Early antiretroviral therapy favors post-treatment SIV control associated with the expansion of enhanced memory CD8+ T-cells.

HIV remission can be achieved in some people, called post-treatment HIV controllers, after antiretroviral treatment discontinuation. Treatment initiation close to the time of infection was suggested to favor post-treatment control, but the circumstances and mechanisms leading to this outcome remain unclear. Here we evaluate the impact of early (week 4) vs. late (week 24 post-infection) treatment initiation in SIVmac251-infected male cynomolgus macaques receiving 2 years of therapy before analytical treatment interruption. We show that early treatment strongly promotes post-treatment control, which is not related to a lower frequency of infected cells at treatment interruption. Rather, early treatment favors the development of long-term memory CD8+ T cells with enhanced proliferative and SIV suppressive capacity that are able to mediate a robust secondary-like response upon viral rebound. Our model allows us to formally demonstrate a link between treatment initiation during primary infection and the promotion of post-treatment control and provides results that may guide the development of new immunotherapies for HIV remission.

Pharmacokinetic Model of Tenofovir and Emtricitabine and Their Intracellular Metabolites in Patients in the ANRS 134-COPHAR 3 Trial Using Dose Records.

Tenofovir (TFV) and emtricitabine (FTC) are part of the recommended highly active antiretroviral therapy (ART). Both molecules show a large interindividual pharmacokinetic (PK) variability. Here, we modeled the concentrations of plasma TFV and FTC and their intracellular metabolites (TFV diphosphate [TFV-DP] and FTC triphosphate [FTC-TP]) collected after 4 and 24 weeks of treatment in 34 patients from the ANRS 134-COPHAR 3 trial. These patients received daily (QD) atazanavir (300 mg), ritonavir (100 mg), and a fixed-dose combination of coformulated TFV disoproxil fumarate (300 mg) and FTC (200 mg). Dosing history was collected using a medication event monitoring system. A three-compartment model with absorption delay (Tlag) was selected to describe the PK of, respectively, TFV/TFV-DP and FTC/FTC-TP. TFV and FTC apparent clearances, 114 L/h (relative standard error [RSE] = 8%) and 18.1 L/h (RSE = 5%), respectively, were found to decrease with age. However, no significant association was found with the polymorphisms ABCC2 rs717620, ABCC4 rs1751034, and ABCB1 rs1045642. The model allows prediction of TFV-DP and FTC-TP concentrations at steady state with alternative regimens.

Pharmacological Validation of Long-Term Treatment with Antiretroviral Drugs in a Model of SIV-Infected Non-Human Primates.

The development of animal models undergoing long-term antiretroviral treatment (ART) makes it possible to understand a number of immunological, virological, and pharmacological issues, key factors in the management of HIV infection. We aimed to pharmacologically validate a non-human primate (NHP) model treated in the long term with antiretroviral drugs after infection with the pathogenic SIVmac251 strain. A single-dose pharmacokinetic study of tenofovir disoproxil fumarate, emtricitabine, and dolutegravir was first conducted on 13 non-infected macaques to compare three different routes of administration. Then, 12 simian immunodeficiency virus (SIV)-infected (SIV+) macaques were treated with the same regimen for two years. Drug monitoring, virological efficacy, and safety were evaluated throughout the study. For the single-dose pharmacokinetic study, 24-h post-dose plasma concentrations for all macaques were above or close to 90% inhibitory concentrations and consistent with human data. During the two-year follow-up, the pharmacological data were consistent with those observed in humans, with low inter- and intra-individual variability. Rapid and sustained virological efficacy was observed for all macaques, with a good safety profile. Overall, our SIV+ NHP model treated with the ART combination over a two-year period is suitable for investigating the question of pharmacological sanctuaries in HIV infection and exploring strategies for an HIV cure.

Prolonged Antiretroviral Treatment Induces Adipose Tissue Remodelling Associated with Mild Inflammation in SIV-Infected Macaques.

