167 Genetically Engineered Bifidobacterium Displaying Salmonella Antigen Protects Mice from Lethal Challenge of Salmonella Typhimurium in a Murine Typhoid Fever Model Molecular Therapy Volume 18, Supp[.]
INFECTIOUS DISEASES AND VACCINES: VACCINE RELATED GENE TRANSFER RESEARCH 165 A Lentiviral HIV Vaccine Candidate Elicits Long-Term Humoral and Cellular HIV-Specic Immunity and Minimal Neutralizing Activity Benyam Asefa,1 Delia Ye,1 Nikolay Korokhov,1 Laurent M Humeau,1 Franck Lemiale.1 VIRxSYS Corporation, Gaithersburg, MD Recombinant viral vectors are considered one of the major means for induction of strong immune responses against recombinant antigens by genetic immunization Recently developed viral-vectored HIV vaccine candidates, despite achieving high levels transgene expression and inducing high magnitude immune responses to HIV, have faced limitations related to anti-vector immunity In contrast, lentiviral vectors (LV) have been shown to be less sensitive to antivector neutralizing activity, while displaying desirable characteristics such as transduction of non-dividing cells, including antigenpresenting cells, and long-term transgene expression We have developed a VSV-G-pseudotyped HIV-based lentiviral vaccine candidate, VRX1023, carrying unmodied HIV Gag, Pol and Rev genes derived from the HIV-1 NL4-3 strain and which expression is driven by the wild-type HIV LTR In mice, this vector was shown to induce signicant mucosal and systemic cellular and humoral responses against HIV after sub-cutaneous injection In the present study, we investigated the ability of this vector to confer durable HIV-specic immunogenicity for a time period of up to year in the murine model HIV-specic immunity was tested in splenocytes from immunized animals, at various timepoints over a 12 month period postimmunization, by IFN-g Elispot Gag and Pol-specific T-cell responses, assessed after stimulation with overlapping HIV-peptides, were sustained in the range of 800 spot forming units/million cells on average for over a year with no diminution over time Rev-specic immunity, although detected at lower levels, was also sustained, for up to months post-immunization Gag p24-specic IgG levels could be detected at remarkably consistent levels from weeks to 12 months post-vector administration LV-specic humoral immunity, specically targeted towards the VSV-G pseudotyping protein, was detected at high level throughout the course of the study, but was found to have minimal neutralizing activity LV was successfully repeatedly administered without any marked increase of anti-vector neutralizing activity In summary, VRX1023 represents a highly attractive HIV vaccine candidate that combines long-term immunogenicity with the ability to elicit weak host neutralizing activity despite repeated immunizations VRX1023, in addition to competing favorably with existing vectors for anti-HIV immune responses, demonstrates unique features likely to address some of the pitfalls of current vector-based HIV vaccine strategies 166 Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Inuenza DNA Vaccine Jeremy M Luke, Clague P Hodgson, James A Williams R&D, Nature Technology Corporation, Lincoln, NE Methods to improve DNA vaccine-induced adaptive immunity are needed This may be accomplished using plasmid vector backboneencoded innate immunity agonists These agonists should not be the target of an adaptive immune response since this would limit repeat usage as well as generate variable results in a population due to attenuated responses in individuals with prior exposure (i.e preexisting immunity) DNA vaccine-induced adaptive immunity can be improved through inclusion of immunostimulatory DNA sequences in the vector backbone For example, plasmid vector encoded unmethylated CpG stimulates innate immune signals through TLR9, resulting in improved adaptive immune responses S64 against the transgene product We report herein that expression of immunostimulatory RNA from the vector backbone may also be used to activate innate immunity and improve adaptive immune responses 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 inammatory cytokine production through IPS-1 activation Since type I interferon enhances antigen-specic 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 were developed One of these, eRNA41H, combines 1) eRNA 11a, an immunostimulatory dsRNA (isRNA) expressed by convergent transcription, with 2) adenoviral VA RNAI (VA1) (Fig 1a) eRNA41H was integrated into the backbone of various DNA vaccine vectors expressing detoxied Inuenza H5N1 A/Vietnam/1203/2004 hemagglutinin (HA) (Fig 1a) 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 IFN activation in a single DNA vaccine vector Antigen-specic immune responses were evaluated in BALB/C mice