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

Objectives:Objectives:

Adenovirus Biology: BasicsAdenovirus Vectors

– Construction– Propagation– Purification

Utilization of Adenovirus Vectors as a Vaccine platform

Limitations of Adenovirus Vectors

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

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

Adenovirus Genomic Adenovirus Genomic Organization reflects life cycleOrganization reflects life cycle

nucleusnucleus

Adenovirus life cycleAdenovirus life cycle

E2 E3 E4E1

E1

ssDBPpol

pTP

E2 E3 E4E1ssDBP

pTPpol

100K

Late gene expressionLate gene expression(structural proteins)(structural proteins)

PreformedPreformedempty capsidsempty capsids

ΨΨΨΨΨΨΨ

Adenovirus as a “work-horse” gene transfer vector:

Your favorite antigen

Construction of [E1-]Ad vectorsConstruction of [E1-]Ad vectors

Gene XGene X

Gene XGene X

ΔΔE1E1Homologous recombinationHomologous recombination

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

{ }

[E1-] Ad Vector Production[E1-] Ad Vector Production

E2 E3 E4Gene X

100K

ssDBPpTPpol

E1

293 cells

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

XXXX

XX

Large-scale purification:Large-scale purification: independentindependent of CsCl2 of CsCl2

centrifugationcentrifugation

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CsClCsCl22 purified Adenovirus vector: purified Adenovirus vector:

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

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

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

Ad induced innate immune responses are Ad induced innate immune responses are in part, MyD88 dependent in part, MyD88 dependent

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

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

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

Wild-type Ad (RCA) reversion Wild-type Ad (RCA) reversion during productionduring production

293 cells: 293 cells:

0 4344E1a and E1bΨ

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

Conclusions:Conclusions:

Great potentialSeveral Obstacles /Limitations must be

considered