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W1805 Entecavir Maintains a High Genetic Barrier to HBV Resistance Through 6 Years in NaïVE Patients
determine the clinical features in HBeAg+ CHB with coexisting PC/BCP Mu. METHODS We identified 368 HBeAg+ CHB cases (211 from China, 157 from US) in 473 consecutive treatment naïve CHB Asian patients followed from 07/04 to 11/08 with obtainable genotypic analysis. PC/BCP Mu were analyzed by Quest Diagnostics, San Juan, CA using a direct sequencing for the US cases and by a Res Lab in Beijing using ABI3730 sequencor as forward primer direct sequencing for the cases in China. RESULTS Of 368 HBeAg+ cases, 83 (23%) were infected with WT+PC/BCP. 72% were male. Mean age 44(18-62), DNA 6.72 (4.0410.07) x log10 c/mL, ALT 145 (21- 1464)IU/L. Genotypes (Gt) were 75% C, 24% B, 1% D. Table 1 shows the frequency of PC/BCP Mu, which increases significantly with age (12% if <30 vs. 88% if ≥30, p <0.0001), and high HBV DNA (6% if < 5.0 x log10 vs. 94% if >5.0 x log10 c/mL, p <0.0001). Most common mutants were in BCP at A1762T(A) with G1764A(G, A/G)=50%, PC at G1698A(A, G/A)=22%, and triple Mu at A1762T(A), G1764A(G, A/G), G1698A(A, G.A)=11%. The frequency of PC Mu was significantly higher in Gt B (82%) vs. C (18%, p=0.02). PC/BCP Mu were not significantly associated with gender or ALT. Liver biopsy obtained in 22/83 cases: 8 in stage 0-I, 15 in stage II-IV. The remaining 285 HBeAg+ WT cases were 55% male, mean age 35(16-60), ALT 119 (13-1216) IU/mL, DNA 6.25(4.80-11.34) x log10 c/mL. The prevalence of PC/BCP Mu was confirmed in 94/105 (89.5%) of HBeAg- CHB treatment naïve cases. CONCLUSIONS In our study, PC/BCP Mu were present in 23% of HBeAg+ treatment naïve cases, which is significantly lower than reported cohort mixed with treatment experienced cases. This indicated that treatment may induce PC/BCP Mu in HBeAg+ patients. Those with PC/BCP Mu tended to be older (> 30) and HBV DNA > 5 x log10 c/ml. PC Mu were more common in those with Gt B infection; while BCP Mu, in Gt C. Further study is needed to determine if co-existing PC/BCP Mu are associated with seroreversion, cirrhosis after HBeAg seroconverion, or increased risk of liver cancer in HBeAg+ CHB. 1. Frequency of PC/BCP in HBeAg+ CHB
CTL Escape Mutation of Core Protein Are More Frequent in Strains of HBeAg Negative Patients with Low Viral Load Hossein Sendi, Marjan Mehrab-Mohseni, Saeid Shahraz, Helene Norder, Seyed-Mayed Alavian, Babak Noorinayer, Mohammad R. Zali, Paul Pumpens, Herbert L. Bonkovsky, Lars Magnius Background: More than 300 million individuals worldwide are chronically infected with hepatitis B virus (HBV). Recent studies in HBV-infected patients have suggested that Cytotoxic T cell (CTL) response is the most important pathway in eliminating HBV infected cells. Although, the T cell response is less vigorous in chronically infected patients, the difference in immune escape mutation profile between different subgroups of CHB patient has been less thoroughly investigated. Aim: To investigate the role of mutations in different immune epitopes of hepatitis B core antigen (HBcAg) among Iranians with HBeAg negative chronic hepatitis B (e-CHB). Methods: The study included 77 patients negative for HBeAg, and positive for anti-HBe. Twenty-nine patients were categorized as e-CHB while 48 were classified as asymptomatic carriers (ASCs). Patients were all treatment naive, and well compensated. All were infected with HBV genotype D and had previously been investigated for the presence of pre-core and basic core promoter (BCP) mutants, and levels of HBV DNA. For core gene sequencing, HBV DNA was extracted from serum and HBcAg was amplified. PCR