Jacques Leibowitch

Restoration of the immune system with anti-retroviral therapy

Clinical benefits of highly active anti-retroviral treatments (HAART) are increasingly evidenced by resolving opportunistic infections and malignancies, as well as declining hospitalization and mortality rates [1]. This suggests that potent and sustained suppression of viral replication, at least to some extent, is associated with reconstitution of the immune system even in adult patients treated at advanced stages of the disease. Increased susceptibility to opportunistic infections and tumors mainly results from the loss of memory CD4+ T cell reactivity against recall antigens which is an early event in HIV disease progression. Primary responses of naive CD4+ T cells against new pathogens are suppressed even earlier in the course of HIV disease, and the progressive depletion in naive CD4+ T cells reflects profound alterations in T cell regeneration capacities. Previous studies revealed that monotherapy with ritonavir, a protease inhibitor, resulted in a slight improvement in memory CD4+ T cell responses to recall Ags only when detectable prior to onset of therapy, suggesting that the loss of CD4+ T cell reactivity might be irreversible at advanced stages of the disease [2]. In contrast our group demonstrated more recently that restoration in CD4+ T cell reactivity to specific antigens was feasible when HAART was administered in progressors [3]. Here we address some of the questions raised by immune restoration with HAART when administered at advanced stages of the disease.

T cell changes after combined nucleoside analogue therapy in HIV primary infection.

OBJECTIVE To characterize the immune changes after treatment of acute HIV-1 infection with triple nucleoside analogue therapy. DESIGN Immunological and virological parameters were monitored from day 0 to weeks 36-44 in eight patients [median CD4 cells = 451 cells/microl (range: 149-624), viral load = 4.8 log10 copies/ml (range: 6.5-3.3)] who started at time of primary HIV infection (PHI) a therapy including zidovudine (ZDV), didanosine (ddl), and lamivudine (3TC). METHODS Lymphoid subsets were evaluated on peripheral blood lymphocytes by four-colour flow cytometry using a panel of mAbs directed against differentiation and activation markers. RESULTS We observed a median -2.1 (range: -1; -3.3) log10 copies/ml viral load decrease and a median +158 cells/microl (range: +7 to +316) CD4 cell count increase at week 4 reaching normal CD4 cell count values of 761 CD4 cells/microl (range: 389-1153) at weeks 36-44. Virus undetectability was obtained at week 24 for all subjects. A rapid CD4 T cell amplification involved both memory and naive CD4 T cells. This was associated with a very rapid and significant decrease in activation markers [human leukocyte antigen-DR (HLA-DR), CD38] on both CD4 and CD8 T cell subsets together with a CD8+CD28+ cell increase as early as week 4. CONCLUSIONS These results show that early therapy with nucleoside analogues can correct the immunological abnormalities observed in CD4 and CD8 T cell subsets at the time of PHI. This early kinetics in T cell recovery appears to be faster than in established disease.

Quantitation of blood lymphocyte mitochondrial DNA for the monitoring of antiretroviral drug-induced mitochondrial DNA depletion.

