The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Pegbelfermin in Patients With Nonalcoholic Steatohepatitis and Stage 3 Fibrosis (FALCON 1): A Randomized Phase 2b Study.

BACKGROUND & AIMS: Pegbelfermin is a polyethlene glycol-conjugated analog of human fibroblast growth factor 21, a nonmitogenic hormone that regulates energy metabolism. This phase 2b study evaluated 48-week pegbelfermin treatment in patients with nonalcoholic steatohepatitis (NASH) and stage 3 (bridging) fibrosis. METHODS: The FALCON 1 study (NCT03486899) was a multicenter, randomized (1:1:1:1), double-blind, placebo-controlled study. Patients with biopsy-confirmed NASH and stage 3 fibrosis (N = 197) received weekly subcutaneous pegbelfermin (10, 20, or 40 mg) or placebo injections for 48 weeks. The week 24 primary endpoint was a ≥1-point decrease in fibrosis score without NASH worsening or NASH improvement without fibrosis worsening; pegbelfermin dose response was assessed using a Cochran-Armitage trend test across proportions (1-sided α = 0.05). Secondary/exploratory endpoints included histological and noninvasive measures of steatosis, fibrosis, and liver injury/inflammation. RESULTS: At week 24, the primary endpoint was met by 14% (placebo) vs 24%-31% (pegbelfermin arms); statistical significance was not reached due to lack of pegbelfermin dose response (P = .134). At weeks 24 and 48, more patients who received pegbelfermin had ≥30% relative reductions in hepatic fat fraction (magnetic resonance imaging-proton density fat fraction) vs placebo, although no differences reached statistical significance. In the pegbelfermin arms, improvements in liver fibrosis (magnetic resonance elastography and N-terminal type III collagen propeptide) and liver injury/inflammation (alanine aminotransferase, aspartate aminotransferase) were observed vs placebo. Adverse events occurred at similar frequencies across arms. No treatment-related serious adverse events were observed. CONCLUSIONS: The FALCON 1 study did not meet its primary endpoint; a ≥1-point decrease in fibrosis score without NASH worsening or NASH improvement without fibrosis worsening assessed via biopsy. Pegbelfermin was generally well tolerated during 48 weeks of treatment.

Pegbelfermin in Patients With Nonalcoholic Steatohepatitis and Compensated Cirrhosis (FALCON 2): A Randomized Phase 2b Study.

BACKGROUND & AIMS: Pegbelfermin is a polyethylene glycol-conjugated analog of human fibroblast growth factor 21, a nonmitogenic hormone that regulates energy metabolism. This phase 2b study evaluated 48-week pegbelfermin treatment in patients with nonalcoholic steatohepatitis (NASH) with compensated cirrhosis. METHODS: FALCON 2 (NCT03486912) was a randomized (1:1:1:1), double-blind, placebo-controlled study. Eligible adults had biopsy-confirmed NASH and stage 4 fibrosis. Pegbelfermin (10, 20, or 40 mg) or placebo was injected subcutaneously once weekly. The primary endpoint was 1 or more stages of improvement in the NASH Clinical Research Network fibrosis score without NASH worsening at week 48; pegbelfermin dose response was assessed using a Cochran-Armitage trend test across proportions (1-sided α = .05). Additional endpoints included histologic and noninvasive measures of steatosis, fibrosis, and liver injury/inflammation. RESULTS: Overall, 155 patients were randomized, and 154 patients received treatment. At week 48, 24% to 28% of the pegbelfermin arms had primary endpoint responses vs 31% of the placebo arm (P = .361). Nonalcoholic fatty liver disease activity score improvements were more frequent with pegbelfermin vs placebo and were driven primarily by reduced lobular inflammation. Numerically higher proportions of the pegbelfermin arms had liver stiffness (magnetic resonance elastography) and steatosis (magnetic resonance imaging-proton density fat fraction) improvements vs placebo; these differences were not statistically significant. Mean N-terminal type III collagen propeptide, alanine aminotransferase, and aspartate aminotransferase values were numerically lower in the 20- and/or 40-mg pegbelfermin arms compared with placebo. Serious adverse events were more frequent with pegbelfermin vs placebo, although none were treatment related. One patient (40-mg pegbelfermin) discontinued treatment because of a treatment-emergent adverse event (worsening ascites). CONCLUSIONS: FALCON 2 did not meet its primary endpoint of 1 or more stages of improvement in the NASH Clinical Research Network fibrosis without NASH worsening assessed via biopsy. Pegbelfermin generally was well tolerated in this advanced NASH population.

