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