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 K 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 K 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.

A Simple Platform for the Rapid Development of Antimicrobials

“…System overview. We have shown that bacteria 18 and viruses 20 can be applied to peptide microarrays to generate synbodies with antibiotic or antiviral activity. The challenge was to create a system to generate the synbodies quickly and provide sufficient quantities of the chosen synbody for in vivo testing.…”

“…In the live bacteria screening assay, the target is labeled with an amine-reactive dye, AlexaFluor (AF), and an internalizing dye, Cell Tracker Orange (CTO), and peptides that bind the bacteria without perturbing the membrane produce fluorescence in both channels while those that disrupt the membrane only produce fluorescence in the AF channel. Viruses were screened by detecting binding to a peptide using a fluorescently labeled virus-specific antibody, or in some cases directly labeling the virus with an Alexa Fluor dye 20 . Antibody detection was favored as it avoids the problems of the dye interfering with virus binding or binding the peptide itself.…”

“…Seven to 10 peptides are selected and the 100 peptide stock used to synthesize all combinations of the peptides into synbodies. We developed a modular and combinatorial approach using maleimide-thiol conjugation to rapidly produce synbody libraries [19][20][21] where selected peptides are conjugated to different peptide scaffolds, each with two conjugation sites, in a combinatorial manner and in parallel. With this approach, dozens or even hundreds of synbodies can be produced in a day.…”

“…We had previously demonstrated that synbodies could be developed with antibiotic activity 18 and shown the value of using D amino acid-containing peptides 19,20 . The challenge we addressed here was whether the process could be done very quickly.…”

“…We used the synbody technology [14][15][16][17][18][19][20][21] as the starting point for developing a platform to meet these specifications. Synbodies are bivalent peptides with antibody like features that are chemically synthesized.…”