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Acute hepatitis B in HCV chronic carriers: virological interaction on clinical course
314A
AASLD ABSTRACTS
HEPATOLOGYO c t o b e r 2 0 0 1
567
568
IN VIVO INHIBITION OF ANTI-HBCIGG P R O D U C T I O N BY HBCAGSPECIFIC C D 4 + TH1-TYPE T CELL CLONES IN A HU-PBL-NOD/SCID MICE MODEL. Tinghua Cao, Philip Meuleman, Isabefle Desombere, G h e n t University, G h e n t Belgium; Matti Saflberg, Karolinska Institute at H u d d i n g e University Hospital, H u d d i n g e Sweden; Geert G Leroux-Roels, Ghent University, G h e n t Belgium
ACUTE HEPATITIS B IN HCV CHRONIC CARRIERS: VIROLOGICAL INTERACTION ON CLINICAL COURSE. Evangelista Sagnelli MD, Division of Infectious Diseases, A O di Caserta, Caserta Italy; Nicola Coppola MD, Cecilia Marrocco PhD, Institute of Infectious Disease, Second University of Naples, Naples Italy; Vincenzo Messina MD, Domenico Di Caprio MD, A n n a Marotta PhD, Division of Infections Diseases, A O di Caserta, Caserta Italy; Mirella Onofrio MD, Carlo Seolastico MD, Pietro Filippini MD, Felice Piccinino MD, Institute of Infectious Disease, Second University of Naples, Naples Italy
Antigen-specific C D 4 + T cell responses are believed to play an i m p o r t a n t role in the control of h u m a n hepatitis B virus (HBV) infection. In the present study, HBcAg-specifie, HLA DR13*-restricted C D 4 + T h l - t y p e T cell clones were generated from the PBL of a subject w h o spontaneously cleared a n acute HBV infection. These HBcAg-specifie C D 4 + T h l - t y p e T cells secreted b o t h 1FN- 7 a n d T N F - a u p o n stimulation with HBcAg a n d were also able to lyse HBe peptide-loaded EBV-transformed lymphoblastoid target cells in vitro. To examine w h e t h e r these HLA DR13*-restricted h u m a n C D 4 + T h l T cells also display the same cytotoxic effects in vivo, we transferred PBL derived from HBV-infected donors or a HBV-naive d o n o r sharing the DR13*, together with the HBcAg-specific C D 4 + T h l - t y p e T cells a n d HBcAg directly into the spleen of Nod/LtSz-PrkdcsCla/Prkdcscid (NOD/SCID) mice that were pretreated with total b o d y irradiation (3Gy) a n d a rat anti mouse IL-2 receptor/3 chain m o n o clonal antibody (TM/31, ling/mouse given intraperitoneally). The p r o d u c t i o n of b o t h secondary anti-HBc-IgG a n d primary HBcAg-Binding IgM in hu-PBLNOD/SCID mice were drastically inhibited by HBcAg-specific C D 4 + T h l - t y p e T cells. No inhibition was observed w h e n C D 4 + T h l cells a n d d o n o r PBL did not share HLA DR13. These results suggest that HBcAg-specific C D 4 + T h l T cells m a y lyse the HBcAg-specific B cells that have taken u p HBcAg in vivo in a n antigen-specific a n d HLA DR13-restricted m a n n e r as was demonstrated in vitro. It is tempting to speculate that HBcAg-specific C D 4 + T h l - t y p e T cells can modulate HBcAg-specific antibody production, exert a regulatory role in deleting HBcAg-specific ( o r - b i n d i n g ) h u m a n B cells in vivo a n d contribute to the recovery of acute HBV infections.
