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Edible Vaccines

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					     Edible Vaccines
             By Varsha Gupta
              COSMOS 2009
     Cluster 1: Biotechnology
       Mentor: Paul Feldstein
Teacher Fellow: Rebecca Sela
  Problems with Traditional
         Vaccines
Vaccines are costly. They cost a
lot not only to produce but also to
constantly ship out from places of
production.
There is a need for personnel to
administer vaccines where doctors
are not plentiful or education is
low.
Third world countries are at a
disadvantage by lack of money or
personnel.                            Figure 1: Child being inoculated


Children hate taking shots.
 Context of Edible Vaccines
Edible vaccines would be cost effective.
There is no need to send personnel to
administer the vaccines.
It’s easier to inoculate children by feeding
them fruits/vegetables rather than having to
give them shots.
Once plants are planted, the cost of
shipment is taken away, reducing the cost
even further.
   Hepatitis B Background
Approximately one third of the world has
the Hepatitis B virus (HBV)
The disease is spread by blood, the
exchange of body fluids, or from mother to
child during pregnancy
The disease is most prominent in Asian
and African countries, especially those with
large populations or those that are
considered Third World
Hepatitis B Virus




      Figure 2: HBV up close
Distribution of HBV in the
          World




                             Figure 3:
                             Spread of HBV
                             around the
                             world
       Hepatitis B Vaccine
The Hepatitis B vaccine was first
introduced in 1981
The process requires between 2-4
doses
Protection is guaranteed for
approximately 25 years, but is thought
to last forever
The vaccine works for 85-90% of
people                                   Figure 4: Vaccine
                                          currently used
Utilizes presence of Hepatitis B
Surface Antigen (HBsAg) to prevent
     Vaccine in Potatoes
The HBV vaccine has successfully been
transplanted into potatoes
Scientists engineered transgenic potatoes
containing the vaccine. The vaccine
expressed the HBsAg in the roots.
The potatoes were tested on mice and
proved to be successful representation of the
original vaccine
So what’s the problem…?
    Figure 5:
    International
    Jainism symbol



      Problems with Potatoes

Potatoes are a food not eaten among
followers of the religion Jainism
This limits out the vaccine for approximately
20 million people in the world. That’s like
limiting out the entire population of New York
state and Washington DC
The solution is to put the vaccine in a more
commonly accessible fruit/vegetable
   The Benefits of Apples
Apples are an easy to grow fruit
35% of apples are produced in China, also a
country with one of the highest prevalence
rates of HBV. This makes it even more cost
effective because the apples would not even
have to be sent to China
Growing season from May to the end of
summer
           More Benefits
Can be stored for several
months
Some areas have two
growing seasons for apples
because of the appropriate
weather
Allergy rate is low for apples
No large religion against
consumption of apples
Many types to appeal to
larger numbers of people
Previous Work with Apples
Apples have been genetically modified in
the past to obtain a preferred
characteristic
In 2008, hypoallergenic apples were
created by the means of inserting genes
which coded against the gene which
triggers an allergic reaction
This proves the workability of apples as
transgenic organisms
 Researchable Question
 Would it be possible to take the
  gene from transgenic potatoes
 with the presence of HBsAg and
genetically engineer it into apples?
     Materials (Part 1 of
        Experiment)
Apple cotyledons                              Figure 6: Scalpel



Tweezers
                         Figure 7: Tweezers

Sterile water
Petri dishes                Parafilm
3mm filter paper            Scalpel
MS agar plates              A. rhizogenes media
                            Pots with soil
             Figure 8:
             Parafilm
                            Kanamycin and
                            cefotaxime
         Procedure
Step 1: Prepare apple cotyledons
Step 2: Transfer cotyledons
Step 3: Growth of the apple




             Figure 9: DNA
 Step 1: Prepare apple
      cotyledons
Obtain an apple plant and cut the cotyledons
from the plant with a scalpel.
Wash the cotyledons by dipping in sterile
water with tweezers.
Culture cotyledons in MS media
Infect with Agrobacterium tumefaciens with
expression vectors pEFEHBS/pEFEHER
which contains the Hepatitis B virus gene that
encodes for the surface antigen.
      Step 2: Transfer
        Cotyledons
Blot the cotyledons on the filter paper and
reculture on MS media for 48 hours
Transfer the cotyledons to MS media that
contains kanamycin and cefotaxime
After growth proceeds, transfer the shoots
onto MS media with supplemental sugar
(store in the dark) to foster growth even
further
Make sure to parafilm the petri dishes shut as
to prevent contamination
  Step 3: Growth of the
         Apples
Take transformed roots and cultivate in
Agrobacterium rhizogenes to produce hairy roots for
expression of HBsAg.
After 3 days shift the cotyledons to MS media with
cefotaxime for the roots to grow
After the roots have established themselves
significantly they can be transferred to pots with
nutrient rich soil.
When the plant begins to bear fruit, the apples can
be used for part 2 of the experiment
     Materials (Part 2 of
        Experiment)
Genetically                Genetically
modified apples            modified potato
(from previous
part)
20 BALB/c mice
                                        Figure 11: Well
ELISA machine                           plate for ELISA


