304    

Background:  HIV-1 persists in a stable latent reservoir in resting memory CD4⁺ T cells even in patients on suppressive HAART regimens. There may be additional reservoirs, and patients on HAART also have free plasma virus at levels below the limit of detection of ultrasensitive clinical assays. We propose that the functionally significant reservoirs are those that contribute directly to this low-level viremia. To define functionally significant reservoirs, we used intensive sampling and ultrasensitive genotyping to accumulate large numbers of pol sequences from the plasma of patients who had suppression of viremia to < 50 copies/mL. These sequences were then directly compared with large sets of sequences from the latent reservoirs of the same patients.

Methods:  Ten patients on HAART underwent intensive sampling (every 2 to 3 days) over a 3-month period. From plasma virus pol sequences were obtained on pelleted virions using an ultrasensitive genotyping method. Latent reservoir sequences were obtained by a novel digital PCR method.

Results:  For most patients, phylogenetic analysis showed extensive commingling of latent reservoir and plasma sequences. In many instances, viruses with identical pol sequences were detected in both compartments. However, in a third of patients, plasma and cellular sequences were intermingled through only a part of the phylogenetic tree, with a large additional group of plasma sequences not found in the resting CD4⁺ T-cell reservoir. Follow-up studies showed that even after 6 to 9 months, this group of plasma viruses did not enter the latent reservoir.

Conclusions:  Two important and disturbing conclusions can be drawn. First, a second major reservoir contributes to persistent viremia in some patients on HAART. Second, the latent reservoir in resting CD4⁺ T cells is not maintained by ongoing viral replication as its composition does not reflect that of the low-level plasma virus in some patients. Thus the reservoir is intrinsically stable, consistent with the biology of memory CD4⁺ T cells, and is unlikely to decay even if low-level viremia is further reduced by intensification of HAART.

Keywords: reservoir; latency; HAART