970 Evaluation of Different Combinations of DNA Vaccines Expressing H5N1 Antigens Following Lethal Challenge with Homologous and Heterologous Avian Influenza Isolates Molecular Therapy Volume 17, Supp[.]
INFECTIOUS DISEASES AND VACCINES II 969 Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza DNA Vaccine James A Williams,1 Jeremy Luke,1 Clague P Hodgson.1 R&D, Nature Technology Corporation, Lincoln, NE Methods to increase DNA vaccine induced innate immune responses to improve adaptive immunity are needed While unmethylated CpG present in the vector backbone of microbial produced plasmids stimulate innate immune signals through TLR9, the overall poor immunological response to DNA vaccines in humans has been attributed, in part, to significantly reduced expression of TLR9 in humans compared to mice For inclusion of an additional innate immunity inducer in a DNA vaccine vector backbone there should be no associated adaptive immune response since this would limit repeat usage and generate variable results in a population due to attenuated responses in individuals with prior exposure (preexisting immunity) This can be accomplished through expression of immunostimulatory RNA from the vector backbone (Fig 1) Retinoic-acid-inducible gene (RIG-I) and melanoma differentiation-associated gene (mda5) are critical cytoplasmic double stranded RNA (dsRNA) pattern receptors required for innate immune activation in response to viral infection Activation of RIG-I and mda5 leads to type I interferon (IFN) and inflammatory cytokine production through IPS-1 activation Since type I interferons enhance antigen-specific immune responses, we hypothesized that DNA vaccines coexpressing a RIG-I RNA agonist (eRNA) would generate superior immune responses to the encoded antigen Plasmid vector backbones expressing various single and double stranded RNAs from DNA polymerase III promoters were screened in a cell culture assay for RIG-I/mda5 agonist activity Optimized, potent RIG-I ligands (eRNAs) were developed (e.g 41H) and integrated into the backbone of various DNA vaccine vectors expressing detoxified Influenza H5N1 A/Vietnam/1203/2004 hemagglutinin (HA) These vectors potently induced Type interferon production in cell culture through RIG-I activation and thus combined high level antigen expression with RNA-mediated type I INF activation in a single DNA vaccine vector Antigen-specific immune responses were evaluated in BALB/C mice after naked DNA intramuscular prime and boost injections The eRNA HA vectors had improved HA-specific serum antibody titers, and improved HA-specific antibody binding avidity, compared to HA vector alone (Fig 2) This demonstrates DNA vaccine potency may be augmented by incorporation of a RIG-I activating agonist into the vector backbone S370 970 Evaluation of Different Combinations of DNA Vaccines Expressing H5N1 Antigens Following Lethal Challenge with Homologous and Heterologous Avian Influenza Isolates Ami Patel,1,2 Michael Gray,1 Tracy Taylor,1 Kaylie N Tran,1 Heinz Feldmann,3 Darwyn Kobasa,1,2 Gary P Kobinger.1,2 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, MT Avian influenza (H5N1) has been associated with pathogenesis in aquatic birds and domestic poultry The recent reemergence of H5N1 in South East Asia and potential for human-to-human transmission has underlined the need to develop newer vaccine platforms that are capable of inducing a broader protection against emerging influenza strains Different approaches have focused on the development of alternative vaccines with better immunogenicity, safety, feasibility for large-scale production, and improved stability As a result, several vaccine platforms targeting different influenza antigens have been developed The current study evaluates the protective efficacy offered by single and combination DNA-based vaccines expressing potential H5N1 antigens against diverging avian influenza subtypes Four potential H5N1 antigens were selected based on surface exposure and ability to generate an antibody response: hemagglutinin (HA), neuraminidase (NA), or based on high sequence conservation and ability to generate a cellular immune response: M2 ion channel, and nucleoprotein (NP) All genes were first generated by gene synthesis and then inserted into a DNA-based platform containing an optimized expression cassette Following immunization, BALB/c mice were challenged with a lethal dose of either homologous or heterologous H5N1 viruses T-cell response was identified post-vaccination through restimulation followed by evaluation of interferon gamma