12 research outputs found
Functional analyses of Nef clones from treatment and placebo arms.
<p>Panel A: Selected Western blot results depict control SF2 Nef, empty (delta-Nef) plasmid and four samples each obtained from participants in the 1% tenofovir gel and placebo study arms. Panel B: Results for the CD4 downregulation activity of participant-derived <i>nef</i> isolates are shown, normalized to control SF2 Nef (which is equal to 1.0). Panel C: Results for the HLA-A*02 downregulation activity of participant-derived <i>nef</i> isolates are shown, normalized to control SF2 Nef (equal to 1.0). No significant differences in Nef-mediated CD4 or HLA downregulation function were observed between study arms (Mann-Whitney, p = 0.2 and p = 0.06, respectively).</p
Sequence analyses of HIV-1 Gag and Nef from treatment and placebo arms.
<p>Maximum-likelihood phylogenetic trees of bulk <i>Gag</i> sequences (Panel A) and clonal <i>Nef</i> sequences (panel B) from participants who received 1% tenofovir gel (red) or placebo (black) demonstrated no major clustering between arms of the trial. HIV-1 subtype B reference sequence HXB2 is shown in green.</p
Replication capacity of recombinant viruses expressing <i>Gag-Protease</i> sequences from 1% tenofovir gel and placebo participants.
<p>Panel A: Replication data are shown for recombinant NL4-3 viruses encoding <i>gag-protease</i> from participants in the 1% tenofovir gel (red) and placebo (black) study arms, expressed as fold-increase in GFP expression over 6 days, relative to day 2. WT NL4-3 control is indicated in blue. Panel B: The replication capacities of recombinant viruses encoding participant-derived <i>gag-protease</i> sequences, calculated as the slope of viral spread normalised to WT NL4-3, are shown. No signifant difference in Gag-Protease function was observed between study arms (Mann-Whitney, p = 0.2).</p
Association between baseline Gag-Protease replication capacity and HIV-1 clinical parameters at 12-months post-infection.
<p>A modest correlation was observed between early Gag-Protease-mediated replication capacity and plasma viral load (Spearman, r = 0.2, p = 0.05; Panel A), but not CD4+ cell count (Spearman, r = 0.1, p = 0.4; Panel B), at 12 months post-infection.</p
Crystal structure of CAP248-2B reveals an unusually long, protruding CDR-L3, with a hydrophobic tip.
<p>(A) Sequence alignment of the CAP248-2B heavy and light chain with their predicted V- and J-gene precursors. The CDRs are shaded, labelled, and colored. The heavy chain FR3 is similarly indicated in blue. (B) Crystal structure of the CAP248-2B Fab. The light and heavy chains are colored olive and forest green respectively, while CDR loops and FR-H3 are colored according to A. Two views are shown around a ~45° axis to highlight the long CDR-L3 (yellow). Insets show the conformational differences between the CDR-H3 and CDR-L3 loops between Fab structure 1 (shown here) and Fab structure 2 (shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.s001" target="_blank">S1 Fig</a>). Two Fabs were present in both asymmetric units, so four loops are shown per inset, two for Fab1 CDR-H3 (red) or L3 (yellow), and two for Fab2 CDR-H3 and L3 (both grey). Asp<sup>100B</sup> and Asp<sup>100C</sup> in the heavy chain and Phe<sup>95C</sup> and Phe<sup>95D</sup> in the light chain are shown with stick representations to highlight the conformational divergence between the two structures. Due to crystal packing all downstream analyses were based on the 3.1 Å Fab1 structure.</p
The CAP248-2B CDR-L3 interacts with the viral membrane.
<p>Two docking orientations for the CAP248-2B Fab are modelled with (A) the CDR-L3 in close proximity to the viral membrane, and (B) the CDR-L3 in close proximity to the fusion peptide. The trimer is coloured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.g004" target="_blank">Fig 4</a>, and the Fab heavy and light chains shown in forest and olive green respectively, and the approximate location of the viral membrane is indicated with dotted lines. In the zoomed panel insets, the CDR-H1 (pink) and CDR-L3 (yellow) are shown in their predicted binding locations for each model. The fusion peptide is colored purple and shown with surface representation. (C) Neutralization of three heterologous viruses by CAP248-2B and related CDR-H1 and CDR-L3 mutants. Percentage inhibition was plotted on the y-axis versus antibody concentration on the x-axis. Dotted lines indicate y-axis intersections for IC<sub>80</sub>, IC<sub>50</sub>, and IC<sub>20</sub>. (D) ELISA showing binding of CAP248-2B and related mutant antibodies to the BG505(gp120)-CAP45(gp41) chimeric SOSIP trimer. Absorbance readings are plotted on the y-axis and antibody concentration on the x-axis. CAP256-VRC26.09 and F105 are used are positive and negative control antibodies. (E) Anti-cardiolipin antibody ELISA, labelled as in D. (F) HEp-2 cell reactivity assays comparing a no antibody control to 50 μg/mL concentrations of either 4E10 (positive control), 35O22 (negative control), or CAP248-2B.</p
Fine mapping of the CAP248-2B epitope.
