Comparison of influenza viral load in nasopharyngeal and midturbinate swabs.

Different specimen types are used for influenza diagnosis but comparative data for viral loads from different swab types are limited. We compared influenza viral loads (determined by quantitative reverse transcription polymerase chain reaction [RT-PCR]) in 93 paired midturbinate and nasopharyngeal swab aliquots from influenza infected patients enrolled in a phase 3 randomized-controlled study with the objective of maximizing the number of swabs available for sequence analysis. Midturbinate swabs yielded a 53% lower viral load versus nasopharyngeal swabs, and this difference was similar for influenza A and B. These data suggest that nasopharyngeal swabs might be preferred in diagnostic settings when obtaining higher viral load is important.

Oral bemnifosbuvir (AT-527) vs placebo in patients with mild-to-moderate COVID-19 in an outpatient setting (MORNINGSKY).

Aim: This phase III study assessed the efficacy/safety/antiviral activity/pharmacokinetics of bemnifosbuvir, a novel, oral nucleotide analog to treat COVID-19. Patients & methods: Outpatient adults/adolescents with mild-to-moderate COVID-19 were randomized 2:1 to bemnifosbuvir/placebo. Time to symptom alleviation/improvement (primary outcome), risk of hospitalization/death, viral load and safety were evaluated. Results: Although the study was discontinued prematurely and did not meet its primary end point, bemnifosbuvir treatment resulted in fewer hospitalizations (71% relative risk reduction), COVID-19-related medically attended hospital visits, and COVID-19-related complications compared with placebo. No reduction in viral load was observed. The proportion of patients with adverse events was similar; no deaths occurred. Conclusion: Bemnifosbuvir showed hospitalization reduction in patients with variable disease progression risk and was well tolerated. Clinical Trial Registration: NCT04889040 (ClinicalTrials.gov).

A Phase 2 Randomized Trial Evaluating the Antiviral Activity and Safety of the Direct-Acting Antiviral Bemnifosbuvir in Ambulatory Patients with Mild or Moderate COVID-19 (MOONSONG Study).

Bemnifosbuvir is an oral antiviral drug with a dual mechanism of action targeting viral RNA polymerase, with in vitro activity against SARS-CoV-2. We conducted a phase 2, double-blind study evaluating the antiviral activity, safety, efficacy, and pharmacokinetics of bemnifosbuvir in ambulatory patients with mild/moderate COVID-19. Patients were randomized 1:1 to bemnifosbuvir 550 mg or placebo (cohort A) and 3:1 to bemnifosbuvir 1,100 mg or placebo (cohort B); all doses were given twice daily for 5 days. The primary endpoint was a change from baseline in the amount of nasopharyngeal SARS-CoV-2 viral RNA by reverse transcription PCR (RT-PCR). The modified intent-to-treat infected population comprised 100 patients (bemnifosbuvir 550 mg, n = 30; bemnifosbuvir 1,100 mg, n = 30; cohort A placebo, n = 30; cohort B placebo, n = 10). The primary endpoint was not met: the difference in viral RNA adjusted means at day 7 was -0.25 log10 copies/mL between bemnifosbuvir 550 mg and cohort A placebo (80% confidence interval [CI], -0.66 to 0.16; P = 0.4260), and -0.08 log10 copies/mL between bemnifosbuvir 1,100 mg and pooled placebo (80% CI, -0.48 to 0.33; P = 0.8083). Bemnifosbuvir 550 mg was well tolerated. Incidence of nausea and vomiting was higher with bemnifosbuvir 1,100 mg (10.0% and 16.7% of patients, respectively) than pooled placebo (2.5% nausea, 2.5% vomiting). In the primary analysis, bemnifosbuvir did not show meaningful antiviral activity on nasopharyngeal viral load as measured by RT-PCR compared with placebo in patients with mild/moderate COVID-19. The trial is registered at ClinicalTrials.gov under registration number NCT04709835. IMPORTANCE COVID-19 continues to be a major global public health challenge, and there remains a need for effective and convenient direct-acting antivirals that can be administered outside health care settings. Bemnifosbuvir is an oral antiviral with a dual mechanism of action and potent in vitro activity against SARS-CoV-2. In this study, we evaluated the antiviral activity, safety, efficacy, and pharmacokinetics of bemnifosbuvir in ambulatory patients with mild/moderate COVID-19. In the primary analysis, bemnifosbuvir did not show meaningful antiviral activity compared with placebo as assessed by nasopharyngeal viral loads. The negative predictive value of nasopharyngeal viral load reduction for clinical outcomes in COVID-19 is currently unclear, and further evaluation of bemnifosbuvir for COVID-19 may be warranted despite the findings observed in this study.

