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P-13 Clinical significance of mutations in the core promoter of hepatitis B virus
540
Posters / International Hepatoiogy Communications 3 Suppl. (1995) $37-S169
P-13 CLINICAL SIGNIFICANCE OF MUTATIONS IN THE CORE PROMOTER OF HEPATITIS B VIRUS Y. Sugai, H. Nakayama Department of Internal Medicine, Iwaki Kyoritsu General Hospital, Japan Core promoter and precore region were sequenced in 3~10 hepatitis B virus(HBV) DNA clones each from individuals with chronic or acute HBV infection. A G-to-A point mutation at nucleotide (nO 1896 in the precore region for the stop codon 28 was observed only in 9 of 10 patients with chronic hepatitis B and 9 of 10 asymptomatic carders,' all of whom were positive for anti-HBe. Clones from the remaining patient and carrier had two point mutations in the core promoter, A-to-T at nt 1762 and G-to-A at nt 1764. A gradual shift from the wild-type HBV to mutants with these two mutations was observed in the asymptomatic carrier as he seroconverted from HBeAg to antiHBe. Clones from two of three patients with fulminant hepatitis B had A1896 in the precore region. Clones from the remaining one patient had T1762 and A1764 in the core promoter. Clones from 8 patients with acute self-limited hepatitis B did not have any such mutations either in the core promoter or precore region. These results indicate mutations in the core promoter, in the absence of precore mutations, would abort the synthesis and secretion of HBeAg at the transcriptional level. They would enhance the seroconversion to anti-HBe in some individuals with persistent HBV infection and induce severe hepatitis in some patients with acute HBV infection.
P-15
HEPATITIS B VIRUS VARIANTS: PROTECTIVE EFFICACY OF LICENSED VACCINES AGAINSTINFECTION WITH A SURFACE MUTANT WHICH EMERGED IN AN IMMUNIZED INFANT Norio Ogatal~,.Alessandro R. Zanetti2, RogerH. Miller3, RobertH. Purcell3 ~Third Department of Internal Medicine, Niigata University, Niigata, Japan, ZInstitutodi Virologia, Universityof Milan, Milan, Italy, 3Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. Emergence in vaccinated individuals of hepatitis B virus (HBV) variants with amino acid substitutionswithin the a determinantof the surfaceprotein has raised a possibility that such variants represent neutralization escape mutants. This possibility may pose a potential threat of spread of such mutants among vaccinated individuals. To test whether this may be the case we evaluated the protective efficacy of licensed HB vaccines against infection with the mutant virus. An HBV surface mutant (subtype ayw ), which emerged in an immunized infant in Italy, contained an argiuine substitutedfor glycine at the 145th codon position of the surface gene. We immunized four chimpanzees in groups of two with either of two licensed recombinant HB vaccines. After serum anti-HBs developed, we inoculated the four animals intravenously with a 103 -diluted serum collectedfrom the infant. We also inoculated two unvaccinated, seronegative chimpanzees with the equivalent dilution of the serum as positive controls. The four vaccinatedchimpanzeesdid not show evidence of HBV infectionor hepatitis during the observationperiod of mole than one year after the virus challenge. In contrast, the two unvaccinated chimpanzees developedclinical hepatitis B. We evaluated evidence of HBV infection in the chimpanzeesby testing HBV-related serum antigens and antibodies and by examiningserum HBV DNA with PCR. We confirmed the presence of the mutant virus in the infected chimpanzeesby direct sequencing of virus DNA amplifiedby PCR from the serum. Thus, immunization of chimpanzees with licensed recombinantHB vaccines affords protection againstinfectionwith a surface mutant of HBV. This suggests that properly immunizedindividualsare not at significantrisk of infection with such variant strainsof virus.
