Development of an influenza pseudovirus based assay for the identification of entry inhibitors by phage display

The threat of a major human influenza pandemic, in particular from highly pathogenic avian influenza viruses (HPAIV) like H5N1,has emphasized the need for new antiviral drugs and therapeutic strategies to combat these pathogens. We have constructed retroviral virus particles pseudotyped with either human or avian influenza hemagglutinin, neuraminidase and matrix proteins in different combinations and investigated their infectivity in a single-round infection assay. The assay is based on the transmission of a marker gene (GFP) to target cells and thereby directly dependent on the function of the envelope proteins enabling the virus to enter the host cell. Therefore, this assay can be ideally used to analyze potential entry inhibitors. Additionally, we expressed the hemagglutinin receptor-binding domain (RBD) of human and avian influenza viruses. The pseudotyped viruses, the RBDs and parts of the HA2 domain are used as targets in phage display library screenings. This method allows us to directly identify peptides interfering with the entry process thereby potentially inhibiting viral entry. First results of these screenings will be presented. To further investigate the specificity and affinity of the selected viral entry inhibitors we developed a solid-phase immunoassay where we use sialic acid coupled to an artificial sequential oligopeptide carrier (SOC). This technique enables us to perform these assays in a small-scale microplate format.