Resistance of HBV covalently closed circular DNA to antiviral therapy

HEPATOLOGYVol. 34, No. 4, Pt. 2, 2001

AASLD ABSTRACTS

317A

579

580

RESISTANCE OF HBV COVALENTLY CLOSED CIRCULAR D N A TO ANTIVIRAL THERAPY. A y m a n M Abdelhamed, Colleen M Kelley, Edward E Cable, Thomas G Miller, Penn State College of Medicine, Hershey, PA; Phillip A Furman, Triangle Pharmaceuticals, Durham, NC; Harriet C Isom, Penn State College of Medicine, Hershey, PA

A VIRAL KINETIC STUDY U S I N G PEGYLATED INTERFERON ALPHA 2B A N D LAM1VUDINE IN NAIVE PATIENTS W I T H HEPATITIS B E ANTIGEN NEGATIVE/HEPATITIS B VIRUS DNA-POSITIVE CHRONIC HEPATITIS B. Vana Sypsa Mrs, Athens Univ Medical Sch, Athens Greece; Nicolaos C Tassopoulos Mr, Western Attica General Hospital, Athens Greece; Dimitris Chrysagis Mr, Loimodon Hospital, Thessaloniki Greece; Maria Raptopoulou Mrs, Aristoteleion Univ of Thessaloniki, Thessaloniki Greece; Ioaunis Ketikoglou Mr, Hippokration General Hospital, Athens Greece; Themistoklis Vassiliadis Mr, Hippokration General Hospital, Thessaloniki Greece; Catherina Haida Mrs, Dimitris Paraskevis Mr, Athens Univ Medical Sch, Athens Greece; Francois Lafleur Mr, Schering Plough, Kemlworth, NJ; Angelos Hatzakis Mr, Athens Univ Medical Sch, Athens Greece

We previously reported a novel transient in vitro system for studying HBV replication using HBV recombinant baculovirus to efficiently deliver the HBV genome to HepG2 cells (Hepatology 28:1134-1146, 1998) that can be used for antiviral testing (Antimicrobial Agents and Chemotherapy 43: 2017-2026, 1999). The HBV recombinant baculovirus system makes it possible to administer antivirals prior to initiation of HBV infection or after HBV infection. The system also yields sufficient replication levels that HBV CCC DNA can be easily detected and HBV DNA species can be quantitatively analyzed separate from DS and RC forms and from total HBV DNA. One of the advantages of the HBV baculovirus/HepG2 system is that the levels of HBV replication are sufficiently high that HBV DNA can be readily detected by Southern blot. In this study, we analyzed Southern blots using a phosphorimager system to measure the effects of antivirals on HBV replication. HepG2 cells were infected one day post seeding with 100 PFU of HBV baculovirus/cell for all experiments. Both pretreatment and post treatment studies were carried out using various antivirals. For pretreatment, drug was added 16 hours prior to HBV baculovirus infection and continued daily for 7 days post infection. For post treatment, drug was added 4 days after HBV baculovirus infection and continued daily for 7 days (11 days post infection). The untreated controls were also fed daily. Cells were treated with thirteen different concentrations of drug whose difference in concentration was a constant log value. To evaluate extracellular HBV DNA, medium from infected cells was harvested, centrifuged and the HBV particles were precipitated. Replicative intermediates (RI) were extracted from cytoplasmic core particles isolated from each culture. To measure HBV CCC DNA, non-protein associated DNA was extracted from the nuclei of infected cells. Extracellular DNA, RI and CCC DNA were all obtained from a single infected 60 m m dish of HepG2 cells. Southern blot analyses of R1, CCC and extracellular HBV DNA were performed. The data were analyzed by correlating the drug concentration to changes in HBV DNA levels using a sigmoidal curve with variable slope. The ICs0 and IC90 values were calculated from the curve using the maximum and minimum responses and the Hill coefficient. We conclude the following from this study. (1) Following antiviral treatment, the levels of HBV RI and extracellular DNA decrease in a concentration dependent fashion. (2) The ICs0 values and the Hill slopes vary for the different HBV DNA species being analyzed and for pretreatment compared to the post treatment protocols as well as for different antivirals. (3) Nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs. (4) Nuclear HBV CCC DNA is more resistant than the nuclear RC form. This study represents the first in vitro comparison of the effects of antivirals administered prior to initiation of HBV infection relative to administration at a time post infection when accumulation of CCC DNA can be readily detected and the first thorough in vitro quantitative study of the antiviral effects on HBV CCC DNA.

