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P-369 Immune response to viral antigens produced by transgene of full-length HBV in purebred mice
Posters/International Hepatology Communications 3 Suppl. (1995) $37-$169
P-369 IIVlVIUNE RESPONSE TO V I R A L ANTIGENS PRODUCED BY TRANSGENE OF F U L L - L E N G T H HBV IN PUREBRED M I C E K. H a s e g a w a , J . K a t o , N . T o r i i , T.Shizuma, Y.Kamogawa, T. K a t e , K. Yamauchi, N.Hayashi. I n s t . of G a s t r o . , Tokyo Women' s Medi cal Co] ] ege, Tokyo, Japan The e s t a b l i s h m e n t of model mouse may p r o v i d e at~eful tool for the study of p a t h o g e n e s i s of HBV v i r u s - induced liver disease. Although transgenic mice expressing viral a n t i g e n have been e s t a b l i s h e d , t h i s model may not be s u i t a b l e f o r the a n a l y s i s of irrmune response to v i r u s , s i n c e they a r e ivr~nunologica]ly t o l e r a n t to viral a n t i g e n . The p r e v i o u s study r e v e a l e d t h a t p a t h o g e n e s i s of hepatitis B was h i g h l y a s s o c i a t e d wi th the i n t e r a c t ion between v i r u s and host irrmune s y s t e m . T h e r e f o r e , d i r e c t d e l i v e r y of HBVDNA into h e p a t o c y t e s of a d u l t purebred mouse may be advantageous for the study in t h i s f i e l d . Based on these f i n d i n g s , we a i m e d to e s t a b l i s h purebred mouse e x p r e s s i n g h e p a t i t i s B v i r a l p r o t e i n in h e p a t o c y t e s u s i n g ] i p o f e c t i n - m e d i a t e d HBV gene t r a n s f e r . The c o m p l e x of l i p o f e e t i n and f u l l - l e n g t h H B V D N A was i n j e c t e d to s u b c a p s u l a r a r e a of the l i v e r of C57 B L / 6 ( H - 2 b ) , C 3 H (H-?_K), BALB/c (H-2d~. HBV t r a n s c r i n t s were d e t e c t e d in the l i v e r by RT-PCR. immunohistochemica] stuby d e m o n s t r a t e d t h a t H B s A g was m a i n l y s t a i n e d in c y t o p l a s m of h e p a t o c y t e and H B c A g l o c a t e d in n u c l e i . HBsAg became d e t e c t a b l e in each mouse. I n c o n t r a s t , l i t t l e a n t i - H B s r a i s e d in C3H d u r i n g the o b s e r v a t i o n the o b s e r v a t i o n p e r i o d , w h i l e B6 and B A L B / c l:roduced high t i t e r of a n t i - H B s . T h e d i f f e r e n t c a p a b i l i t y of a n t i b o d y p r o d u c t i o n among mouse s t r a i n may be due to v a r i e t y of H- 2 response. T h e p r e s e n t s t u b y d e m o n s t r a t e d this system of transgene of HBV into mouse l i v e r p r o v i d e us a useful technique to a n a l y z e the ilrrnune r e s p o n s e to HBV. F u r t h e r m o r e , t h i s model w i l l be a p p l i c a b l e for the study of i n f l u e n c e s fe m u t a t e d HBV on the c] i n i c a l course of v a r i o u s type of HBV induced l i v e r d i s e a s e .
