1. STATENS SERUM INSTITUT DNA Vaccination Anneline Nansen Department of Infectious Disease Immunology Statens Serum Institut (SSI)

2. STATENS SERUM INSTITUT What is a vaccine? A vaccine is a substance that stimulates an immune response that can either prevent an infection or create resistance to an infection

3. STATENS SERUM INSTITUT What are the different types of vaccines? Live vaccines Are able to replicate in the host Attenuated (weakened) so they do not cause disease Whole killed vaccines Subunit vaccines Part of organism (protein, inactivated toxins) Genetic Vaccines Part of genes from organism

4. STATENS SERUM INSTITUT Introduce DNA or RNA into the host Injected (Naked) (Intra muscular, i.m.) Delivered by Gene gun. Naked DNA Coated on gold particles Carried by recombinant live vectors: Vaccinia, adenovirus, or alphaviruses Intracellular bacteria Advantages Easy to produce Induce cellular (CD4 + T cells and CTL’s) and humoral responses Disadvantages Often weak primary responses-need for a boost Genetic Vaccines

5. STATENS SERUM INSTITUT HIV Live-attenuated or killed Vaccines are not applicable Because: If there were a manufacturing error and the HIV is not properly killed or attenuated, the poorly-made vaccine could infect people with HIV Also, because HIV is so highly mutating, there is concern it might be able to mutate out of attenuation and cause disease. Cancer A variety of infectious diseases Tuberculosis Malaria HCV Genetic Vaccines

6. STATENS SERUM INSTITUT Comparative Analysis of various Vaccine formulations

7. STATENS SERUM INSTITUT Properties of Genetic Vaccines

8. STATENS SERUM INSTITUT DNA Vaccine Design Pick Genes, epitope(s), of relevance for protection against the disease of interest Has to be immunogenic in the host Select a plasmid and an expression system Optimize for expression in eukaryotic cells Promotor optimization Synthetic genes with optimized codon usage Optimize immunogenicity Insert multiple CpG motifs (TLR ligand) IL-12, IL-15 others…

9. STATENS SERUM INSTITUT DNA vaccination-Naked plasmid

10. STATENS SERUM INSTITUT Delivery of Naked DNA By Gene Gun Small amounts of DNA Th2 biased immune response i.m injection Large amounts of DNA Th1 biased immune response

11. STATENS SERUM INSTITUT The “gene gun” The Helios Gene Gun is a new way for in vivo transformation of cells or organisms (i.e. gene therapy and genetic immunization (DNA vaccination)). This gun uses Biolistic ® particle bombardment where DNA- or RNA-coated gold particles are loaded into the gun and you pull the trigger. A low pressure helium pulse delivers the coated gold particles into virtually any target cell or tissue. The particles carry the DNA so that you do not have to remove cells from tissue in order to transform the cells.

12. STATENS SERUM INSTITUT

13. Characterization of Gene Expression by Intradermal Administration pcDNA3-LucpcDNA3 One hour after DNA vaccination Gene Gun Modifying the Properties of DCs as Innovative Strategies to Enhance DNA Vaccine Potency Schematic diagram to show DNA vaccination via gene gun

14. STATENS SERUM INSTITUT Employment of intracellular sorting signals to improve antigen processing through MHC class I and II pathways. Employment of intercellular spreading strategies to increase the number of antigen presenting cells that present antigens encoded by DNA vaccines. Employment of Anti-apoptotic strategies to prolong life span of antigen presenting cells that present antigens encoded by DNA vaccines Strategies to Enhance DNA Vaccine Potency

15. STATENS SERUM INSTITUT Th1-Cytokine DNA Chemokine DNA Co-stimulatory molecule DNA Enhancement of DNA vaccine potency Adapted from Calarota SA et al. Immunological Reviews, 2004 Pro-inflammatory DNA

16. STATENS SERUM INSTITUT Kutzler, M. A. et al. J. Clin. Invest. 2004;114:1241-1244 Molecular interactions that contribute to the recruitment, activation, or maturation of DCs in DNA vaccine studies

17. STATENS SERUM INSTITUT Kutzler, M. A. et al. J. Clin. Invest. 2004;114:1241-1244 Proposed schematic of chemokine-induced traffic and activation of DCs following DNA vaccination with plasmid-encoded Flt3L and MIP-1 

18. STATENS SERUM INSTITUT Sumida, S. M. et al. J. Clin. Invest. 2004;114:1334-1342 Immunohistochemistry of injection sites

19. STATENS SERUM INSTITUT Sumida, S. M. et al. J. Clin. Invest. 2004;114:1334-1342 Analysis of DCs extracted from injected muscles

20. STATENS SERUM INSTITUT Sumida, S. M. et al. J. Clin. Invest. 2004;114:1334-1342 Immunogenicity of MIP-1  /Flt3L-augmented DNA vaccines

21. STATENS SERUM INSTITUT DNA vaccination by use of live recombinant viruses

22. STATENS SERUM INSTITUT Examples of live viral vectors Poxviruses Vaccinia Virus (VV) Modified Vaccinia Virus Ankara MVA replication deficient (very safe, even in immodeficient individuals) Pre-existing immunity, because VV is used as vaccine against Small Pox Adenoviruses 49 immunologically distinct adenoviral types (serotypes) Infect many cells types including APC’s Induce potent CTL responses Pre-existing immunity against the vector, because of naturally occuring infections Avipoxviruses Fowlpox Not a natural human pathogen- no pre-existing immunity

23. STATENS SERUM INSTITUT Kinetics of an immune response after a single immunisation with a viral vector or after Prime boost Single prime Homologous Prime-Boost Heterologous Prime-Boost Adapted from Rocha CD et al. Int Microbiol, 2004

24. STATENS SERUM INSTITUT Ligation   BN- Vektor Insert DNA Material e.g.HIV gene The making of recombinant viruses

25. STATENS SERUM INSTITUT Adapted from Rocha CD et al. Int Microbiol, 2004 Homologous Recombination

26. STATENS SERUM INSTITUT Homologous Recombination Fibroblast cell MVA BN Virus 1 hour InfectionTransfection 2 days GFP positive Cells Plasmid DNA MVA BN mit Plasmid DNA

27. STATENS SERUM INSTITUT Prime-boost Vaccination strategies Naked DNA and Protein Possible to prime several times, no immunity Best results if DNA or protein before live viral vector Recombinant Viruses Only one go-because of immunity against the vector after priming Often used as a Booster Vaccine Possible to use different recombinant vectors as prime-boost

28. STATENS SERUM INSTITUT Current and recently completed HIV vaccine clinical trials Adapted from McMichael AJ, ann rev Immunol, 2006

29. STATENS SERUM INSTITUT Skeiky et al. Nature Reviews Microbiology 4, 469–476, 2006 On-going Tuberculosis vaccine clinical trials

30. STATENS SERUM INSTITUT Skeiky et al. Nature Reviews Microbiology 4, 469–476, June 2006 Preventive prime-boost vaccination strategy against Tuberculosis

31. STATENS SERUM INSTITUT THE END