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Duke Researchers Discover Potent Ability of Specific Immune System Cell Against HIV

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Duke Health News 919-660-1306

DURHAM, N.C. - Scientists have long known that immune system
cells known as "killer CD8" cells attack the AIDS virus after
it enters the body by killing virus-infected cells. They also
have known that CD8 cells can stop the virus from infecting new
cells. Researchers now have found that CD8 cells continue to
fight the virus after it enters another kind of immune system
cell and begins to reproduce.

In fact, Duke University Medical Center researchers have
discovered that CD8 cells can stop human immunodeficiency virus
(HIV) in its tracks even when they are added to the immune
cells known as CD4 cells, which are cousins of CD8 cells in the
T-cell family, after HIV has already entered the cells.

Previous work has suggested that the potency of virus
suppression or number of suppressive CD8 cells could determine
how quickly symptoms of AIDS develop. The new findings,
reported in the March 28 issue of the Proceedings of the National Academy of
Science
, might point to novel protective strategies, the
researchers say.

"We have known that CD8 cells are important in controlling
the level of virus in the bloodstream and keeping some patients
in an asymptomatic state," said Dr. Michael Greenberg of the
Duke University Center for AIDS Research. "Now for the first
time, we have shown that CD8 suppressive activity also works
later in the infection process, at the stage of gene expression
during virus replication. Furthermore, this ability is
independent of the HIV protective envelope protein.

"Stimulating the production of these virus-suppressive CD8
cells, by a vaccine or other means, could be a novel way to
keep the spread of the virus under control," he said.

The Duke team's research was supported by numerous grants
from the National Institute of Allergy and Infectious Diseases,
part of the National Institutes of Health (NIH).

An experimental system developed by a young investigator on
Greenberg's team, Dr. Georgia Tomaras, was employed to provide
a clearer timeline of what actually happens during an HIV
infection.

After HIV enters the human body, it infects helper CD4
immune system cells because the viral envelope protein - called
the gp120 glycoprotein - has a special affinity for the CD4
receptor found on these cells.

Once inside a CD4 cell, the virus' genetic material is
copied into DNA and enters the nucleus, where it integrates
into the host cell's DNA. From here the virus takes over the
genetic machinery of the cell, directing it to produce many
copies of HIV that eventually flood into the bloodstream to
infect other CD4 cells. As a result of the destructive nature
of HIV, the number of CD4 cells in the blood drops.

However, not all people who are infected with HIV react the
same way to the virus, and much of the variability may depend
on the activity of these CD8 cells.

"HIV can act very differently from person to person - some
people progress to full-blown disease in a relatively short
period of time, while others remain asymptomatic for many years
after infection," Greenberg said. "In many of these
asymptomatic patients, there appears to be strong CD8 activity.
Similarly, patients who progress quickly to AIDS have very
little CD8 activity."

In their experiments, the Duke researchers used blood
samples taken from asymptomatic patients from Duke's Infectious
Disease Clinic. The researchers developed a unique laboratory
analysis that allowed them to follow in minute detail a single
cycle of infection in individual CD4 cells.

In asymptomatic patients, CD8 cells can identify infected
CD4 cells, latch on to them, and release compounds that cause
the infected cell to burst, killing it. This cytolytic, or
cell-killing, ability has been well documented. Scientists have
also known, since the discovery by Drs. Christopher Walker, Jay
Levy, and colleagues at the University of California-San
Francisco in the mid-1980s, that CD8 cells also possess a
non-cytolytic weapon as well.

However, what scientists did not know was how this
non-cytolytic weapon worked to stop HIV replication.

"Experiments have shown that when the cytolytic action of
CD8 cells has been blocked experimentally, viral replication is
still suppressed, so the CD8 cells are still doing something,"
Greenberg said.

Important work by Dr. Robert Gallo's group at the University
of Maryland-Baltimore demonstrated that CD8 cells release
beta-chemokines that can block entry of HIV into cells.
However, work by Dr. Anthony Fauci at NIH, as well as work by
Levy and Greenberg, have shown that CD8 cells can also suppress
HIV replication by other means. Until now, it was not known how
CD8 cells accomplished this.

Greenberg's new experiments show that CD8 cells affect the
virus after it has already entered the CD4 cell, which is very
different from the way beta-chemokines work. The CD8 cells
somehow stopped HIV from hijacking the CD4 cell's genetic
machinery to reproduce itself.

To further prove this case, the Duke researchers used a
system where the genetic material of HIV was encased with an
envelope protein taken from a very different virus. The CD8
activity was just as strong against this "pseudotyped virus,"
Greenberg said, indicating that CD8's action was independent of
the HIV envelope and specific to the HIV genetic material.

"These experiments are the first to show that the viral
suppression occurs well after the virus enters the cell and is
independent of the entry process," Greenberg explained.

The exact mechanism by which CD8 cells are able to
non-cytolytically suppress viral replication is not known.
According to Greenberg, the agent could be a soluble factor or
a molecule on the surface of CD8 cells that transmits a
biochemical signal to the CD4 cells, or a combination of both.
For the first time researchers now know where in the virus life
cycle to look for it, Greenberg said.

This single-cycle experimental system also yielded a rough
timeline of what happens to a cell infected by HIV, and when
the non-cytolytic activity of CD8 occurs. Prior experiments
used systems with multiple replication cycles, making it
difficult to follow actions within a single round of infection.
The Duke researchers were interested in following a single life
cycle.

They found that within the first two to six hours, HIV entry
into CD4 cells has already been completed. Secondly, reverse
transcription of the viral genetic material was finished by 10
to 14 hours. Finally, expression of early HIV genes occurs
mostly between 14 to 48 hours.

With this life cycle chronology understood, the researchers
could determine when during the infection process the
non-cytolytic activity of the CD8 cells took place. They found
if CD8 cells were added from the time of infection up to six
hours after infection, viral replication was completely halted,
and even after 24 hours, could achieve a significant reduction
in replication.

"These experiments demonstrate that the suppressive activity
of CD8 occurs later in the virus life cycle - after the virus
inserts its genetic material into the genome of the target
cell, but before it is completely expressed," Greenberg said.
"These findings have clarified the protective role of CD8
cells."

The Duke team hopes that these findings will open new doors
for scientists working on developing novel therapeutics and
vaccines for HIV.

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