P-371
ROLE OF 7 -INTERFERON TO OVERCOME IMMUNE RESPONSE DEFECTS IN H E P A T I T I S B V I R U S T R A N S G E N I C MICE. S.M.F.Akbar, K. Kurose, T.Masumoto, K. Michitaka, N. Horiike, H. Iuchi, K. Ohkubo, Y. Itoh, & M. Onji. Third Department of Internal Medicine, Ehime University School of Medicine, Ehime, Japan. We have shown thai hepalitis B virus(HBV)-transgenic mice could not produce anti-KLH Ab in vivo and vitrn being primed with an optimum dose(5 /2 g) of keyhole limpet hemocyanin(KLH) as a result of low expression of la antigen on dendritic cells(Akbar el at. hninunology 1993 ;78:468). The present experiment was designed to evaluate the role of 7 -[FN to overcome the immune response defects in transgenic mice by modulaling the expression of la antigen on dendritic cells. HBV-transgenic mice, produciug HBsAg, HBeAg, & Dane particles in sera and relevent mRNAs in tissues were injected in vivo with an oplimum dose of KLH alone or KLH and 7 -IFN(5X10' U/day,ip,for 6 days). In vitro, dendritic cells isolated from tmnsgenic mice were treated with 7"-IFN(100U/ml for 24 hour). The mean fluorescence intensity of la antigen on dendritic cells from untreated transgenic mice was 70Z 16 and they could not produce anti-KLH Ab when primed with 5 ftg of KLH both in vivo & vitro. But when transgenic mice were treated with 5 .tzg of KLH along with )' -IFN. the mean fluorescence intensity of Ia antigen on dendritic cells were increased significantly to 147 + 24 and most oflhe mice produced anti-KLH Ab both in vivo and vitro. When lransgenic mice derived dendritic cells were isolated and treated in vitro with 7 -[FN, the fluorescence intensity of la antigen on dendritic cells were increased significantly to 166 - 16 and these dendritic cells acted as efficient APC for anti-KLH Ab production from transgenic T/B cells, primed with oplimum dose of KLH. These experiments have shown that, the HBV-carriers are not only incapable to exibit immune response to some of the HBV-related antigens, but also have low responsiveness to other T- cell -dependent antigen, like KLH due to a defect of antigen presentation as a result of low level of erxpression of Ia antigen on dendritic cells & this defect can be overcome by manupulating the [a antigen expression on dendritic cells by cytokine, like 7 -IFN.
S129
P-370
ROLE OF V A C C I N A T I O N ON VIRAL R E P L I C A T I O N I N H E P A T I T I S B VIRUS TRANSGENIC MICE S.M.F.Akbar, K.Kurose, T.Masumolo, K. Michitaka, N. Horiike, H. Iuchi. K. Ohkubo, Y. Itoh, M.Ouji. Thjrd Department of Internal Medicine, Ehime University School of Medicine. Ehime, Japan. This study was undertaken to see the impact of vaccination against hepatitis B virus (HBV) in murine HBV-carriers using our newly developed vaccination protocol. HBV-transgenic mice produced by microinjecling the full genome of HBV inlo the fertilized eggs of C57BL/6 mice and expressing HBsAg, HBeAg, Dane particles in sera and the relevent mRNAs in tissues, without any sign of liver injury were used as an animal model of HBVcarrier state. These mice were injected intraperitoneally with recombinant HBV-vaccine containing 10 g g of HBsAg in CFA/mouse/month, for 8 months and serological markers related to HBV were seen in sera on a monthly basis. As controls, some tmnsgenie mice were injected with PBS & others recieved nothing. HBsAg was estimated by RPHA and ELISA method, HBeAg were estimated by RPHA method and anti-HBs were estimated by EL[SA method. Values ~ 22 and ~ 1.0 were considered as positive in RPHA and ELISA methods, respectively. In all transgenic mice, the HBsAg and HBeAg liters at the begining of the experiment were 2s and 2 ' , respectively by RPHA method and the mean HBsAg level was 25 ± 4.3 by EL[SA. All mice were negalive for antiHBs. A gradual fall of HBsAg and HBeAg liter were detected in most of the mice and at the end of 8 months, HBsAg tilers fell to 2~ in 20% mice, 2= in 40% mice, and 30% mice became negative for HBsAg and 10% mice developed anti-HBs. On the other hand, all mice became negative for HBeAg after the observation period of 8 monlhs. No change in HBV markers were seen in mice after 8 mouths recieving PBS or no special treatment. The repeated vaccination of HBV-lransgenic mice with high doses of HBsAg in CFA have resulted in a gradual fall of HBsAg in sera and have reduced viral replication as evidence by a fall of HBeAg level. Further more. in 10% mice. vaccination have resulted in seroconversion to anti-HBs. This experiment have shown the elhical and scientific basis" for using HBV-vaccine as a therapeutic tool in chronic HBV-carriers.