During chronic SIV/HIV infection, adipose tissue (AT) is the target of both antiretroviral treatment (ART) and the virus. AT might subsequently contribute to the low-grade systemic inflammation observed in patients on ART. To evaluate the inflammatory profile of AT during chronic SIV/HIV infection, we assayed subcutaneous and visceral abdominal AT from non-infected (SIV-, control), ART-naïve SIV-infected (SIV+) and ART-controlled SIV-infected (SIV+ART+) cynomolgus macaques for the mRNA expression of genes coding for factors related to inflammation. Significant differences were observed only when comparing the SIV+ART+ group with the SIV+ and/or SIV- groups. ART-treated infection impacted the metabolic fraction (with elevated expression of PPARγ and CEBPα), the extracellular matrix (with elevated expression of COL1A2 and HIF-1α), and the inflammatory profile. Both pro- and anti-inflammatory signatures were detected in AT, with greater mRNA expression of anti-inflammatory markers (adiponectin and CD163) and markers associated with inflammation (TNF-α, Mx1, CCL5 and CX3CL1). There were no intergroup differences in other markers (IL-6 and MCP-1). In conclusion, we observed marked differences in the immune and metabolic profiles of AT in the context of an ART-treated, chronic SIV infection; these differences were related more to ART than to SIV infection per se.

Inhibition of Adipose Tissue Beiging by HIV Integrase Inhibitors, Dolutegravir and Bictegravir, Is Associated with Adipocyte Hypertrophy, Hypoxia, Elevated Fibrosis, and Insulin Resistance in Simian Adipose Tissue and Human Adipocytes.

For people living with HIV, treatment with integrase-strand-transfer-inhibitors (INSTIs) can promote adipose tissue (AT) gain. We previously demonstrated that INSTIs can induce hypertrophy and fibrosis in AT of macaques and humans. By promoting energy expenditure, the emergence of beige adipocytes in white AT (beiging) could play an important role by limiting excess lipid storage and associated adipocyte dysfunction. We hypothesized that INSTIs could alter AT via beiging inhibition. Fibrosis and gene expression were measured in subcutaneous (SCAT) and visceral AT (VAT) from SIV-infected, dolutegravir-treated (SIVART) macaques. Beiging capacity was assessed in human adipose stromal cells (ASCs) undergoing differentiation and being exposed to dolutegravir, bictegravir, or raltegravir. Expression of beige markers, such as positive-regulatory-domain-containing-16 (PRDM16), were lower in AT of SIVART as compared to control macaques, whereas fibrosis-related genes were higher. Dolutegravir and bictegravir inhibited beige differentiation in ASCs, as shown by lower expression of beige markers and lower cell respiration. INSTIs also induced a hypertrophic insulin-resistant state associated with a pro-fibrotic phenotype. Our results indicate that adipocyte hypertrophy induced by INSTIs is involved via hypoxia (revealed by a greater hypoxia-inducible-factor-1-alpha gene expression) in fat fibrosis, beiging inhibition, and thus (via positive feedback), probably, further hypertrophy and associated insulin resistance.

Pharmacokinetics and tissue distribution of tenofovir, emtricitabine and dolutegravir in mice.

BACKGROUND: Studies of antiretroviral drug (ARV) tissue distribution in preclinical models, such as mice, are key to understanding viral persistence. OBJECTIVES: To determine the plasma and tissue pharmacokinetics and tissue distributions of tenofovir, emtricitabine and dolutegravir in mice. METHODS: ARVs were simultaneously administered to two different strains, and their levels in plasma and tissue samples were determined by a validated LC-MS/MS method. A non-compartmental analysis was performed to estimate the main pharmacokinetic parameters. A tissue penetration factor (TPF) was calculated as the ratio of the concentration in the tissue concerned to that in plasma. RESULTS: ARV plasma pharmacokinetic parameters in both strains were similar to those estimated in the clinical context. Tissue concentrations were highest in the digestive tract, followed by the liver and kidneys, lymphatic system, pancreas, adipose tissue and lungs. Tissue concentrations were lowest in the brain. Triple therapy could not be considered effective in any of the tissues considered. The TPF values obtained showed that tenofovir diffused widely, especially in the digestive tract, liver and kidneys. Emtricitabine had a TPF above 100% in two-thirds of the tissues. Dolutegravir was poorly distributed to all tissues. CONCLUSIONS: Drug specificity was observed, with higher levels of exposure to tenofovir than to emtricitabine or dolutegravir. Tissue specificity was also observed, with strong penetration of the digestive tract and weak penetration of the brain. These data have important implications for future preclinical and clinical studies for developing new HIV therapies with the goal of an HIV cure.

