Kathleen Squires

Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes.

The latency of human immunodeficiency virus type 1 (HIV-1) in resting primary CD4+ T cells is the major barrier for the eradication of the virus in patients on suppressive highly active antiretroviral therapy (HAART). Even with optimal HAART treatment, replication-competent HIV-1 still exists in resting primary CD4+ T cells. Multiple restriction factors that act upon various steps of the viral life cycle could contribute to viral latency. Here we show that cellular microRNAs (miRNAs) potently inhibit HIV-1 production in resting primary CD4+ T cells. We have found that the 3′ ends of HIV-1 messenger RNAs are targeted by a cluster of cellular miRNAs including miR-28, miR-125b, miR-150, miR-223 and miR-382, which are enriched in resting CD4+ T cells as compared to activated CD4+ T cells. Specific inhibitors of these miRNAs substantially counteracted their effects on the target mRNAs, measured either as HIV-1 protein translation in resting CD4+ T cells transfected with HIV-1 infectious clones, or as HIV-1 virus production from resting CD4+ T cells isolated from HIV-1–infected individuals on suppressive HAART. Our data indicate that cellular miRNAs are pivotal in HIV-1 latency and suggest that manipulation of cellular miRNAs could be a novel approach for purging the HIV-1 reservoir.

Initial increase in blood CD4+ lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

Results of a phase 2 clinical trial at 48 weeks (AI424-007): a dose-ranging, safety, and efficacy comparative trial of atazanavir at three doses in combination with didanosine and stavudine in antiretroviral-naive subjects.

Three dose levels of the protease inhibitor (PI) atazanavir (200, 400, and 500 mg once daily) were compared with nelfinavir (750 mg three times daily) when given both as monotherapy and in combination with didanosine and stavudine in 420 antiretroviral-naive subjects infected with HIV-1. Subjects received monotherapy for 2 weeks, followed by combination therapy for 46 weeks. After 48 weeks, mean change from baseline in HIV RNA (−2.57 to −2.33 log10 copies/mL), the proportion of subjects with HIV RNA <400 copies/mL (56%–64%) and <50 copies/mL (28%–42%), and mean increases in CD4 cell count (185–221 cells/mm3) were comparable across treatment groups. Diarrhea was two to three times more common in the nelfinavir group (61% of subjects) than in the atazanavir groups (23%–30% of subjects, p < .0001 versus nelfinavir), and jaundice occurred only in atazanavir-treated subjects (6%, 6%, and 12% in the 200-, 400-, and 500-mg groups, respectively) (p < .03 for all atazanavir regimens vs. nelfinavir). Mean percent change from baseline in fasting low-density lipoprotein (LDL) cholesterol was significantly less in the atazanavir groups (−7% to 4%) than in the nelfinavir group (31%) (p < .0001). In conclusion, once-daily atazanavir is a potent, safe, and well tolerated PI that rapidly and durably suppresses HIV RNA and durably increases CD4 cell count in antiretroviral-naive subjects. Through 48 weeks, atazanavir was not associated with clinically relevant increases in total cholesterol, fasting LDL cholesterol, or fasting triglycerides. In comparison, nelfinavir was associated with prompt, marked, and sustained elevations in these parameters of a magnitude that suggests they are clinically relevant.

State of the Science Conference Initiative to Decrease Cardiovascular Risk and Increase Quality of Care for Patients Living With HIV/AIDS: Executive Summary

