In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

Abstract

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ 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 Qβ 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 Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ 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 Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

The True Story and Advantages of RNA Phage Capsids as Nanotools

“…The A1 protein is incorporated in 3-10 copies per virion, or in 12 copies in accordance with a recent study [32], it is required for infection, but its precise function is not known [for more references, see [8,29,30,31,32,33]]. A recent electron microscopy visualization of foreign epitopes carried by A1 protein within infectious Qβ particles showed that the A1 protein molecules are occupying corners of the Qβ icosahedron [32]. The lysis protein forms pores in the cellular membrane, leading to activation of autolysins and, eventually, cell lysis [34].…”

“…In addition to the standard maturation protein, CP, and replicase subunit, the Allolevivirus genome encodes a C-terminally extended CP known as the minor A1 protein, which appears as a result of ribosomal read-through of a leaky opal termination codon of the CP gene [28] and is essential for the formation of viable Qβ particles in vivo [29,30,31]. The A1 protein is incorporated in 3-10 copies per virion, or in 12 copies in accordance with a recent study [32], it is required for infection, but its precise function is not known [for more references, see [8,29,30,31,32,33]]. A recent electron microscopy visualization of foreign epitopes carried by A1 protein within infectious Qβ particles showed that the A1 protein molecules are occupying corners of the Qβ icosahedron [32].…”

“…MS2 VLPs [160] and Qβ phage [32] offer favorable alternatives to filamentous bacteriophages for the display of immunologically active peptides. Recently, a tumor-associated antigen was identified using MS2 peptide-display technology [282], confirming the importance of such a methodology for future studies.…”

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

“…Phage display is one of the most commonly used methods for in vitro peptide‐screening . Many types of phage can be used in phage display technique, including filamentous bacteriophage, T4 phage, T7 phage, Qβ phage, etc . To be displayed on phage particles, cyclic peptides should be fused to solvent‐exposed surface proteins, such as pIII, pVI, pVII, pVIII, and pIX proteins .…”

Comparative analysis of IgG Responses to recombinant Qβ phage displayed MSP3 and UB05 in Dual HIV-malaria infected adults living in areas differing in Malaria transmission intensities

“…In this study we have determined in low and high malaria transmission areas the impact of dual HIV-malaria infection on IgG subclass responses to two conserved P. falciparum derived asexual blood stage antigens displayed separately upon a recombinant RNA coliphage Qβ as previously described by our group (14, 15). The recombinant phage QβMSP3 displays the conserved C-terminal 88 aa of the merozoite surface protein 3 (16, 17) whilst QβUB05 bears the previously described malaria antigen UBO5 (18).…”