September 29, 2002
The identity of the CD8 antiviral factor (CAF), whose activity was originally described in HIV-infected long-term nonprogressors by Dr. Jay Levy and colleagues in 1986, is a long-lasting mystery of HIV pathogenesis. During this ICAAC symposium, Dr. David Ho presented results of an elegant series of experiments that point to human alpha-defensins as the molecules behind CAF. These findings were recently published in Sciencexpress on September 26.
The original characteristics of CAF described by Dr. Levy over 15 years ago guided the search by Dr. Ho's group, led by Linqui Zhang. They knew that the factor was most likely to be found in long-term nonprogressors, was produced by stimulated CD8+ T-cells, was a small protein less than 20kD in size, and did not require the major histocompatibility complex (MHC) system or cell-cell contact for its activity. Some of this activity has been explained by the presence and activity of the beta-chemokines MIP-1alpha, MIP-1beta, and RANTES, the natural ligands that bind to CCR5, the co-receptor used by R5 strains of HIV, and likely the activity of SDF-1alpha, the ligand of CXCR4. These chemokines do not account for all of the CAF, however, because when they are removed from cell-culture, soluble CAF can still be detected.
Ho's group used protein chip technology developed by Ciphergen, a biotechnology company based in Fremont, California, to identify candidate proteins comparing three long-term nonprogressors, four progressors and 15 HIV-uninfected controls. The protein chip nuclear magnetic resonance technology allowed the investigators to identify three proteins less than 4kD in weight that were secreted by stimulated CD8 cells in all three long-term nonprogressors, eleven of fifteen controls, but none of the four progressors. Upon search of protein databases, the precise weight data learned from the protein chips, along with experiments that demonstrated the number and nature of sulfur bonds within the proteins, identified the candidate proteins as the alpha-defensins.
To confirm this identification, they showed that the addition of monoclonal antibodies to the alpha-defensins eliminated the peaks observed on the protein chips. The supernatant from stimulated CD8+ T cells from long-term nonprogressors inhibited replication of X4 HIV by 50-60 percent in their experiments, but the addition of the monoclonal antibodies to alpha-defensins completely blocked this HIV-inhibition, further confirming their identification. Synthetic alpha-defensins-1 and -2 were noted to have significant HIV inhibitory activity at reasonable concentrations in vitro that did not show toxicity to cells. Defensins purified from donor neutrophils were shown to be even more potent. Dr. Ho also showed pictures of CD8 cells stained for the presence of alpha-defensins, demonstrating that unstimulated CD8 cells had small amounts of these proteins in granules compared to neutrophils, but when stimulated, a small fraction of CD8 cells produced more defensins in their cytoplasmic granules.
Alpha-defensins were described in the 1980s by Robert Lehrer and Tom Ganz, researchers at UCLA. These molecules are secreted primarily by neutrophils and act to puncture the cell membranes of bacteria, helping the immune system to fight these invaders. While the proposed mechanism for CAF is inhibition of HIV transcriptional activity, the major known activity of the defensins does not explain the antiviral activity. While Lehrer, Ganz and others have studied the effect of alpha-defensins on other viruses, no clear mechanism of antiviral activity has been defined.
The role of human alpha-defensins in HIV pathogenesis, as the possible mediators of CAF, is not completely certain, however. Dr. Levy and others question various aspects of the work presented, namely the small number of subjects studied and the relatively modest activity of alpha-defensins against HIV in the test tube. Furthermore, there is some uncertainty about the ability of CD8 cells to produce the defensins in enough quantity to account for all of the observed CAF. The truth probably lies somewhere in the middle, with alpha-defensins playing a role in CAF, but perhaps not accounting for all of the clinical differences observed between long-term nonprogressors and usual progressors.
There is no doubt, however, that the elegant strategy outlined by Dr. Ho in his group's hunt for the identity of CAF, and the use of exciting new technologies in the search, illustrates the best way to clear up these lingering questions. As new pathways and host defenses are identified, new treatments may be developed based on knowledge gained from this thorough and novel approach.