The latent reservoir (LR) for HIV-1 persists in CD4+ T cells and is a barrier to cure. The LR has been well characterized in peripheral blood, but lymph nodes have been proposed as a unique sanctuary with concerns that they harbor a greater proportion of latently infected cells, and that measurements of the LR in peripheral blood would not be representative of other tissue sites. Measuring the LR is complicated by an excess of defective virus that is detected by many assays but is not of concern in cure strategies. Additionally, only a subset of intact proviruses are readily activated in vitro. To resolve these issues we characterized total intact, defective, and inducible virus in paired blood and lymph node samples.
Peripheral blood and lymph node samples were collected from 8 HIV-infected, virally suppressed participants immediately prior to solid organ transplantation. Purified CD4[sup]+[/sup] T cell populations were analyzed using the intact proviral DNA assay (IPDA) to determine the number of intact and defective proviral genomes by ddPCR, and by a novel quantitative viral induction assay (QVIA) to determine the number of inducible proviruses in each sample by quantification of cell-associated RNA at limiting dilution.
No difference in inducible virus was detected between lymph node and blood samples (median= 4.3, 7.9 inducible proviruses per million, respectively). The median number of intact provirus per million CD4+ T cells was 104 (IQR 48-591) in lymph nodes and 61 (IQR=43-440) in blood; this difference did not reach statistical significance (P=0.109). There was also no difference in defective virus per million between these sites (median=789, IQR=357-3642; median=584, IQR=299-1530; P=0.95) The ratio of intact to total virus was median=9.9%. Only a small proportion of the intact provirus population was inducible (median=5.7%, IQR=3.3-10.8%) with an even smaller subset of inducible defective provirus (median=1.3%, IQR=0.9-2.3%). Intact and inducible provirus correlated (r=0.779, P=0.0006).
Using two novel assays to analyze different properties of the HIV-1 LR we found no quantitative differences between the peripheral blood and lymph nodes. Taken together with previously reported data showing no difference in the distribution of HIV-1 proviral sequence variants between these sites, we conclude that levels of intact and inducible HIV-1 in blood provide a reasonable approximation of frequencies in the lymph nodes.