Brian Doehle

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV‐1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV‐1 mutants that do not express the Vif protein are replication incompetent in ‘nonpermissive’ cells, such as primary T cells and the T‐cell line CEM, that express APOBEC3G. In contrast, Vif‐defective HIV‐1 replicates effectively in ‘permissive’ cell lines, such as a derivative of CEM termed CEM‐SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV‐1 virions and inhibits their infectivity. APOBEC3F binds the HIV‐1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM‐SS. Together, these data argue that HIV‐1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA‐editing enzymes.

APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells

While the ability of APOBEC3G to reduce the replication of a range of exogenous retroviruses is now well established, recent evidence has suggested that APOBEC3G can also inhibit the replication of endogenous retrotransposons that bear long terminal repeats. Here, we extend this earlier work by showing that two other members of the human APOBEC3 protein family, APOBEC3B and APOBEC3A, can reduce retrotransposition by the intracisternal A-particle (IAP) retrotransposon in human cells by 20-fold to up to 100-fold, respectively. This compares to an ∼4-fold inhibition in IAP retrotransposition induced by APOBEC3G. While both APOBEC3G and APOBEC3B specifically interact with the IAP Gag protein in co-expressing cells, and induce extensive editing of IAP reverse transcripts, APOBEC3A fails to package detectably into IAP virus-like particles and does not edit IAP reverse transcripts. These data, which identify human APOBEC3A as a highly potent inhibitor of LTR-retrotransposon function, are the first to ascribe a biological activity to APOBEC3A. Moreover, these results argue that APOBEC3A inhibits IAP retrotransposition via a novel mechanism that is distinct from, and in this case more effective than, the DNA editing mechanism characteristic of APOBEC3G and APOBEC3B.

Ledipasvir-sofosbuvir with or without ribavirin to treat patients with HCV genotype 1 infection and cirrhosis non-responsive to previous protease-inhibitor therapy: a randomised, double-blind, phase 2 trial (SIRIUS)

BACKGROUND Patients with cirrhosis resulting from chronic hepatitis C virus (HCV) infection are at risk of life-threatening complications, but consistently achieve lower sustained virological response (SVR) than patients without cirrhosis, especially if treatment has previously failed. We assessed the efficacy and safety of the NS5A inhibitor ledipasvir and the nucleotide polymerase inhibitor sofosbuvir, with and without ribavirin. METHODS In this multicentre, double-blind trial, between Oct 21, 2013, and Oct 30, 2014, we enrolled patients with HCV genotype 1 and compensated cirrhosis who had not achieved SVR after successive treatments with pegylated interferon and protease-inhibitor regimens at 20 sites in France. With a computer-generated randomisation sequence, patients were assigned in a 1:1 ratio to receive placebo matched in appearance to study drugs for 12 weeks followed by once daily combination fixed-dose tablets of 90 mg ledipasvir and 400 mg sofosbuvir plus weight-based ribavirin for 12 weeks, or ledipasvir-sofosbuvir plus placebo once daily for 24 weeks. The primary endpoint was SVR 12 weeks after the end of treatment (SVR12), for which 95% CIs were calculated with the Clopper-Pearson method. This study is registered with ClinicalTrials.gov, number NCT01965535. FINDINGS Of 172 patients screened, 155 entered randomisation, 77 were assigned to receive ledipasvir-sofosbuvir plus ribavirin and 78 ledipasvir-sofosbuvir. 114 (74%) were men, 151 (97%), were white, 98 (63%) had HCV genotype 1a, and 145 (94%) had non-CC IL28B alleles. SVR12 rates were 96% (95% CI 89-99) for patients in the ledipasvir-sofosbuvir plus ribavirin group and 97% (91-100) in the ledipasvir-sofosbuvir group. One patient discontinued treatment because of adverse events while receiving only placebo. The most frequent adverse events were asthenia and headache, pruritus, and fatigue. INTERPRETATION Ledipasvir-sofosbuvir plus ribavirin for 12 weeks and ledipasvir-sofosbuvir for 24 weeks provided similarly high SVR12 rates in previous non-responders with HCV genotype 1 and compensated cirrhosis. The shorter regimen, when given with ribavirin, might, therefore, be useful to treat treatment-experienced patients with cirrhosis if longer-term treatment is not possible. FUNDING Gilead Sciences.