<p>(A) Cartoon of the CAP248-2B paratope (shown as a mirror image of the docked model in B, and colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.g003" target="_blank">Fig 3</a>), showing amino acids that have affinity matured relative to the predicted germline genes with main chain Cα spheres. (B) A surface view schematic of gp41 (dark grey) and proximal regions in gp120 (light grey) showing the predicted location of CAP248-2B CDRs and FW3, coloured as in A. The approximate location of the viral membrane is indicated. (C) The envelope trimer is shown in cartoon view with the viral membrane estimated as in B. Regions of Env predicted to form part of the CAP248-2B epitope are coloured and labelled. Point mutants shown to significantly affect CAP248-2B neutralization are shown with black spheres. (D) Table showing neutralization IC<sub>50</sub> titers for CAP248-2B, PGT151, VRC34, 3BC315, 35O22, 10E8, and 8ANC195 against CAP45 and various mutants. The location of each mutant in either gp41 or the gp120 C terminus is shown on the left and coloured as in C. Fold effects on IC<sub>50</sub> are colored, with warmer colours to indicated increasingly negative effect.</p
Escape mutations from CAP248-2B enhance the neutralization of broadly neutralizing antibodies that bind to gp41.
<p>(A) Neutralization of CAP45 and a mutant variant that includes the six gp120 C-terminal mutations identified in CAP248 autologous sequences (CS-Mut), by broadly neutralizing antibodies with epitopes in V2, V3, the CD4bs, the MPER, and the gp120-gp41 interface. Fold changes less than three are within the variation of the assay (no effect). Fold enhancement in neutralization sensitivity to CAP248-2B is indicated in orange (3–10 fold increased sensitivity) and red (> 10 fold increased sensitivity). Conferred resistance to the mutant virus at IC<sub>50</sub> is shown by grey shading. (B) Neutralization of five paired WT (blue) and CS-Mut (red) viruses by 35O22, 10E8, and 4E10.</p
Escape mutations from CAP248-2B accumulate in the gp120 C terminus.
<p>(A) Sequence alignment of the gp120 C-terminus (positions 500–511) from CAP248 autologous viruses at nine weeks (study enrolment), 1, 2, 3 and 3.5 years post-infection. The primary (position 511) and secondary (position 504) gp160 cleavage sites are indicated with arrows. The total number of viruses with identical amino acid sequence within this region are indicated in brackets to the right. Residues undergoing significant selection pressure are indicated with the asterisks. (B) Sequence logograms showing variation within the gp120 C-terminus for all clades, and clade C only, from the LANL HIV sequence database, as well as from CAP248-2B at 3.5 years post-infection, colored and labelled as in A. The global frequencies for each of the autologous mutations identified in CAP248 sequences were: 500 (E8.88%, K46.56%, G3.56%), 502 (K79.35%, R18.73%, Q1.37%), 505 (V98.98%, A0.55%, M0.08%, L<0.01%), 507 (E47.5%, G3.87%, A0.7%), 508 (R98.24%, K1.49%), 509 (E95.11%, G1.53%, A0.27%). (C) Neutralization by CAP248-2B of the heterologous strain CAP45, when compared to gp120 C-terminal mutant viruses with changes identified from autologous CAP248 Env sequences. Data was plotted as percent inhibition (y-axis) against antibody concentration (x-axis). The wild-type virus is shown in black. Dotted lines indicate y-axis intersections for IC<sub>80</sub>, IC<sub>50</sub>, and IC<sub>20</sub>. (D) Neutralization by CAP248-2B of CAP45 wild-type and mutant viruses with the additional gp41 changes identified from CAP248 autologous sequences, plotted as in C. (E) Binding to cleaved (solid bars) or uncleaved (speckled bars) cell-surface expressed Env measured by flow cytometry. Median fluorescence intensity (MFI) is shown on the y-axis, and Palivizumab was used as an HIV-1 negative control. (F) An SDS-PAGE gel of a single SOSIP trimer sample that was divided into two and subsequently captured from suspension by either CAP248-2B or CAP256-VRC26.09. Samples were run in the presence or absence of dithiothreitol (DTT) to assess the level of furin cleavage.</p
The CAP248-2B epitope is proximal to Env glycans, but not affected by glycan heterogeneity.
<p>Schematic of CAP248-2B bound trimer (colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006074#ppat.1006074.g004" target="_blank">Fig 4</a> with gp120 shown in transparent surface grey scale) including modelled (NAG)<sub>2</sub>MAN<sub>3</sub> basic glycans at epitope proximal N88, N611, N616, N625, and N637 residues (colored brown, yellow, pink, purple, and blue respectively). Two views are shown: (A) Top view, and (B) Side view zoom. Relocation of the N88 glycan between 35O22 and CAP248-2B bound states is indicated. (C) Wong glycan array data for CAP248-2B. Weak binding was detected for two hybrid glycans, but significant binding of >500 a.u. was only detected for one biantennary mono-sialylated complex N-glycan (indicated by the glycan schema). (D) Neutralization of CAP45 by CAP248-2B when compared to epitope proximal glycan mutants. The S613A mutant that removed the glycan at N611 but maintains the amino acid side chain properties is shown with red dashed lines and open circles. Percentage inhibition was plotted on the y-axis versus CAP248-2B antibody concentration on the x-axis. (E) Neutralization of the CAP45 N611D single mutant, and various N611D including double glycan mutants, plotted as in D. (F) Neutralization of heterologous tier-2 strains CAP45, CNE52, CAP228, and ZM249 when grown normally (black lines), with co-transfected furin (dashed pink lines), or in the presence of kifunensine (blue lines), swainsonine (cyan lines), and an O-linked glycosylation inhibitor (dashed orange lines), or in a GnTI deficient cell line (grey lines). Neutralization was also assessed in the presence of sCD4 at predetermined IC<sub>40</sub> concentrations for each virus (dashed green lines), or after a 24 hour virus-target cell incubation period (red lines). Graphs are plotted as in D.</p