P-14 THE STUDY OF THE CORRELATION BETWEEN THE PATHOGENESIS AND THE DELETION MUTANT OF HBV CORE LESION F. Sugata, K. Yasuda,K Shibahara, S. lino. Institute of Medical Science, St. Mar/anna University School of Medicine Previous study showed that there were the deletion mutant of HBV core lesion with the meturecore in the serum of the patient who were chronic hepatltis(CH). But, the ~alysisof the deletion mutant inthe liver tissue and for the correlation between the onset of the mutaganesis were scarcely done. SO, we investigate the deletion mutant not only in the serum but also liver tissue in the HBV carrier. [Method] The temp~at~ DNA for the PCR was isolated from 100 ~ I of the serum or formalin-tixed, paraffin-embedded liver tissue of the pattant by the usual method.The PCR reaction wesdone by using the specitic primers that could amplify HBV core lesion. PCR product wasalectrophoreeed on the agarosegal for detecting the molecular size, and then cloned into the adequate plasmid vector for the purpose of the revealing the ratioot the deletion mutant agalnstthe mature and sequencing. [Res~tlt] The PeR date showed that in ASC patient the deletion mutant wasnot detected both in the serum and the tissue, but in the patients with the chronic livar disease the deletion mutant wasfound as high ratio both in the serum and in the tissue. The cloning date and sequencing indicated that the ratio of the deletion mutant in tissue was higher than in serum and the identical lesion was not round in each deletion mutant. Our date suggested the deletion of the HBV core lesion wasconnected with the onset of the CH and the ,3anomic change occurred in the liver earlier than in the serum.
P-16 MECHANISMS OF CLEARANCE OF HBs A N T I G E N AND FOLLOWING V I R A L PERSISTENCE IN THE PATIENTS WITH CHRONIC H E P A T I T I S B
J. Kato, K. Hcbsegc~wa, N. ToFii,T. St~iz~na K.Yc~mauchi andi~ H~yashi. Ins t . of Gastro., Tokyo Women's Medical Co] lege, Tokyo Tae fa c t that HBsAg convert into anti-HBs has been g e n e r a l l y thought to i n d i c a t e the c e s s a t i o n of viremia and l i v e r dysfunction. Recent development of PCRamplification have allowed us to de t e c t the s a b t l e amount of HBV even in a n t i - H B s p o s i t i v e serum. The c l i n i c a l significance of HBV evading c i r u l a t i u g antibody ren~ains to be solved. However, since the recent study demonstrated the development of hepatoce] l u l a r carcinoma in the p a t i e n t s w i t h chronic h e p a t i t i s B (CHB) a f t e r seroconversion from HBsAg to a n t i - H B s , importance of such a l a t e n t v i r u s should be noted. W i t h these respects, we aimed to identify the mechanisms of the clearance of HBsAg and f o l l o w i n g v i r a l p e r s i s t e n c e in the p a t i e n t s w i t h chronic hepatitisB. Studied were two CHB p a t i e n t s , both of whom showed n o r m a l i z a t i o n of sALT and h i s t o p a t h o l o g i c a l r e s o l u t i o n a f t e r the clearance of HBsAg followed by the appearance of a n t i - H B s , w h i ] e P C R a m p l i f i c a t i o n demonstrated to be p o s i t i v e for HBVDNA in a nt i -H B s phase. We s e r i a l l y sequenced envelope region of HBVDNA in two p a t i e n t s before and a f t e r seroconversion from HBsAg to anti-HBs an t i body. In one p a t i e n t , 16 amino acids d e l e t i o n in preS2 region and three missense mutations wi t hi n "a"-loop were observed before and a f t e r seroconversion, r e s p e c t i v e l y . In the other, a m u t a t i o n in preS] r e s u l t i n g in stop codon and three novel mutations in " a ' - l o o p was found jmt before and a f t e r , seroconversion, r e s p e c t i v e l y . These observations i n d i c a t e that HBV became d e f e c t i v e by mutational change when HBsAg was cleared. However, to survive in the f o l l o w i n g period, v i r a l DNA was sequentially converted to the form capable of evading host immune pressure.