products were purified, and used as templates in the sequencing reaction. Nucleotides 1-450 (amino acid 1-150) of HBcAg were sequenced. Results: Amino acid substitutions were found to cluster in seven different regions: residues 18-27, 35-45, 49-69, 76-87, 91-95, 105-116, and 130-135 of HBcAg. Amino acid mutation rates of HBc protein were higher in strains for ASCs than e-CHB patients (p=0.014). Asn67 mutation was never found with Ile66 and Ser69 mutations (p <0.001). Substitutions for wt Ser21 in the 18-27 CTL epitope were found to be correlated with substitutions for wt Ser87 (p<0.002), and wt Thr12 (p<0.02). Both substitutions for Ser21 and Thr12Ser were associated with low levels of viral load in univariate analysis (p<0.001). None of patients with mutations in an HLA-A2 restricted CTL epitope, 18-27, had viral loads more than 105 cc/mL (p<0.001). Multivariate analysis revealed that high levels (> 105 cc/mL) of HBV were inversely associated with mutations in CTL epitopes of HBc (OR:0.11 , p=0.015), and positively associated with the promoter double T1762A1764 mutation together with G1757 (OR:16.87, p=0.004). Conclusion: Selection of CTL escape mutations consolidates the persistence of HBV infection despite probable reduction of viral fitness, since these mutations are more frequently seen in the context of lower replication of virus. Perhaps similar mechanisms for selection of immune escape mutations also exist in other chronic viral diseases. (e.g., HCV, HIV)
W1805 Entecavir Maintains a High Genetic Barrier to HBV Resistance Through 6 Years in NaïVE Patients Daniel J. Tenney, Kevin A. Pokornowski, Ronald E. Rose, Carl J. Baldick, Betsy J. Eggers, Jie Fang, Michael J. Wichroski, Ulysses A. Diva, Dong Xu, Richard B. Wilber, Helena Brett-Smith, Uchenna H. Iloeje
W1803 3D Structure of Protein-X of Hepatitis B Virus Ashraf Mohammadkhani, Zarrin Minuchehr, Armin Madadkar-Sobhani, Masoud Sotoudeh, Ferdos Rastgar Jazii, Reza Malekzadeh
Background/Aims: Entecavir (ETV) provides both potent viral suppression and a high genetic barrier to resistance. As a result, in nucleoside-naïve patients, ETV resistance (ETVr) was rare through 5 years. The barrier to resistance in lamivudine (LVD)-refractory patients is reduced. Methods: All patients receiving continuous therapy in registrational trials were monitored for resistance through year 6. Sequencing was performed on serum samples with detectable HBV DNA (≥300 copies/mL) at each cross-sectional end-of-year analysis, or with viral breakthrough at anytime, or at discontinuation from study with detectable HBV DNA. Cumulative probabilities of resistance were determined through year 6. Results: In years 1 through 6, respectively, 663, 278, 149, 120, 108 and 99 nucleoside-naïve patients were treated and monitored, with 94% in year 6 having HBV DNA <300 copies/mL. No patient in year 6 showed emerging ETVr at T184, S202 or M250 ± LVD resistance (LVDr) M204I/ V±L180M. The cumulative probability of genotypic ETVr in nucleoside-naïve patients remained at 1.2% through 6 years. Among LVD-refractory patients treated with ETV, 187, 146, 80, 52, 33 and 29 were monitored in years 1 through 6, respectively. The cumulative probabilities of genotypic ETVr at years 1 through 6 were 6%, 15%, 36%, 47%, 51%, and 57% respectively, and of virological breakthrough with ETVr was 50% through year 6. Among the 74 LVD-refractory patients who achieved undetectable HBV DNA on ETV, 5 subsequently developed ETVr. Conclusions: ETVr remains rare (1.2%) in nucleoside-naïve patients through 6 years. LVDr HBV has a reduced resistance barrier to ETV, and patients with LVDr HBV may benefit from add-on or combination therapy.
