document
1
450A AASLD ABSTRACTS HEPATOLOGY October 2001 1111 VACCINATION OF CHIMPANZEES WITH A MULTICOMPONENT DNA- PROTEIN PRIME-BOOST REGIMEN PROVIDES AN ENHANCED RE- COVERY FOLLOWING HETEROLOGOUS HCV CHALLENGE. J Heeney, J Drexhage, B Verstrepen, C Ronier, BPRC, Rijswijk Netherlands; G Mares, D Jacobs, E Depla, G Maertens, Innogenetics, Ghent Belgium; C Brinster, IN- SERM, Lyon France; A Foumfllier, BioMerieux, Lyon France; E Verschoor, BPRC, Rijswijk Netherlands; G Inchauspe, INSERM, Lyon France Vaccine protection from heterologous HCV infection requires immunisation strategies which will elicit immunity to a variety of diverse HCV variants. Towards this goal we have developed a strategy based on the use of multiple structural and nonstructural proteins in an effort to direct immune responses to different stages of virus replication. Furthermore we have used a DNA prime subunit-boost regimen in an effort to modify and increase the magnitude of the immune responses. Two chimpanzees were immunised with four plasmids expressing HCV core, El, E2 and NS3 antigens followed by booster injections performed with the corresponding recombinant proteins. Specific HCV re- sponses were low and in most cases below detection following the DNA prim- ing but were markedly increased after subunit boosting. Antibody responses to E1 and NS3 reached highest titers (up to 1:10 000 and 1 : 50 000 respectively). The fourth protein immunisation was administered after a 4 month interval and had a significant booster effect on antibody responses to all antigens. With regard to T-cell responses, antigen specific proliferation to El, E2 and NS3 became predominant following the second subunit immunisation. Enumera- tion of antigen specific cytokine secreting cells revealed the repeated boosting of both antigen specific IL2 and IL4 secreting cell populations in both immun- ised animals. Upon heterologous challenge (HCV-J strain, genotype lb, 25 CIDS0 infectious dose) a post-challenge peak viremia developed in both ani- mals as well as the control chimpanzee. The control animal remained viremic during the 26 weeks follow-up period with a peak titer of 46 990 UI / ml seen at 4 weeks post-challenge (p.c). Peaks of viremia were 24 580 UI / ml and 17 300 UI / ml in the two vaccinees respectively. One of the vaccinees became negative by the Roche test as early as the 4th week post-challenge (at week 11 by the nested in-house PCR) while RNA was below the level of detection of the Roche assay at week 18 p.c. for the second vaccinee. There was a clear anam- nestic antibody and/or cellular response following challenge in both immun- ised animals. 1112 CO-IMMUNIZATION WITH FLT3-L, A DENDRITIC CELL GROWTH AND STIMULATING FACTOR, ENHANCES HUMORAL AND CELLULAR IMMUNE RESPONSES AFTER GENETIC IMMUNIZATION AGAINST HEPATITIS C VIRUS IN MICE. Jens C Encke, Jomo Bernardin, University of Heidelberg, Heidelberg Germany; Raymond Buidoso, University of Cam- bridge, Cambridge Uk; Wolfgang Stremmel, University of Heidelberg, Heidel- berg Germany Genetic immunization is a promising vaccination technique to induce cellular and humoral immune responses against a specific antigen. In several animal models against a variety of pathogens including HCV, DNA vaccination has been proven to have therapeutic and/or prophylactic potential. Some recent results from preclinical trials with genetic vaccines demonstrate the potency of this vaccination approach in infectious diseases like malaria or HIV. However, success depends largely on the strength and the broadness of the induced immune responses. Unfortunately, the data from HCV DNA vaccines in mice indicate that immune responses after immunization are only weak. Therefore we developed a new co-immunization approach with FLT3-L, the most specific and strongest dendritic cell (DC) growth factor. DC are the target cells and key players in inducing immune responses after genetic vaccination and play a crucial role as central antigen presenting cells. Since recombinant FLT3-L can expand DC in vivo more then 20 times, we used a plasmid expressing FLT3-L to improve the vaccination strategy against HCV. After cloning an immuniza- tion FLT3-L construct into a CMV-promoter-driven expression plasmid, we were able to detect high levels of FLT3-L after transient transfection in the cytoplasm as well as secreted form in the supernatant, shown by Westeru-Blot and ELISA technique. After genetic coimmunization of BALB/c mice with FLT3-L and an HCV NS5 immunization construct a strong B cell activation and antibody production against