biotechnological interventions to animal skin – implications to the … · 2010-01-29 · dna...

Biotechnological interventions to animal

skin – implications to the leather industry

G. Dhinakar Raj

Professor

Department of Animal Biotechnology

Madras Veterinary College

Tamil Nadu Veterinary and Animal Sciences University

Chennai – 600 007

Skin

• Skin is the largest organ of the body

• Functions in protection, sensation,

thermoregulation and metabolism• Primary barrier to pathogens

• Vehicle for delivery of vaccines

• Biological testing

Structure of skin

• Epidermis with its appendices, the hairs.

• It consists of a variety of cell layers produced in the germinative basalcell region located at the base of the dermis. The descendents of thesecells move to the surface of the epidermis while undergoingkeratinization.

• At the final stage they form a horny layer on the surface of theepidermis. This is a continuous process with new cells, produced by thegerminative layer, replacing the horny layer as it breaks into flakes andsloughs off.

• Dermis, a three-dimensional weave of collagen fibers composed ofconnective tissue providing support to the structures associated with theskin, such as hair, sweat glands, nerves and blood vessels.

• It is rich in antigen presenting cells (APCs), called dendritic cells (DCs)and Langerhans cells (LCs) that process and present the antigensdelivered through the skin to initiate immune responses.

• The leather is animal skin treated such that the natural properties of theskin are retained.

Scanning Electron Micrograph of

bovine epidermis; epidermis

loosened after acidic

incubation of skin;

magnification: '2000, length of

bar: 10 mm. Arrow marks

dividing cell.

Scanning Electron Micrograph

of bovine skin, cut after acidic

incubation. Epidermis has

separated from the grain

revealing its surface structure,

magnification: '700, length of

bar: 25 mm.

Epi = epidermis; G = grain.

Skin section of normal sheep.

Normal epidermis without

hyperkeration, parakeration or

asnthosis. Normal hair follicles

(HF) without dermis.

H & E X 160.

Leather grain surface of normal

goat. The surface shows normal

goat grain pattern-undisturbed by

any distortions or contours.

Goat - scabby surface and

unmistakable lichenification

resulting in extensive folds

and wrinkles.

Dermatophilosis/epidermoid

Cyst.

Wool break due to mite

infestation

Biotechnological interventions that could

improve the quality of animal skin

• Diagnosis of diseases

• Vaccines

• Needle free delivery

• Nanotechnology

• Enhancing immunology of the skin

• Alternatives to skin testing methods

PCRPCR• Specifically targets and amplifies a single

sequence from within a complex mixture of

DNA

• In vitro DNA amplification technique invented

by Kary Mullis in 1985

• In the last decade PCR has revolutionized the

detection of infectious pathogens because of

its high sensitivity and specificity

• Automated for routine use in laboratories

worldwide

• PCR is DNA replication in a test tube

Why should we use PCR in diagnosis?

• Very sensitive (1 copy – 10 copies of DNA)

• Can detect organisms that cannot be isolated

• Rapid

Disadvantages of PCR

• Technically demanding

• Can be expensive

• Risk of contamination

• Need rigid QC

BTV PCR

Group-specific primers

BTV S7 (VP7)

BTV S10 (NS3)

BTV S8 (NS2)

BTV S9 (VP 6)

BTV S 6 (NS 1)

Serotype-specific primers

BTV S2 (VP2)

274 bp

101 bp

1156 bpNS1 PCR

VP 7 PCR

274 bp

1156 bp

101 bp

274 bp

1156 bp

Pathogen isolation and identification

Vs

Pathogen identification and isolation

Speed, sensitivity and discriminating

power

Conventional approach – takes a long

time, hence only retrospective diagnosis

Is there a paradigm shift?

