Surface Gene Mutants of Hepatitis B Virus in Infants Who Develop Acute or Chronic Infections Despite Immunoprophylaxis

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1 Surface Gene Mutants of Hepatitis B Virus in Infants Who Develop Acute or Chronic Infections Despite Immunoprophylaxis HONG-YUAN HSU, 1,2 MEI-HWEI CHANG, 2 YEN-HSUAN NI, 2 HO-HSIUNG LIN, 3 SHIH-MING WANG, 1 AND DING-SHINN CHEN 4 Serum hepatitis B virus (HBV) DNA from 4 infants with The appearance of mutations of hepatitis B virus (HBV) fulminant hepatitis B, 3 infants with acute self-limited hepatioccurrence of errors in its replication via the reverse tran- genome during natural infection can be attributed to the tis B, and 15 infants with chronic HBV infection were ampliscription of an RNA intermediate. 1 Various genomic variants fied by polymerase chain reaction followed by direct sequencinvolving the precore or core region of HBV have been deing of the region of HBV genome encoding the major antigenic epitopes of hepatitis B surface antigen (HBsAg). All infants scribed among patients with fulminant, acute self-limited, as were born to carrier mothers and administered immunopro- well as chronic hepatitis B. 2-4 Hepatitis B surface antigen phylaxis from birth. Serum HBV DNA from 13 carrier children (HBsAg)-specific antibody (anti-hbs) is believed to provide born to carrier mothers who did not receive immunoprophytations in the surface (S) gene that result in amino acid protective immunity and aid in clearance of HBV. Point mu- laxis and had comparable length of infection were studied as controls. An S mutant (residue 126, Thr to Ala) initially found substitutions in the common a antigenic determinant, in an infant with fulminant hepatitis was replaced by another which lies between amino acids 124 and 147, can alter B- S mutant (residue 145, Gly to Arg) 4 days later. In a girl with cell epitopes of HBsAg, leading to immunological escape from chronic hepatitis B, Ala-126 variant and Arg-145 variant were the host immunity elicited by either vaccination or previous infection. 5 For instance, HBV escape mutants with a glycine found at 17 and 25 months of age, respectively. The Arg-145 (Gly) to arginine (Arg) substitution at amino acid residue variant persisted for 8 years in an asymptomatic male carrier 145 with a loss in the group specific antigen determinant and for 1 year in an infant with chronic hepatitis B. The Alawere reported to occur in vaccinated Italian infants born to 126 variant persisted for 11 years in one child who had an HBsAg-positive mothers and in liver transplant recipients early loss of hepatitis B e antigen. In the majority of the treated with monoclonal anti-hbs. 6,7 Investigators from Jainfants mothers, corresponding mutations in HBsAg were not pan, Singapore, and Gambia have described similar vaccine detected in serum by direct sequencing. The S mutants deescape mutants with nucleotide change resulting in amino tected in three carrier infants were not found in their mothers acid substitution elsewhere in the a determinant The serum after cloning and sequencing of 10 DNA clones from overall frequency of these mutations is unknown, and their each maternal sample. None of the 13 control patients had biological significance remains speculative. detectable S mutants. These results suggest that S variants In Taiwan, a mass immunization program against hepatitis emerge or are selected under the immune pressure generated B was launched in Studies show that more than 90% by the host or by administration of hepatitis B immune globuof infants born to carrier mothers have been protected against lin and hepatitis B vaccination. An S mutant (residue 129, hepatitis B. The small percentage of babies who still became Gln to Arg) found in one mother-infant pair suggested a direct infected with HBV despite immunoprophylaxis developed maternal-infant transmission, resulting in immunoprophyeither a chronic carrier state, acute self-limited symptomatic laxis failure. None of the family members of children infected hepatitis, or rarely fulminant hepatitis B. 11 The reasons for with Arg-145 variant had the same variant infection, implying a lack of protection after immunization are unclear. One this variant s low transmissability. (HEPATOLOGY 1997; possibility is that the infection was already established in the 26: ) liver at the time of immunization as the result of in utero infection. Alternatively, the emergence of neutralization-resistant variants of HBV might have occurred because of immune selection pressure on the prevailing phenotype of HBV Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; anti-hbs, variants caused by widespread vaccination. 