It's the scourge of daycare, preschools, and playgroups in the U.S., causing more than half a
million annual doctor visits, parent absences from work and tens of thousands of hospitalizations.
In the developing world, it's a killer to be feared, responsible for over
400,000 deaths each year.
It's called rotavirus, and it's the leading worldwide
cause of severe, dehydrating gastroenteritis in children under
age 5, eventually striking nearly all infants and toddlers. In
the U.S., almost all children recover with rehydration therapy
to replace lost fluids and electrolytes, but in developing countries,
where access to care can be limited, rotavirus causes up to 6 percent
of all deaths in young children.
Despite being such a well-known
foe, scientists never knew how rotavirus broke into cells to wreak
its havoc until a Children's Hospital Boston researcher took a
much closer look.
In the 1980s, Phil Dormitzer, MD, PhD, now a
physician and a structural virologist in Children's Laboratory
of Molecular Medicine, was an anthropology student doing fieldwork.
During his travels on the Indian subcontinent, he had several episodes
of diarrhea, some severe, and later learned in one of his classes
that diarrhea is a major cause of global mortality.
"I was an idealistic kid," Dormitzer recalls, "and
it struck me as stupid that in the late 20th century, hundreds
of thousands of children should be dying of diarrhea. I saw solving
the problem as a real opportunity to make an impact on world health."
decided to go to medical school, and at Stanford University met
his mentor Harry Greenberg, MD, who was studying rotavirus. "If
you want to make a vaccine against diarrhea," Greenberg told Dormitzer, "that's
what my lab does."
Vaccines are especially attractive in developing
countries where the health infrastructure is weak. Rehydration
therapy, though simple and effective, requires a health system
that can deliver timely treatment and health education, whereas
with a vaccine, medical workers can sweep through a community and vaccinate
everyone in a military-style operation. Dormitzer began working on rotavirus
vaccines with Greenberg in 1987.
Greenberg was trying to develop a vaccine
based on individual rotavirus proteins. In theory, such vaccines do the same
job as vaccines made from whole viruses, but are easier to manufacture, more
likely to be stable without refrigeration (a boon in developing countries)
and less likely to cause side effects.
|ýWe aim to use this knowledge to
design a cheap, safe and effective vaccine.ţ
Dormitzer's first task was to isolate
and manufacture a single protein on rotavirus's surface, called VP7, and
to try to immunize animals with it. It didn't work, Dormitzer discovered,
because VP7 changes its shape when rotavirus breaks into a cell. The animals' immune
systems weren't "seeing"ˇor making antibodies
againstˇthe right form of the protein.
Dormitzer realized that to build
an effective vaccine, he needed to get a structural understanding of
VP7 so he could see how its parts moved and reoriented during the
process of infection. He decided to relocate to Boston, where Stephen
Harrison, PhD, and the late Don Wiley, PhD, were studying viruses
at the newly formed Children's
Hospital Boston˝Harvard Medical School structural biology unit. Now called
the Laboratory of Molecular Medicine, the unit was, and still is, supported
by the prestigious Howard Hughes Medical Institute. "I came here to learn
how to work on the rotavirus problem structurally," Dormitzer says.
is a large, 20-sided, soccerball-shaped virus. Its surface layer, stripped
off in the course of entry, somehow gets the virus's inner "payload"ˇthe
genes and the replication machineryˇpast the intestinal cell's outer
membrane to the inside, where it can start reproducing.
the surface of rotavirus are 60 "spikes," made up of another protein
known as VP4. Dormitzer has lived and breathed VP4 since arriving
at Children's in 1997,
now shown that the VP4 spikes are what enable rotavirus to enter
intestinal cells, through an elaborate molecular gymnastics and two
consecutive shape changes. In the August 26, 2004, edition of the
Dormitzer published high-resolution images that give evidence of
these maneuvers (see sidebar, below).
First, when rotavirus arrives
in the intestines, digestive enzymes cause some of the VP4 molecules
to rigidify into their spike form. This positions the spike's "head" to
attach to an intestinal cell's outer membrane. Second, the spikes
bend back, and VP4 takes on a folded-umbrella structure. This folding
motion, Dormitzer believes, causes the spike's "body" to punch
through the cell membrane, allowing the virus free passage inside.
head and body proteins contain many of the targets that prime the
immune system to attack rotavirus. As a first step in revealing
that will help in building an optimal vaccineˇDormitzer
and colleagues manipulated bacteria and insect cells into making
large quantities of the proteins in their different configurations.
They then crystallized the proteins in a process akin to making
rock candy: you put the protein in a liquid solution, tinker to
optimize the conditions, then waitˇsometimes
crystals to form.
The researchers then brought the crystals to
be X-rayed at a particle accelerator, of which only a handful exist
in the U.S. As electrons whiz around a track about 1 kilometer
(or 0.6 mile) long, almost at the speed of light, intense X-rays
are given off and sent down pipes into adjoining rooms, where investigators
from around the world plug in and do their experiments. "You
don't sleep very much," Dormitzer says. "Beam time is very valuable."
crystals scatter the X-ray beams in different directions, making
spots on a detector. Back in Boston, these spots were analyzed
to build images of the VP4 spikes and their component parts, precise
down to the atom. Dormitzer's
team then fitted these to reconstructed images of the spikes based
on direct electron microscopy of rotavirus particles. This gave
a more fleshed-out picture of the head and body components.
key for vaccine development is that these components not only trigger
the immune system, but appear to be stable at room temperature
and, Dormitzer believes, could be relatively easy and cheap to
mass-produce. Children's has
applied for patents on each, and Dormitzer is now collaborating
with several other institutions to fashion them into a vaccine.
The VP7 protein may also be used if Dormitzer can get it to hold
a stable shape that the immune system will recognize. "Ultimately,
we'll make a cocktail of
these different components," he predicts.
Other vaccines are in
the pipeline, but Dormitzer and Harrison believe their approachˇengineering
vaccines to have exactly the characteristics needed, based on an
intimate knowledge of a virus's
the wave of the future.
"We've gained important new insights into how this virus attacks cells," says
Dormitzer. "We aim to use this knowledge to design a cheap, safe
and effective vaccine to keep children from dying of an illness
that should be readily preventable."