Gag_206–216, Gag_241–249, and Gag_367–381 epitope-specific CD8⁺ T-cell responses in SIV controllers.

<p>(A) Frequencies of CD8⁺ T cells specific for SIV Gag_206–216, Gag_241–249, and Gag_367–381 epitopes in Group M (upper panels) and Group N (lower) at 4 months (4M), 1 year (1Y), and 2 years (2Y) post-infection. (B) Comparisons of the sum of Gag_206–216-, Gag_241–249-, and Gag_367–381-specific CD8⁺ T-cell frequencies at 4M, 1Y, and 2Y between Groups M and N. No significant difference was observed between the groups.</p

Dominant non-synonymous mutations in proviral <i>gag</i> in SIV controllers.

<p>Amino acid substitutions around SIV Gag_206–216, Gag_241–249, and Gag_367–381 epitopes and in other Gag regions approximately 2 months (2M, top), 1 year (1Y, middle), and 2 years (2Y, bottom) after SIVmac239 challenge are shown. Most of the proviral gag fragments were amplified from CD4⁺ T cells isolated from PBMCs, while those at 2 years in macaques R06-037, R05-005, R07-001, and R07-006 were from cultured CD4⁺ T cells due to limitation of available cell numbers. Mutant sequences shown were completely dominant (i.e., wild-type sequences were undetectable at the residues showing mutant sequences) except for the L216S mutation (the ratio of wild type/mutant: 2/5) in macaque R03-018 at 1 year post-infection. No subdominant mutation was detected.</p

SIV antigen-specific CD8⁺ T-cell responses in SIV controllers.

<p>(A) Frequencies of CD8⁺ T cells specific for Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env in Group M (upper panels) and Group N (lower) at 4 months (4M), 1 year (1Y), and 2 years (2Y) post-infection. Responses were measured by detection of antigen-specific IFN-γ induction using panels of overlapping peptides spanning the entire SIVmac239 Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env amino acid sequences, respectively. (B) Comparisons of CD8⁺ T-cell frequencies specific for SIV antigens other than Gag and Nef at 4M, 1Y, and 2Y between Groups M and N. Group M had significantly higher frequencies of SIV non-Gag/Nef antigen-specific CD8⁺ T cells at 4M (p = 0.0095 by Mann-Whitney U-test) and 1Y (p = 0.0095). (C) Comparisons of the numbers of CD8⁺ T cell-targeted SIV antigens other than Gag and Nef at 4M, 1Y, and 2Y between Groups M and N. The numbers were significantly higher in Group M at 4M (p = 0.0095 by Mann-Whitney U-test) and 1Y (p = 0.0095).</p

SIV controllers analyzed in this study

<p>^aAnimals were unvaccinated or vaccinated with DNA-prime/SeV-boost expressing EGFP (control), Gag_202–216-EGFP and Gag_236–251-EGFP (Gag_{206–216/241–249}), Gag_236–251-EGFP (Gag_241–249), or Gag_202–216-EGFP (Gag_206–216) before SIVmac239 challenge. All of them controlled SIV replication for more than 2 years.</p><p>^bpositive, viral <i>gag</i> fragments were detected by RT-PCR amplification from culture supernatant-derived RNAs of PBMCs at 2 years; negative, those were undetectable.</p><p>^cOn the basis of the data on proviral <i>gag</i> nucleotide sequences at 2 years post-infection, animals were divided into two groups, Group M with multiple mutations and Group N with no mutation.</p><p>SIV controllers analyzed in this study</p

Alleles in the second MHC-I haplotypes in macaques^a

<p>^aDetected alleles not included in the first MHC-I haplotype <i>90-120-Ia</i> are shown.</p><p>Alleles in the second MHC-I haplotypes in macaques<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#t003fn001" target="_blank">^a</a></p

Virological and immunological analyses in macaque R09-009 following CD8⁺ cell depletion.