Posters / International Hepatoiogy Communications 3 Suppl. (1995) $37-S169
P-13 CLINICAL SIGNIFICANCE OF MUTATIONS IN THE CORE PROMOTER OF HEPATITIS B VIRUS Y. Sugai, H. Nakayama Department of Internal Medicine, Iwaki Kyoritsu General Hospital, Japan Core promoter and precore region were sequenced in 3~10 hepatitis B virus(HBV) DNA clones each from individuals with chronic or acute HBV infection. A G-to-A point mutation at nucleotide (nO 1896 in the precore region for the stop codon 28 was observed only in 9 of 10 patients with chronic hepatitis B and 9 of 10 asymptomatic carders,' all of whom were positive for anti-HBe. Clones from the remaining patient and carrier had two point mutations in the core promoter, A-to-T at nt 1762 and G-to-A at nt 1764. A gradual shift from the wild-type HBV to mutants with these two mutations was observed in the asymptomatic carrier as he seroconverted from HBeAg to antiHBe. Clones from two of three patients with fulminant hepatitis B had A1896 in the precore region. Clones from the remaining one patient had T1762 and A1764 in the core promoter. Clones from 8 patients with acute self-limited hepatitis B did not have any such mutations either in the core promoter or precore region. These results indicate mutations in the core promoter, in the absence of precore mutations, would abort the synthesis and secretion of HBeAg at the transcriptional level. They would enhance the seroconversion to anti-HBe in some individuals with persistent HBV infection and induce severe hepatitis in some patients with acute HBV infection.
P-15
HEPATITIS B VIRUS VARIANTS: PROTECTIVE EFFICACY OF LICENSED VACCINES AGAINSTINFECTION WITH A SURFACE MUTANT WHICH EMERGED IN AN IMMUNIZED INFANT Norio Ogatal~,.Alessandro R. Zanetti2, RogerH. Miller3, RobertH. Purcell3 ~Third Department of Internal Medicine, Niigata University, Niigata, Japan, ZInstitutodi Virologia, Universityof Milan, Milan, Italy, 3Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. Emergence in vaccinated individuals of hepatitis B virus (HBV) variants with amino acid substitutionswithin the a determinantof the surfaceprotein has raised a possibility that such variants represent neutralization escape mutants. This possibility may pose a potential threat of spread of such mutants among vaccinated individuals. To test whether this may be the case we evaluated the protective efficacy of licensed HB vaccines against infection with the mutant virus. An HBV surface mutant (subtype ayw ), which emerged in an immunized infant in Italy, contained an argiuine substitutedfor glycine at the 145th codon position of the surface gene. We immunized four chimpanzees in groups of two with either of two licensed recombinant HB vaccines. After serum anti-HBs developed, we inoculated the four animals intravenously with a 103 -diluted serum collectedfrom the infant. We also inoculated two unvaccinated, seronegative chimpanzees with the equivalent dilution of the serum as positive controls. The four vaccinatedchimpanzeesdid not show evidence of HBV infectionor hepatitis during the observationperiod of mole than one year after the virus challenge. In contrast, the two unvaccinated chimpanzees developedclinical hepatitis B. We evaluated evidence of HBV infection in the chimpanzeesby testing HBV-related serum antigens and antibodies and by examiningserum HBV DNA with PCR. We confirmed the presence of the mutant virus in the infected chimpanzeesby direct sequencing of virus DNA amplifiedby PCR from the serum. Thus, immunization of chimpanzees with licensed recombinantHB vaccines affords protection againstinfectionwith a surface mutant of HBV. This suggests that properly immunizedindividualsare not at significantrisk of infection with such variant strainsof virus.
