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757. Generation of Protective Immunity Against 1918 (H1N1) and Divergent Isolates of Avian Influenza (H5N1) Viruses through Different Vaccination Strategies
a single administration of the Vaxfcctin'Pt-formulatcd LEC DNA. Survival and weight loss data indicate that two administrations of the LEC vaccine completely protect mice from the viral challenge. Remarkably, a single administration ofthe LEC DNA H3 HA vaccine at the lowest dose tested (2 Jlg) was sufficient to confer protection to viral challenge. Our data demonstrate the feasibility of linear DNA-based vaccines to protect against a lethal dose of influenza A virus. Efficacy of Vaxfectin'Pt-forrnulatcd LECs at low doses is consistent with previously published data showing enhanced immune responses of cationic lipid-formulated DNA. Efficacy and ease of cell-free manufacture support further development of LEC DNA-based influenza vaccines as a promising approach to rapid vaccine development.
757. Generation of Protective Immunity Against 1918 (H1N1) and Divergent Isolates of Avian Influenza (H5N1) Viruses through Different Vaccination Strategies Darwyn Kobasa,' Ami Patel,2,3 Michael Gray," Gary P. Kobin-
ger. 2.J 'Respiratory Viruses, National Microbiology Laboratory, Public Health Agency ofCanada, Winnipeg, MB, Canada; 2Special Pathogens, National Microbiology Laboratory, Public Health Agency ofCanada. Winnipeg, MB, Canada; 'Medical Microbiology, University 0/Manitoba, Winnipeg, MB, Canada. The influenza A viruses ofthe family orthornyxoviridae comprise a large variety of known animals and human pathogens including avian influenza (I-15N I) and 1918 pandemic (1-11 N I) viruses. Rising concerns of a global pandemic in recent years have been fuelled by an increasing number of fatal human cases caused by poultry to human transmission of the H5N I virus. It is known that the 1918 strain of influenza which killed an estimated 50 millions people is a human-adapted avian influenza. An important concern about present influenza evolution is that the highly virulent 1-15NI virus will develop into a pandemic strain resembling the highly transmissible and virulent 1918 virus. This study aims to evaluate the protective efficacy ofdifferent vaccine strategies in mice against several H5N 1 viruses. The same vaccine strategies were also tested against the 1918 HINI influenza isolate which was used as a prototype tor a highly virulent human strain. Immunization strategies included several DNA and adenovirus-based vaccines encoding for different optimized antigenic expression cassettes. The hemagglutinin (I-IA), the neuraminidase (NA), the ion channel M2 or the nucleoprotein (NP) were evaluated alone or in combinations against homologous or heterologous H5NI or 1918-HINI influenza viruses. Challenge experiments indicate that HA or NA affords better protection than the more conserved M2 or NP antigens against a lethal infection with a homologous H5N I virus. Addition ofNA or NP to a partially protective dose of I-IA substantially improved survival against a homologous challenge. Furthermore, complete protection against a heterologous 1-15N I challenge could be achieved with two sequential immunizations of ONA-based vaccine expressing an optimized I-IA gene. Interestingly, the adaptation of vaccine strategies sufficient for conferring full protection against H5N I viruses induced partial protection against 1918 influenza. Together, these results indicate that simple vaccine strategies can protect against different strains of H5N I viruses yet more aggressive immunization regiments are required for protection against 1918 influenza. Challenge experiments together with T and B cell immune responses following vaccination will be presented.
