persistence of viral replication after anti-hbe seroconversion during antiviral therapy for chronic...

Journal of Hepatology 2000; 32: 307-316 Printed in Denmark . All rights raewed Mmksgaard . Copenhagen

Copyright Q European Association for the Study of the Liver 2000

Journal of Hepatology ISSN 0168-8278

Persistence of viral repletion after anti43Be seroconversion during antiviral therapy for chronic hepatitis B

Christian Pichoud’, Franqoise Berby’, Lieven Stuyver3, Marie-Anne Petit4, Christian Trepo172 and

Fabien Zoulim’>2 ‘~NSERM Unit 271, and 2Liver Unit, H&e1 Dieu, Lyon, Frame, 3~~noge~etics, Ghent, Belgium, and 4Centre Htipato-Biliaire Paul Browse,

CR INSERM 98-04, Villejuif; France

Background/Aims: Hepatitis B virus genome mutants may be selected during the ~rnun~rn~iat~ clearance of infection or during long-term nucleoside analog administration and may escape both antiviral pressures. The pattern of anti-HBe seroconversion was analyzed in patients receiving new nucleoside analogs, l~i~dine or famciclovir, in comparison with patients treated with interferon alpha. M&-M&: Eighteen consecutive patients who seroconverted to anti-HBe were included in the study. Serial serllll;l samples were studied with the quantitative determination of HBV DNA by the branched DNA assay (Chiron) and by a quantitative PCR assay (Roche diagnostics), determination of pre-Sl Ag, the genetic analysis of the viral genome with the determination of pre-core promoter or pre-core region mutations with a line probe assay (Innogenetics) and, in selected sampks of polymerase gene mutations Results: The quantitative PCR assay was found to be

HRONIC C hepatitis B remains a major health prob- lem worldwide, due to a high prevalence (300 mil-

lion) of chronic carriers of hepatitis B virus (HBV) and the clinical consequences of the disease including liver cirrhosis and hepatocellular carcinoma (1). HBV is not a cytopathogenic virus and i~une-mediated viral clearance is frequently associated with a flare of hepatitis, reflecting lysis of infected hepatocytes accompanied by a pronounced decrease of replication

Received 16 April; revised 22 June; accepted 27 June 1999

Correspondence: Fabien Zoulim, INSERM Unit 271, 151 cours Albert Thomas, 69003 Lyon, France. Tel: 33 4 72 68 19 70. Fax: 33 4 72 68 19 71. e-mail: zoulim@lyonl5l .inserm.fr

more sensitive than the bDNA assay, allowing a 2-5- log decrease in viral DNA levels to be demonstrated after anti-HBe seroconversion. Viral persistence after anti-HBe seroconversion induced by interferon, lamivudine or famciclovir, was often associated with circulating HBV genomes harboring mutations in the pre- core promoter. The clinical significance of these findings was demonstrated by the observation of reversion to HBeAg in two patients treated with interferon and one with lamivndine. Conclusion: Persistence of significant levels of viremia that are not detected by the branched DNA assay may be observed after anti-HBe seroconversion. A precise monitoring of viremia levels with more sensitive assays and HBV mutant strains is warranted in patients undergoing antiviral therapy.

Key words: Antiviral therpay; Hepatitis B virus; Im- mune response; Pre-core mutants; Viral resistance.

levels followed by anti-HBe seroconversion and sustained normalization of ALT levels (2). The rate of spontaneous anti-HBe seroconversion is estimated to be approximately 5% per year and to be significantly increased by interferon alpha therapy (3). However, under the pressure of spontaneous or interferon-induced anti-HBe immunity, HBV genomes that harbor nonsense, frameshift, or initiation codon mutations in the viral pre-core region that arrest the expression of HBe Ag without altering the replication capacity of the virus, may be selected (4). In HBe Ag negative carriers, the most frequently detected mutation is a nucleotide transition at the pre-core codon 28 which converts a UGG into a UAG stop codon (M2 mutant) which may also be associated with another mutation at codon 29

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C. Pichoud et al.

that converts GGC to GAC (M4 mutant) (508). Other mutations in the pre-core promoter at nucleotide position 1762 and 1764 that decrease the promoter activity and the expression of HBeAg (9) have been described in both HBeAg positive and negative patients, but are thought to be selected at the time of anti-HBe seroconversion (10,ll). The course of viral infection after the emergence of HBe-minus variants remains a matter of controversy. Some studies showed the progression of chronic hepatitis and resistance of the mutated strains to interferon alpha therapy (12-15), while the results of other studies suggest a link with HBeAg clearance and remission of liver disease (16,17).