after naked DNA intramuscular prime and boost injections The eRNA HA vectors had increased HA-specic serum antibody titers, and improved HA-specic antibody binding avidity, compared to HA vector alone (Fig 1b) A two-fold improvement in hemagglutinin HA518 peptidereactive T-cells was also observed This demonstrates DNA vaccine potency may be augmented by incorporation of a RIG-I activating immunostimulatory RNA into the vector backbone 167 Genetically Engineered Bidobacterium Displaying Salmonella-Antigen Protects Mice from Lethal Challenge of Salmonella Typhimurium in a Murine Typhoid Fever Model Toshiro Shirakawa,1 Sakura Yamamoto,1 Jun Wada,2 Takane Katayama,2 Takumi Jikimoto,3 Masakuni Nakamura,3 Shohiro Kinoshita,3 Kyung-Mi Lee,4 Masato Kawabata.1 Center for Infectious Disease, Kobe University Graduate School of Medicine, Kobe, Japan; 2Ishikawa Prefectural University, Kanazawa, Japan; 3Kobe University Hospital, Kobe, Japan; Korea University School of medicine, Seoul, Korea INTRODUCTION: We have developed a novel vaccine platform utilizing Bidobacterium as an antigen delivery vehicle for mucosal immunization Genetically engineered Bidobacterium displaying Salmonella-agellin on the cell surface was constructed for the oral vaccine of typhoid fever The efciency of this vaccine was evaluated in a murine model of typhoid fever MATERIAL AND METHOD: Salmonella-agellin gene was fused to GL-Binding Protein (ABC transporter) gene for the cell- surface display This antigen-displaying cassette gene was introduced into Bidobacterium Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy INFECTIOUS DISEASES AND VACCINES: VACCINE RELATED GENE TRANSFER RESEARCH longum (B longum) to generate the oral typhoid vaccine 2.5 x 107 CFU of the recombinant B longum (vaccine) or parental B longum, or PBS was orally administrated to BALB/C mice on every other day for two weeks The blood and fecal samples were collected on day 0, 14, and 28, and the splenocytes were isolated on day 14 for the evaluation of the immune responses After the administration of the vaccine, parental B longum, or PBS, totally 42 mice (14 mice in each group) were challenged with Salmonella Typhimurium (1.0 x 107 CFU/mouse) on day 14 RESULTS: The cell-surface display of agellin protein was conrmed by immunocytochemical study and western blotting analysis The induction of anti-agellin specic Ig A antibody in feces and blood serum on day 14 was conrmed by ELISA only in the vaccine administrated mice not in the other mice The isolated splenocytes were incubated with or without agellin protein, the secretions of IFN-γ and IL-12 were stimulated by agellin protein only in the vaccine administrated mice not in the others While 12 of PBS administrated mice, and of parental B longum administrated mice were died (each median survival was 14 and 25 days) due to the challenge with Salmonella Typhimurium, only of the vaccine administrated mice were died in this survival study (Log-rank test, p=0.0022) CONCLUSION: We have generated the novel oral typhoid vaccine utilizing Bidobacterium, and this vaccine efciently induced the mucosal immunity and prevented the death of murine typhoid fever model This genetically engineered Bidobacterium displaying antigen system could be the excellent vaccine platform to induce efcient mucosal immunity 168 Signaling Lymphocytic Activation Molecule (SLAM, CD150)-Blind Measles Virus Is Attenuated and Induces Strong Adaptive Immune Responses in Rhesus Monkeys Vincent Leonard,1 Gregory Hodge,2 Jorge Reyes-del Valle,1 Michael McChesney,2 Roberto Cattaneo.1 Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, MN; California National Primate Research Center and Department of Pathology and Laboratory Medicine, UC Davis School of Medicine, Davis, CA The signaling lymphocytic activation molecule (SLAM, CD150) is the immune cell receptor for measles virus (MV) To assess the importance of the SLAM-MV interactions for virus spread and pathogenesis, we generated a selectively SLAM-blind MV on the wild type IC-B genetic background This virus differs from the fully virulent wild type IC-B strain by a single arginine to alanine substitution at amino acid 533 of the attachment protein hemagglutinin and infects cells through SLAM about 40 times less efciently than the isogenic wild type strain Ex-vivo, this virus infects at low levels primary lymphocytes regardless of SLAM expression When a group of six rhesus monkeys (M mulatta) was inoculated intranasally with the SLAM-blind virus, no clinical symptoms were documented Only one monkey had low-level viremia early after infection, whereas all the hosts in the control group had high viremia levels Despite minimal if any viremia, all six hosts generated neutralizing antibody titers close to those of the control