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

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DNA Vaccination

Anneline Nansen

Department of Infectious Disease ImmunologyStatens Serum Institut (SSI)

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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

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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

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•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

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• 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

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Comparative Analysis of various Vaccine formulations

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Properties of Genetic Vaccines

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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…

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DNA vaccination-Naked plasmid

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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

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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.

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UTCharacterization 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

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

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

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

Strategies to Enhance DNA Vaccine Potency

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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

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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

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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

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

Immunohistochemistry of injection sites

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

Analysis of DCs extracted from injected muscles

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

Immunogenicity of MIP-1/Flt3L-augmented DNA vaccines

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DNA vaccination by use of live recombinant viruses

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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

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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

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Ligation

BN- Vektor Insert

DNA Materiale.g.HIV gene

pCIgB-2

7444 bps

1000

2000

3000

4000

5000

6000

7000

AclNIBcuISpeI

HindIII

XhoI

SanDI

BspLU11I

SrfI

FseI

BbuIPaeISphI

SexAIAcc65I

Asp718IKpnI

XbaICciNI

NotIBseX3I++XmaIII++

MunI

BanIIIBsa29I

BscIBseCIBsiXI

Bsp106IBspDIBspXI

Bsu15IClaI

AdeIDraIII

Asp700IMroXIXmnI

AhdIAspEI

Eam1105IEclHKINruGI

AlwNICaiI

BglII

CMV

intronT7 promoter

'5`UTR

gB

3´UTR

SV40 polyA

f1

Amp r

The making of recombinant viruses

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Adapted from Rocha CD et al. Int Microbiol, 2004

Homologous Recombination

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Homologous Recombination

Fibroblast cell

MVA BN Virus

1 hour

Infection Transfection

2 days

GFP positive Cells

Plasmid DNA MVA BN mit

Plasmid DNA

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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

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Current and recently completed HIV vaccine clinical trials

Adapted from McMichael AJ, ann rev Immunol, 2006

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Skeiky et al. Nature Reviews Microbiology 4, 469–476, 2006

On-going Tuberculosis vaccine clinical trials

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Skeiky et al. Nature Reviews Microbiology 4, 469–476 , June 2006

Preventive prime-boost vaccination strategy against Tuberculosis

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THE END