SIV DNA persistence does not require reinfection.

<p>Several mechanisms have been postulated for the persistence of HIV DNA under prolonged therapy. (A) Infected cells harbouring HIV DNA may be long-lived, or (B) may undergo homeostatic replication, in which the daughter cell carries the HIV DNA. Panel C illustrates one possible mechanism of viral persistence under therapy, where latent cells become reactivated, shed virus and die, leading to the infection of a new generation of cells. However, during active infection (D), activation of latent WT infected cells (red) leads to production of small amounts of WT virus (red) that is effectively diluted by the abundance of EM virus in plasma (blue). Thus, reinfection is predominantly with EM virus, and the WT is effectively removed by activation and replication. The long-term persistence of WT SIV-DNA despite EM dominance in plasma, at levels at or above the AUC estimate (in animals 9021, 9175, 5424, 8020) make reinfection a very unlikely contributor to SIV DNA persistence.</p

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

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

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

SIV DNA half-life decreases with increasing chronic plasma viral load.

<p>The chronic plasma viral load (estimated as the geometric mean viral load from day 100 post-infection) is significantly correlated with the estimated half-life of SIV DNA for each animal (Spearman correlation, r = −0.8358, p<0.0001). The error bars represent confidence intervals (C. I); “*” marks the C. I. limit <0.5 days and “+” marks the C. I. limit >10⁵ days.</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

Additional file 1 of Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Additional file 1