Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.

<p>Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.</p

Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.

<p>Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.</p

Enrichment of phage clones predicted to display authentic NadA fragments on their surface after selection with a serum pool from volunteers immunized with the Bexero vaccine.

<p>Frequency values reported in the vertical axis in panels A–C refer to the occurrence, per single amino acid position, of sequences predicted to express authentic NadA fragments, relative to those predicted to express irrelevant or no polypeptides. The inset in figure A reports the same data with a higher y-axis magnification. The horizontal axis reports the amino acid positions of the translated NadA sequence. A, unselected library; B and C, library outputs after one and two rounds of selection, respectively. D, Cumulative enrichment factors for each amino acid position derived from NadA fragments obtained after one (blue line) and two (red line) rounds of selection; colored bars in the horizontal axis refer to NadA domains; the area between the dashed vertical lines correspond to the cell binding region of NadA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114159#pone.0114159-Tavano1" target="_blank">[18]</a>. E and F, enrichment factors of NadA fragments after one and two rounds of selection, respectively. Only the fragments laying in the upper quartile of enrichment factors values are shown.</p

Properties of the antigen-specific phage library before and after selection with a pool of serum samples from volunteers immunized with the Bexero anti-MenB vaccine.

<p>A–C, abundance of “natural frame” <i>nadA</i> fragments in the library before (A) and after the first and second rounds of selection (B and C, respectively). Each point represents the number of unique fragments (vertical axis) displaying the number of copies indicated in the horizontal axis; D–F, <i>nadA</i> fragment length distribution before (D) and after the first and second rounds of selection (E and F, respectively).</p

Schematic outline of the epitope mapping approach.

<p>The gene encoding the antigen is fragmented by DNAse digestion and the gene fragments are inserted into lambda phage vectors. The phage library is mixed with immune serum and phage particles binding to immunoglobulins are separated using Protein-G coated magnetic beads. The inserts of the phage population obtained after selection are massively sequenced and compared with those of the original unselected library using an <i>ad hoc</i> developed software which identifies the region(s) of the antigen targeted by serum antibodies.</p

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries

<div><p>We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero^® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.</p></div

Enrichment of NHBA fragments after affinity-selection of NHBA libraries with the 31E10/E7 mAb.

<p>Panels A, C and D show the NHBA fragments that were mostly enriched by affinity selection, ranked by frequency. A and C, fragments identified by next-generation sequencing of lambda (A) or M13 filamentous phage (C) libraries; B, cumulative occurrence, per single amino acid, of all fragments identified in the affinity-selected M13 filamentous phage library. D, fragments identified by Sanger sequencing of immunoreactive clones after immunoblotting in the lambda library.</p

HDX-MS analysis.

<p>A, time course of deuterium incorporation for the peptic peptides covering the NHBA sequence. The blue curves correspond to the complex NHBA-mAb 31E10, while the red curves derived from NHBA alone. The peptide showing a significant difference of deuterium uptake between the free and bound forms is highlighted with a red box (peptide Val 56-Met 108). Data herein reported are representative of three independent replicates. Replicates 2 and 3 of the deuterium incorporation time course for peptide 56–108 are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160702#pone.0160702.s003" target="_blank">S3 Fig</a>. B, schematic representation of the overlapping regions identified by the different approaches used in the present study. Shown are data obtained from one experiment, representative of three.</p

Inhibition of binding of mAb 31E10/E7 to NHBA by recombinant NHBA fragments.

<p>Plates were coated with NHBA-NUbp-His and reacted with limiting amounts of mAb in the presence of the inhibitors at the concentrations indicated in the horizontal axis. Fr I, GST-FrI fusion protein; Fr II, GST-FrII fusion protein; Fr III, GST-FrIII fusion protein; Pep 5, Peptide 5; Rev Pep 5, Reversed Peptide 5 (negative control); TPAS, tetrapeptide with the consensus TPAS amino acid sequence. The data in panels A and B represent the means ± SDs of three independent experiments.</p

Binding affinities of mAb 31E10/E7 for representative NHBA fragments.

<p>Binding affinities of mAb 31E10/E7 for representative NHBA fragments.</p