With successful antiretroviral therapy, patients infected with the human immunodeficiency virus (HIV) are living longer; however, recent reports suggest increased rates of coronary heart disease (CHD) among HIV-infected patients,1 and cardiovascular disease has become an important cause of morbidity and mortality in this population.2 Increased CHD rates in the HIV population may relate to traditional risk factors, including advancing age, higher smoking rates, dyslipidemia, insulin resistance, and impaired glucose tolerance. Cardiovascular disease may also be due to nontraditional factors, including changes in body composition with loss of subcutaneous fat and/or accumulation of visceral fat in some patients, inflammation, and direct effects of the virus on the vasculature, as well as to direct effects of specific antiretroviral drugs. Important questions remain as to the pathogenesis, detection, and treatment of cardiovascular disease and related risk factors in HIV-infected patients. These questions concern, among other things, the design of adequate trials to determine CHD incidence and the utility of existing CHD guidelines for screening, prevention, treatment, and risk stratification. To ascertain the state of the science with respect to these and related questions, a multidisciplinary conference with interested HIV specialists, cardiologists, endocrinologists, primary care physicians, National Institutes of Health representatives, and patient advocates was convened June 28–30, 2007, in Chicago, Ill, and chaired by Drs Steven Grinspoon and Robert Eckel. The discussions focused on 6 areas of interest, each with its own working group, including the following: (1) the contribution of metabolic and anthropometric abnormalities to cardiovascular disease risk factors (chaired by Drs Carl Grunfeld and Donald Kotler); (2) the epidemiological evidence for cardiovascular disease and its relationship to highly active antiretroviral therapy (HAART; chaired by Drs Judy Currier and Jens Lundgren); (3) the effects of HIV infection and antiretroviral therapy on the heart and vasculature (chaired by Drs Michael Dube …

Tenofovir Disoproxil Fumarate in Nucleoside-Resistant HIV-1 Infection: A Randomized Trial