Human is the natural host for hepatitis B virus and during viral infection hepatocytes are infected to cause acute and chronic liver disease. The virus genome with 3.2 kb in length is partially double stranded and contains four identified open reading frames (ORFs). The ORFx encodes protein x (HBx) with 154 amino acids in size and a molecular mass of 17.5 kDa which has not been found in mature virions nor associated with nucleocapsid particle. The protein x of hepatitis B virus has been the focus of much attention in recent years, and it is much related with hepatocarcinogenesis. This protein has no counterparts in hosts and is conserved among mammalian hepadnavirus. As no high resolution crystal structure is available, representation of HBx structure has been an interesting area of research to explain its function. The 3D structure of this protein is currently unknown and specific functions of this protein are not well understood. Here we describe a structural model for HBx using computational methods in combination of experimental data as constraints. Material and Methods: Protein-x was translated based on its genome which we have sequenced previously. Bioinformatics tools were employed for searching Hidden Markov Model and conserved motif(s). SSpro program was used to determine the secondary structure of the protein and then ProtScale program was utilized to provide several predefined amino acid scales. Our model was constructed using 1jfua as a template by LOMETS server. Optimization of the model was carried out by GROMACS program. Results: The homology search of HBx against the PDB database using BLAST revealed it as a conserved domain which is found in hepadnaviruses and represented it as a non homologous protein. The propensity of amino acid composition compared to swiss-prot protein release 55.3 described the protein to be positively charged with pI=8.82. The corresponding secondary structure implicates a helixloop-helix region at residues 89-122 with an anti parallel β-sheet at 128-142 in the Cterminal region of the protein. To construct for possible 3D structure a model of HBx was generated with the SPARKS2 threading algorithm using the LOMETS server and membraneanchored thioredoxine-like protein (1jfua) used as a template. Conclusion: Although there is a limited knowledge known about the protein-x of hepatitis B virus, here we modeled its 3D structure for the first time. In this modeled structure there are a combination of βsheets, α-helices and coil motifs. Our model can lead to a better understanding of the function of protein-x and its interaction with the cellular proteins to use it as a target for treatment purposes.
W1806 Two Years Safety and Efficacy of Tenofovir Disoproxil Fumarate (TDF) in Patients with HBV-Induced Cirrhosis Maria Buti, Stephanos J. Hadziyannis, Philippe Mathurin, Petr Urbanek, Morris Sherman, Simone I. Strasser, Chia C. Wang, Joerg Petersen, Jenny Heathcote, Patrick Marcellin, Jeff Sorbel, Elsa Mondou, Jane Anderson, Franck Rousseau Background and aims: Tenofovir disoproxil fumarate (TDF) has activity against hepatitis B virus (HBV) and was recently approved for the treatment of chronic HBV (CHB). An efficacy and safety analysis was performed of the subset of cirrhotic patients receiving TDF for 96 weeks in two Phase 3 CHB registration trials, GS-174-0102 and GS-174-0103. Methods: In study 0102 (HBeAg- CHB) and 0103 (HBeAg+ CHB) patients were randomized 2:1 to double-blind TDF 300 mg or adefovir dipivoxil (ADV) 10 mg once daily for 48 weeks; if Week (W) 48 biopsy was performed patients were eligible to receive open-label TDF for 7 additional years with the option to initiate combination emtricitabine (FTC)+TDF after W72 for confirmed HBV DNA ≥400 copies (c)/mL (69 IU/mL). Entrance criteria included abnormal ALT and viremia HBV DNA >100,000 copies/mL with Roche COBAS TaqMan assay (LLOQ=169 copies/mL). Results: Eighty-one of 426 patients originally randomized to TDF were cirrhotic (19%), 47 HBeAg- and 34 HBeAg+ with median baseline HBV DNA of 7.58 log10 copies/mL and ALT of 92 U/L. Similar proportions of cirrhotic and non-cirrhotic patients suppressed HBV DNA to <400 c/mL (69 IU/mL) at W96: 90% versus 85% (ITT) and 97% versus 95% (observed). Among patients remaining on treatment, 83% of cirrhotic and 78% of non-cirrhotic patients had normal ALT at W96 with median values of 30 and 29 U/L, respectively. Of 29 cirrhotic HBeAg+ patients with W96 serology results 9 seroconverted to anti-HBe (31%) and collectively 2 seroconverted to anti-HBs (both genotype