P-14 THE STUDY OF THE CORRELATION BETWEEN THE PATHOGENESIS AND THE DELETION MUTANT OF HBV CORE LESION F. Sugata, K. Yasuda,K Shibahara, S. lino. Institute of Medical Science, St. Mar/anna University School of Medicine Previous study showed that there were the deletion mutant of HBV core lesion with the meturecore in the serum of the patient who were chronic hepatltis(CH). But, the ~alysisof the deletion mutant inthe liver tissue and for the correlation between the onset of the mutaganesis were scarcely done. SO, we investigate the deletion mutant not only in the serum but also liver tissue in the HBV carrier. [Method] The temp~at~ DNA for the PCR was isolated from 100 ~ I of the serum or formalin-tixed, paraffin-embedded liver tissue of the pattant by the usual method.The PCR reaction wesdone by using the specitic primers that could amplify HBV core lesion. PCR product wasalectrophoreeed on the agarosegal for detecting the molecular size, and then cloned into the adequate plasmid vector for the purpose of the revealing the ratioot the deletion mutant agalnstthe mature and sequencing. [Res~tlt] The PeR date showed that in ASC patient the deletion mutant wasnot detected both in the serum and the tissue, but in the patients with the chronic livar disease the deletion mutant wasfound as high ratio both in the serum and in the tissue. The cloning date and sequencing indicated that the ratio of the deletion mutant in tissue was higher than in serum and the identical lesion was not round in each deletion mutant. Our date suggested the deletion of the HBV core lesion wasconnected with the onset of the CH and the ,3anomic change occurred in the liver earlier than in the serum.
P-16 MECHANISMS OF CLEARANCE OF HBs A N T I G E N AND FOLLOWING V I R A L PERSISTENCE IN THE PATIENTS WITH CHRONIC H E P A T I T I S B
J. Kato, K. Hcbsegc~wa, N. ToFii,T. St~iz~na K.Yc~mauchi andi~ H~yashi. Ins t . of Gastro., Tokyo Women's Medical Co] lege, Tokyo Tae fa c t that HBsAg convert into anti-HBs has been g e n e r a l l y thought to i n d i c a t e the c e s s a t i o n of viremia and l i v e r dysfunction. Recent development of PCRamplification have allowed us to de t e c t the s a b t l e amount of HBV even in a n t i - H B s p o s i t i v e serum. The c l i n i c a l significance of HBV evading c i r u l a t i u g antibody ren~ains to be solved. However, since the recent study demonstrated the development of hepatoce] l u l a r carcinoma in the p a t i e n t s w i t h chronic h e p a t i t i s B (CHB) a f t e r seroconversion from HBsAg to a n t i - H B s , importance of such a l a t e n t v i r u s should be noted. W i t h these respects, we aimed to identify the mechanisms of the clearance of HBsAg and f o l l o w i n g v i r a l p e r s i s t e n c e in the p a t i e n t s w i t h chronic hepatitisB. Studied were two CHB p a t i e n t s , both of whom showed n o r m a l i z a t i o n of sALT and h i s t o p a t h o l o g i c a l r e s o l u t i o n a f t e r the clearance of HBsAg followed by the appearance of a n t i - H B s , w h i ] e P C R a m p l i f i c a t i o n demonstrated to be p o s i t i v e for HBVDNA in a nt i -H B s phase. We s e r i a l l y sequenced envelope region of HBVDNA in two p a t i e n t s before and a f t e r seroconversion from HBsAg to anti-HBs an t i body. In one p a t i e n t , 16 amino acids d e l e t i o n in preS2 region and three missense mutations wi t hi n "a"-loop were observed before and a f t e r seroconversion, r e s p e c t i v e l y . In the other, a m u t a t i o n in preS] r e s u l t i n g in stop codon and three novel mutations in " a ' - l o o p was found jmt before and a f t e r , seroconversion, r e s p e c t i v e l y . These observations i n d i c a t e that HBV became d e f e c t i v e by mutational change when HBsAg was cleared. However, to survive in the f o l l o w i n g period, v i r a l DNA was sequentially converted to the form capable of evading host immune pressure.