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206. Enhanced Efficacy of Improved Adenovirus-Based Ebola Virus Vaccine
INFECTIOUS DISEASES AND VACCINES I IL-28B to boost immune responses to a multi-clade consensus HIV Gag plasmid during DNA vaccination. We show here for the first time that IL-28B robustly enhances immune responses during in an adjuvant setting. Moreover, we describe for the first time how IL28B reduces Regulatory T cell populations during DNA vaccination while increasing the percentage of splenic CD8+ T cells in vaccinated animals. These cells are more granular and have higher antigenspecific cytolytic degranulation when compared to cells taken from animals that received antigen alone. Lastly, we are the first to report that plasmid encoded IL-28B can induce 100% protection from mortality after a lethal influenza challenge. These data suggest that IL-28B is a strong candidate for further studies of vaccine or immunotherapy protocols.
205. Evaluation of AAV-Mediated IFNα-Fene Therapy Efficacy in HBV Transgenic Mice: Constitutive Versus Inducible Expression
Lucia Vanrell,1 Cristina Olague,1 Africa Vales,1 Lilian Tenembaum,2 Gloria Gonzalez-Aseguinolaza.1 1 Division of Gene Therapy, School of Medicine, Center for Applied Medical Research, University of Navarra, Pamplona, Navarra, Spain; 2Laboratory for Experimental Neurosurgery, Université Libre de Bruxelles, Brussels, Belgium. Background: Hepatitis B virus (HBV) and hepatitis C virus (HCV) constitute the main etiologic factors for the chronic viral hepatitis affecting more than 550 million people worldwide. Interferon alpha (IFNα) has demonstrated therapeutic efficacy in both chronic hepatitis B and C. However, the monotherapy response rate (25%35%) can be increased, and side effects limited, by improving the pharmacokinetic of IFNalpha therapy with stabilising ligands. Gene therapy allows a continuous in vivo expression of the transgene at the desired site. AAV vectors lack pathogenicity in humans and have demonstrated prolonged expression of numerous transgenes, in several tissues and immunocompetent animal models. Aims: Therapeutic efficacy evaluation of a recombinant AAV expressing murine IFN alpha (AAVIFN) under the control of a constitutive or an inducible promoter in HBV transgenic mice. Methods: We have produced AAV vectors expressing luciferase or murine IFNalpha1 into two different expression cassettes (AAVIFN). The first one contains the promoter for the human Elongation Factor-1 alpha (hEF1alpfa), the transgene, and the SV40 polyadenilation signal sequence. The second one contains a CMV-TetON promoter, the transgene, and the SV40 polyadenilation signal. AAV serotype 8 virus production was performed by cotransfection into 293 T cells of the plasmid containing the expression cassette flanked by the AAV ITRs, and the plasmids containing the necessary AAV and adenoviral proteins, using linear polyethylenimine (25 kDa). Viruses were purified by iodixanol centrifugation gradient and viral titers were determined by real time PCR. IFN-alpha concentration was determined by the cytopathic effect (CPE) reduction assay. Serum viral load was determined by qPCR after viral DNA purification from serum samples. Results AAV constitutively expressing murine IFNalpha was able to inhibit HBV viral replication to undetectable levels in HBV transgenic mice, while the administration of AAV expressing luciferase had no effect over viral replication. However, prolonged IFN-alpha expression resulted in severe side effects as shown by a profound pancytopenia that led to animal’s death. Experiments are now being performed to determine the antiviral efficacy and safety of the AAV vector expressing IFN-alpha in an inducible manner. Conclusion This report highlights the promises and the limitations of IFNα-gene therapy in viral hepatitis.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
206. Enhanced Efficacy of Improved Adenovirus-Based Ebola Virus Vaccine