Background: Chronic hepatitis due to HBV/HCV coinfection is characterized by a reciprocal inhibition of viral genomes and by a more severe clinical presentation. However, the interaction between HBV and HCV in HBV acute hepatitis occurring in chronic HCV carriers has not been so far investigated. Patients and methods: We studied 19 cases of HBV acute icteric hepatitis (Group A) occurred in HCV chronic carriers in comparison with a control group of 19 cases of HBV acute hepatitis observed in the same period in subjects with no sign of HCV infection (Group B). All patients in group A were male drug addicts, median age 29 years (range 21-36), showing circulating HBsAg, high titres oflgM anti-HBc and anti-HCV. In group B only 7 patients were drug addicts, 13 were males and 6 females, median age 26 years (range 21-40). All patients were hospitahsed in our ward fromjanuary 2000 to april 2001; no patient had received HBV vaccine. As a second control group for patients in group A, 19 drug addicts with chronic HCV infection and no sign of HBV infection were selected (pair-matched by age, +/- 5 years, and sex) among those observed in the same period in the same ward (Group C). Patients in different groups were followed-up from 5 14 months. Samples of plasma (59 from patients in group A and 65 from those in group B) obtained at the time of first observation and during the foUow-up were stored frozen at 80°C. HBV-DNAwas performed by a quantitative PCR in all samples from groups A and B. HCV-RNA was determined by qualitative PCR on samples from groups A and C. Results. By comparing the 3 groups we observed: A) similar patterns of HBV replication with HBV-DNA become negative in 14-24 days in group A and B; B) marked inhibition of HBV on HCV genome since, in the acute phase of the disease, HCV-RNA was found in the plasma of only one patient in group A (5.2%) and in 84% of patients in group C (p<0.000): the only positive patient in group A became negative in a few days; C) a severe clinical presentation (P.T. < 25% or development of ascites or signs of portosystemic encephalopaty) was observed in 5 patients in group A (26,3%)and in none in group B (p<0.05). At present, only 9 patients in group A and 11 in group B were followed-up for at least 5 months. All the 11 patients followed-up in group B and 8 of the 9 in group A recovered from acute hepatitis B with HBsAg negative within the 5th month and one developed HBV and HCV related chronic hepatitis. Of the 9 patients followed-up in group A, 4 became HCV-RNA positive in a few months and 5 remained negative throughout the observation (one cleared also serum auti-HCV). The data from this study allow these considerations: A) acute hepatitis B is more severe in patients with an underlying HCV chronic hepatitis than in those without, indicating the need of HBV vaccination in the latter. B) in acute hepatitis B occurred in chronic HCV carriers there is a marked inhibition exerted by HBV on HCV genome that may persist for several months in about a half of cases.
569
57O
PRIMARY HBV ANTIGEN SPECIFIC T HELPER CELL A N D CYTOTOXIC T LYMPHOCATE RESPONSE IN THE TRIMERA MOUSE MODEL AFTER VACCIANTION W I T H PROTEIN ANTIGENS A N D D N A VECTORS. W u l f O Boecher, W i b k e Schwerin, I Dept of Internal Medicine, University Hospital, Mainz Germany; Michael Geissler, Dept of Internal Medicine, Universitay Hospital, Freiburg Germany; Sina Hoffmann, 1 Dept of Internal Medicine, University Hospital, Mainz Germany; A Cooper, Dept of Virology, W e i z m a n n Institute of Schience, Rehovot Israel; Peter R Galle, I Dept of Internal Medicine, University Hospital, Mainz Germany; H a n n s F Loehr, I Dept of Internal Medicine, University Hospital, Maiz G e r m a n y
RELATEDNESS OF NUCLEOTIDE MUTATION OF X-GENE W I T H HISTO-PATHOLOGIC IN CHRONIC HEPATITIS B VIRUS CARRIERS. Haak-Cheoul Kim, K w o n - M o o k Chae, Ki-Jung Yun, Tae-Hyeon Kim, W o o G u n Song, W o n k w a n g University School of Medicine, [ksan South Korea
Introduction: H u m a n i z e d Balb/c mice (Trimera mice) conditioned b y lethal total b o d y irradiation a n d b o n e m a r r o w transplantation from SCID mice have been described to s u p p o r t rapid engraftment of h u m a n peripheral blood m o n o nuclear cells (PBMC). Using Balb/c recipient mice s p e c i f c B a n d Th cell responses were stimulated in vivo a n d detected ex vivo quantitatively o n a single cell level. In the current study, it is s h o w n that b y the use of a viral antigen (HBV core (HBc) antigen) a Th cell stimulation failure should be overcome b y vaccination with high doses of the antigen or with DNA plasmids encoding for the HBc antigen. M a t e r i a l a n d M e t h o d s : Balb/c mice were lethally irradiated a n d bone m a r r o w transplanted from NOD/SCID Balb/c mice. H u m a n PBMC from HBV-naive donors were infused intraperitoneally a n d mice were vaccinated i.p. with purified a n d r e c o m b i n a n t antigens (HBcAg, TT a n d HBcDNA). Cells from trimera mice were collected ten days after vaccination by peritoneal lavage. Analysis of antigen-specific h u m a n T a n d B cells was performed by Elispot analysis for IFN- T. Results: The induction of strong p r i m a r y Th cell responses against a viral neo-antigen is observed. Strong HBc specific Th cell responses were i n d u c e d b y different doses of HBcAg a n d b y HBc-DNA vaccination. In contrast the vaccination with TT (positive control) induces only high TT specific Th cell frequencies while the EBV-vaccination (negative control) reveals no detectable Th cells. C o n c l u s i o n : Since the trimera can be experimentally infected with HBV, the trimera mice model will enable studies of new vaccination strategies s u c h as peptide a n d DNA vaccination against HBV infection and also against different other h u m a n infections.