New hope in the treatment of chronic hepatitis B has been provided with the development of new antivirals which have shown a very potent antiviral effect in clinical trials. However, the kinetics of viral replication and the lack of anti-HBe response in the majority of the patients imply a prolonged administration of antivirals to eradicate or control viral infection (18,19). Such long-term antiviral treatment with a single nucleoside analog has been associated with the selection of polymerase gene-resistant mutants that are resistant to the polymerase inhibitor in 23% of the patients after 12 months of therapy and in 38% after 24 months (18). La~vudine-~sistant mutants harbor mutations within the very conserved C domain in the YMDD motif of the reverse transcriptase (M552V or M5521) that con- fer a decreased sensitivity to therapy (20). These are very frequently associated with mutations in the B domain of the reverse transcriptase L528M (21). Fam- ciclovir-resistant strains mainly harbor mutations in the B domain of the reverse transcriptase which may be cross resistant with lamivudine (22). The selection of such mutants is often associated with viral persistence and the progression of liver disease (23,24). In a group of patients who have the capacity to mount a vigorous antiviral immune response, anti-HBe seroconversion may be favored by antiviral therapy. This anti- HBV immune response may help to withdraw antiviral therapy prior to the emergence of viral polymerase gene-resistant mutants.

We therefore decided to study the molecular patterns of anti-HBe seroconversion in 18 consecutive patients who seroconverted during lamivudine, famciclovir, or interferon therapy. We focused our attention on the determination of viremia levels with sensitive methods and on the determination of pre-core and polymerase genes sequences to survey the selection of pre-core and/or polymerase gene mutants that may escape both the anti-HBe immune response and the antiviral pressure of drug therapy. Interestingly, the results of our investigation revealed new features of anti-HBe

sero~onversion in patients treated with nucleoside analogs. Indeed, pre-core promoter mutants were found to persist at low levels despite this combined antiviral pressure which may favor the emergence of polymerase mutants that are resistant to antiviral agents.

Patients and Methods Patients Eighteen consecutive patients who were followed in our liver depart- ment and seroconverted to anti-HBe were studied. All patients were positive for HBeAg in serum at the beginning of the study and suffer- ed from liver biopsy-proven chronic hepatitis B (12 with chronic active hepatitis, and six with active cirrhosis). The mode of transmission was sexual in two, professional in two, IV drug addiction in one, in- trafamilial or vertical in five, nosocomial or transfusion in three, and unknown in five. Patients were followed monthly for serum ALT levels (expressed in comparison with the upper limit of normal values (ULN)), and bi-monthly for HBeAg, anti-HBe, and HBV DNA in serum. Anti-HBe seroconversion was demonstrated by virological monitoring of the patients during follow-up. Among these 18 seroconverters, six patients received famciclovir in phase II and phase III clinical trials, five received lamivudine for compassionate use, five received interferon alpha, one patient received interferon alpha followed by Intone, and,one patient received no treatment and spontaneously seroconverted to anti-HBe. Serum samples were prospect- ively stored at -20°C. Samples were then retrospectively selected for further virological studies at relevant time points, i.e., prior to therapy, at the time of anti-HBe seroconversion, after seroconversion and in some patients at the time of reversion to HBeAg. A total of 79 samples (mean of 4.4 samples per patient; range: 3 to 7 per patient) were submitted to detailed virological analysis including the quantitative determination of serum viral DNA and pre-SlAg, and the detection of pre-core mutants. Analysis of the viral polymerase gene sequence was performed on selected samples.

Viral markers in serum and assessment of response to therapy Viral markers including HBsAg, HBeAg and anti-HBe were determined routinely in the serum of the patients by standard procedure (Monolisa, Pasteur, France and EBK, Sorin, Italy, respectively). Quantitative determination of viral DNA in serum was performed by the branched DNA assay (Quantiplex, Chiron, USA) according to manufacturer’s instructions. The limit of detection of this assay is 0.7 106 viral genomes equivalent per mL (25,26).