6 Naturally ocantibody to HBsAg; HBeAg, hepatitis B e antigen; anti-hbe, antibody to HBeAg; HBIG, curring escape mutants have also been reported in chronic hepatitis B immune globulin; PCR, polymerase chain reaction; RPHA, reversed passive hemagglutination test; ALT, alanine aminotransferase. carriers who became seropositive for anti-hbs in the presence From the Departments of 1 Emergency Medicine; 2 Pediatrics; and 3 Obstetrics and of HBsAg after long-term follow-up. 12 Furthermore, a recent Gynecology; and 4 Hepatitis Research Center, College of Medicine, National Taiwan study showed that vaccine escape mutants are infectious and University, Taipei, Taiwan. can cause hepatic injury. 13 Thus, immunized Taiwanese in- Received August 27, 1996; accepted March 31, Supported by grant NSC B MH from the National Science Council fants who develop acute or chronic HBV infection may have of the Republic of China. acquired these variants from their mothers, who had carried Address reprint requests to: Mei-Hwei Chang, M.D., Department of Pediatrics, HBV for at least one to two decades. These mothers could National Taiwan University Hospital, No.7, Chung-Shan South Road, Taipei, Taiwan. be infected with an escape mutant through other sources or Fax: Copyright 1997 by the American Association for the Study of Liver Diseases. through natural long-term evolution. The current study was /97/ $3.00/0 designed to assess the relationship between HBV vaccine es- 786

2 HEPATOLOGY Vol. 26, No. 3, 1997 HSU ET AL. 787 PATIENTS AND METHODS cape mutants and the clinical and virological outcome of HBV infections that occur in newborns despite immunopro- phylaxis. uct was treated with chloroform, precipitated by ethanol in the presence of ammonium acetate, dried, redissolved in 50 ml of water, and subjected to sequencing by the dideoxy chain termination method using [a-p 33 ]datp (AmpliCycle sequencing kit; Perkin Elmer Corp., Branchburg, NJ). Appropriate negative controls and positive controls using DNA from HBeAg-positive carriers were included in each reaction mixture. In addition, all negative controls from the first round of amplification were included in the second amplification step. The S gene nucleotide sequence and amino acid sequences of HBV derived from all the subjects and their mothers were compared with a published sequence of adw subtype (the most prevalent strain in Taiwan) 15 and with a wild strain from a HBeAg- positive carrier child who was followed up long-term. Cloning and Sequencing of S Gene of HBV DNA From Maternal Sera. To evaluate whether the S variants found in immunized infants were present as quasispecies in the maternal serum, we performed clon- ing and sequencing of HBV DNA from the mothers of three carrier infants (patients 9, 10, and 11) using the Original TA Cloning Kit (Invitrogen Corp., San Diego, CA). Briefly, amplified products by nested PCR were cloned into a pcr 2.1 vector by T4 DNA ligase. The ligated vector was transformed into One Shot competent cells, and these were plated onto Luria-Bertani (LB) agar plates containing ampicillin. Single colonies were picked and grown in SOC medium with ampicillin for 16 hours. Minipreparations for DNA were done and digested to test for the correct insert size. Finally, 10 individual positive clones were sequenced for each sample by the dideoxy chain termination method using [a-p 33 ]datp. Subjects. Four infants with fulminant hepatitis B and three with acute self-limited hepatitis B who presented with symptoms of acute hepatitis between 1990 and 1995 were studied. All seven were born to hepatitis B e antigen (HBeAg)-negative carrier mothers, and plasma-derived hepatitis B vaccine had been administered intramuscularly three times at 0, 1, and 2 months of age. All mothers were asymptomatic throughout pregnancy, as well as after delivery. Sera were taken from the patients and their carrier mothers at presenta- tion or at delivery. After being assayed for HBV markers, the sera were stored frozen at 70 C until they were analyzed. The other 15 infants (cases 8-22) were born to HBeAg-positive HBsAg carrier mothers. Mothers attending routine prenatal clinics in the Department of Obstetrics