S T A T E N S S E R U M I N S T I T U T 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
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