Recombinant peptide vaccine
description
Transcript of Recombinant peptide vaccine
*Recombinant
Peptide vaccine
ANUJ KUMAR RAJAPhD. 1st yearAnimal Biotechnology centerNDRI, INDIA
*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.
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 killed forms of the microbe
its toxins Surface proteins
*
Live, attenuated vaccines
Inactivated vaccines
Subunit vaccines
Recombinant peptide vaccines
Conjugate vaccines
Toxoid vaccines
Recombinant live vector vaccines
Virus-like particles (VLPs)
“Naked” DNA vaccines
Edible vaccinesDendritic cell vaccines
*
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.
Hepatitis B Virus
Viral coat protein
surface antigen,
HBsAg
Highly immunogenic
particles have been
isolated from
infected persons
and used as a
vaccine.
Virus cannot be grown
in to a culture to
produce the protein.
Serious
limitationRisk of final
preparation being
contaminated
with active virion
and other type of
pathogens.
Constant
supply of
plasma from
infected
individuals
Therefore
efforts has
been made to
produce
HBsAg by
recombinant
means
Why there is a need to form recombinant peptide
vaccine ?
1
2
Procedure for development of recombinant peptide vaccine
Pathogenic
microorganismEpitope
cDNA
Expression
Vector
transfection
Selection of
recombinant
Production of
recombinant
peptides
Purification of
recombinant
peptide Purified peptide
VaccinationChecking for
immunogenecity
Presentation of peptides by MHC molecule
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.
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.
Extraction of Viral RNA & cDNApreparation
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
Steps involved in preparation of recombinant peptide vaccines
The viral genome was reverse transcribed using the primer E5R.
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
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 BamHIdigestion and their identity was confirmed by sequence analysis.
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.
Production and purification of HEV peptides
*
*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).
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.
*
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.
Feasible even if virus cannot be cultivated
Requires primary course of injections followed by boosters.
Advantages
Production and quality control simpler
No other viral or external proteins, therefore less toxic.
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
*Hepatitis B. The vaccine uses hepatitis B
surface antigen produced in yeast.
*B subunit of cholera toxin.
*Vaccine against TB.
Examples of vaccine produced by Recombinant means.