and Gynecology were routinely tested for serum HBsAg, typically during the first visit in the first trimester. Mothers who were found to be seropositive for HBsAg were further tested for HBeAg and antibody to HBeAg (anti-hbe). HBsAg was screened in the serum of newborns of HBsAg carrier mothers delivered in our hospital during the period between 1983 and For the chronic carrier infants, the available first (at birth, infancy, or early childhood) and final serum samples during follow-up as well as maternal serum (obtained at delivery or later time) were examined for HBV surface mutants. Serum HBV markers were measured by radioimmunoassay (Abbott Laboratories, North RESULTS Chicago, IL). Clinical Features. Table 1 shows the clinical features of the Thirteen HBsAg carrier children (9 male and 4 female) who were identified during our previous seroepidemiologic survey in 1984, 14 seven infants with acute HBV infection (patients 1-7). All before mass hepatitis B vaccination in Taiwan, were selected as a four infants with fulminant hepatitis died, and all three with control group. They were selected because 1) they were not vaccianti-hbs. Sera from 14 infants (patients 9-22) were obtained acute, self-limited disease survived with seroconversion to nated and did not receive hepatitis B immune globulin (HBIG); 2) they were born to HBeAg-positive HBsAg carrier mothers, sug- at birth, before immunization, and tested for HBsAg by regesting a perinatal transmission of HBV from mother to infant; and versed passive hemagglutination test (RPHA); all 14 were 3) the tested sera from these 13 children were obtained at a mean positive for HBsAg. Regardless of the HBsAg seropositivity, age (63.0 { 39.4 months; range, months) comparable to the these newborns received HBIG at birth and vaccination at 0, mean final follow-up age of the 15 carrier children who received 1, 2, and 12 months of age and were followed up at a 6-month hepatitis B vaccine and HBIG (67.6 { 40.5 months; range, intervals. Table 2 lists their serological and biochemical data months), thereby minimizing the influence of different durations of infection. 12 on follow-up. Three infants had evidence of chronic hepatitis Polymerase Chain Reaction Amplification of HBV DNA and Direct Se- with fluctuating levels of alanine transferase (patients 8-10). quencing of S Region. The region encoding the a determinant and In follow-up, two of these children seroconverted from flanking sequence (nucleotide position 425 to position 840) of S HBeAg to anti-hbe and had improvements in ALT levels. gene was amplified by nested polymerase chain reaction (PCR). The remaining 12 children had normal aminotransferase lev- Briefly, HBV DNA extracted from 20 ml of serum by phenol-chloro- els. Of the 13 tested sera from controls who did not receive form was amplified in a 50-mL reaction mixture containing 10 immunoprophylaxis and who all developed chronic HBV inmmol/l Tris-HCl (ph 8.8), 50 mmol/l KCl, 1.5 mmol/l MgCl 2, fection, 11 were HBeAg-positive and two were negative for mmol/l each of deoxynucleotide triphosphate, 0.1 mg of each HBeAg but positive for anti-hbe. The ALT levels ranged beprimer, and 2 U of Taq DNA polymerase (DyNAZyme II; Finntween 3 and 68 IU/L, with a mean of 17.3 { 17.8 IU/L. zymes Oy, Finland) and overlaid with 40 ml of mineral oil. The first amplification step was performed for 30 cycles with the outer Direct Sequencing of S Gene of HBV DNA in Sera From Patients, primers A1: 5 -ATCCGC,TGGAT,GTGTC,TGCGG-3 and A2: 5 of sequencing the S gene of HBV DNA from each subject. Their Mothers, and Controls. Tables 1 and 2 shows the results GGCAA,CGGGG,TAAAG,GTTCA-3 (position 369 to 388 and position , respectively) in the HBV genome. Of the first Surface mutants were found in one of seven infants with amplification product, 5 ml was then used as the template in a acute hepatitis and one of their seven mothers. Patient 4 was second amplification step performed for 30 cycles with inner primer a 2-month-old child with fulminant hepatitis who initially pairs B1: 5 -TTAGG,GTTTA,AATGT,ATACC,C-3 (position 822- had a S mutant with amino acid substitution (threonine to 842 and B2: 5 -CATCT,TCTTG,TTGGT,TCTTC,TG-3 (position alanine) at residue 126 (named Ala-126 variant) which was ). Thermal cycles were performed using a DNA Thermal replaced by another S mutant with a conversion from glycine Cycler 480 (Perkin-Elmer Cetus, Norwalk, CT) with denaturing at to