A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Generation of a VeroE6 Pgp gene knock out cell line and its use in SARS-CoV-2 antiviral study.

Vero cells are widely used for antiviral tests and virology research for SARS-CoV-2 as well as viruses from various other families. However, Vero cells generally express high levels of multi-drug resistance 1 (MDR1) or Pgp protein, the efflux transporter of foreign substances including many antiviral compounds, affecting the antiviral activity as well as interpretation of data. To address this, a Pgp gene knockout VeroE6 cell line (VeroE6-Pgp-KO) was generated using CRISPR-CAS9 technology. These cells no longer expressed the Pgp protein as indicated by flow cytometry analysis following staining with a Pgp-specific monoclonal antibody. They also showed significantly reduced efflux transporter activity in the calcein acetoxymethyl ester (calcein AM) assay. The VeroE6-Pgp-KO cells and the parental VeroE6 cells were each infected with SARS-CoV-2 to test antiviral activities of remdesivir and nirmatrelvir, two known Pgp substrates, in the presence or absence of a Pgp inhibitor. The compounds showed antiviral activities in VeroE6-Pgp-KO cells similar to that observed in the presence of the Pgp inhibitor. Thus, the newly established VeroE6-Pgp-KO cell line adds a new in vitro virus infection system for SARS-CoV-2 and possibly other viruses to test antiviral therapies without a need to control the Pgp activity. Removal of the Pgp inhibitor for antiviral assays will lead to less data variation and prevent failed assays.

Genetic Surveillance of SARS-CoV-2 Mpro Reveals High Sequence and Structural Conservation Prior to the Introduction of Protease Inhibitor Paxlovid.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (Mpro). Nirmatrelvir is a potent Mpro inhibitor and the antiviral component of Paxlovid. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of Mpro prior to the introduction of Mpro inhibitors, Mpro sequences and its cleavage junction regions were retrieved from ~4,892,000 high-quality SARS-CoV-2 genomes in the open-access Global Initiative on Sharing Avian Influenza Data (GISAID) database. Any mutations identified from comparison to the reference sequence (Wuhan-Hu-1) were catalogued and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of Mpro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (α-, ß-, and γ-coronaviruses). Additionally, we showed that over time, the SARS-CoV-2 Mpro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the emergency use authorization (EUA) of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 Mpro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals. IMPORTANCE The recent authorization of oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. The emergence of new variants, as well as the selective pressure imposed by antiviral drugs themselves, raises concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of Mpro evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and Mpro inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 Mpro mutational landscape.

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants.

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the Mpro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (ß, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the Mpro specifically for α, ß, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and ß Mpro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Persistent Bacterial Coinfection of a COVID-19 Patient Caused by a Genetically Adapted Pseudomonas aeruginosa Chronic Colonizer.

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen which causes chronic infections in immunocompromised patients and leads to high mortality rate. It is identified as a common coinfecting pathogen in COVID-19 patients causing exacerbation of illness. In our hospital, P. aeruginosa is one of the top coinfecting bacteria identified among COVID-19 patients. We collected a strong biofilm-forming P. aeruginosa strain displaying small colony variant morphology from a severe COVID-19 patient. Genomic and transcriptomic sequencing analyses were performed with phenotypic validation to investigate its adaptation in SARS-CoV-2 infected environment. Genomic characterization predicted specific genomic islands highly associated with virulence, transcriptional regulation, and DNA restriction-modification systems. Epigenetic analysis revealed a specific N6-methyl adenine (m6A) methylating pattern including methylation of alginate, flagellar and quorum sensing associated genes. Differential gene expression analysis indicated that this isolate formed excessive biofilm by reducing flagellar formation (7.4 to 1,624.1 folds) and overproducing extracellular matrix components including CdrA (4.4 folds), alginate (5.2 to 29.1 folds) and Pel (4.8-5.5 folds). In summary, we demonstrated that P. aeuginosa clinical isolates with novel epigenetic markers could form excessive biofilm, which might enhance its antibiotic resistance and in vivo colonization in COVID-19 patients.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

Genotype and subtype profiling of PSI-7977 as a nucleotide inhibitor of hepatitis C virus.

PSI-7977, a prodrug of 2'-F-2'-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC(50)) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.