Molecular Therapy Volume 15.Supplem ent I. M;'l"2007
Coprright © The American Socie ty o f G ene Th erapy
CANCER GENE THERAPY
758. Adenoviral-Mediated Delivery of Pseudomonas Exotoxin (PE) Fused to IL-13 Induces Regression of Intracranial Human Glioblastoma Xenografts in Mice M. Candolfi,' W. Xiong,' M. Puntel,' A. K. M. O. Muhammad,' Chunyan Liu, I Kurt M. Kroeger, I Sonali Monkar, I Ron Rodriguez,' Pedro R. Lowenstein,' Maria O. Castro.' 'Gene Therapeutics Research Institute, Cedars Sinai Medical Center-UCLA, Los Angeles, CA; "Department ofUrology, Medical Oncology, Radiation Oncology and Molecular Radiation Sciences, Viral Oncology, Cellular & Molecular Medicine, Johns Hopkins University School ofMedicine, Baltimore, MD. Human Glioblastomas (GBM) overexpress an IL-13 receptor (IL13a2R) that is absent in the normal brain. Thus, attempts have been made to target toxic molecules to glioma cells by fusing them with IL-13 . However, protein preparations have a short half life, requiring frequent administrations. Thus, we constructed an adenoviral vector (Ad) to transfer a chimeric toxin composed by IL-13 and Pseudomonas exotoxin A (IL 13-PE) to preclinical GBMs. We constructed Ad-IL4-TRE-ILl3-PE that expresses IL-13-PE, and, as a safety feature, it also expresses a mutated form of IL4 that blocks the physiological receptor, i.e., ILI3/IL4R. Transgene expression is driven by the bidirectional TRE promoter, which is activated by the transactivator (TetON, expressed within Ad-TetON), in the presence of the inducer doxycyline (Dox). IL4 ands ILl3-PE expression was detected using human U251 glioma cells infected with Ad-IL4-TRE-IL I3-PE+Ad-TetON, which reduced cell viability 70% in the "ON" state (+Dox). Human glioma cell viability remained unaffected in the "OFF" state, indicating that the expression of the chimeric toxin can be tightly regulated. Transgene expression from Ad-IL4-TRE-ILl3-PE was also detected in COS-7 cells. Howe ver, COS-7 cells, which do not express the IL 13alpha2R, did not undergo cell death in the presence of the therapeutic virus , suggesting that 1L13-PE cytotoxicity is specific to glioma cells. We also administered Ad-IL4-TRE-ILI3-PE+Ad-TetON in the striatum of nude mice, which were fed chow containing Dox. IL-4 and 1L13-PE toxin were readily detected in the mouse brain , with no signs of toxicity. We then administered Ad-IL4-TRE-ILl3-PE+Ad-TetON intratumorally into intracranial human U251 OBM xenografts in nude mice. While saline-treated mice (median survival : 45 days) all the animals treated with Ad-I L4-TRE-1L 13-PE survived for over 100 days post-tumor implantation. Our results suggest that Ad-mediated intratumoral expression ofiL 13-PE toxin will lead to effective cytotoxicity oflL-l3a2R expressing-GBM cells without side effects to the surrounding normal brain and warrant further development of this approach for the implementation ofa elinical trial tor OBM. Supported by: National institutes of Health /National Institute of Neurological Disorders & Stroke (NIH/N/NDS) Grants IROi NS44556.0/, Minority Supplement NS445561; I R21 NS054/43-0/ and I R03 TIV006273-01to N/H/N/NDSGrants / ROI NS 42893.0/; U54 NS045309-01, and / R2/ NS047298-01 to PRL; the Bram and Elaine Goldsmith and the Medallions Group Endowed Chairs in Gene Therapeutics to PRL and MGC, respectively, and The Linda Tallen & David Paul Kane Foundation and the Board ofGovernors at CSMC.
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757. Generation of Protective Immunity Against 1918 (H1N1) and Divergent Isolates of Avian Influenza (H5N1) Viruses through Different Vaccination Strategies Darwyn Kobasa,' Ami Patel,2,3 Michael Gray," Gary P. Kobin-
ger. 2.J 'Respiratory Viruses, National Microbiology Laboratory, Public Health Agency ofCanada, Winnipeg, MB, Canada; 2Special Pathogens, National Microbiology Laboratory, Public Health Agency ofCanada. Winnipeg, MB, Canada; 'Medical Microbiology, University 0/Manitoba, Winnipeg, MB, Canada. The influenza A viruses ofthe family orthornyxoviridae comprise a large variety of known animals and human pathogens including avian influenza (I-15N I) and 1918 pandemic (1-11 N I) viruses. Rising concerns of a global pandemic in recent years have been fuelled by an increasing number of fatal human cases caused by poultry to human transmission of the H5N I virus. It is known that the 1918 strain of influenza which killed an estimated 50 millions people is a human-adapted avian influenza. An important concern about present influenza evolution is that the highly virulent 1-15NI virus will develop into a pandemic strain resembling the highly transmissible and virulent 1918 virus. This study aims to evaluate the protective efficacy ofdifferent vaccine strategies in mice against several H5N 1 viruses. The same vaccine strategies were also tested against the 1918 HINI influenza isolate which was used as a prototype tor a highly virulent human strain. Immunization strategies included several DNA and adenovirus-based vaccines encoding for different optimized antigenic expression cassettes. The hemagglutinin (I-IA), the neuraminidase (NA), the ion channel M2 or the nucleoprotein (NP) were evaluated alone or in combinations against homologous or heterologous H5NI or 1918-HINI influenza viruses. Challenge experiments indicate that HA or NA affords better protection than the more conserved M2 or NP antigens against a lethal infection with a homologous H5N I virus. Addition ofNA or NP to a partially protective dose of I-IA substantially improved survival against a homologous challenge. Furthermore, complete protection against a heterologous 1-15N I challenge could be achieved with two sequential immunizations of ONA-based vaccine expressing an optimized I-IA gene. Interestingly, the adaptation of vaccine strategies sufficient for conferring full protection against H5N I viruses induced partial protection against 1918 influenza. Together, these results indicate that simple vaccine strategies can protect against different strains of H5N I viruses yet more aggressive immunization regiments are required for protection against 1918 influenza. Challenge experiments together with T and B cell immune responses following vaccination will be presented.
