Persistence of Unintegrated HIV DNA Associates With Ongoing NK Cell Activation and CD34+DNAM-1brightCXCR4+ Precursor Turnover in Vertically Infected Patients Despite Successful Antiretroviral Treatment

Abstract

The quantification of proviral DNA is raising interest in view of clinical management and functional HIV eradication. Measures of all unintegrated HIV DNA (uDNA) forms in infected reservoir cells provides information on recent replication events that is not found from other proviral DNA assays. To evaluate its actual relevance in a cohort of perinatally-infected adult HIV patients (PHIV), we studied how peripheral blood mononuclear cell uDNA levels correlated with total HIV DNA (tDNA) and with overall replication or innate immune control parameters including NK cell activation/exhaustion and lymphoid turnover. Twenty-two PHIV were included, with successfully controlled HIV (HIV RNA <50 copies/mL) on combined antiretroviral therapy for mean of 8.7 ± 3.9 years. uDNA accounted for 16 [5.2-83.5] copies/µg and was strongly correlated with tDNA (ρ=0.700, p=0.001). Flow cytometric analysis of peripheral NK cells showed that CD69 expression was directly correlated uDNA (p=0.0412), but not with tDNA. Interestingly, CD56^-CD16⁺NK cells which include newly described inflammatory precursors and terminally differentiated cells were directly correlated with uDNA levels (p<0.001), but not with tDNA, and an inverse association was observed between the proportion of NKG2D⁺ NK cells and uDNA (ρ=-0.548, p=0.015). In addition, CD34⁺DNAM-1^brightCXCR4⁺ inflammatory precursor frequency correlated directly with uDNA levels (ρ=0.579, p=0.0075). The frequencies of CD56^-CD16⁺ and CD34⁺DNAM-1^brightCXCR4⁺ cells maintained association with uDNA levels in a multivariable analysis (p=0.045 and p=0.168, respectively). Thus, control of HIV-1 reservoir in aviremic patients on ART is an active process associated with continuous NK cell intervention and turnover, even after many years of treatment. Quantification of linear and circular uDNA provides relevant information on the requirement for ongoing innate immune control in addition to ART, on recent replication history and may help stratify patients for functional HIV eradication protocols with targeted options.

Keywords: ART; CD34; HIV; inflammatory precursor; natural killer; reservoir; residual replication; unintegrated HIV-DNA.

Figure 1

HIV-RNA and CD4+ T-cell count (CD4) trends over the years and total HIV DNA at study time in three representative patients. Treatments are shown on the top, bars indicate start and stop of relative antiretroviral medication. 3TC, lamivudine; ABC, abacavir; ATV, atazanavir; AZT, zidovudine; D4T, stavudine; DDC, zalcitabine; EFV, efavirenz; EVG/c, elvitegravir/cobicistat; FTC, emtricitabine; FPV, fosamprenavir; TDF, tenofovir disoproxil fumarate; NVP, nevirapine; RTV, ritonavir Top panel, Patient ID=10: multiple virological failures over the years due to low adherence to antiretroviral therapy (ART); Middle panel, patient ID=13: successfully treated from birth while maintaining good adherence to ART; Bottom panel, patient ID=14: treated late after birth, although with very good adherence to ART over the years.

Figure 2

Measurements of HIV DNA reservoir in the study population. (A) Levels of total (tDNA), unintegrated (uDNA) and integrated (iDNA) HIV DNA. Each dot represents one patient sample and the white histogram represents the mean. Two patients’ samples did not yield sufficient cellular DNA for uDNA and iDNA analysis (open circles). (B) Levels of tDNA, uDNA and iDNA. Data represent the median [IQR]. (C) Percentages of uDNA and iDNA among tDNA in each study participant. When uDNA copy numbers were < QL of the assay, the % were reduced to 0% (ID 6, 10, 13, 17, 21, 22). When tDNA, uDNA and iDNA copy numbers were < QL, the sample is reported as TND (target not detected; ID 9, 11, 12, 15). Samples, ID 19 and ID 20, were not analyzed for uDNA and iDNA (n/a, not applicable).

Figure 3

Flow cytometric analysis of peripheral NK cells in the cohort of PHIV. (A) Flowcytometric analysis of PBMC in a representative patient. Dot plots show gating strategy of PBMC to assess NK cell frequency. (B) Flowcytometric analysis of NK cells in a representative patient. CD3-14-19-56+ cells are stained for different NK cell surface antigens using directly labelled specific mAbs. (C) Flowcytometric analysis of a representative patient showing gating strategy and labelling to assess the frequency of (CD3-14-19-56-) and of CD34+CDNAM-1bright inflammatory cell precursors and of (CD3-14-19-56-). “Lineage” labels cells as lineage positive (CD3+14+19+56+) and lineage negative (CD3-14-19-56-)for gating and analysis of inflammatory precursors. (D-G) Histograms indicate mean ± SD of NK cells in PBMC of patients at the time of HIV reservoir determination. Frequencies are shown for the three major circulating NK cell subsets (D), for NK cells expressing activating receptors (E), inhibitory NK cell receptors (F), and activation molecules (G).

Figure 4

Natural killer and inflammatory precursor cell frequencies in PHIV patients after stratification for presence (1 = uDNA+) or absence (0 =uDNA-) of detectable uDNA. Lateral histograms (Green) show individual measurement frequencies *p<0.05; **p<0.01; #p<0.001; CD34+IP= CD34⁺ inflammatory precursors (CD34⁺DNAM-1^brightCXCR4⁺).

Figure 5

IFN-γ production is conserved in peripheral blood Natural Killer cells from cART-treated and plasma HIV-RNA suppressed patients with presence of uDNA. Flow cytometric analysis of IFN-γ production by CD56+ NK cells upon 8h- and 16h-stimulation in vitro with IL-12+IL-15. Medium alone as negative control (CNT) or maximal stimulus as positive control (PMA+IONO) are also shown. Upper row: uDNA positive patient (uDNA +), lower row: uDNA negative patient (uDNA -).