HLA haplotypes, longitudinal plasma viruses and inferred transmitted HIV.

<p>Suggested HLA haplotypes and longitudinal plasma viruses of the study patients are shown. Transmitted viruses were inferred based on viral sequence and immune response. LNR, Low Number of Reads; ND, Not Determined; NA: Not applicable.</p

Differential responses to Nef134-8(Y135) and Nef134-10(Y135) in patient groups 1 and 2.

<p>% live CD8⁺ cells reactive to Tet-8(Y135) (x-axis) and Tet-10(Y135) (y-axis) after stimulation. Horizontal and vertical dashed lines denote the cut off value for defining tetramer reactivity (mean + 2SD of 5 healthy volunteers). Circles, subgroup 1A and 2A; triangles, subgroup 1B and 2B. Red, Y135-provirus-positive; green, Y135-provirus-negative Tet-8-positive; blue, Y135-provirus-negative Tet-8-negative. Single open circle, Y-135-provirus-LNR, Tet-8-negative.</p

Baseline clinical data of the studied patients.

<p>Baseline clinical data of the studied patients.</p

Longitudinal comparison of clinical markers between HLA-A*24:02-positive patients inferred to have acquired the Y135F-containing virus and others.

<p>Plasma viral loads (copies/ml; A) and CD4 T cell counts (cells/μl; B) at the first visit and 50 week intervals afterwards are shown for patients for whom de novo infection with HIV-1 containing Y135 could not be ruled out and patients inferred to have acquired Y135F at transmission. Symbols reflect the median of 3 (IQR 2–4) measurements recorded per patient in each period. Symbols and color codes are the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150397#pone.0150397.g004" target="_blank">Fig 4</a>. Horizontal lines and error bars denote the median and IQR for each group. P-values were calculated using the Mann-Whitney test.</p

Phylogenetic tree of the <i>nef</i> gene nucleotide sequences of HIV-1 in the plasma.

<p>HIV-1 <i>nef</i> gene in the plasma from the 31 patients in the study was amplified by the RT-PCR and directly sequenced. Published <i>nef</i> sequences from SF2 strain (GenBank accession number: K02007) and global subtype B consensus in 2004 (provided at HIV database; <a href="http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html" target="_blank">http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html</a>) were included as controls.</p

A*24:02 stabilization and endogenous antigen presentation assays.

<p>(A) A*24:02 stabilization assay. Binding of Nef134-8 (blue circles) and Nef134-10 (red circles) and their Y135F-containing peptide variants (blue and red triangles, respectively) to T2 cells expressing A*24:02 was measured. Symbols and error bars denote the mean and SEM, respectively. A peptide derived from an HLA-B*35-restricted epitope in HIV-1 reverse transcriptase (B35-14) served as a negative control. % maximum fluorescence was calculated by dividing the MFI of the sample by the maximal MFI in all measured samples in each experiment in each independent experiment. (B) Renilla luciferase (hRluc) activity after transfection of the minigene expressing Nef(Y135), Nef(Y135F), Gag18-46 fused to hRluc or mock controls (culture without transfection). Histograms and error bars denote the mean and SD of triplicate cocultures performed in 3 independent experiments. RLU: Relative light units. (C) Recognition of endogenously expressed epitopes derived from Nef(Y135), Nef(Y135F) and Gag18-46 minigenes by dually specific CTL clones T26-102 or C1-28. Histograms and error bars denote the mean and SEM of replicate IFN-γ ELISA measurements derived from triplicate co-cultures performed in 3 independent experiments. Results are normalized to the mean IFN-γ concentration generated by CTL clones upon co-culture with Nef(Y135)-expressing cells. Asterisks (***) denote p < 0.001 calculated using the Tukey post-test after one-way ANOVA. (D) Double-tetramer staining of CTL clone T26-102 with A*24:02/Nef134-8(Y135) tetramer-allophycocyanin (Tet-8(Y135)-APC) and A*24:02/Nef134-8(Y135F) tetramer-phycoerythrin (Tet-8(Y135F)-PE) (left) and CTL clone C1-28 with A*24:02/Nef134-10(Y135) tetramer-allophycocyanin (Tet-10(Y135)-APC) and A*24:02/Nef134-10(Y135F) tetramer-phycoerythrin (Tet-10(Y135F)-PE) (right).</p