A randomizedtrial was conducted to assess the viraldynamicsof HBVunder the inhibitory effects of pegylatedinterferon alpha 2b (PEGqNTRON) and lamivudine on HBV replication in vivo. Patients were randomizedin 3 groups,Arm A: PEG-1NTRON100 mcg QW for 48 weeks,ArmB: PEG-INTRON200 mcgQW for 4 weeksfollowedby PEG-INTRON100 mcgQW for 44 weeksand Arm C: PEG-INTRON100 mcg QW and Lamivudine100 mg QD for 48 weeks. Eighteenpatients who had completed16 weeks of therapywere included in the analysis (5, 7 and 6 from arms A, B and C respectively).Blood sampleswere collectedfrequentlyduring the first48 hours (hours: 0, 4, 8, 16, 20, 24, 28, 36, 48) and then at days 4, 5, 7, 9, 11, 12, 14, 21, 28, 42, 56, 84 and 112.Patients were serum HBeAgnegativewith HBVDNA higher than 10~copies/mhThe mean (SD) baseline log~0HBV-DNAwas 6.82 (1.06) copies/ml. Data were analyzed using a standard model of viral infection involvingthree equations on the changeof viral load and of the number of infected and target cells. Due to the frequent early blood sampling, it was possible to estimate the virion clearancerate (c) with higher accuracythan other studies (1-3). The antiviralefficacy(e) of the treamtents and the death rate of infected cells (8) were estimatedby fitting viral load data up to week 16 using a model assumingthat the generationof new productivelyinfected cells during therapy is completelyinhibited while virus production is incompletelyinhibited (2). A mixedeffects approachwas used to fit the model.Mean (SD)c was 1.920 (0.903) dayl and did not differ amongthe three arms.Thisvaluecorresponds to an averageHBVvirion half-lifeof tl/2= 8.7 hours. The averagetotal virion production was estimatedas 1.5 x 101z copies/day.The mean valuesof 8 were0.020, 0.044 and 0.044 day1 for arms A, Band C, respectivelyand did not differsignificantly amongthe three groups.The meanvaluesof • weresignificantlylowerin armsA (29.6%,p<0.001) and B (75.8%, p=0.023) comparedto arm C (92.6%).Furthermore, ~ was found to be negatively associatedwith baselineviral load (p = 0.015). In conclusion, 1) the half-lifeof freeHBVvirus was found approximatelyone-third of that previouslyestimated,2) the death rate of infected cellswas not found to differbetweenPEG-INTRON200 mcgand PEG-INTRON100 mcg + Lamivudine,3) the combinationof PEG-INTRON+ Lamivudineis superior to the monotherapyof PEG-INTRON 100 or 200 mcg in terms of antiviral efficacy, and 4) the combination of PEGINTRON+Lamivudmehas favorableviral dynamicparametersand should be widelyassessedin clinical trials. 1NowakMA, Bonhoeffer S, Hill AM, et al. Viral dynamics in hepatitis B virus infection. Proc. Natl. Acad. Sci. USA 1996, 93:4398-4402. ZTsiangM, RooneyJF, TooleJJ, et al. Biphasic clearanceof hepatitis Bvirus frompatients during adefovirdipivoxiltherapy.Hepatology 1999~ 29(6): 1863-1869.3LauGKK,TsiangM, Yuen ST, et al. Combination therapywith Lamivudine and Famciclovirfor chronic hepatitis B-infectedChinese patients: a virai dynamicsstudy. Hepatology2000; 32(2): 394 399. Supported by"Schering-Plough.

581

582

HBSAG VACCINE FAILURE SHOWS A STRONG HLA CLASS II INDEPENDENT ASSOCIATION W I T H C4AQ0 ALLELES. Thomas Hoehler, Johannes Gutenberg University, Mainz Germany; Beate Stradmann-Bellinghausen, Institute of Legal Medicine, Mainz Germany; Roland Saenger, Glaxo SmithKline, Munich Germany; Christian Rittner, Peter M Schneider, Institute of Legal Medicine, Mainz Germany

ANTISENSE PEPTIDE NUCLEIC ACIDS (PNAS): A N E W CLASS OF HBV REVERSE TRANSCRIPTASE INItlBITORS. Magdalena Robaczewska, B~atrice Seigneres, INSERM, Lyon France; Anna J. Podhajska, UG-AMG, Gdansk Poland; Christian Trepo, INSERM, Lyon France; Peter E. Nielsen, Panum Institute, Copenhagen Denmark; Fabien Zoufim, Lucyna Cova, INSERM, Lyon France