Cerebrospinal fluid exposure to bictegravir/emtricitabine/tenofovir in HIV-1-infected patients with CNS impairment.

OBJECTIVES: The penetration of antiretroviral drugs into deep compartments, such as the CNS, is a crucial component of strategies towards an HIV cure. This study aimed to determine CSF concentrations of bictegravir, emtricitabine and tenofovir in patients with HIV-related CNS impairment (HCI) enrolled in a real-life observational study. METHODS: Patients with HCI treated by optimized ART, including bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF) for at least 1 month were enrolled. Plasma and CSF concentrations were measured by quality control-validated assays (LC-MS/MS). The inhibitory quotient (IQARV) was calculated as the ratio of unbound (bictegravir) or total (emtricitabine and tenofovir) concentration to half (or 90%) maximal inhibitory concentration for bictegravir (or emtricitabine and tenofovir). All numerical variables are expressed as median (range). RESULTS: Twenty-four patients (nine women) were enrolled. The age was 45 (26-68) years. Unbound bictegravir and total emtricitabine and tenofovir CSF concentrations were 4.4 (1.6-9.6), 84.4 (28.6-337.4) and 1.6 (0.7-4.3) ng/mL, respectively. The unbound bictegravir CSF fraction was 34% (15%-82%) versus 0.33% (0.11%-0.92%) in plasma. Three patients had an IQARV above unity for the three antiretrovirals. Factors positively associated with the CSF concentration (unbound for bictegravir) were age and total plasma concentration for the three antiretrovirals. Patients aged over 51 years had higher CSF concentrations (unbound for bictegravir). CONCLUSIONS: We observed low CSF exposure to bictegravir, emtricitabine and tenofovir. These results suggest that BIC/FTC/TAF should be used with caution as first-line treatment for people living with HIV with HCI under 51 years of age.

Population Pharmacokinetic Model of Plasma and Cellular Mycophenolic Acid in Kidney Transplant Patients from the CIMTRE Study.

BACKGROUND AND OBJECTIVE: Mycophenolate mofetil is widely used in kidney transplant recipients. Mycophenolate mofetil is hydrolysed by blood esterases to mycophenolic acid (MPA), the active drug. Although MPA therapeutic drug monitoring has been recommended to optimise the treatment efficacy by the area under the plasma concentration vs time curve, little is known regarding MPA concentrations in peripheral blood mononuclear cells, where MPA inhibits inosine monophosphate dehydrogenase. This study aimed to build a pharmacokinetic model using a population approach to describe MPA total and unbound concentrations in plasma and into peripheral blood mononuclear cells in 78 adult kidney transplant recipients receiving mycophenolate mofetil therapy combined with tacrolimus and prednisone. METHODS: Total and unbound plasma concentrations and peripheral blood mononuclear cell concentrations were assayed. A three-compartment model, two for plasma MPA and one for peripheral blood mononuclear cell MPA, with a zero-order absorption and a first-order elimination was used to describe the data. RESULTS: Mycophenolic acid average concentrations in peripheral blood mononuclear cells were well above half-maximal effective concentration for inosine monophosphate dehydrogenase and no relationship was found with the occurrence of graft rejection. Three covariates affected unbound and intracellular MPA pharmacokinetics: creatinine clearance, which has an effect on unbound MPA clearance, human serum albumin, which influences fraction unbound MPA and the ABCB1 3435 C>T (rs1045642) genetic polymorphism, which has an effect on MPA efflux transport from peripheral blood mononuclear cells. CONCLUSION: This population pharmacokinetic model demonstrated the intracellular accumulation of MPA, the efflux of MPA out of the cells being dependent on P-glycoprotein transporters. Nevertheless, further studies are warranted to investigate the relevance of MPA concentrations in peripheral blood mononuclear cells to dosing regimen optimisation.