the target NS5 HCV protein was found. Further- more, a TH1 directed induction of a T cell response was observed measured by T cell proliferation assays and ELISPOT. These results were compared to sim- ilar experiments with a GM-CSF vector, which is known to be a strong prolif- eration and differentiation factor of hematopoetic cells. Combinations of FLT3-L/GM-CSF/HCV-NS5 demonstrated so far the strongest immune re- sponses. Studies are underway to evaluate the prophylactic and therapeutic potential of this co-immunization approach in an HCV tumor model. Our results clearly indicate that a combination vaccination strategy with FLT3-L and a HCV antigen can significandy improve the B and T cell response and has important implications for immunotherapeutic approaches against HCV as well as other infectious diseases or malignancies. 1113 A NOVEL, DIRECT METHOD FOR MEASURING HCV KINETICS IN VIVO BASED ON IMMUNOAFFINITY CHROMATOGRAPHY AND MASS SPEC- TROMETRY. Raghavakaimal Sreekumar, Deborah Rasmussen, John Poter- ucha, John Gross, Michael Charlton, Division of Gastroenterology and Hepa- tology, Mayo Clinic, Rochester, MN Background: To date, studies of HCV kinetics have all relied upon indirect measures of viral turnover, incorporating mathematical models that are depen- dent on assumptions of viral kinetics, number of infected hepatocytes, inter- feron mechanisms of action and efficacy. The estimated half life (tl/2) of hepatitis C virions has varied greatly among such studies. Aims: To directly measure the fractional synthesis rate (FSR) of HCV ill chronically infected patients. Methods: A modified envelope protein, based on a conserved region of E2, was used to generate a monoclonal murine (Balb/c) anti-HCV antibody for immunoaffinity chromatography. Specificity of the anti-E2 antibody for HCV was determined by silver stain and by direct sequencing of peptides eluted from the immunoaffinity column. The fractional synthesis rate of HCV was measured in six subjects with chronic HCV infection by determining the rate of incorporation of 13C-leucine into HCV during a continuous infusion of L-[1-13C]-leucine. Study subjects were receiving no regular medications. En- richments of 13C-leucine in the hydrolysates of HCV were measured by gas chromatography mass spectrometry. Abundance of 13C-leucine in ketoisoca- proic acid was used for estimation of precursor pool enrichment. Results: Four participants with HCV genotype 1 and two with non-1 genotypes were studied. The enrichment (atoms percent excess) of 13C-leucine in hydrolysed HCV increased linearly between 120 and 360 minutes of isotope infusion. All cal- culations of synthesis rates of HCV were made during this linear phase of increment in enrichment. Using this method the fractional synthesis rate of HCV (mean -+ SD) was calculated to be 57.5 ± 9.8%/day, (range 46.2-64.2°£/ day). As the level of HCV viremia was steady, as measured by serial HCV RNA quantitation, clearance of HCV must equal the rate of synthesis. In these study subjects, HCV viral load is replaced every 41.7hrs. Conclusions: A novel method for directly measuring the synthesis rate of HCV has been developed. This method will allow the relative impact of antiviral agents, immunosuppres- sive agents, HCV genotype and host variables on HCV production to be deter- mined. By combining HCV synthesis rates with levels of HCV viremia, non- derivative calculations of HCV kinetics, incorporating rates of synthesis and clearance, can be made. 1114 THE ACTIN BINDING PROTEIN CORTACTIN MEDIATES CLATHRIN- BASED ENDOCYTOSIS IN HEPATOCYTES. Hong Cat, James D Orth, Jing Chen, Mark A McNiven, Mayo Clinic, Rochester, MN Endocytosis of plasma proteins is a major function of the hepatocyte although the molecular machinery that supports this essential process is undefined. Further, the actiI1 cytoskeleton is believed to contribute to endocytic pit for- mation, but the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an 80-kDa F-acdn associated protein and a prominent substrate of src kinases. This protein has been shown to localize within membrane ruffles in cultured hepatocytes and is a direct binding partner of the large GTPase, mechanoenzyme, dynamin (McNiven et al., 2000). This previous study demonstrated that the SH3 domain of cortactin interacts directly with the proline rich domain (PRD) of dynamin 2 (Dyn2) and regulates actin cytoskeleton reorganization and subsequently hepatocellular shape. Because of these characteristics, cortactin is a likely candidate to link the actin cytoskeleton to endocytosis. Therefore, the