Whole-Virus Screening to Develop Synbodies for the Influenza Virus.

ABSTRACT

There is an ongoing need for affinity agents for emerging viruses and new strains of current human viruses. We therefore developed a robust and modular system for engineering high-affinity synbody ligands for the influenza A/Puerto Rico/8/1934 H1N1 virus as a model system. Whole-virus screening against a peptide microarray was used to identify binding peptides. Candidate peptides were linked to bis-maleimide peptide scaffolds to produce a library of candidate influenza-binding synbodies. From this library, a candidate synbody, ASU1060, was selected and affinity-improved via positional substitution using d-amino acids to produce a new synbody, ASU1061, that bound H1N1 in an ELISA assay with a KD of <1 nM, comparable to that of a monoclonal antibody for neuraminidase (NA). We prepared a modified version of ASU1061 that contained an additional C-terminal peptide to simulate conjugation of the synbody to a carrier protein, called ASU1063, and found that H1N1 binding was unchanged. Subsequent work identified the synbody target as nucleoprotein (NP), a highly conserved protein in influenza, with a KD of <1 nM for ASU1063. This suggests that virus-binding synbodies can be conjugated to carrier proteins or other moieties that could improve the therapeutic profile of the resulting synbody. This method is a rapid process that offers a means of developing new affinity ligands to influenza and other viruses.