In the spring of 1855, Father Edward Sorin, CSC, must have cast a troubled gaze upon the
marshy land surrounding Saint Mary's and Saint Joseph's lakes. Only the year before a typhoid
epidemic had devastated the Notre Dame campus, and now two early deaths in March suggested
the fever would rise again. The likely cause, the University's founder believed, was high lake
levels caused by a dam on a neighboring farm. Malaria, cholera and yellow fever plagued Notre
Dame's early years, and Sorin blamed the over-full lakes. When the landowner reneged on
negotiations to sell, Sorin famously took matters into hand and sent a half dozen men over to tear
down the dam.
It would be a few more decades before Sorin's instincts were confirmed by science.
Swamp-born mosquitoes were a major culprit, lively vectors of malaria and yellow fever. But
more than 150 years later, his solution still qualifies as state-of-the-art, particularly for malaria,
which remains one of the world's most intractable health problems. Up to 500 million cases
occur annually, and the most virulent form -- Plasmodium falciparum -- takes more than 1
million lives each year. Some estimates put the yearly toll of malaria well beyond 3 million.
To make matters worse, the malarial parasite is increasingly drug resistant. In the same
way, the mosquito vectors are adapting to insecticides and repellants. Environmental and social
changes are expanding the disease's range and compounding its impact. The technologies that
work -- insecticides, bed nets, habitat modification -- are ancient concepts.
"There really hasn't been anything new developed for dealing with most vector-borne
diseases," says Frank Collins of Notre Dame's Center for Global Health and Infectious Diseases.
A new movement aims to change that. With new tools, a renewed global commitment, and a
fresh infusion of capital and strategic thinking, scientists at Notre Dame and beyond are working
especially hard to make a target of malaria and other intransigent infectious agents.
Notre Dame's role is both next generation and back-to-basics. Researchers are using the
genome -- the high-tech, wholesale reading of the genetic code -- to provide new clues to the
basic biology. "Let's just understand these organisms better," says Collins. "If we can learn
enough about these mosquitoes or sand flies or ticks, we might be able to devise an entirely new
approach."
For example: Redesign the mosquito.
George Craig, a leading mosquito researcher who worked at Notre Dame until his death
in 1995, imagined using genetic engineering to simply eliminate the mosquito. This generation
has a more subtle approach. Scientists already know how to reprogram mosquito DNA. They are
confident that they can soon figure out what changes would render a mosquito "vector
incompetent" -- unable to carry deadly malaria or dengue fever. And they believe they can
convince nature to incorporate this "improvement." Their hardest job may just be convincing the
public.
The book of genes
"The mosquito is both an elegant, exquisitely adapted organism and a scourge of humanity."
Those words capture the tricky problem of malaria and marked the beginning of a new era for
vector biologists. They lead off the October 2002 publication in Science magazine of "The
Genome Sequence of the Malaria Mosquito Anopheles gambiae." It's an understated title: Anopheles gambiae is the most important carrier of malaria in the world, and reading the genome
opened new paths for malaria researchers. Nature published the genome of the malarial parasite Plasmodium falciparum that same month, raising hopes that the new science of genomics would
quickly banish the ancient malady.
It was the kind of result that warranted celebrations, but the accomplishment was more
start than finish. The published genomes hadn't done much more than force open the library
door. Once inside, the real work began. "It's like walking into the Library of Congress without
any kind of card catalog," says Collins, a contributor to the decoding process.
Collins was an English graduate student before taking up mosquito biology, and he likens
understanding the genome to reading a great novel. "At the most simplistic layer, you're reading
words. The words don't necessarily mean a lot. They have some kind of general meaning: noun,
verb, or a preposition or conjunction. When you put them together they form sentences and
paragraphs, which themselves specify higher levels of meaning and complexity."
So far, a lot of meaning escapes scientists. "We have a pretty good handle on the
spelling," he says. "We know the letters." In A. gambiae researchers recognize probably 90
percent of the words. Each word is a gene, and it carries the instructions to make a single protein.
The general meaning of probably about half of those words is known. Scientists can read some
sentences. Occasionally they can even read several sentences together: How a dozen or more
proteins all interact to do something. "But how do all 500,000 words in this book go together to
make a novel that means something? That's pretty far off."
Decoding the genome was automated, enabled by fabulously expensive machines that
break up long chains of DNA into readable segments. A computer then puts the whole puzzle
together again.