Prognostic value of severe acute respiratory syndrome coronavirus-2 viral load and antibodies in patients hospitalized with COVID-19.

Observational studies have identified the potential prognostic value for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load and anti-SARS-CoV-2 antibodies in coronavirus disease 2019 (COVID-19). However, viral load in nasopharyngeal (NP) swabs produced inconsistent results in prognostic analyses, and the prognostic value of viral load or antibodies has not been confirmed in large clinical trials. COVACTA and REMDACTA were double-blind, randomized, controlled trials with a combined enrollment of 1078 patients hospitalized with COVID-19 treated with tocilizumab or placebo in COVACTA or tocilizumab plus remdesivir or placebo plus remdesivir in REMDACTA. We assessed the potential prognostic value of NP and serum SARS-CoV-2 viral load and serum anti-SARS-CoV-2 antibodies at baseline as biomarkers for clinical outcomes in patients enrolled in these trials. In adjusted Cox proportional hazard models, serum viral load was a more reliable predictor of clinical outcomes than NP viral load; high serum viral load was associated with higher risk for death and mechanical ventilation/death and lower likelihood of hospital discharge (high vs. negative viral load hazard ratios [95% confidence interval {CI}] were 2.87 [1.57-5.25], 3.86 [2.23-6.68], and 0.23 [0.14-0.36], respectively, in COVACTA and 8.11 [2.95-22.26], 10.29 [4.5-23.55], and 0.21 [0.15-0.29], respectively, in REMDACTA) and high serum viral load correlated with levels of inflammatory cytokines and lung damage biomarkers. High anti-SARS-CoV-2 spike protein antibody (ACOV2S) levels were associated with higher likelihood of hospital discharge (high vs. below the limit of quantification hazard ratios [95% CI] were 2.55 [1.59-4.08] for COVACTA and 1.54 [1.13-2.09] for REMDACTA). These results support the role of baseline SARS-CoV-2 serum viral load and ACOV2S antibody titers in predicting clinical outcomes for patients hospitalized with COVID-19.

Investigating the transmission of baloxavir-resistant influenza viruses from treated index patients to untreated household contacts in the BLOCKSTONE study.

In a post-hoc analysis of the phase 3 BLOCKSTONE study (JapicCTI-184180), we investigated household transmission of baloxavir-resistant (PA/I38X) influenza viruses. Using baloxavir resistance rates from prior clinical trials and the rate of influenza transmission observed in the study, the predicted number of PA/I38X transmission events was 4.8, assuming wild type and PA/I38X viruses were equally transmissible. However, no PA/I38X viruses were observed. These results suggest a low potential for baloxavir-resistant influenza virus transmission from treated to untreated individuals, potentially due to reduced viral/transmission fitness for PA/I38X viruses and/or low viral titres at the time when resistant viruses arise.

Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model.

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.

Toll-Like Receptor 7 Agonist RG7854 Mediates Therapeutic Efficacy and Seroconversion in Woodchucks With Chronic Hepatitis B.