In parallel, in selected stored serum samples, HBV DNA levels were assessed with a quantitative PCR assay (~plicor~ HBV monitor test, Roche Diagnostics). Briefly, 50 ~1 of serum were pre- pared by p~trea~ent with pol~thylene glycol, alkaline lysis of the pelleted viral particles, and neutralization of the lysate. After adding a fixed amount of internal standard and a PCR mix, 30 cycles of PCR amplification were performed following the manufacturer’s instructions. Biotinylat~ ampiicons were captured on streptavidine- coated microwells and hybridized with specific dinitrophenyl-labeled oiigonucl~tide probes. Following incubation with alkaline phospha- tase conjugated anti-DNP antibodies and a calorimetric substrate, a kinetic of 0.D. determination together with a calibration curve allowed a quantitative determination of HBV DNA levels. The limit of detection of this PCR assay is 400 copies of viral genome per ml, and quantitation is linear up to 4.107 copies per ml (27,28).

Pm-SlAg levels in serum were assessed with a previously published radioimmunoassay using a polyclonai anti-HBsAg for capture of HBV particles and either a monoclonal antibody against pre-SlAg or against HBsAg. A semi-quantitative determination of the pre- SlAg was allowed by the calculation of the pre-SlAg/HBsAg signals as described in detail elsewhere (29,30).

Analysis of HBV genome sequences Isolation of viral DNA for sequence analysis was performed with standard procedures. Two hundred microliter serum samples were in-

308

New patterns of anti-HBe seroncanversion

cubated for 4 h at 37°C in a buffer (50 mM Tris, 25 mM NaCI, 2.5 mM EDTA, 0.5% SDS) contai~ng 0.8 mglml Proteinase K, followed by phenol-chloroform extraction and precipitation with ethanol. The pellet was resuspended in 20 ~1 Hz0 and used for PCR amplification.

Defection of pre-core mutants and determination of viral genotypes In order to detect the variability of the viral genome at codon 28 and 29 of the pre-core region, a research line probe assay (LiPA) was designed with specific probes covering the following motifs: (i) 28W- 29G (wild type); (ii) 28X-29G (mutant M2); (iii) 28W-29D (mutant M4); and (iv) 28X-29D (double mutant M2-M4). In addition, two specific probes for the pre-core promoter region covering the important variability at nucleotides 1762 and 1764 were also designed. Probes were applied on a nitrocellulose membrane as described (31). HBV pre-core region was amplified from a 10 pl aliquot of serum DNA extract, with biotinylated primers HBPr8: S-GAA GGA AAG AAG TCA GAA GGC-3’ (antisense) and HBPr69: 5’-ACA TAA GAG GAC TCT TGG AC-3’ (sense). Primer pair HBPr70: 5’- TAC TTC AAA GAC TGT GTG TTT A-3’ (sense), and HBPr7: S-CTC CAC AGT AGC TCC AAA TTC-3’ (antisense) was used in a set- ound round of amplification for nested PCR when required. LiPA test performance was identical as described for INNO-LiPA HCV II (31). Viral genotypes were also determined by LiPA after amplification of the S gene and hybridization of the PCR products on 18 specific probes allowing the determination of the 6 HBV genotypes {A to F) as described in detail elsewhere (32). In nine patients, genotype determination was confirmed by sequence analysis of the viral polymerase gene.

Determination of viral polymerase gene sequence The DNA sequence of the polymerase gene from selected patients, who received either famciclovir or lamivudine therapy and remained positive for serum HBV DNA after anti-HBe seroconversion by the quantitative PCR assay, was determined directly from PCR products. HBV polymerase sequence from a.a. 490 to 631 (nucleotide position 596 to 1016), which includes domains B and C of the viral reverse transcriptase, was analyzed as described elsewhere (33). One tenth of extracted DNA was amplified in a 50 ~1 reaction containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgClz, 0.2 mM of each dNTP 1 unit of Taq polymerase (Perkin Elmer), and 1 pM of each primer (gl: 5’- CCT GCT GGT GGC TCC AGT TC-3’ at nucleotide oosition 55- 76, and primer pR5: 5’-GGT TGC GTC AGC AAA dAC TTG-3’ at position 1197-l 178, were used for amplification of the conserved domains A to E of the viral polymerase gene. A nested PCR was performed on some samples, that did not give a successful amplification, using primers p4 5’-CTC ACA ATA CCC CAG AGT CTA GAC T-3’ at nucleotide position 23&254 and pR2: 5’-AAA GCC CAA AAG ACC CAC AAT’-3’ at position 1017-997. For each round of PCR, 30 cycles of amplification were performed with denaturation at 95°C for 1 min, annealing at 5O’C for 1 min, and extension at 72’C for 2 min. Viral DNA sequence analysis was performed directly on amplification products using primer p4 and automated sequence ana- lyzer.