GOAL of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis in hepatocytes. METHODS: Immunofluores- cence microscopy was used to test if cortactin was associated with elathrin- coated pits in a cultured hepatocyte cell line (Clone 9). Functional studies were performed by microinjection of purified, concentrated, cortactin-specific anti- bodies or transient expression of a mutant cortactin construct lacking the interactive C-terminal SH3 domain (CortactinASH3). Manipulated cells were either processed for electron microscopy using the ruthenium red technique to visualize membrane invaginations or tested for the ability to internalize labeled transferrin (receptor-mediated) or dextran (fluid-phase). RESULTS: Immuno- fluorescence experiments revealed a striking colocalization of cortaetin and clathrin at the plasma membrane (PM) in discrete puncta representing endo- cytic invaginations. Consistent with this observation, cells microinjected with anti-cortactin antibodies or the cortactinASH3 construct displayed a dramatic decrease in the uptake of labeled transferrin but exhibited normal internaliza- tion of dextran. Interestingly, antibody injection markedly altered the cyto- plasmic distribution of clathrin, inducing large amounts of this polymer to associate with the PM compared to control injected cells. This increased mem- brane affinity suggests that normal cortactin function is required to support the formation and vesiculation of clathrin-coated pits from the PM. EM of these inhibited cells was consistent with this finding and showed little, if any, endo- cytic membrane invaginations, suggesting that clathrin-coated pit formation is prevented at early stages by the cortactin antibodies. CONCLUSIONS: These observations are the first to link a specific component of the actin cytoskeleton, cortactin, to the endocytic machinery in hepatocytes or any other mammalian cell.
Transcript of document
450A AASLD ABSTRACTS HEPATOLOGY October 2001
1111
VACCINATION OF CHIMPANZEES WITH A MULTICOMPONENT DNA- PROTEIN PRIME-BOOST REGIMEN PROVIDES AN ENHANCED RE- COVERY FOLLOWING HETEROLOGOUS HCV CHALLENGE. J Heeney, J Drexhage, B Verstrepen, C Ronier, BPRC, Rijswijk Netherlands; G Mares, D Jacobs, E Depla, G Maertens, Innogenetics, Ghent Belgium; C Brinster, IN- SERM, Lyon France; A Foumfllier, BioMerieux, Lyon France; E Verschoor, BPRC, Rijswijk Netherlands; G Inchauspe, INSERM, Lyon France
Vaccine protection from heterologous HCV infection requires immunisation strategies which will elicit immunity to a variety of diverse HCV variants. Towards this goal we have developed a strategy based on the use of multiple structural and nonstructural proteins in an effort to direct immune responses to different stages of virus replication. Furthermore we have used a DNA prime subunit-boost regimen in an effort to modify and increase the magnitude of the immune responses. Two chimpanzees were immunised with four plasmids expressing HCV core, El, E2 and NS3 antigens followed by booster injections performed with the corresponding recombinant proteins. Specific HCV re- sponses were low and in most cases below detection following the DNA prim- ing but were markedly increased after subunit boosting. Antibody responses to E1 and NS3 reached highest titers (up to 1:10 000 and 1 : 50 000 respectively). The fourth protein immunisation was administered after a 4 month interval and had a significant booster effect on antibody responses to all antigens. With regard to T-cell responses, antigen specific proliferation to El, E2 and NS3 became predominant following the second subunit immunisation. Enumera- tion of antigen specific cytokine secreting cells revealed the repeated boosting of both antigen specific IL2 and IL4 secreting cell populations in both immun- ised animals. Upon heterologous challenge (HCV-J strain, genotype lb, 25 CIDS0 infectious dose) a post-challenge peak viremia developed in both ani- mals as well as the control chimpanzee. The control animal remained viremic during the 26 weeks follow-up period with a peak titer of 46 990 UI / ml seen at 4 weeks post-challenge (p.c). Peaks of viremia were 24 580 UI / ml and 17 300 UI / ml in the two vaccinees respectively. One of the vaccinees became negative by the Roche test as early as the 4th week post-challenge (at week 11 by the nested in-house PCR) while RNA was below the level of detection of the Roche assay at week 18 p.c. for the second vaccinee. There was a clear anam- nestic antibody and/or cellular response following challenge in both immun- ised animals.
