UNIT-V

VACCINE [ Plant -Agrobacterium tumefaciens]

Vaccine:
A vaccine is a suspension of weakened, killed, or fragmented microorganisms or toxins or other
biological preparation, such as those consisting of antibodies, lymphocytes, or mRNA, that is
administered primarily to prevent disease.

Or

A vaccine is defined as any substance which is used to stimulate the production of antibodies, in
turn providing immunity against one or a few diseases.

The process of administering the vaccine is called vaccination or immunization. It is

responsible for the eradication of many diseases – especially infectious diseases such as
smallpox and chickenpox. The term “vaccine” is derived from the Latin word “vaccinus”, from
“vacca”, which means “from cows”. Edward Jenner coined the term vaccine for the technique
where, he injected individuals with a mild form of cowpox, thereby rendering them immune to
smallpox.
HISTORY OF VACCINE:
Type of vaccine:

 Inactivated vaccines.
 Live-attenuated vaccines.
 Messenger RNA (mRNA) vaccines.
 Subunit, recombinant, polysaccharide, and conjugate vaccines.
 Toxoid vaccines.
 Viral vector vaccines
Process of vaccine production.

Vaccination is based on the memory of the immune system. Vaccines contain attenuated disease-
causing organisms or toxins that initiate an immune response in the body.

 The first step in vaccine production is to prepare the antigen. Viruses are grown in the
primary or cultured cells; bacteria are grown in the bioreactors.
 With the advent of biotechnology, recombinant antigenic protein is produced in bacteria or
yeast.
 Then antigen is isolated and inactivated, the recombinant protein is processed and ultra-
filtrated.
 Then vaccine is prepared by adding adjuvant, preservatives and stabilizers.
 Adjuvants enhance the immune response and stabilizers increase the shelf life.
 The final step in vaccine production is packaging.
 Every vaccine has to be licensed by FDA before being brought into use.
 A vaccine needs to go through extensive tests and clinical trials to confirm its safety before
approval by the FDA.
Vaccine produce by using Agrobacterium:
Agrobacterium:
Agrobacterium is a Gram-negative soil pathogenic bacterium that naturally will infect the
plants and transfer their genes (T-DNA) to the nucleus of the plant cells.
Strains used:
Two strains of Agrobacterium species that have been commonly used as a biological
vector are Agrobacterium tumefaciens (A. tumefaciens) and Agrobacterium
rhizogenes (A. rhizogenes). The main difference between these two species is the plasmid
that they carry. A. tumefaciens carries tumour-inducing plasmid (Ti-plasmid), while A.
rhizogenes carries root-inducing plasmid (Ri-plasmid).
For vaccine production:
A.tumefaciens is the most preferred strain by researchers for stable expression of the desired
protein. In the Ti-plasmid, there are genes encoding for plant hormones such as auxin and
cytokinin synthesis, which will induce tumour tissue in plants.
However, for vaccine production,
 These genes will be deleted to form disarmed Ti-plasmid and heterologous gene is
inserted forming a recombinant plasmid vector.
 The recombinant plasmid vector is transformed into A. tumefaciens and with the help
from vir gene of the bacterium, the introduced heterologous gene is transferred by the
transformed bacterium and integrated into the host plant nuclear genomic DNA by
nonhomologous recombination at random sites.
 The transformed bacteria are transferred into the plant leaves by soaking the leaves in
the A. tumefaciens culture.
 This method is able to yield a stable integration of the transgene into the genome of the
plant.

Step :1

The T-DNA plasmid removed from bacterium

Step:2

Plasmid is cut using a restriction enzyme

Step:3
Vaccine fragment DNA cut with same restriction enzyme and inserted into plasmid

Step:4

T -DNA carrying inserted vaccine DNA incorporated into plant cell chromosome

Step:5

Plant cell cultured

Step:6

A transgenic plant expressing vaccine DNA is generated

 Some various vaccines produce through Agrobacterium-mediated
transformation system in dicotyledonous plant models.

Example:

 Successfully expressed gene encoding for cholera toxin B subunit protein in potato
leaf explants using A. tumefaciens.
 Similarly, potato has been transformed to produce VP60 protein against rabbit
hemorrhagic disease virus, in which rabbits immunized with the potato’s leaf extract
showed increased anti-VP60 antibody titers and were protected against the
hemorrhagic disease. These discoveries have led to the production of more diverse
antigens in various crop species.
 Helicobacter pylori TonB protein was expressed in transgenic Arabidopsis
thaliana through this method. The antigen produced was recognizable by rabbit anti-
TonB antiserum and suitable to be used as vaccine against Helicobacter infections by
oral administration.
 Hepatitis B surface antigen gene was able to be introduced into tomato plants
mediated by A. tumefaciens.