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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 Surfacekilled forms of its toxins proteins the microbe* The agent stimulates the bodys 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.
*Recombinant peptide vaccines consist of proteinantigens that have been produced in a heterologousexpression system (e.g., bacteria or yeast). The vaccinated person produces antibodies to theprotein antigen, thus protecting him/her fromdisease.
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 toHighly immunogenicparticles have been produce Serious HBsAg byisolated from limitationinfected persons Risk of final recombinantand used as a preparation being meansvaccine. contaminated 2 with active virion and other type of pathogens.
Procedure for development of recombinant peptide vaccine Pathogenic Epitope Expression microorganism Vector cDNA Production of Selection of recombinant recombinant peptides transfection
Purification of recombinant peptide Purified peptide Checking for Vaccination immunogenecity
Presentation of peptides by MHC molecule
Example… Hepatitis E VirusHEV has emerged as a significant cause of sporadic cases andextended outbreaks of acute hepatitis in many parts of theworld.The 7.5 kb single stranded positive sense RNA genome ispredicted to contain 3 open reading frames (ORF).ORF1 - Non-structural viral proteinsORF 2 - major structural protein.
It is suggested that product of ORF 2 gene may be antigenicdeterminants and raised the possibility of bacterially expressedpeptide as an HEV vaccine candidate.The dimeric form of the peptide elicited a vigorous antibodyresponse in experimental animals and the resulting antiserawere found to cross-react against HEV, effecting an efficientimmune capture of the virus.A 23 kDa peptide locating to amino acid residues 394 to 604 ofthe major Hepatitis E Virus (HEV) structural protein wasexpressed in E. coli.
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
The viral genome was reverse transcribed using the primer E5R.
Vector for Expression of peptide fragmentThe clonedsequence wasligated to theBamH1 and EcoR1cloning sites onthe pGEX vectorand expressed asGST fusionpeptide 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 sequenceanalysis.
Production and purification of HEV peptidesAn 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.
*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 Freunds adjuvant, and subsequent doses were mixed with incomplete Freunds 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).
Serially diluted rabbit pE2 antiserum was titrated by Westernblotting against an equal mixture of a heated and an unheatedsample of purified E2.Limiting dilution of the serum reactive against the 42 kDa E2dimer was 1:6,400 and that against the 23 kDa E2 monomer was1:800.
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.
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.