167 genetically engineered bifidobacterium displaying salmonella antigen protects mice from lethal challenge of salmonella typhimurium in a murine typhoid fever model

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[.]

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INFECTIOUS DISEASES AND VACCINES: VACCINE

RELATED GENE TRANSFER RESEARCH

165 A Lentiviral HIV Vaccine Candidate Elicits

Long-Term Humoral and Cellular HIV-Speci c

Immunity and Minimal Neutralizing Activity

Benyam Asefa,1 Delia Ye,1 Nikolay Korokhov,1 Laurent M

Humeau,1 Franck Lemiale.1

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

anti-vector neutralizing activity, while displaying desirable characteristics

such as transduction of non-dividing cells, including

antigen-presenting cells, and long-term transgene expression

We have developed a VSV-G-pseudotyped HIV-based lentiviral

vaccine candidate, VRX1023, carrying unmodi ed 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 signi cant 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-speci c immunogenicity for a time period of up to 1

year in the murine model

HIV-speci c immunity was tested in splenocytes from immunized

animals, at various timepoints over a 12 month period

post-immunization, 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-speci c

immunity, although detected at lower levels, was also sustained,

for up to 6 months post-immunization Gag p24-speci c IgG levels

could be detected at remarkably consistent levels from 2 weeks to

12 months post-vector administration

LV-speci c humoral immunity, speci cally 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 In uenza 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

backbone-encoded 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

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 1 (RIG-I) and melanoma differentiation-associated gene 5 (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 in ammatory cytokine production through IPS-1 activation Since type I interferon enhances antigen-speci c 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 detoxi ed In uenza H5N1 A/Vietnam/1203/2004

hemagglutinin (HA) (Fig 1a) These vectors potently induced Type

1 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-speci c immune responses were evaluated in BALB/C mice after naked DNA intramuscular prime and boost injections The eRNA HA vectors had increased HA-speci c serum antibody titers, and improved HA-speci c antibody binding avidity, compared to HA vector alone

(Fig 1b) A two-fold improvement in hemagglutinin HA518 peptide-

reactive 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 Bi dobacterium 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

1 Center for Infectious Disease, Kobe University Graduate School

of Medicine, Kobe, Japan; 2 Ishikawa Prefectural University, Kanazawa, Japan; 3 Kobe University Hospital, Kobe, Japan;

4 Korea University School of medicine, Seoul, Korea.

INTRODUCTION: We have developed a novel vaccine platform

utilizing Bi dobacterium as an antigen delivery vehicle for mucosal immunization Genetically engineered Bi dobacterium displaying

Salmonella- agellin on the cell surface was constructed for the

oral vaccine of typhoid fever The ef ciency 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 Bi dobacterium

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 con rmed by immunocytochemical study and western blotting analysis The induction of anti- agellin speci c Ig A antibody in feces and blood serum on day 14 was con rmed 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 9 of parental B longum administrated

mice were died (each median survival was 14 and 25 days) due to

the challenge with Salmonella Typhimurium, only 2 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 Bi dobacterium, and this vaccine ef ciently induced

the mucosal immunity and prevented the death of murine typhoid

fever model This genetically engineered Bi dobacterium displaying

antigen system could be the excellent vaccine platform to induce ef cient 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

1 Department of Molecular Medicine and Virology and Gene Therapy Track, Mayo Clinic College of Medicine, Rochester, MN;

2 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 ef ciently 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 ef cient 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

169 Evaluation of Mismatched Immunity To Expand Cross-Protection Against In uenza 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

1 National Microbiology Laboratory/Public Health Agency of Canada, Winnipeg, MB, Canada; 2 University of Manitoba, Winnipeg, MB, Canada; 3 INRS-Institut Armand-Frappier/

University of Quebec, Laval, QC, Canada.

Conventional inactivated and live-attenuated vaccines are effective against seasonal and sporadic in uenza 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 in uenza 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 ef cacy against heterosubtypic in uenza infections The current study evaluates different strategies for expanding cross-protection against in uenza 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 bene cial 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 cross-protection 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 in uenza viruses Understanding the relationship between mismatched immunity and protection is of critical value in order to achieve wider cross-protection against similar and divergent in uenza 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 in ammation, 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

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