Bridging and Validation of the Specific Graves Ophthalmopathy Quality of Life Questionnaire With Health State Utility Values.

OBJECTIVE: In thyroid eye disease (TED), inflammation and expansion of orbital muscle and periorbital fat result in diplopia and proptosis, severely impacting patient quality of life (QOL). The reported health state utility (HSU) scores, which are QOL measures, allow quantification of TED impact and improvement with therapies; however, no current QOL instrument has been validated with HSU scores for TED. Here, we used the disease-specific Graves Ophthalmopathy Quality of Life (GO-QOL) questionnaire and HSU scores to validate QOL impact. METHODS: The GO-QOL scores from patients in 2 randomized, masked, placebo-controlled teprotumumab trials (N=171) were compared with 6 HSU values based on severity of proptosis/diplopia in those studies. Patient GO-QOL and HSU scores were compared at baseline and after 6-month treatment via regression analyses. GO-QOL and HSU scores were correlated for validation and quantification of QOL impact by severity state and to estimate quality-adjusted life year improvement. RESULTS: GO-QOL scores were correlated with TED severity, indicating that worse severity was associated with lower (worse) GO-QOL scores. Less severe health states were represented by higher (better) GO-QOL scores. Importantly, GO-QOL scores were positively correlated with utility scores of the 6 health states, allowing for conversion of the GO-QOL scores to utility scores. A positive (improved) 0.013 utility change was found for each 1-point (positive) improvement in GO-QOL score produced by teprotumumab versus placebo. CONCLUSION: Patients with moderate-to-severe active TED health states demonstrate increasing TED severity associated with declining utility values and worsening GO-QOL scores. These results indicate that the GO-QOL scores can be used to bridge to the HSU scores for benefit quantification.

Determining the involvement and therapeutic implications of host cellular factors in hepatitis C virus cell-to-cell spread.

UNLABELLED: Hepatitis C virus (HCV) infects 180 million people worldwide and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. It has been shown that HCV can spread to naive cells using two distinct entry mechanisms, "cell-free" entry of infectious extracellular virions that have been released by infected cells and direct "cell-to-cell" transmission. Here, we examined host cell requirements for HCV spread and found that the cholesterol uptake receptor NPC1L1, which we recently identified as being an antiviral target involved in HCV cell-free entry/spread, is also required for the cell-to-cell spread. In contrast, the very low density lipoprotein (VLDL) pathway, which is required for the secretion of cell-free infectious virus and thus has been identified as an antiviral target for blocking cell-free virus secretion/spread, is not required for cell-to-cell spread. Noting that HCV cell-free and cell-to-cell spread share some common factors but not others, we tested the therapeutic implications of these observations and demonstrate that inhibitors that target cell factors required for both forms of HCV spread exhibit synergy when used in combination with interferon (a representative inhibitor of intracellular HCV production), while inhibitors that block only cell-free spread do not. This provides insight into the mechanistic basis of synergy between interferon and HCV entry inhibitors and highlights the broader, previously unappreciated impact blocking HCV cell-to-cell spread can have on the efficacy of HCV combination therapies. IMPORTANCE: HCV can spread to naive cells using distinct mechanisms: "cell-free" entry of extracellular virus and direct "cell-to-cell" transmission. Herein, we identify the host cell HCV entry factor NPC1L1 as also being required for HCV cell-to-cell spread, while showing that the VLDL pathway, which is required for the secretion of cell-free infectious virus, is not required for cell-to-cell spread. While both these host factors are considered viable antiviral targets, we demonstrate that only inhibitors that block factors required for both forms of HCV entry/spread (i.e., NPC1L1) exhibit synergy when used in combination with interferon, while inhibitors that block factors required only for cell-free spread (i.e., VLDL pathway components) do not. Thus, this study advances our understanding of HCV cell-to-cell spread, provides mechanistic insight into the basis of drug synergy, and highlights inhibition of HCV spread as a previously unappreciated consideration in HCV therapy design.

Assessment of hearing dysfunction in patients with Graves' disease and thyroid eye disease without or with teprotumumab.

