CD8ɑ+ cells suppress SIV replication without evidence of viral immune escape during post treatment control

Abstract

Post-treatment control (PTC) is a rare phenomenon in which people living with HIV (PLWH) maintain viral control following ART interruption. Characterizing virus populations present in PTCs may help elucidate mechanisms of immunologic control, but this is challenging without detectable plasma viremia. To model PTC, eight Mauritian cynomolgus macaques (MCM) were infected with barcoded SIVmac239M and began an 8-month ART regimen two weeks post-infection (wpi). Six months following ART interruption, all MCM were rechallenged with non-barcoded SIVmac239 followed by CD8ɑ+ cell depletion two months later. Animals were grouped as viremic (n=5) or aviremic (n=3) based on the detection of plasma viremia between ART interruption and CD8ɑ+ cell depletion; all animals became viremic post-depletion. Barcode sequencing of plasma virus revealed that lineages with high pre-ART viral loads dominated the rebounding populations post-depletion and detectable rechallenge virus in two animals post-depletion. Additional sequencing of three CD8+ T cell epitopes within plasma viruses identified point mutations only in viruses isolated from the viremic group post-depletion. A second cohort of 5 MCM who initiated ART 8 wpi was examined to identify the impact of the timing of ART initiation on viral epitope diversity and showed increased diversity prior to ART initiation and following ART interruption. These results suggest that early ART initiation is associated with reduced diversity within cytotoxic T lymphocyte (CTL) epitopes and a longer time to rebound, as well as highlight an important role of restricting the emergence of CTL immune escape variants in increasing the likelihood of PTC.

Figure 1.

(A) Study design and timeline. Intravenous SIVmac239M infection is indicated by the white virion, ART administration is indicated by the grey bar, intravenous SIVmac239 rechallenge is indicated by the pink virion, CD8ɑ+ cell depletion is indicated by the green x-ed out cell, and necropsy is indicated by “Nx.” Animals with no detectable viremia between ART interruption and CD8ɑ+ cell depletion are referred to as “Early ART aviremic” (n=3, blue). The remaining animals with detectable viremia above 10³ copies/mL of plasma for at least two consecutive time points between ART interruption and CD8ɑ+ cell depletion are referred to as the “Early ART viremic” (n=5, black). (B) SIV viral loads over the course of the animal study, with each animal indicated by a unique color and symbol combination, with blue representing aviremic animals and black representing viremic animals. ART was administered between days 14 and 268 post-SIVmac239M infection and indicated by the grey shaded area. Intravenous SIVmac239 rechallenge occurred on day 442 post-SIVmac239M infection. Administration of a CD8ɑ+ cell-depleting antibody occurred on day 497 post-SIVma239M infection. Necropsies were conducted between days 539 and 556 post-SIVmac239M infection.

Figure 2.

Number of unique SIVmac239M lineages detected in the plasma during the first two weeks of SIVmac239M infection, prior to ART initiation (A) and following CD8ɑ+ cell depletion (B). Animals are coded by unique color and symbol combinations, with blue representing aviremic animals and black representing viremic animals. Only barcodes present at a frequency of 0.002 or greater, which corresponds to 1/minimum input templates of time points included in this analysis, were included. Significance is determined by Mann-Whitney test.

Figure 3.

Distribution of SIVmac239M lineages throughout time for the viremic (A, cy1037) and aviremic (B, cy1044) animals which had detectable rechallenge virus following intravenous rechallenge. Each unique lineage detected post-ART or post-depletion is indicated by color, with the rechallenge virus indicated in bright pink. In the case of cy1044 (B), only the top ten lineages detected post-depletion are shown due to color and space constraints; the remaining lineages are show in shades of grey.

Figure 4.

Detection of SIVmac239M lineages following ART interruption (blue), SIVmac239 rechallenge (pink) and after CD8a+ cell depletion (green). Grey dots represent the peak pre-ART viral load of each lineage, with lineages ranked from highest pre-ART replication to lowest pre-ART replication. The rechallenge virus is present on the far right of graphs B (cy1037) and H (cy1044), as this lineage was absent prior to ART initiation but was detected post-rechallenge and depletion.

Figure 5.

Proportion of variant Gag_386–398GW9 (blue), Nef_103–111RM9 (teal), and Rev_59–68SP10 (purple) epitope sequences in viremic (left, black) and aviremic (right, blue) animals prior to ART initiation (A), 7–10 days following CD8ɑ+ cell depletion (B), and 21–28 days post-CD8ɑ+ cell depletion (C).

Figure 6.

Study design and timeline for the Late ART animals (A). All 5 animals were infected intrarectally with SIVmac239 and began ART 8 weeks post-infection. ART was withdrawn after approximately 22 months. SIV plasma viral loads for the Late ART animals (B). ART administration is shown by the grey shaded bar. Animals are indicated by unique symbols.

Figure 7.

Gag_386–398GW9 (A), Nef_103–111RM9 (B), and Rev_59–68SP10 (C) epitope sequences pre- and post-ART for Late ART (left, red) and Early ART viremic (right, black) animals. The “pre-ART” time point was between 11 and 14 days post-SIVmac239M infection for the Early ART cohort and 49 days post-SIVmac239 for the Late ART cohort. The “post-ART” time point was the first time point following ART interruption with a viral load greater than 10³ copies/mL. Wild type epitope sequences are in pink bars. Epitope sequences that comprised less than 5% of the total population are in the striped grey and black bars. Variant epitope sequences are shown in different colors. Residues matching the wild-type epitope sequences are indicated by dots and nonsynonymous residues are indicated by letters.