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From the Marrow of a Survivor
Antibodies to Ebola virus appear 10 days to two weeks after
the infection, which is bad timing for the infected person
as the virus has more often than not run its lethal course
by then.
"We got bone marrow from two survivors [of the 1995 Ebola
hemorrhagic fever epidemic in Kikwit, Democratic Republic
of Congo]," says Burton. Case workers for the U.S. Centers
for Disease Control and Prevention (CDC) provided the marrow.
"We made phage display libraries from that bone marrow."
Phage display is a method for selecting from billions of
protein variants those that bind to a particular target. In
the technique, libraries of antibodies are fused to the viral
coat protein of the phage—a filamentous virus that infects
bacteria. Then the virus is allowed to reproduce in culture,
where it copiously makes new copies of itself and the antibody
library.
"In effect, we reconstituted the antibody response [the
survivors] made in Africa six months later in the laboratory,"
says Burton.
Since the phage virus displays these proteins on the surface
of the virions, a scientist can easily select for antibodies
to them in vitro by passing the viral stew over a stationary
phase containing the target substrate—in this case, irradiated,
inactivated Ebola virions. Those that can bind do, and the
best antibodies are those that bind the tightest and resist
being washed off the stationary phase.
"You are left with the ones you are interested in," explains
Burton. Then these antibodies of interest can be sequenced
and placed into an expression system where they can be mass
produced—Burton has produced gram quantities of one such
antibody in the past few years.
This antibody reacted particularly strongly against the
viral coat glycoprotein on inactivated Ebola virus. Subsequent
tests carried out by Burton's collaborators in BioSafety Level
4 laboratories have shown the antibody to be reactive against
live Ebola virus in cell culture and in live models. Promising
results so far.
Burton and his colleagues are interested in looking at the
possibility of using the antibody derived from this patient
as a serum that might be used to treat patients, particularly
as a first-line defense for laboratory workers who accidentally
receive a needle prick injury.
He also is developing a colorimetric assay to test samples
taken from animals in the wild to look for evidence of Ebola
virus. This assay might then be made into a field-ready kit
so health care workers have better odds of identifying Ebola
virus's animal reservoir.
"If the animal has antibodies against Ebola virus in its
serum, then you can see that in this color test," says Burton.
Such a detection method would also prove invaluable for
safeguarding against the accidental import of Ebola virus
into the United States or other countries through monkeys,
as has happened on several occasions. In 1989, for instance,
an outbreak of Ebola hemorrhagic fever in Reston, Virginia
killed several monkeys that had been imported from the Philippines.
And Burton uses his antibodies as probes to study the basic
science of Ebola, an important advance, because much about
the virus is unknown.
Dangerous Mystery
There is much about the Ebola virus that is still a mystery.
Replication strategies are poorly understood. The mechanism
for Ebola entry into a cell is not known. We do know that
once Ebola virus is inside cells, it goes about replicating
itself, and we know that the virus requires the recognition
of a receptor on the surface of a cell to enter that cell.
But we do not know for certain what that receptor is.
Ebola forms long filamentous virions inside infected cells.
When a virion is made, the structural proteins associate with
the RNA strand, packaging it in a capsid that then associates
with viral proteins that insert into the cell membrane, which
allows the whole package to bud off from the infected cell
and form a new virion. The genetic material is a single strand
of antisense (-) RNA of about 20,000 nucleotides. When transcribed
by its own polymerase enzyme, the viral RNA codes for a nucleoprotein,
a few structural proteins, the polymerase, and the glycoprotein
target of Burton's antibody.
"We're interested in the function of the glycoprotein,"
says Buchmeier, though he adds that he works mostly with the
related family of arenaviruses, which like the filovirus family
to which Ebola belongs, cause hemmoragic fever in humans.
The glycoprotein forms spikes, approximately seven nanometers
long, on the virion surface. These glycoproteins define the
receptor specificity, mediate the cell fusion and cell entry,
and may have certain domains that interfere with other cell
functions. Like all viruses, Ebola has a certain cell specificity—it
targets endothelial cells and macrophages. Ebola may even
use its spikes to spread from cell to cell, thus evading the
immune system and increasing its virulence.
"That," says Burton, "probably has something to do with
its extreme pathogenicity and the fact that the immune response
to it is so slow."
Once inside a cell, the virion uncoats and the polymerase
transcribes the viral (-) RNA into a (+) sense strand inside
a host cell's cytoplasm. There, the sense strand, and at some
point, the polymerase switch into replication mode and copy
the (+) sense strand into an anti-(-) sense strand. These
are packaged with other virus components and released, along
with components of infected cells.
"[Infected cells] release a storm of early cytokines, like
TNF-alpha, interleukin-6, and the interferons alpha and beta,"
says Buchmeier. "These cytokines are very toxic and cause
shock and damage to the body."
Death comes from a combination of dehydration, massive hemmoraging,
and shock, which results from this massive release of cytokines.
Though there are vaccines in trial, there is currently no
cure for Ebola hemorrhagic fever. The best treatment consists
of administering fluids and taking protective measures to
ensure containment, like isolating the patient and washing
sheets with bleach.
The Once and Future Virus?
The timing of the appearance of Ebola hemorrhagic fever
in Africa 25 years ago was a case of epidemiological irony.
Even as this new threat was emerging, another deadly virus
was being cornered there. In 1976, the World Health Organization
was monitoring progress on its global smallpox eradication
effort started a decade earlier. This effort was to be successful
within a year—the last case of smallpox on earth occurred
in Somalia in 1977.
Today it is Ebola virus that looms large, though perhaps
not in numbers. Ebola hemorrhagic fever has killed hundreds.
Smallpox hundreds of millions. Still, what will eventually
become of Ebola virus is impossible to say. In the latest
outbreak, the World Health Organization was reporting, as
of last week, 33 confirmed cases of Ebola and 24 deaths in
the countries of Gabon and the Democratic Republic of Congo.
For his part, Burton is interested in how the antibody he
has isolated might be used as a possible treatment.
"You cannot be complacent about something like Ebola virus,"
says Burton. "You have to watch out for [it]."
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