Drug resistance mutation detection in limiting dilution series.

<p>Two HIV-1 subtype B samples were used for the dilution series (A, samples 40–44; B, samples 45–49). The four groups shown in the histogram are based on the median frequency of each drug resistance mutation in its respective <i>undiluted</i> sample: >20% (white bars), 10–20% (bright grey bars), 2–10% (dark grey bars), and 1–2% (black bars). The means and standard deviations are given of the percentage of sites reporting drug-resistance mutations in these categories. The number of mutations in each category is represented by n.</p

Triplicate sample analysis.

<p>A set of three samples was measured in triplicate at each of the 11 sites. Variant percentages measured in the triplicate samples are shown for each mutation.</p

Neutralization assays on JR-FL glycan mutants indicate binding of inferred intermediate antibodies to both N301 and N332 on HIV-1 Env.

<p>(<b>A</b>) Neutralization curves of inferred intermediate antibodies on wild-type JR-FL virus (<b>B</b>) JR-FL N301A mutant virus, and (<b>C</b>) JR-FL N332A mutant virus. (<b>D</b>) Neutralization curves of 3H+3L and (<b>E</b>) PGT121 on wild-type JR-FL virus compared to single and double glycan mutant viruses. (<b>F</b>) Neutralization curves of PGT121 on wild-type 92BR020 virus compared to single and double glycan mutant viruses.</p

Antibody 3H+3L likely crosslinks between trimers.

<p>(<b>A</b>) 3H+3L binds an epitope overlapping with those of PGT121, PGT128, and 2G12 as shown by competition of biotinylated antibody 3H+3L with an antibody panel. Binding assays were performed by flow cytometry on JR-FLΔCT isolate transfected in 293T cells. (<b>B</b>) IgG and Fab fragments were tested for binding on JR-FLΔCT isolate expressed on transfected 293T cells and no substantial differences in avidity were observed. Solid lines represent IgG and dashed lines represent Fab fragments. (<b>C</b>) Purified IgGs of 3H+3L and PGT121 were digested into Fab fragments using Lys-C, purified, and tested for neutralization on a cross-clade panel. Loss of neutralization was found for 3H+3L Fab, but not for PGT121 Fab. Reported values are IC₅₀ ratios of Fab compared to IgG using the equation: (IC₅₀ Fab)∶(IC₅₀ IgG).</p

Putative germline of PGT121 does not bind monomeric gp120 or cell surface Env.

<p>(<b>A</b>) PGT121germline does not bind recombinant gp120 (92BR020). Recombinant gp120s were produced in 293F cells and purified by lectin column before use in ELISA binding assays. ELISA values are reported in optical density at 405 nm (OD405). (<b>B</b>) PGT121germline does not cell surface Env (92BR020). Cell surface Env was produced by transfecting pseudovirus in 293T cells and binding was measured by flow cytometry (reported in mean fluorescence intensity or MFI).</p

Selected heavy and light chain clones were paired and tested for neutralization breadth and potency on a cross-clade 6-virus panel.

<p>(<b>A</b>) Heavy and light chain nodes leading to mAb PGT121 and (<b>B</b>) PGT124 were paired and tested on a 74-virus panel of PGT121- or PGT124-sensitive viruses. Boxes are colored by IC₅₀ values (µg/ml) of each isolate neutralized.</p

Higher levels of somatic hypermutation correlates with greater neutralization breadth and potency.

<p>(<b>A</b>) Summary of neutralization data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003754#ppat-1003754-g004" target="_blank">Figure 4A–B</a> by each clade neutralized. Listed in colored boxes are percentage values. (<b>B</b>) Overall mutation frequencies of mAb combinations, which were calculated by overall number of nucleotide mutations in both heavy and light chains divided by combined heavy and light chain lengths. The mutation frequency of each heavy and light chain was calculated over both V- and J-genes.</p

PGT121–123 variants identified by deep sequencing were used to build phylogenetic trees using ImmuniTree.

<p>(<b>A</b>) An example identity (y-axis) and mutation (x-axis) plot for PGT121 used to identify PGT121-variants for heavy chain and for (<b>B</b>) light chain; each color represents a unique clone. (<b>C</b>) Heavy chain and (<b>D</b>) light chain SHM phylogenies inferred by the ImmuniTree algorithm. Nodes that were inferred by the algorithm are represented as small circles. Nodes representing observed sequences are depicted as larger circles; node size is proportional to the number of reads assigned to that node. The trees are colored based on level of mutation from germline. Previously isolated affinity matured mAbs are labeled (e.g. PGT122) and nodes selected for synthesis and characterization are labeled blue.</p

Inferred intermediate antibodies preferentially bind native Env relative to monomeric gp120.

<p>mAbs 3H+3L (blue) and PGT121 (red) were tested for binding by ELISA to monomeric gp120, which was extracted from lysed virus supernatants: (<b>A</b>) 92BR020, (<b>C</b>) 92RW020, (<b>E</b>) JR-FL E168K/N192A, (<b>G</b>) IAVI C22. Antibodies were also tested for cell surface Env binding by flow cytometry: (<b>B</b>) 92BR020, (<b>D</b>) 92RW020, (<b>F</b>) JR-FL E168K/N192A, (<b>H</b>) IAVI C22. mAb 2G12 was included as a control (gray). ELISA values are reported as optical density at 405 nm (OD405) and flow cytometry values are reported as mean fluorescence intensity (MFI).</p

Mutation summary of selected heavy and light chain clones.

<p>Mutation frequency was calculated over the V-gene and J-gene as nucleotides or amino acids differing from the putative germline sequence for (<b>A</b>) heavy chain sequences and (<b>B</b>) light chain sequences. The CDR3 regions and insertions and deletions were excluded from the analysis. CDR3 lengths were determined according to the IMGT definition. Analyses were performed with the SciPy stack <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003754#ppat.1003754-Jones1" target="_blank">[53]</a> and figures were generated using matplotlib <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003754#ppat.1003754-Hunter1" target="_blank">[54]</a> and graphviz <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003754#ppat.1003754-Ellson1" target="_blank">[55]</a>.</p