Results Basefine characteristics of the seroconverters Virological findings at baseline are described in detail in Table 1. ALT levels were elevated in all cases (mean ALT levels: 4.76xULN; range: 1.429.8xULN) at baseline. HBeAg was positive in all patients while anti- HBe could be detected together with HBeAg in two. The pre-SlAg/HBsAg ratio was found to be higher than 7% in all but one patient who harbored a low level viremia at baseline (case #14). The mean serum viral DNA levels were 1071.28X lo6 equivalent ge-

names/ml (range: CO.7 to 5000X106 equivalent ge- name/ml) with the branched DNA assay Viral DNA levels ranged from 20844 to >40X106 copies/ml with the quantitative PCR assay. Distribution of viral genotypes was comparable to that of a large cohort of HBeAg positive patients followed in our liver depart- ment (unpublished results). Genotype A was more prevalent (nine patients), followed by genotype D (six patients), genotype C (two patients) and genotype B (one patient). The geographical origin of the patients was Asia in four (infected with genotype B in one, genotype C in two and genotype D in one), Mediter- anean Basin in one (genotype D) and France in 13 (genotype D in four and genotype A in nine). Determi- nation of pre-core mutant showed the presence of a pre-core promoter mutant (either pure or as a mixture) in nine patients, and of pre-core M2 mutant in two. The seven other patients were infected with “pure” wild-type sequences in the pre-core region.

Immure-mediated anti-HBe seroconversion Six seroconverters received interferon alpha therapy at a dose of 5 megaunits thrice weekly for 6 months (five patients) or 18 months (one patient); one patient seroconverted spontaneously. Anti-HBe seroconversion occurred after 2 (one patient), 4 (two patients), and 6 (one patient) months of interferon therapy while in two patients it occurred 4 and 12 months after cessation of therapy (Table 2 and Fig. 1). Anti-HBe seroconversion was associated with a return of ALT to normal values in all seven cases. A drop in viral DNA titers was observed in all cases, viral DNA became undetectable by the bDNA assay in each case, and decreased dramati- cally in the quantitative PCR assay by 2 to 5 logs. However, in all cases serum viral DNA remained positive at relatively low titers ranging from 829 to 257 143 copies/ml in the PCR assay. In four patients initially infected with a pre-core promoter mutant, a progressive replacement of mutant virus populations by wild- type viruses occurred after anti-HBe seroconversion in 2 (cases #4 and 6) (Fig. 1), while in the two others mutant virus persisted after seroconversion (cases #5 and 7) (Table 2).

In patient #5, interferon therapy was followed by a maintenance therapy with lamivudine. Seven months after anti-HBe seroconversion, while the patient was still under lamivudine maintenance therapy, low levels of viremia were still detectable by the quantitative PCR assay. Analysis of the pre-core region showed a mixture of pre-core promoter mutant and wild-type genomes. Viral polymerase gene sequence by direct sequencing of PCR products, after seroconversion while the patient was still under lamivudine treatment, showed 3

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C. Pichoud et al.

TABLE 1

Baseline characteristics of the patients

Patient Treatment ALT HBeAglHBeAb Pre-SlIHBs bDNAa PCRa Genotype Promoter cd28-29 ratio