P-372
CLASSIFICATION OF HBV PRE $2 PEPTIDE TYPES BY USING PCR TECHNIQUE AND ITS CLINICAL SIGNIFICANCE M. Nishikawa, H. Yonemitsu, T. Kimura, K. Ryo, H. Sekiya, I. Haruta, Y. Kamogawa, T. Kato, K. Yamauchi, N. Hayashi Division of Medicine, Institute of Gastroenterology, Tokyo Women's Medical College, Tokyo, Japan We have already reported that the first 39 nucleotide sequences of the pre $2 region of HBV are quite variable, and these sequences can be classified into three groups; adr-, adwand ayw- type pre $2. And we have also reported that the adrtype pre $2 HBV infection might have an important role in pathogenesis of chronic hepatitis B (CH-B) in HLA-A24 positive patients. Utilizing specific primers, three pre $2 types can be detected by polymerase chain reaction (PCR). Here, we determined the pre $2 types derived from 12 HLA-A24 positive CH-B patients at the point of both high and normal serum ALT levels by PCR and examined the relevance between pre $2 changes and serum ALT levels. We detected adr-type of pre $2 at active stage (=high ALT), however this type disappeared or converted into other types when serum ALT were normalized. In 2 patients (Case 1 and Case 2), we have followed for 3 years at 3 months intervals. In Case 1, serum ALT level was normalized when adr-type of pre $2 was disappeared. And in Case 2, serum ALT level was normalized after disappearance of adr-type of pre $2. This results indicate that the complex of pre $2 peptide and HLA-A24 molecule may affect hepatocyte injury, and the first 13 amino acid of pre $2 may be recognized as one of the critical epitope for cytotoxic T cell which cause hepatocyte damage.
P-369 IIVlVIUNE RESPONSE TO V I R A L ANTIGENS PRODUCED BY TRANSGENE OF F U L L - L E N G T H HBV IN PUREBRED M I C E K. H a s e g a w a , J . K a t o , N . T o r i i , T.Shizuma, Y.Kamogawa, T. K a t e , K. Yamauchi, N.Hayashi. I n s t . of G a s t r o . , Tokyo Women' s Medi cal Co] ] ege, Tokyo, Japan The e s t a b l i s h m e n t of model mouse may p r o v i d e at~eful tool for the study of p a t h o g e n e s i s of HBV v i r u s - induced liver disease. Although transgenic mice expressing viral a n t i g e n have been e s t a b l i s h e d , t h i s model may not be s u i t a b l e f o r the a n a l y s i s of irrmune response to v i r u s , s i n c e they a r e ivr~nunologica]ly t o l e r a n t to viral a n t i g e n . The p r e v i o u s study r e v e a l e d t h a t p a t h o g e n e s i s of hepatitis B was h i g h l y a s s o c i a t e d wi th the i n t e r a c t ion between v i r u s and host irrmune s y s t e m . T h e r e f o r e , d i r e c t d e l i v e r y of HBVDNA into h e p a t o c y t e s of a d u l t purebred mouse may be advantageous for the study in t h i s f i e l d . Based on these f i n d i n g s , we a i m e d to e s t a b l i s h purebred mouse e x p r e s s i n g h e p a t i t i s B v i r a l p r o t e i n in h e p a t o c y t e s u s i n g ] i p o f e c t i n - m e d i a t e d HBV gene t r a n s f e r . The c o m p l e x of l i p o f e e t i n and f u l l - l e n g t h H B V D N A was i n j e c t e d to s u b c a p s u l a r a r e a of the l i v e r of C57 B L / 6 ( H - 2 b ) , C 3 H (H-?