As most people who follow HIV therapies have learned by now, HIV infects a new target cell through the process of entry, which can be described as having three basic stages. First, a protein on the surface of HIV attaches to a CD4 protein on the surface of a target cell. This step, attachment, allows the viral protein to change its shape so that it can bind with a different kind of protein on the cell's surface, called a co-receptor. Then, after co-receptor binding and a few more intermediate steps have been accomplished, the virus can finally pull itself into contact with the cell's surface, where the two merge in a process called fusion. After fusion is complete, the viral payload can be delivered and the process of hijacking the cell and turning it into an HIV factory is well on the way.
Fuzeon, the only approved entry inhibitor, acts to block the fusion process at the point when the virus is pulled into contact with the cell. As for the other steps, several experimental drugs are in development to block attachment and co-receptor binding. As a number of orally available entry inhibitor candidates move through the drug development pipeline (injectable Fuzeon was approved in 2003), concerns are being raised about a subset of the class, known as CCR5 blockers. Because there are two basic types of cellular co-receptors that HIV can use, drugs are being developed that block both kinds. The most common co-receptor protein that HIV uses for entry is called CCR5 or R5, for short. In someone who is newly infected, R5 is often the only kind of virus that can be found. But in about half of the people who develop advanced HIV disease, the virus begins to use another co-receptor called CXCR4, or X4. The shift to using X4 is considered a bad sign because it is often accompanied by a dramatic increase in the rate of T-cell depletion. It is not entirely clear if HIV with the X4 phenotype causes accelerated T-cell loss or if it is only a symptom of some other shift in the T-cell ecology, but everyone agrees: you want to avoid developing an X4-using virus.
This means there is a critical open question about using the new R5 blocking drugs: will they cause HIV to start using X4? And will that be worse than letting the R5-using virus chug along at its own, slower, but no less dangerous pace? So far there's no solid evidence that blocking R5 will lead to HIV mutations that prefer using X4. But there is increasing evidence that some people may have small amounts of X4 virus in their bodies that could be given a green light to take over if their R5-using cousins are shut down. Again, it's not clear if these were acquired at the time of infection or if HIV can mutate step-by-step from exclusively using R5, to using both R5/X4, to using only X4. While there is now an experimental phenotype assay that can detect R5, X4 and dual R5/X4-using virus in a person's blood, it may not be sensitive enough in all cases to identify X4-using variants that are hiding in tissues or are only present in very small numbers. Now, as several pharmaceutical companies are getting ready to start large phase III trials for their R5 blocking drugs, discussion of the X4 problem is heating up.
It's recognized that HIV with the X4 phenotype is rarely, if ever, transmitted -- even when the donor predominantly carries X4 virus. In sexually transmitted infections, it is thought, dendritic cells (DC) patrolling the body's mucosal frontier are the first immune cells to contact HIV. However, instead of infecting the DC, HIV is internalized into a bubble-like vesicle and eventually carried to a lymph node, where it is introduced to circulating T-cells, normally the next line of defense in the immune response to foreign viruses. Unfortunately, these T-cells are the very cells that HIV prefers to infect. This is where HIV actually takes root in a new host.
So, why do R5 viruses seem to be favored for starting new infections? In a laboratory-based study by J. Alcami and colleagues in Madrid, infections of T-cells by X4-using HIV were observed to be greatly reduced in the presence of dendritic cells, while infections by R5 strains were enhanced. This, the authors say, suggests that dendritic cells may produce a chemokine, or signaling factor, that effectively prevents X4 HIV from establishing a new infection -- while having the opposite effect on R5 virus. In other words, if dendritic cells are the gatekeepers to HIV infection, they may routinely filter out X4 virus, which allows R5 viruses to initially become the dominant strain. (Bermejo, A1043)
In the Harrigan cohort, detection of the R5/X4 or X4 phenotype increased from 6% in people with CD4 counts above 500 cells/mm3 to over 50% in those with CD4 counts below 25 cells/mm3. There was only one exclusively X4 phenotype sample in the cohort. The odds of having an X4-using virus increased by about 1.5-fold in those with CD4 counts between 200 and 500 compared to those above 500 cells/mm3; the odds were 5- to 7-fold greater in those with CD4 counts between 25 and 200 cells/mm3; and jumped to 17-fold greater in those with fewer than 25 CD4 cells/mm3. (Harrigan, B3117)
In the Moyle study, detection of the R5/X4 phenotype ranged from about 7% in samples with CD4 counts above 300 cells/mm3 to 46% in those with CD4 counts below 100 cells/mm3. There were no exclusively X4 phenotype samples. The mean CD4 count for the R5 samples was 307 versus 117 cells/mm3 for the R5/X4 samples. (Moyle, B5725)
In neither study was viral load a significant predictor of co-receptor usage phenotype. In the Harrigan cohort, injection drug use was not correlated with having R5 or X4 HIV; in the Moyle study there was no difference between B and non-B HIV subtypes.
Researchers from the lab of Jay Levy in San Francisco investigated the patterns of gene expression in T-cells stimulated by infection with two different phenotypes of HIV, the old-school SI and NSI variants. The quantities of various RNA gene products from the test cells were analyzed using microarrays that can detect thousands of known gene products to see which were upregulated and which were downregulated when compared to uninfected cells. They found that SI HIV tends to upregulate genes involved with production of a cellular factor called tumor necrosis factor (TNF), which is associated with immune hyperstimulation, a state often implicated in T-cell depletion. (Bonneau, A4381)
The observation that people with AIDS have high levels of TNF in the blood was also made very early in the epidemic. At Bangkok, a group led by A. Valentin of the National Cancer Institute in Maryland reported on their recent studies of TNF levels in 63 patients (30 controls) and the impact that TNF has on virus production and co-receptor availability. They found that TNF inhibited replication of R5-using HIV strains while having no effect on X4 HIV. TNF was also observed to downregulate the number of CCR5 co-receptors that appear the surface of T-cells, which could explain why R5-using viruses have such a hard time of it when TNF levels are increased. (Valentin, A1048)
Couple this with Bonneau's finding that X4 HIV stimulates TNF production, and you have one possible explanation for how the shift from R5 to X4 predominance occurs. Of course, these shifts may be happening all the time on a small scale in isolated tissue compartments. What causes the overall population of HIV to shift? And why does the appearance of X4 virus signal the demise of so many T-cells?
One theory for the accelerated CD4 cell depletion associated with X4 HIV maintains that the virus is capable of killing T-cells while they are still in their thymic cradle. In the experiment, thymocytes were infected with HIV and microarray technology was used to monitor changes in the expression of 22,000 gene products. They also assayed for various indicators of apoptosis. The most significant finding was that HIV infection induced apoptosis through a pathway involving a protein called caspase. This was confirmed by artificially inhibiting caspase, which had the effect of blocking apoptosis in infected cells. These results suggest that if the war between R5- and X4-using HIV comes to the thymus, the impact on CD4 cell production could be very dramatic indeed. There is still much to be learned about how and where HIV causes all the harm it does. Fortunately, we don't need to understand every detail of HIV pathogenesis to continue developing newer and better therapies.
Back to the GMHC Treatment Issues July/August 2004 contents page.