#I

#2 #3 #4 #5 #6 #I

#8 #9 #lO #I1 #12

#13 #14 #15

#16 #17 #18

NO 2.3 +I-

IFN 2.6 i-l- IFN 2.9 +I- IFN 2.0 -+-I-+ IFN-+LAM 29.8 +I- IFN 3.9 +I- IFN 1.7 +I-

LAM 2.4 +I- LAM 1.4 +I- LAM 3.6 +I+ LAM 5.2 +I- LAM 2.6 +I-

FAM 2.3 +I- FAM 2.3 +I- FAM 7.1 +I-

FAM 1.7 +I- FAM 2.4 +I- FAM 9.6 +I-

44% 22.8

58% 833.1 30% 19.4 ND 1.1 11% 3459 18% 1767.0 ND 1040.0

6 384 336 D

>40000000 A 1038655 C

237911 C 39272 851 D

>40000000 A >40000000 B

INDb WT+M2

WT WT MT MT MT MT

WT w-r w WT w-r WT

9% 313.7 4820860 D WT WT 29% 22.0 3 597948 A w-r WT

1% 119.0 >40000000 D WT WT+MZ 17% 5000 >40000000 D MT WT 23% 46.0 33 904460 A MT WT

28% 4960.0 3% co.7

28% 1188.0

26 796478 A 20844 D

>40 000 000 A

1225 198 A 337637 A

>40000000 A

WT+MT WT+MT WT

15% 15% 33%

co.1 co.7 490.0

WT WT MT

w w-r WT

WT wl- WI-

a Results of HBV DNA detection by the bDNA assay are expressed in lo6 equivalent genome per ml and those obtained with the quantitative PCR assay are expressed in genome copies per ml. b IND indicates an indeterminate reaction (no hybridization) on probes for the promoter region. Mutations in the pre-core region or promoter are indicated in italics.

TABLE 2

Characteristics of the patients at the end of follow-up

Patient Treat- Time after ALT HBeAg/ Pre-Sll bDNA” PCRa Promoter cd28-29 Polymerase ment seroconversion HBeAb HBs ratio

#l

#2 #3 #4 #5

#6 #I

#8 #9 #lO #ll #12 #12

#13 #I4 #15

#16 #17 #18

NO 52 MO

IFN 12 MO IFN 19 MO IFN 15 MO IFN 26 MO -+LAM IFN 3 MO IFN 15 MO

LAM 10 MO LAM 5Mo LAM 12 MO LAM 13 MO LAM 5 MO LAM reversion

FAM 13 MO FAM 7Mo FAM 5Mo

FAM 10 MO FAM 9Mo FAM 6Mo

1.0 -1-k 12% co.7

1.0 -I+ 13% co.7 1.0 -I+ 8% <0.7 1.0 -/+ ND co.7 1.0 -1-k 3% <0.7

1.0 1.0

1.0 1.0 1.0 1.0 1 2.54

1.0 1.0 1.0

1.0 1.0 1.0

-1-k 7% +I+ ND

-Ii- 5% -I+ 0% -1-k 0% -I+ 21% -I+ 30% +-I- 18%

-1-k 18% -I+ 0% -I+ 11%

-1-k 7% -1-t 4% -I-+ 18%

co.7 17 568 WT WT

523.2 13272607 MT WT

co.7 co.7 co.7 co.7 co.7

8.6

CO.7 co.7 co.7

<0.7 CO.1 co.7

6523 WT+MT WT wild type <400 WI-+MT WT ND 2471 WI- WT+M2 wild type

888 WT+MT WT wild type 412 W’C+MT WT ND

4 192 671 MT WT L528MIM552 V

48 231 W+MT WT wild type <4Oo w WT wild type

922 WF+MT WT+MZ wild type

7899 w-r WT+M4 R471K 1694 WT+MT WT wild type

<400 w-r WT ND

<400 WT WT+M2

194 199 WT WT 8385 WT WT

829 WT WT 894 W-I-+-MT w-r S441CN483Dl

S565A

a Results of HBV DNA detection by the bDNA assay are expressed in lo6 equivalent genome per ml and those obtained with the quantitative PCR assay are expressed in genome copies per ml. ND: not determined. Mutations in the pre-core region or promoter as well as in the polymerase gene are indicated in italics.

amino acid changes S441C, Y4834 S565A by com- rus and stable seroconversion, no selection of pre-core

parison with the consensus sequence of genotype D. mutant was observed after anti-HBe seroconversion

In the two patients infected with pure wild-type vi- (cases #2 and 3). In the remaining patient infected with

310

a M2 mutant who spontaneously seroconverted to anti-HBe, the M2 mutant persisted after seroconversion (case #l). The semi-quantitative detection of pre- Sl Ag in the serum was performed on serial samples in five of these patients (#l, 2, 3, 5 and 6) and showed a significant decrease of pre-Sl Ag levels after anti-HBe seroconversion.