The YMDD and rtA194T mutations result in decreased replication capacity in wild-type HBV as well as in HBV with precore and basal core promoter mutations.

BACKGROUND: A recent study indicated that addition of the hepatitis B e antigen (HBeAg) precore (PC) or basal core promoter (BCP) mutations to wild-type HBV offset the reduced replication of the HBV polymerase rtA194T±rtL180M+rtM204V mutations. rtA194T was reportedly associated with tenofovir resistance. We investigated these findings in genotype D HBV, where both PC and BCP naturally occur in vivo. METHODS: A plasmid containing a wild-type 1.3 genome length genotype D HBV laboratory strain was used as a parent for PC, BCP, rtA194T±rtL180M+rtM204V, rtL180M+rtM204V and rtM204I mutants. Viral replication was evaluated by Southern blot analysis of intracellular HBV core DNA following transient transfection of HepG2 cells. Drug susceptibility was evaluated by quantitative PCR of intracellular HBV DNA. RESULTS: PC and BCP mutations each increased HBV DNA replication by approximately 200% over wild-type. rtA194T reduced replication by <40%, whereas rtL180M+rtM204V, rtL180M+rtA194T+rtM204V or rtM204I each reduced by >75% from their respective wild-type, PC or BCP genome backbone (P<0.05). The enhanced replication by PC or BCP offset the reduction by rtA194T; however, the other reverse transcriptase (RT) mutations in PC or BCP backbones remained significantly lower than wild-type (P<0.05). Regardless of the backbone, rtA194T±rtL180M+rtM204V remained susceptible to tenofovir in vitro. rtA194T alone remained susceptible to lamivudine, while rtL180M+rtM204V and rtL180M+rtA194T+rtM204V were resistant. CONCLUSIONS: PC or BCP mutations increased HBV DNA replication, offset the decreased replication by rtA194T alone, but they did not fully rescue the impaired replication conferred by other RT mutations as compared with wild-type. rtA194T±rtL180M+rtM204V did not confer tenofovir resistance.

HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A-H and its use in antiviral phenotyping assays.

The aim of this study is to establish a phenotyping assay to analyze patient HBV polymerase/reverse transcriptase (RT) sequences for potential drug resistance against RT inhibitors. HBV RT (pol aa 304-715, including the entire RT) from clinical isolates were amplified and ligated into a plasmid vector (pRTAN) expressing a genotype A HBV genome lacking the RT region. HBV DNA replication of the recombinants and their drug susceptibilities were assessed by transient transfection into HepG2 cells and intracellular core DNA was analyzed either by Southern blot or using a 96-well format and quantification by qPCR. Cloning of the HBV RT gene from clinical isolates representing genotypes A-H was successful and led to virus DNA replication. Recombinants expressing patient derived RT genes containing the rtL180M+M204V lamivudine resistance (LAM-R) mutations demonstrated a LAM-R phenotype. Similarly, patient derived RT genes containing the adefovir resistance (ADV-R) mutations rtA181V or rtN236T demonstrated an ADV-R phenotype. Recombinants containing HBV RT from paired patient samples without genotypic changes had similar EC(50) values. In conclusion, a phenotyping assay for HBV RT gene was developed, allowing evaluation of patient-derived HBV RT from genotypes A-H, and confirmed the drug resistance phenotype in samples containing LAM-R or ADV-R mutations.

No resistance to tenofovir disoproxil fumarate detected after up to 144 weeks of therapy in patients monoinfected with chronic hepatitis B virus.

