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Concepts Regarding Adenovirus based vaccine systems. By: Andrea Amalfitano D.O., Ph.D. Osteopathic Heritage Foundation Professor Dept of Microbiology and.

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Presentation on theme: "Concepts Regarding Adenovirus based vaccine systems. By: Andrea Amalfitano D.O., Ph.D. Osteopathic Heritage Foundation Professor Dept of Microbiology and."— Presentation transcript:

1 Concepts Regarding Adenovirus based vaccine systems. By: Andrea Amalfitano D.O., Ph.D. Osteopathic Heritage Foundation Professor Dept of Microbiology and Molecular Genetics and Pediatrics Michigan State University

2 Objectives: Adenovirus Biology: Basics Adenovirus Vectors – Construction – Propagation – Purification Utilization of Adenovirus Vectors as a Vaccine platform Limitations of Adenovirus Vectors

3 Adenovirus: -7 Human subgroups, ~42 serotypes - primate, porcine, ovine, avian, murine nanometers -36 kb dsDNA linear genome

4 Adenovirus –Pathology/Facts 5% of all Acute respiratory illnesses- (~30,000,000 cases/yr) Pharyngitis-infants Gastroenteritis-infants Conjunctivitis-All Pneumonia- Infants, Military Recruits – (Ad 4, 7,21) – Literally millions vaccinated with large oral doses of these wild-type viruses

5 Adenovirus Genomic Organization reflects life cycle

6 nucleus Adenovirus life cycle E2E3E4E1 ssDBP pol pTP

7 E2E3E4E1 ssDBP pTP pol 100K Late gene expression (structural proteins) Preformed empty capsids Ψ Ψ Ψ Ψ Ψ Ψ Ψ

8 Adenovirus as a “work-horse” gene transfer vector: Your favorite antigen

9 Construction of [E1-]Ad vectors  Gene X   ΔE1 Homologous recombination

10 Newer methods for creating modified Ad vectors- bacterial recombination  E1E3 Gene X Ad[E1-]ANTIGEN X Viral Particles  ori Kan GENE X Pac I pAd[E1-] GENE X Amp r Kan ori  Pac I Pme I GENE X pAd[E1-] Enhancer/Promoter E1 E3 E1 E3 E2b PacI E1+,E2b+ cells Poly A { }

11 [E1-] Ad Vector Production E2E3E4 Gene X 100K ssDBP pTP pol E1 293 cells

12 Ease of scale-up for production of modified Ad vectors Gene X X X X X X X X X X X Large-scale purification: independent of CsCl2 centrifugation independent of CsCl2 centrifugation x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

13 CsCl 2 purified Adenovirus vector:

14 Adenovirus vector production, 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: : 1996 Green et.al.: Human Gene Therapy 13: : 2002 Stability and long term storage – Long term stability and lyophilization capability Croyle et.al.: Gene Therapy 8(17): : 2001

15 Efficacy of Adenovirus based Vaccine: Humoral and Cellular Immune response 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): : 2006 “Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus-based immunization” – Gao et.al.: J. Virology 80(4): :2006 “Safety and immunogenicity of Adenovirus vectored nasal and epicutaneous influenza vaccines in humans.” – Van Kampen et. al.: Vaccine 23: : 2005

16 Why are Adenoviruses so 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

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

18 Ad dyregulated Liver Transcriptome is significantly impacted upon by lack of MyD88 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 infected Wt mice 6 hpi

19 Adenovirus Limitations: “Toxicity” due to continued expression of multiple Ad encoded genes present in [E1-]Ad based vectors: Gene X “E1 - like ” E2E3E4 ssDBP pTP pol 100K ProteinX

20 Pre-existing Immunity hampers efficacy and might increase toxicity Upwards of 50-85% of adults have some pre-existing immunity to Adenoviruses Piedra et.al.: Pediatrics 101: : 1998 Chirmule et.al.: Gene Therapy 6: : 1999 Varnavski et.al.: J. Virology 76(11): :2002

21 Wild-type Ad (RCA) reversion during production

22 293 cells: E1a and E1b Ψ

23 RCA due to recombination with Ad sequences present in 293 cells Your Favorite Antigen Ψ E1a and E1b Ψ E1a and E1b Ψ Wild-type Ad=RCA 480

24 Conclusions: Great potential Several Obstacles /Limitations must be considered


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