CREATING TRANSGENIC

BANANA PLANTS THAT

PROVIDE IMMUNITY AGAINST

MALARIA Rena Jiang, COSMOS 2014, Cluster 1

WHAT IS MALARIA?

● Plasmodium falciparum is a parasite

transmitted by mosquitoes

● Mainly occurs in tropical and

subtropical regions near the

equator o South America

o Subsaharan africa

● Effects o fever, weakness, vomitting, ruptured

spleen, kidney failure, anemia,

seizures, cardiovascular collapse

WHY DOES IT MATTER?

● There are treatments, but there aren’t enough

resources to provide them to all the people in

third world countries

● Malaria causes families to experience high

economic losses due to costs of treatment

● 600,000+ people die every year from malaria

● There is currently no FDA approved malaria

vaccine to provide an easy fix -- until 2015, that is.

What can be done to change that?

EDIBLE VACCINES

● Transgenic plants are grown to carry antigens that

trigger an immune response from the body

● Cloaking antigens in fruits allow them to survive the

gastric pH

● It provides readily available options for third world

countries

● It does not require trained doctors to administer

● It is possible in a variety of plants, including ones

native to the problem-areas

BANANAS

● Cavendish banana plants

o disadvantages takes 10 months to bear

fruit

o advantages most popular species of

banana

children are willing to eat

them

doesn’t need to be

cooked to be eaten

grows in problem-areas

Distribution of Banana Plants

Global Malaria Transmission

MALARIA LIFE CYCLE

● 3 phases o Mosquito

Malaria zygotes develop and

migrate to the salivary glands to

be injected into human hosts

o Exoerythrocytic Parasite grows and multiplies in

liver

o Erythrocytic Parasite attacks red blood cells

Causes the clinical symptoms of

malaria

ERYTHROCYTIC STAGE ● Merozoites from liver enter

RBC to become

trophozoites

● Trophozoites become

schizonts that rupture the

RBC to infect more cells

● Some trophozoites

become gametocytes,

which can be taken up by

mosquitoes that transmit

the parasite to other hosts

APICAL COMPLEX

● When the parasite is

entering a host cell, it must

reorient itself so that the

apical end is juxtapose to

the cell membrane

● Surface proteins allow the

apical end to attach to the

membrane o one of these proteins is

apical membrane antigen 1,

or AMA-1

AMA-1

AMA-1

● Of the many apical complex proteins, AMA-1

plays a significant role in the parasite’s ability to

attach to the host cell membrane o according to The Walter & Eliza Hall Institute, AMA-1

antibodies significantly inhibit host cell invasion

● Although a malaria vaccine currently does not

exist, AMA-1 is already established as an

important candidate.

RESEARCHABLE QUESTION: HOW

CAN AMA-1 BE EXPRESSED IN

TRANSGENIC BANANA PLANTS TO

PRODUCE AN EDIBLE VACCINE THAT

REDUCES THE CHANCES OF PEOPLE

BECOMING INFECTED BY

PLASMODIUM FALCIPARUM?

PLAN OF ATTACK

● Transform banana plants to express AMA-1

antigens by using agrobacterium tumefaciens

● Check for transformation with PCR analysis

● When banana fruits grow, use northern and

western blotting to check for presence of RNA

and protein

● Humanized mouse trial: ELISA after ingesting

bananas

● Human trial: test for dosage and efficacy

TRANSFORMATION: CLONING AMA-1

● PCR to amplify DNA

● Use restriction

enzymes to insert

AMA-1 and

kanamycin

resistance gene

into pBI121 with

BanLec promoter

(banana fruit-

specific)

TRANSFORMATION: AGROBACTERIUM TUMEFACIENS

● Transform Agrobacterium tumefaciens strain EHA 105

to contain the pBI121 plasmid using electroporation o add plasmid to bacterium, shock, add 1ml liquid medium,

incubate for recovery, spread on plates with kanamycin to select transformed bacterium

TRANSFORMATION: CAVENDISH BANANA PLANTS

● Experimental: o Cut 5mm banana shoot tips

o Dip wounded shoots in agrobacterium and blot dry

o Plate on MS with acetosyringone

o Incubate for three days

o Transfer shoots to MS plate containing kanamycin and

cefotaxime to select transformed cells and eliminate

bacterium growth

o Incubate for two to four weeks to regenerate shoots

● Control: o transform using agrobacterium without AMA-1 gene

TRANSFORMATION: CAVENDISH BANANA PLANTS

CHECK FOR TRANSFORMATION

● Perform PCR on the regenerated

shoots to check that AMA-1 gene

has been incorporated

● When banana fruits grow in ~10

months, perform northern and

western blots to check that the

AMA-1 gene is being expressed

correctly

CHECK FOR TRANSFORMATION

● Northern: confirm that AMA-1 is being

transcribed in the fruit o RNA probe: complementary to AMA-1 RNA,

labeled with chemiluminescence

● Western: confirm the presence of AMA-1 in

the fruit o Primary antibody: Rat anti AMA-1 (4G2dc1) that

only binds to correctly folded AMA-1

o Secondary antibody: Goat anti rat conjugated

to HRP

TESTING IN HBLT MICE

TESTING IN HBLT MICE

● hBLT mice are ‘humanized’ in that they have

human immune systems and livers o malaria infection can be successfully simulated