Characteristics of Dolutegravir and Bictegravir Plasma Protein Binding: a First Approach for the Study of Pharmacologic Sanctuaries.

This study aimed to characterize in vitro dolutegravir (DTG) and bictegravir (BIC) binding. They had a preferential binding to human serum albumin (HSA) with two classes of albumin sites. Human alpha-1-acid glycoprotein (HAAG) binding of DTG and BIC showed an atypical nonlinear binding. The low-affinity site on HSA, the main plasma binding protein, suggests that the high protein binding rate should not impair passive diffusion.

Effect of high-dose rifampicin on efavirenz pharmacokinetics: drug-drug interaction randomized trial.

BACKGROUND: High-dose rifampicin is considered to shorten anti-TB treatment duration but its effect on antiretroviral metabolism is unknown. OBJECTIVES: To assess the effect of doubling the rifampicin dose (to 20 mg/kg/day, R20) on efavirenz pharmacokinetics (PK) in HIV/TB coinfected patients. METHODS: Open-label Phase 2 drug-drug interaction randomized trial. Pulmonary TB, ART-naive adults were randomized to R20 and either efavirenz 600 mg (EFV600) or 800 mg (EFV800), or rifampicin 10 mg/kg/day (R10) and EFV600 with a 1:1:1 ratio. Patients were first started on TB treatment and 2-4 weeks later started on ART. They were switched to R10 and EFV600 after 8 weeks. Full PK sampling was done 4 weeks (on rifampicin) and 24 weeks (off rifampicin) after ART initiation. Transaminases, plasma HIV-1 RNA and sputum cultures were monitored. The efavirenz geometric mean ratio (GMR) of AUC at 4 and 24 weeks after ART initiation within the same patient was calculated in each arm and its 90% CI was compared with a preset range (0.70-1.43). RESULTS: Of 98 enrolled patients (32 in the R20EFV600 arm, 33 in the R20EFV800 arm and 33 in the R10EFV600 arm), 87 had full PK sampling. For the R20EFV600, R20EFV800 and R10EFV600 arms, GMRs of efavirenz AUC were 0.87 (90% CI: 0.75-1.00), 1.12 (90% CI: 0.96-1.30) and 0.96 (90% CI: 0.84-1.10). Twelve weeks after ART initiation, 78.6%, 77.4% and 72.4% of patients had HIV-1 RNA below 100 copies/mL and 85.7%, 86.7% and 80.0% had Week 8 culture conversion, respectively. Two patients per arm experienced a severe increase in transaminases. CONCLUSIONS: Doubling the rifampicin dose had a small effect on efavirenz concentrations and was well tolerated.

UPLC-MS/MS method for the simultaneous quantification of bictegravir and 13 others antiretroviral drugs plus cobicistat and ritonavir boosters in human plasma.

A sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for 14 antiretroviral drugs and 2 boosters in human plasma. Plasma (100 µL) was precipitated with a solution of acetonitrile containing labelled internal standards. The compounds were separated with a total chromatic run time of 6 min using an Acclaim TM RSLC 120 C18 column (2.1 × 100 mm, 2.2 µm). The method was fully validated according to the European Medecines Agency guidelines. Linearity of all analytes concentrations was validated up to 5000 ng/mL. Lower limits of quantification were ranged from 2.5 ng/mL to 10 ng/mL according to compounds. Intra-day and inter-day precision ranged from 0.2% to 8.9% and accuracies were below 13%. This UPLC-MS/MS method can be applied to clinical pharmacology research and therapeutic drug monitoring in patients living with HIV.

A 2.5-years within-patient evolution of a Pseudomonas aeruginosa with in vivo acquisition of ceftolozane-tazobactam and ceftazidime-avibactam resistance upon treatment.

