Dynamics of HIV-1 DNA Levels
The "PINT" Study published ahead of print in "AIDS" shows the analysis of plasma viral load and different forms of cell-associated HIV DNA (integrated, unintegrated linear, episomal: 1-LRT, 2-LTR) in 8 patients treated at primary HIV infection and 8 at chronic infection with TDF/FTC/Raltegravir. The results support continued HIV transmission after one year of ART and show that 2-LTR circles exhibit an initial increase peaking at 3 weeks followed by biphasic decay with a half-life of 29 days.
This study was designed to analyse viral dynamics after starting ART in terms of plasma HIV RNA and cellular-associated HIV DNA.
HIV-1 DNA is expressed in a variety of forms including integrated and productive, integrated but latent, an unintegrated linear form, and circular episomal forms of 1-LTR and 2-LTR HIV DNA that represent aborted integration events.
The “PINT” study treated 8 acute primary HIV infection (PHI) or early PHI patients and 8 chronically infected patients (CHI) with Truvada and raltegravir for 1 year.
HIV RNA copies/ml of plasma were determined by an assay with a quantification limit of 0.3 copies per ml. HIV DNA was extracted from purified peripheral blood CD4 T cells. Total HIV DNA was quantified by a real-time PCR assay specific for HIV-gag, while integrated HIV DNA was quantified using a nested realtime PCR. Quantification of episomal HIV DNA was based on a real-time PCR assay specific for the 2-LTR junction which incorporated a sequence-specific, dual-labeled fluorogenic TaqMan probe.
Viremia was not different between groups during the final phase (p=0.9) settling at a mean of 9 HIV RNA copies/ml by approximately Week 16.
There was less than a 10-fold change over the year in HIV DNA while HIV RNA decreased 10 000 fold. Total HIV DNA levels per CD4 T cell were significantly lower for PHI compared to CHI at 6 of 11 time points over the course of the year, exhibiting two phases of decay, but decay rates did not differ between groups. The mean decay rates of total HIV DNA for the 2 phases corresponded to half-lives of 31 and 1,206 days respectively.
Levels of integrated HIV DNA/106 CD4 T cells showed a similar degree of variation as total HIV DNA copies and were significantly higher for CHI at all time points.
Integrated HIV DNA exhibited a biphasic decay with a significantly faster first phase in PHI than in CHI (mean half-life of 10 days versus 43 days, p<0.04), reaching its second phase within 63 and 172 days respectively.
At no time were 2-LTR copies/106 CD4 T cells different between groups (figure 1). Furthermore, their early dynamics were very different to other components of viral DNA.
Figure 1: dynamics of 2-LTR in PHI and CHI patients treated by TruvadaTM and raltegravir (median levels)
Median 2-LTR levels increased for a period of 2 to 4 weeks (doubling time 11.3 days). After this time they decayed rapidly over the following 4–6 weeks with a half-life of 24.6 days, before settling into a slower decay rate. This profile occurred in both groups. Excluding this transient behavior, during the period from baseline to day 50, there was a slow decay in 2-LTR numbers with a half-life of 169 days. This rate of loss was faster than the second phases of each of the other viral DNA measurements.
The authors were unable to directly measure linear unintegrated HIV DNA which was calculated as total minus 2-LTR minus integrated. At baseline median levels of 2-LTR circles plus integrated HIV DNA were 38% of median levels of total HIV DNA for the PHI group and 31% for the CHI group. From week 4 onwards in PHI median total HIV DNA was almost identical to median copies of 2-LTR circles which indicates that 1-LTR circles were a minor component. Integrated DNA was approximately 1/10th as prevalent as total DNA.
The presence of higher levels of integrated HIV DNA in CHI may be due to its accumulation over the course of infection, as absolute levels significantly correlated with duration of infection. This integrated latent pool, however, contained both replication competent and defective forms.
Nonlinear regression fitting of this model shows that this latent pool accumulates with time achieving 50% of its maximum after 2 years. After starting ART, this pool decreases with a half-life of approximately 2 years.
The mathematical model used determined half-lives of 18 days for linear unintegrated HIV DNA, 29 days for 2-LTR circles or the cells containing them, 24 days for cells with integrated HIV DNA in the first phase loss, and 656 days for the half-life of the source of infection, and rates of progression from unintegrated linear to integrated with a half-life of 3.2 days.
This is consistent with a relatively short-half life of 2-LTR circles or the cells that contain them. Their high levels after one year of therapy also indicates substantial new infection of cells.
In conclusion, the contribution of the latent pool of linear unintegrated HIV DNA to new rounds of productive infection may be considerably higher than previously supposed. These calculations also challenge the viewpoint that the reservoir is completely established early after primary infection. Although the accumulation slows with time, the reservoir may continue to increase. The picture is a fast, but progressive build-up of the reservoir. The dynamics of 2-LTR circles favour the continued entry of virus into cells.
Murray JM et al. Integrated HIV DNA accumulates prior to treatment while episomal HIV DNA records ongoing transmission afterwards. AIDS 2012, epub ahead of print, doi: 10.1097/QAD.0b013e328350fb3c
Key words: HIV DNA, HIV dynamics, HIV raltegravir, HIV reservoirs, HIV é-LTR