Pegbelfermin (BMS-986036), a PEGylated fibroblast growth factor 21 analogue, in patients with non-alcoholic steatohepatitis: a randomised, double-blind, placebo-controlled, phase 2a trial.

BACKGROUND: Pegbelfermin (BMS-986036), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. In this phase 2a study, we aimed to evaluate the safety and efficacy of pegbelfermin in patients with non-alcoholic steatohepatitis. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2a study, we recruited adults (aged 21-75 years) with a body-mass index of at least 25 kg/m2, biopsy-confirmed non-alcoholic steatohepatitis (fibrosis stage 1-3), and a hepatic fat fraction of at least 10% when assessed by magnetic resonance imaging-proton density fat fraction. These patients were enrolled at 17 medical centres in the USA. Eligible patients were stratified by type 2 diabetes status and they were randomly assigned (1:1:1) by a computer-based system to receive subcutaneous injections of placebo once a day, 10 mg pegbelfermin once a day, or 20 mg pegbelfermin once a week, all for 16 weeks. Participants, the study team administering treatment, and investigators analysing outcomes (who were independent of the study team and had no further involvement) were masked to treatment groups. The primary outcomes were safety and the absolute change in hepatic fat fraction after 16 weeks of treatment. All patients who were randomly assigned to groups and received the study drug or placebo were included in the primary analyses. This trial was registered with ClinicalTrials.gov, number NCT02413372. FINDINGS: Between May 12, 2015, and Aug 4, 2016, 184 overweight or obese patients with non-alcoholic steatohepatitis were screened for study inclusion. Of these, 95 (52%) patients were excluded because they no longer met study criteria and 80 (43%) patients entered the placebo lead-in phase. After further exclusions, 75 (94%) patients were randomly assigned to groups, received at least one dose of treatment (25 patients to receive 10 mg pegbelfermin once a day; 24 patients to receive 20 mg pegbelfermin once a week, and 26 patients to receive placebo), and were included in the primary analysis. A prespecified interim analysis at week 8 showed a greater than expected change in the primary outcome and supported early closing of patient enrolment, since this analysis indicated that the full planned sample size was not needed. We observed a significant decrease in absolute hepatic fat fraction in the group receiving 10 mg pegbelfermin daily (-6·8% vs -1·3%; p=0·0004) and in the group receiving 20 mg pegbelfermin weekly (-5·2% vs -1·3%; p=0·008) compared with the placebo group. Most adverse events were mild; the most common events were diarrhoea in eight (16%) of 49 patients treated with pegbelfermin and two (8%) of 26 patients treated with placebo and nausea in seven (14%) patients treated with pegbelfermin and two (8%) patients treated with placebo. There were no deaths, discontinuations due to adverse events, or treatment-related serious adverse events. INTERPRETATION: Treatment with subcutaneously administered pegbelfermin for 16 weeks was generally well tolerated and significantly reduced hepatic fat fraction in patients with non-alcoholic steatohepatitis. Further study of pegbelfermin is warranted in patients with non-alcoholic steatohepatitis. Additional studies that use liver biopsies would allow for the assessment of pegbelfermin's effects on liver histology. Moreover, further studies should allow assessments of the safety and effectiveness of pegbelfermin in a larger number of patients. FUNDING: Bristol-Myers Squibb.

Daclatasvir and Peginterferon/Ribavirin for Black/African-American and Latino Patients with HCV infection.

