- Email: [email protected]
695. Generation of DNA Plasmid-Encoded Broadly Neutralizing Antibodies In Vivo
VACCINES AND IMMUNOTHERAPY without FGF9, there was significantly less Ad3D generated in FGF9treated cell. In contrast, the PtDd-producing Ad3, was not affected by FGF9-mediated HD5 expression. There was no effect of FGF9 on the titers of both viruses in HeLa cells, which do not produce HNP1 and HD5. These data indicate a functional link between PtDd in defensin inactivation. We are currently screening our collection of human cancer cell lines for defensin expression to correlate defensin levels with Ad3 spread in cultures and in xenograft tumors. Considering that oncolytic vectors based on Ad3 and the DSG2-interacting Ad11 have shown promise for cancer therapy in clinical trials, our studies on the role of PtDd and defensins in viral spread are relevant for oncolytic virotherapy of cancer.
695. Generation of DNA Plasmid-Encoded Broadly Neutralizing Antibodies In Vivo
Seleeke Flingai,1 Kar Muthumani,1 Megan Wise,1 Colleen Tingey,1 Kenneth Ugen,2,3 David B. Weiner.1 1 Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 2Morsani College of Medicine, University of South Florida, Tampa, FL; 3Center for Molecular Delivery, University of South Florida, Tampa, FL. The development of vaccines against viral pathogens such as HIV-1 and Dengue virus (DENV) has been wrought with difficulties. Recent advances in human antibody isolation have uncovered broadly neutralizing antibodies (bNAbs), which are capable of preventing infection against a wide array of viral strains. Yet generating and delivering biologically-relevant levels of such antibodies using conventional monoclonal antibody methodology is impractical, often requiring huge expenses and repeated administrations for clinical benefit. Creating new methods of delivering monoclonal antibodies could drastically tip the scale in the fight against a number of devastating pathogens. In this study, we have constructed optimized DNA plasmid formulations capable of expressing Fab fragments of the HIV-1 bNAb VRC01. When delivered with adaptive in vivo electroporation, a single administration of the optimized plasmid constructs resulted in generation of Fab molecules in mouse sera possessing gp120-binding and HIV-1 neutralizing activity against diverse HIV-1 isolates for at least 7 days. Importantly, this delivery method resulted in a more rapid increase (i.e. in as little as 48 hours) in gp120-binding Fab levels compared to immunization with recombinant gp120 protein. Further study of this technology has resulted in the in vivo generation of human IgG1 neutralizing antibodies targeting all four serotypes of DENV. This platform establishes a novel approach for delivering protective monoclonal antibodies in vivo.
696. Lentiviral-Based Anti-HIV Therapeutic Vaccine: Design, Preclinical Studies and PhaseI/II Clinical Trial Preliminary Results
Cécile Bauche,1 Emeline Sarry,1 Ana Bejanariu,1 Marie Rodriguez,1 Emmanuelle Sabbah-Petrover.1 1 THERAVECTYS, Villejuif, France. THERAVECTYS, develops a new generation of prophylactic and therapeutic vaccines using optimized lentiviral vectors. It’s most advanced product, a therapeutic anti-HIV vaccine treatment, has entered clinical Phase I/II end of 2012. This vaccination should allow seropositive patients to gain an immunological status identical to the so-called “Functional Cured” patients who develop an efficient immunological response capable of controlling the infection without therapy. Vaccine candidates are integrative and self-inactivated liverecombinant lentiviral vectors. They encode an HIV antigen, under the regulation of a patented promoter that is preferentially induced Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
in APC (generating the specific immune response), and showing a basal level expression in all cells (allowing their elimination by the settled immune response).These vaccine candidates are classified as “Live recombinant vectored vaccines” (EMA, 2011). Preclinical studies demonstrated i)the generation of a strong, specific and very long lasting T-cell immune response (up to 2 years in murine animal models), ii) the restricted diffusion of the vaccine candidates after injection and iii)their fast disappearance within few weeks, correlated with an absence of macroscopic and microscopic toxicity. These data allowed the settlement of the anti-HIV therapeutic Phase I/II clinical trial, that is held in France and Belgium and is actually enrolling 36 HIV-1 infected patients. Theravectys’ anti-HIV vaccine treatment is assessed at three doses and safety, tolerability and immunogenicity compared to a placebo group. Furthermore, vaccine efficiency will be evaluated by the interruption of the HAART treatment in all patients, including placebo. Results are expected in October 2014 with intermediary analysis in April 2013.
