Recent trends

1. DNA VACCINE
Presented by:
Pradip k. Chaudhary
CDBT,T.U.
1

2. Introduction
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• 3rd generation vaccine, contains DNA coding for sp.
Protiens froms pathogen.
• genetically engineered DNA so cells directly produce an
antigen, resulting in a protective immunological response.
• Injected into host cells, inner machinery of host cells
reads DNA and synthesize pathogen protiens
• Recognise and processed by host cells and displays on
their surface.
• Immune system is alerted and triggered immune response
• As of June 2015 only one human DNA vaccine has been
approved for human use, the single-dose Japanese
encephalitis vaccine called IMOJEV, released in 2010.

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Figure : A schematic representation of a simple DNA plasmid

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• When applied to Human subjects is still biggest challenge
for practical DNA vaccine to use. ?????
• Many different strategies have been tested in preclinical
models to address this problem,
o Novel plasmid vectors
o Codon optimization to enhance antigen expression
o New gene transfections systems to increase delivery
o Protein or live virus vector to boosting to maximise immune
stimulation
o DNA vaccine with traditional or molecular adjuvants
• Traditional DNA vaccine based on Bacterial Plasmid and by
efficient eukaryotic promoters.
• Delivered through different routes
o intramuscular, subcutaneous, mucosal or transdermal delivery

5. Mechanism of action of DNA vaccine
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Mechanisms of antigen presentation following DNA immunisation
Antigen presentation mediated directly by transfected myocytes; b:
Transfection of professional APCs; c: Cross priming.

