"CAF" protein mystery solved by AIDS researchers

Blood proteins shown to thwart HIV provide clues to potential new treatment

Rockefeller professor David Ho and assistant professor Linqi Zhang, of the Aaron Diamond AIDS Research Center, discovered that the killer T cells of "long-term non-progressors" of HIV, people who test positive for the virus but don't get sick, produce proteins that inhibit HIV.

Acclaimed AIDS researcher David Ho, M.D., a Rockefeller University Irene Diamond Professor who heads the Aaron Diamond AIDS Research Center (ADARC), and his research team, have discovered that several natural proteins -alpha-defensins 1, 2 and 3 – can be manufactured and released by killer T cells to inhibit HIV.

The research, reported in the September 26 issue ofScience Express, begins to unravel a 16-year mystery in the field of HIV research about how a small proportion of HIV-positive individuals avert the virus’s destructive effects.

The elusive substance in the blood of “long-term non-progressors”- people who test positive for the virus, but who do not get sick – has been studied since 1986, when it was discovered by leading scientist Jay Levy of UCSF. Since then, HIV research teams have tried to identify the proteins. But the CD8+ Antiviral Factor (CAF) proteins, as they are called, yielded little information to scientists, until now.

“Many renowned scientists have tried to clone and express these proteins,” says lead scientist for the study Linqi Zhang, Ph.D., assistant professor at Rockefeller and an ADARC staff scientist. “But they’ve been unsuccessful because not every cell has the correct machinery to process the protein once it’s translated.” The proteins are hard to identify using traditional approaches.

“Alpha-defensins are promising as a future addition to the HIV treatment arsenal,” says Ho. “Researchers at ADARC are already pursuing new therapeutic approaches based on the current research results.”

Rockefeller University/ADARC scientists have recently shown that CD8+ T cells produce alpha-defensins. Here two immune system cells, a neutrophil (left) and a CD8+ T cell (right), produce alpha-defensin proteins (green). Neutrophils have long been known to produce the proteins.

Alpha-defensins 1, 2, and 3, members of a family of so-called “natural peptide antibiotics,” work in combination – if the host immune system can produce them – to prohibit the virus’s replication, or copying, in the body.

Defensins, which come in approximately a dozen known forms, were first described by scientists Tomas Ganz and Robert Lehrer in 1985. The proteins were thought to be made exclusively by neutrophils, a specialized immune system cell, to kill bacteria. The Rockefeller/ADARC researchers have learned that they are also made by CD8+ T cells and inhibit the replication of HIV by an as-yet-undefined mechanism.

In the Science study, Zhang, Ho and their ADARC colleagues Winjie Yu, Ph.D., Tian He, Ph.D., Jian Yu, Ph.D., and Wenyong Zhang, Ph.D., used traditional biochemical methods combined with a novel, chip-based protein analysis system, called ProteinChip, to compare CD8+ cells from long-term non-progressors with cells from HIV patients whose immune systems had begun to fail. Ciphergen Biosystems, Inc. of Fremont, California developed ProteinChip.

The technology, which measures the qualities of a protein via simple mass spectroscopy analysis, yielded immediate results, and brought the first tangible sense of direction to identifying CAF proteins.

“Until that point, we and all the other scientists working on the project were almost blind to the proteins’ identity,” says Zhang. “ProteinChip suddenly put a mountain range in our line of sight.”

Ho, Zhang and their colleagues scoured those “mountains” – literally the peaks of spectroscopy read-out from six different kinds of analysis – to make molecular sense of the data.

The scientists then compared their data with that of known proteins in publicly available databases, hoping for clues that would help them define the CAF proteins. To their surprise, the Rockefeller/ADARC scientists came up with an exact match -alpha-defensins 1, 2, and 3.

The remaining months of research were spent studying the proteins’ efficacy. Since only very small quantities of the proteins exist, obtained from samples of human blood, Zhang’s research team used synthetic defensin, alone and in combination, to try to further understand why the T cells that do manufacture the proteins make three versions.

In their experiments, the scientists learned that individual defensins alone don’t perform well. A combination of at least two, and possibly all three, is required to confer inhibitory effects.

The three alpha-defensins published in the Science study are active against all strains of the virus.

“This discovery is a major step forward in our understanding of how the body fights HIV,” says Zhang. “By understanding how some people’s immune systems are able to control HIV infection, we may be able to develop new treatments that take advantage of this phenomenon.”

Research in 1995 found a family of proteins called beta-chemokines that could account for some of the viral suppression in non-progressors, but beta-chemokines were ineffective against many strains of the virus and could not fully account for the CAF activity.

Additional authors of the Science paper include Rebecca Cafferey, Enrique Dalmasso, Siyu Fu, Thang Pham and Jianfeng Mei, all Ciphergen researchers or technicians.

This research was supported by the Campbell Foundation and the Irene Diamond Fund.