W1804 Clinical Features of Chronic Hepatitis B (CHB) in Treatment Naïve Asian Patients with Positive HBeAg and Co-Existing Precore and/or Basal Core Promoter (PC/BCP) Mutations Calvin Pan, Zheng Zeng, Ke-Qin Hu BACKGROUND HBeAg+ CHB is traditionally considered as wild type(WT) infection. Recent studies suggested a frequent coexistence of WT with PC/BCP Mutations(Mu) in this population. HBeAg seroreversion is common in Asian patients after achieving seroconversion with or without therapy. The PC/BCP Mu may predispose to HBeAg seroreverion, but the disease features remain to be determined in treatment naïve patients with WT+PC/BCP. AIMS To
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W1802
CTL Escape Mutation of Core Protein Are More Frequent in Strains of HBeAg Negative Patients with Low Viral Load Hossein Sendi, Marjan Mehrab-Mohseni, Saeid Shahraz, Helene Norder, Seyed-Mayed Alavian, Babak Noorinayer, Mohammad R. Zali, Paul Pumpens, Herbert L. Bonkovsky, Lars Magnius Background: More than 300 million individuals worldwide are chronically infected with hepatitis B virus (HBV). Recent studies in HBV-infected patients have suggested that Cytotoxic T cell (CTL) response is the most important pathway in eliminating HBV infected cells. Although, the T cell response is less vigorous in chronically infected patients, the difference in immune escape mutation profile between different subgroups of CHB patient has been less thoroughly investigated. Aim: To investigate the role of mutations in different immune epitopes of hepatitis B core antigen (HBcAg) among Iranians with HBeAg negative chronic hepatitis B (e-CHB). Methods: The study included 77 patients negative for HBeAg, and positive for anti-HBe. Twenty-nine patients were categorized as e-CHB while 48 were classified as asymptomatic carriers (ASCs). Patients were all treatment naive, and well compensated. All were infected with HBV genotype D and had previously been investigated for the presence of pre-core and basic core promoter (BCP) mutants, and levels of HBV DNA. For core gene sequencing, HBV DNA was extracted from serum and HBcAg was amplified. PCR products were purified, and used as templates in the sequencing reaction. Nucleotides 1-450 (amino acid 1-150) of HBcAg were sequenced. Results: Amino acid substitutions were found to cluster in seven different regions: residues 18-27, 35-45, 49-69, 76-87, 91-95, 105-116, and 130-135 of HBcAg. Amino acid mutation rates of HBc protein were higher in strains for ASCs than e-CHB patients (p=0.014). Asn67 mutation was never found with Ile66 and Ser69 mutations (p <0.001). Substitutions for wt Ser21 in the 18-27 CTL epitope were found to be correlated with substitutions for wt Ser87 (p<0.002), and wt Thr12 (p<0.02). Both substitutions for Ser21 and Thr12Ser were associated with low levels of viral load in univariate analysis (p<0.001). None of patients with mutations in an HLA-A2 restricted CTL epitope, 18-27, had viral loads more than 105 cc/mL (p<0.001). Multivariate analysis revealed that high levels (> 105 cc/mL) of HBV were inversely associated with mutations in CTL epitopes of HBc (OR:0.11 , p=0.015), and positively associated with the promoter double T1762A1764 mutation together with G1757 (OR:16.87, p=0.004). Conclusion: Selection of CTL escape mutations consolidates the persistence of HBV infection despite probable reduction of viral fitness, since these mutations are more frequently seen in the context of lower replication of virus. Perhaps similar mechanisms for selection of immune escape mutations also exist in other chronic viral diseases. (e.g., HCV, HIV)
W1805 Entecavir Maintains a High Genetic Barrier to HBV Resistance Through 6 Years in NaïVE Patients Daniel J. Tenney, Kevin A. Pokornowski, Ronald E. Rose, Carl J. Baldick, Betsy J. Eggers, Jie Fang, Michael J. Wichroski, Ulysses A. Diva, Dong Xu, Richard B. Wilber, Helena Brett-Smith, Uchenna H. Iloeje
W1803 3D Structure of Protein-X of Hepatitis B Virus Ashraf Mohammadkhani, Zarrin Minuchehr, Armin Madadkar-Sobhani, Masoud Sotoudeh, Ferdos Rastgar Jazii, Reza Malekzadeh
