dna tattooing and its role for bacterial vaccines
DESCRIPTION
DNA vaccine, TattooingTRANSCRIPT
K.KARTHIK,
5070
OVERVIEW
DNA TATTOOING & ITS ROLE FOR BACTERIAL VACCINES
BACTERIAL DNA
VACCINES
DNA VACCINES
TATTOOING
INTRODUCTION
Edward Jenner – James Phipps – small pox – 1796
1990, Wolff – naked DNA leads to expression when
transfected
60 licensed vaccines present day
( Mariana et al., 2010)
VACCINE PLATFORM IN USE NOW
• LIVE – ATTENUATED :
-Repeated serial passaging- attenuation of organism
-Elicit both CMI & humoral
-Disadvantage : reversion to virulance
-e.g.: BCG
• KILLED VACCINES:
-No risk of reversion
-Only humoral immunity
-Requires boosters
-e.g.: typhoid vaccine
Cont..
• SUBUNIT VACCINE:
-Purified antigenic proteins ( flagella , capsule )
-Poorly immunogenic
- Requires boosters
- e.g.: Haemophilus influenza (Hib)
• TOXOIDS:
-Inactivated bacterial toxins
-Needs boosters
-Development of adverse reactions
-e.g.: TT vaccine
•3rd generation vaccine
• Recombinant DNA technology in which a DNA transgene coded into
plasmid
•Other names : polynucleotide , genetic , somatic transgene , DNA based
vaccines
NUCLEIC ACID VACCINE – Official term by WHO
( Robinson et al., 1997)
DNA VACCINES????
CONSTRUCTION – IS IT A CAKE
WALK ?
DNA VACCINE
GENE OF INTEREST
PLASMID BACKBONE
EUKARYOTIC EXPRESSION
VECTOR
• VR1012 (Hartikka et al., 1996) and pVAX1
ADVANTAGES 0F DNA VACCINE
Commendable qualities Attributes
Time to manufacture Rapid production
Safety Unable to revert to virulent form
Stability More temperature-stable
Mobility Ease of storage and transport
Immunogenicity T and B cell responses
Autoimmunity against own DNA
Insertionalmutagenesis
Antibiotic resistance transfer
First generation DNA vaccine –
induce low immunity
DISADVANTAGES
DELIVERY METHODSIntra dermal or S/c injection ( Rollier et al., 2008)
Dermal patch with nano particles
-Derma vir against HIV ( Lori et al., 2007)
Painting – stripping few layers of skin then applying
vaccine ( Watabe et al., 2001)
DNA tattooing ????
Electroporation – applying high voltage pulse – disrupt
cell membrane , generates pores – allow macromolecules
(Barr et al., 2002)
Gene gun ( Fuller et al., 2006)
- First reported by Tang et al., 1992
- Directly transfects dendritic cells
( Condon et al., 1996)
- Bombordment with gold particles (1 micron)
propelled with helium
Advantages:
• Requires less DNA
• Coated particles are stable
• Avoids injuries
COMPARISON OF COMMONLY USED
DNA VACCINE DELIVERY METHODS
MODES ADVANTAGES DISADVANTAGES
Intra muscular/dermal •No special delivery
mechanism
•pDNA spreads rapidly
throughout the body
•Relatively large amounts of
DNA used
•Th1 response may not be
present
Gene gun •DNA bombarded directly
into cells
•Small amounts DNA
•Th2 response may not be
present
•Requires inert particles as
carrier
Jet injection •No particles required
•DNA can be delivered to
cells mm to cm below skin
surface
•Significant shearing of DNA
after high-pressure expulsion
•Requires large amounts of
DNA (up to 300 μg)
TATTOOING ????? ?????
SMALL AMOUNT OF VACCINE
SUFFICIENT?
ENTRY
• Protein synthesis
• Cross presentation
LYMPH
• Reach lymph glands
• Activate B & T cells
ARGUMENT
• Production of immunity even after removal of injection site
WHAT FOR TATTOOING?
• Skin has been a successful delivery route for DNA vaccine
(Mitragotri, 2005)
• Higher immunogenicity is due to high APCs
- Langerhan cells- epidermis
-Dendritic cells – dermis
(Larregina et al., 2006)
TECHNIQUES FOR
INTRADERMAL VACCINATION
• Gene gun and particle injection systems
(Mitragotri, 2005; Steitzet al., 2006)
• Jet injectors (Mitragotri, 2005; Bahloul et al., 2006)
• Electroporators ( Hirao et al., 2008)
• DNA tattooing
(Bins et al., 2005; Pokorna et al., 2008)
HISTORY OF ITS USE
• Used in medical research to deliver various materials like:
1. Bleomycin – treatment of hypertrophic scars
(Naeini et al., 2006)
2. Viruses to induce papillomas in mice
(Gomez et al., 2001)
3. Pigments to study processes associated with cosmetic tattooing
(Miller et al., 2005)
4. DNA for gene therapy
(Hogan et al.,1996)
PROBLEMS IN DNA TATTOOING
• Induce strong immune response in murine models
(Bins et al., 2005)
• Superiority to i/m vaccine in non human primates
(Verstrepen et al., 2008)
• No results in humans
• Mouse & macaque skin – high hair follicles than human
(Godin et al., 2007)
FIRST OF ALL
• Trails with “ex vivo” human skin
• Pvax:Luc & Pvax:GFP
WHAT IS DNA TATTOOING• Invasive procedure
• Delivers naked plasmid DNA into the skin through thousands of
punctures using a multiple needle tattoo device.