Jason S. Richardson,1 Michel K. Yao,1 Kaylie N. Tran,1,2 Maria A. Croyle,5,6 James E. Strong,1,3,4 Heinz Feldmann,1,3 Gary P. Kobinger.1,3 1 Special Pathogens, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, MB, Canada; 2Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3 Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 4Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; 5Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, TX; 6Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX. Background: Zaire ebolavirus (ZEBOV) causes hemorrhagic fever with an associated death rate estimated as high as 90% in infected humans. Vaccine based on virus-like particles, vesicular stomatitis virus, human parainfluenza virus type 3 or adenovirus containing or expressing the ZEBOV envelope glycoprotein (ZGP) have successfully protected nonhuman primates from an otherwise lethal challenge of Ebola virus. Currently Phase I testing in humans using a recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZGP sequence from a human cytomegalovirus immediate early gene (CMV) promoter (Ad-CMVZGP) Ebola vaccine is underway. Objective: 1) Increase the expression of the ZGP antigen from an adenovirus serotype 5 vector. Improvements of the expression cassette included codon optimization for translation in mammalian cells, inclusion of a consensus Kozak sequence and engineering of a hybrid CAG promoter which combines the human cytomegalovirus immediate early gene enhancer and a modified chicken beta-actin promoter (Ad-CAGoptZGP). 2) Evaluate protection efficacy and T and B cell immune response elicited by Ad-CAGoptZGP compared to the standard Ad-CMVZGP vector on a dose-to-dose basis against lethal ZEBOV challenge in mice. Significant findings: In vitro, evaluation of the Ad-CAGoptZGP demonstrated a 3 to 7-fold increase in the expression of the ZGP antigen when compared to the Ad-CMVZGP vaccine. The Ad-CAGoptZGP vaccine fully protected mice against a lethal challenge with mouse-adapted ZEBOV at a dose 100 times lower than the minimal dose required to attain full protection with the Ad-CMV/ZGP vaccine. Mice administered the Ad-CAGoptZGP vaccine resulted in improved immune responses even at a dose 10 times lower for the T cell response and 100 times lower for the B cell response than with the Ad-CMVZGP vaccine. Unexpectedly, the improved Ad-CAGoptZGP vaccine afforded 100% protection for mice when administered 30 minutes post-exposure with mouseadapted ZEBOV, although weight loss was observed. Performance of the improved Ad-CAGoptZGP vaccine in the presence of pre-exisitng immunity to the vaccine carrier is currently being evaluated in guinea pigs and results will be presented. Understanding and improving the molecular components of adenovirus-based vaccines may be useful for vaccination and post-exposure therapy.
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205. Evaluation of AAV-Mediated IFNα-Fene Therapy Efficacy in HBV Transgenic Mice: Constitutive Versus Inducible Expression
Lucia Vanrell,1 Cristina Olague,1 Africa Vales,1 Lilian Tenembaum,2 Gloria Gonzalez-Aseguinolaza.1 1 Division of Gene Therapy, School of Medicine, Center for Applied Medical Research, University of Navarra, Pamplona, Navarra, Spain; 2Laboratory for Experimental Neurosurgery, Université Libre de Bruxelles, Brussels, Belgium. Background: Hepatitis B virus (HBV) and hepatitis C virus (HCV) constitute the main etiologic factors for the chronic viral hepatitis affecting more than 550 million people worldwide. Interferon alpha (IFNα) has demonstrated therapeutic efficacy in both chronic hepatitis B and C. However, the monotherapy response rate (25%35%) can be increased, and side effects limited, by improving the pharmacokinetic of IFNalpha therapy with stabilising ligands. Gene therapy allows a continuous in vivo expression of the transgene at the desired site. AAV vectors lack pathogenicity in humans and have demonstrated prolonged expression of numerous transgenes, in several tissues and immunocompetent animal models. Aims: Therapeutic efficacy evaluation of a recombinant AAV expressing murine IFN alpha (AAVIFN) under the control of a constitutive or an inducible promoter in HBV transgenic mice. Methods: We have produced AAV vectors expressing luciferase or murine IFNalpha1 into two different expression cassettes (AAVIFN). The first one contains the promoter for the human Elongation Factor-1 alpha (hEF1alpfa), the transgene, and the SV40 polyadenilation signal sequence. The second one contains a CMV-TetON