Objective: To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV‐1‐infected patients. Design: As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patients first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient. Methods: mtDNA and nuclear DNA quantitation per cell (&bgr;‐globin gene copies) were both measured by real‐time polymerase chain reaction analysis of whole DNA extracts of 361 serial live‐cryopreserved PBMCs collected in former trials and clinical follow‐ups from 60 individuals with established or recently acquired HIV‐1 infections before and during administration of various antiviral combination therapies. Results: mtDNA amounts were stable or increasing over years of natural HIV‐1 infection in untreated patients (n = 7), consistent with our finding of a lack of differences in mtDNA copy numbers in patients with either a long established or recent lentivirus infection. Our quantitation system revealed significant changes in mtDNA copy number depending on the designated triple, quadruple, or quintuple anti‐HIV drug combinations. Zidovudine + zalcitabine + ritonavir and zidovudine + lamivudine + didanosine regularly lead to mtDNA depletion in each of the treated patients, whereas none of 7 patients (and 35 cell specimens) receiving a stavudine + lamivudine + indinavir combination had any significant mtDNA content variations. In 7 patients, mtDNA copy numbers returned to pretreatment levels and/or higher levels without any interruption of the previously mtDNA‐depleting antiretroviral drug combination. Conclusion: Our assay system allowed the detection of significant changes in the mtDNA content of PBMCs from HIV‐1‐infected patients taking antiretroviral drugs, as has been reported in the literature with other detection systems. Yet, mtDNA copy numbers regularly diminished during administration of some but not all nucleoside analog‐containing combinations. This, plus the occasional finding that depleted mtDNA contents spontaneously increased to baseline levels and/or higher levels during uninterrupted treatment, should raise a note of caution about resorting to the PBMC mtDNA marker for monitoring of antiretroviral drug‐related mitochondrial toxicities.

Is oxygen a key factor in the lipodystrophy phenotype

BackgroundThe lipodystrophic syndrome (LD) is a disorder resulting from selective damage of adipose tissue by antiretroviral drugs included in therapy controlling human-immunodeficiency-virus-1. In the therapy cocktail the nucleoside reverse transcriptase inhibitors (NRTI) contribute to the development of this syndrome. Cellular target of NRTI was identified as the mitochondrial polymerase-gamma and their toxicity described as a mitochondrial DNA (mtDNA) depletion resulting in a mitochondrial cytopathy and involved in fat redistribution. No mechanisms offer explanation whatsoever for the lipo-atrophic and lipo-hypertrophic phenotype of LD. To understand the occurrence we proposed that the pO2 (oxygen partial pressure) could be a key factor in the development of the LD. For the first time, we report here differential effects of NRTIs on human adipose cells depending on pO2 conditions.Results and discussionWe showed that the hypoxia conditions could alter adipogenesis process by modifying expression of adipocyte makers as leptin and the peroxisome proliferator-activated receptor PPARgamma and inhibiting triglyceride (TG) accumulation in adipocytes. Toxicity of NRTI followed on adipose cells in culture under normoxia versus hypoxia conditions showed, differential effects of drugs on mtDNA of these cells depending on pO2 conditions. Moreover, NRTI-treated adipocytes were refractory to the inhibition of adipogenesis under hypoxia. Finally, our hypothesis that variations of pO2 could exist between adipose tissue from anatomical origins was supported by staining of the hypoxic-induced angiopoietin ANGPTL4 depended on the location of fat.ConclusionToxicity of NRTIs have been shown to be opposite on human adipose cells depending on the oxygen availability. These data suggest that the LD phenotype may be a differential consequence of NRTI effects, depending on the metabolic status of the targeted adipose tissues and provide new insights into the opposite effects of antiretroviral treatment, as observed for the lipo-atrophic and lipo-hypertrophic phenotype characteristic of LD.

Frequency of Cytokine-Producing T Cells in HIV-Infected Patients Treated with Stavudine, Didanosine, and Ritonavir

To assess prospectively the influence of the control of viral replication on the frequency of cytokine-producing T cells, and to correlate these changes with immune activation, we conducted a 15-month follow-up study of IFN-gamma- and IL-2-producing CD4+ and CD8+ T cells at a single-cell level in 12 previously untreated patients receiving highly active antiretroviral therapy (HAART). At baseline we observed a strikingly high proportion of IFN-gamma-producing CD8+ T cells. The treatment-induced decrease in the proportion of IFN-gamma-producing CD8+ T cells ran parallel to the decrease in HLA-DR+ and CD38+CD8+ T cell subsets and was associated with the reduction in HIV RNA level. IL-2-producing cells were mainly CD4+. As a consequence of CD4+ T cell loss, the number of IL-2-producing CD4+ T cells was lower in patients than in control subjects (52 vs. 171 cells/microl), but the proportion of these cells was unchanged (22.4 vs. 19.3). During therapy the proportion of CD4+ IL-2-producing cells was initially stable and then fell markedly at month 5, followed by a gradual return to previous values. The reduction in viral load was associated with the fall in the proportion of CD4+ activated subsets. Intracellular cytokine assays are a new approach to the assessment of T cell function in HIV infection. Our results suggest that the functional capacity of CD4+ T cells is probably less severely altered than previously thought on the basis of conventional assays. CD8+ T cells exhibit an increased capacity to produce IFN-gamma that is associated with an increase in activation marker expression. These alterations decrease partially and in parallel under treatment.

