the production and mechanism of action of recombinant peptide vaccine.

1. * Recombinant
Peptide vaccine
ANUJ KUMAR RAJA
PhD. 1st year
Animal Biotechnology center
NDRI, INDIA

2. VACCINE ???? * A vaccine is a biological preparation that
improves immunity to a particular disease.
A vaccine typically contains an agent that resembles
a disease-causing microorganism
Weakened or
Surface
killed forms of its toxins
proteins
the microbe
* The agent stimulates the body's immune system to recognize
the agent as foreign, destroy it, and "remember" it, so that the
immune system can more easily recognize and destroy any of
these microorganisms that it later encounters.

3. *
Live, attenuated Inactivated
Subunit vaccines
vaccines vaccines
Recombinant Conjugate
Toxoid vaccines
peptide vaccines vaccines
Recombinant live Virus-like “Naked” DNA
vector vaccines particles (VLPs) vaccines
Dendritic cell
Edible vaccines
vaccines

4. *
Recombinant peptide vaccines consist of protein
antigens that have been produced in a heterologous
expression system (e.g., bacteria or yeast).
The vaccinated person produces antibodies to the
protein antigen, thus protecting him/her from
disease.

5. Why there is a need to form recombinant peptide
vaccine ?
Viral coat protein
surface antigen,
Hepatitis B Virus HBsAg
Virus cannot be grown
in to a culture to
produce the protein. Constant
supply of
1 plasma from Therefore
infected efforts has
individuals been made to
Highly immunogenic
particles have been produce
Serious HBsAg by
isolated from limitation
infected persons Risk of final recombinant
and used as a preparation being means
vaccine. contaminated
2 with active virion
and other type of
pathogens.

6. Procedure for development of recombinant peptide vaccine
Pathogenic Epitope Expression
microorganism Vector
cDNA
Production of
Selection of
recombinant
recombinant
peptides
transfection

7. Purification of
recombinant
peptide Purified peptide
Checking for
Vaccination
immunogenecity

8. Presentation of peptides by MHC molecule

9. Example… Hepatitis E Virus
HEV has emerged as a significant cause of sporadic cases and
extended outbreaks of acute hepatitis in many parts of the
world.
The 7.5 kb single stranded positive sense RNA genome is
predicted to contain 3 open reading frames (ORF).
ORF1 - Non-structural viral proteins
ORF 2 - major structural protein.

10. It is suggested that product of ORF 2 gene may be antigenic
determinants and raised the possibility of bacterially expressed
peptide as an HEV vaccine candidate.
The dimeric form of the peptide elicited a vigorous antibody
response in experimental animals and the resulting antisera
were found to cross-react against HEV, effecting an efficient
immune capture of the virus.
A 23 kDa peptide locating to amino acid residues 394 to 604 of
the major Hepatitis E Virus (HEV) structural protein was
expressed in E. coli.

11. Steps involved in preparation of recombinant peptide vaccines
Extraction of Viral RNA & cDNA
preparation
Cloning of HEV sequence in
pGEX expression vector
Production and purification of
HEV peptides.
Immune capture of HEV
Reactivity of human sera against
purified pE2

12. The viral genome was reverse transcribed using the primer E5R.

13. Vector for Expression of peptide
fragment
The cloned
sequence was
ligated to the
BamH1 and EcoR1
cloning sites on
the pGEX vector
and expressed as
GST fusion
peptide in E coli.
Bacterial cytosol

14. Recombinant plasmids were transferred into E. coli.
Transformants were selected as ampicillin resistant clones
in LB agar.
Plasmid was extract from these transformants.
The cloned sequences were recovered by EcoRI and BamHI
digestion and their identity was confirmed by sequence
analysis.

15. Production and purification of HEV peptides
An overnight culture of the transformant was grown.
Bacterial cytosol containing the soluble fusion peptide was allowed to
bind with glutathione conjugated sepharose 4B and the purified GST
fusion peptide was eluted with glutathione.
Purified fusion protein was designated GE2.
Alternatively, the moiety of HEV peptide was obtained by thrombin
cleavage.
This purified HEV peptide was denoted as pE2.

16. *
Hyperimmune sera against GE2 were raised in rabbits.
The animals were given four bi-weekly intramuscular doses of the
purified peptide.
The first dose was mixed with complete Freund's adjuvant, and
subsequent doses were mixed with incomplete Freund's adjuvant.
The animals were bled on the 9th week.
* Polystyrene paddles were coated with rabbit anti-GE2 to capture
the HEV.
* Nested RT-PCR for detection of HEV RNA.
* The outer primer pair was A5F and A3R and the inner primer pair
was B5F and B3R (Table I).

17. *

18. Serially diluted rabbit pE2 antiserum was titrated by Western
blotting against an equal mixture of a heated and an unheated
sample of purified E2.
Limiting dilution of the serum reactive against the 42 kDa E2
dimer was 1:6,400 and that against the 23 kDa E2 monomer was
1:800.

19. Advantages
Production and quality control simpler
No other viral or external proteins, therefore less toxic.
Feasible even if virus cannot be cultivated
Safer in cases where viruses are oncogenic or establish a
persistent infection.
Limitation
May be less immunogenic than conventional inactivated
whole-virus vaccines.
Requires adjuvant
Requires primary course of injections followed by boosters.

20. Examples of vaccine produced by Recombinant means.
* Hepatitis B.The vaccine uses hepatitis B
surface antigen produced in yeast.
* B subunit of cholera toxin.
* Vaccine against TB.