concepts regarding adenovirus based vaccine systems. by: andrea amalfitano d.o., ph.d. osteopathic...
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Concepts Regarding Adenovirus based vaccine Concepts Regarding Adenovirus based vaccine systems.systems.
By:By:Andrea Amalfitano D.O., Ph.D.Andrea Amalfitano D.O., Ph.D.
Osteopathic Heritage Foundation Professor Osteopathic Heritage Foundation Professor Dept of Microbiology and Molecular Genetics and PediatricsDept of Microbiology and Molecular Genetics and Pediatrics
Michigan State UniversityMichigan State University
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Objectives:Objectives:
Adenovirus Biology: BasicsAdenovirus Vectors
– Construction– Propagation– Purification
Utilization of Adenovirus Vectors as a Vaccine platform
Limitations of Adenovirus Vectors
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Adenovirus:Adenovirus:
-7 Human subgroups , ~42 serotypes-7 Human subgroups , ~42 serotypes- primate, porcine, ovine, avian, - primate, porcine, ovine, avian, murine murine -70-90 nanometers-70-90 nanometers-36 kb dsDNA linear genome-36 kb dsDNA linear genome
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Adenovirus –Pathology/FactsAdenovirus –Pathology/Facts5% of all Acute respiratory illnesses-(~30,000,000
cases/yr)Pharyngitis-infantsGastroenteritis-infantsConjunctivitis-AllPneumonia- Infants, Military Recruits
– (Ad 4, 7,21)– Literally millions vaccinated with large oral doses of Literally millions vaccinated with large oral doses of
these wild-type virusesthese wild-type viruses
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Adenovirus Genomic Adenovirus Genomic Organization reflects life cycleOrganization reflects life cycle
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nucleusnucleus
Adenovirus life cycleAdenovirus life cycle
E2 E3 E4E1
E1
ssDBPpol
pTP
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E2 E3 E4E1ssDBP
pTPpol
100K
Late gene expressionLate gene expression(structural proteins)(structural proteins)
PreformedPreformedempty capsidsempty capsids
ΨΨΨΨΨΨΨ
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Adenovirus as a “work-horse” gene transfer vector:
Your favorite antigen
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Construction of [E1-]Ad vectorsConstruction of [E1-]Ad vectors
Gene XGene X
Gene XGene X
ΔΔE1E1Homologous recombinationHomologous recombination
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Newer methods for Newer methods for creating modified Ad creating modified Ad
vectors-vectors-bacterial recombinationbacterial recombination
E1 E3
Gene X
Ad[E1-]ANTIGEN XAd[E1-]ANTIGEN XViral ParticlesViral Particles
oriKan
GENE X
Pac IPac I
pAd[E1-]pAd[E1-]GENE XGENE X
Ampr
Kanori
Pac I
Pac I
Pac I
Pme IPme I
GENE X
pAd[E1-]pAd[E1-]
Enhancer/Promoter
E1
E3
E1
E3
E2b
E2b
E2b
PacI E1+,E2b+cells
Poly A
{ }
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[E1-] Ad Vector Production[E1-] Ad Vector Production
E2 E3 E4Gene X
100K
ssDBPpTPpol
E1
293 cells
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Ease of scale-up for production of Ease of scale-up for production of modified Ad vectorsmodified Ad vectors
Gene Gene XX
XX
XX
XXXX
XX
XX
XX
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XX
Large-scale purification:Large-scale purification: independentindependent of CsCl2 of CsCl2
centrifugationcentrifugation
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CsClCsCl22 purified Adenovirus vector: purified Adenovirus vector:
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Adenovirus vector production, Adenovirus vector production, purification, and storage:purification, and storage:
Serum Free Cell Culture Large Scale Cultivation Systems:
– cell suspension based systems– Micro-carrier bead based– Hollow fiber
Isayeva et.al.: BioProcessing Journal p.64-70: 2003 Anion Exchange Based Column Chromatography
Purification Huyghe et.al.: Human Gene Therapy 6:11403-1416: 1996 Green et.al.: Human Gene Therapy 13:1921-1934: 2002
Stability and long term storage– Long term stability and lyophilization capability
Croyle et.al.: Gene Therapy 8(17):1281-90: 2001
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Efficacy of Adenovirus based Vaccine: Efficacy of Adenovirus based Vaccine: Humoral and Cellular Immune Humoral and Cellular Immune
response inductionresponse induction “Phase 1 safety and immunogenicity evaluation of a
multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector”
– Catamzaro et.al.: J. Infectious Diseases: 194(12):1638-1649: 2006
“Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization”
– Gao et.al.: J. Virology 80(4): 1959-1964:2006
“Safety and immunogenicity of Adenovirus vectored nasal and epicutaneous influenza vaccines in humans.”
– Van Kampen et. al.: Vaccine 23: 1029-1036: 2005
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Why are Adenoviruses so Why are Adenoviruses so potent?potent?
The Ad capsid is intrinsically capable of inducing potent innate immune responses.– In part, these responses are TLR mediated.
Harman et.al. J. Virology : in press
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Ad induced innate immune responses are Ad induced innate immune responses are in part, MyD88 dependent in part, MyD88 dependent
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Ad dyregulated Liver Transcriptome is Ad dyregulated Liver Transcriptome is significantly impacted upon by lack of MyD88 significantly impacted upon by lack of MyD88
functionality:functionality:
Ad treated Wild-Type mice exhibit significantly higher activation of immune response, nucleotide binding, RNA processing, and Extracellular and Adhesion Genes relative to Ad treated, MyD88KO mice.
Ad treated Wild-Type mice exhibit significantly greater repression of mitochondria-related genes as well as cell cycle and growth gene groups relative to Ad treated, MyD88KO mice.
– Hartman et.al.: J. Virology: in press.
Statistically different genes between Ad and mock infected samples assessed using a 1-way ANOVA, p = .05 with Benjamini and Hochberg Multiple Testing Correction.
Ad infectedWt mice
6 hpi
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Adenovirus Limitations: Adenovirus Limitations: ““Toxicity” due to continued expression of multiple Ad Toxicity” due to continued expression of multiple Ad
encoded genes present in [E1-]Ad based vectors:encoded genes present in [E1-]Ad based vectors:
Gene X
“E1-
like”
E2 E3 E4ssDBPpTPpol
100KProteinProteinXX
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Pre-existing Immunity hampers Pre-existing Immunity hampers efficacy and might increase efficacy and might increase
toxicitytoxicity
Upwards of 50-85% of adults have some pre-existing immunity to Adenoviruses
Piedra et.al.: Pediatrics 101:1013-1019: 1998 Chirmule et.al.: Gene Therapy 6: 1574-1583: 1999 Varnavski et.al.: J. Virology 76(11): 5711-
5719:2002
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Wild-type Ad (RCA) reversion Wild-type Ad (RCA) reversion during productionduring production
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293 cells: 293 cells:
0 4344E1a and E1bΨ
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RCA due to recombination with RCA due to recombination with Ad sequences present in 293 Ad sequences present in 293
cellscells
0 4344Your Favorite AntigenΨ
0 4344E1a and E1bΨ
0 4344E1a and E1bΨ
Wild-type Ad=RCA
480
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Conclusions:Conclusions:
Great potentialSeveral Obstacles /Limitations must be
considered