Four days a week or less on appropriate anti-HIV drug combinations provided long-term optimal maintenance in 94 patients: the ICCARRE project

Short, intraweekly cycles of anti‐HIV combinations have provided intermittent, effective therapy (on 48 patients) (1). The concept is now extended to 94 patients on treatment, 4 days per week or less, over a median of 2.7 discontinuous treatment years per patient. On suppressive combinations, 94 patients volunteered to treatment, 5 and 4 days per week, or reduced stepwise to 4,3,2, and 1 days per week in 94, 84, 66, and 12 patients, respectively, on various triple, standard, antiviral combinations, or nonregistered, quadruple, antiviral combinations. Ninety‐four patients on treatment 4 days per week aggregated 165 intermittent treatment years; no viral breakthrough was observed over 87 average treatment weeks per patient, 63 of 94 having passed 2.5 intermittent treatment years on any of the antiviral combinations prescribed. On the hyperintermittent treatment of 3, 2, and 1 days per week, HIV RNA surged >50 copies, 4 weeks apart, in 18 instances (6.8 viral escapes/100 hyperdiscontinuous maintenance years). Viral escapes could have been a result of erratic adherence (EA) to regimen or follow‐up (3 patients)—drug taken at half of the daily recommended dosage (8 patients) and/or overlooked archival‐resistant HIVs from antecedent treatment failures (6 patients). Aside from the above circumstances, HIV unexpectedly rebounded in 3 patients on 2 days per week treatment and 1 patient on 1 day per week treatment, posting 2.2 intrinsic viral escapes/100 highly discontinuous treatment years. All 18 escapes were eventually reversed by 7 days per week salvage combinations, and 11 of 18 patients have been back for a second course of intermittent therapy, 4 days per week or less. Both cell‐activation markers on the surface of T lymphocytes and cell‐bound HIV DNA levels remained stable or declined. CD4/CD8 ratios rose to ≤1 in 35% of patients, whereas CD4 counts went ≤500/μl in 75%. These values were previously 7 and 40%, respectively, on 7 days per week therapy. In our aging, long, HIV‐enduring, multitreated patient cohort, treatment 4 days per week and less over 421 intermittent treatment years reduced prescription medicines by 60%—equivalent to 3 drug‐free/3 virus‐free remission year per patient—actually sparing €3 million on just 94 patients at the cost of 2.2 intrinsic viral failure/100 hyperintermittent treatment years. At no risk of viral escape, maintenance therapy, 4 days per week, would quasiuniversally offer 40% cuts off of current overprescriptions.—Leibowitch, J., Mathez, D., de Truchis, P., Ledu, D., Melchior, J. C., Carcelain, G., Izopet, J., Perronne, C., David, J. R. Four days a week or less on appropriate anti‐HIV drug combinations provided long‐term optimal maintenance in 94 patients: the ICCARRE project. FASEB J. 29, 2223‐2234 (2015). www.fasebj.org

Short cycles of antiretroviral drugs provide intermittent yet effective therapy: a pilot study in 48 patients with chronic HIV infection

