8 research outputs found

    Rapidly expanding sequence diversity during HIV-1 infection.

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    <p>Heat maps illustrate sites exhibiting amino acid sequence diversity at days 0, 3, 59, 165, 476 and 1543 post-presentation. Plotted is the percentage of amino acid diversity at each position with respect to the dominant baseline (day 0) amino acid residue. All 3174 amino acids of HIV-1 are represented, with the first amino acid of Gag located in the top left corner of the grid and the last amino acid of Nef located in the bottom right corner. Completely conserved residues are <i>dark blue</i>, low-level variant residues (<10% divergent from baseline) are <i>light blue</i>, moderately variable residues (10–50%) in <i>orange</i>, and highly variant residues (>50%) in <i>red</i>. (<b>A</b>) 0 days p.p., (<b>B</b>) 3 days p.p., (<b>C</b>) 59 days p.p., (<b>D</b>) 165 days p.p., (<b>E</b>) 476 days p.p., (<b>F</b>) 1543 days p.p..</p

    Comparison of sequence variant quantification by 454 deep sequencing and by PCR cloning/sequencing.

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    <p>Orthogonal regression of variant frequency estimates obtained by 454 and clonal sequence data across the highly variable 1544 nucleotide region spanning Vif to Tat in subject 9213 (slope = 1.01; 95% CI, 0.73 to 1.40).</p

    Viral escape from acute and chronic phase CD8+ T cell responses.

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    <p>Stacked heat-maps illustrate variant codon frequencies over time for each residue of the CD8 epitopes targeted by subject 9213. Shown are epitopes targeted during the acute (Day 59) and chronic (Day 476) phases of HIV-1 infection. The baseline sequence is shown at the top of each epitope, with non-HIV-1B consensus residues highlighted in <i>blue</i>. The magnitude of each response is shown in SFC per million PBMC.</p

    Limited evolution in the HIV-1 proteome prior to establishment of viral set point.

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    <p>Sequence diversity is plotted for all evolving codons in each HIV-1 protein as the percent of sequences with an amino acid residue different from the dominant baseline residue. Colored lines denote individual evolving amino acid residues within each protein. The time of infection prior to the establishment of viral set point (day 165) is highlighted in <i>grey</i>.</p

    Cellular immune responses drive early low-frequency quasispecies diversity.

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    <p>(<b>A</b>) For each protein, the average frequency of non-dominant baseline residues of positions within the 19 described CD8 epitopes restricted by subject 9213's HLA alleles (<i>left</i>) and outside of the 19 described epitopes (<i>right</i>) is plotted for each time point sequenced. <i>Colored lines</i> denote the proteins for which diversity was substantially higher inside of CD8 epitopes versus outside CD8 epitopes. (<b>B</b>) To determine rates of viral escape for each epitope escape mutations were defined as any amino acid substitution within the epitope. <i>Symbols</i> denote the cumulative observed frequency of all escape mutations, and lines depict the best fit by non-linear regression of the observed frequency data to the CTL escape model of Asquith et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002529#ppat.1002529-Asquith1" target="_blank">[65]</a>. <i>Open symbols</i> and dashed lines denote epitopes for which evolution was consistent with reversion. <i>Black symbols</i> and <i>dotted lines</i> denote epitopes for which there was no evidence of escape. CD8 responses against each epitope are shown in parentheses in the legend and were measured by IFN-gamma Elispot assay (Spot Forming Cells/Mill PBMC (SFC)). (<b>C</b>) Frequency of wild-type (<i>black</i>) and variant (<i>red</i>) haplotypes of the Vif B38-WI9 epitope and flanking regions over time. Shown at the top is the clade B consensus sequence for reference. (<b>D</b>) Frequency of wild-type (<i>black</i>) and variant (<i>red</i>) haplotypes of the Nef A24-RW8 B38-WI9 epitope and flanking regions over time. <i>Blue</i> residues highlight differences between the day 0 transmitted sequence and HIV-1B consensus sequence.</p

    Clinical course and whole genome deep sequence coverage for subject 9213.

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    <p>(<b>A</b>) Clinical course of infection in subject 9213 shown as days post-presentation. Plasma viral load (copies/ml) is shown in <i>red</i> and CD4+ T cell count (cells/ml) in <i>blue</i>. Estimated acute/early Fiebig stages are shown and <i>arrows</i> indicate time points sequenced on the deep sequencing platform. (<b>B</b>) High-quality sequencing reads per site across the HIV-1 genome for subject 9213 at six time points (days post presentation). Reads are aligned to the consensus assembly of their respective time point using <i>Mosaik v1.0</i> (<b>Table S9</b> in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002529#ppat.1002529.s001" target="_blank">Text S1</a></b>) and coverage (sequencing reads per site) calculated from bases that pass the defined Neighbor Quality Standard (NQS, see Supplementary Methods in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002529#ppat.1002529.s001" target="_blank">Text S1</a></b>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002529#ppat.1002529-Altshuler1" target="_blank">[28]</a>. PCR amplicon locations are denoted by horizontal bars under the x-axis.</p

    Variant-specific CD8+ T cell Elispot responses.

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    <p>Elispot responses in Spot Forming Cells (SFC) per million PBMC to wild-type and variant peptides for the two dominant epitopes (<b>A</b>) Vif B38-WI9 (WHLGQGVSI) and (<b>B</b>) Nef A24-RW8 (RYPLTFGW). Bars in <i>black</i> denote responses to clade B consensus epitopes. Bars in <i>red, orange, and pink</i> denote responses to epitopes containing escape variants.</p

    Reversion of transmitted mutations over the course of infection.

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    <p>(<b>A</b>) Rates of reversion of transmitted mutations within both restricted and unrestricted CD8 epitopes in subject 9213. Reversion was defined as the replacement of a transmitted non-consensus residue by the HIV-1B consensus residue. <i>Symbols</i> denote the observed frequency of viruses expressing the consensus residue and <i>lines</i> depict the best fit by non-linear regression of the observed frequency data to the CTL escape model of Asquith et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002529#ppat.1002529-Asquith1" target="_blank">[65]</a>. <i>Closed symbols</i> and <i>solid lines</i> denote epitopes restricted by subject 9213, while <i>open symbols</i> and <i>dashed lines</i> denote epitopes not restricted by 9213. Listed in <i>parentheses</i> are the mutations listed by the consensus residue, HXB2 position, followed by the transmitted mutation, i.e., F197S. (<b>B</b>) Rates of reversion of all transmitted mutations exhibiting sequence variation over the course of infection. Each line represents a different mutation.</p
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