Reversion to HBeAg was observed in two cases (patients #6 and 7, see Fig. 1). Both cases were infected with a pre-core promoter mutant prior to therapy. In

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these two cases, HBV DNA was undetectable by the bDNA assay prior reversion to HBeAg, while it remained positive at relatively high levels by the quantitative PCR assay (1.4X lo5 and 2.5X lo5 copies/ml). At the time of HBeAg reactivation, viral DNA was detectable in both the bDNA and the PCR assays. In case #6 (Fig. l), this episode of reactivation was associated with an ALT flare (4XULN) and followed by a sustained anti-HBe seroconversion accompanied by a sustained normalization of ALT levels, a significant decrease of HBV DNA titers below the limit of detection of the bDNA assay and to 17 568 copies/ml in the PCR assay. This was also associated with the disappearance of the pre-core promoter mutant which was replaced by wild-type virus sequences. In case #7, there was no increase in ALT levels, and anti-HBe remained positive together with HBeAg. Persistence of high titers of serum viral DNA and pre-core promoter mutant was observed in this patient.

Anti-HBe seroconversion during lamivudine therapy Five patients received lamivudine monotherapy at a dose of 100 mg/day (cases #8 to 12) (see Table 2 and Fig. 2). Anti-HBe seroconversion occurred after 6 (two patients), 7, 8, and 10 months of therapy, respectively. ALT levels returned to normal values in all patients after anti-HBe seroconversion. Serum HBV DNA levels became lower than the limit of detection of the bDNA assay at the time of seroconversion in all cases. Analysis of viremia levels after anti-HBe seroconversion with the quantitative PCR assay showed a significant decrease of circulating HBV DNA titers, by at least 3 to 5 logs, being lower than the limit of detection in one patient, and persisting at low levels in three patients (#lo, 11 and 12) (412 to 2471 copies/ml).

Analysis of the pre-core region of HBV after anti-

Fig. 1. Interferon-mediated anti-HBe seroconversion. In case #4, interferon alpha therapy was associated with a stable anti-HBe seroconversion. In case #6, a reversion to HBeAg was observed. ALT levels are expressed in comparison with the upper limit of normal (0) and the pre-SlAg levels as a pre-SlIHBsAg ratio in percentage (m). The results qf the quantitative determination of HBV DNA levels are presented with a logarithmic scale: l bDNA assay, n PCR assay. The detection limit for both assays is also presented (bDNA: . . . . . ; PCR: *-*-a-). The left axis corresponds to pre-SlAg and ALT levels while the left axis corresponds to HBV DNA determination. The results of the determination of the pre-core mutation pattern are presented under the graphs. The arrows indicate the time of anti-HBe seroconversion and the star the time of reversion to HBeAg.

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C. Pichoud et al.

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Fig. 2. Lamivudine-associated anti-HBe seroconversion. In case #9, lamivudine therapy was associated with a stable anti-HBe seroconversion. In case #12, a reversion to HBeAg was observed while the patient was still receiving lamivudine. The legend is the same as in Fig. 1. Results of polymerase gene sequencing at the time of viral breakthrough are also indicated.

HBe seroconversion showed: (i) the co-existence of wild-type and pre-core promoter mutant viruses, suggesting a progressive replacement of mutant virus by wild-type sequence genomes (patients #ll and 12); (ii) and the slow appearance of pre-core promoter mutant virus population together with wild-type virus in the

two patients initially infected with “pure wild type virus” (cases #8 and 9). In patient #lo, wild-type promoter sequence and a mixture of wild type and M2 sequences in the pre-core region were found to be stable after anti-HBe seroconversion.

Determination of pre-Sl Ag levels in the serum of these five patients showed a significant decrease of pre- Sl Ag in cases #8, 9, and 10 after seroconversion and the persistence of stable levels in cases #l 1 and 12. The persistence of high levels of pre-SlAg was associated with the persistence of low levels viremia in case #ll and with a reversion to HBeAg in case #12.

One case of reversion to HBeAg was observed al- though lamivudine therapy was maintained (case #12, Fig. 2). In this patient reversion was observed 10 months after anti-HBe seroconversion. It was associated with the persistence of pre-core promoter mutant viruses and a double mutation in the viral polymerase with a L528M and a M552V changes. These data indicate that viral strains harboring mutations in the pre- core promoter and the viral polymerase gene can be selected in the context of anti-HBe immune response and lamivudine therapy.

The viral polymerase gene sequence was also analyzed in two other patients in whom viremia was still detectable after seroconversion (patients #lO and 11). Direct sequencing of PCR products showed a wild-type sequence in domains B and C of the viral polymerase, indicating the absence of selection of lamivudine resistant strains at the time of the study.

