SHIV Plasma Viral Loads in MVC-treated and comparator macaques.

<p>SHIV Plasma Viral Loads in MVC-treated and comparator macaques.</p

The diversity of the infecting SHIV-162P3 inoculum.

<p>(A) A maximum likelihood tree constructed with 42 independent full-length clones isolated from the infecting SHIV-162P3 inoculum. An unrooted tree layout is displayed. The horizontal scale bar represents genetic distance. (B) Entropy plot of inoculum diversity as a function of nucleotide position.</p

Maraviroc susceptibilities of pseudoviruses with full-length T1 envelopes.

<p>In each graph, the percentages of inhibition relative to the extent of virus replication in the no-MVC control at various MVC concentrations are shown. The MVC susceptibilities of the following clones are shown: (A) Mac46–19, the most prevalent Mac46 day 14 env clone, (B) Mac463–20, a minority day 14 Mac46 env clone, (C) Mac73–34, the most prevalent day 21 Mac73 env clone, (D) Mac803–28, the most prevalent day 14 Mac80 env clone, (E) Mac803–30, a minority day 14 Mac80 env clone, (F) SHIV stock and CR02, the most prevalent env clones from the pre-infection SHIV-162P3 stock and day 14 control macaque CR02, respectively. Error bars represent the standard errors of the means of results from at least two experiments, each performed in triplicate. Nonlinear regression with a variable slope was used to estimate a fitted curve. MVC, maraviroc.</p

Alignment of gp120 V3 and gp41 fusion peptide sequences obtained pre-challenge and from post-challenge time points 1 and 2.

<p>Independent clonal sequences isolated from three MVC-exposed, SHIV-162P3 infected macaques are shown. The pre-challenge sequences are the same for each macaque as they were derived from the SHIV-162P3 challenge stock. Predicted amino acid differences are shown and similarities indicated with dashes. The number of independent clones with the identical sequence is indicated to the left of each sequence. (A) Mac46, (B) Mac73, (C) Mac80. FP, fusion peptide.</p

The relationship between SHIV-162P3 stock full-length <i>env</i> obtained by standard cloning and single genome amplification.

<p>(A) An unrooted maximum likelihood tree constructed with standard clones and 17 previously reported sequences generated by single genome amplification. Blue circles, SGA clones; Yellow circles; standard clones. The horizontal bar represents genetic distance. (B) Highlighter plot indicates the gp160 nucleotide variation between clones. Clones are numbered sequentially; SGA clones are depicted with “sga” after the clone number. Adenine, green; Cytosine, aqua; Thymine, red; Guanine, orange. Grey bars indicate missing sequence.</p

Phylogenetic analysis of T₁ and T₂ gp160 <i>env</i> sequences.

<p>Composite tree of 169 complete gp160 sequences that includes independent sequences isolated from the infecting SHIV-162P3 challenge stock and MVC-exposed and control macaques. Black star, SHIV-162P3 consensus <i>env</i> sequence; open stars, clonal SHIV-162P3 isolates; orange diamonds, day 14 Mac80; light green circles, day 21 Mac73; light blue squares, day 14 Mac46; red diamonds, day 70 Mac80; dark green circles, day 42 Mac73; dark blue squares, day 56 Mac46; grey triangles, day 14 control macaque CR02; black triangles, day 42 CR02; inverted black triangles, day 42 control macaque L375. Numerals indicate posterior probabilities of node support. The horizontal scale bar represents genetic distance.</p

Correlations between trimer-binding and neutralizing antibody titers.

<p>The scatterplots show neutralization titers on the y-axes and the trimer-binding antibody titers on the x-axes. Within each plot the symbols corresponding to neutralization of the BG505.T332N and B41 viruses are color-coded as indicated on the figure panels. Spearman correlation coefficients (r-values) and the corresponding significances (p-values) are color-coded analogously. Correlation analyses were performed for both BG505 and B41 NAb and binding antibody titers for sera from the early monovalent immunogen regimens during weeks 4–36 (top panel, group 1; lower panel, group 3).</p

Mapping autologous NAb specificities using BG505.T332N, B41 and CZA97 virus mutants.

<p><b>A</b>, Neutralization of BG505.T332N virus mutants. The values recorded for the various mutant viruses are the percentage neutralization at a dilution of 1/50, relative to the BG505.T332N parental virus (labeled WT and defined as 100%), and are the averages of 2 replicates ± s.e.m. Red boxes highlight fully or substantially resistant viruses (<25% neutralization); yellow, moderately resistant viruses (25–75% neutralization); green, sensitive viruses (>75% neutralization). The Q130N, S241N and P291T changes introduce N-linked glycans at positions 130, 241 and 289, respectively. The S241N+P291T double mutant contains glycans at both positions 241 and 289. The MG505 cl.A2 and cl.H3 viruses differ from BG505.T332N at several positions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s002" target="_blank">S1 Fig</a>). Of note is that MG505 cl.A2 has a lysine residue at position-241 (i.e., as per the S241K mutant), whereas cl.H3 has a glycan site (i.e., as per the S241N mutant). A glycan is present at position-289 in both MG505 clones. The K241S change in MG505 cl.A2 restores the Ser residue that is present at position-241 in the BG505.T332N virus. Full titration curves for the sera showed only a single example of reduced neutralization of the glycan knock-in mutants, compared with BG505.T332N, that was not evident at the standard test dilution of 1/50 (rabbit #5739 at week 62; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s005" target="_blank">S4 Fig</a>). Overall, the extent of neutralization of the glycan knock-in mutants did not correlate well with the titers against the wild-type BG505.T332N virus (Spearman rank correlation for the 241-glycan knock-in: r = 0.30, p = 0.099; for the 289-glycan knock-in: r = 0.21, p = 0.25; for the double mutant: r = 0.27, p = 0.15). <b>B</b>, Neutralization of B41 virus mutants. The organization is the same as in panel-A. The B41 parental virus is labeled WT and its neutralization defined as 100%. The N132T and A291T changes into this virus introduce N-linked glycans at positions 130 and 289, respectively. The CH01 and VRC01 bNAbs were used as control reagents for assessing overall neutralization sensitivity (only shown for B41 mutants for which a partial effect on CH01 neutralization was observed). <b>C</b>, Neutralization of CZA97 virus mutants. The organization is the same as in panel-A. The serum dilution used was 1/60. The CZA97 cl.12 parental virus is labeled cl.12 and its neutralization defined as 100%. The CZA97 cl.29 virus differs from cl.12 at several positions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s002" target="_blank">S1 Fig</a>) but of note is that it contains a glycan at position-411. The CZA97 cl.12-D411N and cl.29-N411D mutants contain and lack glycans at position-411, respectively.</p

Individual rabbits immunized with three different trimers can develop moderate to strong autologous NAb responses to each of them^a.

<p>Individual rabbits immunized with three different trimers can develop moderate to strong autologous NAb responses to each of them<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#t001fn001" target="_blank">^a</a>.</p

Cross-boosting of clade A and clade B autologous NAb responses by clade C trimers.

<p>NAb titers at the indicated time points for all five animals in the listed groups are compared. Bars show arithmetic means + s.e.m. The clade C trimer boosts occurred at weeks 36 and 48. <b>A</b> and <b>C</b>, BG505.T332N titers; <b>B</b> and <b>D</b>, B41 titers.</p