An “Escape Clock” for Estimating the Turnover of SIV DNA in Resting CD4+ T Cells

Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an “escape clock” approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication

Data from: Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in Simian Immunodeficiency Virus infection

CD8+ T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires “escape mutations” that reduce CD8+ T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8+ T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8+ T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8+ T cells reach 0.5% of total CD8+ T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8+ T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8+ T cells

Tetramer staining data

Tetramer staining dat

Reversion in animal 1.7105.

<p>Mane-A*10 negative animal 1.7105 was challenged with SHIV_mn229, which is a viral stock containing 10% WT and 90% K165R escape mutation at the Gag KP9 epitope. In the absence of KP9-specific immune response, virus quickly reverts to WT. During reversion, the fraction of WT in resting CD4+ T cells closely follows the fraction in plasma, similarly to the escape pattern in Mane-A*10 positive animals with high chronic viral loads challenged with WT SIV₂₅₁.</p

Estimating the half-life of SIV DNA in resting CD4+ T cells.

<p>The proportion of WT virus in plasma (green circles), the fraction of WT virus estimated from the area under the curve (AUC) of viral load (blue circles) and the experimentally observed fraction of WT virus SIV DNA in resting CD4+ T cells (red squares) are shown for each animal in the top part of each panel. The black line illustrates the best-fit SIV DNA half-life to the observed fraction of WT virus in resting CD4+ T cells for each animal. Animals are arranged in order of increasing half-life of SIV DNA. The bottom part of each panel (black triangles) represents total plasma viral load. Viral loads are all on the same log₁₀ scale, from 10–10⁹. The bottom right two panels (B0517 and B0547) illustrate two animals in which EM appeared only transiently in plasma. In this case, the fraction WT virus is nearly 100% in both plasma and AUC estimates at the time points where DNA was measured, so the ‘escape clock’ fits equally well with a half-life of 1 day or 100,000 days.</p

Macaques studied.

*<p>Flu-KP9 is recombinant Influenza viruses expressing SIV Gag KP9 CTL epitope; Flu-SIV is recombinant influenza viruses expressing SIV Gag KP9 CTL epitope and 2 SIV Tat CLT epitopes (KSA10 and KVA10).</p

Measuring WT and EM virus in plasma and resting CD4+ T cells.

<p>(A) The levels of WT and EM virus in plasma were estimated using a variant-specific real-time PCR assay, shown here for animal 1335. In order to estimate the proportion of WT virus in resting CD4+ T cells, cells were first sorted (B), and then DNA extracted and the levels of WT and EM virus measured using the variant-specific PCR (C). Combining this data, we can plot the fraction of WT virus in the plasma (closed squares) and resting CD4+ T cells (open squares) over time (D).</p

Estimating the half-life of SIV-DNA in resting cells using the ‘escape clock.’

<p>The fitting of the experimental data for animal 1335 is shown to illustrate the modelling approach. Panel A illustrates the approach using a single timepoint late in infection. In the top figure in panel A, the levels of WT (red) and total (WT + EM, blue) virus are plotted over time (on a linear scale). The shaded rectangle indicates the current fraction of WT virus on day 105 post-infection in plasma (pink for WT and light blue for EM). If viral DNA turned over fast (with half-life of half a day or less), both WT and EM would follow the ratio in plasma and we would expect nearly 100% EM SIV DNA in resting CD4+ T cells on day 105. The bottom half of panel A represents the case when viral DNA does not decay (has infinite half-life). In this case, viral DNA accumulates all the time since inoculation, and the amount of each virus type follows the area under the curve (AUC) of WT (∫Wdt) and EM (∫Edt) viral load. The scale of viral DNA is linear in arbitrary units. In this case we would expect a much higher WT fraction in viral DNA in resting cells on day 105 (rectangle). The box in the middle on the right of panel A shows the experimentally measured fraction of WT DNA in resting cells, which is between the two extremes. This implies that the half-life of viral DNA lies between 0.5 days and infinite time. Panel B illustrates the fitting of the optimal half-life of SIV-DNA using the longitudinal data for animal 1335. The green circles are the experimental values of WT virus fraction in plasma over time; the blue circles are the fraction of WT virus estimated from the AUC of viral load. The red squares show the experimentally observed fraction of WT virus SIV DNA in resting CD4+ T cells. The black line illustrates the fraction of WT virus expected from the model (Eq.2) with different values of SIV DNA half-life. The top left figure shows the estimated DNA fraction for half-life of 0.5 days, which follows the experimental plasma fraction closely, but is considerably below the observed DNA fraction. The top right figure shows the DNA fraction estimated from infinite lifetime (area under the curve), which is higher than the observed fraction in the later stage of infection. The larger figure at the bottom shows the estimate using the best-fit lifetime of 21.1 days, which closely follows the observed variation of WT DNA.</p