Molecular Therapy Volume 15.Supplem ent I. M;'l"2007
Coprright © The American Socie ty o f G ene Th erapy
CANCER GENE THERAPY
758. Adenoviral-Mediated Delivery of Pseudomonas Exotoxin (PE) Fused to IL-13 Induces Regression of Intracranial Human Glioblastoma Xenografts in Mice M. Candolfi,' W. Xiong,' M. Puntel,' A. K. M. O. Muhammad,' Chunyan Liu, I Kurt M. Kroeger, I Sonali Monkar, I Ron Rodriguez,' Pedro R. Lowenstein,' Maria O. Castro.' 'Gene Therapeutics Research Institute, Cedars Sinai Medical Center-UCLA, Los Angeles, CA; "Department ofUrology, Medical Oncology, Radiation Oncology and Molecular Radiation Sciences, Viral Oncology, Cellular & Molecular Medicine, Johns Hopkins University School ofMedicine, Baltimore, MD. Human Glioblastomas (GBM) overexpress an IL-13 receptor (IL13a2R) that is absent in the normal brain. Thus, attempts have been made to target toxic molecules to glioma cells by fusing them with IL-13 . However, protein preparations have a short half life, requiring frequent administrations. Thus, we constructed an adenoviral vector (Ad) to transfer a chimeric toxin composed by IL-13 and Pseudomonas exotoxin A (IL 13-PE) to preclinical GBMs. We constructed Ad-IL4-TRE-ILl3-PE that expresses IL-13-PE, and, as a safety feature, it also expresses a mutated form of IL4 that blocks the physiological receptor, i.e., ILI3/IL4R. Transgene expression is driven by the bidirectional TRE promoter, which is activated by the transactivator (TetON, expressed within Ad-TetON), in the presence of the inducer doxycyline (Dox). IL4 ands ILl3-PE expression was detected using human U251 glioma cells infected with Ad-IL4-TRE-IL I3-PE+Ad-TetON, which reduced cell viability 70% in the "ON" state (+Dox). Human glioma cell viability remained unaffected in the "OFF" state, indicating that the expression of the chimeric toxin can be tightly regulated. Transgene expression from Ad-IL4-TRE-ILl3-PE was also detected in COS-7 cells. Howe ver, COS-7 cells, which do not express the IL 13alpha2R, did not undergo cell death in the presence of the therapeutic virus , suggesting that 1L13-PE cytotoxicity is specific to glioma cells. We also administered Ad-IL4-TRE-ILI3-PE+Ad-TetON in the striatum of nude mice, which were fed chow containing Dox. IL-4 and 1L13-PE toxin were readily detected in the mouse brain , with no signs of toxicity. We then administered Ad-IL4-TRE-ILl3-PE+Ad-TetON intratumorally into intracranial human U251 OBM xenografts in nude mice. While saline-treated mice (median survival : 45 days) all the animals treated with Ad-I L4-TRE-1L 13-PE survived for over 100 days post-tumor implantation. Our results suggest that Ad-mediated intratumoral expression ofiL 13-PE toxin will lead to effective cytotoxicity oflL-l3a2R expressing-GBM cells without side effects to the surrounding normal brain and warrant further development of this approach for the implementation ofa elinical trial tor OBM. Supported by: National institutes of Health /National Institute of Neurological Disorders & Stroke (NIH/N/NDS) Grants IROi NS44556.0/, Minority Supplement NS445561; I R21 NS054/43-0/ and I R03 TIV006273-01to N/H/N/NDSGrants / ROI NS 42893.0/; U54 NS045309-01, and / R2/ NS047298-01 to PRL; the Bram and Elaine Goldsmith and the Medallions Group Endowed Chairs in Gene Therapeutics to PRL and MGC, respectively, and The Linda Tallen & David Paul Kane Foundation and the Board ofGovernors at CSMC.
u.o.c:
S291