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Prolonged expression of bilirubin-UDP-glucuronosyl transferase-1 after adenovirus-mediated gene transfer into newborn Gunn rats
220A 453
AASLD
ABSTRACTS
PROLONGED EXPRESSION OF BILIRUBIN-UDP-GLUCURONOSYL TRANSFERASE-I AFTER ADENOVIRUS-MEDIATED GENE TRANSFER INTO NEWBORN GUNN RATS. M Takahashi, K Sen,2uota, N Roy Chowdhurv. JRov
454
EXPRESSION AND IMMUNE RESPONSE TO HEPATITIS C VIRUS DNA VACCINE CONSTRUCTS Katsutoshi Tokushige r Taka}i Wakita, Darius Moradpour. Jack R. Wands. Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129 Development of a vaccine against HCV is complicated by the high mutation rate, the presence of different genotypes and the high variability of the envelope region. Recently, the injection of naked plasmid DNA encoding viral genes has been shown to induce humeral and cellular immune responses in the host. The HCV core region is highly conserved among the various genotypes and was selected as a potential viral target region for this approach. Methock Plasmids expressing HCV core were constructed in the presence and absence of 5'-NC region(pHCV4-2 and pHCV2-2, respectively) and placed under the control of a CMV promoter. In addition, genes encoding the small(S), middle(preS2 and S), and large(preS1, preS2 and S) envelope proteins of hepatitis B vires were added to the HCV core gone as fusion constructs. In order to investigate in vivo expression of HCV core protein in muscle cells, HCV-hciferase fusion constructs were also developed. Humoral immunity to HCV core protein was assayed in the serum of mice by Western blot analysis and RIA following intramuscular vaccination. Cellular immunity was assessed by a lymphocyte proliferation assay in the presense of recombinant HCV core protein. Resti~ All constructs expressed HCV core antigen by Western blot analysis and immunofluorescence studies in vitro. HCV core antigen expression was greatest in cells transfected with the HCV-DNA construct lacking the 5' NCR. After the injection of HCV-luciferase constructs in the muscle of mice, luciferase activity was detected by enzymatic assay for 2 weeks. Of ten mice injected with the HCV vaccine constructs (pHCV4-2 or 22), 5 mice seroconverted with anti-core antibodies as measured by RIA and Western blot analysis. Spleen cells derived from mice immunized with the DNA vaccine proliferated when incubated with HCV core protein, compared to splenocytes derived from mice immunized with mock ]3NA. The immune response to the various HBV-HCV chimeric fusion constructs was also studied. Condmi3~ By using HCV-luciferase constructs, in vivo expression of HCV core antigen was confirmed in mouse muscle cells. HCV core DNA-based vaccines induced both humoral and cellular immune responses in vivo. The role of protein secretion with regard to stimulation of the optimal immune response in the host has been analyzed with HBV-HCV fusion constructs. These results suggest that HCV DNA constructs are promising candidates as prophylactic and therapeutic vaccines.
456
COMPARISON OF ALBUMIN, DEXTRAN-70 AND H ~ C C E L IN THE PREVENTION OF EFFECTIVE HYPOVOLEMIA IN CIRRHOTIC PATIENTS WITH ASCITES TREATED WITH TOTAL PARACENTESIS. A RANDOMIZED M U L T I C E N T E R STUDY. International Group for the Study of Ascites in Cirrhosis.
