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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
● 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
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
● 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.
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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