Background
HIV-1 latency is the hallmark of persistent infection, because silently infected cells can escape antiretrovirals or immune attack. The use of compounds aimed at reversing HIV-1 latency coupled with fully suppressive antiretrovirals is the basis for the shock and kill strategy. Recent studies suggested that maraviroc (MVC), the only anti-HIV-1 agent targeting a cellular factor, may exert an HIV-1 latency reversal effect. The aim of this study was to evaluate MVC mediated induction of HIV-1 in two in vitro immortalized cell models of HIV-1 latency.
Materials and Methods
HIV-1 promoter activation was evaluated in TZM-bl cells expressing luciferase under the control of HIV-1 LTR. Induction of extracellular and cell-associated HIV-1 RNA (CAR) was evaluated by real time PCR in U1/HIV-1, an HIV-1 latently infected promonocyte cell line, which can be induced to produce virus upon stimulation with transcription activators. NF-kβ induction was evaluated in TZM-bl and U1/HIV-1 nuclear extracts by the NF-kB (p65) Transcription Factor Assay Kit. Expression of CCR5 in the cell lines tested was assessed by flow cytometric analysis.
Cell lines were treated with 4-fold dilutions of MVC (from 80 to 0.31 µM) for 24 hours. Phytohemagglutinin (PHA, 10 μg/ml) and Ionomycin (ION, 1 μg/ml) in combination with Phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) were used as control latency reversal agents (LRA). Induction was expressed as fold-activation (FA) with respect to untreated cells. MVC cytotoxicity was measured by the Cell Titer-Glo cell viability assay.
Results
Cell surface CCR5 was detectable in 40% and 94% of TZM-bl and U1/HIV-1 cells, respectively. MVC was not cytotoxic in the tested range (from 160 to 0.31 µM). No HIV-1 promoter activation was observed in TZM-bl cells at any MVC concentration (0.93±0.07 FA), nor with PHA (1.04±0.06 FA). ION+PMA induced luciferase expression by 4.51±0.15 FA.
HIV-1 CAR was weakly and equally induced in U1/HIV-1 cells at different MVC concentration (80, 20, 5 and 1.25 µM) with values (1.28±0.08 FA) comparable to PHA (1.30±0.37 FA), but considerably lower than ION+PMA (317.53±120.32 FA). Extracellular HIV-1 RNA FA was 3.11±0.92, 1.35±0.50, 1.90±0.46 at 80, 20 and 5 µM MVC, respectively, higher than PHA (0.83±0.19) but much lower than ION+PMA (1768.03±1055.31). NF-kB expression was not upregulated at any MVC concentration in either U1-HIV-1 (0.74±0.16 FA) or TZM-bl (1.11±0.05 FA) cells.
Conclusions
We showed a weak induction of extracellular HIV-1 RNA in U1/HIV-1 cells at MVC concentration ranging 5 to 80 µM, comparable to standard 10 μg/ml PHA. However, MVC failed to activate the HIV-1 promoter in the TZM-bl model and NF-kB expression in TZM-bl and U1/HIV-1 cells. Based on this and previous studies, MVC appears to act as a weak LRA in some but not all cell line models. While further investigation could unveil the reasons for such differential effects, ex-vivo studies of patient derived latently infected CD4 cells are required to define a possible role of MVC as clinically relevant LRA.

Background:
With increasing long-term control of viral replication and with HIV-related health issues rising globally, new approaches for sustainable therapy and efforts towards a cure have reached research agendas worldwide. Cure strategies like latency reversal have garnered much interest, but largely lack selectivity.
Our research strategy takes a different approach by following the changing viral properties of HIV-1 over the course of infection and during times of suppressive therapy. Recently, our laboratory demonstrated that envelope properties of HIV correlate with immunological recovery and disease outcome. In particular, while the presence of CXCR4-tropic (X4) virus correlates with poorer outcomes in therapy-naives, effective therapy seems to facilitate a superior control of X4 viruses¹.
Based on this unexpected activity, this study aims at a detailed characterization of HIV inside the key T-cell populations during ART to identify critical lymphoid compartments responsible for the selective elimination of X4-tropic proviruses and cells.
Materials and methods:
Peripheral blood from HIV-positive patients within the Swiss HIV cohort study was used for selective cell fractionation, applying MACS technology. Non-relevant CD8+(CTLs) and CD19+(B-cells) were depleted, and CD8-CD19- cells were selected for CD4 and Integrin B7 (gut homing) or CCR7 (lymph node homing). Proviral load (pVL) was determined by qPCR. For multi-dimensional data visualization a customized tSNE tool was applied.
Results:
Taking total HIV DNA per 10e6 cells as a proxy for reservoir size, MACSorted fractions achieved the expected significant proviral enrichment in CCR7+/ß7+(CD8-CD19-) cell fractions, enabling a detailed analysis of CD4+ and CD4- fractions. Our study focuses in the retention/re-establishment of crucial immune compartments.
Selective FACS sorts, using highly-specific marker antibodies, revealed that ≥90% of cells with gut homing also have properties compatible for Lymph node homing. First analyses applying tSNE identify homing properties in detail and a depth down to individual cells where we find a significant tissue contribution beyond the circulation, evidencing active compartments.
Conclusions:
Only a small fraction of the total HIV RNA-positive cells are present in the peripheral blood at any time², highlighting the importance of assessing the viral distribution in lymphoid compartments like lymph node or gut. Our first cell selection approach demonstrates the ability to assess central markers for viral sanctuaries and cell-homing. Currently, we are expanding the marker panel to co-analyze HIV Envelope (surface) and intracellular Gag-expression as proxies for viral intactness, and by longitudinally following patient samples (suppressed but with high pVL) during cART therapy-episodes. Relevant markers identified by tSNE are being used for live cell sorts. Virus reactivation from sorted cell fractions will determine viral phylogeny and tropism and potential links to compartmentalization and viral dynamics. We believe that our strategy will contribute to the formulation of new strategies towards targeted cellular virus elimination and ultimately HIV eradication.
¹ Bader, J. et al. Therapeutic immune recovery prevents emergence of CXCR4-tropic HIV-1. Clin. Infect. Dis. ciw737 (2016). doi:10.1093/cid/ciw737
² Buggert, M., Japp, A. S. & Betts, M. R. Everything in its right place: resident memory CD8: +: T cell immunosurveillance of HIV infection. Curr. Opin. HIV AIDS 14, (2019).