Background/Aims: Entecavir (ETV) provides both potent viral suppression and a high genetic barrier to resistance. As a result, in nucleoside-naïve patients, ETV resistance (ETVr) was rare through 5 years. The barrier to resistance in lamivudine (LVD)-refractory patients is reduced. Methods: All patients receiving continuous therapy in registrational trials were monitored for resistance through year 6. Sequencing was performed on serum samples with detectable HBV DNA (≥300 copies/mL) at each cross-sectional end-of-year analysis, or with viral breakthrough at anytime, or at discontinuation from study with detectable HBV DNA. Cumulative probabilities of resistance were determined through year 6. Results: In years 1 through 6, respectively, 663, 278, 149, 120, 108 and 99 nucleoside-naïve patients were treated and monitored, with 94% in year 6 having HBV DNA <300 copies/mL. No patient in year 6 showed emerging ETVr at T184, S202 or M250 ± LVD resistance (LVDr) M204I/ V±L180M. The cumulative probability of genotypic ETVr in nucleoside-naïve patients remained at 1.2% through 6 years. Among LVD-refractory patients treated with ETV, 187, 146, 80, 52, 33 and 29 were monitored in years 1 through 6, respectively. The cumulative probabilities of genotypic ETVr at years 1 through 6 were 6%, 15%, 36%, 47%, 51%, and 57% respectively, and of virological breakthrough with ETVr was 50% through year 6. Among the 74 LVD-refractory patients who achieved undetectable HBV DNA on ETV, 5 subsequently developed ETVr. Conclusions: ETVr remains rare (1.2%) in nucleoside-naïve patients through 6 years. LVDr HBV has a reduced resistance barrier to ETV, and patients with LVDr HBV may benefit from add-on or combination therapy.
Human is the natural host for hepatitis B virus and during viral infection hepatocytes are infected to cause acute and chronic liver disease. The virus genome with 3.2 kb in length is partially double stranded and contains four identified open reading frames (ORFs). The ORFx encodes protein x (HBx) with 154 amino acids in size and a molecular mass of 17.5 kDa which has not been found in mature virions nor associated with nucleocapsid particle. The protein x of hepatitis B virus has been the focus of much attention in recent years, and it is much related with hepatocarcinogenesis. This protein has no counterparts in hosts and is conserved among mammalian hepadnavirus. As no high resolution crystal structure is available, representation of HBx structure has been an interesting area of research to explain its function. The 3D structure of this protein is currently unknown and specific functions of this protein are not well understood. Here we describe a structural model for HBx using computational methods in combination of experimental data as constraints. Material and Methods: Protein-x was translated based on its genome which we have sequenced previously. Bioinformatics tools were employed for searching Hidden Markov Model and conserved motif(s). SSpro program was used to determine the secondary structure of the protein and then ProtScale program was utilized to provide several predefined amino acid scales. Our model was constructed using 1jfua as a template by LOMETS server. Optimization of the model was carried out by GROMACS program. Results: The homology search of HBx against the PDB database using BLAST revealed it as a conserved domain which is found in hepadnaviruses and represented it as a non homologous protein. The propensity of amino acid composition compared to swiss-prot protein release 55.3 described the protein to be positively charged with pI=8.82. The corresponding secondary structure implicates a helixloop-helix region at residues 89-122 with an anti parallel β-sheet at 128-142 in the Cterminal region of the protein. To construct for possible 3D structure a model of HBx was generated with the SPARKS2 threading algorithm using the LOMETS server and membraneanchored thioredoxine-like protein (1jfua) used as a template. Conclusion: Although there is a limited knowledge known about the protein-x of hepatitis B virus, here we modeled its 3D structure for the first time. In this modeled structure there are a combination of βsheets, α-helices and coil motifs. Our model can lead to a better understanding of the function of protein-x and its interaction with the cellular proteins to use it as a target for treatment purposes.