arginine at amino acid position 145 (named Arg-145 vari- 94 C for 1 minute, annealing at 55 C for 1 minute, and extension at 72 C for 2 minutes. Next, a final denaturation step was undertaken for 7 minutes at 94 C and a final extension step for 7 minutes at 72 C. Ten microliters of nested PCR product (415 bp) was ana- lyzed by electrophoresis on a 2% agarose gel; the expected band was visualized after ethidium-bromide staining. An amplified prod- ant) 4 days later. In five (patients 8, 9, 10, 11, and 14) of 15 carrier children who received immunoprophylaxis, S mutants were detected in the first available serum samples obtained at different times after birth (Table 2). Among them, the Ala-126 variant

3 788 HSU ET AL. HEPATOLOGY September 1997 TABLE 1. Clinical, Serological, and Virological Features in Mothers and Their Infants Who Developed Fulminant or Acute Self-Limited Hepatitis B Despite Immunization Patient s Viral Strain Maternal Viral Strain Age ALT* HBsAg/ HBeAg/ Point Mutation Amino Acid Conversion Point Mutation Amino Acid Conversion Patient Sex (mo) Diagnosis (IU/L) Anti-HBs Anti-HBe (Nucleotide Position) (Amino Acid Position) (Nucleotide Position) (Amino Acid Position) 1 F 3 Acute 196 //0 0// Wild type Wild type 2 F 4 Acute 329 //0 0// Wild type ArG(530) ThrrAla 3 F 2.5 Acute 1,071 //0 0// Wild type Wild type 4 M 2 Fulminant 122 0/0 0// ArG(530) ThrrAla(126) Wild type 11 0/0 0// GrA(587) GlyrArg(145) 5 M 3 Fulminant 83 0/0 0// Wild type Wild type 6 F 3 Fulminant 1,842 0/0 0// Wild type Wild type 7 M 4 Fulminant 916 //0 0// Wild type Wild type NOTE. All patients had been given HBIG at birth and were immunized with three doses of hepatitis B vaccine at 0, 1, and 2 months of age. All mothers were seropositive for HBsAg and anti-hbe but seronegative for HBeAg. * Normal, õ30 IU/L. Serum obtained 4 days apart from first serum. DISCUSSION In this study, 6 of 22 infants (27%) who developed acute or chronic HBV infections despite immunoprophylaxis were infected by HBV with amino acid changes within the a determinant. The corresponding mutations were not detected in four of the six infants mothers by direct sequencing. The emergence and takeover by different S mutant strains at dif- ferent times during the course of evolution were found in some infants who were studied. These results indicate that most infants who develop HBV infection from maternal-infant transmission despite standard immunoprophylaxis be- come infected with the wild type strain from their mothers, at least wild type in regard to the HBsAg gene. There was some evidence that the S mutants were more pathogenic than wild type. The Ala-126 variant was found in one infant with fulminant hepatitis, an infant with chronic hepatitis, and an infant with normal ALT levels with early e seroconversion. One of the two carriers infected with Arg- 145 variant and the only one infected with Arg-129 variant also showed ALT elevations. However, these variants were absent in most of those who were chronically infected but had normal aminotransferase levels during follow-up. The possibility that these HBsAg mutants play some role in the induction of hepatitis activity needs to be evaluated further. The finding that S mutants were present in one of seven infants with acute hepatitis B and one of their seven mothers implies that the appearance of S mutants may have occurred by chance unrelated to immunoprophylaxis. However, 5 of 15 carrier infants who received immunoprophylaxis had S mutants, and none of 13 control carrier children without immunoprophylaxis had such mutants. Because the duration of infection in the two groups of patients was similar, the emergence of S mutants in the indexed carrier children might have been induced by the immunoprophylaxis. Nevertheless, we cannot completely exclude the possibility that children who harbored a variant had been infected from sources other than their mothers. A lack of significant difference in the prevalence of S mutants among our 15 indexed carrier infants (5 of 15) and their mothers (2 of 13) does not support a causal relationship between the appearance of S mutants and emerged in two (patients 10 and 14) and the Arg-145 variant emerged in two (as a single strain in patient 11 and as a mixture in patient 9). The remaining child (patient 8) had a unique variant with a change of amino acid from glutamine to arginine at residue 129 (named Arg-129 variant). A shift of predominant viral