Background/Aim: The main funtion ofHBV X-gene is trans-/or cis-activaturs of a n u m b e r of cellular a n d viral genes. At genomic level, there are m a n y functional regions, a n d those appear to be important for viral life cycle. As mutation of nucleotide sequence influence the replication of HBV, a n d m i g h t be influence the activity of liver disease. But there are controversy of the severity of liver disease to the relatedness with the mutation of HBV X-gene. Thus we investigated the nucleotide mutation of X-gene on the aspect of mutation point a n d severity of liver disease in chronic HBV carriers. Method: W e selected only 48 subjects those liver biopsy a n d their blood samples were collected at same time. And they were divided by 2 groups according to histologic status. G r o u p A were 11 patients w h o s e histologic finding were nonspecific or minimal hepatitis. W h e r e g r o u p B were 37 patients, a n d those were subdivided to 2 subgroups. Those have a n y portal or lobular inflammation without any fibrosis (B1) were 27 patients, a n d the other with fibrosis(B2) were 10 patients. Polymerase chain reaction (PCR) for X-gene a n d direct sequencing were performed. O n the analytic sequence, the mutation were compared to the severity of liver disease. Result: The clinical status was similar to b o t h group, except transaminase was higher in g r o u p B. They were all included to genotype C, a n d there were m a n y point mutations with 1.12% variability. H o t spots were f o u n d frequently in 2 n d TATA-box, NRE, ¥fi-box a n d mid-portion of N-terminal. The nucleotide mutations were occurred frequently at 1762 a n d 1764 with accompanied each other. The accompanied mutations were f o u n d in 10 patients of 48 chronic HBV carriers. The accompanied mutations were f o u n d only 1 case in g r o u p A, b u t 9 cases in g r o u p B. A n d also there was 5 cases in g r o u p Bt, b u t 4 cases in g r o u p B2. Conclusion: The mutation of HBV in X gene, especially 1762 a n d 1764, m i g h t be related to the chronic severity of liver disease due to HBV.
AASLD ABSTRACTS
HEPATOLOGYO c t o b e r 2 0 0 1
567
568
IN VIVO INHIBITION OF ANTI-HBCIGG P R O D U C T I O N BY HBCAGSPECIFIC C D 4 + TH1-TYPE T CELL CLONES IN A HU-PBL-NOD/SCID MICE MODEL. Tinghua Cao, Philip Meuleman, Isabefle Desombere, G h e n t University, G h e n t Belgium; Matti Saflberg, Karolinska Institute at H u d d i n g e University Hospital, H u d d i n g e Sweden; Geert G Leroux-Roels, Ghent University, G h e n t Belgium
ACUTE HEPATITIS B IN HCV CHRONIC CARRIERS: VIROLOGICAL INTERACTION ON CLINICAL COURSE. Evangelista Sagnelli MD, Division of Infectious Diseases, A O di Caserta, Caserta Italy; Nicola Coppola MD, Cecilia Marrocco PhD, Institute of Infectious Disease, Second University of Naples, Naples Italy; Vincenzo Messina MD, Domenico Di Caprio MD, A n n a Marotta PhD, Division of Infections Diseases, A O di Caserta, Caserta Italy; Mirella Onofrio MD, Carlo Seolastico MD, Pietro Filippini MD, Felice Piccinino MD, Institute of Infectious Disease, Second University of Naples, Naples Italy
Antigen-specific C D 4 + T cell responses are believed to play an i m p o r t a n t role in the control of h u m a n hepatitis B virus (HBV) infection. In the present study, HBcAg-specifie, HLA DR13*-restricted C D 4 + T h l - t y p e T cell clones were generated from the PBL of a subject w h o spontaneously cleared a n acute HBV infection. These HBcAg-specifie C D 4 + T h l - t y p e T cells secreted b o t h 1FN- 7 a n d T N F - a u p o n stimulation with HBcAg a n d were also able to lyse HBe peptide-loaded EBV-transformed lymphoblastoid target cells in vitro. To examine w h e t h e r these HLA DR13*-restricted h u m a n C D 4 + T h l T cells also display the same cytotoxic effects in vivo, we transferred PBL derived from HBV-infected donors or a HBV-naive d o n o r sharing the DR13*, together with the HBcAg-specific C D 4 + T h l - t y p e T cells a n d HBcAg directly into the spleen of Nod/LtSz-PrkdcsCla/Prkdcscid (NOD/SCID) mice that were pretreated with total b o d y irradiation (3Gy) a n d a rat anti mouse IL-2 receptor/3 chain m o n o clonal antibody (TM/31, ling/mouse given intraperitoneally). The p r o d u c t i o n of b o t h secondary anti-HBc-IgG a n d primary HBcAg-Binding IgM in hu-PBLNOD/SCID mice were drastically inhibited by HBcAg-specific C D 4 + T h l - t y p e T cells. No inhibition was observed w h e n C D 4 + T h l cells a n d d o n o r PBL did not share HLA DR13. These results suggest that HBcAg-specific C D 4 + T h l T cells m a y lyse the HBcAg-specific B cells that have taken u p HBcAg in vivo in a n antigen-specific a n d HLA DR13-restricted m a n n e r as was demonstrated in vitro. It is tempting to speculate that HBcAg-specific C D 4 + T h l - t y p e T cells can modulate HBcAg-specific antibody production, exert a regulatory role in deleting HBcAg-specific ( o r - b i n d i n g ) h u m a n B cells in vivo a n d contribute to the recovery of acute HBV infections.