• Solid vibrating needle cause wound – leads to inflammation
( Gopee et al., 2005)
• Tattoo machine – 7 linear needles with which vaccines can be
administered
• Oscillating frequency of 145Hz
COMMERCIAL TATTOO MACHINES
• Aella (now called
Permanent Make Up
[PMU])
• Cheyenne tattoo
machine
• Marketed by
MT.DERM,
Berlin,Germany
PROCEDURE
• Skin cleaned with phosphate buffer saline
• Chess board pattern marking with marker to represent
tattooing area
• 10μl DNA solution applied over 50mm2
• Needle size – 29 gauge
NO PAIN NO GAIN
• Depth of puncture 1-2 mm
• Surface area of tattoo – 2×1cm
• Voltage – 17.4 v
• 145 punctures / s
• For injecting 10 micro litre – 60 900 solid punctures
RESULTS
• Histology
• Flow cytometry
• Confocal laser scanning microscopy
Expression of LacZ in a cryosection
CONCENTRATION MATTERS
AN
TIG
EN
E
XP
RE
SS
ION
SPECULATIONS
• Better uptake of DNA by APCs
• Better uptake of DNA by non APCs
• Trauma accompanying tattoo – attracts leucocytes
(Bins et al., 2005)
??????
• Whether observed luciferase from keratinocyte form good
measure of vaccine efficacy?
• Can expression in keratinocyte be correlated with
immunogenicity?
• Cross presentation plays a major role in immunogenicity
(Bins et al., 2007)
CURE or CURSE?
DisadvantagesAdvantages
(Genetic Vaccines and Therapy 2008)
“ELECTROTATTOOING”
• Transfection efficiency of naked DNA by tattooing is
extremely low
• So to increase the efficacy- combining both
ELECTROPORATION & TATTOOING
(Van den Berg et al., 2009)
WILL TATTOOING AFFECTS
SUPERCOILING?
• Yes, It affects upto 3% (Koen et al., 2009 )
BUT
• Higher % in jet injection due to high pressure
(Walther et al., 2001)
• Due to high voltage in electroporation
(Heller et al., 2007)
“This ( punctures) is probably what makes
it work better than normal injection
because the tissue is damaged & this
affect the immune cells which then look
out for antigen “
- Martin muller
WHAT NEXT?
• Tattooing of DNA vaccine for melanoma
antigen/other disease to patients
• Estimating the dose
• DNA tattooing for veterinary use
BACTERIAL DNA VACCINESBacteria Targeted antigens
Mycobacterium tuberculosis Ag85A, Ag85B,
FbpA, Htpx,
Rv3407, Hsp65,
Mtb75F, ESAT-6,
MPT64
Streptococcus pneumoniae PspA
Clostridium tetani TetC
Clostridium botulinum AHc
Salmonella typhi OmpC
Listeria monocytogenes Listeriolysin O, p60
Helicobacter pylori HP-NAP, Ure-B,
HP-kat, HspA,
HspB
Bacteria Targeted antigens
Brucella abortus SOD
Mycoplasma agalactiae P48
Staphylococcus PBP2a, pCI, pClfa,
pSrt
Bacillus anthracis PA
Yersinia enterocolitica Hsp
Pseudomonas aeruginosa OprF, FliC R90A
Expert rev vaccines. 2010
APPROVED DNA VACCINESVaccine target Product
name
Company
involved
Date licensed
and country
Target
West Nile virus West Nile
Innovator
Fort Dodge 2005 USA Horses
Infectious
haematopoietic
necrosis virus
Apex-IHN Novartis 2005 Canada Salmon
Growth
hormone
releasing
hormone
LifeTide-SW5 VGX Animal
Health
2007 Australia Swine
Melanoma Canine
Melanoma
Vaccine
Merial 2007 USA,
conditional
license
Dogs
Biotechnol Adv. 2009 ; 27(4): 353–370
HOME TRACK RECORDS
• Immune response studies of DNA vaccine construct encoding
- Lipoprotein B of Pasteurella multocida serotype B 2 in mice
- catalytically inactivated lethal factor ( lef) & protective
antigen 63 (PA 63) gene of Bacillus anthracis strain 34 F2 in
mice
- encoding ompH gene of Pasteurella multocida serotype B2
- encoding epsilon toxin (Etx) gene of Clostridium perfringens
type D in mice
FUTURE PROSPECTS
• BICISTRONIC / POLYCISTRONIC VACCINES
can be available for different antigens
• DNA Vaccines against TB, HIV , malaria may be
available in the near future
IS THIS THE END?
• DNA vaccines can be need of the hour to prevent lot of
diseases
• People won’t take up tattooing of DNA vaccines for cold or
some simple diseases but they will take for some serious
diseases
• DNA tattooing will soon may become one of the better
delivery technique and the dose of DNA used should be
optimized
• This might be cure for some disease like HIV, TB etc