Context Resistance to antiretroviral drugs is a leading cause of treatment failure in HIV-1infected patients. Contribution In this multicenter, double-blind trial, 552 treatment-experienced patients with detectable RNA levels despite continuing antiretroviral therapy were randomly assigned to receive tenofovir DF (a nucleotide analogue) or placebo. At 6 months, patients given tenofovir DF had greater reductions in HIV-1 RNA levels than those given placebo. Nearly 15% of patients in both groups had clinical adverse events, such as severe diarrhea, pain, or depression. Implications In HIV-infected patients with suboptimal viral suppression, adding tenofovir DF to ongoing antiretroviral therapy reduces viral loads. The Editors Combination antiretroviral therapy has decreased mortality rates for patients with HIV-1 infection (1, 2). Suppression of HIV-1 viral load has been shown to be highly predictive of slower clinical disease progression (3). However, the clinical utility of a combination antiretroviral regimen typically wanes, often because of one or more factors such as drug resistance or poor adherence (4). These factors are even more important for patients who have previously used antiretroviral drugs. This difficult-to-treat population has limited treatment options and often experiences drug-related adverse effects due to previous antiretroviral therapy regimens (5-7). Given these limitations, novel potent agents that combine ease of dosing with favorable safety and resistance profiles are needed to increase the long-term durability of combination antiretroviral therapy (8, 9). Simplifying HIV treatment regimens using once-daily antiretroviral drugs may improve adherence and therapeutic outcomes (10, 11). Tenofovir disoproxil fumarate (tenofovir DF) is an oral prodrug of tenofovir, a novel, acyclic nucleotide analogue with in vitro activity against HIV-1 and HIV-2 (12, 13). Unlike nucleoside analogues, tenofovir is active in both active and resting lymphoid cells and macrophages (13). In rhesus macaques acutely infected with simian immunodeficiency virus, tenofovir administered as monotherapy 24 hours after inoculation eradicated viral DNA from lymph nodes, plasma, and leukocytes (14). Tenofovir has also demonstrated in vitro activity against wild-type and lamivudine-resistant hepatitis B virus (15). In a 186-patient phase II dose-ranging study, Schooley and colleagues (16) demonstrated the potency and favorable safety profile of tenofovir DF, 300 mg, highlighting its potential for further study in larger phase III trials. Tenofovir has a favorable resistance profile with activity against most nucleoside analogueresistant viruses and infrequent (3%) emergence of the K65R resistance mutation through 96 weeks (17-21). Our study was designed to confirm the antiviral efficacy and safety of tenofovir DF compared with placebo in treatment-experienced patients who had detectable HIV-1 RNA levels despite combination antiretroviral therapy. Methods Study Sample Institutional review boards at all study sites approved the study protocol and informed consent statements. Recruitment began in October 1999 and continued until June 2000 at 75 HIV clinics in western Europe, North America, and Australia. All patients gave written informed consent. Patients 18 to 65 years of age were eligible if they had received antiretroviral therapy (four agents or fewer) for at least 8 weeks before randomization and had stable plasma HIV-1 RNA levels of 400 to 10 000 copies/mL on the Roche Amplicor HIV-1 Monitor UltraSensitive test, version 1.0 (Roche Diagnostics, Branchburg, New Jersey). The lower limit of quantification for this test was 50 copies/mL. We placed no entry restrictions on CD4 cell count. Additional inclusion criteria were serum creatinine concentration of 133 mol/L or less ( 1.5 mg/dL), calculated creatinine clearance (using the CockcroftGault formula [22]) of at least 1.00 mL/s ( 60 mL/min), absolute neutrophil count of at least 1.000 109 cells/L, platelet count of at least 50.0 109 cells/L, hemoglobin level of at least 80 g/L, total bilirubin level of 26 mol/L or less ( 1.5 mg/dL), serum phosphorus level of at least 0.71 mmol/L ( 2.2 mg/dL), alanine aminotransferase level less than 108 U/L, aspartate aminotransferase level less than 90 U/L, negative results on a serum pregnancy test for women of childbearing potential, and life expectancy of more than 1 year. We excluded patients who had previously participated in clinical trials with intravenous tenofovir, tenofovir DF, or adefovir dipivoxil; had been immunized within 30 days of baseline; had an active AIDS-defining condition within 30 days of baseline; or were receiving aminoglycoside antibiotics, amphotericin B, cidofovir, cisplatin, foscarnet, intravenous pentamidine, vancomycin, ganciclovir, systemic chemotherapeutic agents, oral corticosteroids, probenecid, or other investigational agents on an ongoing basis. Patients who were receiving their first antiretroviral regimen were not excluded. We assessed 976 patients for eligibility and randomly assigned 552 to study groups (Figure). Four hundred twenty-four patients were excluded. Of the 380 who did not meet inclusion criteria, the most common reason (n = 269) was HIV-1 RNA levels outside the range of 400 to 10 000 copies/mL. Among the remaining 44 patients, 22 elected not to participate in the study and the other 22 were excluded because of investigator decision, concomitant participation in other investigational studies, screening after the cutoff date for enrollment, incomplete laboratory values, lack of follow-up, or family emergency. No statistical analyses were performed for patients who were not randomly assigned to a study group. Figure. Recruitment and disposition flow chart. Design Through an interactive voice response system that centralized patient randomization, Interactive Clinical Technologies, Inc. (San Francisco, California), generated the random allocation sequence, assigned patients to their treatment groups, and blinded kit numbers to conceal the allocation sequence. Patients were stratified according to HIV-1 RNA level (<5000 copies/mL or 5000 copies/mL), CD4 cell count (<0.2 109 cells/L or 0.2 109 cells/L), and number of antiretroviral drugs taken before study entry (fewer than four or at least four). Patients were then randomly assigned in a 2-to-1 ratio to add tenofovir DF, 300 mg, or identical-appearing placebo to their existing antiretroviral regimen. Randomization was not stratified by study center. Changes to the background regimen were discouraged during the first 24 weeks. After week 24, all patients received open-label tenofovir DF for the remainder of the 48-week study. As predefined in both the study protocol and the statistical analysis plan, the single end point for primary efficacy was the time-weighted average change in HIV-1 RNA level from baseline to week 24. Secondary efficacy end points included the proportion of patients with HIV-1 RNA levels of 50 copies/mL or less and 400 copies/mL or less at weeks 24 and 48, the time-weighted average change in HIV-1 RNA level from baseline to week 48, and CD4 cell count at weeks 24 and 48. Adherence to treatment was measured by using pill counts at each study visit, but pill counts were not formally analyzed and drug concentrations were not measured during routine study visits. Approximately half of the patients (n = 274) were randomly assigned to a virologic genotyping substudy at baseline. HIV-1 reverse transcriptase nucleotides 1 to 1200 and all of the HIV-1 protease gene were sequenced by following the reverse transcriptase polymerase chain reaction from plasma HIV-1 RNA (Vircogen, Virco, Mechelen, Belgium). All plasma HIV-1 samples that were obtained at baseline, at week 24 or 48, or at early study termination and had more than 50 copies of HIV-1 RNA per mL were analyzed in a blinded fashion. A polymerase chain reaction product could not be obtained from 21 patients at baseline (14 in the tenofovir DF group, 7 in the placebo group). Plasma HIV-1 RNA was insufficient for genotypic analysis in 58 patients at week 24 (43 patients in the tenofovir DF group, 15 in the placebo group) and 129 patients at week 48. Assessments The incidences of grade 3 or 4 clinical adverse events or laboratory abnormalities were evaluated as safety end points. At each postbaseline study visit (weeks 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48), the investigator assessed and recorded all adverse events found during history and physical examination as well as laboratory toxicities found on chemical and hematologic tests and urinalysis, including date of onset and resolution, severity, relationship to study drug, outcome, and action taken with study medication. Severity was recorded and graded according to the modified severity grading for adult adverse experiences outlined by the National Institutes of Allergy and Infectious Diseases, Division of AIDS. Plasma levels of HIV-1 RNA and CD4 cell counts were measured at all study visits except weeks 28, 36, and 44. Statistical Analysis Data were analyzed for the intention-to-treat sample and the as-treated sample. The intention-to-treat sample included all enrolled patients who received at least one dose of study medication and was the primary sample for analyses of efficacy. The as-treated sample included patients who received at least one dose of study medication but excluded all data obtained after permanent discontinuation of assigned study medication or addition of other antiretroviral medication. The safety analysis sample included all patients who received at least one dose of study medication. As the primary efficacy end point, the time-weighted average change in HIV-1 RNA level from baseline to week 24 represents the difference between the time-weighted average postbaseline values and values at baseline. This difference is defined as follows (23, 24). If the area under the curve (AUC) at week 24 (