W1806 Two Years Safety and Efficacy of Tenofovir Disoproxil Fumarate (TDF) in Patients with HBV-Induced Cirrhosis Maria Buti, Stephanos J. Hadziyannis, Philippe Mathurin, Petr Urbanek, Morris Sherman, Simone I. Strasser, Chia C. Wang, Joerg Petersen, Jenny Heathcote, Patrick Marcellin, Jeff Sorbel, Elsa Mondou, Jane Anderson, Franck Rousseau Background and aims: Tenofovir disoproxil fumarate (TDF) has activity against hepatitis B virus (HBV) and was recently approved for the treatment of chronic HBV (CHB). An efficacy and safety analysis was performed of the subset of cirrhotic patients receiving TDF for 96 weeks in two Phase 3 CHB registration trials, GS-174-0102 and GS-174-0103. Methods: In study 0102 (HBeAg- CHB) and 0103 (HBeAg+ CHB) patients were randomized 2:1 to double-blind TDF 300 mg or adefovir dipivoxil (ADV) 10 mg once daily for 48 weeks; if Week (W) 48 biopsy was performed patients were eligible to receive open-label TDF for 7 additional years with the option to initiate combination emtricitabine (FTC)+TDF after W72 for confirmed HBV DNA ≥400 copies (c)/mL (69 IU/mL). Entrance criteria included abnormal ALT and viremia HBV DNA >100,000 copies/mL with Roche COBAS TaqMan assay (LLOQ=169 copies/mL). Results: Eighty-one of 426 patients originally randomized to TDF were cirrhotic (19%), 47 HBeAg- and 34 HBeAg+ with median baseline HBV DNA of 7.58 log10 copies/mL and ALT of 92 U/L. Similar proportions of cirrhotic and non-cirrhotic patients suppressed HBV DNA to <400 c/mL (69 IU/mL) at W96: 90% versus 85% (ITT) and 97% versus 95% (observed). Among patients remaining on treatment, 83% of cirrhotic and 78% of non-cirrhotic patients had normal ALT at W96 with median values of 30 and 29 U/L, respectively. Of 29 cirrhotic HBeAg+ patients with W96 serology results 9 seroconverted to anti-HBe (31%) and collectively 2 seroconverted to anti-HBs (both genotype
W1804 Clinical Features of Chronic Hepatitis B (CHB) in Treatment Naïve Asian Patients with Positive HBeAg and Co-Existing Precore and/or Basal Core Promoter (PC/BCP) Mutations Calvin Pan, Zheng Zeng, Ke-Qin Hu BACKGROUND HBeAg+ CHB is traditionally considered as wild type(WT) infection. Recent studies suggested a frequent coexistence of WT with PC/BCP Mutations(Mu) in this population. HBeAg seroreversion is common in Asian patients after achieving seroconversion with or without therapy. The PC/BCP Mu may predispose to HBeAg seroreverion, but the disease features remain to be determined in treatment naïve patients with WT+PC/BCP. AIMS To
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