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

2. 


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).

3. 1.
2.
3.
Dead bacteria or inactivated viruses.
Live non virulent or weakened (attenuated)
bacteria/ or viruses.
Viral fragments or bacterial molecules
(subunit vaccines).

4. 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.

5. 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.


6. Advantages
 Purity in preparation.
 Stability
 Safe use
Disadvantages
 High cost factor
 Possible alteration in native conformation

7. 



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.

8. 

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.

9. 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.

10. 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


11. 






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.

12. 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.

13. 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.

14. Advantages
 Immune response is substantially high.
 Stable
Disadvantages
 Side effects

15. 




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.

16. 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.

17. 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)

18. 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.

19. 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.

20. 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.

22. 






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.