<p>(A) Changes in peripheral CD8⁺ T-cell counts after the initial anti-CD8 antibody administration. Group N macaque, R09-009, was administered anti-CD8 antibody at week 108 post-infection and on days 3, 7, and 10 after the first administration. (B) Changes in plasma viral loads. (C) Changes in CD8⁺ T-cell responses specific for SIV Gag, Nef, Vif, Vpx, Vpr, Tat, Rev, Pol, and Env. (D) CD8⁺ T-cell responses specific for SIV Gag_206–216, Gag_241–249, Gag_367–381, Vif_114–124, Nef_9–19, Nef_89–97, and Nef_193–203 epitopes at week 113 post-infection. (E) Dominant non-synonymous mutations in plasma viral cDNA regions encoding Gag, Vif, and Nef epitopes. Viral <i>gag</i>, <i>vif</i>, and <i>nef</i> cDNA fragments were amplified from plasma RNA obtained at weeks 110 and 118 post-infection. Amino acid substitutions around SIV Gag_206–216, Gag_241–249, Gag_367–381, Vif_114–124, Nef_9–19, Nef_89–97, and Nef_193–203 epitopes are shown.</p

Plasma viral loads determined by using fivefold-concentrated plasma^a

<p>^aAfter centrifugation of 1 ml of plasma at 25,000 x <i>g</i> for 2 hours, 0.8 ml of its supernatant was discarded for fivefold concentration of plasma. The remaining 0.2 ml was subjected to RNA extraction for quantitation of viral loads (VL) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#ppat.1005247.ref025" target="_blank">25</a>]. Plasma samples of R06-037 and R07-006 at 2 years post-infection (pi) were unavailable (ND, not determined).</p><p>Plasma viral loads determined by using fivefold-concentrated plasma<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#t002fn001" target="_blank">^a</a></p

Nef_9–19, Nef_89–97, Nef_193–203, and Vif_114–124 epitope-specific CD8⁺ T-cell responses in SIV controllers.

<p>(A) Frequencies of CD8⁺ T cells specific for SIV Nef_9–19, Nef_89–97, Nef_193–203, and Vif_114–124 epitopes in Group M (upper panels) and Group N (lower) at 4 months (4M), 1 year (1Y), and 2 years (2Y) post-infection. (B) Comparisons of the sum of Nef_9–19-, Nef_89–97-, Nef_193–203-, and Vif_114–124-specific CD8⁺ T-cell frequencies at 4M, 1Y, and 2Y between Groups M and N. The sum of CD8⁺ T-cell frequencies specific for these epitopes in Group M was significantly higher compared to Group N at 2Y post-infection (p = 0.0190 by Mann-Whitney U-test).</p

Dominant non-synonymous mutations in proviral <i>vif</i> and <i>nef</i> in SIV controllers.

<p>In the upper panel, amino acid substitutions around SIV Vif_114–124 epitope and in other Vif regions approximately 2 years after SIVmac239 challenge are shown. In the lower panel, amino acid substitutions around SIV Nef_9–19, Nef_89–97, and Nef_193–203 epitopes and in other Nef regions approximately 2 years after SIVmac239 challenge are shown. Sequences of <i>vif</i> in macaques R03-018 and R07-008 and <i>nef</i> in macaques R07-008 were not determined because these cDNA fragments could not be amplified. Macaques R07-001 and R07-003 had multiple G-to-A mutations in <i>nef</i> (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#ppat.1005247.s001" target="_blank">S1 Fig</a>). Mutant sequences shown were completely dominant except for <i>vif</i> P138L (the ratio of wild type/mutant: 1/2) in R05-005, <i>vif</i> Q162R (1/1) in R07-006, <i>nef</i> P012T (1/10) in R06-037, and <i>nef</i> S013P (3/10), I090T (1/5), D096N (1/5), and E191K (1/5) in R07-002. In addition, subdominant <i>nef</i> mutations resulting in P012S (5/2) and G044E (5/2) were detected in macaque R07-002, while the wild-type sequences were dominant at these positions.</p

Plasma viral loads and peripheral %CD4 in SIV controllers.

<p>(A) Plasma viral loads (SIV <i>gag</i> RNA copies/ml plasma) determined as described previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005247#ppat.1005247.ref026" target="_blank">26</a>]. The lower limit of detection is approximately 4 x 10² copies/ml. On the basis of the data on proviral <i>gag</i> nucleotide sequences at 2 years post-infection, animals were divided into two groups, Group M (M) with multiple CD8⁺ T-cell escape mutations and Group N (N) with no mutation. (B) Percentage of CD4⁺ T cells in PBMCs.</p