● Experimental: Ingest an unknown amount of

transgenic banana that contains AMA-1

● Control: Ingest the same unknown amount of

banana without AMA-1

TESTING IN HBLT MICE

● Perform ELISA to

detect antibodies in

mice after ingesting

transgenic bananas

● Infect mice with

malaria and

observe differences

in the two groups

HUMAN TRIALS: PEOPLE FROM HIGH-RISK AREAS

● Experimental: Ingest an unknown amount

transgenic bananas with AMA-1

● Control: Ingest same unknown amount of

transgenic bananas without AMA-1

● Test for antibodies with ELISA

● After ~1 year, bring both groups back to

determine efficacy

● How many bananas provide enough antigens

to trigger a sufficient response?

PREDICTED RESULTS

● Banana transformation will be successful: it

will effectively produce AMA-1 antigens

● AMA-1 antigen will survive the pH of the

digestive system and trigger antibodies to

form

● Antibodies will provide immunity against

parasites attacking red blood cells

POTENTIAL PROBLEMS

● Too few antigens may be expressed in the fruit, if

any at all

● Antigens produced will be destroyed by the

human digestive system before a response can be

triggered

● AMA-1 only interrupts part of the malaria cycle,

which is very complex. Some forms of the parasite

may still survive in the host in other parts of its cycle

● How can quality control be implemented to ensure

safety?

FUTURE AREAS OF RESEARCH

● AMA-1 antigens may need to be expressed in

combination with other surface antigens to

fully protect someone from malaria in all parts

of its cycle o PfEMP1, for example, is an antigen that allows

the parasite to avoid being destroyed by the

spleen when it filters the blood. This would make

it a strong vaccine candidate as well.

QUESTIONS?

BIBLIOGRAPHY "Anti-AMA1 monoclonal antibody 4G2dc1." . Center of Bioinformatics, n.d. Web. 25 July 2014.

"Apical Membrane Antigen 1 as an Anti-malarial Drug Target." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 25 July 2014.

Casey, Joanne L., Andrew M. Coley, Robin F. Anders, Vince J. Murphy, Karen S. Humberstone, Alan W. Thomas, and Michael Foley. "Antibodies to Malaria Peptide Mimics Inhibit Plasmodium falciparum Invasion of Erythrocytes." Infection and Immunity 72: n. pag. American Society for Microbiology. Web. 25 July 2014.

Chowdhury, Kamal, and Omar Bagasra. "An edible vaccine for malaria using transgenic tomatoes of varying sizes, shapes and colors to carry different antigens." . ScienceDirect, n.d. Web.

Mandal, Ananya, MD. "Malaria Mechanism." News Medical. AZoM, n.d. Web. 25 July 2014.

May, Gregory D., Rownak Afza, Hugh S. Mason, Allcja Wiecko, Frantisek J. Novak, and Charles J. Amtzen. "Generation of Transgenic Banana (Musa Acuminata) Plants via Agrobacterium-Mediated Transformation." Nature (1995): n. pag. Web. 24 July 2014.

"Plasmodium Falciparum - Malaria." Plasmodium Falciparum - Malaria. Parasites In Humans, n.d. Web. 25 July 2014.

Sunil Kumar, G. B., T. R. Ganapathi, and V. A. Bapat. "Expression of Hepatitis B Surface Antigen in Transgenic Banana Plants and NT- I Cell Line of Tobacco." Barc Newsletter (2002): n. pag. Web. 24 July 2014.

Tyler, Jessica S., Moritz Treeck, and John C. Boothroyd. "Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication." . Science DIrect, n.d. Web. 25 July 2014.

Wang, Lina. "Oral Vaccine Delivery: Can it Protect Against Non-mucosal Pathogens?." Medscape Log In. Medscape, n.d. Web. 25 July 2014.

IMAGE SOURCES

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http://jaxservices.jax.org/invivo/images/humanized-blt-process.png

http://lifesciencesfoundation.org/content/media/2013/06/24/PPlantBiotech-

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http://www.cell.com/cms/attachment/610112/4877277/gr1.jpg

http://upload.wikimedia.org/wikipedia/en/c/c2/Malaria_Enters_Erythrocyte.JPG

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