Anti-HBe seroconversion associated with famciclovir treatment Anti-HBe seroconversion was observed in six patients who received famciclovir therapy (Fig. 3 and Table 2). Seroconversion occurred after 4, 11 or 12 months of treatment during a 12.month administration of the antiviral in phase III clinical trials in three patients. In three other patients included in a phase II or III clinical trial, seroconversion was observed 2, 11 and 23 months after cessation of therapy.

In all three patients (cases #13 to 15) who seroconverted during therapy, ALT levels returned to normal values after anti-HBe seroconversion, while HBV DNA titers were below the limit of detection of the bDNA assay and decreased by 3 to 5 logs with the PCR assay (HBV DNA range: <400 to 48 231 copies/ ml). At the end of follow-up two patients were infected with a mixture of wild-type and pre-core promoter mutants, and a mixture of M2 and wild-type strains at pre-core codon 28 was found in patient #15. Since low- level viremia persisted despite anti-HBe seroconversion and antiviral treatment in all three patients, sequence

312


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Fig. 3. F~c~c~ovir therapy and anti-HBe seroconversion. Patient #15 seroconverted to anti-HBe during antiviral therapy. Patient #17 seroconverted after cessation of treatment. The legend is the same as in Fig. 1. Results of polymerase gene sequencing at the time of viral breakthrough are also indicated.

analysis of the viral polymerase gene was performed but did not reveal any mutation in the conserved domains of the reverse transcriptase.

Among the three patients (#16 to 18) who seroconverted 2, 11 and 23 months after cessation of therapy, no ALT flare was observed during clinical monitoring

in 2 (cases #16 and 17). HBV DNA levels were below the limit of detection of the bDNA assay post-treatment. With the PCR assay the levels of viremia were 1225 198 and 337637 copies/ml. Both patients were infected with wild-type pre-core viruses. After anti-HBe seroconversion a significant decline in HBV DNA levels by 2 to 3 logs was observed with the PCR assay. In both cases a selection of a pre-core promoter mutant (case #17) or a M4 mutant (case #16) was observed alter anti-HBe seroconversion. In case #18, high levels of viremia were observed after cessation of famciclovir therapy associated with elevated ALT (9.6xULN). Anti-HBe seroconversion was observed 2 months after cessation of therapy and accompanied by a rapid decline of viral DNA levels, by more than 5 logs, below the limit of detection of the bDNA and the PCR assays. Pre-core promoter mutants that were present prior to seroconversion were replaced by wild-type sequences after anti-HBe seroconversion. Analysis of the evolution of pre-SlAg in these six patients showed a significant decrease of the pre-Sl AglHBsAg ratio after seroconversion.

Discussion In patients with chronic hepatitis B, anti-HBe seroconversion usually reflects an immune-mediated clearance of infected hepatocytes that actively replicate HBV and is associated with the control of viral replication and a remission of liver disease. The rate of spontaneous anti-HBe seroconversion is approximately 5% each year, and is significantly increased by interferon therapy (34) as well as therapy with new nucleoside analogs such as lamivudine (18) or famciclovir (35). However, the selection of viral pre-core promoter and/or pre-core region mutants may allow the persistence of viral replication after anti-HBe seroconversion (4,6,17).

In our study of 18 consecutive seroconverters, using new quantitative methods for the detection of HBV DNA and the molecular characterization of circulating HBV genomes, we have observed new molecular features of anti-HBe seroconversion. Using a quantitative PCR assay (27,28), we could confirm that viral DNA titers not only drop after anti-HBe seroconversion by more than 2 logs but more ~portantly may persist below the detection limit of the bDNA assay at significant levels. Indeed, persistence of circulating viral DNA titers higher than 400 copies/ml was observed in 7/7 patients treated with interferon, in 4/5 treated with lamivudine, and 4/6 patients treated with famciclovir, while the bDNA assay could detect HBV genomes in only two patients after anti-HBe seroconversion. The discrepancy observed between the quantitative data in