 Background. Patient race and ethnicity have historically impacted HCV treatment response. This phase 3 study evaluated daclatasvir with peginterferon-alfa-2a/ribavirin (pegIFN alfa-2a/RBV) in treatment-naive black/African American (AA), Latino, and white non-Latino patients with chronic HCV genotype 1 infection. MATERIAL AND METHODS: In this single-arm, open-label study, 246 patients received daclatasvir plus pegIFN alfa-2a and weight-based RBV. Patients with an extended rapid virologic response (eRVR; undetectable HCV-RNA at treatment weeks 4 and 12) received 24 weeks of treatment; those without eRVR received an additional 24 weeks of treatment with pegIFN alfa-2a/RBV. The primary endpoint was sustained virologic response at post-treatment week 12 (SVR12; HCV-RNA < 25 IU/mL) compared with the cohort historical rate. RESULTS: Most patients were IL28B non-CC (84.4% black/AA; 77.6% Latino) genotype 1a-infected (72.7%; 81.3%), with HCV-RNA ≥ 800,000 IU/mL (81.3%; 64.5%). SVR12 rates were 50.8% (65/128; 95% confidence interval [CI], 42.1-59.4) for black/AA and 58.9% (63/107; 95% CI, 49.6-68.2) for Latino patients. The majority (55.5%; 58.9%) received 24 weeks treatment; rapid reductions (> 4-log10) in HCV-RNA levels were observed. Only 60.9% (78/128) of black/AA and 63.6% (68/107) of Latino patients completed treatment. On-treatment serious adverse events (SAEs) occurred in 21 patients. Discontinuations due to adverse events (AEs) occurred in 9 black/AA and 6 Latino patients. CONCLUSION: SVR12 rates for black/AA (50.8%) and Latino (58.9%) cohorts treated with daclatasvir plus pegIFN alfa-2a/RBV and the lower bound of the 95% CIs were higher than the estimated historical control (black/AA, 26% SVR; Latino, 36% SVR) treated with pegIFN alfa-2a/RBV. These data support daclatasvir use in all-oral direct-acting antiviral combinations.

Small-molecule inhibitors of dengue-virus entry.

Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal "stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC90s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a "pocket-open" state, which has lost affinity for the stem peptide and cannot support the final "zipping up" of the stem.

Anemia during treatment with peginterferon Alfa-2b/ribavirin and boceprevir: Analysis from the serine protease inhibitor therapy 2 (SPRINT-2) trial.

UNLABELLED: Boceprevir (BOC) added to peginterferon alfa-2b (PegIFN) and ribavirin (RBV) significantly increases sustained virologic response (SVR) rates over PegIFN/RBV alone in previously untreated adults with chronic hepatitis C genotype 1. We evaluate the relationship of incident anemia with triple therapy. A total of 1,097 patients received a 4-week lead-in of PegIFN/RBV followed by: (1) placebo plus PegIFN/RBV for 44 weeks (PR48); (2) BOC plus PegIFN/RBV using response-guided therapy (BOC/RGT); and (3) BOC plus PegIFN/RBV for 44 weeks (BOC/PR48). The management of anemia (hemoglobin [Hb]<10 g/dL) included RBV dose reduction and/or erythropoietin (EPO) use. A total of 1,080 patients had ≥1 Hb measurement during treatment. The incidence of anemia was 50% in the BOC arms combined (363/726) and 31% in the PR48 arm (108/354, P<0.001). Among BOC recipients, lower baseline Hb and creatinine clearance were associated with incident anemia. In the BOC-containing arms, anemia was managed by the site investigators as follows: EPO without RBV dose reduction, 38%; RBV dose reduction without EPO, 8%; EPO with RBV dose reduction, 40%; and neither RBV dose reduction nor EPO, 14%. SVR rates were not significantly affected by management strategy (70%-74%), and overall patients with anemia had higher rates of SVR than those who did not develop anemia (58%). Serious and life-threatening adverse events (AEs) and discontinuations due to AEs among BOC-treated patients did not differ by EPO use. CONCLUSION: With BOC/PR therapy, SVR rates in patients with incident anemia were higher than nonanemic patients and did not vary significantly according to the investigator-selected approach for anemia management. Prospective studies are needed to confirm this observation.

Chronic hepatitis C genotype 1 patients with insulin resistance treated with pioglitazone and peginterferon alpha-2a plus ribavirin.