Chowdhurv. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York. In Criglcr-Najjar syndrome type I and its animal model the Guun rat, a genetic deficiency ofbilirubin-UDP-glucuronosyltransferase-1 (B-UGT~), causes severe hyperbilirubinemia, resulting in neurotoxicity. We showed previously that injection of a haman B-UGTt-recombinant adenovirus in adult Gunn rats temporarily corrects the metabolic defect, ameliorating jaundice. The adenovirally transferred genes disappear because of host immune response, and reinjection of the virus fails to cause transgene expression. Here, we explored the possibility of achieving long-term expression of adenovirally transferred genes by inducing tolerance to the recombinant virus by injection into newborn rats. Two replication-defective recombinant adenoviruses were used: Ad-hBUGTt expressing human B-UGT~ and Ad-LacZ, expressing bacterial 13-galactosidase, both from a cytomegaiovirus promoter. Newborn Gunn rats (1-3 day old), or eongeneic normal Wistar rats were injected 20 ~1 of the purified recombinant viurses ( 2x108 pfu) into jugular veins. Seven days aRer injection of Ad-LacZ, nearly all hepatocytes were positive for 13-galactosidase activity ; 1-2% of the cells remained positive at 9 weeks. In Ad-hBUGT:-injected Gann rat pups, 4 weeks aRer injection, serum bilirubin concentrations were reduced to 65 to 83 panel/liter (n=4), compared to 134 tmaol/liter in AdLacZ injected controls. Two of these pups were followed for 9 weeks without further manipulation, and their serum bilirubin levels remained unchanged. The two other Ad-hBUGT~-treated pups were reinjected with AdhBUGT I (6xl0 s pfu i.e.) 4 weeks after initial injection. In these rats serum bilirubin levels were further reduced to 3.9 to 24 pruol/liter, and remained at that level throughout the study period (9 weeks). HPLC of the bile showed excretion of significant amounts of bilirubin ghcuronides 9 weeks after injection. Haman BUGT~ was detected in liver biopsy samples by immunoblot. CONCLUSION: I.V~ injection during the newborn period tolerizes rats to recombinant adenoviruses, whereby prolonged transgene expression can be achieved by repeated injection. In view of the increasing fi'equency of prenatal diagnosis of inherited diseases, this finding is potentially important in haman gene therapy.
455
PREVENTION OF HCV INFECTION IN CHIMPANZEES BY HYPERIMMUNE SERUM AGAINSTTHE HYPERVARIABLE REGION I (HVR1): EMERGENCE OF NEUTRALIZATION ESCAPE MUTANTS IN VIVO. P Farci. A Shimoda. D Won~. T Cabezon. D De Gioannis. A Strazzera. M Shaoiro. HJ Alter and RH Purcell. Lab. of Infectious Diseases, NIAID, Bethesda, SmithKline Beecham, Belgium, Dept. Internal Medicine, Univ. of Cagliari, Italy, Transfusion Medicine, NIH, Bethesda, Bioqual, Rockville, MD. To investigate whether the HVR1 of HCV is a critical neutralization domain, we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to the sequence of the HVR1 (anti-HVR1) of strain H77, a pedigreed virus titered in chimpanzees. The serum was reactive in ELISA against the homologous peptide and the recombinant El/E2 protein complex. The neutralization test was performed by mixing the virus inoeulum (64 CID~0) in vitro either with the anti- HVR1 serum, with the pre-immune rabbit serum, with an anti-HCV-negative human plasma or with the H79 plasma which was previously shown to contain neutralizing antibodies against H77, each at a final dilution of 1: l 0 and previously inactivated at 56°C for 30 min. The mixtures were kept overnight at 4°C and the residual infectivity was tested by IN. inoculation into seronegative chimpanzees. Five chimpanzees were studied: two received the H77 virus plus anti-HVR 1, one H77 plus the preimmune rabbit serum, one H77 plus the normal plasma and one I-I77plus H79 plasma. Serum I-ICVRNA was tested by PCR with primers from the 5'NC region. Selected PCR-positive samples were amplified with primers from the El/E2 genes and analyzed both by direct sequencing and by sequencing of u 10 molecular clones each. Sequence analysis of 104 clones from the H77 viral inoculum revealed at least 19 viral variants. The synthetic peptide used for immunization was based on the predominant variant (70/104 clones). Three minor variants were represented by 6, 5 and 4 clones, respectively, 4 by 2 clones and I 1 by a single clone. Following challenge, HCV infection was not detected in one of the 2 animals inoculated with H77 mixed with anti-HVRl. However, the other animal developed HCV viremia, hepatitis and antibody seroconversion. Interestingly, none of the 10 HCV molecular clones derived from this animal had the same sequence as the predominant viral strain, but, rather, that of two minor variants (one represented by 6/104 and the other by 2/t04 of the original clones). In contrast, the predominant viral strain (70/104), together with a minor variant (6/104) was consistently detected in the other three animals. All three developed hepatitis C regardless of the plasma used for neutralization. Similar data were obtained by direct sequencing. Conclusions: Complete protection from HCV infection in one animal and protection against the predominant HCV strain in a second animal suggests that a hyperimune serum against HVRI can neutralize HCV and that HVRI is a critical neutralization domain. The study also provides evidence for the emergence of neutralization escape mutants in vivo, a strategy whereby HCV can evade the host's immune response and establish persistent infection.