Strengths Vs Weaknesses of PCR in

diagnosis

• Speed

• Sensitivity

• Specificity

• Origin of virus in a

outbreak (topotype)

• Sometimes differentiation

between vaccine and field

virus

• Phylogenetic relationships

• Does not detect live virus

per se

• Need live virus for cell

culture, animals and

vaccine studies

• Problems of high

sensitive tests in relation

to laboratory

contamination and GLP

Real time PCR

• Monitor the accumulation of the PCR

product while amplification is

occurring

• Analyze cycle to cycle changes in

fluorescent signal generated during the

three phases of PCR

• The fewer cycles to see detectable

fluorescence signal the greater the

amount of template DNA

Real-Time PCR

Real-time PCR monitors the

fluorescence emitted during the reaction

as an indicator of amplicon production

at each PCR cycle (in real time) as

opposed to the endpoint detection

GAPDH standards GAPDH standard

curve; Y=0.24x + 8.19;

r2=0.994

GAPDH sample curves

IFN g - StandardsIFN g standard curve

Y=0.29x +11.20

r2=0.987

IFN g sample curves

Melting curve –

standard, sample,

control

Vaccination

• Oral or injected vaccine

– induces the host to generate antibodies

against the disease-causing organism

– subsequent infections do not establish the

disease

– infectious agent is inactivated by the

antibodies and immune response

Conventional Vaccines

• Inactivated agent

– killed organism used

• Attenuated agent

– live, nonvirulent form of organism

New generation vaccines

• Subunit Vaccines

• Peptide Vaccines

• Genetic Immunization (DNA Vaccines)

• Vector Vaccines

• Anti-idiotype vaccines

• Edible vaccines

Subunit Vaccines

• Generally whole pathogenic agent is

used to construct attenuated or

inactivated vaccine

• Immune response generally elicited by

interaction with proteins on outer

surface of pathogen

Schematic of an animal virus

Envelope

Proteins

Envelope

Nucleic Acid

Capsid

Subunit Vaccine

• So, is the entire pathogen required?

Subunit Vaccine

• No, only outer surface proteins are

needed to elicit an immune response

• Vaccines that use components of a

pathogen rather than the whole

organism are “subunit” vaccines

Subunit Vaccine

• Advantages

– Using a purified protein ensures that thevaccine is safe and stable

• Disadvantages

– Purification may be costly

– Isolated protein may not have the sameconformation as in the pathogen, so maynot have the same antigenicity

Development of Subunit

Vaccine against HSV

HSV

Not Protected Protected

clone gD

gene transfect

inject

purify &

concentrateinfect

infect

CHO cell secreted protein

Multiprotein Subunit Vaccine

Vaccines

• Subunit Vaccines

• Peptide Vaccines

• Genetic Immunization (DNA Vaccines)

• Vector Vaccines

• Anti-idiotype vaccines

• Edible vaccines

Peptide Vaccines

• Subunit vaccine uses entire protein

– Contains several antigenic determinants

• Peptide Vaccine

– vaccine from a specific domain of an

antigenic protein

– single epitope or antigenic determinant

Generalized membrane-bound

protein with external epitopes

5 possible

antigenic

epitopes

Structure of a peptide

vaccine

Linker

Short

PeptidesCarrier

Protein

KLH or HBcAg

Limitations of Peptide

Vaccines– Epitope must consist of a contiguous stretch of

amino acids

– Not all peptides are effective in eliciting an

immune response (may need 2 or more)

– Peptide must have the same conformation as in

pathogen

– Amount of peptide required to elicit an immune

response may be 1000X more than for inactivated

pathogen

Vaccines

• Subunit Vaccines

• Peptide Vaccines

• Genetic Immunization (DNA Vaccines)

• Vector Vaccines

• Anti-idiotype vaccines

• Edible vaccines

DNA Vaccines

plasmidMuscle cell

Gene

for

antigen

Muscle cell

expresses protein -

antibody made

CTL response

DNA Vaccines

• Plasmids are easily manufactured in large amounts

• DNA is very stable

• Storage and transport are straight forward

• DNA sequence can be changed easily in the laboratory.

This means that we can respond to changes in the

infectious agent

• By using the plasmid in the vaccinee to code for antigen

synthesis, the antigenic protein(s) that are produced are

processed (post-translationally modified) in the same

way as the proteins of the virus against which protection

is to be produced. This makes a far better antigen than

purifying that protein and using it as an immunogen.