UNLABELLED: Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue with potent activity against human immunodeficiency virus type 1 and hepatitis B virus (HBV). To date, no reports of HBV clinical resistance to TDF have been confirmed. In two phase 3 studies (GS-US-174-0102 and GS-US-174-0103), 375 hepatitis B e antigen-negative (HBeAg(-) ) patients and 266 HBeAg(+) patients with chronic hepatitis B (some nucleoside-naive and some lamivudine-experienced) were randomized 2:1 to receive TDF (n = 426) or adefovir dipivoxil (ADV; n = 215) for 48 weeks. After week 48, eligible patients received open-label TDF with no interruption. The studies are being continued through week 384/year 8; week 144 data are presented here. Per protocol, viremic patients (HBV DNA level ≥ 400 copies/mL or 69 IU/mL) had the option of adding emtricitabine (FTC) at or after week 72. Resistance analyses of HBV polymerase/reverse transcriptase (pol/RT) were based on population dideoxy sequencing. Phenotypic analyses were conducted in HepG2 cells with recombinant HBV derived from patient serum. Most patients maintained TDF monotherapy treatment across both studies (607/641, 95%). A resistance analysis of HBV pol/RT was performed at the baseline for all patients, for viremic patients at week 144 or at the last time when they were on TDF monotherapy (34 on TDF and 19 on ADV-TDF), and for patients who remained viremic after the addition of FTC (7/20 on TDF and 5/14 on ADV-TDF). No patient developed amino acid substitutions associated with resistance to TDF. Virological breakthrough on TDF monotherapy was infrequent over 144 weeks (13/426, 3%) and was attributed to documented nonadherence in most cases (11/13, 85%). Persistent viremia (≥400 copies/mL) through week 144 was rare (5/641, 0.8%) and was not associated with virological resistance to TDF by population or clonal analyses. CONCLUSION: No nucleoside-naive or nucleoside-experienced patient developed HBV pol/RT mutations associated with TDF resistance after up to 144 weeks of exposure to TDF monotherapy.

Anti-hepatitis B virus activity in vitro of combinations of tenofovir with nucleoside/nucleotide analogues.

BACKGROUND: Long-term management of some chronic hepatitis B patients might require combination therapy using drugs with distinct resistance profiles to sustain viral suppression and to reduce the resistance-associated failure. Tenofovir disoproxil fumarate (TDF), approved for hepatitis B virus (HBV) and HIV-1 treatment, is active against wildtype HBV and HBV containing YMDD mutations, which confer resistance to emtricitabine (FTC), lamivudine (3TC) and telbivudine (LdT) and contribute to entecavir (ETV) resistance. We therefore evaluated the in vitro anti-HBV activity of tenofovir (TFV), the active parent drug of TDF, combined with FTC, 3TC, ETV, LdT and adefovir (AFV). METHODS: The anti-HBV activities of the compounds were tested using the AD38 cell line that expresses wild-type HBV from a tetracycline-controllable promoter. Intracellular HBV DNA levels were quantified using real-time PCR assay and cytotoxicities were assessed with XTT assays. The antiviral data of the drug combinations were evaluated using MacSynergy analyses on the basis of the Bliss independence model as well as isobologram analyses on the basis of the Loewe additivity theory. RESULTS: All drug combinations tested, FTC+TFV, 3TC+TFV, ETV+TFV, LdT+TFV and AFV+TFV, showed additive antiviral interactions as analysed by MacSynergy. Isobologram analyses revealed that these combination pairs were additive, with the exception of FTC+TFV, which demonstrated slight synergistic activity. No cytotoxic or antagonistic effects were observed with any of the combinations tested. CONCLUSIONS: The combination of TFV with FTC, 3TC, ETV, LdT or AFV had additive to slightly synergistic anti-HBV effects in vitro. These results support the use of TDF as a component in combination regimens with currently available anti-HBV nucleoside analogues.

Antiviral effects of lamivudine, emtricitabine, adefovir dipivoxil, and tenofovir disoproxil fumarate administered orally alone and in combination to woodchucks with chronic woodchuck hepatitis virus infection.

Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit the replication of wild-type hepatitis B virus (HBV) and lamivudine (3TC)-resistant virus in HBV-infected patients, including those who are coinfected with human immunodeficiency virus. The combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during the treatment of chronic HBV infection. The antiviral effect of oral ADV or TDF, alone or in combination with 3TC or emtricitabine (FTC), against chronic woodchuck hepatitis virus (WHV) infection was evaluated in a placebo-controlled study in the woodchuck, an established and predictive model for antiviral therapy. Once-daily treatment for 48 weeks with ADV plus 3TC or TDF plus FTC significantly reduced serum WHV viremia levels from the pretreatment level by 6.2 log(10) and 6.1 log(10) genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log(10) genome equivalents/ml), ADV alone (4.8 log(10) genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log(10) genome equivalents/ml), TDF alone (2.9 log(10) genome equivalents/ml), 3TC alone (2.7 log(10) genome equivalents/ml), and FTC alone (2.0 log(10) genome equivalents/ml). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen, characteristically accompanied by declines in hepatic WHV replication and the hepatic expression of WHV antigens. Most woodchucks had prompt recrudescence of WHV replication after drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs or drug combinations administered. In conclusion, the oral administration of 3TC, FTC, ADV, and TDF alone and in combination was safe and effective in the woodchuck model of HBV infection.