Differential Sensitivity of Murine Leukemia Virus to APOBEC3-Mediated Inhibition Is Governed by Virion Exclusion

ABSTRACT While members of the APOBEC3 family of human intrinsic resistance factors are able to restrict the replication of Vif-deficient forms of human immunodeficiency virus type 1 (HIV-1), they are unable to block replication of wild-type HIV-1 due to the action of Vif, which induces their degradation. In contrast, HIV-1 Vif is unable to block inhibition mediated by APOBEC3 proteins expressed by several heterologous species, including mice. Here, we have asked whether the simple retrovirus murine leukemia virus (MLV) is sensitive to restriction by the cognate murine or heterologous, human APOBEC3 proteins. We demonstrate that MLV is highly sensitive to inhibition by human APOBEC3G and APOBEC3B but resistant to inhibition by murine APOBEC3 or by other human APOBEC3 proteins, including APOBEC3F. This sensitivity fully correlates with the ability of these proteins to be packaged into MLV virion particles: i.e., human APOBEC3G and APOBEC3B are packaged while murine APOBEC3 and human APOBEC3F are excluded. Moreover, this packaging in turn correlates with the differential ability of these APOBEC3 proteins to bind MLV Gag. Together, these data suggest that MLV Gag has evolved to avoid binding, and hence virion packaging, of the cognate murine APOBEC3 protein but that MLV infectivity is still restricted by certain heterologous APOBEC3 proteins that retain this ability. Moreover, these results suggest that APOBEC3 proteins may help prevent the zoonotic infection of humans by simple retroviruses and provide a mechanism for how simple retroviruses can avoid inhibition by APOBEC3 family members.

Sofosbuvir plus ribavirin for the treatment of chronic genotype 4 hepatitis C virus infection in patients of Egyptian ancestry

BACKGROUND & AIMS We conducted an open-label phase 2 study to assess the efficacy and safety of the oral nucleotide polymerase inhibitor sofosbuvir in combination with ribavirin in patients of Egyptian ancestry, chronically infected with genotype 4 hepatitis C virus (HCV). METHODS Treatment-naive and previously treated patients with genotype 4 HCV were randomly allocated in a 1:1 ratio to receive sofosbuvir 400mg and weight-based ribavirin, for 12 or 24 weeks. The primary efficacy endpoint was the proportion of patients with sustained virologic response (HCV RNA <25IU/ml) 12 weeks after cessation of therapy (SVR12). RESULTS Thirty treatment-naive and thirty previously treated patients were enrolled and treated for 12 weeks (n=31) or 24 weeks (n=29). Overall, 23% of patients had cirrhosis and 38% had diabetes. 14% of treatment-naive patients were interferon ineligible and 63% of treatment-experienced patients had prior non-response. SVR12 was achieved by 68% of patients (95% CI, 49-83%) in the 12-week group, and by 93% of patients (95% CI, 77-99%) in the 24-week group. The most common adverse events were headache, insomnia, and fatigue. No patient discontinued treatment due to an adverse event. CONCLUSIONS The findings from the present study suggest that 24 weeks of sofosbuvir plus ribavirin is an efficacious and well tolerated treatment in patients with HCV genotype 4 infection.

Sofosbuvir plus ribavirin for treating Egyptian patients with hepatitis C genotype 4

BACKGROUND & AIMS Egypt has the highest prevalence of chronic hepatitis C virus (HCV) infection in the world, and more than 90% of patients are infected with genotype 4 virus. We evaluated the efficacy and safety of the HCV polymerase inhibitor sofosbuvir in combination with ribavirin in HCV genotype 4 patients in Egypt. METHODS Treatment-naïve or treatment-experienced patients with genotype 4 HCV infection (n=103) were randomly assigned to receive either 12 or 24 weeks of sofosbuvir 400 mg and ribavirin 1000-1200 mg daily. Randomization was stratified by prior treatment experience and by presence or absence of cirrhosis. The primary endpoint was the percentage of patients with HCV RNA <25 IU/ml 12 weeks after therapy (SVR12). RESULTS Among all patients, 52% had received prior HCV treatment and 17% had cirrhosis at baseline. SVR12 rates were 90% (46/51) with 24 weeks and 77% (40/52) with 12 weeks of sofosbuvir and ribavirin therapy. Patients with cirrhosis at baseline had lower rates of SVR12 (63% 12 weeks, 78% 24 weeks) than those without cirrhosis (80% 12 weeks, 93% 24 weeks). The most common adverse events were fatigue, headache, insomnia, and anemia. Two patients experienced serious adverse events (cerebral ischemia, dyspnea). No adverse events resulted in treatment discontinuation. CONCLUSION Sofosbuvir plus ribavirin for 12 or 24 weeks is effective in treating both treatment-naïve and treatment-experienced Egyptian patients with genotype 4 HCV.

NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome

BACKGROUND & AIMS The efficacy of NS5A inhibitors for the treatment of patients chronically infected with hepatitis C virus (HCV) can be affected by the presence of NS5A resistance-associated substitutions (RASs). We analyzed data from 35 phase I, II, and III studies in 22 countries to determine the pretreatment prevalence of various NS5A RASs, and their effect on outcomes of treatment with ledipasvir-sofosbuvir in patients with genotype 1 HCV. METHODS NS5A gene deep sequencing analysis was performed on samples from 5397 patients in Gilead clinical trials. The effect of baseline RASs on sustained virologic response (SVR) rates was assessed in the 1765 patients treated with regimens containing ledipasvir-sofosbuvir. RESULTS Using a 15% cut-off, pretreatment NS5A and ledipasvir-specific RASs were detected in 13% and 8% of genotype 1a patients, respectively, and in 18% and 16% of patients with genotype 1b. Among genotype 1a treatment-naïve patients, SVR rates were 91% (42/46) vs. 99% (539/546) for those with and without ledipasvir-specific RASs, respectively. Among treatment-experienced genotype 1a patients, SVR rates were 76% (22/29) vs. 97% (409/420) for those with and without ledipasvir-specific RASs, respectively. Among treatment-naïve genotype 1b patients, SVR rates were 99% for both those with and without ledipasvir-specific RASs (71/72 vs. 331/334), and among treatment-experienced genotype 1b patients, SVR rates were 89% (41/46) vs. 98% (267/272) for those with and without ledipasvir-specific RASs, respectively. CONCLUSIONS Pretreatment ledipasvir-specific RASs that were present in 8-16% of patients have an impact on treatment outcome in some patient groups, particularly treatment-experienced patients with genotype 1a HCV. LAY SUMMARY The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection received treatments that included ledipasvir-sofosbuvir to determine how prevalent NS5A RASs are in patients at baseline, and found that ledipasvir-specific RASs were present in 8-16% of patients prior to treatment and had a negative impact on treatment outcome in subset of patient groups, particularly treatment-experienced patients with genotype 1a HCV.

L159F and V321A Sofosbuvir-Associated Hepatitis C Virus NS5B Substitutions

BACKGROUND Sofosbuvir (SOF) exhibits a high barrier to resistance, with no S282T NS5B substitution or phenotypic resistance detected in phase 3 registration studies. METHODS Here, emergence of the NS5B variants L159F and V321A and possible association with resistance was evaluated in 8 studies of SOF (NEUTRINO, FISSION, POSITRON, FUSION, VALENCE, PHOTON-1, PHOTON-2, and P7977-2025) and 5 studies of combination ledipasvir (LDV) and SOF (LDV/SOF; LONESTAR, ELECTRON [LDV/SOF arms], ION1, ION2, and ION3), using deep sequencing. RESULTS Deep sequencing detected L159F in 15% (53 of 353) and V321A in 5% (17 of 353) of patients with virologic failure in the SOF studies. Intensification of SOF treatment with LDV reduced the emergence of L159F or V321A to 2% (1 of 50 each) at virologic failure. L159F and V321A did not influence the outcome of retreatment with SOF, ribavirin, and pegylated interferon. At baseline, L159F was detected only in genotype 1-infected patients (1%) and was only associated with increased virologic failure in patients treated for short durations with SOF and ribavirin. CONCLUSIONS Deep-sequencing analysis confirmed that NS5B variants L159F and V321A emerged in a subset of patients treated with SOF at virologic failure. These variants had no impact on retreatment outcome with SOF, ribavirin, and pegylated interferon. Baseline L159F in genotype 1 did not affect the treatment outcome with LDV/SOF.