Interpretation takes a bit more work, but comparison is critical. For example, A. gambiae is actually one of seven species that look identical under the microscope. A. gambiae prefers to
bite humans, but another, A. quadriannulatus, prefers animals. Comparing the genomes might
allow researchers to figure out why A. gambiae is so partial to humans. That insight might in
turn allow the design of a better repellant or yield a target for genetic modification. To make this
kind of comparison easier, researchers can use VectorBase, a new database of genomes for
disease vectors directed by Collins. Already in its library is A. gambiae. The genome of another
mosquito, Aedes aegypti, will be completed this fall; the work is being coordinated at Notre
Dame by David Severson, professor of biological sciences. Decoding sand flies, ticks and
several other mosquito species is also in the works.
Collins is confident that scientists will be able to genetically engineer the mosquito so
diseases aren't spread so easily. "Not only is it doable, it is in some respects a much more
reasonable approach to dealing with problems of disease transmission," he says. Much of Africa,
where malaria looms largest, lacks the infrastructure to deliver modern medical solutions. "A lot
of people still die of measles because we can't get vaccine to them."
If you can "improve" the mosquito, then harness this genetic legerdemain to a natural,
infectious process, you don't need infrastructure. Nature will finish the job.
Morphing mosquitoes
Two doors down from Collins' office sits Nora Besansky, his wife and one of many scientific
partners in this endeavor. On her door are taped two golden pipette awards, good-natured pokes
from Notre Dame graduate students for being the most stressed out.
The biological sciences professor is trying to solve one of the most important riddles of
mosquito ecology and malaria transmission. Anopheles gambiae is the predominant malarial
vector in Africa, but it rarely works alone. Over eons, the species has evolved into those seven
different identical-looking mosquitoes. In such West African nations as Burkina Faso and Mali,
however, Anopheles gambiae appears to be evolving again, splitting into two forms known
simply as M and S.
The traditional A. gambiae mosquito breeds in pools and puddles. It requires a rainy
season, along with animals and people tromping through the mud, digging ditches, making
bricks. During the dry season A. gambiae dies back, and malaria transmission subsides.
Twentieth-century engineering has brought a new habitat to West Africa, particularly in
the thirsty southern semi-desert area of the Sahara known as the Sahel. Rice fields, irrigation
ditches and impoundments are now spread over vast regions of previously arid land. Apparently Anopheles gambiae abhors a vacuum, because a subtle variant called M has begun to exploit this
new habitat. Rice fields are flooded, and with two rice crops a year there is no more dry season.
"There are greater numbers, and it's found in places it never used to be found before and at times
it never used to be found before," says Besansky. More mosquitoes means more malaria.
Under a microscope it is impossible to tell the M form from S, but when scientists
examine the sperm deposited in the females, it shows that the male and female match forms
about 99 percent of the time. Even though the remaining 1 percent is enough to keep the species
genetically mixed, Besansky believes they are on independent evolutionary tracks. Her mission
is to find out how. There have to be genes underlying the mating preference, but it is difficult to
study mating behavior. "They mate at dusk, in swarms," she laughs. "Hello, you can't do this in
the lab. You can't tell them apart."
But the genomics toolbox can. A team at the University of California, Davis, transferred
DNA from A. gambiae M and S to computer chips. A comparison showed three small regions of
difference, and now Besansky and others are racing to decipher the details. That means going
beyond the genome and back into the field. Her collaborators in Burkina Faso are doing the
ecological work, trying to figure out how M and S interact on the ground. Funding from
Besansky's own grants and from the National Institutes of Health has helped build high-end
malaria labs in Burkina Faso and neighboring Mali, which have been critical to the work. "This
is a highly interdisciplinary project," she says. "It would be impossible for me to do what I do
without those good collaborators in Africa."
The intricacy of the Anopheles gambiae complex, especially the M and S enigma, shows
just how difficult even basic malarial biology can be. Will it be necessary to modify each species
in the complex, or will it be enough to knock out the major human vectors, A. gambiae and its
close relative, A. arabiensis? Or will other species fill the void? "Transgenic mosquitoes are not
a magic bullet," concludes Besansky. "There has to be a whole suite of tools. We are still going
to need the insecticides, we are still going to need the bed nets. We're going to need the
antimalarial drugs and vaccines."
