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Vaccines
 

Vaccines

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Different vaccines description, how they are produced, their advantages and disadvantages.

Different vaccines description, how they are produced, their advantages and disadvantages.

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    Vaccines Vaccines Presentation Transcript

    •    Vaccination is the phenomenon of preventive immunization. Vaccination involves the administration(oral or injection) of an antigen to elicit an antibody response that will protect the organism against future infections. History of vaccinia --- the cow pox viruses inoculated by Jenner, stimulate the body’s immune system to produce antibodies which neutralize the cow pox as well as small pox viruses. (small pox- virulent disease).
    • 1. 2. 3. Dead bacteria or inactivated viruses. Live non virulent or weakened (attenuated) bacteria/ or viruses. Viral fragments or bacterial molecules (subunit vaccines).
    • 1. 2. 3. Subunit R.V : These are components of the pathogenic organisms. They include proteins, peptides and DNA. Attenuated R.V: These are genetically modified pathogenic organisms (bacteria or viruses) that are made non- pathogenic and are used as vaccines. Vector R.V : These are the genetically modified viral vectors that can be used as vaccines against certain pathogens.
    • Components of pathogenic organism’s proteins, peptides and DNA. Eg: Hepatitis B vaccine.  While proteins must be produced in a cellular environment, peptides can be synthesized.  However, peptides are often only weakly immunogenic.  For this reason, a mixture of peptides may be utilized, although whole recombinant proteins are most often used. 
    • Advantages  Purity in preparation.  Stability  Safe use Disadvantages  High cost factor  Possible alteration in native conformation
    •     Primarily affects liver causing chronic hepatitis, cirrhosis and liver cancer. Hepatitis B virus is a 42nm particle, called Dane particle. Consists of a core containing viral genome (DNA) surrounded by a phospholipid envelope carrying surface antigens. The gene encoding for hepatitis B surface antigen (HBsAg) has been identified.
    •   Recombinant hepatitis B vaccine as a subunit vaccine, is produced by cloning HbsAg gene in yeast cells. Saccharomyces cerevisiae, a harmless baking and brewing yeast, is used for this purpose.
    • 1. 2. 3. 4. 5. 6. The gene foe HbsAg is inserted (pMA 56) which is linked to the alcohol dehydrogenase promoter. These plasmids are then transferred and cultured. The cells grown in tryptophan, free medium are selected and cloned. The yeast cells are cultured. The HbsAg gene is expressed to produce 2nm sized particles similar to those found in patients infected with hepatits B. (these particles are immunoreactive with anti- HbsAg antibodies) The subunit HbsAg as 22nm particles can be isolated and used to immunize individuals against hepatits B.
    • These are genetically modified pathogenic organisms (bacteria or viruses) that are made non- pathogenic and are used as vaccines.  The genetic manipulations for the production of these vaccines are broadly of 2 types: 1. Deletion or modification of virulence genes of the pathogenic organisms. 2. Genetic manipulation of non- pathogenic organisms to carry and express antigen determinants from pathogenic organisms. Eg. Cholera vaccine 
    •        Vibrio cholerae On entering the small intestine, V.cholerae colonises and starts producing large amounts of toxic protein, a hexameric enterotoxin. Currently used cholera vaccine is composed of phenol-killed V. cholerae. Enterotoxin, a hexamer, consists of one A subunit and 5 identical B subunits. The A subunit has 2 functional domains- the A1 peptide which possesses the toxic activity and A2 peptide that joins A subunits to B subunits. By genetic engg, it was possible to delete the DNA sequence encoding A1 peptide that joins A subunit to B subunits. By genetic engg., it was possible to delete the DNA sequence encoding A1 peptide and create a new strain of V. cholerae.
    • 1. 2. A tertracycline resistance gene was inserted into the A1 peptide sequence of V.cholerae chromosome. This destroys the DNA sequence encoding for A1 peptide, besides making the strain resistant to tetracycline. Unfortunately, the tetracycline resistant gene is easily lost and the enterotoxin activity is restored. Because of this, the new strain of V.cholerae as such cannot be used as a vaccine. The DNA sequence of A1 peptide is incorporated into the plasmid, cloned and digested with restriction enzymes (Cla1 and Xba1). In this manner, the A1 peptide coding sequence is deleted (the DNA encoding for 183 of the 194 amino acids of the A1 peptide is actually removed. By using T4 DNA ligase, the plasmid is recircularized. This plasmid contains a small portion of A1 peptide coding sequence.