DNA Vaccines

• Mixtures of plasmids could be used that encode many

protein fragments from a virus/viruses so that a broad

spectrum vaccine could be produced

• The plasmid does not replicate and encodes only the

proteins of interest

• No protein component so there will be no immune

response against the vector itself

• Because of the way the antigen is presented, there is a

CTL response that may be directed against any antigen in

the pathogen.

DNA Vaccines

Possible Problems

• Potential integration of plasmid into host

genome leading to insertional mutagenesis

• Induction of autoimmune responses (e.g.

pathogenic anti-DNA antibodies)

• Induction of immunologic tolerance (e.g. where

the expression of the antigen in the host may

lead to specific non-responsiveness to that

antigen)

Vaccines

• Subunit Vaccines

• Peptide Vaccines

• Genetic Immunization (DNA Vaccines)

• Vector Vaccines

• Anti-idiotype vaccines

• Edible vaccines

Transfer vector for

Homologous Recombination

Plasmid

Cloned Antigen

Gene

Vaccinia virus

thymidine kinase

DNA

Vaccinia virus

promoter

Vaccinia virus

thymidine kinase

DNA

Homologous Recombination

into the Vaccinia Genome

Plasmid

Vaccinia virus

DNA

Cloned antigen

gene

Vaccinia virus DNA

Vaccinia

virus promoter

Vaccines

• Subunit Vaccines

• Peptide Vaccines

• Genetic Immunization (DNA Vaccines)

• Vector Vaccines

• Anti-idiotype vaccines

• Edible vaccines

antibody

Anti-idiotype vaccine

epitope

Antibody

with

epitope

binding site

Virus

antibody

Anti-idiotype vaccine cont

Make antibody

against antibody

idiotype

Anti-

idiotype

antibody

Anti-idiotype

antibody mimics

the epitope

Anti-anti-idiotype

antibody

Anti-idiotype antibodycont 2Use anti-idiotype antibody as

injectable vaccine

Binds and

neutralizes virus

Anti-idiotype

antibody

Anti-anti-idiotype

antibody

Anti-anti-idiotype

antibody

Use as

vaccine

Vaccines

• Subunit Vaccines

• Peptide Vaccines

• Genetic Immunization (DNA Vaccines)

• Vector Vaccines

• Anti-idiotype vaccines

• Edible vaccines

A subunit based edible vaccine?

• economical

• heat stable

• oral administration

– no more needles

– mucosal immune response

• sub-unit vaccine

– unable to replicate

– overcome maternal antibodies

Needle-free technology – Why?

• Better quality of hides and skin

• Consistent vaccine delivery

• Lower vaccine volume

• Higher antigen dispersion

• Elimination of broken needles

• Elimination of needle disposal

• Less pain and stress to animal

The skin is rich in antigen-presenting cells. Dendritic cells (DCs) can be

found at high density in the dermis whereas Langerhans cells (LCs) are

mostly localized in the epidermis.

• Comparative MRI’s show good subcutaneous

delivery

• Slightly broader dispersion than conventional needle

Needle & SyringeNeedle-Free

Needle free technologies available

• Spring loaded jet injector

• Battery powered jet injector

• Gas powered jet injector

Schematic overview of the main cellular mechanisms

involved in nasal vaccination.

NANOTECHNOLOGY

Nanotechnology is science of seeing, measuringand manipulating matter at atomic, molecularand supramolecular levels.

At this scale, the physical, chemical andbiological properties of materials differfundamentally and often unexpectedly fromthose of the corresponding bulk materials.

Nanobiotechnology, is the biomedicalapplications of nano-sized systems

Nanoparticles are defined as particles whichmeasures 1-100 nm

Nano…• Nanotechnology (Greek word nano means ‘dwarf’) is the

creation and utilization of materials, devices, and systemsthrough the control of matter on the nanometer length scale

• It is the popular term for the construction and utilization offunctional structures with at least one characteristicdimension measured in nanometer scale –a nanometer(nm) is one billionth of a meter (10 –9 m).

• This is roughly four times the diameter of an individual atom.

• Width of DNA is approximately 2.5 nm and proteinmolecules measure 1–20 nm.