Jumping genes
How do you make a transgenic mosquito?
No single mad scientist is likely to retreat to the lab to create one. Hundreds of
investigators following dozens of lines of inquiry are engaged in this project. The paper
enumerating the Anopheles gambiae genome, for example, has more than 100 authors.
For Malcom Fraser, Notre Dame professor of biological sciences, the quest began with a
completely different organism. In the early 1980s Fraser was a graduate student working on
baculoviruses, which inhabit insects and other invertebrates. His professor at Ohio State had
noticed a mutation, a tiny change in the trail left by the virus. It suggested a little more DNA
than expected. Fraser spent weeks at the microscope learning to spot the slight difference
between the normal virus and the mutation.
Through years of fancy labwork, Fraser maintained his fascination with the mutation.
Eventually he worked out the source of that extra DNA: It was a transposon, also known as a
jumping gene. Transposons are virus-like segments of DNA that have the ability to move
around, causing mutations. Most of the mutations are deleterious, but organisms have evolved
ways to resist the influence of transposons. The resulting give-and-take can leave a hefty trail;
some 45 percent of human DNA, for example, is thought to be made up of transposons and their
inactive remains.
Because transposons move around so easily, geneticists have harnessed them to
manipulate DNA. Fraser saw this potential and named the genetic sequence he had found
piggyBac. He began exploring the transposon's potential for re-engineering insects, with the
ultimate goal of remaking the mosquito. By attaching a desirable gene to piggyBac -- let's say
one that would frustrate the malaria parasite's effort to enter the salivary glands of the mosquito
-- it could potentially deliver it into the mosquito. Even better, because of the infectious, viral
behavior of transposons, they could perhaps penetrate a gene pool far faster than the simple laws
of heredity would normally allow.
But the more species Fraser examined, the more evidence he discovered of piggyBac --
or something very similar--all over the natural world. It suggested that at some point in
evolutionary time piggyBac had moved between species. "That's dangerous if you're going to
use them for transgenic engineering of insects that you intend to release in the field," Fraser says.
"These invasive DNAs can have a significant impact on new species when they get in and start
jumping around and causing mutations. We're talking about consequences that we can't really
predict. I don't want to be known as the guy who released piggyBac and killed half the insects in
a particular region of the world."
There are other ways to engineer a mosquito, and Fraser is still working toward that goal.
He's received a Grand Challenge grant from the Bill and Melinda Gates Foundation to explore
what he hopes will be a more stable method of genetic tinkering. Rather than tackle the
monstrous complexity of the malaria parasite, he's looking at dengue fever first. Dengue is a
virus carried by the Aedes aegypti mosquito. It kills between 25,000 and 50,000 people a year,
and in the last few decades a new and deadly hemorrhagic form has emerged.
Despite Fraser's reluctance to use transposons, the method is by no means dead. Last
spring Tony James at the University of California, Irvine, successfully used transposons to
engineer a dengue-resistant mosquito. Severson, the Notre Dame professor who specializes in Aedes aegypti, will be working with James on the next stage of the project. The researchers hope
to try large-scale cage trials in Mexico that would simulate ecological conditions in an endemic
area.
No genetically modified mosquitoes will be released into the wild. "We are 10 or 20
years away from doing something like that, at least," estimates Severson. The cage trials,
however, can test both biological and social questions. Biologically, the scientists need to find
out how the modified mosquito works outside the lab. Will it survive? Perhaps even more
important, says Severson, is public awareness and education, and the ability of the scientists to
work with the public and government agencies that will ultimately decide whether such a project
should proceed.
"A large segment of the population has to be on board with this," says Nora Besansky.
What's more, scientists have to find a way to make genetic modification work faster than the
natural pace of evolution; otherwise it won't seem practical. "I think we have to harness some of
these genetic cheaters to make it happen in a frame that's relevant for public health," she says.
"In fact, probably in a frame that's relevant for a politician -- five or 10 years."
Besansky understands Fraser's concern about transposons. "There are advantages and
disadvantages to just about every one of the mechanisms that has been proposed," she says.
Disease and development
Father Thomas G. Streit, CSC, '80, '85M.Div., '94Ph.D. tells a more intimate tale of his work in
Haiti, where he spends eight months of the year. Of the 65 employees he's had under the age of
30, 10 have died. Several were lost to that nation's general chaos and violence, but six have died
of infectious diseases and one of prenatal eclampsia. None would have died in the United States.
