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

2. A vaccine is a substance that stimulates
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. 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. Introduce DNA or RNA into the host
Genetic Vaccines
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

5. It has it all!

6. A variety of infectious diseases
Genetic Vaccines
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

7. Comparative Analysis of various Vaccine formulations

8. Properties of Genetic Vaccines

9. 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…

10. DNA vaccination-Naked plasmid

11. 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

12. 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.

13. Guns are good for something!!
Gene gun: 1 µm DNA per shot
Intra muscular: 100 µg of pCMV-S,
Mice: 20g, Human: g, 5000*100 µg = 0.5g DNA

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

17. Strategies to Enhance DNA Vaccine Potency
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

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

19. Molecular interactions that contribute to the recruitment, activation, or maturation of DCs in DNA vaccine studies
Kutzler, M. A. et al. J. Clin. Invest. 2004;114:

20. Proposed schematic of chemokine-induced traffic and activation of DCs following DNA vaccination with plasmid-encoded Flt3L and MIP-1a
Growth factor
Chemokine
Kutzler, M. A. et al. J. Clin. Invest. 2004;114:

21. Immunohistochemistry of injection sites
Sumida, S. M. et al. J. Clin. Invest. 2004;114:

22. Analysis of DCs extracted from injected muscles
Sumida, S. M. et al. J. Clin. Invest. 2004;114:

23. Immunogenicity of MIP-1a/Flt3L-augmented DNA vaccines
Sumida, S. M. et al. J. Clin. Invest. 2004;114:

24. DNA vaccination by use of live recombinant viruses

25. 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

26. 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

27. The making of recombinant viruses
DNA Material e.g.HIV gene
Ligation
Insert
BN- Vektor  

28. 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

29. Current and recently completed HIV vaccine clinical trials
Adapted from McMichael AJ, ann rev Immunol, 2006

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

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