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. Children hate taking shots.

Figure 1: Child being inoculated

Context of Edible Vaccines

Edible vaccines would be cost effective. There is no need to send personnel to administer the vaccines. Its 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 Utilizes presence of Hepatitis B Surface Antigen (HBsAg) to prevent

Figure 4: Vaccine currently used

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 whats 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. Thats 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 Tweezers Sterile water Petri dishes 3mm filter paper MS agar plates
Figure 8: Parafilm Figure 6: Scalpel Figure 7: Tweezers

Parafilm Scalpel A. rhizogenes media Pots with soil 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 modified apples (from previous part) 20 BALB/c mice ELISA machine Syringes with needles HBV vaccine Genetically modified potato

Figure 11: Well plate for ELISA

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 wash so that nonspecific proteins are removed

Figure 13: Blood Serum

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 apples 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>