The present study evaluated the efficacy of intermittent antiviral treatment administered to HIV‐infected patients under stepwise reductions in weekly medication. Forty‐eight patients were invited to reduce their antiviral medication to 5 consecutive days per week; after control over HIV activity was ascertained, antiviral drugs were cut to 4 consecutive days per week. Of the 48, 39 then reduced medicines further to 3 d, and 12 of those eventually undertook a 2 d/wk schedule. Clinical and immunological status and plasma HIV load were repeatedly monitored. HIV was unremittingly maintained below detection levels in all patients under either 5‐ or 4‐d/wk treatment regimens, for a mean 56 ± 40 wk/patient (5‐d regimen) and 84 ± 46 wk/patient (4‐d regimen). Of the 39 patients under 3‐d regimens, 35 maintained optimal control over HIV activity for a mean 50 ± 32 wk, as did 10 of the 12 under 2‐d regimens, for 24 ± 10.5 wk. Summing up treatment ≤ 5 d/wk, plasma HIV remained below detection levels for a cumulative 8895 wk (170 patientyr). No major HIV‐related clinical event was reported. and CD4+ T‐cell counts and percentages readily increased over the last value noted under the 7‐d treatment course. Viral failure was documented in 6 of the 48 patients: 4 under a 3‐d/wk regimen, 2 under a 2‐d/wk regimen. All 6 patients had their treatment swiftly set back to a 7‐d/wk regimen, resulting in rapid control over HIV replication. In summary, intermittent antiretroviral regimens optimally suppressed HIV in patients taking antiviral medicines 5 and 4 d/wk, as well as in a substantial proportion of patients under 3‐ or 2‐d/wk antiviral regimens, reducing both expenses and, possibly, drug toxicity. Controlled prospective clinical trials are warranted before considering short weekly cycles of antiretroviral medicines an alternative in the management of chronically HIV‐infected patients.—Leibowitch, J., Mathez, D., de Truchis, P., Perronne, C., Melchior, J. C. Short cycles of antiretroviral drugs provide intermittent yet effective therapy: a pilot study in 48 patients with chronic HIV infection. FASEB J. 24, 1649–1655 (2010). www.fasebj.org

Lymphadenopathy-Associated Virus/Human T-Lymphotropic Virus Type III in Allogeneic Bone Marrow Transplantation

Excerpt To the editor: The acquired immunodeficiency seen after bone marrow transplantation and that due to infection with the lymphadenopathy-associated virus/human T-lymphotropic virus type HI (L...

Plasma and Intracellular Antiretroviral Concentrations in HIV-Infected Patients under Short Cycles of Antiretroviral Therapy

Study of plasma and intracellular concentrations of atazanavir, lopinavir, nevirapine, and efavirenz was conducted on 48 patients under short cycles of antiretroviral therapy. Intracellular concentrations (IC) were still measurable for all drugs after 85 h or 110 h drug intake despite the absence of drug in plasma for atazanavir and lopinavir. A linear relationship between plasma and intracellular efavirenz was observed. Further studies to fully understand the impact of IC in the intermittent antiviral treatment are required.

Four days a week and less on appropriate antiviral combinations provided long-term optimal control over HIV-1 in 92 patients.

Patients and treatments 92 volunteer patients on optimally suppressive antiviral combinations for 5 months or more consented to step wisely reduce their weekly treatment from 7 to 5 to 4 days/wk, or directly from 7 to 4 d/wk after bi-monthly checks on HIV plasma levels at < 50 copies. Weekly treatment was further reduced to 3, 2, and 1 d/wk for respectively 72, 59 and 12 pts. Antiviral combinations included : one integrase inhibitor-base + 2 or 3 NRTIs, or 1 NNRTI and 1 PI (for 4 d/wk regimens, (Rx); standard triple combinations of 1 PI or 1 NNRTI + 2 NRTIs (for 4, 3 , 2 d/wk Rx); novel quadruple antiviral compositions of 1 NNRTI + 3 NRTIs (for 4, 3 , 2, 1 d/wk Rx).