UNLABELLED: Patients with chronic hepatitis C and insulin resistance are less likely to respond to anti-hepatitis C virus (HCV) therapy and are at risk for more rapid fibrosis progression. Coadministration of pioglitazone with peginterferon/ribavirin improves insulin sensitivity and increases virologic response rates in insulin-resistant HCV genotype 4 patients, but it is unclear whether this finding applies to genotype 1 patients. For this reason we randomized treatment-naive HCV genotype 1 patients with insulin resistance to receive either standard care (peginterferon alpha-2a plus ribavirin for 48 weeks, n = 73) or pioglitazone 30-45 mg/day plus standard care (n = 77) in an open-label multicenter trial. Patients randomized to pioglitazone received the drug during a 16-week run-in phase, the 48-week standard-care phase, and the 24-week untreated follow-up phase. Pioglitazone treatment improved hemoglobin A1c (HbA1c), plasma glucose, insulin levels, and homeostasis model assessment of insulin resistance score and increased serum adiponectin levels during the 16-week run-in phase and maintained these improvements during the standard-care phase. However, we observed no statistically significant difference between the two groups in the primary efficacy endpoint, the decrease from baseline to Week 12 of peginterferon alpha-2a/ribavirin treatment in mean log(10) HCV RNA titer (-3.5 ± 1.71 and -3.7 ± 1.62 IU/mL in the pioglitazone and standard-care groups, respectively, Δ = 0.21 IU/mL, P = 0.4394). CONCLUSION: Treatment with pioglitazone before and during treatment with peginterferon alpha-2a plus ribavirin improved several indices of glycemic control in patients with chronic hepatitis C and insulin resistance, but did not improve virologic response rates compared with peginterferon alpha-2a plus ribavirin alone.

Intensified peginterferon α-2a dosing increases sustained virologic response rates in heavy, high viral load hepatitis C genotype 1 patients with high low-density lipoprotein.

BACKGROUND AND GOAL: Patients infected with hepatitis C virus (HCV) with elevated low-density lipoprotein (LDL) levels achieve higher sustained virologic response (SVR) rates after peginterferon (PegIFN)/ribavirin treatment versus patients with lower LDL. Our aim was to determine whether SVR rates in patients with low/elevated LDL can be improved by dose intensification. STUDY: In PROGRESS, genotype 1 patients with baseline HCV RNA ≥ 400,000 IU/mL and body weight ≥ 85 kg were randomized to 48 weeks of 180 µg/wk PegIFN α-2a (40 kDa) plus ribavirin (A: 1200 mg/d; B: 1400/1600 mg/d) or 12 weeks of 360 µg/wk PegIFN α-2a followed by 36 weeks of 180 µg/wk, plus ribavirin (C: 1200 mg/d; D: 1400/1600 mg/d). This retrospective analysis assessed SVR rates among patients with low (<100 mg/dL) or elevated (≥ 100 mg/dL) LDL. Patients with high LDL (n=256) had higher baseline HCV RNA (5.86 × 10(6) IU/mL) versus patients with low LDL (n=262; 4.02 × 10(6) IU/mL; P=0.0003). RESULTS: Multiple logistic regression analysis identified a significant interaction between PegIFN α-2a dose and LDL levels on SVR (P=0.0193). The only treatment-related SVR predictor in the nested multiple logistic regression was PegIFN α-2a dose among patients with elevated LDL (P=0.0074); therefore, data from the standard (A+B) and induction (C+D) dose arms were pooled. Among patients with low LDL, SVR rates were 40% and 35% in the standard and induction-dose groups, respectively; SVR rates in patients with high LDL were 44% and 60% (P=0.014), respectively. CONCLUSIONS: Intensified dosing of PegIFN α-2a increases SVR rates in patients with elevated LDL even with the difficult-to-cure characteristics of genotype 1, high baseline viral load, and high body weight.

Role of HIV membrane in neutralization by two broadly neutralizing antibodies.

Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.

MiraLAX is not as effective as GoLytely in bowel cleansing before screening colonoscopies.

BACKGROUND & AIMS: Successful colonoscopies require good bowel preparations-poor bowel preparations can increase medical costs, rates of missed lesions, and procedure duration. The combination of polyethylene glycol (PEG) 3350 without electrolytes (MiraLAX; Schering-Plough Healthcare Products, Inc, Kenilworth, NJ) and 64 oz of Gatorade (PepsiCo, Inc, Purchase, NY) has gained popularity as a bowel preparation regimen. However, the efficacy and tolerability of this approach has not been compared with standard bowel preparations in clinical trials. We compared split-dose (PEG) 3350 with electrolytes (GoLytely; Braintree Laboratories, Inc, Braintree, MA) with split-dose MiraLAX alone and in combination with pretreatment medications (bisacodyl or lubiprostone) to determine the efficacy and patient tolerability of MiraLAX as an agent for bowel preparation. METHODS: We performed a prospective, randomized, blinded, controlled trial at a tertiary care center. Patients (n=403) were randomly assigned to groups given GoLytely, MiraLAX, MiraLAX with bisacodyl (10 mg), or MiraLAX with lubiprostone (24 µg). MiraLAX was combined with 64 oz of Gatorade. All patients were surveyed regarding preparation satisfaction and tolerability. The Ottawa bowel preparation scale was used to grade colon cleanliness. RESULTS: GoLytely was more effective at bowel cleansing (average Ottawa score, 5.1) than MiraLAX alone (average Ottawa score, 6.9) or in combination with lubiprostone (average Ottawa score, 6.8), or bisacodyl (average Ottawa score, 6.3) (P<.001). MiraLAX was associated with a trend toward longer procedure duration (P=.096). Groups given MiraLAX rated the overall experience as more satisfactory than those given GoLytely (P<.001). There were no differences between polyp detection rates (P=.346) or adverse events (P=.823). CONCLUSIONS: Split-dose MiraLAX in 64 oz of Gatorade is not as effective as 4 L split-dose GoLytely in bowel cleansing for screening colonoscopies.

