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 -70-90 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 6:11403-1416: 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

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):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

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 101:1013-1019: 1998 Chirmule et.al.: Gene Therapy 6: 1574-1583: 1999 Varnavski et.al.: J. Virology 76(11): 5711- 5719:2002

21. Wild-type Ad (RCA) reversion during production

22. 293 cells: 0 4344 E1a and E1b Ψ

23. RCA due to recombination with Ad sequences present in 293 cells 0 4344 Your Favorite Antigen Ψ 0 4344 E1a and E1b Ψ 0 4344 E1a and E1b Ψ Wild-type Ad=RCA 480

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