Conventional treatment of chronic hepatitis B (CHB) is rarely curative due to the immunotolerant status of patients. RG7854 is an oral double prodrug of a toll-like receptor 7 (TLR7) agonist that is developed for the treatment of CHB. The therapeutic efficacy, host immune response, and safety of RG7854 were evaluated in the woodchuck model of CHB. Monotreatment with the two highest RG7854 doses and combination treatment with the highest RG7854 dose and entecavir (ETV) suppressed viral replication, led to loss of viral antigens, and induced seroconversion in responder woodchucks. Since viral suppression and high-titer antibodies persisted after treatment ended, this suggested that a sustained antiviral response (SVR) was induced by RG7854 in a subset of animals. The SVR rate, however, was comparable between both treatment regimens, suggesting that the addition of ETV did not enhance the therapeutic efficacy of RG7854 although it augmented the proliferation of blood cells in response to viral antigens and magnitude of antibody titers. The induction of interferon-stimulated genes in blood by RG7854/ETV combination treatment demonstrated on-target activation of TLR7. Together with the virus-specific blood cell proliferation and the transient elevations in liver enzymes and inflammation, this suggested that cytokine-mediated non-cytolytic and T-cell mediated cytolytic mechanisms contributed to the SVR, in addition to the virus-neutralizing effects by antibody-producing plasma cells. Both RG7854 regimens were not associated with treatment-limiting adverse effects but accompanied by dose-dependent, transient neutropenia and thrombocytopenia. The study concluded that finite, oral RG7854 treatment can induce a SVR in woodchucks that is based on the retrieval of antiviral innate and adaptive immune responses. This supports future investigation of the TLR7 agonist as an immunotherapeutic approach for achieving functional cure in patients with CHB.

Tocilizumab in patients hospitalised with COVID-19 pneumonia: Efficacy, safety, viral clearance, and antibody response from a randomised controlled trial (COVACTA).

Background: In COVACTA, a randomised, placebo-controlled trial in patients hospitalised with coronavirus disease-19 (COVID-19), tocilizumab did not improve 28-day mortality, but shortened hospital and intensive care unit stay. Longer-term effects of tocilizumab in patients with COVID-19 are unknown. Therefore, the efficacy and safety of tocilizumab in COVID-19 beyond day 28 and its impact on Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) clearance and antibody response in COVACTA were investigated. Methods: Adults in Europe and North America hospitalised with COVID-19 (N = 452) between April 3, 2020 and May 28, 2020 were randomly assigned (2:1) to double-blind intravenous tocilizumab or placebo and assessed for efficacy and safety through day 60. Assessments included mortality, time to hospital discharge, SARS-CoV-2 viral load in nasopharyngeal swab and serum samples, and neutralising anti-SARS-CoV-2 antibodies in serum. ClinicalTrials.gov registration: NCT04320615. Findings: By day 60, 24·5% (72/294) of patients in the tocilizumab arm and 25·0% (36/144) in the placebo arm died (weighted difference -0·5% [95% CI -9·1 to 8·0]), and 67·0% (197/294) in the tocilizumab arm and 63·9% (92/144) in the placebo arm were discharged from the hospital. Serious infections occurred in 24·1% (71/295) of patients in the tocilizumab arm and 29·4% (42/143) in the placebo arm. Median time to negative reverse transcriptase-quantitative polymerase chain reaction result in nasopharyngeal/oropharyngeal samples was 15·0 days (95% CI 14·0 to 21·0) in the tocilizumab arm and 21·0 days (95% CI 14·0 to 28·0) in the placebo arm. All tested patients had positive test results for neutralising anti-SARS-CoV-2 antibodies at day 60. Interpretation: There was no mortality benefit with tocilizumab through day 60. Tocilizumab did not impair viral clearance or host immune response, and no new safety signals were observed. Future investigations may explore potential biomarkers to optimize patient selection for tocilizumab treatment and combination therapy with other treatments. Funding: F. Hoffmann-La Roche Ltd and the US Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under OT number HHSO100201800036C.

Combining baloxavir marboxil with standard-of-care neuraminidase inhibitor in patients hospitalised with severe influenza (FLAGSTONE): a randomised, parallel-group, double-blind, placebo-controlled, superiority trial.