Approximately 5 percent of healthy Caucasian adults fail to develop anti-HBs antibodies after a regular vaccine course. Vaccination failure has been linked to the presence of the HLA-class II alleles DRBI*0301, DRBI*0701 and DQBI*0201. However, since the initial description of these associations the functional background has remained unclear. Complement component C 4 is encoded within the MHC telomeric to the HLA-class II genes and essential for classical pathway activation. C4 deficiencies are c o m m o n in Western Caucasians populations and arise due to gene deletion, conversions or mutations. 4269 heahhy healthy individuals were vaccinated in a prospective trial with Engerix B (20 m g major HBsAg; at 0, 1 and 6 month). Nonresponse was classified as an anti-HBs < 10 Ufl after the last vaccination. 73 nonresponders (NR) (1.7%) were identified. For comparison 53 responders (R) (anti-HBs > 1 0 IU/1) were drawn randomly from the same cohort. C4 typing was carried out by high voltage agarose gel electrophoresis after desialation of the samples with neuraminidase, followed by immunofixation or hemolytic overlay. C4AQ0 alleles were confirmed by C4a-chain typing using SDS-PAGE. C4 Deletions were studied by Southern blot. HLA-class I and II antigens had been previously typed. Statistical analysis was performed using chi-square test. C4AQ0 alleles were observed in 44/73 (60.3%) of NR compared to 17/53 (32%) of R (p<0.001). The c o m m o n haplotype DRBI*0301/C4ADel/B8 was observed in 23/73 (31.5%) NR compared to 6/53 (11.3%) R (p<0.01). C4AQO alleles not caused by C4Del were present in 21/50 (42%) NR without C4Del compared to 11/47 (23.4%) in R (p<0.01). DRBI*0301 alleles not linked to C4ADel (7/73 (10%) NR vs. 6/53 (11.3%) R and C4 B deletions (7/73 (9.6%) NR vs. 5/53 (9%) R) were observed with similar frequencies in the 2 groups. These data show a markedly increased prevalence of C4A deletions and of nonexpressed C4AQ0 alleles in HBsAg NR. This association is independent of the reported association of DRBI*0301 with vaccine failure, since only DRBI*0301 positive haplotypes carrying the C4A deletion are associated with nonresponse. Co-ligation of complement receptors on B-cells dramatically lowers the threshold for B cell activation. C4AQ0 alleles could lead to inefficient complement activation and a failure of B-cells to secrete anti-HBs.

Peptide nucleic acids (PNAs), a new class of oligonucleotide (ODN) mimics in which the bases are attached to a pseudo-peptide backbone, appear as particularly attractive antisense agents, since they are resistant to nuclease and protease digestion and have high affinity for RNA and DNA. However, their antiHBV activity has not yet been investigated. The aim of this study was to target the antisense PNA to the stem-loop structure of DHBV encapsidation signal e in order to inhibit reverse transcription (RT). In addition, the inhibitory activity of PNAs was compared with phosphorothioate-modified ODNs (S-ODNs). Two different, partly overlapping antisense PNAs (PNA2 and PNA3) targeting e sequence were tested. In addition, PNA CTRL2 and CTRL3, of the same sequence, except 2 nucleotide (nt) mismatch, were used as controls. The inhibitory activities of PNAs and S-ODNs were investigated in rabbit reticulocyte lysate assay for the expression of enzymatically active DHBV polymerase. Analysis of different doses of antisense PNAs on RT reaction revealed that low concentrations of both PNA2 and PNA3 strongly inhibited (-) strand DNA priming and elongation without modification ofDHBV polymerase expression. In addition, the inhibitory effect of PNAs on (-) strand DNA synthesis was more efficient as compared with S-ODNs of the same sequence. The inhibition was specific since the same concentration of control PNAs (CTRL2 and CTRL3), differing only by 2 nt, had no effect on RT reaction. Moreover, PNAs, but not S-ODNs, co-translationally competed with viral polymerase for the RT initiation site on e, probably via steric hindrance. Ongoing studies in primary duck hepatocyte culture will be informative on PNAs efficacy on DHBV replication cycle inhibition. In conclusion, we provide here the first evidence that PNAs, a new class of antisense agents, are strong and specific inhibitors of hepadnaviral reverse transcriptase.