Phage escape libraries for checkmate analysis

Abstract

Ligand-binding epitopes of proteins can mutate rapidly, as shown by viral mutations that lead to escape from neutralizing antibodies. We have undertaken to recreate in vitro the evolutionary competition between viral mutations that allow escape from antibody binding and host mutations that generate new neutralizing antibodies to the mutated viral antigen. To examine this vital race, we describe a phage-based method that allows rapid analysis of molecules that perturb the binding of proteins to their ligands. Because the system can amplify by replication, single-molecule sensitivity can be achieved. When combinatorial protein or smallmolecule libraries are studied, large numbers of binding events can be analyzed simultaneously. Such libraries may be used in a sequential phage escape format, where cycles of phage binding and release of mutants are driven by antibodies or small molecules and the difficulty of escape increases at each cycle. Ultimately, the sequencing of the viral mutants allows annotation of the allowed trajectory of escape. Likewise, sequencing of the antibody perturbants charts the chemistry of the immune system response to the viral challenge. We have termed such analysis of competing mutations a ''checkmate analysis.'' When viral systems are studied, a checkmate analysis allows experimental evaluation of the evolutionary contest between viruses and the immune system and may predict which antibodies and small-molecule ligands should be generated in anticipation of viral mutations before these mutations create viral epidemics.high-throughput screening ͉ influenza ͉ single-molecule detection ͉ small-molecule inhibitors ͉ combinatorial antibody libraries

Textbook of Personalized Medicine

“…An approach, dubbed "checkmate analysis," may predict which antibodies or small molecule therapeutics will best neutralize these viral mutations before they can develop into global epidemics (Dickerson et al 2007 ). This is phage-based method that allows rapid analysis of molecules that perturb the binding of proteins to their ligands.…”

Predicting protein evolution in vitro by phage escape technology

“…Although methods exist to study protein evolution in hindsight, [31][32][33][34][35] the introduction of a technology that can predict protein evolution prior to the emergence of quasi species and subsequent resistance to the immune system or therapeutics would lead to a paradigm shift in the study of evolving disease. To this end, phage escape technology 36 has been developed as a means to analyze protein-protein interactions and can be applied to study protein evolution or to screen large libraries of molecules for their modulation of protein-ligand interactions. This phage-based method permits the rapid in vitro analysis of hundreds of millions of binding events simultaneously, while also having the advantage of single molecule resolution due to the exponential replication of phage-infected bacteria.…”

“…Recently, we have validated this hypothesis and have shown that functional, trimeric HA can be expressed in E. coli at the macroscopic, microscopic and ultrastructural levels. 36 Additionally, influenza has been postulated to use a glycan shield to hide antigenic epitopes from the host immune system in a similar manner to the HIV gp120. 6,51,52 However, studies have shown that glycosylation of HA does not modify protein antigenicity 53,54 or viral virulence; 55 instead, the main function of glycosylation may to be to facilitate the proper folding and association of the monomers into a functional trimer when monomers are present in relatively dilute concentrations.…”