Ceftolozane-tazobactam is considered to be a last resort treatment for infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa Although, resistance to this antimicrobial have been described in vitro, development of resistance in vivo was rarely reported. Here, we described the evolution of resistance to ceftolozane-tazobactam of P. aeruginosa isolates recovered from the same patient during recurrent infections over 2.5 years.Antimicrobial susceptibility testing results showed that 24 of the 27 P. aeruginosa isolates recovered from blood (n=18), wound (n=2), pulmonary sample (n=1), bile (n=2) and stools (n=4) of the same patient were susceptible to ceftolozane-tazobactam and ceftazidime-avibactam but resistant to ceftazidime, piperacillin-tazobactam, imipenem and meropenem. Three clinical isolates acquired resistance to ceftolozane-tazobactam and ceftazidime-avibactam along with a partial restoration of piperacillin-tazobactam and carbapenems susceptibilities. Whole genome sequencing analysis reveals that all isolates were clonally related (ST-111) with a median of 24.9 single nucleotide polymorphisms (SNPs) (range 8-48). The ceftolozane-tazobactam and ceftazidime-avibactam resistance was likely linked to the same G183D substitution in the chromosome-encoded cephalosporinase.Our results suggest resistance to ceftolozane-tazobactam in P. aeruginosa might occur in vivo upon treatment through amino-acid substitution in the intrinsic AmpC leading to ceftolozane-tazobactam and ceftazidime-avibactam resistance accompanied by re-sensitization to piperacillin-tazobactam and carbapenems.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing.

Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556-1566.

Comparison of the effect of direct-acting antiviral with and without ribavirin on cyclosporine and tacrolimus clearance values: results from the ANRS CO23 CUPILT cohort.

PURPOSE: Direct-acting antiviral agents have demonstrated their efficacy in treating HCV recurrence after liver transplantation and particularly the sofosbuvir/daclatasvir combination. Pharmacokinetic data on both calcineurin inhibitors and direct-acting antiviral exposure in liver transplant recipients remain sparse. METHODS: Patients were enrolled from the ANRS CO23 CUPILT cohort. All patients treated with sofosbuvir/daclatasvir with or without ribavirin were included in this study when blood samples were available to estimate the clearance of immunosuppressive therapy before direct-acting antiviral initiation and during follow-up. Apparent tacrolimus and cyclosporine clearances were estimated from trough concentrations measured using validated quality control assays. RESULTS: Sixty-seven mainly male patients (79%) were included, with a mean age of 57 years and mean MELD score of 8.2; 50 were on tacrolimus, 17 on cyclosporine. Ribavirin was combined with sofosbuvir/daclatasvir in 52% of patients. Cyclosporine clearance remained unchanged as well as tacrolimus clearance under the ribavirin-free regimen. Tacrolimus clearance increased 4 weeks after direct-acting antivirals and ribavirin initiation versus baseline (geometric mean ratio 1.81; 90% CI 1.30-2.52). Patients under ribavirin had a significantly higher fibrosis stage (> 2) (p = 0.02) and lower haemoglobin during direct-acting antiviral treatment (p = 0.02) which impacted tacrolimus measurements. Direct-acting antiviral exposure was within the expected range. CONCLUSION: Our study demonstrated that liver transplant patients with a recurrence of hepatitis C who are initiating ribavirin combined with a sofosbuvir-daclatasvir direct-acting antiviral regimen may be at risk of lower tacrolimus concentrations because of probable ribavirin-induced anaemia and higher fibrosis score, although there are no effects on cyclosporine levels. TRIAL REGISTRATION: NCT01944527.

Dolutegravir Cerebrospinal Fluid Diffusion in HIV-1-Infected Patients with Central Nervous System Impairment.

This study aimed to determine dolutegravir cerebrospinal fluid (CSF) diffusion in 13 patients with HIV-related cerebral impairment enrolled in a real-life observational study. Dolutegravir median (range) CSF concentration [9.6 (3.6-22.8) ng/mL] reached CSF therapeutic concentrations whatever the blood-brain barrier status and diffused in correlation with the albumin quotient (P = .0186).

Specific Biological Features of Adipose Tissue, and Their Impact on HIV Persistence.