1112
CO-IMMUNIZATION WITH FLT3-L, A DENDRITIC CELL GROWTH AND STIMULATING FACTOR, ENHANCES HUMORAL AND CELLULAR IMMUNE RESPONSES AFTER GENETIC IMMUNIZATION AGAINST HEPATITIS C VIRUS IN MICE. Jens C Encke, Jomo Bernardin, University of Heidelberg, Heidelberg Germany; Raymond Buidoso, University of Cam- bridge, Cambridge Uk; Wolfgang Stremmel, University of Heidelberg, Heidel- berg Germany
Genetic immunization is a promising vaccination technique to induce cellular and humoral immune responses against a specific antigen. In several animal models against a variety of pathogens including HCV, DNA vaccination has been proven to have therapeutic and/or prophylactic potential. Some recent results from preclinical trials with genetic vaccines demonstrate the potency of this vaccination approach in infectious diseases like malaria or HIV. However, success depends largely on the strength and the broadness of the induced immune responses. Unfortunately, the data from HCV DNA vaccines in mice indicate that immune responses after immunization are only weak. Therefore we developed a new co-immunization approach with FLT3-L, the most specific and strongest dendritic cell (DC) growth factor. DC are the target cells and key players in inducing immune responses after genetic vaccination and play a crucial role as central antigen presenting cells. Since recombinant FLT3-L can expand DC in vivo more then 20 times, we used a plasmid expressing FLT3-L to improve the vaccination strategy against HCV. After cloning an immuniza- tion FLT3-L construct into a CMV-promoter-driven expression plasmid, we were able to detect high levels of FLT3-L after transient transfection in the cytoplasm as well as secreted form in the supernatant, shown by Westeru-Blot and ELISA technique. After genetic coimmunization of BALB/c mice with FLT3-L and an HCV NS5 immunization construct a strong B cell activation and antibody production against the target NS5 HCV protein was found. Further- more, a TH1 directed induction of a T cell response was observed measured by T cell proliferation assays and ELISPOT. These results were compared to sim- ilar experiments with a GM-CSF vector, which is known to be a strong prolif- eration and differentiation factor of hematopoetic cells. Combinations of FLT3-L/GM-CSF/HCV-NS5 demonstrated so far the strongest immune re- sponses. Studies are underway to evaluate the prophylactic and therapeutic potential of this co-immunization approach in an HCV tumor model. Our results clearly indicate that a combination vaccination strategy with FLT3-L and a HCV antigen can significandy improve the B and T cell response and has important implications for immunotherapeutic approaches against HCV as well as other infectious diseases or malignancies.
