204 IL 28B Enhances Cellular Immunity by Reduction of T Regulatory Cell Populations during DNA Vaccination Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene[.]
INFECTIOUS DISEASES AND VACCINES I resistant to stretch, or more stiff, compared to BL10 mice In summary, the absence of dystrophin increases stiffness (decreases compliance) at the MTJ The decreased mechanical compliance of the MTJ may explain the reduced mobility seen in DMD patients Gene therapy for DMD should consider both the muscle and the MTJ 202 Transgene Expression over a Prolonged Observation Period after bFGF Gene Therapy To Promote Healing of Injured Flexor Tendons Jin Bo Tang,1,2 Yi Cao,1 Chuan Hao Chen,1 Ya Fang Wu,1 Xiao Tian Wang,2 Paul Y Liu.2 Hand Surgery Research Center, Department of Hand Surgery, Affiliated Hospital of Nantong University, Nantong, Jingsu, China; Gene Therapy, Department of Surgery, Roger Williams Medical Center, Boston University School of Medicine, Providence, RI Introduction Previously, we demonstrated that AAV2-bFGF gene therapy significantly increases the strength of flexor tendons but does not increase adhesions An ideal gene therapy for wound healing should produce an increase in transgene expression during the healing process, but “shut off” of this expression when healing is achieved For this reason, we investigated expression of the transgene—bFGF gene—over a prolonged period (16 weeks) in injured flexor tendons Methods 50 long toes of 25 chickens were used The flexor digitorum profundus (FDP) tendons of 20 toes were transected completely, and were repaired surgically with modified Kessler repair method The tendons were subsequently injected with AAV2-bFGF at 2´109 viral particles/tendon, into sites in the proximal and distal tendon stumps The AAV2-bFGF harbors bFGF gene of rat origin 20 FDP tendons in 20 long toes of 10 chickens received no injection 10 tendons from 10 long toes of the remaining chickens were not injured, serving as normal controls At postoperative weeks 2, 4, 8, 12, and 16, tendons were harvested and stained immunohistochemically to determine the presence of bFGF Real-time PCR analysis was performed to determine levels of overall bFGF gene expression Results Compared with the controls, compound expression of bFGF and expression of delivered bFGF genes was drastically increased at to weeks in the tendons that had undergone bFGF gene therapy From weeks to 16, the amount of bFGF in the AAV2-bFGF treated tendon declined At 16 weeks, the amount of bFGF in the gene therapy tendons was identical to that in the controls Expression of the bFGF gene decreased from week 12 to 16 At week 16, compound expression level of the endogenous and delivered bFGF gene returned to the level identical to that in the uninjured tendon The expression of delivered rat bFGF was not detectable in real-time PCR analysis Discussion: In a clinical relevant animal model involving complete cut and surgical repairs of the tendon, our investigation showed that the compound expression of bFGF gene in the AAV2-bFGF treated tendon peaks in the most critical period of the tendon healing The transgene expression peaks in that healing period However, supernormal expression of the bFGF transgene, and excessive production of bFGF, cease when tendon healing is completed This information is important in that it shows that bFGF gene therapy does not result in problems of bFGF overproduction after tendon wounds have healed 203 An Investigation of Efficiency of Gene Delivery Methods and Time-Course of Transgene Expression in Injured Tendons and Tissue Reactions Caused by Different Vectors Jin Bo Tang,1 Chuan Hua Chen,1 Ya Fang Wu,1 Bella Avanessian,2 Xiao Tian Wang,2 Paul Y Liu.2 Department of Hand Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; 2Gene Therapy, Department of Surgery, Roger Williams Medical Center, Providence, RI Introduction Comparisons of efficiency of gene delivery—through different vector systems—for in vivo tendon healing have not been reported previously We investigated efficiency of gene delivery to injured tendons and tissue reactions caused by different vectors Methods Plasmid, adeno-associated viral (AAV), and adenoviral vectors were used to transfect the injured tendon using 72 digital flexor tendons of bilateral toes of 18 white leghorn chickens After transverse tendon cuts, pCMV-EGFP, pCAG-EGFP, rAAV2-EGFP, and Ad5-EGFP were injected into the tendons At 3, 7, 14, and 21 days, the tendons were subjected to examination under a fluorescence microscope for GFP expression to determine the efficiency of transgene delivery by different vectors These sections were also stained with DAPI, and the percent of GFP-positive cells in the sections were recorded The tendon sections were also stained with hematoxylin and eosin to examine the inflammation caused by these vectors Inflammatory cells were counted under a microscope and were compared statistically The results of cell counts were analyzed statistically using ANOVA followed by post hoc Tukey test Results, The GFP expression, compared with normal tendons, was observed in experimental tendons at 3, 7, 14 and 21 days post-injection; for all vectors, it was highest at days At 14 days, a marked decrease in GFP expression was observed The GFP expression in the tendons injected with either rAAV2-EGFP or Ad5-EGFP was higher than in those injected with pCMV-EGFP or pCAG-EGFP vectors At days 3, 7, and 14, the percentage of GFP-positive cells in AAV2-GFP and Ad5GFP treated tendons was significantly higher than that in the plasmid vector treated tendons (p < 0.05 or p < 0.001) At days and 14, the AAV2-GFP treated tendons had the highest percentage of the cells showing positive GFP (p < 0.05 or p < 0.01) Tissue reactions of the tendons induced by the liposome-plasmid vectors (including pCMVEGFP and pCAG-EGFP) were the most prominent Inflammatory reactions were least severe in tendons injected with AAV2 vector Conclusions Among the vectors tested, efficiency of gene delivery was highest using AAV2 and Ad5 vectors The AAV2 vector caused the mildest tissue reaction This study suggests that the AAV2 vector is a promising gene delivery vector for tendon healing gene therapy Its time-course of transgene expression the expression starts very early in the tendon healing process and its expression drops as the healing proceeds can be favorable to the tendon healing, and avoids potentially detrimental effects of prolonged gene expression Infectious Diseases and Vaccines I 204 IL-28B Enhances Cellular Immunity by Reduction of T Regulatory Cell Populations during DNA Vaccination Matthew P Morrow, Panyupanok Pankhong, Dominick J Laddy, Kimberly A Kraynyak, Jian Yan, Neil Cisper, David B Weiner Pathology and Laboratory Medicine, The University of Pennsylvania, Philadelphia, PA Improving the potency of immune response is paramount among issues concerning vaccines to deadly pathogens, such as HIV IL-28B belongs to the newly described Interferon Lambda (IFNλ) family of cytokines, and has not yet been assessed for its potential ability to act as a vaccine adjuvant We tested the ability of plasmid encoded S80 Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 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 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 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 Special Pathogens, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, MB, Canada; 2Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada; 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 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 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 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 S81 ... during in an adjuvant setting Moreover, we describe for the first time how IL2 8B reduces Regulatory T cell populations during DNA vaccination while increasing the percentage of splenic CD8+ T. .. 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...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