APPLICATION OF NANOPARTICLES

Vaccine delivery

Carriers for various proteins

Targeted drug delivery

Gene therapy using vectors

Gene delivery

Transfection of genes to cells

Tumor imaging, in vivo biomolecular profiling

of cancer biomarkers

Therapeutic use

Morphology of CaP nanoparticles in

SEM

• Spherical, smooth and tendency to

agglomerate

Morphology of CaP nanoparticles in TEM

CaP nanoparticles

appeared spherical,

smooth and

uniformly

distributed

Size of CaP nanoparticles using SEM

• The mean (±

SE) size of

CaP

nanoparticles

was 557.44 ±

18.62 nm

(n=40).

Humoral immunity - Mean (± SE) HI titers (log2)

in the serum of chickens vaccinated with CaP

nanoparticles coupled NDV

2.00a ±0.001.67a ±0.684.00 b ±0.584

2.17a ± 0.314.17b ± 0.314.20b ± 0.543

2.33a ± 0.214.00b ± 0.454.20b ±0.712

2.75 a ± 0484.00 a ± 0.413.00 a ± 0.411

IIIIII

Treatment GroupsWeeks

PV

Prolonged immunity in CaP coupled NDV vaccinated chickens (Group 1)

Cell mediated immunity - Mean (± SE) stimulation index of

splenocytes stimulated with antigen (NDV) following

vaccination of chickens with CaP nanoparticles coupled

NDV

0.75a ±0.03001.29 b ±0.01001.41b ±0.07004

0.52a± 0.00200.87 b±0.01701.38 b ±0.0363

0.50 a± 0.00050.99 b±0.00061.11b±0.00042

0.59a ±0.00210.72a ±0.00261.08b ±0.00211

IIIIII

Treatment Groups

Weeks

PV

Early increase in stimulation index in CaP groupcompared to conventional vaccine

Gelatin nanoparticles

Components of a modern vaccine

Caudal Fold Tuberculin

Test• The Purified Protein

Derivative (PPD)

tuberculin is injected

intradermally

(between the layers

of skin) of the caudal

tail fold, under the

animal’s tail

• The injection site is examined 72 hours

later, plus or minus 6 hours

• If there is any response at the injection

site

– swelling

– redness

– hardness

• The animal is considered suspect

(responder) and further testing is required

Comparative

Cervical

Tuberculin (CCT)

Test

• This test is only done on animals that respond to

the caudal fold test (CFT)

• Once the animal is secured a section of the neck

is shaved in two places before administering the

CCT test between (intradermally) the skin layers

on the animal’s neck

• Skin thickness is measured using a special

caliper and then the veterinarian will inject avian

(bird) tuberculin and bovine (cattle) tuberculin

into two shaved sites on the neck

• Swelling is often the reaction to the avian

TB injection.

– Producers should not be alarmed

• Test results are plotted on a scattergram.

Based on where the results fit on the

graph, the animals will be classified as

negative, suspect or reactor

Alternative to skin testing

• Interferon assay

• Component of cell mediated

immune response

• Antigen specific secretion

• Measurable

• Associated with TB exposure

QuantiFERON® kitWhole Blood CultureWhole Blood Culture

Measure [IFN- ] & Interpret] & Interpret

Centrifuge 5 minutes to

separate plasma above gelCollect 1mL of

blood in 3

tubes

Incubate at

37ºC for 16-24

hours.

Nil

Mtb

PHA

TMBTMB

COLORCOLOR

Measure [ IFN-Measure [ IFN- ] in] in

‘‘SandwichSandwich’’ ELISA ELISASoftware calculates

results and prints

report

Collect 50 L of

plasma for

ELISA

Nil

Mb

PHA

TB diagnosis using ELISPOT

• Collect blood

• Recover, wash, & countPBMCs

• Aliquot 250,000 PBMCs to4 wells with anti-IFN-

• Add saline, PHA, ESAT-6 orCFP-10 & incubate

• Wash away cells

• Develop & count spotswhere cells produced IFN-

INF-

Antibody

Sensitized T

cell

INF-

Captured

Detection

Antibody

Chromogen

Spot

Saline ESAT-6 CFP-10 PHA

Sum up….

• Biotechnological methods offer ways and

means for improving the overall health of

animals and in particular the quality of skin

?