Background: Chronic hepatitis due to HBV/HCV coinfection is characterized by a reciprocal inhibition of viral genomes and by a more severe clinical presentation. However, the interaction between HBV and HCV in HBV acute hepatitis occurring in chronic HCV carriers has not been so far investigated. Patients and methods: We studied 19 cases of HBV acute icteric hepatitis (Group A) occurred in HCV chronic carriers in comparison with a control group of 19 cases of HBV acute hepatitis observed in the same period in subjects with no sign of HCV infection (Group B). All patients in group A were male drug addicts, median age 29 years (range 21-36), showing circulating HBsAg, high titres oflgM anti-HBc and anti-HCV. In group B only 7 patients were drug addicts, 13 were males and 6 females, median age 26 years (range 21-40). All patients were hospitahsed in our ward fromjanuary 2000 to april 2001; no patient had received HBV vaccine. As a second control group for patients in group A, 19 drug addicts with chronic HCV infection and no sign of HBV infection were selected (pair-matched by age, +/- 5 years, and sex) among those observed in the same period in the same ward (Group C). Patients in different groups were followed-up from 5 14 months. Samples of plasma (59 from patients in group A and 65 from those in group B) obtained at the time of first observation and during the foUow-up were stored frozen at 80°C. HBV-DNAwas performed by a quantitative PCR in all samples from groups A and B. HCV-RNA was determined by qualitative PCR on samples from groups A and C. Results. By comparing the 3 groups we observed: A) similar patterns of HBV replication with HBV-DNA become negative in 14-24 days in group A and B; B) marked inhibition of HBV on HCV genome since, in the acute phase of the disease, HCV-RNA was found in the plasma of only one patient in group A (5.2%) and in 84% of patients in group C (p<0.000): the only positive patient in group A became negative in a few days; C) a severe clinical presentation (P.T. < 25% or development of ascites or signs of portosystemic encephalopaty) was observed in 5 patients in group A (26,3%)and in none in group B (p<0.05). At present, only 9 patients in group A and 11 in group B were followed-up for at least 5 months. All the 11 patients followed-up in group B and 8 of the 9 in group A recovered from acute hepatitis B with HBsAg negative within the 5th month and one developed HBV and HCV related chronic hepatitis. Of the 9 patients followed-up in group A, 4 became HCV-RNA positive in a few months and 5 remained negative throughout the observation (one cleared also serum auti-HCV). The data from this study allow these considerations: A) acute hepatitis B is more severe in patients with an underlying HCV chronic hepatitis than in those without, indicating the need of HBV vaccination in the latter. B) in acute hepatitis B occurred in chronic HCV carriers there is a marked inhibition exerted by HBV on HCV genome that may persist for several months in about a half of cases.