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8. • Intrinsic elements of plasmid DNA can also activate innate immune responses, thereby
enhance adaptive immune response.
• toll-like receptor-9 (TLR9) is a cytosolic PRR that binds DNA sequences containing
unmethylated cytosine-guanine (CpG) motifs leading to activation of MyD88 dependent
signaling pathways
• cyclic-GMP-AMP (cGAMP) synthase (cGAS) which, after recognition of dsDNA, induces
cGAMP to activate the stimulator of interferon genes (STING).
• DAI (DLM-1/ZBP1) also activates STING and induces type I interferon expression.
• TBK1, downstream of cGAS and DAI, is important to enhancement of DNA vaccine
action
• DNA sensor is AIM2, which induces inflammasome activation and inflammatory
cytokine production.
• The helicase proteins, DHX29 and RIG-I, sense cytosolic nucleic acids and may
contribute to DNA vaccine action
• Other helicase DDX41, IFI16, DNA-PK and MRE11.
• Molecular adjuvants that represent ligands of the above sensors and signaling proteins
are currently being tested for their ability to improve DNA vaccine immunogenicity.
IFN: interferon; NF: nuclear factor
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9. DNA vaccine constructs design
Codon optimization
• codon optimization is generally required to achieve
efficient mammalian expression of pathogen proteins.
• codon optimization results in enhanced CD8 T-cell
responses
• codon optimization does not always positively
correlate with DNA vaccine efficacy
• For eg. Malaria DNA vaccine, Schistosoma mansoni
Sm14 protein
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10. Promoter selection
• DNA vaccine gene expression is normally driven by a
polymerase II type promoter but not strong.
• CMV is the first choice for most DNA vaccines and
strongest activity(high gene expression) in most cell
types.
o HIV-1 Env DNA vaccines have shown that stronger promoters
induced higher protein expression and immune responses.
• viral promoters being sensitive to inhibition by
inflammatory cytokines eg. TNF-α and IFN-γ
• MHC class II promoter overcome this problems
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11. Optimization of plasmid vector backbone
• sucrose selection construct
o 72 bp SV40 enhancer at the 5ʹ of CMV promoter to increase the
extra-chromosomal transgene expression of the human T-
lymphotropic virus type I (HTLV-I) R region
o at the 3ʹ of a CMV promoter to increase translation efficiency.
o HIV-1 gp120 DNA vaccination; increased
neutralizing antibody titers
• Minicircle DNA (mcDNA) technology
o episomal DNA vectors, small mol. size
o mcDNA is superior to plasmid DNA in eliciting antigen-specific
CD8+ T-cell responses
• modified novel mini-intronic plasmid system was robustly
expressed in vivo and in vitro
• Multicistronic vectors are sometimes constructed to
express more than one immunogen
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12. Traditional adjuvants for DNA vaccines
• Addition of alum adjuvant to a DNA vaccine encoding
HBsAg increased antibody responses in mice, guinea
pigs and nonhuman primates.
• Polysaccharide mediated
• cationic liposome encapsulated pcDNA3.1-based
influenza A virus M1 gene induced both humoral and
cellular immune responses and protected the mice
against respiratory infection.
o Liposome effective in intranasal DNA vaccination
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13. Molecular adjuvants for DNA vaccines
plasmid-encoded immune-stimulatory molecules including various
cytokine genes or PRR ligands
Ligands of PRRs
o TLR3 and TLR9 recognize dsRNA and ssDNA;
• TLR3 their ligands act to enhanced responses to a HPV-16 E7 DNA
vaccine
o The RIG-I ligand, eRNA41H, enhanced the humoral immune response to an
influenza DNA vaccine
Plasmid-encoded cytokines
o IL-2 induces the proliferation of T and NK cells
o A fusion construct of Mycoplasma pneumoniae p1 gene carboxy terminal
region with IL-2 resulted in enhanced vaccine responses
Plasmid-encoded signaling molecules
o Programmed cell death-1 (PD-1)-based plasmids were shown to enhance DNA-
vaccine-induced CD8+ T-cell responses against HIV.
shRNA or siRNA as molecular adjuvants
o RNAi can be used to downregulate genes that suppress DNA vaccine
action
o use of shRNA to knock down caspase 12, a cell death mediator
that is upregulated after DNA vaccination, increased plasmid
gene expression and T-cell and antibody responses
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14. Prime-boost strategies
• boosted by the administration of recombinant protein
or recombinant poxviruse.
• “Prime-boost” strategies with recombinant protein
have successfully increased both neutralising
antibody titre, and antibody avidity and persistence,
for weak immunogens, such as HIV-1 envelope protein.
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15. Gene delivery:
Several methods to improve delivery of DNA vaccine;
• Mechanical delivery consisting microinjection by various
types of needles including pressure injection
• Electrical delivery eg. electroporation, ionophoresis
• chemical (liposomes and various polymers)
• mucosal delivery
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16. • Needle injection is effective in intramuscular
injection; provoke strong, antigen-specific Th1biased,
humoral and cellular immune responses.
• Gene gun delivery of DNA which propels the DNA-
coated gold particles into the epidermis; Th2-bias
response
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Fig: a: Intramuscular injection; b: Electroporation; c: Transient
increased permeability of cell membrane (yellow arrows) results in
plasmid transfer into the cell; d: Resting of cell membrane (red
arrow).

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18. Advantages over traditional vaccines
• Generally only requiring one-step cloning into plasmid vector,
thereby reducing cost and production time
• IN vivo expression of an antigen gene by eukaryotic promoter
and endogenous post-translational modification results in native
protein structure ensuring appropriate immune response.
• Plasmid DNA is stable in RT
• Triggered both CMI (MHC I and II ) and HI
• Highly specific
Disadvantages
 Mild inflammation in the injection site
 Activation of oncogenes (genomic incorporation )
 Eliciting anti-DNA antibodies
• plasmid vaccines is the reduced level of immunogenicity
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Summary of human clinical trials
involving DNA vaccines. Number
of clinical trials of DNA vaccines
carried out in different time
periods, clinical trial phases and
the diseases being targeted are
summarized for all 162 DNA
vaccine trials registered in the
ClinicalTrial.gov database.

20. Reference:
1. Li Lei, and Petrovsky Nikolai(2015) “Molecular
mechanisms for enhanced DNA vaccine
immunogenicity” EXPERT REVIEW OF VACCINES,
14760584.2016.1124762.
2. Sidgi Syed Anwer Abdo Hasson1, Juma Khalifa Zayid
Al-Busaidi , Talal Abdulmalek Sallam (2015) “The
past, current and future trends in DNA vaccine
immunisations” Asian Pacific Journal of Tropical
Biomedicine.
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