IMPORTANCE: Thyroid eye disease (TED) negatively impacts quality of life. TED occurs predominantly in Graves' disease (GD). Teprotumumab improves TED but concern for hearing adverse events (AEs) has emerged. Hearing dysfunction is reported in thyroid autoimmune disease but the background prevalence in GD/TED without teprotumumab remains uncertain. OBJECTIVE: To quantify ear-related diagnostic codes/hearing AEs in GD, TED, and patients receiving teprotumumab by examining medical claims and clinical trials. DESIGN AND PARTICIPANTS: Deidentified claims for ear/labyrinth-related ICD-10 codes (KOMODO®) were examined in GD patients without TED, and TED patients without/with teprotumumab treatment. Hearing AE incidence/severity was evaluated in teprotumumab clinical trials. Graves' Ophthalmopathy QOL (GO-QOL) scores were compared in teprotumumab TED trial patients without/with hearing AEs. RESULTS: GD (469,720), TED (38,566) and teprotumumab-treated (967) patients were identified in the claims database. Ear-related codes (including those not specific for hearing) occurred in 24% GD, 33% TED, and 32% teprotumumab-treated patients. "Sensorineural hearing loss bilateral" was most frequent: 32,961/469,720 (7%) GD, 4,279/38,566 (11.1%) TED, and 104/967 (10.8%) teprotumumab patients. Pre-teprotumumab use,165 (17.1%) patients had ear-related codes while 98 (10.1%) had new ear-related codes post-treatment.Eight teprotumumab oncology trials revealed 8.1% (51/633) had Ear/Labyrinth Disorders with 2.1% (13) considered study-drug-related and 3.8% (24) hearing impairment/tinnitus-related AEs, with 1.3% (8) considered teprotumumab-related. Similar rates occurred in TED trials.GO-QOL improved in teprotumumab-treated patients without/with hearing AEs. Incidence/severity was consistent across patients with chronic and acute TED. CONCLUSIONS: These analyses indicate similar occurrence of hearing claims in patients with GD/TED alone as following teprotumumab treatment. Future analyses of incremental hearing risk from teprotumumab should utilize a priori study designs accounting for background hearing dysfunction in patients with GD/TED.

Utility Assessment of Moderate to Severe Thyroid Eye Disease Health States.

Importance: Thyroid eye disease (TED) results in varying degrees of proptosis and diplopia negatively affecting quality of life (QoL), producing possibly substantial visual changes, disfigurement, and disability. Objective: To determine the association of varying TED severities with QoL in a non-TED population by assessing health state utility scores. Design, Setting, and Participants: This qualitative study, conducted from April 20, 2020, to April 29, 2021, assessed health states for active, moderate-severe TED, and values were elicited using time trade-off methods. Six health states of varying severity were determined from 2 placebo-controlled clinical trials (171 patients with TED and clinical activity score ≥4, ±diplopia/proptosis) and refined using interviews with US patients with TED (n = 6). Each health state description was validated by interviews with additional TED patient advocates (n = 3) and physician experts (n = 3). Health state descriptions and a QOL questionnaire were piloted and administered to a general population. Visual analog scales (VASs) were also administered to detect concurrence of the findings. Main Outcomes and Measures: TED health state utility scores and whether they differ from one another were assessed using Shapiro-Wilk, Kruskal-Wallis, pairwise Wilcoxon rank sum, and paired t tests. Results: A total of 111 participants completed time trade-off interviews. The mean (SD) utility value was 0.44 (0.34). The lowest (worse) mean utility value was observed in the most severe disease state (constant diplopia/large proptosis) with 0.30 (95% CI, 0.24-0.36), followed by constant diplopia/small proptosis (0.34; 95% CI, 0.29-0.40), intermittent or inconstant diplopia/large proptosis (0.43; 95% CI, 0.36-0.49), no diplopia/large proptosis (0.46; 95% CI, 0.40-0.52), and intermittent or inconstant diplopia/small proptosis (0.52; 95% CI, 0.45-0.58). The highest (best) mean value, 0.60 (95% CI, 0.54-0.67), was observed for the least severe disease state (no diplopia/small proptosis). Conclusions and Relevance: These findings suggest that patients with active, moderate-severe TED may have substantial disutility, with increasing severity of proptosis/diplopia more likely to have detrimental associations with QoL. These health state scores may provide a baseline for determining QoL improvement in these TED health states (utility gains) treated with new therapies.

Permissiveness of human hepatoma cell lines for HCV infection.

BACKGROUND: Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). RESULTS: We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. CONCLUSIONS: We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.

Hepatitis C virus Cell-to-cell Spread Assay.

