Two-dimensional histograms illustrating the distributions of g2p FPR predictions across all replicate ancestral reconstructions on the maximum credibility tree.

<p>The -axis corresponds to time intervals from to the first positive MT-2 assay (), rescaled for each subject. The -axis corresponds to the log-transformed FPR predictions for the ancestral sequences. Both axes were partitioned into 25 bins. Each cell is coloured with respect to its FPR value with opacity proportional to the square root of the number of data points in the corresponding bins, normalized by the total number of points in the time interval.</p

Simulated trajectories of genotype frequencies (solid and dashed lines) and population-level coreceptor usage phenotype (shaded regions) under the fitness valley and gradual models of HIV coreceptor usage evolution.

<p>Simulations were generated under a five-allele Moran model with mortality selection <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002753#pcbi.1002753-Muirhead1" target="_blank">[42]</a>, effective population size , forward mutation rate of per replication, and fitness vectors of (1, 1.025, 1.05, 1.075, 1.1) and (1, 0.999, 0.999, 0.999, 1.1) corresponding to gradual and valley landscapes, respectively. Note that the relatively rapid and complete fixation of the fifth variant is partly due to the model assumption of no back mutation, and is not consistent with the observation that CXCR4-using variants tend to remain a minority species in HIV infections.</p

Mutations within the V3 loop comprising the predominant pathway for each subject, stratified by time of emergence.

<p>The MARCS V3 sequence reconstructed at the MRCA of the maximum credibility tree is shown at the top of each plot. Residues highlighted in red correspond to mutations that arose in a CXCR4-using background (FPR3.5). The duration of HIV coreceptor evolution from to the first CXCR4-using ancestor is indicated in months alongside each plot (see text).</p

Excerpts from the maximum credibility trees for HIV evolution within subjects DS2 and DS7 with reconstructed mutations mapped to individual branches (labels comprising the ancestral residue, position in the V3 loop, and the derived residue).

<p>These excerpts emphasize the lineages that attained a CXCR4-using ancestral genotype (FPR3.5). Branches are coloured with respect to the predicted FPR value (see legend inset). Vertical lines indicate the times of the first serum sample (dashed) and first positive MT-2 assay (solid), respectively. Open circles indicate the start of the branch carrying mutations promoting CXCR4 usage, which otherwise cannot be distinguished because other branches that do not carry such mutations have been collapsed. Percentiles indicate the fraction of the most recent sample that descend from the corresponding lineage.</p

Membrane-associated (ma) IL-15 of DC and CD40L expression of CD4⁺ T cells in 5 groups of macaques.

<p>(A) Membrane-associated (ma) IL-15 of DC and (B) CD40L expression on CD4⁺ T cells in 5 groups of macaques pre- and post-4^th immunization, presented as % mean (±sem). The significance between pre- and post-immunization was analysed by paired “t” test and differences between the 5 groups after immunization was analysed by ANOVA.</p

Correlation between DC maIL-15 and A3G mRNA or protein in CD4⁺ central and effector T or B memory cells.

<p>Correlation between maIL-15 on DC and A3G mRNA in PBMC (A<b>–</b>C), intracellular A3G protein in CD4⁺CD95⁺CCR7⁺ central memory cells (D<b>–</b>F), CD4⁺CD95⁺CCR7⁻ effector memory cells (G,I), CD20⁺ B cells (J<b>–</b>L) or CD20⁺CD27⁺ memory B cells (M<b>–</b>O) in the combined groups (1<b>–</b>5), group 1 or group 3 macaques, respectively. IL-15 and A3G were assayed after the last immunization and before the animals were challenged with SHIV SF162.P4. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p

AID and A3G expression in CD20⁺ B cells pre- and post-2^nd immunization and their correlation.

<p>Comparative investigation of (A) AID and (B) A3G expression in CD20⁺ B cells before and after the 2^nd immunization in the 4 groups of immunized macaques; group 5 unimmunized controls remained unchanged (data not presented). Correlation between A3G and AID expression in CD20⁺ B cells in the 5 groups after 2^nd immunization is presented in (C). Representative flow cytometry of AID and A3G in pre- and post 2^nd immunization is shown in (D). * p<0.05 and ** p<0.01. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p

A3G in CD20⁺ and CD27⁺ memory B cells pre- and post-immunization in the 4 groups.

<p>Expression of A3G in (A) CD20⁺ B cells and (B) CD20⁺CD27⁺ memory B cells in 5 groups of macaques before and after the 4^th immunization assayed by flow cytometry with MAb to A3G, CD20 and CD27 and (C) representative illustration; (n = 8 per group, except gp4 n = 6). * p<0.05. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p

Correlation between the viral load and A3G mRNA, protein in B cells or AID.

<p>Correlation between PVL or CVL and A3G mRNA in PBMC (A,B), AID (C, D), A3G proteins in CD20⁺ B cells (E,F) and A3G in CD4⁺CD95⁺CCR7⁻ effector memory T cells (G,H). AID and A3G were assayed after the 4^th immunization, whereas CVL was calculated as the “area under the curve”. The two protected macaques are shown by open circles. Pearson's correlation coefficient was used for statistical analysis. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p

Correlation between A3G memory B and CD4⁺ T cells, and A3G mRNA with HLA or neutralizing antibodies.

<p>Correlation between A3G in CD20⁺CD27⁺ memory B cells and CD4⁺CD95⁺CCR7⁻ effector memory T cells (A) in all 5 groups, (B) in group 1 (without SHIV antigens) and (C) in group 3 macaques. Correlation between A3G mRNA in PBMC and serum anti-HLA class I antibodies (D<b>–</b>F), anti-HLA class II antibodies (MFI) (G<b>–</b>I) assayed by the Luminex HLA antibody method and neutralizing activity (J<b>–</b>L) determined by using a TZM-b1 assay in the corresponding groups. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p