Fluconazole compared with ketoconazole for the treatment of Candida esophagitis in AIDS. A randomized trial.

OBJECTIVE To determine the clinical and endoscopic response of candida esophagitis to antifungal therapy and to compare the two oral antifungal agents, fluconazole and ketoconazole. DESIGN Multicenter, randomized, double-blind trial. SETTING Fifteen U.S. centers including university, private practice, and county hospital settings. PATIENTS A total of 169 patients with the acquired immunodeficiency syndrome (AIDS); odynophagia, dysphagia, or retrosternal pain; white esophageal plaques at endoscopy; and pseudohyphae on esophageal brushings or biopsies. INTERVENTION Patients were randomly assigned to fluconazole (100 mg/d) or ketoconazole (200 mg/d). Doses were doubled at week 1 or 2 if no symptomatic improvement had occurred during the preceding week. Therapy was continued for 2 weeks after resolution of symptoms or for a maximum of 8 weeks. MEASUREMENTS Patients were clinically evaluated weekly, and laboratory tests were done every 2 weeks. Endoscopy was repeated within 5 days after the end of therapy. RESULTS A total of 143 patients were clinically evaluable (assessed within 7 days after therapy), and 129 patients were endoscopically evaluable (endoscopy repeated after therapy). Endoscopic cure occurred in 91% of patients treated with fluconazole and in 52% of those given ketoconazole for a difference of 39% (95% Cl, 24% to 52%; P less than 0.001). Esophageal symptoms resolved in 85% of fluconazole-treated patients and in 65% of ketoconazole-treated patients for a difference of 20% (Cl, 6% to 34%; P = 0.006). Intention-to-treat analyses also yielded statistically significant differences for the comparisons listed above. Side effects were minimal and comparable in the two groups; only one patient in each group had therapy discontinued for adverse effects that were possibly related to the study medications. CONCLUSIONS Fluconazole is associated with significantly greater rates of endoscopic and clinical cure than ketoconazole in patients with AIDS and candida esophagitis. Both drugs appear to be safe and well tolerated.

