In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

The Escherichia coli bacteriophage, Q beta (Coliphage Q beta), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qb RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qb display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qb resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Q beta icosahedral shell by electron microscopy. Evolution of Q beta-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Q beta selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Q beta-G-H loop, validating the requirement of the tandem pair epitope. Q beta-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.