BACKGROUND: Neuraminidase inhibitors (NAIs) are considered the standard of care for hospitalised patients with influenza. We aimed to test whether combining the cap-dependent endonuclease inhibitor baloxavir marboxil (hereafter baloxavir) with standard-of-care NAIs would result in improved clinical outcomes compared with NAI monotherapy in hospitalised patients with severe influenza. METHODS: We did a randomised, parallel-group, double-blind, placebo-controlled, superiority trial. Patients aged 12 years or older who were hospitalised with laboratory-confirmed influenza (by RT-PCR or a rapid test) and had a National Early Warning Score 2 (NEWS2) of 4 or greater were included. Recruitment took place in 124 centres across 25 countries. Using a permuted-block method and an interactive response system, patients were randomly assigned (2:1) to receive either baloxavir plus NAIs (hereafter the baloxavir group) or placebo plus NAIs (hereafter the control group). Participants, investigators, and those assessing outcomes were masked to group assignment. Baloxavir was administered orally on day 1 and day 4 (40 mg for bodyweight <80 kg, or 80 mg for ≥80 kg), and on day 7 if no clinical improvement had occurred by day 5. The NAIs included in this study were oseltamivir, zanamivir, and peramivir, which were selected and administered according to local standard practice. The primary endpoint was time to clinical improvement, defined as time to a NEWS2 of 2 or lower for 24 h or hospital discharge, whichever came first, based on daily assessments over the study duration of 35 days. Secondary endpoints included safety analyses. The modified intention-to-treat infected (mITTI) population (ie, all patients who were randomly assigned to treatment, received a dose of study drug, and were RT-PCR-positive for influenza at any timepoint according to the treatment assigned at randomisation) was used in all efficacy analyses. The safety population (ie, all patients who received at least one dose of study treatment, according to the treatment received) was used in the safety analyses. The trial is registered with ClinicalTrials.gov, NCT03684044. FINDINGS: Overall, 366 patients were enrolled between Jan 8, 2019, and March 16, 2020, of whom 241 were assigned to the baloxavir group and 125 to the control group. The mITTI population comprised 322 patients, 208 in the baloxivir group and 114 in the control group. In total, 280 (87%) of these patients had influenza A infections. Median time to clinical improvement was 97·5 h (95% CI 75·9 to 117·2) in the baloxavir group and 100·2 h (75·9 to 144·4) in the control group (median difference -2·7 h [95% CI -53·4 to 25·9], p=0·467). Baloxavir plus NAI was well tolerated, and no new safety signals were observed; serious adverse events occurred in 29 (12%) of 239 patients in the baloxavir group versus 19 (15%) of 124 patients in the control group, of which one was considered related to treatment (orthostatic hypotension in a patient in the control group). Overall, four deaths (2%) occurred in the baloxavir group and seven (6%) in the control group; none were considered related to treatment. INTERPRETATION: Combining baloxavir with NAIs did not result in superior clinical outcomes compared with NAIs alone. The combination of baloxavir plus NAI was well tolerated. The findings suggest that combination antivirals would not be routinely indicated in clinical practice for hospitalised patients with severe influenza. FUNDING: F Hoffmann-La Roche and the Biomedical Advanced Research and Development Authority.

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model.

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil.

BACKGROUND: Baloxavir marboxil (BXM) is an approved drug that selectively targets cap-dependent endonuclease on PA subunit in the RNA polymerase complex of influenza A and B viruses. Amino acid substitutions at position 38 in the PA subunit were identified as a major pathway for reduced susceptibility to baloxavir acid (BXA), the active form of BXM. Additionally, substitutions found at positions E23, A37, and E199 in the PA subunit impact BXA susceptibility by less than 10-fold. METHODS: We comprehensively evaluated the impact of novel amino acid substitutions identified in PA, PB1, and PB2 subunits in BXM clinical trials and influenza sequence databases by means of drug susceptibility and replicative capacity. RESULTS: PA/I38N in A(H1N1)pdm09 and PA/I38R in A(H3N2) were newly identified as treatment-emergent substitutions in the CAPSTONE-2 study. The I38N substitution conferred reduced susceptibility by 24-fold, whereas replicative capacity of the I38N-substituted virus was impaired compared with the wild-type. The I38R-substituted virus was not viable in cell culture. All other mutations assessed in this extensive study did not significantly affect BXA susceptibility (< 2.4-fold change). CONCLUSION: These results provide additional information on the impact of amino acid substitutions in the trimeric viral polymerase complex to BXA susceptibility and will further support influenza surveillance.

Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon-α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus.

RG7834 is a small-molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon-α (wIFN-α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log10 from baseline and WHV DNA by a mean of 1.71 log10. ETV + wIFN-α reduced WHsAg and WHV DNA by means of 2.40 log10 and 6.70 log10, respectively. The combination of RG7834, ETV, and wIFN-α profoundly reduced WHsAg and WHV DNA levels by 5.00 log10 and 7.46 log10, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off-treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV-specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.

Baloxavir Marboxil Single-dose Treatment in Influenza-infected Children: A Randomized, Double-blind, Active Controlled Phase 3 Safety and Efficacy Trial (miniSTONE-2).

BACKGROUND: Baloxavir marboxil (baloxavir) is a novel, cap-dependent endonuclease inhibitor that has previously demonstrated efficacy in the treatment of influenza in adults and adolescents. We assessed the safety and efficacy of baloxavir in otherwise healthy children with acute influenza. METHODS: MiniSTONE-2 (Clinicaltrials.gov: NCT03629184) was a double-blind, randomized, active controlled trial enrolling children 1-<12 years old with a clinical diagnosis of influenza. Children were randomized 2:1 to receive either a single dose of oral baloxavir or oral oseltamivir twice daily for 5 days. The primary endpoint was incidence, severity and timing of adverse events (AEs); efficacy was a secondary endpoint. RESULTS: In total, 173 children were randomized and dosed, 115 to the baloxavir group and 58 to the oseltamivir group. Characteristics of participants were similar between treatment groups. Overall, 122 AEs were reported in 84 (48.6%) children. Incidence of AEs was similar between baloxavir and oseltamivir groups (46.1% vs. 53.4%, respectively). The most common AEs were gastrointestinal (vomiting/diarrhea) in both groups [baloxavir: 12 children (10.4%); oseltamivir: 10 children (17.2%)]. No deaths, serious AEs or hospitalizations were reported. Median time (95% confidence interval) to alleviation of signs and symptoms of influenza was similar between groups: 138.1 (116.6-163.2) hours with baloxavir versus 150.0 (115.0-165.7) hours with oseltamivir. CONCLUSIONS: Oral baloxavir is well tolerated and effective at alleviating symptoms in otherwise healthy children with acute influenza. Baloxavir provides a new therapeutic option with a simple oral dosing regimen.

Baloxavir treatment of ferrets infected with influenza A(H1N1)pdm09 virus reduces onward transmission.

Influenza viruses cause seasonal outbreaks and pose a continuous pandemic threat. Although vaccines are available for influenza control, their efficacy varies each season and a vaccine for a novel pandemic virus manufactured using current technology will not be available fast enough to mitigate the effect of the first pandemic wave. Antivirals can be effective against many different influenza viruses but have not thus far been used extensively for outbreak control. Baloxavir, a recently licensed antiviral drug that targets the influenza virus endonuclease, has been shown to reduce virus shedding more effectively than oseltamivir, a widely used neuraminidase inhibitor drug. Thus it is possible that treatment with baloxavir might also interrupt onward virus transmission. To test this, we utilized the ferret model, which is the most commonly used animal model to study influenza virus transmission. We established a subcutaneous baloxavir administration method in ferrets which achieved similar pharmacokinetics to the approved human oral dose. Transmission studies were then conducted in two different locations with different experimental setups to compare the onward transmission of A(H1N1)pdm09 virus from infected ferrets treated with baloxavir, oseltamivir or placebo to naïve sentinel ferrets exposed either indirectly in adjacent cages or directly by co-housing. We found that baloxavir treatment reduced infectious viral shedding in the upper respiratory tract of ferrets compared to placebo, and reduced the frequency of transmission amongst sentinels in both experimental setups, even when treatment was delayed until 2 days post-infection. In contrast, oseltamivir treatment did not substantially affect viral shedding or transmission compared to placebo. We did not detect the emergence of baloxavir-resistant variants in treated animals or in untreated sentinels. Our results support the concept that antivirals which decrease viral shedding could also reduce influenza transmission in the community.