HEPATOLOGY October 1995
Large volume paracentesis without plasma volume expansion is constantly associated with an impairment in effective intravascular volume, as indicated by marked activation of endogenous vasoconstrictor systems. Albumin is highly effective in preventing this abnormality. However, it is not well established whether other plasma expanders are also effective. To investigate the efficacy of three different plasma expanders in the prevention of paracentesis-induoed effective hypovolemia, 289 cirrhotics with ascites were randomly allocated to treatment with total paracentesis and intravenous album/n (8 g/l of ascites removed, n=97), dextran-70 (8 g/l, n=93) or hemaccel (8 g/l, n=99). The effect of treatment on effective intravascular volume was assessed by measuring plasma renin activity (PRA) before and 6 and 30 days after treatment. The incidence of effective hypovolentia (~50% increase in PRA over baseline values to a level greater than 4 ng/ml.h at day 6) was markedly lower in patients treated with albumin (18%) than in those treated with dextran-70 or hemaccel (34 and 38%, respectively, p<0.01). In patients who developed effective hypovolemia pRA remained markedly increased over baseline values 30 days after treatment. Variables independently associated with a greater risk of developing effective hypovolemia were volume of ascitic fluid removed and type of plasma expander used. Patients who developed effective hypovolemia had significantly higher probability of readmission for ascites during follow-up (51% at 6 months)and lower probability of survival (57% at 6 months) as compared to patients who did not develop effective hypovolemia (32% and 75%, respectively). Effective hypovolemia after treatment was an independent predictor of survival. In summary, albumin is more effective than dextran-70 and hemaccel in preventing effective hypovolenda after paracentesis. This abnormality is not spontaneously reversible and is associated with a greater risk of ascites recurrence and shorter survival. Albumin should be considered the plasm~ expander of choice in cirrhotic patients treated with paraoentesis.
AASLD
ABSTRACTS
PROLONGED EXPRESSION OF BILIRUBIN-UDP-GLUCURONOSYL TRANSFERASE-I AFTER ADENOVIRUS-MEDIATED GENE TRANSFER INTO NEWBORN GUNN RATS. M Takahashi, K Sen,2uota, N Roy Chowdhurv. JRov
454
EXPRESSION AND IMMUNE RESPONSE TO HEPATITIS C VIRUS DNA VACCINE CONSTRUCTS Katsutoshi Tokushige r Taka}i Wakita, Darius Moradpour. Jack R. Wands. Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129 Development of a vaccine against HCV is complicated by the high mutation rate, the presence of different genotypes and the high variability of the envelope region. Recently, the injection of naked plasmid DNA encoding viral genes has been shown to induce humeral and cellular immune responses in the host. The HCV core region is highly conserved among the various genotypes and was selected as a potential viral target region for this approach. Methock Plasmids expressing HCV core were constructed in the presence and absence of 5'-NC region(pHCV4-2 and pHCV2-2, respectively) and placed under the control of a CMV promoter. In addition, genes encoding the small(S), middle(preS2 and S), and large(preS1, preS2 and S) envelope proteins of hepatitis B vires were added to the HCV core gone as fusion constructs. In order to investigate in vivo expression of HCV core protein in muscle cells, HCV-hciferase fusion constructs were also developed. Humoral immunity to HCV core protein was assayed in the serum of mice by Western blot analysis and RIA following intramuscular vaccination. Cellular immunity was assessed by a lymphocyte proliferation assay in the presense of recombinant HCV core protein. Resti~ All constructs expressed HCV core antigen by Western blot analysis and immunofluorescence studies in vitro. HCV core antigen expression was greatest in cells transfected with the HCV-DNA construct lacking the 5' NCR. After the injection of HCV-luciferase constructs in the muscle of mice, luciferase activity was detected by enzymatic assay for 2 weeks. Of ten mice injected with the HCV vaccine constructs (pHCV4-2 or 22), 5 mice seroconverted with anti-core antibodies as measured by RIA and Western blot analysis. Spleen cells derived from mice immunized with the DNA vaccine proliferated when incubated with HCV core protein, compared to splenocytes derived from mice immunized with mock ]3NA. The immune response to the various HBV-HCV chimeric fusion constructs was also studied. Condmi3~ By using HCV-luciferase constructs, in vivo expression of HCV core antigen was confirmed in mouse muscle cells. HCV core DNA-based vaccines induced both humoral and cellular immune responses in vivo. The role of protein secretion with regard to stimulation of the optimal immune response in the host has been analyzed with HBV-HCV fusion constructs. These results suggest that HCV DNA constructs are promising candidates as prophylactic and therapeutic vaccines.
