Comprehensive monitoring of genotypic and phenotypic antiviral resistance was performed on 673 entecavir (ETV)-treated nucleoside naïve hepatitis B virus (HBV) patients. ETV reduced HBV DNA levels to undetectable by PCR (<300 copies/mL, <57 IU/mL) in 91% of hepatitis B e antigen (HBeAg)-positive and -negative patients by Week 96. Thirteen percent (n ‫؍‬ 88) of the comparator lamivudine (LVD)-treated patients experienced a virologic rebound (>1 log increase from nadir by PCR) in the first year, with 74% of these having LVD resistance (LVDr) substitutions evident. In contrast, only 3% (n ‫؍‬ 22) of ETV-treated patients exhibited virologic rebound by Week 96. Three ETV rebounds were attributable to LVDr virus present at baseline, with one having a S202G ETV resistance (ETVr) substitution emerge at Week 48. None of the other rebounding patients had emerging genotypic resistance or loss of ETV susceptibility. Genotyping all additional ETV patients with PCR-detectable HBV DNA at Weeks 48, 96, or end of dosing identified seven additional patients with LVDr substitutions, including one with simultaneous emergence of LVDr/ETVr. Generally, ETV patients with LVDr were detectable at baseline (8/10) and most subsequently achieved undetectable HBV DNA levels on ETV therapy (7/10). No other emerging substitutions identified decreased ETV susceptibility. In conclusion, ETVr emergence in ETV-treated nucleoside naïve patients over a 2-year period is rare, occurring in two patients with LVDr variants. These findings suggest that the rapid, sustained suppression of HBV replication, combined with a requirement for multiple substitutions, creates a high genetic barrier to ETVr in nucleoside naïve patients.

Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV).In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV) (32); and many will ultimately develop severe liver disease, including cirrhosis, hepatocellular carcinoma, and liver failure. Significant improvements in patient outcomes have been realized since the use of antiviral therapy for HBV. Due to the poor efficacies of these therapies and the emergence of viral resistance, however, additional therapies are needed (16). Prior to 2005, HBV therapies included parenteral regimens containing interferon alfa and the oral nucleoside/nucleotide analogs lamivudine (LVD) and adefovir dipivoxil (ADV). However, interferon alfa shows poor response rates and poor sustained efficacy (ϳ30 to 40% [reviewed in reference 18]), has low tolerability, and is contraindicated in patients with decompensated liver disease. LVD and ADV are associated with the development of viral resistance. LVD resistance (LVDr) is reported to occur in 24% of patients treated for 1 year, and this rate increases to 70% after 4 years (19). The rate of ADV resistance (ADVr) in nucleoside-naïve HBeAg-negative HBV patients has been r...

Deoxyspergualin (DSG) is a potent immunosuppressant whose mechanism of action remains unknown. To elucidate its mechanism of action, an intracellular DSG binding protein was identified. DSG has now been shown to bind specifically to Hsc70, the constitutive or cognate member of the heat shock protein 70 (Hsp70) protein family. The members of the Hsp70 family of heat shock proteins are important for many cellular processes, including immune responses, and this finding suggests that heat shock proteins may represent a class of immunosuppressant binding proteins, or immunophilins, distinct from the previously identified cis-trans proline isomerases. DSG may provide a tool for understanding the function of heat shock proteins in immunological processes.

Virologic resistance emerging during entecavir (ETV) therapy for hepatitis B virus (HBV)requiresT he genetic changes that impart resistance to the four approved nucleoside analogs used for the treatment of chronic hepatitis B virus (HBV) have been described (Table 1). Lamivudine (LVD) resistance (LVDr) arises from substitutions of the methionine (rtM204) to valine (rtM204V), isoleucine (rtM204I), or rarely serine (rtM204S) in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV reverse transcriptase (RT). The rtM204V change is always accompanied by an rtL180M growth adaptive substitution, 1 whereas the rtM204I substitution is found with or without rtL180M. Additionally, the rtV173L substitution has been found in some LVDr isolates and increases HBV replication capacity in vitro. 2 Resistance to telbivudine also arises from changes of the YMDD motif rtM204. 3 In contrast, resistance to adefovir (ADV) arises through rtN236T or rtA181V changes, whereas recent studies indicate that the rtA181T change often associated with ADV is more likely related to LVD therapy. 4,5 Entecavir (ETV), a novel deoxyguanosine analog, has demonstrated efficacy for treating HBV patients infected with wild-type or LVDr HBV. [6][7][8][9] Comprehensive resistance monitoring during clinical studies has revealed less than 1% viral resistance in nucleoside-treatment-naive (nucleoside-naive) patients through four years of therapy, reflecting a high genetic barrier to resistance that likely

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.