697. Vectored Expression of the BroadlyNeutralizing Anti-Influenza Antibody, FI6, in Airway Protects Mice Against Influenza Despite Preexisting Serum AAV Antibodies
Maria P. Limberis,1 Marco Crosariol,1 Julio Sanmiguel,1 Christine Draper,1 Roberto Calcedo,1 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. Vectored delivery of antibodies against infectious viruses is a novel strategy to achieve protection without requiring the mounting of an immune response that is traditionally generated by active immunization. We demonstrated that localized expression of protective anti-influenza antibodies at the airway mucosal surface where replication of airborne viruses initiates, is a safe and effective strategy for preventing influenza infection. Here we investigated the potential of an AAV-vectored vaccine to be effectively re-administered and protect mice against lethal challenge with H1N1. BALB/c mice were given AAV9 vector expressing either the anti-respiratory syncytial virus (RSV) antibody palivizumab or the reporter transgene firefly luciferase in half log increments ranging from 5E10 GC/kg to 5E12 GC/kg. Twenty-eight days later, mice were readministered intranasally with 5E12 GG/kg of AAV9 vector expressing the broadlyneutralizing anti-influenza antibody, FI6. At the time of AAV9 vector re-administration, the level of serum-circulating AAV9-specific neutralizing antibody ranged from 1:80 in mice dosed with 5E10 GC/ kg to 1:2,560 in mice dosed with 5E12 GC/kg of AAV9 expressing either palivizumab or firefly luciferase. Fourteen days later, mice were challenged intranasally with 20 LD50 of mouse-adapted H1N1 (PR8) and monitored daily for signs of disease. Mice that exhibited behavioral signs of distress or lost 30% of their initial body weight were euthanized. Naïve mice and mice that were administered AAV9 expressing palivizumab or firefly luciferase were not protected against challenge with PR8. In contrast, mice re-administered with AAV9 vector expressing FI6 were protected against PR8 despite the presence of serum circulating NAB titer that ranged from 1:80 to 1:5,120. Despite the presence of high level serum circulating AAV9 NAb (1:20) which blocks systemic administration of AAV9, intranasal AAV9 re-administration was successful in protecting against PR8 challenge. Our data demonstrate the potential of our approach to sequentially protect the host against different airborne pathogens by virtue of vector re-administration.
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695. Generation of DNA Plasmid-Encoded Broadly Neutralizing Antibodies In Vivo
Seleeke Flingai,1 Kar Muthumani,1 Megan Wise,1 Colleen Tingey,1 Kenneth Ugen,2,3 David B. Weiner.1 1 Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 2Morsani College of Medicine, University of South Florida, Tampa, FL; 3Center for Molecular Delivery, University of South Florida, Tampa, FL. The development of vaccines against viral pathogens such as HIV-1 and Dengue virus (DENV) has been wrought with difficulties. Recent advances in human antibody isolation have uncovered broadly neutralizing antibodies (bNAbs), which are capable of preventing infection against a wide array of viral strains. Yet generating and delivering biologically-relevant levels of such antibodies using conventional monoclonal antibody methodology is impractical, often requiring huge expenses and repeated administrations for clinical benefit. Creating new methods of delivering monoclonal antibodies could drastically tip the scale in the fight against a number of devastating pathogens. In this study, we have constructed optimized DNA plasmid formulations capable of expressing Fab fragments of the HIV-1 bNAb VRC01. When delivered with adaptive in vivo electroporation, a single administration of the optimized plasmid constructs resulted in generation of Fab molecules in mouse sera possessing gp120-binding and HIV-1 neutralizing activity against diverse HIV-1 isolates for at least 7 days. Importantly, this delivery method resulted in a more rapid increase (i.e. in as little as 48 hours) in gp120-binding Fab levels compared to immunization with recombinant gp120 protein. Further study of this technology has resulted in the in vivo generation of human IgG1 neutralizing antibodies targeting all four serotypes of DENV. This platform establishes a novel approach for delivering protective monoclonal antibodies in vivo.