Machine-learning based patient classification using Hepatitis B virus full-length genome quasispecies from Asian and European cohorts.

Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Variable viral mutant signatures develop within individual patients due to the low-fidelity replication of the viral polymerase creating 'quasispecies' populations. Here we present the first comprehensive survey of the diversity of HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct European and Asian patient populations. Seroconversion to the HBV e antigen (HBeAg) represents a critical clinical waymark in infected individuals. Using a machine learning approach, a model was developed to determine the viral variants that accurately classify HBeAg status. Serial surveys of patient quasispecies populations and advanced analytics will facilitate clinical decision support for chronic HBV infection and direct therapeutic strategies through improved patient stratification.

PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization.

RG7834 is a potent, orally bioavailable small-molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three-hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies that phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure.

A novel orally available small molecule that inhibits hepatitis B virus expression.

BACKGROUND & AIMS: The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834). METHODS: RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir. RESULTS: Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice. CONCLUSION: We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues. LAY SUMMARY: We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms.

Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) are promising non-nucleos(t)ide HBV replication inhibitors. HAPs are known to promote core protein mis-assembly, but the molecular mechanism of abnormal assembly is still elusive. Likewise, the assembly status of core protein induced by SBA remains unknown. Here we show that SBA, unlike HAP, does not promote core protein mis-assembly. Interestingly, two reference compounds HAP_R01 and SBA_R01 bind to the same pocket at the dimer-dimer interface in the crystal structures of core protein Y132A hexamer. The striking difference lies in a unique hydrophobic subpocket that is occupied by the thiazole group of HAP_R01, but is unperturbed by SBA_R01. Photoaffinity labeling confirms the HAP_R01 binding pose at the dimer-dimer interface on capsid and suggests a new mechanism of HAP-induced mis-assembly. Based on the common features in crystal structures we predict that T33 mutations generate similar susceptibility changes to both compounds. In contrast, mutations at positions in close contact with HAP-specific groups (P25A, P25S, or V124F) only reduce susceptibility to HAP_R01, but not to SBA_R01. Thus, HAP and SBA are likely to have distinctive resistance profiles. Notably, P25S and V124F substitutions exist in low-abundance quasispecies in treatment-naïve patients, suggesting potential clinical relevance.

In vitro drug combination studies of Letermovir (AIC246, MK-8228) with approved anti-human cytomegalovirus (HCMV) and anti-HIV compounds in inhibition of HCMV and HIV replication.

Despite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Letermovir (AIC246, MK-8228) is a new anti-HCMV agent in clinical development that acts via a novel mode of action and has demonstrated anti-HCMV activity in vitro and in vivo. For the future, drug combination therapies, including letermovir, might be indicated under special medical conditions, such as the emergence of multidrug-resistant virus strains in transplant recipients or in HCMV-HIV-coinfected patients. Accordingly, knowledge of the compatibility of letermovir with other HCMV or HIV antivirals is of medical importance. Here, we evaluated the inhibition of HCMV replication by letermovir in combination with all currently approved HCMV antivirals using cell culture checkerboard assays. In addition, the effects of letermovir on the antiviral activities of selected HIV drugs, and vice versa, were analyzed. Using two different mathematical techniques to analyze the experimental data, (i) additive effects were observed for the combination of letermovir with anti-HCMV drugs and (ii) no interaction was found between letermovir and anti-HIV drugs. Since none of the tested drug combinations significantly antagonized letermovir efficacy (or vice versa), our findings suggest that letermovir may offer the potential for combination therapy with the tested HCMV and HIV drugs.

Differential regulation of NF-κB-mediated proviral and antiviral host gene expression by primate lentiviral Nef and Vpu proteins.

NF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with ß-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs) in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages.