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DNA Vaccination
Anneline Nansen
Department of Infectious Disease Immunology
Statens Serum Institut (SSI)
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
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!
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
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
Chemokine DNA
Th1-Cytokine DNA
Co-stimulatory molecule 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
BN- Vektor
BglII
CaiI
AlwNI
NruGI
EclHKI
Eam1105I
AspEI
AhdI
XmnI
MroXI
Asp700I
AclNI
BcuI
SpeI
HindIII
CMV
intron
T7 promoter
'5`UTR
7000
Amp r
XhoI
SanDI
1000
6000
pCIgB-2
2000
7444 bps
5000
f1
3000
4000
ClaI
Bsu15I
BspXI
BspDI
Bsp106I
BsiXI
BseCI
BscI
Bsa29I
BanIII
DraIII
AdeI
gB
SV40 polyA
MunI
++ XmaIII
++ BseX3I
NotI
CciNI
XbaI
KpnI
Asp718I
Acc65I
SexAI
3´UTR
FseI
SphI
PaeI
BbuI
BspLU11I
SrfI
Insert
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
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