Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

Abstract

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1–2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10–15 years on therapy, there were as many as 3.5–5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.

Antigen-driven clonal selection shapes the persistence of HIV-1 infected CD4+T cellsin vivo

“…To investigate the contribution of T-cell differentiation to the persistence of these Agspecific clones, we applied the ddPCR assay to CD4 + T cells subsets sorted based on the expression of CCR7 and CD45RA (Supplementary Figure S10). Although we observed the expected proportions of naïve (TNa), central memory (TCM), effector memory (TEM) and effector memory CD45RA + (TEMRA) cells within total CD4 + T cells, most proviral copies were found in TCM and TEM subsets (Figures 6C and Figure S10), in accordance with previous studies based on HIV-1 DNA (19,26,27), HIV-RNA (57) and on specific expanded clones carrying proviruses (58,59). However, we observed wide variation in the relative contribution of TCM or TEM cells for different proviruses (from 0 to 100%), suggesting adaptive immune responses to individual epitopes are heterogeneous and likely influenced by other factors, like frequency of peptide-MHC stimulation, TCR affinity (60) and T-cell activation.…”

HIV Populations Shift After Tuberculosis Associated Immune Reconstitution Inflammatory Syndrome: Implications For HIV Remission

“…After cART initiation, and as expected (29,38,41,42), CA HIV DNA levels (37,39) decreased in all evaluable study participants ( Figure S5) as infected cells were eliminated. CA HIV DNA levels in the TBI study group remained comparable to the TBNI and NTBNI study groups after short-term cART and with prolonged cART (TBI vs TBNI and NTBNI; p > 0.05, linear mixed-effects model).…”

Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy

“…Interestingly, but perhaps coincidentally, a provirus in this gene (in the form of a solo LTR) is in one of the largest clones we have seen in any on-ART donor [21,45].…”

“…HORMAD2 , whose protein product is involved in meiosis, is a good example (Figure S5), in that it had the largest number of proviral ISs of any non-expressed gene in both the in vitro-infected PBMC and on-ART datasets. Interestingly, but perhaps coincidentally, a provirus in this gene (in the form of a solo LTR) is in one of the largest clones we have seen in any on-ART donor [21, 45].…”

“…Turning the problem around, if the cells that carry proviruses that are not expressed have a survival advantage, and if the location of the proviruses in the genome was a substantial factor in whether the proviruses were expressed, we would have expected to see a larger shift in the distribution of the IS on ART. Although there is, with time on ART, a very slow and erratic selective loss of relatively intact or inducible proviruses [47, 60, 61] which leads to an enrichment in the fraction of defective proviruses [45], there is no evidence that the location of the provirus plays a role in this selection. Our conclusion that the location of an HIV provirus in the genome has little, if any, effect on its expression in infected individuals is supported by data, obtained from cells that were infected with HIV-based vectors in vitro, that the distributions, in the host genome, of the vector proviruses that are and are not expressed are quite similar [59].…”