ART Fails to Reach and Block HIV in Lymphoid Tissues
This report is part of a series of focused summaries from the “5th International Workshop on HIV Persistence, Reservoirs & Eradication Strategies” held in St Maarten, December 6-9, 2011. This article recaps 3 presentations given in a row by Professors Timothy Schacker (University of Minnesota), Mario Stevenson (University of Miami) and Courtney Fletcher (University of Nebraska) during the session “Anatomic Reservoirs of HIV” on Thursday December 8, 2011.
The Most Interesting Presentations of the Workshop
According to David Margolis comments on www.natap.org, “Perhaps the most interesting presentation (abstr. 35) of the meeting was a trio of talks by Timothy Schacker (U Minnesota), Courtney Fletcher (U Nebraska), and Mario Stevenson (U Miami), analyzing virions by in situ hybridization, intracellular drug levels, and HIV DNA forms in the lymph node tissue of patients on ART“ (1).
Obviously, it was also the opinion of John Cohen from “Science” who dedicated his workshop report to these data: “Tissue Says Blood Is Misleading, Confusing HIV Cure Effort” (2).
Although we previously published a video interview of Professor Mario Stevenson on this topic, this report wants to give further details about this study.
An open Debate for More than 10 Years
Since the advent of ART, it has been discussed whether these combination were potent enough to completely block HIV replication beyond the blood compartment, even when plasma HIV RNA is below the detection limit.
This is difficult to address, as longitudinal tissue sampling in patients on effective ART regimens is difficult to obtain.
In 2002 and 2003 we published different studies analysing the distribution of anti-HIV Protease Inhibitors in different non-blood compartments and found differential penetration into lymphoid tissue with concentrations in lymph node tissue/concentrations in plasma ratios of 2.07, 0.58, and 0.21 for indinavir, nelfinavir, and lopinavir, respectively (3, 4).
However, all these studies used total lymph node tissues and not cells isolated from lymph nodes, for pharmacologic evaluation. Furthermore, the techniques used to measure ARV drugs levels were not what they are today in terms of sensitivity.
In 2005, we reported at the Rio IAS conference that some kind of “cellular ARV resistance” exists in lymph node cells compared to PBMCs, and was correlated with the expression of multi-drug resistance proteins (5).
The study presented this year at the HIV Persistence workshop extends and confirms these previous findings.
There are several potential sources of HIV in lymphoid tissues (picture 1), including lymph nodes and gut-associated lymphoid cells.
In this prospective study, the authors plan to include 12 patients (mean age: 33 years, mean years since diagnosis: 5.8, mean nadir CD4: 381, mean viral load at entry 34,784 copies/ml) before and after starting a ART regimen. Patients are analysed in plasma, PBMC, lymphoid cells taken from inguinal peripheral lymph nodes, terminal ileum and rectal cells obtained by biopsy at baseline, month 1, 3 and 6. The measures done include viral, histologic and pharmacological tests performed in different laboratories (Table 1).
The characteristics of the 5 first included patients are shown in table 2.
PBMCs are isolated by density centrifugation of venous blood; cells are counted, pelleted, lysed with cold methanol and stored for analysis. Inguinal lymph nodes are biopsied; lymphocytes are isolated from lymph node stromal cells and processed similar to PBMCs. GALT lymphoid tissue is obtained via colonoscopy to the terminal ileum; after surgical removal of the tissue, lymphocytes are isolated, counted, pelleted, lysed and stored for analysis.
TFV-DP, FTC-TP, ATV, DRV, and EFV concentrations are quantified by ultra high pressure liquid chromatography/tandem mass spectrometry (UPLC/MS/MS); the limit of sensitivity is approximately 2.5 fmol/106 cells.
Nine subjects have currently completed pharmacologic protocol evaluations:
-4 on TDF/FTC, ATV/RTV
-1 on TDF/FTC, DRV/RTV
-4 on TDF/FTC, EFV
Intra-cellular concentrations by compartment are shown in picture 2.
TFV-DP, FTC-TP, ATV, DRV and EFV were quantifiable in plasma, PBMCs and cells from LN, ileal and rectal tissue.
The preliminary data support unique compartmentalization for TFV and FTC, and ATV and EFV:
-TFV: Ileal ≈ rectal > PBMC > LN
-FTC: PBMC > LN >> ileal > rectal
-ATV: PBMC > rectal >> ileal > LN
-EFV: PBMC > ileal ≈ rectal >> LN
They also allow the hypothesis that fully suppressive ARV concentrations are not uniformly achieved in cells of the GALT and LN, and may contribute to cryptic HIV replication.
Picture 3 shows the ratios of intracellular TFV-diphosphate, FTC-triphosphate, atazanavir and efavirenz in lymph nodes, ileum and rectum compared with those concentrations in PBMCs. The data are four 4 patients who were receiving TDF/FTC/ATV/RTV and 4 patients who were receiving TDF/FTC/EFV. The panel shows the data with the scale reduced to a maximum of 1 to better illustrate the low ratios. A ratio of less than one indicates that concentrations in that compartment are lower than those in PBMCs.
Factors possibly associated with the penetration, activation and persistence of ARVs into cells of the GALT and LN include (picture 4):
-Susceptibility for influx and efflux membrane transporters and for drug metabolizing enzymes,
-Constitutive expression of phosphorylation enzymes,
-Activation state of PBMC, GALT and LN compartments and dependence of the NRTI on cellular activation for phosphorylation,
-Host genetic traits.
Detailing virologic results in these patients, Mario Stevenson showed that tissue analysis reveals continued de novo infection in lymphoid tissue despite suppression of plasma viremia.
A representative patient is shown in picture 5.
In situ studies of HIV RNA in lymphoid tissue were equally striking. Abundant but widely distributed virion particles were seen in one lymphoid follicle, but an adjacent one seemed completely devoid of virus.
The presenters concluded that tissue analysis revealed discordant virologic response to ART in lymphoid tissue despite effective suppression of plasma viremia. Continued de novo infection may be a consequence of suboptimal antiretroviral concentrations in cells of lymphoid tissue. While de novo infection does not denote ongoing replication, it provides conditions for reservoir replenishment (picture 6).
These results expand recent data showing that cell-to-cell HIV propagation, in particular in tissue, might be a major component of the mechanisms allowing the maintenance for the reservoirs (6)
1-Margolis DM. HIV Persistence during Therapy 5th International Workshop, Dec 6-9 2011 St Martin, West Indies. Natap.org
2-Cohen J. HIV/AIDS research. Tissue says blood is misleading, confusing HIV cure efforts. Science 2011; Dec 23;334(6063):1614.
3-Solas C et al. Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients. antimicrob Agents Chemother 2003 Jan;47(1):238-43.
4-Lafeuillade A et al. Differences in the detection of three HIV-1 protease inhibitors in non-blood compartments: clinical correlations. HIV Clin Trials 2002 Jan-Feb;3(1):27-35.
5-Lafeuillade A et al. Investigating cellular antiretroviral resistance: preliminary results of the “ICARE” study. 3th IAS Conference on HIV Pathogenesis and treatment, Rio de Janeiro, 24-27 July 2005, abstract WeOa0201
6-Sigal A et al. Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 2011 Aug 17;477(7362):95-8. doi: 10.1038/nature10347.
Key words: HIV lymph nodes, HIV lymphoid tissue, HIV pharmacologic sanctuaries