First large, multicenter, open-label study utilizing Hla-b*5701 screening for abacavir hypersensitivity in North America

A hypersensitivity reaction is associated with abacavir in approximately 2–8% of exposed patients. The frequency of the HLA-B*5701 allele varies across racial groups and significantly correlates with risk of hypersensitivity. Studies in Europe and Western Australia demonstrated that prospective screening can significantly reduce the rate of hypersensitivity by avoiding the use of abacavir in patients carrying the HLA-B*5701 allele. Prospective HLA-B*5701 screening in a large, racially diverse North American population resulted in less than 1% of individuals diagnosed with a suspected abacavir hypersensitivity reaction (ABC HSR) and no positive skin patch test through 30 weeks among HLA-B*5701-negative individuals.

Phase I/II study of the pharmacokinetics, safety and antiretroviral activity of tenofovir alafenamide, a new prodrug of the HIV reverse transcriptase inhibitor tenofovir, in HIV-infected adults

BACKGROUND Tenofovir alafenamide (formerly GS-7340) is a new oral prodrug of tenofovir, a nucleotide analogue that inhibits HIV-1 reverse transcription. Unlike the currently marketed tenofovir prodrug, tenofovir disoproxil fumarate, tenofovir alafenamide is stable in plasma and then rapidly converted into tenofovir once inside cells. METHODS The pharmacokinetics, safety and antiviral activity of 40 or 120 mg of tenofovir alafenamide compared with 300 mg of tenofovir disoproxil fumarate when administered as monotherapy once daily for 14 days in HIV-1-infected, treatment-naive subjects was studied. RESULTS Administration of 40 mg of tenofovir alafenamide for 14 days resulted in lower tenofovir Cmax (13 versus 207 ng/mL) and lower systemic exposures (AUC0-t, 383 versus 1810 ng · h/mL) compared with subjects who received tenofovir disoproxil fumarate. There were higher intracellular tenofovir concentrations within peripheral blood mononuclear cells with both 40 mg of tenofovir alafenamide (8.2 μM) and 120 mg of tenofovir alafenamide (16.9 μM) compared with 300 mg of tenofovir disoproxil fumarate (0.9 μM). The most commonly observed adverse events were headache, nausea and flatulence, which occurred similarly across the three groups. After 14 days, the mean changes in HIV-1 RNA were -0.94 log₁₀copies/mL for the tenofovir disoproxil fumarate group, -1.57 log₁₀ copies/mL for the 40 mg of tenofovir alafenamide group and -1.71 log₁₀ copies/mL for the 120 mg of tenofovir alafenamide group. The mean first-phase HIV-1 RNA decay slopes were -0.36, -0.63 and -0.64 for the tenofovir disoproxil fumarate group, the 40 mg of tenofovir alafenamide group and the 120 mg of tenofovir alafenamide group, respectively. No resistance mutations to either tenofovir alafenamide or tenofovir disoproxil fumarate were detected. CONCLUSIONS Tenofovir alafenamide, a new once-daily oral prodrug of tenofovir, showed more potent anti-HIV-1 activity and higher intracellular tenofovir levels compared with tenofovir disoproxil fumarate, while maintaining lower plasma tenofovir exposure at 40 mg with good tolerability over 14 days of monotherapy.