Induction pegylated interferon alfa-2a and high dose ribavirin do not increase SVR in heavy patients with HCV genotype 1 and high viral loads.

BACKGROUND & AIMS: Patients infected with hepatitis C virus (HCV) genotype 1, body weight ≥85 kg, and high baseline viral load respond poorly to standard doses of pegylated interferon (peginterferon) and ribavirin. We evaluated intensified therapy with peginterferon alfa-2a plus ribavirin. METHODS: This double-blind randomized trial included HCV genotype 1-infected outpatients from hepatology clinics with body weight ≥85 kg and HCV RNA titer ≥400,000 IU/mL. Patients were randomized to 180 µg/wk peginterferon alfa-2a for 48 weeks plus 1200 mg/day ribavirin (standard of care) (group A, n = 191) or 1400/1600 mg/day ribavirin (group B, n = 189). Additional groups included 360 µg/wk peginterferon alfa-2a for 12 weeks then 180 µg/wk peginterferon alfa-2a for 36 weeks plus 1200 mg/day ribavirin (group C, n = 382) or 1400/1600 mg/day ribavirin (group D, n = 383). Follow-up lasted 24 weeks after treatment. RESULTS: Sustained virologic response rates (HCV RNA level <15 IU/mL at end of follow-up) in groups A, B, C, and D were 38%, 43%, 44%, and 41%, respectively. There were no significant differences among the 4 groups or between pooled peginterferon alfa-2a regimens (A + B vs C + D: odds ratio [OR], 1.08; 95% confidence interval [CI], 0.83-1.39; P = .584) or pooled ribavirin regimens (A + C vs B + D: OR, 1.00; 95% CI, 0.79-1.28; P = .974). CONCLUSIONS: In patients infected with HCV genotype 1 who are difficult to treat (high viral load, body weight ≥85 kg), a 12-week induction regimen of peginterferon alfa-2a and/or higher-dose ribavirin is not more effective than the standard regimen.

Hepatitis C virus treatment-related anemia is associated with higher sustained virologic response rate.

BACKGROUND & AIMS: Hepatitis C virus (HCV) treatment is frequently complicated by anemia from ribavirin (RBV)-related hemolysis and peginterferon-alfa (PEG-IFN)-related bone marrow suppression. We investigated the relationships among treatment outcomes, anemia, and their management with RBV dose reduction and/or erythropoiesis-stimulating agents (ESAs). METHODS: We analyzed data from a trial conducted at 118 United States academic and community centers in treatment-naïve patients with HCV genotype 1. Patients were treated for as many as 48 weeks with 1 of 3 PEG-IFN/RBV regimens. ESAs were permitted for anemic patients (hemoglobin [Hb] <10 g/dL) after RBV dose reduction. Sustained virologic responses (SVR) were assessed based on decreases in Hb, anemia, and ESA use. RESULTS: While patients received treatment, 3023 had their Hb levels measured at least once. An SVR was associated with the magnitude of Hb decrease: >3 g/dL, 43.7%; ≤3 g/dL, 29.9% (P < .001). Anemia occurred in 865 patients (28.6%); 449 of these (51.9%) used ESAs. In patients with early-onset anemia (≤ 8 weeks of treatment), ESAs were associated with higher SVR rate (45.0% vs 25.9%; P < .001) and reduced discontinuation of treatment because of adverse events (12.6% vs 30.1%, P < .001). ESAs did not affect SVR or discontinuation rates among patients with late-stage anemia. CONCLUSIONS: Among HCV genotype 1-infected patients treated with PEG-IFN/RBV, anemia was associated with higher rates of SVR. The effect of ESAs varied by time to anemia; patients with early-onset anemia had higher rates of SVR with ESA use, whereas no effect was observed in those with late-onset anemia. Prospective trials are needed to assess the role of ESAs in HCV treatment.