monkeys, while MV-directed cellular immunity reached levels at least as high as in wild type-infected monkeys These ndings prove formally that efcient SLAM recognition is necessary for MV virulence and pathogenesis They also suggest that the selectively SLAM-blind wild type MV can be developed into a vaccine vector Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 169 Evaluation of Mismatched Immunity To Expand Cross-Protection Against Inuenza Virus Ami Patel,1,2 Isabelle Meunier,3 Kaylie N Tran,1 Anders Leung,1 Stephane Pillet,3 Darwyn Kobasa,1,2 Veronika von Messling,3 Gary P Kobinger.1,2 National Microbiology Laboratory/Public Health Agency of Canada, Winnipeg, MB, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3INRS-Institut Armand-Frappier/ University of Quebec, Laval, QC, Canada Conventional inactivated and live-attenuated vaccines are effective against seasonal and sporadic inuenza outbreaks however these approaches require yearly reformulation and may not extend protection against divergent isolates or other subtypes The emergence of the novel swine-like pandemic H1N1/2009 and avian inuenza H5N1 highlights the need to improve vaccine cross-protection through re-evaluation of current strategies as well as alternative vaccine platforms In particular, DNA-based expression systems are being evaluated in order to improve vaccine efcacy against heterosubtypic inuenza infections The current study evaluates different strategies for expanding cross-protection against inuenza viruses through conventional vaccine candidates as well as experimental DNA-based subunit vaccines A single H5N1 A/Hanoi/30408/2005 hemagglutinin (H5-HA)-based DNA vaccine was protective against homologous challenge however complete protection was not achievable against heterologous A/Hong Kong/483/1997 challenge Different H5N1 A/ Hanoi/30408/2005 antigens (hemagglutinin H5-HA, neuraminidase H5-NA, M2 protein H5-M2, or nucleoprotein H5-NP) were mixed and evaluated to determine an optimal combination which would offer the greatest range of protection against divergent challenge In BALB/c mice, the combination of H5-HA+H5-NA offered the best protection against homologous challenge and the H5-HA+H5-NP combination was optimal against a heterologous A/Hong Kong/483/1997 challenge Interestingly, the addition of three or more related and unrelated antigens reduced cellular immune responses and survival against heterologous challenge This suggests that mismatched immunity may be both benecial as well as disadvantageous Similar ndings were observed in ferrets immunized against H1N1/2009 by matched inactivated or seasonal H1N1 inactivated and live-attenuated vaccines Animals immunized with the seasonal vaccines displayed increased clinical symptoms, lung pathology, and reduced survival compared to unvaccinated vehicle treated control ferrets To the opposite, strong immunity against a H1N1 seasonal virus led to complete crossprotection against H5N1-induced mortality and morbidity in ferrets These data will be presented and discussed with the focus on immune parameters that could reduce or more desirably enhance heterotypic protection against a wide range of inuenza viruses Understanding the relationship between mismatched immunity and protection is of critical value in order to achieve wider cross-protection against similar and divergent inuenza viruses 170 Immune Response Against Measles Virus Encoding Helicobacter pylori Neutrophil-Activating Protein Antigen Ianko D Iankov, Iana Haralambieva, Evanthia Galanis Mayo Clinic College of Medicine, Rochester, MN Background: Helicobacter pylori is a spiral-shaped microorganism associated with chronic gastritis, peptic ulcer, stomach cancer and gastric MALT lymphomas in humans H pylori neutrophil-activating protein (HP-NAP) is a major virulence factor and protective antigen with a central role in pathogenesis of the chronic mucosal inammation, attracting neutrophils and other immune cells at infection site HP-NAP is potent inducer of cytokine release and Th1 polarization of immune response Attenuated measles virus (MV) Edmoston strain is one of the components of the live vaccine against S65 ... Agency of Canada, Winnipeg, MB, Canada; 2University of Manitoba, Winnipeg, MB, Canada; 3INRS-Institut Armand-Frappier/ University of Quebec, Laval, QC, Canada Conventional inactivated and live-attenuated... Response Against Measles Virus Encoding Helicobacter pylori Neutrophil-Activating Protein Antigen Ianko D Iankov, Iana Haralambieva, Evanthia Galanis Mayo Clinic College of Medicine, Rochester, MN Background:... matched inactivated or seasonal H1N1 inactivated and live-attenuated vaccines Animals immunized with the seasonal vaccines displayed increased clinical symptoms, lung pathology, and reduced survival