"These are people of some means because they had a job," he says.
Streit works on lymphatic filariasis in Haiti. The parasite is also transmitted by
mosquitoes and damages the human lymphatic system, causing swelling such as elephantiasis.
The World Health Organziation has targeted filariasis for elimination in Haiti by 2012. It's a far
easier task than malaria, but the planned solution shows just how tightly bound poverty and
disease can be.
Filariasis can be eliminated through simple economic development. Its mosquito host
breeds in raw sewage, so screens and basic investments in sanitation can accomplish a lot. But in
Haiti even the simplest development goals can't be taken for granted, so the preferred approach
here is to fortify the salt supply with diethylcarbamazine, a drug that kills juvenile parasites. It's
the same idea as adding iodine to salt, but Streit found there was not even an iodization program
in Haiti. Iodine is critical for brain development, and deficiencies can drop IQ by as much as 15
points. "Without adequate dietary iodine, these kids aren't as smart as they should be," says
Streit. "Talk about handicaps to development! If we help them with these diseases, we're going
to give them a better chance to develop their country."
For years, the disease and development dynamic has been a classic chicken-and-egg
debate. Are some countries poor because of disease? Or is disease another symptom of poverty?
In the end the problem cuts both ways, but that hasn't stopped Columbia economist Jeffrey
Sachs, director of the U.N. Millennium Project, from calling for a fundamental change in the
economic strategies of wealthy nations toward the poor. In The End of Poverty, for example,
Sachs examines the investment by rich nations in malaria and AIDS -- diseases that particularly
stunt African growth -- and calls for "an end to the international community's gross negligence
regarding the diseases ravaging Africa."
The Notre Dame biologists agree that simple economic development would go a long
way toward eradicating disease, but Collins says development alone is not enough. "There are
still some parts of the world where the intensity of infectious transmission of some of these
diseases is so high that economics isn't going to eliminate them," he says. "There is still dengue
in Singapore. Not a lot, but it's there. You can't get more economically well-developed and
regulated than a place like Singapore."
Meanwhile malaria in Africa enjoys an almost perfect storm of reinforcing factors. "The
vectors are incredibly efficient," says Collins. "Even putting screens in everybody's houses isn't
going to stop people who are out of doors after dark from getting bitten. We're not going to get
rid of all the mosquitos in Africa just by economic development."
Despite a festering public discomfort with genetically modified organisms, Collins thinks
it's important to remember that public health is a completely different motive than most of the
agribusiness examples of genetic modification.
"Every 30 seconds someone dies from malaria, and the majority of those are children
under the age of 5," says David Severson. "Look a child in the eye in Western Kenya and say we
can't explore these options because we have issues with transgenic organisms. I think we have
an obligation to pursue these things."
Signs of hope
"I've been working in parasitology for 30 years. We're worse off now than when I started," says
John Adams, Notre Dame professor of biological sciences. "People should be worried," he adds,
as he enumerates the landscape of disease from the resurgence of malaria to the accelerating
emergence of new maladies such as AIDS and avian flu. "These are all signs that we're not
taking care of our environment in a way that's conducive to good health."
But there are also signs of hope. Sachs and other like-minded crusaders have put public
health back on the map, leading to the Global Fund to Fight AIDS, Tuberculosis and Malaria.
The Gates Foundation has energized the scientific community with a series of creative and
effective grant programs.
New fields such as genomics are just beginning to reach their potential and spawn the
kinds of serendipitous solutions the world needs. For example, Adams has spent years struggling
with the virulent malarial parasite Plasmodium falciparum, which, despite its resilience in the
wild, is extremely difficult to work with in the lab. It has been particularly resistant to the kinds
of genetic manipulation typically used to unlock the final meaning of genes: what proteins they
make.
But Fraser and Adams have discovered that piggyBac seems perfectly suited to breaking
down falciparum. "With most things being difficult with falciparum, you couldn't ask for more,"
says Adams.
You could call it luck, but the discovery demonstrates just how collaborative research on
these problems is, both within Notre Dame and beyond. "Even though we may be working on
different diseases and different organisms, tropical diseases share many common features," says
Adams. "A whole group of people looking at different aspects of the same diseases are more
likely to come up with novel ideas."
(October 2006)