    • 3. The plasmid, containing the deleted A1 peptide sequence is transferred by conjugation into the V. cholerae strain carrying a tetracycline resistance gene. 4. Recombination can occur b/w the plasmid (containing a small portion of peptide A1 coding sequence) and the chromosome of V. cholerae (carrying tetracycline resistance gene). The result of this double crossover is the formation of V. cholerae containing a chromosomal DNA lacking A1 peptide DNA sequence. As the bacterium multiplies, the plasmids are lost in the next few generations. 5. The V. cholerae cells defective in A1 peptide are selected, based on tetracycline sensitivity. It may be noted that this new strain lacks tetracycline resistance gene.
    • Advantages  Immune response is substantially high.  Stable Disadvantages  Side effects
    •      These are the genetically modified viral vectors that can be used as vaccines against certain pathogens. Vaccinia viruse (Jenner) Genes encoding significant Ag’s from pathogens may be transferred to attenuated viruses/bacteria Vectors include: vaccinia, polio, adenoviruses Salmonella, BCG strain of M. bovis, oral Strep Other vectors may prove to be safer.
    • Vaccinia virus       Contains a double stranded DNA that encodes about 200 different proteins. The genome of this virus can accommodate stretches of foreign DNA which can be expressed along with the viral genes. It can replicate in the host cell cytoplasm(of the infected cells) rather than nucleus. [E] The foreign DNA is under the control of the virus, and is expressed independantly from the host cell genome. Harmless, easy to cultivate and stable for years after lyophilization. All these features make it strong candidates for vector vaccine.
    • Advantages 1. It stimulates B-lymphocytes (to produce antibodies) and T-lymphocytes (to kill virus infected cells). [E]. Thus vaccinia can produce a high level of immunoprotection. 2. Can vaccinate different different individuals against different diseases simultaneously. (genes encoding different antigens)
    • Procedure 1.      Assembly of plasmid insertion vector: Fresh vaccinia viruses are processed to release hteir DNAs. Now genes from hepatitis B virus, herpes simplex virus and influenza virus are added one after the other and inserted into vaccinia virus genome. These DNA clusters are cloned in E.coli for increasing their number and to produce plasmid insertion vectors. The plasmid contains the foreign subunit genes, the natural vaccinia genes, including the promoter genes. The recombinant plasmids are isolated and purified and serve as plasmid insertion vectors.
    • Procedure 2.       Production of recombinant vaccinia viruses The animal cells are infected with plasmid insertion vectors and normal vaccinia viruses. As the viral replication occurs, the plasmids are taken upto produce recombinant vaccinia viruses. The plasmid insertion vector incorporates its gene into vaccinia virus genome at a place that encodes fot the enzyme thymidine kinase (TK). Thus the recombinant viruses have lost their ability to produce TK. These are 2 advantages of loss of TK gene – one, is that it is easy to select recombined vaccinia viruses that lack TK gene and the second, is that these viruses are less infectious than normal viruses. The recombinant vaccinia viruses, released from the cultured animal cells, can be successfully used as vaccines.
    • Advantages 1. 2. 3. Authenticated antigens that closely resemble natural antigens can be produced. The virus can replicate in the host cells. This enables the amplification of the antigens for their action on Blymphocytes and T- lymphocytes. There is a possibility of vaccinating several diseases with one recombinant vaccinia virus. Disadvantages 1. 2. The most important limitation is the yet known risks of using these vaccines in humans. There may be serious complications of using recombinant viral vaccines in immunosuppressed individuals such as AIDS patients.
    •        It is known that the antigens located on the surface of a bacterial cell are more immunogenic than the antigens in the cytoplasm. Based on this observation, scientists have developed strategies to coat the surfaces of non-pathogenic organisms with antigens of pathigenic bacteria. Flagellin is a protein present in the flagella (thread like filaments)of Salmonella. A synthetic oligonucleotide encoding the epitope of cholera toxin B subunit was inserted into Salmonella flagellin gene. This epitope was in fact found on the flagellum surface. These flagella engineered bacteria, when administered to mice, raised antibodies against the cholera B toxin subunit peptide. It may be possible in future to incorporate multiple epitopes (2 or 3) into the flagellin gene to create multivalent bacterial vaccines.