Immunogenicity and Safety of the Human Papillomavirus 6, 11, 16, 18 Vaccine in HIV-Infected Young Women

BACKGROUND The objective of this study was to determine whether the 3-dose quadrivalent human papillomavirus (HPV) vaccine series (HPV-6, -11, -16, -18) is immunogenic and safe in young women infected with human immunodeficiency virus (HIV). METHODS We enrolled 99 women aged 16-23 years in a phase 2, open-label, multicenter trial, conducted from 2008 to 2011 by the Adolescent Medicine Trials Network for HIV/AIDS Interventions. Outcome measures were immunogenicity 4 weeks after dose 3, measured by (1) geometric mean titers (GMTs) and (2) seroconversion rates for HPV-6, -11, -16, and -18, among those seronegative and HPV DNA negative for each type. Immune responses were compared to those of a historical comparison group of HIV-negative women (n = 267) using univariate methods. Clinical and laboratory adverse events were assessed after each dose. RESULTS The mean age of subjects was 21.4 years; 80% were non-Hispanic black, 69 were not taking antiretroviral therapy (ART), and 30 were taking ART. No differences in GMTs were noted among participants taking ART vs the comparison group, but GMTs were lower in participants not taking ART vs the comparison group for HPV-16 (2393 vs 3892 milli-Merck units per milliliter [mMU/mL], P = .012) and HPV-18 (463 vs 801 mMU/mL, P = .003). Seroconversion rates were 100% for HPV-6, -11, -16, and -18 among participants taking ART. Rates ranged from 92.3% (for HPV-18) to 100.0% (for HPV-6) among participants not taking ART. One severe adverse event (fatigue) was noted. CONCLUSIONS In a sample of HIV-infected women who were HPV DNA and HPV seronegative, immune responses to HPV vaccination were generally robust and the vaccine was well tolerated.

Sex-Based Outcomes of Darunavir–Ritonavir Therapy: A Single-Group Trial

BACKGROUND Women account for an increasing proportion of patients with HIV-1 but remain underrepresented in antiretroviral clinical trials. OBJECTIVE To evaluate sex-based differences in efficacy and adverse events in treatment-experienced, HIV-positive women and men receiving darunavir-ritonavir therapy over 48 weeks. DESIGN Multicenter, open-label, phase 3b study designed to enroll a high proportion of women, with sample size determined on the basis of a noninferiority design with a maximum allowable difference of 15% in virologic response favoring men. (ClinicalTrials.gov registration number: NCT00381303) SETTING 65 sites in the United States, Puerto Rico, and Canada. PATIENTS 287 women and 142 men. INTERVENTION Patients received darunavir-ritonavir, 600/100 mg twice daily, plus an investigator-selected optimized background regimen. MEASUREMENTS Virologic response (HIV RNA <50 copies/mL using a time-to-loss of virologic response [TLOVR] algorithm) and adverse events were assessed over 48 weeks. RESULTS 67% of patients were women; 84% of patients were black or Hispanic. A higher proportion of women discontinued treatment than men (32.8% vs. 23.2%; P = 0.042); more women than men discontinued treatment for reasons other than virologic failure. Response rates in women and men at week 48 were 50.9% and 58.5%, respectively (intention-to-treat TLOVR), and 73.0% and 73.5%, respectively (TLOVR censored for patients who withdrew for reasons other than virologic failure). The absolute difference in response, based on logistic regression and adjusted for baseline log(10) viral load and CD4(+) cell count, was -9.6 percentage points (95% CI, -19.9 to 0.7 percentage points; P = 0.067) for intention-to-treat TLOVR and -3.9 percentage points (CI, -13.9 to 6.0 percentage points; P = 0.438) for TLOVR population that censored patients who withdrew for reasons other than virologic failure. Adverse events were similar between the sexes. The most common grade 2 to 4 adverse events that were considered at least possibly treatment related in women and men were nausea (5.2% and 2.8%, respectively), diarrhea (4.5% and 4.9%, respectively), and rash (2.1% and 2.8%, respectively). LIMITATION Baseline characteristics differed between sexes. CONCLUSION Nonsignificant, sex-based differences in response were found during the 48-week study; however, these differences were probably due to higher discontinuation rates in women, suggesting that additional efforts are needed to retain women in clinical trials.