Hepatitis C virus (HCV) can infect naïve cells via entry of "cell-free" extracellular virus or direct "cell-to-cell" transmission. Here, we describe an assay for detecting HCV cell-to-cell transmission, using a non-growing cell culture system that avoids confounding effects of cell growth. The assay consists of infecting a small number of cells in a confluent monolayer and then blocking subsequent cell-free extracellular virions with a neutralizing antibody such that only cell-to-cell transmission may occur. Under these conditions, incubation at 37 °C results in the formation of infected cell foci. The extent of cell-to-cell spread can then be determined by counting the number of cells in each focus. The assay may be modified to assess the effects of inhibitors and/or specific cellular genes on cell-to-cell spread of HCV.

New hepatitis C virus drug discovery strategies and model systems.

INTRODUCTION: Hepatitis C virus (HCV) is a major cause of liver disease worldwide and the leading indication for liver transplantation in the United States. Current treatment options are expensive, not effective in all patients and are associated with serious side effects. Although preclinical, anti-HCV drug screening is still hampered by the lack of readily infectable small animal models, the development of cell culture HCV experimental model systems has driven a promising new wave of HCV antiviral drug discovery. AREAS COVERED: This review contains a concise overview of current HCV treatment options and limitations with a subsequent in-depth focus on the available experimental models and novel strategies that have, and continue to enable, important advances in HCV drug development. EXPERT OPINION: With a large cohort of chronically HCV-infected patients progressively developing liver disease that puts them at risk for hepatocellular carcinoma and hepatic decompensation, there is an urgent need to develop effective therapeutics that are well tolerated and effective in all patients and against all HCV genotypes. Significant advances in HCV experimental model development have expedited drug discovery; however, additional progress is needed. Importantly, the current trends and momentum in the field suggests that we will continue to overcome critical experimental challenges to reach this end goal.

Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ∼170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture-derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol-dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.

Hepatitis C virus infection in phenotypically distinct Huh7 cell lines.

In 2005, the first robust hepatitis C virus (HCV) infectious cell culture system was developed based on the HCV genotype 2a JFH-1 molecular clone and the human-derived hepatoma cell line Huh7. Although much effort has been made to dissect and expand the repertoire of JFH-1-derived clones, less attention has been given to the host cell despite the intriguing facts that thus far only Huh7 cells have been found to be highly permissive for HCV infection and furthermore only a limited number of Huh7 cell lines/stocks appear to be fully permissive. As such, we compiled a panel of Huh7 lines from disparate sources and evaluated their permissiveness for HCV infection. We found that although Huh7 lines from different laboratories do vary in morphology and cell growth, the majority (8 out of 9) were highly permissive for infection, as demonstrated by robust HCV RNA and de novo infectious virion production following infection. While HCV RNA levels achieved in the 8 permissive cell lines were relatively equivalent, three Huh7 lines demonstrated higher infectious virion production suggesting these cell lines more efficiently support post-replication event(s) in the viral life cycle. Consistent with previous studies, the single Huh7 line found to be relatively resistant to infection demonstrated a block in HCV entry. These studies not only suggest that the majority of Huh7 cell lines in different laboratories are in fact highly permissive for HCV infection, but also identify phenotypically distinct Huh7 lines, which may facilitate studies investigating the cellular determinants of HCV infection.

Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a novel coronavirus that causes a highly contagious respiratory disease, SARS, with significant mortality. Although factors contributing to the highly pathogenic nature of SARS-CoV remain poorly understood, it has been reported that SARS-CoV infection does not induce type I interferons (IFNs) in cell culture. However, it is uncertain whether SARS-CoV evades host detection or has evolved mechanisms to counteract innate host defenses. We show here that infection of SARS-CoV triggers a weak IFN response in cultured human lung/bronchial epithelial cells without inducing the phosphorylation of IFN-regulatory factor 3 (IRF-3), a latent cellular transcription factor that is pivotal for type I IFN synthesis. Furthermore, SARS-CoV infection blocked the induction of IFN antiviral activity and the up-regulation of protein expression of a subset of IFN-stimulated genes triggered by double-stranded RNA or an unrelated paramyxovirus. In searching for a SARS-CoV protein capable of counteracting innate immunity, we identified the papain-like protease (PLpro) domain as a potent IFN antagonist. The inhibition of the IFN response does not require the protease activity of PLpro. Rather, PLpro interacts with IRF-3 and inhibits the phosphorylation and nuclear translocation of IRF-3, thereby disrupting the activation of type I IFN responses through either Toll-like receptor 3 or retinoic acid-inducible gene I/melanoma differentiation-associated gene 5 pathways. Our data suggest that regulation of IRF-3-dependent innate antiviral defenses by PLpro may contribute to the establishment of SARS-CoV infection.

Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme.

Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-A crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitin-like N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitin-aldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity.

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.

Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection.

Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target.