1. Creating a potato that provides immunity against measles
by Andrew Yoo Cluster 1

2. Measles Background information
Highly contagious virus disease
Incubation period of days
Symptoms include high fever, dry cough, runny nose, sore throat, and rashes all over skin

3. Background Information continued….
Measles can be prevented with a vaccine called MMR, which uses live measles viruses.
The MMR vaccine has decreased measles deaths by 75% since 2000.
However, measles remains one of the leading causes of death in young children worldwide, with 145,700 measles deaths in 2013.
Measles is common in third-world countries where vaccines are not readily available, such as in regions in Asia and Africa.

4. Benefits of an edible vaccine for measles
-Edible vaccines are less expensive and do not require storage, such as refrigeration, as is the case for classic vaccines.
-Edible vaccines are more accessible because they can be grown locally, whereas traditional vaccines must be transported long distances.
-Edible vaccines do not require needles, so there is no risk of contamination.
Edible vaccines also stimulate mucosal immunity, which is an immune response in the mucous membranes of the body, including airways and digestive tract. This is because edible vaccines come into contact with lining of digestive tract.
Antigens of edible vaccines are not damaged by enzymes in digestive tract due to protection from cell walls.

5. Structure of Measles Virus
Hemagglutinin and the fusion glycoproteins are on the surface of the measles virus.
Hemagglutinin proteins bind to cell surface receptors on the host cell, mostly CD46 and SLAM(CD150), which is a signaling lymphocyte activation molecule found in immune system cells.
Fusion proteins trigger membrane fusion between the viral envelope and host cell membrane.
Edible vaccine for measles can be created by making the crop express hemagglutinin and fusion antigens.

6. Why we should use potatoes for the edible vaccine
Potato allergies are very rare.
Potatoes are a very popular source of carbs for many people around the world, and are found in many everyday foods, including french fries and chips.
Potatoes have successfully been genetically engineered to express antigens for the hepatitis B virus.

7. Researchable Question
Can an edible vaccine for measles be created by genetically engineering a potato to express the hemaglutinin and fusion protein antigens?

8. Experiment Design
Transform plant to express genes for measles antigens and kanamycin resistance.
Use western blotting on fully grown potatoes to check for presence of hemaglutinin and fusion proteins.
Run trials of the potato vaccine using humanized mice

9. Creating plasmid to insert into potato
hemaglutinin
1. Use restriction enzymes to insert hemaglutinin, fusion protein, and kanamycin resistance genes into pHB114 plasmid.
2. Add the CaMV 35S promoter in front of each of the newly inserted gene sequences.
CaMV 35S promoter
pHB114 plasmid
Fusion protein
Kanamycin resistance

10. Transforming Agrobacterium tumefaciens
1. Use electroporation to transform the Agrobacterium tumefaciens with the pHB114 plasmid.
- In electroporation, an electric shock creates temporary pores in the cell membrane of bacterium, allowing plasmids to enter.
2. Grow the bacteria on media containing kanamycin in order to select transformed bacteria containing the pHB114 plasmid.

11. Transformation of Potato Plant
Cut potato leaves and expose them to agrobacterium.
Transfer leaves to media containing kanamycin to select successfully transformed cells. Wait for the cells to multiply and form a callus.
Wait to the calluses to grow shoots and roots, and put
them in soil to grow.

12. Western Blotting
1. Extract protein from potato tubers and separate them using gel electrophoresis
2. Transfer proteins to nitrocellulose blotting paper.

13. Western Blotting 3. Incubate blotting paper in nonfat dried milk.
-The milk proteins bind to remaining sticky places on the nitrocellulose and prevent antibodies from sticking to the membrane nonspecifically.
4. Add solution containing hemaglutinin and fusion-protein specific enzyme-labeled anitbodies to the blotting paper.
5. Add chemiluminescent substrate to the blotting paper.
- The substrate will bind to the enzymes on the antibodies and help to detect the antibodies. This way, we can determine the presence of measles antigens in the potato plant.

14. Using hBLT Mice to Test Vaccine
-Humanized BLT mice have human immune systems and are commonly used to test drugs and vaccines.
- To create hBLT mice, transplant human liver and thymus tissues, as well as CD34+ hematopoietic stem cells into mice.
- The mice later start producing human white blood cells.

15. Using hBLT Mice to Test Vaccine
Create three groups of 10 mice with similar size, weight, and living conditions
- Group 1: Mice are vaccinated with the MMR vaccine, and are later infected with the measles virus.
Group 2: Mice are fed with the genetically modified potato, and are later infected with measles virus.
Group 3: Mice are fed with normal potatoes and are later infected with measles virus

16. Expected Results
Some potatoes will be able to be successfully transformed and will produce the measles antigens.
The measles antigens will be detected in the Western blot
The mice that ingest the potato vaccine will start producing antibodies for the measles antigens and will gain immunity to measles

17. Potential Problems
The genes for the measles antigens might not be expressed in the potato tubers.
The measles antigens might be degraded by digestive enzymes in the stomach.
The antigens alone might not create a sufficient immune response.
The antigens might not be able to activate the immune system and instead pass out of the body unused.
The potato might not be able to grow well if it produces too much antigen
The potato cells not successfully glycosylate the hemaglutinin antigens.

18. Future Areas of Research
Increasing the amount of antigens produced by edible vaccines by linking antigen genes with regulatory elements.
Enhancing the odds that antigens will activate the immune system, such as by linking antigens to molecules that bind well with components of immune system

19. Works Cited
Colf, Leremy A., Sean Juo, and Christopher Garcia. "Structure of the Measles Virus Hemagglutinin." Nature Structural & Molecular Biology 14 (2007): Web. 28 July 2015.
Guan, Zheng-jun, Bin Guo, and Yan-lin Huo. "Overview of Expression of Hepatitis B Surface Antigen in Transgenic Plants." Vaccine 28.46 (2010): n. pag. U.S. National Library of Medicine, 28 Oct Web. 28 July 2015.
Mahmood, Tahrin, and Ping-Chang Yang. "Western Blot: Technique, Theory, and Trouble Shooting." North American Journal of Medical Sciences (2012): n. pag. Medknow Publications & Media Pvt Ltd, 4 Sept Web. 28 July 2015.
"Measles." Symptoms of Measles. Mayoclinic, n.d. Web. 28 July 2015.
"Measles." WHO, n.d. Web. 28 July 2015.
Ota, Martin O., Zaza Ndhlovu, SangKon Oh, and Willian J. Moss. "Hemagglutinin Protein Is a Primary Target of the Measles Virus—Specific HLA-A2—Restricted CD8+ T Cell Response during Measles and after Vaccination." Journal of Infectious Diseases  (2007): Oxford Journals, Web. 28 July 2015.
Thanavala, Yasmin, Martin Mahoney, Sribani Pal, Adrienne Scott, and Liz Ritcher. "Immunogenicity in Humans of an Edible Vaccine for Hepatitis B." Proc Natl Acad Sci U S A 102.9 (2005): n. pag. Proc Natl Acad Sci U S A, 22 Feb Web. 28 July 2015.
Wells, Peter. "BLT Humanized Mice -." The Jackson Laboratory, Web. 28 July 2015.
"Western Blot Procedure." Western Blot Method. Davidson College, n.d. Web. 28 July 2015.

20. Images Cited

21. Questions?