Dna vaccine

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DNA VACCINE

Presented by:Pradip k. Chaudhary

CDBT,T.U.

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Introduction• 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 expressiono New gene transfections systems to increase deliveryo Protein or live virus vector to boosting to maximise immune

stimulationo DNA vaccine with traditional or molecular adjuvants

• Traditional DNA vaccine based on Bacterial Plasmid and by efficient eukaryotic promoters.

• Delivered through different routeso intramuscular, subcutaneous, mucosal or transdermal delivery

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Mechanism of action of DNA vaccine

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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• 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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DNA vaccine constructs designCodon 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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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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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) technologyo episomal DNA vectors, small mol. sizeo 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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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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Molecular adjuvants for DNA vaccinesplasmid-encoded immune-stimulatory molecules including various cytokine genes or PRR ligands

Ligands of PRRso TLR3 and TLR9 recognize dsRNA and ssDNA;

• TLR3 their ligands act to enhanced responses to a HPV-16 E7 DNA vaccineo The RIG-I ligand, eRNA41H, enhanced the humoral immune response to an

influenza DNA vaccinePlasmid-encoded cytokines

o IL-2 induces the proliferation of T and NK cellso A fusion construct of Mycoplasma pneumoniae p1 gene carboxy terminal region

with IL-2 resulted in enhanced vaccine responsesPlasmid-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 adjuvantso RNAi can be used to downregulate genes that suppress DNA vaccine actiono 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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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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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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• 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

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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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 theClinicalTrial.gov database.

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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.