expression The B-cell responses against five related or distant influenza A virus were evaluated through detection of hemagglutinination inhibition (HI) and neutralizing (NAB) antibody titers Full protection was achieved against homologous challenge using a single HA DNA vaccine, however a combination of HA+NA conferred optimal protection, minimizing the dose of DNA required for 100% survival Against heterologous challenge, HA+NP afforded complete protection, while the HA single vaccine was only partially protective The immunodominant T-cell responses against each antigen corresponded with vaccine efficacy as determined through survival HI and NAB titers were highest against similar H5N1 viruses, but interestingly were also detectable against more Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy INFECTIOUS DISEASES AND VACCINES II divergent influenza viruses The data suggests that cross-protection may be achieved against diverging H5N1 subtypes through optimal combination of antigens 971 Reduced Cellular Response Following Immunization with Multivalent DNA Vaccines Against Avian Influenza H5N1 Suggests Antigenic Interference Ami Patel,1,2 Michael Gray,1 Tracy Taylor,1 Kaylie N Tran,1 Darwyn Kobasa,1,2 Gary P Kobinger.1,2 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada Avian influenza (H5N1) infects primarily aquatic birds and domestic poultry, however the potential for human-to-human transmission raises concerns that an H5N1 virus will be the next pandemic strain Conventional inactivated and live-attenuated vaccine strategies have been effective against seasonal influenza outbreaks, but they may not provide optimal protection against emerging H5N1 isolates This has led to the development of experimental vaccines with more appropriate efficacy The current study evaluates protection and the generation of immune responses following immunization with single or multivalent DNA-based vaccines against H5N1 Single DNA vaccines containing one of four potential H5N1 antigens were generated and different combinations were mixed for each multivalent vaccine The hemagglutinin (HA) and neuraminidase (NA) antigens were selected based on their potential to generate strong antibody responses The M2 ion channel and nucleoprotein (NP) were selected since they share high sequence conservation among diverging H5N1 isolates and have the potential to elicit a cellular immune response All genes were initially generated using gene synthesis and inserted into an optimized DNA-based platform BALB/c mice were immunized with a single or combination vaccine and challenged with a lethal dose of either homologous or heterologous H5N1 virus The cellular immune response following vaccination was identified through expression of interferon gamma following restimulation with peptide pools from each of the four antigens Although complete protection against homologous challenge was observed for the HA single DNA vaccine, optimal protection against homologous and heterologous challenges was better achieved using different multivalent combinations Interestingly, while a bivalent vaccine (HA+NP) was fully protective against heterologous virus, a tetravalent vaccine (HA+NA+NP+M2) had reduced efficacy against the same challenge The T-cell response against the HA was similar following single and multivalent vaccination, however further analysis indicated that the responses against NA and NP in multivalent vaccines was significantly reduced The data suggests that the cellular immune response may favour HA, resulting in a significant dilution of T-cell responses targeting additional antigens The addition of superfluous antigen may contribute negatively towards vaccine efficacy and the generation of a broad immune response against divergent H5N1 challenge 972 Meganucleases: A Novel Anti-Viral Strategy Roman Galetto, Julianne Smith, Philippe Duchateau, Agnès Gouble, Christophe Perez, Jean Pierre Cabaniols, Sylvestre Grizot, Fayza Daboussi, Fabien Delacôte, Frédéric Cedrone, Jean Charles Epinat, Sylvain Arnould, Aymeric Duclert, Frédéric Pâques Cellectis S.A., Romainville, Ỵle de France, France Meganucleases belong to a large family of sequence specific endonucleases recognizing large cleavage sites (>12bp) in living cells We have shown that the expression of the I-SceI meganuclease can prevent the infection with a recombinant Herpes Simplex Virus (HSV) containing the endonuclease recognition site, based on the dramatic reduction of viral DNA detected in I-SceI trasnfected Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy cells The majority