Although white AT can contribute to anti-infectious immune responses, it can also be targeted and perturbed by pathogens. The AT's immune involvement is primarily due to strong pro-inflammatory responses (with both local and paracrine effects), and the large number of fat-resident macrophages. Adipocytes also exert direct antimicrobial responses. In recent years, it has been found that memory T cells accumulate in AT, where they provide efficient secondary responses against viral pathogens. These observations have prompted researchers to re-evaluate the links between obesity and susceptibility to infections. In contrast, AT serves as a reservoir for several persistence pathogens, such as human adenovirus Ad-36, Trypanosoma gondii, Mycobacterium tuberculosis, influenza A virus, and cytomegalovirus (CMV). The presence and persistence of bacterial DNA in AT has led to the concept of a tissue-specific microbiota. The unexpected coexistence of immune cells and pathogens within the specific AT environment is intriguing, and its impact on anti-infectious immune responses requires further evaluation. AT has been recently identified as a site of HIV persistence. In the context of HIV infection, AT is targeted by both the virus and the antiretroviral drugs. AT's intrinsic metabolic features, large overall mass, and wide distribution make it a major tissue reservoir, and one that may contribute to the pathophysiology of chronic HIV infections. Here, we review the immune, metabolic, viral, and pharmacological aspects that contribute to HIV persistence in AT. We also evaluate the respective impacts of both intrinsic and HIV-induced factors on AT's involvement as a viral reservoir. Lastly, we examine the potential consequences of HIV persistence on the metabolic and immune activities of AT.

Estimated glomerular filtration rate but not solute carrier polymorphisms influences anemia in HIV-hepatitis C virus coinfected patients treated with boceprevir or telaprevir-based therapy.

OBJECTIVES: Ribavirin (RBV) induced anemia may be influenced by host genetic factors affecting RBV transport solute carrier (SLC) or metabolism inosine triphosphatase (ITPA), as already reported. We investigated the influence of single nucleotide polymorphisms (SNPs) on SLC genes on anemia, RBV trough concentration (Ctrough) and response in HIV-hepatitis C virus coinfected patients receiving triple therapy with boceprevir or telaprevir. METHODS: Patients from the ANRS HC26/HC27 studies were genotyped for SLC28A3 SNPs (rs10868138 and rs56350726) and SL29A1 SNPs (rs760370). Hemoglobin (Hb) decline was collected at baseline day 0 (D0), week 4 (W4) and week 8 (W8), and RBV Ctrough was measured at W4 and W8 by HPLC. A multivariate analysis including SLC SNPs, estimated glomerular filtration rate (eGFR), ITPA deficiency and RBV Ctrough was performed to determine predictive factors of anemia and response. RESULTS: SLC genotyping was performed in 130 patients. Neither SLC28A3 nor SLC29A1 SNPs were associated with Hb decline both at W4 and W8. No association was found between SLC polymorphisms and RBV Ctrough. Independent predictive factors of Hb decline at W4 were D0 Hb, ITPA deficiency and W4 RBV Ctrough in the multivariate analysis (P < 0.05). Only D0 Hb, W4 RBV Ctrough and eGFRD0-W8 were predictive of anemia at W8 (P < 0.05). Response was not influenced by SLC SNPs. CONCLUSION: eGFR, but not SLC polymorphisms, influences anemia in HIV-hepatitis C virus coinfected patients receiving boceprevir-based or telaprevir-based therapy. RBV is still a cornerstone of hepatitis C treatment, thus renal function and RBV Ctrough should be monitored in patients receiving RBV regimen combined with first-generation direct-acting antiviral agent.

Raltegravir Pharmacokinetics in Patients on Asunaprevir-Daclatasvir.

Raltegravir pharmacokinetics was studied in 20 patients included in the ANRS HC30 QUADRIH Study before and after addition of anti-hepatitis C virus (anti-HCV) quadritherapy, including pegylated-interferon-ribavirin and asunaprevir plus daclatasvir. Raltegravir pharmacokinetic parameters remained unchanged whether administered on or off anti-HCV therapy. In addition, concentrations of raltegravir, asunaprevir, and daclatasvir were not affected by liver cirrhosis. These data suggest that in human immunodeficiency virus (HIV)-HCV-coinfected patients, whether cirrhotic or not, asunaprevir and daclatasvir could be administered safely with raltegravir.