_K), BALB/c (H-2d~. HBV t r a n s c r i n t s were d e t e c t e d in the l i v e r by RT-PCR. immunohistochemica] stuby d e m o n s t r a t e d t h a t H B s A g was m a i n l y s t a i n e d in c y t o p l a s m of h e p a t o c y t e and H B c A g l o c a t e d in n u c l e i . HBsAg became d e t e c t a b l e in each mouse. I n c o n t r a s t , l i t t l e a n t i - H B s r a i s e d in C3H d u r i n g the o b s e r v a t i o n the o b s e r v a t i o n p e r i o d , w h i l e B6 and B A L B / c l:roduced high t i t e r of a n t i - H B s . T h e d i f f e r e n t c a p a b i l i t y of a n t i b o d y p r o d u c t i o n among mouse s t r a i n may be due to v a r i e t y of H- 2 response. T h e p r e s e n t s t u b y d e m o n s t r a t e d this system of transgene of HBV into mouse l i v e r p r o v i d e us a useful technique to a n a l y z e the ilrrnune r e s p o n s e to HBV. F u r t h e r m o r e , t h i s model w i l l be a p p l i c a b l e for the study of i n f l u e n c e s fe m u t a t e d HBV on the c] i n i c a l course of v a r i o u s type of HBV induced l i v e r d i s e a s e .
P-371
ROLE OF 7 -INTERFERON TO OVERCOME IMMUNE RESPONSE DEFECTS IN H E P A T I T I S B V I R U S T R A N S G E N I C MICE. S.M.F.Akbar, K. Kurose, T.Masumoto, K. Michitaka, N. Horiike, H. Iuchi, K. Ohkubo, Y. Itoh, & M. Onji. Third Department of Internal Medicine, Ehime University School of Medicine, Ehime, Japan. We have shown thai hepalitis B virus(HBV)-transgenic mice could not produce anti-KLH Ab in vivo and vitrn being primed with an optimum dose(5 /2 g) of keyhole limpet hemocyanin(KLH) as a result of low expression of la antigen on dendritic cells(Akbar el at. hninunology 1993 ;78:468). The present experiment was designed to evaluate the role of 7 -[FN to overcome the immune response defects in transgenic mice by modulaling the expression of la antigen on dendritic cells. HBV-transgenic mice, produciug HBsAg, HBeAg, & Dane particles in sera and relevent mRNAs in tissues were injected in vivo with an oplimum dose of KLH alone or KLH and 7 -IFN(5X10' U/day,ip,for 6 days). In vitro, dendritic cells isolated from tmnsgenic mice were treated with 7"-IFN(100U/ml for 24 hour). The mean fluorescence intensity of la antigen on dendritic cells from untreated transgenic mice was 70Z 16 and they could not produce anti-KLH Ab when primed with 5 ftg of KLH both in vivo & vitro. But when transgenic mice were treated with 5 .tzg of KLH along with )' -IFN. the mean fluorescence intensity of Ia antigen on dendritic cells were increased significantly to 147 + 24 and most oflhe mice produced anti-KLH Ab both in vivo and vitro. When lransgenic mice derived dendritic cells were isolated and treated in vitro with 7 -[FN, the fluorescence intensity of la antigen on dendritic cells were increased significantly to 166 - 16 and these dendritic cells acted as efficient APC for anti-KLH Ab production from transgenic T/B cells, primed with oplimum dose of KLH. These experiments have shown that, the HBV-carriers are not only incapable to exibit immune response to some of the HBV-related antigens, but also have low responsiveness to other T- cell -dependent antigen, like KLH due to a defect of antigen presentation as a result of low level of erxpression of Ia antigen on dendritic cells & this defect can be overcome by manupulating the [a antigen expression on dendritic cells by cytokine, like 7 -IFN.
