A major challenge in measuring the reservoir of HIV that persists despite antiretroviral therapy (ART) is that many of the virus genomes that can be found integrated into the DNA of CD4 T cells are incomplete or mutated in ways that preclude further rounds of replication. For researchers aiming to develop a cure, it is important to try and distinguish between defective virus copies and intact viruses capable of rekindling the infection when ART is interrupted. A new study from the laboratory of Robert Siliciano, published in the latest issue of Nature Medicine, attempts to assess the proportions of defective and replication competent HIV in people on ART, comparing individuals who began treatment very early after infection to those who started later.
Siliciano and colleagues used genetic sequencing techniques to measure the number of intact HIV DNA sequences in ten individuals who started ART more than 180 days after infection, and compared the results to a cohort of nine people who started within 100 days of infection (most had begun within 60 days). The researchers were surprised to find that defective copies of HIV accumulated rapidly: in the early-treated individuals, only 7% of the HIV DNA copies that could be detected were intact. In the cohort that initiated ART during chronic infection, the proportion of intact viruses was expected to be lower and that proved to be the case: only 2% of virus genomes were complete, and 98% of the HIV DNA in this group was defective.
The average size of the replication competent HIV reservoir was estimated to be 12 infectious proviruses per million resting CD4 T cells in the early-treated group compared to 37 infectious proviruses per million resting CD4 T cells in the individuals who began ART later, although the researchers note there was “substantial person-to-person variation.” These data appear to suggest that the impact of early ART on the size of the HIV reservoir may not be as dramatic as had been thought, but there are some caveats:
- While most of the early-treated cohort began ART within three months of infection, other studies attempting to ascertain the effect of early treatment on the size of the HIV reservoir have included individuals who initiated ART within a matter of days. The techniques used in this paper have yet to be applied to measuring the reservoir in such ultra-early-treated people.
- A difference of 12 vs. 37 infectious proviruses per million resting CD4 T cells may seem relatively slight, but a threefold larger HIV reservoir may be more significant when you consider the whole body (which has been estimated to contain 1.9 – 3.5 trillion lymphocytes).
- The differences in the total amount of HIV DNA detectable in the early vs. later treated cohorts are far greater than was observed for intact proviruses. In the examples provided in the paper, the amount of HIV DNA detectable in the early-treated group ranges from 72-315 copies per million resting CD4 T cells, compared to 1,333-9,785 copies per million resting CD4 T cells in the individuals treated later. While the DNA mostly represents defective virus copies, they are not necessarily benign. A recent study from the laboratory of Anthony Fauci at NIAID found that at least some defective proviruses can still produce HIV proteins and therefore potentially contribute to persistent inflammation and immune activation in HIV-positive people. A separate study, also published recently, found HIV DNA in multiple tissues evaluated at autopsy and reported that there appeared to be an association with tissue pathology. Measures of total HIV DNA have also shown correlations with various clinical parameters in HIV-positive people, as outlined in a new review by Christine Rouzioux and colleagues.
The study results underscore that, despite a great deal of effort, the optimum method for measuring the replication-competent HIV reservoir remains unknown. The current gold standard is the quantitative virus outgrowth assay (QVOA), but this requires the sampling and activation of very large numbers of cells and is expensive and time consuming. Furthermore, the Siliciano laboratory found that levels of intact HIV DNA sequences (which they suggest represent “probably the closest estimate of the true size of the latent reservoir”) did not correlate with results obtained by QVOA.
The development of accurate and reliable tests for measuring the HIV reservoir continues to be a major priority for the HIV cure research field.