of current antiviral treatments are based on the hindrance of productive viral replication by agents that inhibit virally encoded proteins Many chronic viral infections are due to double-stranded DNA viruses or viruses that involve a doublestranded DNA intermediate during their replicative cycle Thus, meganucleases may represent a novel class of antiviral agents that could cleave and either partially excise or eliminate viral DNA from infected cells, rendering them virus free This strategy mimics the cleavage of foreign DNA by restriction endonucleases in bacteria However, developing the full potential of this new approach depends on the assembly of artificial meganucleases distinctively cleaving the viral sequences We have developed a semi-rational approach to redesign the DNA-binding interface of the I-CreI meganuclease, enabling us to tailor novel endonucleases cleaving chosen sequences with high specificity This approach allowed us to generate several meganucleases cleaving human and viral sequences to date These proteins keep the essential properties of natural meganucleases in terms of activity and specificity, with no detectable toxicity even in excess dose We will describe the making and characterization of these molecules, as well as the potential of engineered meganucleases as antiviral therapeutic strategies 973 Comparative Performance of Electroporation Mediated DNA Immunization Versus Classical Vaccine Approaches Brian D Livingston,1 Stephen Little,2 Lillian Chau,1 Barry Ellefsen,1 Alain Luxembourg,1 Claire Evans,1 Drew Hannaman.1 Ichor Medical Systems, San Diego, CA; 2USAMRIID, Fort Deterick, MD DNA vaccines are a promising immunization approach capable of inducing antigen specific cellular and humoral immune responses with the potential to enable the development of novel vaccines for a range of applications However, DNA vaccines have generally elicited suboptimal immune responses in clinical trials In vivo electroporation (EP) is a promising DNA delivery method wherein brief electrical fields are applied in a target tissue in the presence of the DNA of interest to increase intracellular uptake When used to delivery DNA vaccines, EP has been demonstrated in animal models to increase the potency of DNA vaccines by up to 1000-fold compared to DNA injection alone, enhancing both antibody and T-cell responses Although extensively compared against other methods of DNA vaccine delivery, there has been more limited comparison of the immunogenicity of EP mediated DNA vaccine delivery with classical vaccine approaches (e.g., live attenuated, killed, and subunit vaccines) Using Bacillus anthracis protective antigen (PA) as a model antigen a comparative immunogenicity and bacterial challenge study was conducted in non-human primates Rhesus macaques were immunized with a PA encoding DNA vaccine administered by EP based delivery or conventional intramuscular injection Additional macaques immunized with the currently licensed anthrax vaccine, Biothrax, an aluminum adjuvanted subunit vaccine of which PA is the primary immunogenic component EP based delivery of the DNA vaccine at both dose levels elicited anti-PA IgG titers and neutralizing antibody responses that were 10-100 fold greater than those observed in rhesus macaques vaccinated with DNA using conventional intramuscular injection Importantly, electroporation delivery of the DNA vaccine induced anti-PA antibody responses that were comparable in magnitude and kinetics to those achieved with the currently licensed vaccine The protective efficacy conferred by the varied vaccine strategies was evaluated by aerosol challenge with a lethal dose of anthrax spores nearly a year after immunization While all of the macaques immunized with DNA in the absence of EP succumbed to infection, EP delivery of the DNA conferred protection comparable to those observed in the Biothrax positive control group These results indicate not only that EP based delivery enhances S371 ... following immunization with single or multivalent DNA- based vaccines against H5N1 Single DNA vaccines containing one of four potential H5N1 antigens were generated and different combinations were... BALB/c mice were immunized with a single or combination vaccine and challenged with a lethal dose of either homologous or heterologous H5N1 virus The cellular immune response following vaccination... aerosol challenge with a lethal dose of anthrax spores nearly a year after immunization While all of the macaques immunized with DNA in the absence of EP succumbed to infection, EP delivery of the DNA