456
COMPARISON OF ALBUMIN, DEXTRAN-70 AND H ~ C C E L IN THE PREVENTION OF EFFECTIVE HYPOVOLEMIA IN CIRRHOTIC PATIENTS WITH ASCITES TREATED WITH TOTAL PARACENTESIS. A RANDOMIZED M U L T I C E N T E R STUDY. International Group for the Study of Ascites in Cirrhosis.
Chowdhurv. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York. In Criglcr-Najjar syndrome type I and its animal model the Guun rat, a genetic deficiency ofbilirubin-UDP-glucuronosyltransferase-1 (B-UGT~), causes severe hyperbilirubinemia, resulting in neurotoxicity. We showed previously that injection of a haman B-UGTt-recombinant adenovirus in adult Gunn rats temporarily corrects the metabolic defect, ameliorating jaundice. The adenovirally transferred genes disappear because of host immune response, and reinjection of the virus fails to cause transgene expression. Here, we explored the possibility of achieving long-term expression of adenovirally transferred genes by inducing tolerance to the recombinant virus by injection into newborn rats. Two replication-defective recombinant adenoviruses were used: Ad-hBUGTt expressing human B-UGT~ and Ad-LacZ, expressing bacterial 13-galactosidase, both from a cytomegaiovirus promoter. Newborn Gunn rats (1-3 day old), or eongeneic normal Wistar rats were injected 20 ~1 of the purified recombinant viurses ( 2x108 pfu) into jugular veins. Seven days aRer injection of Ad-LacZ, nearly all hepatocytes were positive for 13-galactosidase activity ; 1-2% of the cells remained positive at 9 weeks. In Ad-hBUGT:-injected Gann rat pups, 4 weeks aRer injection, serum bilirubin concentrations were reduced to 65 to 83 panel/liter (n=4), compared to 134 tmaol/liter in AdLacZ injected controls. Two of these pups were followed for 9 weeks without further manipulation, and their serum bilirubin levels remained unchanged. The two other Ad-hBUGT~-treated pups were reinjected with AdhBUGT I (6xl0 s pfu i.e.) 4 weeks after initial injection. In these rats serum bilirubin levels were further reduced to 3.9 to 24 pruol/liter, and remained at that level throughout the study period (9 weeks). HPLC of the bile showed excretion of significant amounts of bilirubin ghcuronides 9 weeks after injection. Haman BUGT~ was detected in liver biopsy samples by immunoblot. CONCLUSION: I.V~ injection during the newborn period tolerizes rats to recombinant adenoviruses, whereby prolonged transgene expression can be achieved by repeated injection. In view of the increasing fi'equency of prenatal diagnosis of inherited diseases, this finding is potentially important in haman gene therapy.