S129
P-370
ROLE OF V A C C I N A T I O N ON VIRAL R E P L I C A T I O N I N H E P A T I T I S B VIRUS TRANSGENIC MICE S.M.F.Akbar, K.Kurose, T.Masumolo, K. Michitaka, N. Horiike, H. Iuchi. K. Ohkubo, Y. Itoh, M.Ouji. Thjrd Department of Internal Medicine, Ehime University School of Medicine. Ehime, Japan. This study was undertaken to see the impact of vaccination against hepatitis B virus (HBV) in murine HBV-carriers using our newly developed vaccination protocol. HBV-transgenic mice produced by microinjecling the full genome of HBV inlo the fertilized eggs of C57BL/6 mice and expressing HBsAg, HBeAg, Dane particles in sera and the relevent mRNAs in tissues, without any sign of liver injury were used as an animal model of HBVcarrier state. These mice were injected intraperitoneally with recombinant HBV-vaccine containing 10 g g of HBsAg in CFA/mouse/month, for 8 months and serological markers related to HBV were seen in sera on a monthly basis. As controls, some tmnsgenie mice were injected with PBS & others recieved nothing. HBsAg was estimated by RPHA and ELISA method, HBeAg were estimated by RPHA method and anti-HBs were estimated by EL[SA method. Values ~ 22 and ~ 1.0 were considered as positive in RPHA and ELISA methods, respectively. In all transgenic mice, the HBsAg and HBeAg liters at the begining of the experiment were 2s and 2 ' , respectively by RPHA method and the mean HBsAg level was 25 ± 4.3 by EL[SA. All mice were negalive for antiHBs. A gradual fall of HBsAg and HBeAg liter were detected in most of the mice and at the end of 8 months, HBsAg tilers fell to 2~ in 20% mice, 2= in 40% mice, and 30% mice became negative for HBsAg and 10% mice developed anti-HBs. On the other hand, all mice became negative for HBeAg after the observation period of 8 monlhs. No change in HBV markers were seen in mice after 8 mouths recieving PBS or no special treatment. The repeated vaccination of HBV-lransgenic mice with high doses of HBsAg in CFA have resulted in a gradual fall of HBsAg in sera and have reduced viral replication as evidence by a fall of HBeAg level. Further more. in 10% mice. vaccination have resulted in seroconversion to anti-HBs. This experiment have shown the elhical and scientific basis" for using HBV-vaccine as a therapeutic tool in chronic HBV-carriers.
P-372
CLASSIFICATION OF HBV PRE $2 PEPTIDE TYPES BY USING PCR TECHNIQUE AND ITS CLINICAL SIGNIFICANCE M. Nishikawa, H. Yonemitsu, T. Kimura, K. Ryo, H. Sekiya, I. Haruta, Y. Kamogawa, T. Kato, K. Yamauchi, N. Hayashi Division of Medicine, Institute of Gastroenterology, Tokyo Women's Medical College, Tokyo, Japan We have already reported that the first 39 nucleotide sequences of the pre $2 region of HBV are quite variable, and these sequences can be classified into three groups; adr-, adwand ayw- type pre $2. And we have also reported that the adrtype pre $2 HBV infection might have an important role in pathogenesis of chronic hepatitis B (CH-B) in HLA-A24 positive patients. Utilizing specific primers, three pre $2 types can be detected by polymerase chain reaction (PCR). Here, we determined the pre $2 types derived from 12 HLA-A24 positive CH-B patients at the point of both high and normal serum ALT levels by PCR and examined the relevance between pre $2 changes and serum ALT levels. We detected adr-type of pre $2 at active stage (=high ALT), however this type disappeared or converted into other types when serum ALT were normalized. In 2 patients (Case 1 and Case 2), we have followed for 3 years at 3 months intervals. In Case 1, serum ALT level was normalized when adr-type of pre $2 was disappeared. And in Case 2, serum ALT level was normalized after disappearance of adr-type of pre $2. This results indicate that the complex of pre $2 peptide and HLA-A24 molecule may affect hepatocyte injury, and the first 13 amino acid of pre $2 may be recognized as one of the critical epitope for cytotoxic T cell which cause hepatocyte damage.