Validating the structural role of Ser52 in HV1-69-sBnAbs.

<p><b>A</b>) F10 V-segment germline variants were analyzed for H5VN04 binding in the phage-Ab (5 scFv/phage) format by MSD ELISA <i>(left)</i> and for their ability to activate B-cell when expressed as B-cell receptors in the presence of H5VN04 <i>(right)</i>. <b>B</b>) HV1-69-sBnAb variants of S52I in F10 and A66, G52aP in CR6331, G17 and D8 were analyzed for H5VN04 reactivity by ELISA. <b>C</b>) Kinetic analysis by Biacore of F10 and A66 CDR-H2 variants against purified H5VN04. Residues colored in blue are non-germline amino acids. <b>D</b>) Circular dichroism measurement of F10 and the non-H5 reactive variant characterized by a germline configured CDR-H2 shows a highly similar CD profile for both constructs.</p

Characterization of HV1-69-sBnAbs VH domain.

<p><b>A</b>) Alignment of 38 published HV1-69-sBnAbs is shown with highlights referring to hydrophobic residues at position 53 (light plum), the conserved Phe54 (dark plum), the occurrence of CDR-H3-Tyr (pink) residues. Other highlights refer to panel <b>B</b>), which describes the result of a Fisher's exact test with Bonferroni adjustment that compared V-segment amino acid substitutions diversity and frequency of the 37 51p1 allele related HV1-69-sBnAbs with that of a reference <i>IGHV1-69</i> 51p1 allele related Ab dataset. 13 amino acid substitutions were determined to uniquely associate with the HV1-69-sBnAb dataset (P<0.05).</p

Understanding the structural role of the distinctive CDR-H2 amino acid substitutions in HV1-69-sBnAbs.

<p>A) VDW contact analysis (black lines) shows that Ser52 of F10 and CR9114 (orange), and Ile52 of CR6261(gray) make only intramolecular contacts; i.e., do not form contacts with their respective H5VN04s. Antibodies are shown in color; HA is in light gray. At far right, steric consequences of the germline Ile52 and the Ile52Ser substitutions are shown when the Abs are overlaid on their framework residues (RMSD ∼0.5 Å). Comparing structures of the HV1-69-sBnAbs, centered on Ile52 of CR6261 (green), with F10 (yellow) and CR9114 (cyan), the Ile52Ser mutation in F10 and CR9114 enables the 2 strands to come closer together, as indicated by the yellow and cyan arrows. Distances in red indicate hypothetical steric clashes (<3 Å) that would be created if Ile52 were present in CR9114 and F10. B) Comparison between the unbound (PDB 4FQH, left) and H5VN04-bound structures (PDB 4FQI, right) of CR9114, colored according to the magnitude of structural change after superposition on the main-chain of the VH domain (from blue = 0 Å, through white = 1 Å, to red = 1.8 Å). CDRs and side-chains of the major contact residues are shown, as depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004103#ppat-1004103-g001" target="_blank">Figure 1A</a>. Distances between the Cα and Cβ atoms of Phe54 and the Cα atom of CDR-H3 Tyr98 (shown as dashed lines) are indicated. Large rotations of the side chains of CDR-H3 Tyr98, CDR-H2 Phe54 and CDR-H2 Ile53 are also evident, as previously noted <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004103#ppat.1004103-Dreyfus1" target="_blank">[7]</a>.</p

Studying the location of SHM hotspots and nucleotide substitutions frequencies in the <i>IGHV1-69</i> reference Ab dataset.

<p><b>A</b>) Number of V-segment substitutions observed in 38 HV1-69-sBnAbs with color notations that designate the HV1-69-sBnAbs characterized by the distinctive CDR-H2 Ser52 or Ala/Gly52a and “other” which do not contain CDR-H2 Ser52 or Ala/Gly52a. (<b>B–D</b>) <b>Upper panel</b> – the common nucleotide substitutions that generated the distinctive amino acid substitutions in the respective CDR domain. <b>Main panel</b> - The <i>IGHV1-69</i> 51p1 Ab reference dataset was studied for substitution frequency of nucleotides in the V-segment CDRs and for location of AID and polη hotspots (AID = WR<u>C</u>Y yellow solid triangle/R<u>G</u>YW dark red solid triangle, Polη W<u>A</u> brown open triangle/<u>T</u>W blue empty triangle. The horizontal line shows the mean±SD (6.44±7.23) of non-germline nucleotide substitution frequency observed for FR regions to serve as a reference.</p

Semi-synthetic HV1-69 phage-Ab library yields potent anti-H5VN04/H1CA0409 Abs characterized by a minimal V-segment amino acid substitutions.

<p><b>A</b>) Characterization of binding activities of anti-H5VN04 and anti-H1CA0409 phage-Abs isolated from the semi-synthetic HV1-69 phage-display library. Sequences are detailed in <b>Figure S4 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004103#ppat.1004103.s001" target="_blank">Text S1</a></b>. <b>B</b>) Heavy chain CDR sequences of anti-H5VN04 phage Abs characterized by >95% neutralization activity against both H5VN04 and H1PR8 pseudotyped viruses. The 5 highlighted residues in the CDRs refer to panel <b>C</b>) which describes the result of a Chi square statistical analysis approach used to identify residues that were significantly enriched as compared to their frequency in the library (P<0.05). Also highlighted are Tyr99 and position73 in the <i>IGHV1-69</i> germline sequences.</p