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the bDNA and the PCR assays is due to the absence of a universal standard for the detection and quantifi- cation of HBV DNA (36). Since the linearity of the PCR assay is observed in samples containing HBV DNA levels below 100 000 copies/ml (27,28), its clinical value depends mainly on the detection of low levels of viremia (Pawlotsky et al., submitted manuscript). The clinical relevance of the detection of relatively low levels of viral replication was emphasized by the de- scription of reversion to HBeAg in two patients who received interferon therapy and one patient receiving lamivudine treatment. Interestingly, the significant decrease of viral DNA titers after seroconversion was also associated with a relative decline in pre-SlAg levels in serum in patients with sustained anti-HBe seroconversion and control of viral replication, consist- ent with our previous observations (29,30,37). This indicates that the detection of pre-SlAg after anti-HBe seroconversion may be a marker of the persistence of a pool of infected hepatocytes that still harbor viral CCC DNA. In agreement with this hypothesis was the finding that pre-Sl Ag persisted at relatively high levels in two patients who reverted to HBeAg (#6 and 12), in one patient who experienced an increase in viral DNA titers together with the persistence of anti-HBe (#13), and in one patient whose viral DNA levels remained detectable after seroconversion despite lamivudine therapy (#ll).

Another particular finding came from the characterization of viral genomes after anti-HBe seroconversion with a line probe assay for the study of the pre-core region and by sequencing of the viral polymerase gene in patients receiving nucleoside analog therapy In patients receiving interferon alpha, the persistence of a pre-core promoter mutant was associated with a reversion to HBeAg in two cases, which may suggest that these mutants may escape the anti-HBe immune response (10) by modulating the level of HBeAg expression (9) and may re-emerge with an HBeAg positive phenotype (4). In patients who seroconverted during lamivudine therapy the persistence of pre-core promoter mutants in four out of five patients may suggest that these mutants do not only escape the anti-HBe immune response but may also be recalcitrant to nucleoside analog therapy. In the three patients who seroconverted during famciclovir therapy, the same observation of the persistence of pre-core promoter mutants was made, confirming the previous hypothesis. With regard to pre-core stop codon mutants (M2), no mutant was selected after seroconversion in this small series of interferon seroconverters, while M2 mutants persisted or were selected by the anti-HBe immune response in one case of lamivudine treatment and one

case of famciclovir treatment. Another interesting finding was the possibility of anti-HBe seroconversion after cessation of famciclovir therapy which was not associated with ALT flare in two of these three patients. It remains to determine whether this phenom- enon of delayed anti-HBe seroconversion is indeed a specific feature of famciclovir treatment. Furthermore, in an additional group of 32 HBeAg positive patients who did not seroconvert to anti-HBe, no change in the pre-core sequence and pre-Sl Ag detection was observed (data not shown) (38), confirming the clinical relevance of our findings.

Reversion to HBeAg positivity was observed in three cases among this group of 18 consecutive seroconverters, which underlines the need for careful monitoring of patients, even after anti-HBe seroconversion. HBV reactivation with the loss of anti-HBe and a reversion to HBeAg has been described in patients without immune suppression (39) and has been observed in clinical trials after cessation of lamivudine therapy (40,41). In our series, this serological event was observed in one patient during interferon treatment, and in one patient after cessation of interferon. More interestingly, we described the first case of HBeAg reversion in a patient who was still undergoing lamivudine therapy. In two of these cases HBV DNA was still detectable by the quantitative PCR assay, in two of two tested cases pre- SlAg levels remained at significant levels suggesting the persistence of a pool of infected hepatocytes despite anti-HBe immune response, and in the three cases a persistence of pre-core promoter mutants was observed suggesting that these mutants can escape the anti-HBe immune response. In the case treated with lamivudine, analysis of HBV polymerase gene showed a selection of a double mutant L528M + M552V (20). These findings may indicate a progressive and sequential selection of a pre-core promoter mutant after anti- HBe seroconversion followed by the selection of polymerase mutant resistant to lamivudine.

Altogether our findings emphasize the need to monitor viral replication after anti-HBe seroconversion with more sensitive and quantitative assays. Furthermore, during nucleoside analog therapy, not only sensitive assays to measure the level of viremia but assays to detect viral mutants are required to predict the selection of viral strains that may escape both the immune response and the inhibition by antiviral therapy. These assays will be important to tailor antiviral strategies to the virological status of the patients.

Acknowledgements We are grateful to Dr Frederic Eberle (Roche Dia- gnostics, France) for providing the quantitative PCR

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assays. This work was supported by grants from IN- SERM, the “Hospices Civils de Lyon”, and Smith Kline Beecham Pharmaceuticals.

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