Remdesivir inhibits the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp; Nsp12). Here, we conducted viral resistance analyses from the Phase 3 PINETREE trial of remdesivir in nonhospitalized participants at risk of severe COVID-19. Nasopharyngeal swabs (collected at baseline [Day 1], Days 2, 3, 7, and 14) were eligible for analysis if their viral load was above the lower limit of quantification for the RT-qPCR assay (2228 copies/mL). The SARS-CoV-2 genome was sequenced for all remdesivir participants and 50% of placebo participants (baseline, Days 3, 7, and 14) and for participants who progressed to COVID-19-related hospitalization or all-cause death (all time points). Emergent substitutions in Nsp12 and other replication complex proteins were phenotyped using site-directed mutagenesis in a SARS-CoV-2 subgenomic replicon system. Overall, emergent Nsp12 substitutions were detected in 8/115 (7.0%) remdesivir participants and 7/129 (5.4%) placebo participants (1 substitution overlap between groups). Based on a structural analysis, none of the emergent Nsp12 substitutions were in direct contact with the incoming nucleoside triphosphate substrate, the RNA, or the RNA template 5 ' overhang. One substitution (A376V) showed reduced susceptibility to remdesivir (12.6-fold change in remdesivir half-maximal concentration [EC50]); it also showed reduced fitness when introduced in the SARS-CoV-2 replicon and virus in vitro. Other substitutions had <1.1-fold change in remdesivir EC50. None of the emergent substitutions in Nsp8, Nsp10, Nsp13, or Nsp14 (remdesivir, 10/115 [8.7%]; placebo, 10/129 [7.8%]) showed reduced remdesivir susceptibility. In conclusion, emergent substitutions in the SARS-CoV-2 RdRp complex with reduced remdesivir susceptibility were uncommon, indicating a high barrier to remdesivir resistance.

Background TAF resistance has not been detected after up to three years of treatment in CHB patients. Here, we report results from annual resistance surveillance from years 3 through 8 of TAF treatment. Methods Two randomized, double-blind (DB), active-controlled trials to evaluate TAF treatment of hepatitis e antigen (HBeAg)-negative and HBeAg-positive participants with CHB were conducted over 8 years (384 weeks). Sequence analysis of the pol/RT region was attempted for any participant who experienced a viral breakthrough, viral blip, or persistent viremia with HBV DNA ≥69 IU/mL at annual intervals and for any participant who discontinued the study drug with HBV DNA ≥69 IU/mL. Participants that developed substitutions at conserved pol/RT sites or at polymorphic residues (if observed in ≥2 participants within the study) were also phenotyped against TAF. Results Out of 1298 participants, the percentage of participants who qualified for resistance analysis annually from Week 144 to Week 384 remained low (range 1.7 – 8.4%). Among those qualifying for sequencing, the proportions with persistent viremia progressively declined with time, with only 3 participants being persistently viremic by Year 8. The viral load of these 3 participants at baseline was >108 log10 IU/mL and declined over time but did not reach 69 IU/mL. During the 5-year open-label period where all participants received TAF, conserved site substitutions in the HBV viral pol/RT were observed in 13 participants. Conclusions Overall, no resistance to TAF was detected in adult CHB participants with positive or negative HBeAg who received TAF therapy for up to 8 years.

Abstract Background Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in nonhospitalized and hospitalized adult and pediatric patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 REDPINE study, which demonstrated the safety of RDV in participants with severely reduced kidney function. Methods REDPINE (NCT04745351) was a double-blind, placebo-controlled trial in participants hospitalized for COVID-19 with severely reduced kidney function who were randomized 2:1 to receive RDV or placebo for 5 days. Full-genome deep sequencing of SARS-CoV-2 was performed on nasopharyngeal swab samples collected on Days 1 (baseline), 3, 5, 7, 14, 21, and/or 29. Emergent amino acid substitutions from RDV-treated participants were tested for RDV susceptibility in a SARS-CoV-2 replicon system. Results Of the 243 participants randomized and treated, 82 (RDV, 55; placebo, 27) met the resistance analysis criteria (all participants with all-cause death or invasive mechanical ventilation [IMV] through Day 29 or with SARS-CoV-2 RNA above the viral load assay lower limit of quantitation on Day 14). Sequencing data at both baseline and postbaseline timepoints were obtained from 60 participants (RDV, 41; placebo, 19). Among these, emergent amino acid substitutions in Nsp12 were observed in 8/41 (19.5%) in the RDV group and 1/19 (5.3%) in the placebo group. The proportions of participants with all-cause death or IMV through Day 29 were comparable between participants in the RDV group with emergent Nsp12 substitutions (5/8, 62.5%) or without emergent Nsp12 substitutions (30/33, 90.9%). In 4 participants in the RDV group, Nsp12 substitutions with low-level reduced susceptibility to RDV were identified in samples collected 9 days after cessation of RDV treatment (2.9- to 3.4-fold change in EC50): M794I (n = 2), C799F (n = 1), and E136V (n = 1). Three of these 4 participants had received solid organ transplants and were receiving concomitant immunosuppressive therapies during the study. Conclusion The results of resistance analyses in participants with severely reduced kidney function hospitalized for COVID-19 confirm a high barrier to clinically meaningful resistance to RDV in COVID-19 patients. Disclosures Kristen Andreatta, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jiani Li, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Lauren Rodriguez, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Dong Han, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Simin Xu, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jason K. Perry, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Yiannis Koullias, MD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Robert H. Hyland, DPhil, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Charlotte Hedskog, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds

Background & Aims: Bulevirtide (BLV) is a HDV/HBV entry inhibitor that is associated with virologic response (responders, HDV-RNA undetectable or >= 2 log(10) IU/ml decrease from baseline) in >50% of patients after a 24-week treatment. However, some patients only achieve a <1 log(10) IU/ml decline in HDV-RNA after the 24-week treatment (non-responders). Here, we report a viral resistance analysis in participants receiving BLV monotherapy who were non-responders or experienced virologic breakthrough (VB, i.e., two consecutive increases in HDV-RNA of >= 1 log(10) IU/ml from nadir or two consecutive HDV-RNA detectable results if previously undetectable) from the phase II MYR202 and phase III MYR301 study. Methods: Deep-sequencing of the BLV-corresponding region in HBV PreS1 and of the HDV HDAg gene, as well as in vitro phenotypic testing, were performed for the participant with VB (n = 1) and non-responders (n = 20) at baseline (BL) and Week 24 (WK24). Results: No amino acid exchanges associated with reduced susceptibility to BLV within the BLV-corresponding region or within HDAg were identified in isolates from any of the 21 participants at BL or at WK24. Although variants (HBV n = 1; HDV n = 13) were detected at BL in some non-responders or in the participant with VB, none were associated with reduced sensitivity to BLV in vitro. Furthermore, the same variant was detected in virologic responders. A comprehensive phenotypic analysis demonstrated that the BLV EC50 values from 116 BL samples were similar across non-responders, partial responders (HDV RNA decline >= 1 but <2 log10 IU/ml), and responders regardless of the presence of HBV and/or HDV polymorphisms. Conclusions: No amino acid substitutions associated with reduced sensitivity to BLV monotherapy were detected at BL or WK24 in non-responders or the participant with VB after 24-week BLV treatment. (c) 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Background & Aims: Bulevirtide (BLV) is a small lipopeptide agent that specifically binds to the sodium taurocholate cotransporting polypeptide (NTCP) bile salt transporter and HBV/HDV receptor on the surface of human hepatocytes and inhibits HDV and HBV entry. As a satellite virus of HBV, HDV virions are formed after assembly of HDV RNA with the HBV envelope proteins (HBsAg). Because both viruses exist as eight different genotypes, this creates a potential for high diversity in the HBV/HDV combinations. To investigate the sensitivity of various combinations of HBV/HDV genotypes to BLV, clinical and laboratory strains were assessed.Methods: For the laboratory strains, the different envelopes from HBV genotypes A through H were combined with HDV genotypes 1-8 in cotransfection assays. Clinical plasma isolates were obtained from clinical studies and academic collaborations to maximise the diversity of HBV/HDV genotypes tested.Results: The mean BLV EC50 against HDV laboratory strains ranged from 0.44 to 0.64 nM. Regardless of HBV and HDV genotypes, the clinical isolates showed similar sensitivities to BLV with mean values that ranged from 0.2 to 0.73 nM.Conclusions: These data support the use of BLV in patients infected with any HBV/HDV genotypes. Impact and implications: This study describes the potent activity of BLV against multiple laboratory strains spanning all HBV/ HDV A-H/1-8 genotype combinations and the most diverse collection of HDV clinical samples tested to date, including HBV/HDV genotype combinations less frequently observed in the clinic. Overall, all isolates and laboratory strains displayed similar in vitro nanomolar sensitivity to BLV. This broad-spectrum antiviral activity of BLV has direct implications on potential simplified treatment for any patient infected with HDV, regardless of genotype, and supports the new 2023 EASL Clinical Practice Guidelines on HDV that recommend antiviral treatment for all patients with CHD.(c) 2023 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Persistence of chronic hepatitis B (CHB) is attributed to main-tenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus produc-tion and spread but have no direct impact on cccDNA or expres-sion of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human he-patocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models contain-ing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing AR-CUS-POL mRNA. In both mouse and NHP, we observed a sig-nificant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Conclusion: 4334 is a potent CI with broad coverage across CI binding pocket variants and NrtI mutants.The activity of 4334 against pgRNA encapsidation is consistent with a class II CI. 4334 treatment results in melting of cytoplasmic HBV capsids, which prevents cccDNA formation.A Phase 1a first-in-human study with 4334 is planned for 2022.

Background and aims: The increased global incidence of hepatitis E virus (HEV) infections, warrants accurate and affordable diagnostics across different geographical regions.The soluble and highly conserved HEV open reading frame 2 (ORF2) capsid antigen (HEV-Ag) is detectable in self-limited acute enteric hepatitis by HEV-Ag ELISA which is a promising serological assay in settings where HEV-RNA testing is not feasible.Our aim was to assess the HEV-Ag biomarker in an HEV outbreak in a low income country.Method: A prospective single center longitudinal study during HEV outbreaks in the Chittagong, Bangladesh region between October 2018 and October 2019 was conducted based on recruitment of acute jaundice cases with clinical signs and symptoms of suspect HEV infections.Acute HEV infection was defined as a positive test result for anti-HEV IgM antibodies.Results: Forty four of the 51 enrolled enteric hepatitis cases (86%) were confirmed HEV by anti-HEV IgM ELISA at day 0 hospital entry.The anti-HEV-IgM and IgG were positive in all patients and did not reveal significant differences; neither between the time points day 0 and follow-up hospitalization on day 2-6 or day 7-10 nor between RNA-positive (n = 36) versus RNAnegative (n = 8) HEV groups.The HEV-Ag positivity was higher in viral RNA-positive (29/36, 81%) than the viral RNA-negative (1/8, 12%) group, p <0.001 and the HEV-Ag levels positively correlated with viremia, r = 0.77, p <0.0001.All non-HEV cases; n = 7 tested negative anti-HEV IgM and HEV-Ag and 5 of 7 (71%) tested anti-HAV IgM positive.