recombinant vaccine 11

8/12/2019 Recombinant Vaccine 11

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DESIGN AND PRODUCTION

OF RECOMBINANT

SUBUNIT VACCINES


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The advancement of genomics,

proteomics and biotechnology provide

us the opportunity to develop safe and

more effective vaccines


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ACTIVE VACCINE

Stimulates Humoral Immune Response,Cellular

Immune Response or Both, with the aim of

protecting against or eliminating a pathogen

PASSIVE VACCINE

Preparation of Abs, Protect against a pathogen or

disease and is administered before, at or around

the time of known or potential exposure


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comparison of different vaccine types

LIVE VACCINES (ATTENUATED)

(MMR, Oral Polio)

Advantages:

One or few doses required

Long lasting protection

Both humoral and cellular responses

Disadvantages:

Controlled attenuation normally required

Poorly defined composition

Risk of reversion to pathogenicity

Certain risk of transmission


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KILLED VACCINE

(Polio and Influenza)

Advantages:

No risk of reversion to pathogenicity

No risk of transmission

Disadvantages:

Multiple dose typically required

Poorly defined composition

Antigen produced by cultivation of a pathogen

Mainly humoral responses

Ad uvants normall needed


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TOXOID

(Tetanus and Diphtheria)

Advantages:

Product is devoid of live organism

Implies greater safety

Disadvantages:

Multiple dose typically required

Relatively expensive to manufacture

Cultivation of a pathogen for toxin production


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SUBUNIT VACCINES

SUBUNIT VACCINES ARE DEFINED AS THOSE

CONTAINING ONE OR MORE

PURE OR SEMI-PURE

ANTIGENS


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RECOMBINANT SUBUNIT VACCINES

Identify and isolate a specific gene fromvirulent bacteria or virus (gene that codes

immuno protective protein).

Gene is inserted into plasmid DNA and

ligated with ligase.New (engineered) plasmid inserted into

another bacterium (transform).

Allowed to grow and actually produce

the antigenic protein.The vaccine is comprised of purified

proteins recovered from the expression vector.

Target gene


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RECOMBINANT SUBUNIT VACCINES

Advantages:

No risk of pathogenicity

Defined composition

Various delivery systems

Simplified large scale production

Further engineering possible

Disadvantages:

Multiple doses typically require

Adjuvants needed


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recent development in vaccinology

POLYNUCLEOTIDE VACCINATION

This technology has been referred to as genetic

immunization or DNA immunization

The basis for this approach to immunization is that cells

can take-up laboratory made DNA and express the genes

within the transfected cells

Thus, the animal acts as a bioreactor to produce the

vaccine


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recent development in vaccinology

ADVANTAGES OF POLYNUCLEOTIDE IMMUNIZATION

Safe and long lived immunity

inexpensive

can induce immune responses in the presence of maternal

antibodies

Most recently, it has also been used for immunizing fetuses. Thus,

animals are born immune to the pathogens and have life longprotection

Also being tested in humans against malaria, influenza, and HIV


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TARGETS FOR THE

DEVELOPMENT OF SUBUNIT VACCINE

Identifying genes

Isolating genes

Modifying genes

Re-expressing genes in other hosts or organisms


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Recombinant Protein Expression Systems

Bacteria (Escherichia coli)

Yeast (Pichia pastoris)

Virus (Baculovirus)

Animal cell culture (CHO)

Plants

Sheep/Cows


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Bacterial Systems

Grow quickly (8-12 hrs

to produce protein)

High yields (50-500

mg/L)

Low cost of media

(simple mediaconstituents)

Low fermentor costs

Difficulty expressing large

proteins (>50 kD)

No glycosylation or signal

peptide removal

Eukaryotic proteins are

sometimes toxic Cant handle S-S rich

proteins

Advantages Disadvantages


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Yeast Systems

Grow quickly (12-24

hrs to produce protein)

Very high yields (50-5000 mg/L)

Low cost of media

(simple mediaconstituents)

Low fermentor costs

Can express large proteins

(>50 kD)

Glycosylation & signalpeptide removal

Has chaperonins to help

fold tough prtns Can handle S-S rich

proteins

Advantages More advantages


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Baculovirus Systems


Grow very slowly (10-12

days for set-up) Cell culture is only

sustainable for 4-5 days

Set-up is time consuming,

not as simple as yeast


(>50 kD) Correct glycosylation &

signal peptide removal

Has chaperonins to helpfold tough proteins

Very high yields, cheap


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Mammalian Systems

Selection takes time

(weeks for set-up)

Cell culture is only

sustainable for limited

period of time

Set-up is very time

consuming, costly, modest

yields


(>50 kD) Correct glycosylation &

signal peptide removal,

generates authentic proteins Has chaperonins to help

fold tough proteins



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DEVELOPMENT OF

RECOMBINANT HBV

SUBUNIT VACCINE


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Marker +VE -VE HB6 HB7 HB14 HB15 HB19

PCR AMPLIFICATION OF PRE-S1 REGION

1.4 Kb


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PCR confirmation of T-A clones

EcoR1 Restriction Digestion for

confirmation of T-A clones


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PCR confirmation of positive clones

Vector product without insert

PCR product

PCR Confirmation of HBsAg Positive Clones


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Expression optimization of HBsAg in P.pastoris

M S14 S15 S16 S21 S25 GS115 M

25 kDa


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Western Blotting With Anti-HBsAg

kDa

115

96

65

50

35

25

15

10

1 2 3 -Ve M

15% SDSPAGEWestern Blot

25kDa HBsAg

Induced


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Elisa With Anti-HBsAg

PBS +Ve Recombinant clone with HBsAg GS115(Host)


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PRODUCTION OF SUBUNIT VACCINE

SEED CULTURE BANK

FERMENTATION

HARVESTING

CELL LYSIS

PROTEIN SOLUBLIZATION / REFOLDING

FILTRATION / CONCENTRATION

PROTEIN PURIFICATION

FORMULATION

PACKAGING / QC

MARKETING


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THANKYOU

Biotechnology offers new approaches to animal production and health

which could benefit the region

recombinant vaccine 11

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