1113
A NOVEL, DIRECT METHOD FOR MEASURING HCV KINETICS IN VIVO BASED ON IMMUNOAFFINITY CHROMATOGRAPHY AND MASS SPEC- TROMETRY. Raghavakaimal Sreekumar, Deborah Rasmussen, John Poter- ucha, John Gross, Michael Charlton, Division of Gastroenterology and Hepa- tology, Mayo Clinic, Rochester, MN
Background: To date, studies of HCV kinetics have all relied upon indirect measures of viral turnover, incorporating mathematical models that are depen- dent on assumptions of viral kinetics, number of infected hepatocytes, inter- feron mechanisms of action and efficacy. The estimated half life (tl/2) of hepatitis C virions has varied greatly among such studies. Aims: To directly measure the fractional synthesis rate (FSR) of HCV ill chronically infected patients. Methods: A modified envelope protein, based on a conserved region of E2, was used to generate a monoclonal murine (Balb/c) anti-HCV antibody for immunoaffinity chromatography. Specificity of the anti-E2 antibody for HCV was determined by silver stain and by direct sequencing of peptides eluted from the immunoaffinity column. The fractional synthesis rate of HCV was measured in six subjects with chronic HCV infection by determining the rate of incorporation of 13C-leucine into HCV during a continuous infusion of L-[1-13C]-leucine. Study subjects were receiving no regular medications. En- richments of 13C-leucine in the hydrolysates of HCV were measured by gas chromatography mass spectrometry. Abundance of 13C-leucine in ketoisoca- proic acid was used for estimation of precursor pool enrichment. Results: Four participants with HCV genotype 1 and two with non-1 genotypes were studied. The enrichment (atoms percent excess) of 13C-leucine in hydrolysed HCV increased linearly between 120 and 360 minutes of isotope infusion. All cal- culations of synthesis rates of HCV were made during this linear phase of increment in enrichment. Using this method the fractional synthesis rate of HCV (mean -+ SD) was calculated to be 57.5 ± 9.8%/day, (range 46.2-64.2°£/ day). As the level of HCV viremia was steady, as measured by serial HCV RNA quantitation, clearance of HCV must equal the rate of synthesis. In these study subjects, HCV viral load is replaced every 41.7hrs. Conclusions: A novel method for directly measuring the synthesis rate of HCV has been developed. This method will allow the relative impact of antiviral agents, immunosuppres- sive agents, HCV genotype and host variables on HCV production to be deter- mined. By combining HCV synthesis rates with levels of HCV viremia, non- derivative calculations of HCV kinetics, incorporating rates of synthesis and clearance, can be made.
1114
THE ACTIN BINDING PROTEIN CORTACTIN MEDIATES CLATHRIN- BASED ENDOCYTOSIS IN HEPATOCYTES. Hong Cat, James D Orth, Jing Chen, Mark A McNiven, Mayo Clinic, Rochester, MN
Endocytosis of plasma proteins is a major function of the hepatocyte although the molecular machinery that supports this essential process is undefined. Further, the actiI1 cytoskeleton is believed to contribute to endocytic pit for- mation, but the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an 80-kDa F-acdn associated protein and a prominent substrate of src kinases. This protein has been shown to localize within membrane ruffles in cultured hepatocytes and is a direct binding partner of the large GTPase, mechanoenzyme, dynamin (McNiven et al., 2000). This previous study demonstrated that the SH3 domain of cortactin interacts directly with the proline rich domain (PRD) of dynamin 2 (Dyn2) and regulates actin cytoskeleton reorganization and subsequently hepatocellular shape. Because of these characteristics, cortactin is a likely candidate to link the actin cytoskeleton to endocytosis. Therefore, the GOAL of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis in hepatocytes. METHODS: Immunofluores- cence microscopy was used to test if cortactin was associated with elathrin- coated pits in a cultured hepatocyte cell line (Clone 9). Functional studies were performed by microinjection of purified, concentrated, cortactin-specific anti- bodies or transient expression of a mutant cortactin construct lacking the interactive C-terminal SH3 domain (CortactinASH3). Manipulated cells were either processed for electron microscopy using the ruthenium red technique to visualize membrane invaginations or tested for the ability to internalize labeled transferrin (receptor-mediated) or dextran (fluid-phase). RESULTS: Immuno- fluorescence experiments revealed a striking colocalization of cortaetin and clathrin at the plasma membrane (PM) in discrete puncta representing endo- cytic invaginations. Consistent with this observation, cells microinjected with anti-cortactin antibodies or the cortactinASH3 construct displayed a dramatic decrease in the uptake of labeled transferrin but exhibited normal internaliza- tion of dextran. Interestingly, antibody injection markedly altered the cyto- plasmic distribution of clathrin, inducing large amounts of this polymer to associate with the PM compared to control injected cells. This increased mem- brane affinity suggests that normal cortactin function is required to support the formation and vesiculation of clathrin-coated pits from the PM. EM of these inhibited cells was consistent with this finding and showed little, if any, endo- cytic membrane invaginations, suggesting that clathrin-coated pit formation is prevented at early stages by the cortactin antibodies. CONCLUSIONS: These observations are the first to link a specific component of the actin cytoskeleton, cortactin, to the endocytic machinery in hepatocytes or any other mammalian cell.