Sofosbuvir Plus Velpatasvir Combination Therapy for Treatment-Experienced Patients With Genotype 1 or 3 Hepatitis C Virus Infection: A Randomized Trial

Context Options are needed when initial therapy fails for hepatitis C virus (HCV) infection. Contribution In a phase 2 trial, treatment-experienced patients with genotype 3 HCV infection who either did not have cirrhosis or had compensated cirrhosis, as well as patients with genotype 1 HCV infection that was unsuccessfully treated with a protease inhibitor plus peginterferon or ribavirin, received sofosbuvir plus 1 of 2 doses of velpatasvir with or without ribavirin. Sustained virologic response rates 12 weeks after treatment were high in all groups that received sofosbuvir plus 100 mg of velpatasvir. Treatment was well-tolerated. Caution The number of patients was small. Implication Larger trials of sofosbuvir plus velpatasvir are indicated for patients with HCV infection for whom initial therapy fails. Of the 6 hepatitis C virus (HCV) genotypes, 1 and 3 are the most common and account for approximately 46% and 22% of all global infections, respectively (1). Chronic infection with genotype 1 HCV is most prevalent in the Americas, Europe, and China, and genotype 3 HCV infection is most prevalent in India, Pakistan, and Southeast Asia (1). With the advent of direct-acting antiviral agents, effective interferon-free combination regimens are now available for most patients chronically infected with genotype 1 or 3 HCV (2, 3). However, some subgroups of patients do not achieve optimal rates of sustained virologic response (SVR) with existing 12-week regimensin particular, cirrhotic patients with genotype 1 or 3 HCV infection who previously received unsuccessful treatment of HCV infection (46). These patients, who are at increased risk for progression to decompensated cirrhosis, hepatocellular carcinoma, and other liver complications, have a medical need for more effective and well-tolerated treatment (7, 8). Velpatasvir (Gilead Sciences) is a novel inhibitor of the HCV NS5A protein, which is involved in HCV replication, virion assembly, and modulation of host cellular response. Velpatasvir (formerly GS-5816) has demonstrated potent pangenotypic antiviral activity in vitro (9) and in a 3-day monotherapy study in patients with genotype 1, 2, 3, or 4 HCV infection (10, 11). Pharmacology studies showed no clinically important drug interactions between sofosbuvir and velpatasvir (12). A recent phase 2 study demonstrated the safety and efficacy of 12 weeks of the combination of sofosbuvir and velpatasvir in treatment-naive patients with genotype 1 to 6 HCV infection, with rates of SVR at week 12 after treatment (SVR12) of 93% to 100% (13). We evaluated the antiviral activity, safety, and tolerability of sofosbuvir administered with 25 or 100 mg of velpatasvir with and without ribavirin for 12 weeks in treatment-experienced patients with genotype 1 or 3 HCV infection. Because previously treated patients have historically had a poorer response than treatment-naive patients, we chose to evaluate sofosbuvir plus velpatasvir with ribavirin, as well as sofosbuvir plus velpatasvir alone. Methods Design Overview This phase 2, multicenter, randomized, open-label study was conducted from June 2013 (when the first patient was enrolled) to August 2014 (when the last patient completed follow-up). The study was originally designed to enroll 2 cohorts of patients with chronic genotype 3 HCV infection who had not achieved SVR after previous therapy with an interferon-based regimenapproximately 100 patients without cirrhosis and 100 patients with compensated cirrhosis. Favorable results in treatment-naive patients with genotype 1 HCV infection in a phase 2 trial of similar design (13) prompted us to amend our protocol to enroll a third cohort of approximately 100 patients with chronic genotype 1 HCV infection who did not achieve SVR after previous therapy with an approved or experimental NS3/4A protease inhibitor in combination with peginterferon and ribavirin. Up to 50% of patients with genotype 1 HCV infection could have compensated cirrhosis. The protocol was approved by the institutional ethics committees at all sites, and the study was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. Setting and Participants The study was conducted at 58 clinical sites: 7 in Australia, 2 in New Zealand, and 49 in the United States. Some patients were recruited partly through a posting of study details on ClinicalTrials.gov and others through referral by their treating physicians. Adults (aged 18 years) with HCV RNA levels greater than 10000 IU/mL were eligible. The HCV RNA genotype was determined by the central laboratory using the Versant HCV Genotype 2.0 Assay (LiPA) (Siemens). If results were inconclusive, we used the TruGene HCV 5NC Genotyping Kit (Siemens) with the OpenGene DNA Sequencing System (Siemens). The presence of cirrhosis was established by liver biopsy, a FibroTest score greater than 0.75 and an aspartate aminotransferaseplatelet ratio index greater than 2 during screening, or a FibroScan