population during the course of chronic HBV infection was found in two cases. In patient 9, a viral mixture with predominance of the Arg-145 variant accompanied by hepatitis activity persisted until 17 months of age, when he became seronegative for HBsAg and only the Arg-145 variant was detectable. These findings suggested that the amount of HBsAg produced was too low to be detected or that the mutant influenced the antigenicity and detectability of HBsAg in the commercial immunoassay. Serum from patient 10 contained the Ala-126 variant at 17 months of age but contained the Arg-145 variant at a later time; the latest serum was free of virus (Figs. 1 and 2 and Table 2). Three of 20 mothers (15%) examined had S mutants. Although the mother of patient 2 harbored Ala-126 variant, no S mutants could be detected in the infant. The mothers of patients 8 and 14 had the same S mutants as their infants. Notably, the mother of patient 8 had Arg-129 variant, not only at the time that the infant presented with HBsAg positivity but also during her pregnancy with this child (Fig. 1 and Table 2). None of the 13 control carrier children had HBV DNA mutations in the sequenced region within the S gene. The prevalence of S mutants among the 15 indexed carrier infants with immunoprophylaxis was significantly different from that of 13 controls (5 of 15 vs. 0 of 13; P Å.03, Fisher s exact test). Cloning and Sequencing of Maternal Samples. In the three maternal sera (mothers of patients 9, 10, and 11) examined, no surface mutant was identified in 10 individual clones from each maternal sample, suggesting that the S mutants increased de novo and were not present as a minor quasispecies in the mother. Transmission of Arginine-145 Variant Among Family Members. To assess the transmissability of the arginine-145 variant, serum from family members of two infants with this variant were studied. The elder sister of patient 10 was an HBsAg carrier and harbored the wild type strain, whereas her father was not a carrier. The father of patient 11 was an HBsAg carrier infected with wild strain, and his brother was not a carrier.

4 HEPATOLOGY Vol. 26, No. 3, 1997 HSU ET AL. 789 TABLE 2. Clinical, Serological, and Virological Features in Mothers and Infants Who Developed Chronic HBV Infection Despite Immunoprophylaxis Patient s Viral Strain Maternal Viral Strain ALT HBsAg/ HBeAg/ Point Mutation Amino Acid Conversion Point Mutation Amino Acid Conversion Patient Sex Age (IU/L) Anti-HBs Anti-HBe (Nucleotide Position) (Amino Acid Position) (Nucleotide Position) (Amino Acid Position) 8 M 1yr,6mo 53 //0 //0 ArG (540) GlnrArg(129) ArG(540)* GlnrArg(129)* 2yr 17 //0 //0 ArG(540) GlnrArg(129) 9 M 6.5 mo 134 //0 //0 GrA(587) GlyrArg(145) Wild type 9.5 mo 50 //0 /// GrA(587) GlyrArg(145) 1yr 97 //0 /// GrA(587) GlyrArg(145) 1yr,5mo 57 0/0 //0 GrA(587) GlyrArg(145) 10 F 1 yr, 5 mo 102 /// //0 ArG(530) ThrrAla(126) Wild type 2yr,1mo ND ND 0// GrA(587) GlyrArg(145) 3yr,6mo 56 /// 0// GrA(587) GlyrArg(145) 3yr,8mo 68 /// 0// GrA(587) GlyrArg(145) 7yr,8mo 13 0// 0// PCR(0) 11 M 2 yr 7 //0 //0 GrA(587) GlyrArg(145) Wild type 8yr 23 //0 //0 GrA(587) GlyrArg(145) 12 M 1 yr, //0 //0 Wild type Wild type mo 7 yr, //0 //0 Wild type mo 13 F 2 yr, 8 mo 9 //0 //0 Wild type Wild type 11 yr 13 //0 //0 Wild type 14 M 8 days 10 //0 //0 ArG(530) ThrrAla(126) ArG(530) ThrrAla(126) 10 yr, //0 0// ArG(530) ThrrAla(126) mo 15 M 5 mo 17 //0 //0 Wild type ND 7yr,7mo 7 //0 //0 Wild type 16 F 3 yr, 9 mo 11 //0 0// Wild type Wild type 6yr 2 //0 0// Wild type 17 F 1 yr, 9 mo ND //0 //0 Wild type Wild type 4yr,1mo 7 //0 //0 Wild type 18 M 1 yr 19 //0 //0 Wild type Wild type 7yr,9mo 10 //0 //0 Wild type 19 M 2 mo ND //0 //0 Wild type Wild type 5yr 15 //0 //0 Wild type 20 M 1 day ND //0 //0 Wild type Wild type 3yr,1mo 7 //0 //0 Wild type 21 M 1 day ND //0 //0 Wild type Wild type 10 mo 37 //0 //0 Wild type 22 F 1 day ND //0 //0 Wild type ND 1yr,6mo 10 //0 //0 Wild type NOTE. All patients received HBIG at birth and were immunized with three to four doses of HBV vaccine. All carrier mothers were seropositive for HBeAg but seronegative for anti-hbe. Abbreviation: ND, not done. * The mutant existed in maternal serum both at delivery and first trimester of pregnancy. Viral mixture with predominance of Arg-145 variant and a minority of wild type virus. immunoprophylaxis. However, such a comparison is unfair minor quasispecies in the maternal sera and was transmitted because carrier mothers in Taiwan were infected in early from mother to infant and selected in the baby. For instance, childhood and had been infected for much longer. 