DNA Vaccination

Anneline Nansen

Department of Infectious Disease ImmunologyStatens Serum Institut (SSI)

What is a vaccine?

A vaccine is a substance that stimulates

an immune response that can eitherprevent 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

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!

• 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

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 shotIntra 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

pcDNA3-Luc pcDNA3

One hour after DNA vaccination

Gene Gun

Modifying the Properties of DCs as Innovative Modifying the Properties of DCs as Innovative Strategies to Enhance DNA Vaccine PotencyStrategies to Enhance DNA Vaccine Potency

Schematic diagram to show DNA vaccination via gene gun

Employment of intracellular sorting signals Employment of intracellular sorting signals to improve antigen processing through to improve antigen processing through MHC class I and II pathways.MHC class I and II pathways.

Employment of intercellular spreading Employment of intercellular spreading strategies to increase the number of strategies to increase the number of antigen presenting cells that present antigen presenting cells that present antigens encoded by DNA vaccines.antigens encoded by DNA vaccines.

Employment of Anti-apoptotic strategies to Employment of Anti-apoptotic strategies to prolong life span of antigen presenting prolong life span of antigen presenting cells that present antigens encoded by cells that present antigens encoded by DNA vaccinesDNA vaccines

Strategies to Enhance DNA Vaccine Potency

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

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

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

Growth factorChemokine

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

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-1/Flt3L-augmented DNA vaccines

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

Ligation

BN- Vektor Insert

DNA Materiale.g.HIV gene

The making of recombinant viruses

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 , June 2006

Preventive prime-boost vaccination strategy against Tuberculosis