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

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

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

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

It has it all!

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

Comparative Analysis of various Vaccine formulations

Properties of Genetic Vaccines

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…

DNA vaccination-Naked plasmid

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

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.

Guns are good for something!! Gene gun: 1 µm DNA per shot Intra muscular: 100 µg of pCMV-S, Mice: 20g, Human: 80.000 g, 5000*100 µg = 0.5g DNA

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

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

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

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;114:1241-1244

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;114:1241-1244

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

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

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

DNA vaccination by use of live recombinant viruses

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

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

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

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

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

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

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