Correction: In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display.

[This corrects the article DOI: 10.1371/journal.pone.0113069.]

In vitro evolution and affinity-maturation with Coliphage qβ display.

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

Morphology of wild type vs. hybrid Qβ phage plaques.

<p>Panel A) wild type Qβ phages; Panel B) Qβ-FMDV VP1 G-H loop phages; Panel C) Qβ-GFP rescued phages from <i>E. coli</i> SURE (expression host with F⁺) over-expressing A1-GFP protein infected with wild type Qβ. Panel D) QβΔA1 phages. All at very low multiplicity of infection (MOI), and all plates are exactly 1 day (24 hours) old when photographed.</p

Field light micrograph of modified Qβ.

<p>A) Unlabelled Qβ virion: the original particle projection obtained with conventional transmission electron microscopy of a negatively stained sample was treated by Marham rotation 3 times 120\grad\intervals and printed at reversed contrast; Magnification Bar: 50 nm. B) Negatively stained Qβ, modified at A1 gene products by additional of FMDV G-H loop motif decorating the corners by IgG of mAb SD6 against VP1 G-H loop motif at 120\grad\intervals (arrows); Magnification Bar 25 nm. C) Phage particles projection depicted in B was treated by Makham rotation and printed at reversed contrast; arrows showing antibody against integrin motif attached to the corners of the virus particle; Magnification 25 nm.</p

Schematic representation of the RNA phage display vector construction.

<p>General cloning procedure from PCR fragments to pBRT7Qβ with transient cloning in the pUC18-cassette working plasmid. Step 1: cloning of PCR fragment into pCR2.1 vector; Step 2: cloning of the foreign gene from PCR into the pUC-cassette (with <i>Nsi</i>I) using <i>Af</i>lII and <i>Esp</i>I sites; Step 3: Cloning of the foreign gene into pBRT7Qβ using <i>Afl</i>II and <i>Nsi</i>I. P: promoter; <i>Amp</i>: ampicillinase gene; <i>Kan</i>: kanamycin resistance gene; ori: origin of replication.</p

Table adapted from Drake [29].

a<p>In the cases where multiple targets were measured, the average is presented.</p>b<p>Taken from Domingo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113069#pone.0113069-Domingo3" target="_blank">[48]</a>.</p><p>Table adapted from Drake <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113069#pone.0113069-Drake1" target="_blank">[29]</a>.</p

RT-PCR of RNA purified from Qβ-phage plaques.

<p>Panel A) Lane 2: Qβ-HisJ; lane 3: Qβ-tHisF; lane 4: soft agar stab from HisJ plate; lane 4: soft agar stab from tHisF. Panel B) Lanes 2 and 4: wild type Qβ; Lanes 3 and 5: Qβ-GFP. Panel C) Lanes 2 and 4: Qβ-FMDV; Lanes 1 and 5: wild type Qβ (positive control). The 100 bp and 1 kb DNA ladder were used.</p

Agarose gel electrophoresis of the RNA display system vector construction.

<p>Panel A) Lanes 1–3: positive recombinant pUCHisJ plasmid clone (cl) restricted with <i>Afl</i>II and <i>Nsi</i>I; Lanes 5–7: positive pUCtHisF and Lane 8: negative clone. Panel B) Lanes 2–6: positive recombinants pBRT7QβHisJ restricted with <i>Afl</i>II and <i>Nsi</i>I; Lane 7: negative clone. Panel C) Lanes 2–7: positive recombinants pBRT7QβtHisF restricted with <i>Afl</i>II and <i>Nsi</i>I. Panel D): Lane 1: pQβ8 negative control; Lanes 2 and 3: positive recombinants pQβ8ΔA1; Lanes 4 and 5: positive recombinants pBRT7Qβ-FMDV; Lanes 6 and 7: positive recombinants pBRT7QβΔA1 all restricted with <i>Nhe</i>I. Lanes “ladder” were loaded with the 100 bp or 1 kb DNA ladder.</p

Ouchterlony double diffusion assay.

<p>A) Wells 1 and 2 represent Qβ-FMDV phages; wells 3 and 4 represent QβΔA1 phages; wells 5 and 6 represent wild-type Qβ; center well contains polyclonal serum from immunized guinea pig (labeled “Ab”). B) Same as panel A but with 1/3 of the serum concentration. C) Wells 1 and 2 Qβ-FMDV are the same as panel A; wells 5 and 6 contain half the phage titer of wells 1 and 2; well 3 represents phages from pBRT7QβΔA1 and well 4 represents phages from pQβ8ΔA1; center well contains IgGs purified from serum panel A and B (labeled “Ab”). The line of precipitation is visible as a white haze forming a half-circle around some of the wells in the experiments.</p