569
57O
PRIMARY HBV ANTIGEN SPECIFIC T HELPER CELL A N D CYTOTOXIC T LYMPHOCATE RESPONSE IN THE TRIMERA MOUSE MODEL AFTER VACCIANTION W I T H PROTEIN ANTIGENS A N D D N A VECTORS. W u l f O Boecher, W i b k e Schwerin, I Dept of Internal Medicine, University Hospital, Mainz Germany; Michael Geissler, Dept of Internal Medicine, Universitay Hospital, Freiburg Germany; Sina Hoffmann, 1 Dept of Internal Medicine, University Hospital, Mainz Germany; A Cooper, Dept of Virology, W e i z m a n n Institute of Schience, Rehovot Israel; Peter R Galle, I Dept of Internal Medicine, University Hospital, Mainz Germany; H a n n s F Loehr, I Dept of Internal Medicine, University Hospital, Maiz G e r m a n y
RELATEDNESS OF NUCLEOTIDE MUTATION OF X-GENE W I T H HISTO-PATHOLOGIC IN CHRONIC HEPATITIS B VIRUS CARRIERS. Haak-Cheoul Kim, K w o n - M o o k Chae, Ki-Jung Yun, Tae-Hyeon Kim, W o o G u n Song, W o n k w a n g University School of Medicine, [ksan South Korea
Introduction: H u m a n i z e d Balb/c mice (Trimera mice) conditioned b y lethal total b o d y irradiation a n d b o n e m a r r o w transplantation from SCID mice have been described to s u p p o r t rapid engraftment of h u m a n peripheral blood m o n o nuclear cells (PBMC). Using Balb/c recipient mice s p e c i f c B a n d Th cell responses were stimulated in vivo a n d detected ex vivo quantitatively o n a single cell level. In the current study, it is s h o w n that b y the use of a viral antigen (HBV core (HBc) antigen) a Th cell stimulation failure should be overcome b y vaccination with high doses of the antigen or with DNA plasmids encoding for the HBc antigen. M a t e r i a l a n d M e t h o d s : Balb/c mice were lethally irradiated a n d bone m a r r o w transplanted from NOD/SCID Balb/c mice. H u m a n PBMC from HBV-naive donors were infused intraperitoneally a n d mice were vaccinated i.p. with purified a n d r e c o m b i n a n t antigens (HBcAg, TT a n d HBcDNA). Cells from trimera mice were collected ten days after vaccination by peritoneal lavage. Analysis of antigen-specific h u m a n T a n d B cells was performed by Elispot analysis for IFN- T. Results: The induction of strong p r i m a r y Th cell responses against a viral neo-antigen is observed. Strong HBc specific Th cell responses were i n d u c e d b y different doses of HBcAg a n d b y HBc-DNA vaccination. In contrast the vaccination with TT (positive control) induces only high TT specific Th cell frequencies while the EBV-vaccination (negative control) reveals no detectable Th cells. C o n c l u s i o n : Since the trimera can be experimentally infected with HBV, the trimera mice model will enable studies of new vaccination strategies s u c h as peptide a n d DNA vaccination against HBV infection and also against different other h u m a n infections.
Background/Aim: The main funtion ofHBV X-gene is trans-/or cis-activaturs of a n u m b e r of cellular a n d viral genes. At genomic level, there are m a n y functional regions, a n d those appear to be important for viral life cycle. As mutation of nucleotide sequence influence the replication of HBV, a n d m i g h t be influence the activity of liver disease. But there are controversy of the severity of liver disease to the relatedness with the mutation of HBV X-gene. Thus we investigated the nucleotide mutation of X-gene on the aspect of mutation point a n d severity of liver disease in chronic HBV carriers. Method: W e selected only 48 subjects those liver biopsy a n d their blood samples were collected at same time. And they were divided by 2 groups according to histologic status. G r o u p A were 11 patients w h o s e histologic finding were nonspecific or minimal hepatitis. W h e r e g r o u p B were 37 patients, a n d those were subdivided to 2 subgroups. Those have a n y portal or lobular inflammation without any fibrosis (B1) were 27 patients, a n d the other with fibrosis(B2) were 10 patients. Polymerase chain reaction (PCR) for X-gene a n d direct sequencing were performed. O n the analytic sequence, the mutation were compared to the severity of liver disease. Result: The clinical status was similar to b o t h group, except transaminase was higher in g r o u p B. They were all included to genotype C, a n d there were m a n y point mutations with 1.12% variability. H o t spots were f o u n d frequently in 2 n d TATA-box, NRE, ¥fi-box a n d mid-portion of N-terminal. The nucleotide mutations were occurred frequently at 1762 a n d 1764 with accompanied each other. The accompanied mutations were f o u n d in 10 patients of 48 chronic HBV carriers. The accompanied mutations were f o u n d only 1 case in g r o u p A, b u t 9 cases in g r o u p B. A n d also there was 5 cases in g r o u p Bt, b u t 4 cases in g r o u p B2. Conclusion: The mutation of HBV in X gene, especially 1762 a n d 1764, m i g h t be related to the chronic severity of liver disease due to HBV.