously from complex serum samples. The results show that a relatively small number of sites on the virus bind a large number of independently generated antibodies, suggesting that immunodominance plays a role in the humoral immune response to NoV infections.