455
PREVENTION OF HCV INFECTION IN CHIMPANZEES BY HYPERIMMUNE SERUM AGAINSTTHE HYPERVARIABLE REGION I (HVR1): EMERGENCE OF NEUTRALIZATION ESCAPE MUTANTS IN VIVO. P Farci. A Shimoda. D Won~. T Cabezon. D De Gioannis. A Strazzera. M Shaoiro. HJ Alter and RH Purcell. Lab. of Infectious Diseases, NIAID, Bethesda, SmithKline Beecham, Belgium, Dept. Internal Medicine, Univ. of Cagliari, Italy, Transfusion Medicine, NIH, Bethesda, Bioqual, Rockville, MD. To investigate whether the HVR1 of HCV is a critical neutralization domain, we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to the sequence of the HVR1 (anti-HVR1) of strain H77, a pedigreed virus titered in chimpanzees. The serum was reactive in ELISA against the homologous peptide and the recombinant El/E2 protein complex. The neutralization test was performed by mixing the virus inoeulum (64 CID~0) in vitro either with the anti- HVR1 serum, with the pre-immune rabbit serum, with an anti-HCV-negative human plasma or with the H79 plasma which was previously shown to contain neutralizing antibodies against H77, each at a final dilution of 1: l 0 and previously inactivated at 56°C for 30 min. The mixtures were kept overnight at 4°C and the residual infectivity was tested by IN. inoculation into seronegative chimpanzees. Five chimpanzees were studied: two received the H77 virus plus anti-HVR 1, one H77 plus the preimmune rabbit serum, one H77 plus the normal plasma and one I-I77plus H79 plasma. Serum I-ICVRNA was tested by PCR with primers from the 5'NC region. Selected PCR-positive samples were amplified with primers from the El/E2 genes and analyzed both by direct sequencing and by sequencing of u 10 molecular clones each. Sequence analysis of 104 clones from the H77 viral inoculum revealed at least 19 viral variants. The synthetic peptide used for immunization was based on the predominant variant (70/104 clones). Three minor variants were represented by 6, 5 and 4 clones, respectively, 4 by 2 clones and I 1 by a single clone. Following challenge, HCV infection was not detected in one of the 2 animals inoculated with H77 mixed with anti-HVRl. However, the other animal developed HCV viremia, hepatitis and antibody seroconversion. Interestingly, none of the 10 HCV molecular clones derived from this animal had the same sequence as the predominant viral strain, but, rather, that of two minor variants (one represented by 6/104 and the other by 2/t04 of the original clones). In contrast, the predominant viral strain (70/104), together with a minor variant (6/104) was consistently detected in the other three animals. All three developed hepatitis C regardless of the plasma used for neutralization. Similar data were obtained by direct sequencing. Conclusions: Complete protection from HCV infection in one animal and protection against the predominant HCV strain in a second animal suggests that a hyperimune serum against HVRI can neutralize HCV and that HVRI is a critical neutralization domain. The study also provides evidence for the emergence of neutralization escape mutants in vivo, a strategy whereby HCV can evade the host's immune response and establish persistent infection.
HEPATOLOGY October 1995
Large volume paracentesis without plasma volume expansion is constantly associated with an impairment in effective intravascular volume, as indicated by marked activation of endogenous vasoconstrictor systems. Albumin is highly effective in preventing this abnormality. However, it is not well established whether other plasma expanders are also effective. To investigate the efficacy of three different plasma expanders in the prevention of paracentesis-induoed effective hypovolemia, 289 cirrhotics with ascites were randomly allocated to treatment with total paracentesis and intravenous album/n (8 g/l of ascites removed, n=97), dextran-70 (8 g/l, n=93) or hemaccel (8 g/l, n=99). The effect of treatment on effective intravascular volume was assessed by measuring plasma renin activity (PRA) before and 6 and 30 days after treatment. The incidence of effective hypovolentia (~50% increase in PRA over baseline values to a level greater than 4 ng/ml.h at day 6) was markedly lower in patients treated with albumin (18%) than in those treated with dextran-70 or hemaccel (34 and 38%, respectively, p<0.01). In patients who developed effective hypovolemia pRA remained markedly increased over baseline values 30 days after treatment. Variables independently associated with a greater risk of developing effective hypovolemia were volume of ascitic fluid removed and type of plasma expander used. Patients who developed effective hypovolemia had significantly higher probability of readmission for ascites during follow-up (51% at 6 months)and lower probability of survival (57% at 6 months) as compared to patients who did not develop effective hypovolemia (32% and 75%, respectively). Effective hypovolemia after treatment was an independent predictor of survival. In summary, albumin is more effective than dextran-70 and hemaccel in preventing effective hypovolenda after paracentesis. This abnormality is not spontaneously reversible and is associated with a greater risk of ascites recurrence and shorter survival. Albumin should be considered the plasm~ expander of choice in cirrhotic patients treated with paraoentesis.