Syringes with
needles
HBV vaccine         Figure 10:
                  BALB/c mouse
         Procedure
Step 1: Obtain BALB/c mice and
prepare them for the clinical trial
Step 2: Inoculate the mice and
observe
Step 3: Draw blood samples from
mice
Step 4: Use ELISA process to
determine the presence of HBsAg
Step 1: Obtain BALB/c mice and
prepare them for the clinical trial
 Control the mice for the experiment
 -Control factors should include gender, age, weight, diet, and living
 conditions.
 20 mice should be split into 4 groups of 5
 mice each
 -Group 1: This group will be the negative control. These mice will not
 receive any type of vaccination for HBV.
 -Group 2: This group will be the positive control and receive booster
 shots in the correct dosage of the HBV vaccine used today.
 -Group 3: This group will be the other positive control and receive
 the previously tested genetically modified potatoes with the vaccine.
 -Group 4: This group will be the experimental group. They will be
 receiving the newly created apples with the vaccine.
  Step 2: Inoculate the
   mice and observe
Draw blood from all mice for preliminary sample
Group 1 should not receive any treatment and
should continue with the daily ritual
Group 2 will be injected with the traditional vaccine
by means of a syringe and needle.
Group 3 will be fed the transgenic potatoes. Each
mouse will receive 5 g of potato.*
Group 4 will be fed the transgenic apples. Each
mouse will receive 5 g of the modified apples.*
Immunize at weekly intervals for three weeks.
*The mice will have to be observed to ensure the
complete consumption of the food products given to
them. This may take up to 24 hours.
   Step 3: Draw blood
   samples from mice
 Blood must be drawn at weekly intervals for
the testing period (about 40 weeks)
Needles must be inserted at the orbital
plexus (vein that drains from near the eye) of
the mice for greatest efficiency and least pain
for mice.
Complete extraction from mice and store
samples until tested with ELISA
                              Figure 12:
                              Orbital plexus
                              on a mouse
  Step 4: ELISA process
Apply HBsAg to well plate to serve
as a standard
Add bovine serum albumin (BSA)
to the plate so that the absorption
of other proteins is inhibited.
Add serum (blood plasma without
clotting factors) from the mice
samples to the plates and then        Figure 13: Blood

wash so that nonspecific proteins          Serum


are removed
Step 4: ELISA process
         cont.
Add primary antibodies that will bind only to
the Hepatitis B antigen
Add enzyme secondary antibodies to support
the breakdown of nonbinding proteins
Wash the well plates to clear out the particles
Shoot a current through the well plate with
the samples and the control and compare to
see how effective the genes in the apple
were for the groups of mice.
           ELISA process




Figure 14: A sandwich ELISA. (1) Plate is coated with a capture
antibody; (2) sample is added, and any antigen present binds to
capture antibody; (3) detecting antibody is added, and binds to antigen;
(4) enzyme-linked secondary antibody is added, and binds to detecting
antibody; (5) substrate is added, and is converted by enzyme to
detectable form.
         Potential Problems
The genes may not transform and incorporate into
the apple’s gene properly. The gene may only work
for a potato or another fruit/vegetable we are not
studying.
With all the transferring of the cotyledons between
petri dishes, it may face too much contamination.
There could be a hidden variable which affects the
outcome of the experiment with the mice.
With the ELISA process, foreign proteins may
remain on the well plate, contaminating our sample
and making the results appear in an incorrect
manner.
                  Works Cited
Feldstein, Paul. Personal Interview. July 2009.
Han, Mei, et al. “Research Advances on Transgenic Plant
   Vaccines.” Acta Genetica Sinica 33.4 (2006): 285-293.
Kumar, Sunil, et al. “Expression of hepatitis B surface antigen in
   potato hairy roots.” Plant Science 170.5 (2006): 918-925.
Lee, Beom Seok, et al. "A Fully Automated Immunoassay from
   Whole Blood on a Disc." Lab on a Chip 9.11 (2009): 1548-1555.
Schmidt, Georg, et al. “Production of recombinant allergens in
   plants.” Phytochemistry Reviews 7.3 (2008): 539-552.
Youm, Jung-Won, et al. “Oral immunogenicity of potato-derived
   HBsAg middle protein in BALB/c mice.” Vaccine 25.3 (2007):
   577-584.
                    Images Cited
Figure1:<http://antipemurtadan.files.wordpress.com/2009/05/suntik.jpg>
Figure2:<http://people.rit.edu/japfaa/HBV.jpg>
Figure3:<http://www.library.northwestern.edu/govinfo/news/090519.jpg >
Figure4:<http://www.wallcur.com/images/products/PractiVaccineB_big.jpg>
Figure5:<http://www.yorkshireandhumberfaiths.org.uk/contentimages/symbol_jainis
   m_1.gif >
Figure6:<http://www.wintools.ch/images/Scalpel_03.jpg>
Figure7:<http://www.gamart.com.au/cms/assets/image/Tweezers.jpg>
Figure8:<http://biotek.com.au/products/catalog/images/parafilm.jpeg>
Figure9:<http://www.theory-of-evolution.net/chap8/dna-1.GIF>
Animation:<http://www.animationlibrary.com/animation/29905/Tree_grows/>
Figure10:<http://www.scanbur.eu/images/products/Lab_animals_BalbC.jpg>
Figure11:<http://agri.nv.gov/EnzymeLinkedImmunosorbent.jpg>
Figure12:<http://www.medaille.edu/vmacer/120_lab_rodent3_saphenous2.jpg>
Figure13:<http://homepage.usask.ca/~vim458/virology/studpages2007/Tara_Alycia/
   serum.gif>
Figure14:<http://en.wikipedia.org/wiki/File:ELISA-sandwich.svg>

				
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