Hepatitis B virus containing the I233V mutation in the polymerase reverse-transcriptase domain remains sensitive to inhibition by adefovir.

An isoleucine-to-valine change at position 233 (rtI233V) of hepatitis B virus (HBV) polymerase was recently reported to cause decreased in vitro susceptibility to, and treatment failure of, adefovir dipivoxil (ADV). To further evaluate these findings, we screened our ADV clinical-study sequence database of 853 patients and identified 4 who, at baseline, had HBV with this mutation. All 4 patients responded to treatment with ADV, with a median change in HBV DNA levels of 4.0 log(10) copies/mL after 48 weeks of treatment. Phenotypic evaluation of clinical isolates and of a laboratory strain with the rtI233V mutation demonstrated their full susceptibility to adefovir in vitro, and HBV with the rtI233V mutation developed in none of the patients.

In vitro drug susceptibility analysis of hepatitis B virus clinical quasispecies populations.

Analysis of the replication and drug resistance of patient serum hepatitis B virus (HBV) populations can contribute to the therapeutic management of chronic hepatitis B. We developed a procedure for cloning serum HBV quasispecies populations and for phenotypic analysis of the cloned populations for in vitro drug susceptibility. Equivalent sequences were compared to the respective serum HBV DNAs of the cloned quasispecies by population sequencing. Analysis of individual clones revealed that each population contained a diversity of HBV quasispecies. Furthermore, secreted HBV in the supernatant following transfection of the quasispecies populations remained mostly unchanged from the respective input populations. HBV obtained from patients who had developed resistance to adefovir or lamivudine, as demonstrated by development of the rtA181V or rtL180M/M204V mutations in HBV polymerase, respectively, were tested. Phenotypic analysis demonstrated that a population containing the HBV rtA181V mutation showed a 2.9-fold increase in the 50% effective concentration (EC(50)) for adefovir compared to the wild-type baseline isolate, while the lamivudine-resistant HBV quasispecies population showed a >1,000-fold increase in the lamivudine EC(50). In summary, a strategy of cloning full genome HBV quasispecies populations from patient sera was developed, which could provide a useful tool in clinical HBV drug resistance phenotyping and studies of the evolution of clinical viral species.

Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus.

Tenofovir is an acyclic nucleotide analog with activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Tenofovir disoproxil fumarate (tenofovir DF), a bis-alkoxyester prodrug of tenofovir, is approved for the treatment of HIV and is currently being developed to treat chronic hepatitis B. In this report, we further characterize the in vitro activity of tenofovir against HBV as well as its metabolism in hepatic cells. We show that tenofovir is efficiently phosphorylated to tenofovir diphosphate (TFV-DP) in both HepG2 cells and primary human hepatocytes. TFV-DP has a long intracellular half-life (95 h) and is a potent and competitive inhibitor of HBV polymerase (Ki = 0.18 microM). Tenofovir has a 50% effective concentration of 1.1 microM against HBV in cell-based assays, and potency is improved > 50-fold by the addition of bis-isoproxil progroups. Tenofovir has previously demonstrated full activity against lamivudine-resistant HBV in vitro and clinically. Here we show that the major adefovir resistance mutation, rtN236T, confers three- to fourfold-reduced susceptibility to tenofovir in cell culture; the clinical significance of this susceptibility shift has not yet been determined. The rtA194T HBV polymerase mutation recently identified in tenofovir DF-treated HIV/HBV-coinfected patients did not confer in vitro resistance to tenofovir as a single mutation or in a lamivudine-resistant viral background. Overall, the antiviral and metabolic profile of tenofovir supports its development for the treatment of chronic hepatitis B.

Adenovirus-based gene therapy during clevudine treatment of woodchucks chronically infected with woodchuck hepatitis virus.

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.

Mutations of the woodchuck hepatitis virus polymerase gene that confer resistance to lamivudine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil.

Administration of either lamivudine (2'-deoxy-3'-thiacytidine) or L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947-1954, 1999; Y. Zhu et al., J. Virol. 75:311-322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to L-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.