A rotavirus spike protein conformational intermediate binds lipid bilayers.

During rotavirus entry, a virion penetrates a host cell membrane, sheds its outer capsid proteins, and releases a transcriptionally active subviral particle into the cytoplasm. VP5, the rotavirus protein believed to interact with the membrane bilayer, is a tryptic cleavage product of the outer capsid spike protein, VP4. When a rotavirus particle uncoats, VP5 folds back, in a rearrangement that resembles the fusogenic conformational changes in enveloped-virus fusion proteins. We present direct experimental evidence that this rearrangement leads to membrane binding. VP5 does not associate with liposomes when mounted as part of the trypsin-primed spikes on intact virions, nor does it do so after it has folded back into a stably trimeric, low-energy state. But it does bind liposomes when they are added to virions before uncoating, and VP5 rearrangement is then triggered by addition of EDTA. The presence of liposomes during the rearrangement enhances the otherwise inefficient VP5 conformational change. A VP5 fragment, VP5CT, produced from monomeric recombinant VP4 by successive treatments with chymotrypsin and trypsin, also binds liposomes only when the proteolysis proceeds in their presence. A monoclonal antibody that neutralizes infectivity by blocking a postattachment entry event also blocks VP5 liposome association. We propose that VP5 binds lipid bilayers in an intermediate conformational state, analogous to the extended intermediate conformation of enveloped-virus fusion proteins.

Serum cholesterol and statin use predict virological response to peginterferon and ribavirin therapy.

UNLABELLED: Elevated low-density lipoprotein (LDL) levels and statin use have been associated with higher sustained virological response (SVR) rates in patients receiving chronic hepatitis C therapy. However, these relationships have not been well characterized in randomized controlled trials. Furthermore, little is known about the relationship between high-density lipoprotein (HDL) and virological response. To determine whether baseline LDL or HDL levels and statin use affect SVR rates, we retrospectively evaluated the IDEAL (Individualized Dosing Efficacy Versus Flat Dosing to Assess Optimal Pegylated Interferon Therapy) trial, in which 3070 treatment-naive, hepatitis C virus (HCV) genotype 1-infected patients were treated for up to 48 weeks in one of the following arms: (1) peginterferon (PEG-IFN) alfa-2b at 1.5 microg/kg/week with ribavirin (RBV) at 800 to 1400 mg/day, (2) PEG-IFN alfa-2b at 1.0 microg/kg/week with RBV at 800 to 1400 mg/day, or (3) PEG-IFN alfa-2a at 180 microg/week with RBV at 1000 to 1200 mg/day. Virological responses were assessed by pretreatment statin use and baseline elevated LDL levels (> or =130 mg/dL) or low HDL levels (<40 mg/dL for men and <50 mg/dL for women). In 1464 patients with baseline elevated LDL levels or low HDL levels, the SVR rate was significantly higher than that in patients with normal levels (44.9% versus 34.0%, P < 0.001). In 66 patients receiving a statin pretreatment, the SVR rate was higher than the rate of those not receiving it (53.0% versus 39.3%, P = 0.02). In a multivariate logistic regression analysis using the stepwise selection method with baseline characteristics, a high LDL level [odds ratio (OR) = 1.6, 95% confidence interval (CI) = 1.4-1.8, P < 0.001], a low HDL level (OR = 0.5, 95% CI = 0.3-0.8, P = 0.004), and statin use (OR = 2.0, 95% CI = 1.1-3.7, P = 0.02) were independently associated with SVR. CONCLUSION: Baseline elevated LDL levels or low HDL levels and preemptive statin usage were associated with higher SVR rates. Prospective studies may be considered to explore the biological impact of these factors on HCV RNA replication and treatment response.

The FALCON program: Two phase 2b randomized, double-blind, placebo-controlled studies to assess the efficacy and safety of pegbelfermin in the treatment of patients with nonalcoholic steatohepatitis and bridging fibrosis or compensated cirrhosis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD); no approved therapies for NASH currently exist. Pegbelfermin (PGBF), a human fibroblast growth factor 21 analog, has metabolic effects that may provide benefit for patients with NASH. DESIGN: The FALCON 1 and 2 studies are phase 2b, multicenter, double-blind, placebo-controlled, randomized trials to assess safety and efficacy of PGBF treatment in patients who have histologically-confirmed NASH with stage 3 liver fibrosis (FALCON 1; NCT03486899) or compensated cirrhosis (FALCON 2; NCT03486912). In both studies, randomized patients receive once weekly subcutaneous injections of PGBF (10, 20, or 40 mg) or placebo during a 48-week treatment period and are then followed for an additional 4 weeks. ENDPOINTS: The primary efficacy endpoint for FALCON 1 is the proportion of patients who achieve ≥1 stage improvement in fibrosis (by NASH CRN fibrosis score) without NASH worsening or NASH improvement (≥2 point decrease in NAFLD Activity Score) without fibrosis worsening at Week 24. For FALCON 2, the primary efficacy endpoint is ≥1 stage improvement in fibrosis without NASH worsening at Week 48. Key safety endpoints for both studies include incidence and frequency of adverse events, bone mineral density and immunogenicity. SUMMARY: Previous clinical trial data show that PGBF can reduce hepatic fat and improve metabolic factors and biomarkers of hepatic injury and fibrosis. The FALCON studies aim to evaluate PGBF treatment specifically in patients with NASH and advanced fibrosis, who are at greatest risk of poor clinical outcomes over time.

Induction pegylated interferon alfa-2b in combination with ribavirin in patients with genotypes 1 and 4 chronic hepatitis C: a prospective, randomized, multicenter, open-label study.

BACKGROUND & AIMS: Standard of care (SOC) treatment for chronic hepatitis C (CHC) involves weekly pegylated (PEG) interferon plus weight-based ribavirin with resultant sustained virologic response (SVR) rates at or near 50% for genotypes 1 and 4 virus. Induction therapy with higher doses of PEG interferon may improve first-phase viral kinetics and thus improve the overall SVR in genotypes 1 and 4 patients. METHODS: This multicenter, randomized, open-label trial enrolled treatment-naive genotypes 1- and 4-infected CHC patients to either initial induction therapy versus SOC. The induction group received PEG interferon alfa-2b 3.0 mcg/kg/wk for 12 weeks followed by PEG interferon alfa-2b 1.5 mcg/kg/wk for 36 weeks and 13 +/- 2 mg/kg ribavirin daily for 48 weeks. SOC patients received PEG interferon alfa-2b 1.5 mcg/kg weekly for 48 weeks and 13 +/- 2 mg/kg ribavirin daily for 48 weeks. The primary end point was SVR. RESULTS: There were 610 patients enrolled throughout the United States. Complete early virologic response was 62.6% versus 57.7% in induction versus SOC (NS). Overall SVR was 32% in induction versus 29% in SOC group (NS). Dose reduction of either PEG interferon (24.1% vs 23.8%) or ribavirin (26.8% vs 25.1%) was similar between the 2 groups. There was a trend toward a significant difference when comparing the SVR in induction therapy in patients weighing more than 85 kg versus those receiving SOC (38% vs 28%; P = .08). CONCLUSIONS: Induction therapy does not enhance complete early virologic response or SVR rates in a predominantly genotype 1 CHC population compared with SOC therapy.

Requirements for the formation of membrane pores by the reovirus myristoylated micro1N peptide.

The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the micro1 protein, each complexed with three copies of the protector protein sigma3. Conformational changes in micro1 following the proteolytic removal of sigma3 lead to release of the myristoylated N-terminal cleavage fragment micro1N and ultimately to membrane penetration. The micro1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant micro1 trimers and synthetic micro1N peptides with both RBCs and liposomes. The micro1 trimer mediates hemolysis and liposome disruption under conditions that promote the micro1 conformational change, and mutations that inhibit micro1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free micro1 trimer. Autolytic cleavage to form micro1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that micro1N cleavage is not required when steric barriers are removed. Synthetic myristoylated micro1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of beta-strand secondary structure, suggest a model in which micro1N beta-hairpins assemble in the membrane to form a beta-barrel pore.

Structure of the dengue virus envelope protein after membrane fusion.

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three 'fusion loops' at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.