696. Lentiviral-Based Anti-HIV Therapeutic Vaccine: Design, Preclinical Studies and PhaseI/II Clinical Trial Preliminary Results
Cécile Bauche,1 Emeline Sarry,1 Ana Bejanariu,1 Marie Rodriguez,1 Emmanuelle Sabbah-Petrover.1 1 THERAVECTYS, Villejuif, France. THERAVECTYS, develops a new generation of prophylactic and therapeutic vaccines using optimized lentiviral vectors. It’s most advanced product, a therapeutic anti-HIV vaccine treatment, has entered clinical Phase I/II end of 2012. This vaccination should allow seropositive patients to gain an immunological status identical to the so-called “Functional Cured” patients who develop an efficient immunological response capable of controlling the infection without therapy. Vaccine candidates are integrative and self-inactivated liverecombinant lentiviral vectors. They encode an HIV antigen, under the regulation of a patented promoter that is preferentially induced Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
in APC (generating the specific immune response), and showing a basal level expression in all cells (allowing their elimination by the settled immune response).These vaccine candidates are classified as “Live recombinant vectored vaccines” (EMA, 2011). Preclinical studies demonstrated i)the generation of a strong, specific and very long lasting T-cell immune response (up to 2 years in murine animal models), ii) the restricted diffusion of the vaccine candidates after injection and iii)their fast disappearance within few weeks, correlated with an absence of macroscopic and microscopic toxicity. These data allowed the settlement of the anti-HIV therapeutic Phase I/II clinical trial, that is held in France and Belgium and is actually enrolling 36 HIV-1 infected patients. Theravectys’ anti-HIV vaccine treatment is assessed at three doses and safety, tolerability and immunogenicity compared to a placebo group. Furthermore, vaccine efficiency will be evaluated by the interruption of the HAART treatment in all patients, including placebo. Results are expected in October 2014 with intermediary analysis in April 2013.
697. Vectored Expression of the BroadlyNeutralizing Anti-Influenza Antibody, FI6, in Airway Protects Mice Against Influenza Despite Preexisting Serum AAV Antibodies
Maria P. Limberis,1 Marco Crosariol,1 Julio Sanmiguel,1 Christine Draper,1 Roberto Calcedo,1 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA. Vectored delivery of antibodies against infectious viruses is a novel strategy to achieve protection without requiring the mounting of an immune response that is traditionally generated by active immunization. We demonstrated that localized expression of protective anti-influenza antibodies at the airway mucosal surface where replication of airborne viruses initiates, is a safe and effective strategy for preventing influenza infection. Here we investigated the potential of an AAV-vectored vaccine to be effectively re-administered and protect mice against lethal challenge with H1N1. BALB/c mice were given AAV9 vector expressing either the anti-respiratory syncytial virus (RSV) antibody palivizumab or the reporter transgene firefly luciferase in half log increments ranging from 5E10 GC/kg to 5E12 GC/kg. Twenty-eight days later, mice were readministered intranasally with 5E12 GG/kg of AAV9 vector expressing the broadlyneutralizing anti-influenza antibody, FI6. At the time of AAV9 vector re-administration, the level of serum-circulating AAV9-specific neutralizing antibody ranged from 1:80 in mice dosed with 5E10 GC/ kg to 1:2,560 in mice dosed with 5E12 GC/kg of AAV9 expressing either palivizumab or firefly luciferase. Fourteen days later, mice were challenged intranasally with 20 LD50 of mouse-adapted H1N1 (PR8) and monitored daily for signs of disease. Mice that exhibited behavioral signs of distress or lost 30% of their initial body weight were euthanized. Naïve mice and mice that were administered AAV9 expressing palivizumab or firefly luciferase were not protected against challenge with PR8. In contrast, mice re-administered with AAV9 vector expressing FI6 were protected against PR8 despite the presence of serum circulating NAB titer that ranged from 1:80 to 1:5,120. Despite the presence of high level serum circulating AAV9 NAb (1:20) which blocks systemic administration of AAV9, intranasal AAV9 re-administration was successful in protecting against PR8 challenge. Our data demonstrate the potential of our approach to sequentially protect the host against different airborne pathogens by virtue of vector re-administration.
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