Acute phase CTL responses against ARF-10 in two SIV-infected Rhesus macaques.

<p>A) and B) Magnitude of CTL responses against 15-mers in ARF-10 in Rhesus macaques rh2261, A, and r04028, B, as measured by IFN–γ ELISPOT using cryopreserved PBMC harvested 3 weeks after initial SIV infection. The SIV infection history of the animals is described in the text. Bars are color coded to match with remaining panels in the figure. C) and D) Peptide dilutions to fine map the minimal optimal epitopes targeted in ARF-10 by rh2261, C, and r04028, D. For rh2261, we used PBMC harvested in the acute phase of infection. For animal r04028, due to PBMC sample limitations, we expanded antigen-specific CTL by exposing PBMC to autologous irradiated BLCL pulsed with the responsive 15-mer for several weeks and used these cells in an ELISPOT. For panels A–C, we used 100,000 PBMC per well, in duplicate, in ELISPOT plates. For the epitope mapping with r04028, we used 20,000 antigen-specific cells per well combined with 10,000 autologous BLCL per well as antigen presenting cells. Peptides tested are shown at right of each mapping panel and the peptides we determined represented the minimal epitopes are shown in color to match panels A and B. E) The mapped epitopes within ARF-10 are boxed using the color scheme described above.</p

Acute phase CTL responses against ARF-1 in two SIV-infected Rhesus macaques.

<p>A) and B) Similar to panels A and B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g002" target="_blank">figure 2</a>. Here, we depict the week 3 responses to 15-mers within ARF-1 in two animals, r97111, A, and r97035. The infection history of these animals is described in the text. C) and D) The minimal epitope was mapped to the RP9 peptide in both animals using serial 10-fold dilutions of peptide in IFN–γ ELISPOT assays. The peptides used are shown at right and the RP9 peptide, which was determined to be the minimal epitope is shown in orange. E) The location of the RP9 epitope within ARF-1.</p

Viral sequence evolution in overlapping reading frames encoding Env and ARFs -1 and -10.

<p>Sequence evolution in overlapping reading frames in two animals that targeted ARF-1 at three weeks post-infection; r97111, A, and r97035, B. The targeted epitope is shown boxed in orange for each animal. Data from acute phase time points are from deep pyrosequencing, while the single chronic time point assayed in each animal was sequenced with Sanger sequencing. The key is in the upper right and is identical to that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g003" target="_blank">figure 3</a> and details of sequence methods and analysis are depicted in the caption for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g003" target="_blank">figure 3</a> and in the Materials and Methods section. C) The portion of sequence reads matching inoculum within targeted epitopes (solid shapes) contrasted with the portion matching inoculum outside the epitope in ARF-1. D) IFN-γ ELISPOT assay to measure the ability of PBMC from r97111 to recognize dilutions of the wild type RP9 peptide (orange) versus escape mutant (blue) peptide.</p

The genomic locations of ARFs -1 and -10 as well as the primers used to amplify this region for pyrosequencing, within the SIVmac239 genome.

<p>The SIVmac239 genome is 10,535 nt in length. The ORFs encoding the classical viral proteins are shown in blue and the ORFs encoding the ARFs studied in this report are shown in orange. Note that ARF-10 is depicted as separate from Rev exon 1 to emphasize its independent translation. However, ARF-10 is a composite of the first exon of Rev and the first 50 amino acids translated from the Rev intron. The relative locations of the forward and reverse primers used to amplify viral RNA for pyrosequencing are shown as labeled black arrows. This image was created using the software Geneious version 5.6.4 created by Biomatters, available from <a href="http://www.geneious.com" target="_blank">www.geneious.com</a>.</p

MID-tagged oligonucleotides used to generate ARF1/ARF10/Env specific amplicons.

<p>MID tags are in bold italics. Sequence-specific primer is 3′ to the MID tag while adaptor sequence is 5′ to the tag in each oligonucleotide. The number of MID-tagged primers is less than the number of samples as some primers were re-used in separate runs.</p

Viral sequence evolution in overlapping reading frames encoding Env and ARFs -1 and -10.

<p>We used next generation pyrosequencing (454 Life Sciences) of the amplicon depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g001" target="_blank">figure 1</a> to sequence the portion of SIV encoding ARF-10, ARF-1 and the first 89 amino acids of Env. A) Kinetics of viral evolution at weeks 4, 8 and 12 from animal rh2261. Mutations synonymous in a given reading frame are boxed in green and non-synonymous changes are boxed in yellow. Matching residues are depicted as “.”. Mutations are depicted as “X” when nucleotide mutations in a given codon could give rise to more than one amino acid. The shade of the box surrounding a mutation represents the frequency of underlying mutations at that codon. The key is in the upper right of the figure. Only mutations present at >1% are shown. The minimal epitope mapped in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g002" target="_blank">figure 2</a> is shown boxed in the same animal-specific color coding as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g002" target="_blank">figure 2</a>; The QW9 epitope for animal rh2261, A, and the AF8 epitope for animal r04028, B. We used simple Sanger sequencing of virus derived from each animal at their individual times of euthanasia to determine the consensus escape patterns in this region after the resolution of the acute phase of infection. C) The frequency of amino acid sequences representing the inoculum within the targeted epitope (intra-epitope, solid shapes) and outside the targeted epitopes (extra-epitope, open shapes) within ARF-10 plotted against weeks post infection. D) IFN-γ ELISPOT assay of recognition of the peptide representing the wild type QW9 sequence (orange) versus the escape variant (orange) by PBMC isolated from animal rh2261 from 4 weeks post infection. E) Intracellular cytokine staining (ICS) assay to measure recognition of a CTL line from r04028 against the wild type AF8 peptide (orange) versus the escape variant (blue). Details of the CTL line are described in the text.</p