12 Notably, patient 10 might have been infected in utero predominantly S mutants in the carrier infants who received immunoprophylaxis with the wild strain but with a few mutants as well. The emerged early in life (between 8 days and 2 years), administration of HBIG or vaccine may have allowed for although it is not known when S mutants emerged in the the selection and gradual multiplication of the variant. The mothers. On the other hand, the Arg-129 variant was present cellular immune response to variant S might develop, resulting in maternal blood of case 8 throughout pregnancy and might in hepatitis activity. The Arg-145 variant might have have been transmitted directly from mother to baby and es- been selected because the substitution of amino acid 145 caped the immunoprophylaxis. would have more reduced binding ability to anti-hbs than The S mutants found in three carrier infants were not that of the Ala-126 variant. 8 Similarly, change of viral population found after cloning and sequencing of HBV DNA in sera from in case 4 might have been due to vigorous immune their carrier mothers, suggesting that S mutants arose de novo. response during the course of fulminant hepatitis. The appearance Because only 10 clones were sequenced from each mother, it of cellular immune responses to S antigen during remains possible that the mutant strain was present as a the incubation period of acute HBV infection and the early

5 790 HSU ET AL. HEPATOLOGY September 1997 appearance of anti-hbs humoral immune response during the course of fulminant hepatitis 16,17 may have created conditions required for selection and escape and the mutants to rapidly outgrow the wild strain. The Arg-145 variant circulated as a stable strain and retained its ability to replicate at high titer for 8 years in patient 11, consistent with previous findings. 6 Furthermore, the transmissability of Arg-145 variant did not seem to be higher than that of the wild type virus because other family members of patient 10 or patient 11 were not infected by this variant. Antigenic changes in HBsAg reactivity could be expected in the Ala-126 and Arg-129 variants because threonine is a polar amino acid, whereas alanine is a hydrophobic amino acid and arginine is a larger residue than glutamine and is charged. This issue could be resolved by cloning these variant S antigen genes into an in vitro expression system and testing these translation products by their reactivity with the currently used HBsAg test. In the initial years of the vaccination program, blood taken from the infants (patients 9-22) at birth before the administration of HBIG or vaccines was routinely tested for HBsAg to help identify in utero infection with HBV. Immunoprophylaxis for such infants appeared to be of no benefit but apparently was not harmful. The findings in this study suggest FIG. 1. Sequence gel of negative strand of S gene of HBV DNA from that use of HBIG plus subsequent vaccination in patients patients 12, 14, 8, and 10 (left to right, arrowhead indicates nucleotide change, resulting in amino acid substitution). Patient 12 (A) had a classical infected in utero (patients 9-22) might inadvertently serve to HBV sequence for this region; patient 14 (B) had S mutant with T-to-C select antibody neutralization-resistant variants. mutation at nucleotide position 530, resulting in amino acid change from In summary, examination of maternal serum and serial threonine to alanine at residue 126; patient 8 (C) had S mutant with T-to- serum samples from their infants who developed acute or C mutation at nucleotide position 540, leading to amino acid change from chronic HBV infection despite immunoprophylaxis showed glycine to arginine at residue 129; and patient 10 (D) had S mutant with C-to-T at nucleotide position 587, converting glycine to arginine at amino acid position 145. that HBV inoculum transmitted from mother to infant was generally the wild type strain. S variants with mutations within the a determinant emerged or were selected later under the immune pressure generated by the host per se FIG. 2. Serial ALT level, HBV markers, and HBV strain in sera from patient 10. M1, alanine-126 variant; M2, arginine-145 variant; ND, not done.

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