DESIGN AND PRODUCTION

OF RECOMBINANT
SUBUNIT VACCINES


The advancement of genomics,
proteomics and biotechnology provide
us the opportunity to develop safe and
more effective vaccines



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

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
comparison of different vaccine types
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
Adjuvants normally needed
comparison of different vaccine types
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




SUBUNIT VACCINES

SUBUNIT VACCINES ARE DEFINED AS THOSE
CONTAINING ONE OR MORE
PURE OR SEMI-PURE
ANTIGENS


comparison of different vaccine types
SUBUNIT VACCINES (NON-RECOMBINANT)
Constituent proteins of bacteria or virus are isolated and purified
Advantages:
Defined Composition
Various delivery systems available

Disadvantages:
Antigens must be produced and purified by cultivation of a
pathogen
Multiple doses typically required
Adjuvant needed
RECOMBINANT SUBUNIT VACCINES


Identify and isolate a specific gene from
virulent 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
comparison of different vaccine types
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

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

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 long
protection

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


TARGETS FOR THE
DEVELOPMENT OF SUBUNIT VACCINE

Identifying genes
Isolating genes
Modifying genes
Re-expressing genes in other hosts or organisms



Recombinant Protein Expression Systems

Bacteria (Escherichia coli)
Yeast (Pichia pastoris)
Virus (Baculovirus)
Animal cell culture (CHO)
Plants
Sheep/Cows

Bacterial Systems
Grow quickly (8-12 hrs
to produce protein)
High yields (50-500
mg/L)
Low cost of media
(simple media
constituents)
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
Yeast Systems
Grow quickly (12-24
hrs to produce protein)
Very high yields (50-
5000 mg/L)
Low cost of media
(simple media
constituents)
Low fermentor costs
Can express large proteins
(>50 kD)
Glycosylation & signal
peptide removal
Has chaperonins to help
fold tough prtns
Can handle S-S rich
proteins
Advantages More advantages
Baculovirus Systems
Advantages Disadvantages
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

Can express large proteins
(>50 kD)
Correct glycosylation &
signal peptide removal
Has chaperonins to help
fold tough proteins
Very high yields, cheap
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

Can express large proteins
(>50 kD)
Correct glycosylation &
signal peptide removal,
generates authentic proteins
Has chaperonins to help
fold tough proteins

Advantages Disadvantages
DEVELOPMENT OF
RECOMBINANT HBV
SUBUNIT VACCINE
Marker +VE -VE HB6 HB7 HB14 HB15 HB19
PCR AMPLIFICATION OF PRE-S1 REGION
1.4 Kb
PCR confirmation of T-A clones
EcoR1 Restriction Digestion for
confirmation of T-A clones
PCR confirmation of positive clones
Vector product without insert
PCR product
PCR Confirmation of HBsAg Positive Clones

Expression optimization of HBsAg in P.pastoris
M S14 S15 S16 S21 S25 GS115 M
25 kDa
Western Blotting With Anti-HBsAg
kDa
115
96
65
50
35
25

15
10
1 2 3 -Ve M
15% SDSPAGE
Western Blot
25kDa HBsAg
Induced
Elisa With Anti-HBsAg


PBS +Ve Recombinant clone with HBsAg GS115(Host)
PRODUCTION OF SUBUNIT VACCINE
SEED CULTURE BANK
FERMENTATION
HARVESTING
CELL LYSIS
PROTEIN SOLUBLIZATION / REFOLDING
FILTRATION / CONCENTRATION
PROTEIN PURIFICATION
FORMULATION
PACKAGING / QC
MARKETING

THANKYOU
Biotechnology offers new approaches to animal production and health
which could benefit the region