value greater than 12.5 kPa. Exclusion criteria were hepatic decompensation or co-infection with hepatitis B virus or HIV; aminotransferase level more than 10 times the upper limit of normal; direct bilirubin level more than 1.5 times the upper limit of normal; platelet count less than 90109 cells/L; hemoglobin A1c level greater than 8.5%; creatinine clearance rate less than 60 mL/min/1.73 m2, as calculated by the CockcroftGault equation; hemoglobin level less than 11 g/dL for female patients or less than 12 g/dL for male patients; albumin level less than 454.55 mol/L; and prothrombin time (international normalized ratio) greater than 1.5 times the upper limit of normal. To be eligible, patients must not have achieved SVR after previous treatment of HCV infection and must not have discontinued the previous regimen due to an adverse event. Patients with genotype 3 HCV infection with previous exposure to an approved or experimental HCV-specific direct-acting antiviral agent were excluded. Full eligibility criteria are listed in the study protocol (Supplement). All patients provided written informed consent before screening. Supplement. Study Protocol Randomization and Interventions Three cohorts of eligible patients were enrolled: treatment-experienced patients with genotype 3 HCV infection without cirrhosis, treatment-experienced patients with genotype 3 HCV infection with compensated cirrhosis, and patients with genotype 1 HCV infection whose previous treatment with a protease inhibitor and interferon-based regimen was unsuccessful. Within each cohort, patients were randomly assigned to 4 groups (by a 1:1:1:1 ratio), each of which received 12 weeks of a single oral daily dose of 400 mg of sofosbuvir plus 25 mg of velpatasvir, 25 mg of velpatasvir with ribavirin, 100 mg of velpatasvir, or 100 mg of velpatasvir with ribavirin (Figure). Velpatasvir was orally administered in single daily doses of either 25 or 100 mg. Ribavirin was administered orally in a divided daily dose determined by body weight: 1000 mg daily in patients weighing less than 75 kg and 1200 mg daily in patients weighing 75 kg or greater. Figure. Study flow diagram. Patients in groups 9 to 12 were enrolled after patients in groups 1 to 8 had completed the trial and were assessed for efficacy. HCV = hepatitis C virus; RBV = ribavirin; SOF = sofosbuvir; VEL = velpatasvir. Random assignment of patients was managed with an interactive Web-response system (Bracket). A statistician employed by the sponsor (L.H.) generated the randomization code using SAS, version 9.2 (SAS Institute), which was validated by another statistician employed by the sponsor. The randomization was stratified by cohort. Within cohort 3 (patients with genotype 1 HCV infection), randomization was stratified by genotype 1 subtype (1a or 1b) and cirrhosis status (presence or absence). Investigators (Appendix), patients, and trial personnel were not blinded to treatment assignment. Outcomes and Follow-up The primary efficacy outcome measure was SVR12, defined as a serum HCV RNA level below the lower limit of quantification (LLOQ) 12 weeks after completion of treatment. We measured HCV RNA levels with the Cobas TaqMan HCV Quantitative Test, v2.0 (Roche Diagnostics), in combination with the High Pure System Viral Nucleic Acid Kit (Roche Diagnostics), and used an LLOQ of 25 IU/mL. Secondary efficacy outcome measures included the proportion of patients with virologic failure, which was defined as either on-treatment virologic failure (HCV RNA level at or above the LLOQ after 8 weeks of therapy, confirmed >1 log10 increase in HCV RNA level from nadir, or confirmed HCV RNA level at or above the LLOQ after 2 consecutive HCV RNA levels less than the LLOQ) or relapse (HCV RNA level at or above the LLOQ during the posttreatment period or HCV RNA levels less than the LLOQ at the end of treatment). Other secondary efficacy outcome measures (SVR4, SVR24, and HCV RNA levels less than the LLOQ by study visit and HCV RNA levels and change from baseline in HCV RNA levels through week 12) are not reported here. The primary safety outcome measure was any adverse event leading to permanent withdrawal of study drugs. Safety assessments during treatment and up to 30 days after treatment included reports of adverse events, standard laboratory testing, 12-lead electrocardiography, assessment of vital signs, and symptom-driven physical examinations. Adverse events were coded using the Medical Dictionary for Regulatory Activities and graded by severity by the investigator according to the Gilead Sciences Grading Scale for Severity of Adverse Events and Laboratory Abnormalities. Deep sequencing of the HCV NS5A and NS5B gene was done from pretreatment plasma samples from all enrolled patients and from posttreatment samples from all patients with virologic failure. The HCV NS5A and NS5B coding regions were amplified by DDL Diagnostic Laboratory (Rijswijk, The Netherlands) with standard reverse transcr

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

ABSTRACT Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type.