promoter, the transgene, and the SV40 polyadenilation signal. AAV serotype 8 virus production was performed by cotransfection into 293 T cells of the plasmid containing the expression cassette flanked by the AAV ITRs, and the plasmids containing the necessary AAV and adenoviral proteins, using linear polyethylenimine (25 kDa). Viruses were purified by iodixanol centrifugation gradient and viral titers were determined by real time PCR. IFN-alpha concentration was determined by the cytopathic effect (CPE) reduction assay. Serum viral load was determined by qPCR after viral DNA purification from serum samples. Results AAV constitutively expressing murine IFNalpha was able to inhibit HBV viral replication to undetectable levels in HBV transgenic mice, while the administration of AAV expressing luciferase had no effect over viral replication. However, prolonged IFN-alpha expression resulted in severe side effects as shown by a profound pancytopenia that led to animal’s death. Experiments are now being performed to determine the antiviral efficacy and safety of the AAV vector expressing IFN-alpha in an inducible manner. Conclusion This report highlights the promises and the limitations of IFNα-gene therapy in viral hepatitis.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
206. Enhanced Efficacy of Improved Adenovirus-Based Ebola Virus Vaccine
Jason S. Richardson,1 Michel K. Yao,1 Kaylie N. Tran,1,2 Maria A. Croyle,5,6 James E. Strong,1,3,4 Heinz Feldmann,1,3 Gary P. Kobinger.1,3 1 Special Pathogens, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, MB, Canada; 2Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3 Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 4Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; 5Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, TX; 6Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX. Background: Zaire ebolavirus (ZEBOV) causes hemorrhagic fever with an associated death rate estimated as high as 90% in infected humans. Vaccine based on virus-like particles, vesicular stomatitis virus, human parainfluenza virus type 3 or adenovirus containing or expressing the ZEBOV envelope glycoprotein (ZGP) have successfully protected nonhuman primates from an otherwise lethal challenge of Ebola virus. Currently Phase I testing in humans using a recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZGP sequence from a human cytomegalovirus immediate early gene (CMV) promoter (Ad-CMVZGP) Ebola vaccine is underway. Objective: 1) Increase the expression of the ZGP antigen from an adenovirus serotype 5 vector. Improvements of the expression cassette included codon optimization for translation in mammalian cells, inclusion of a consensus Kozak sequence and engineering of a hybrid CAG promoter which combines the human cytomegalovirus immediate early gene enhancer and a modified chicken beta-actin promoter (Ad-CAGoptZGP). 2) Evaluate protection efficacy and T and B cell immune response elicited by Ad-CAGoptZGP compared to the standard Ad-CMVZGP vector on a dose-to-dose basis against lethal ZEBOV challenge in mice. Significant findings: In vitro, evaluation of the Ad-CAGoptZGP demonstrated a 3 to 7-fold increase in the expression of the ZGP antigen when compared to the Ad-CMVZGP vaccine. The Ad-CAGoptZGP vaccine fully protected mice against a lethal challenge with mouse-adapted ZEBOV at a dose 100 times lower than the minimal dose required to attain full protection with the Ad-CMV/ZGP vaccine. Mice administered the Ad-CAGoptZGP vaccine resulted in improved immune responses even at a dose 10 times lower for the T cell response and 100 times lower for the B cell response than with the Ad-CMVZGP vaccine. Unexpectedly, the improved Ad-CAGoptZGP vaccine afforded 100% protection for mice when administered 30 minutes post-exposure with mouseadapted ZEBOV, although weight loss was observed. Performance of the improved Ad-CAGoptZGP vaccine in the presence of pre-exisitng immunity to the vaccine carrier is currently being evaluated in guinea pigs and results will be presented. Understanding and improving the molecular components of adenovirus-based vaccines may be useful for vaccination and post-exposure therapy.
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