The X4 files: sampling the science on HIV co-receptors in Bangkok
Bob Huff, for GMHC
It has become conventional wisdom that the International AIDS Conference (IAC) is no longer the place to find cutting-edge research on HIV science. And although Track A, the basic science track at the conference, had fewer posters and presentations than the tracks for clinical research, prevention, social issues and policy, a respectable 618 basic science presentations were submitted and 439 accepted to the 2004 IAC in Bangkok. Here’s a selection of abstracts covering one aspect of HIV basic research of emerging importance.
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.
A better blocker?
One attractive feature of blocking CCR5 is that there may be little or no toxicity penalty to pay, since some people are born without CCR5 proteins on their cells and seem to suffer no ill effects. (Yes, it is rare for these people to become HIV infected, and when they do — via other co-receptors, no doubt — they tend to have a very slow course of progression.) But blocking CXCR4 may not be as trouble-free, since a lack of the protein has been shown to lethally prevent infant mice from developing normally. Nevertheless, a couple of X4-blockers have been used in human safety studies with no disastrous results. Ideally, it seems, one would want to use an X4-blocker in tandem with an R5-blocker to prevent the possibility of an X4-using HIV variant escaping and causing accelerated immune damage. But, in the first few upcoming trials, at least, the R5 blockers may have to work without a safety net as researchers rely on the imperfect assay to screen out those at risk for switching to an X4-using virus. If all goes according to schedule and no unforeseen problems with toxicity arise (and there is no guarantee that they won’t), the first oral entry inhibitors could possibly appear in expanded access studies by late 2006 and be approved for sale in 2007.
Here’s a look at some of what we learned about the R5 and X4 co-receptors at the International AIDS Conference in Bangkok.
It is recognised 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 internalised 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 defence 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 favoured 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. 
Although most people’s HIV infections start out using the R5 co-receptor, in about half the X4 strain will eventually appear at some point during their lives. But how quickly does this happen, and to whom? In Bangkok, two studies presented retrospective analyses of co-receptor usage in a large number of samples and correlated the R5 phenotype with viral load, CD4 count and other variables. A study by Harrigan retroactively evaluated samples and records from 806 participants in a cohort of treatment naive-adults in British Columbia, and a study by Moyle evaluated data and co-receptor phenotype in a collection of 169 stored samples from treatment-naive individuals. In general, both studies confirm that, within a given sample population, as the duration of HIV infection increases and CD4 counts decline, the prevalence of the exclusive R5 phenotype decreases and X4 and dual R5/X4 phenotypes become more common.
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. 
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. 
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.
A study by Ito and colleagues of the National Institutes of Health in Bethesda, Maryland, investigated how competing “swarms” of HIV with different co-receptor usage phenotypes might interact within lymphoid tissue. They found that X4-using HIV variants — including R5/X4-using strains — were able to suppress replication of their R5-using competitors. Meanwhile, the R5 viruses had no effect on the X4 interlopers. The researchers identified several chemokines stimulated by X4 HIV that seemed to be responsible for suppressing the R5 variants. When a cocktail of these chemokines was added to lymphoid tissue with only R5-using HIV present, the same suppressive effects were observed. The authors suggest that a better understanding of these chemokine-induced effects could possibly lead to the development of a new approach to anti-HIV therapy. 
Early in the history of the epidemic one of the first distinguishing phenotypes of HIV described by scientists was the ability of some strains to cause groups of infected cells to fuse into clumps called syncytia. Syncytium inducing (SI) HIV was recognised as a highly virulent form and it became associated with rapid progression to terminal AIDS. At one point it was thought that syncytium formation was HIV’s main mechanism for killing T-cells; to this day it is not entirely clear what is responsible for T-cell death in AIDS and multiple effects are suspected, ranging from apoptosis (cell suicide), to immune exhaustion through overstimulation to direct killing. Eventually, SI and non-syncytium-inducing (NSI) HIV were correlated with the X4- and R5-using phenotypes and the SI and NSI nomenclature was generally put on the shelf.
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 analysed 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. 
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 on the surface of T-cells, which could explain why R5-using viruses have such a hard time of it when TNF levels are increased. 
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?
From cradle to grave
Choudhary and colleagues from the University of California, Irvine, investigated the effect of HIV infection with an X4 strain on thymocytes, precursors to T-cells that reside in the thymus, a kind of incubator for immature immune 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.
In vivo studies at Bangkok
At Bangkok, Pfizer presented five posters on its CCR5 blocker, UK-427,857, including results from a 10-day proof-of-concept study. Patients with CD4 counts above 250 cells/mm3 (currently off treatment or treatment-naïve) were randomised to a series of escalating doses or to placebo. Dosing began at 25mg once-a-day (QD) and increased to 300mg twice-a-day (BID), including one arm at 150mg BID taken with food. Monitoring continued for 30 days after treatment was stopped at day 10.
All doses from 100mg QD onward produced mean viral load reductions better than -1.0 log copies/mL. At the 150mg BID dose, food reduced the peak concentration and total exposure (AUC) to the drug by about half, although there was no difference in viral load reduction at that dose whether taken with food or not. This may be because blood concentrations of CCR5 blockers are not as important as how many R5 receptors become occupied by drug molecules and how long they stay. It seems that these drugs tend to stick to their targets and not let go, a quality that could extend the effective potency of a dose by several days. In this study, high levels of receptor saturation were achieved at every dose except 25mg QD.
All persons enrolled were assayed for HIV co-receptor usage phenotype and were required to have an exclusively R5-using strain at time of enrollment.
Of the 66 patients exposed to the drug, 2 experienced an emergence of a dual R5/X4 strain by day 11 of the study. One of these individuals reverted to R5 phenotype by day 40, but the other’s dual phenotype HIV persisted throughout the first 6 months of follow-up. Because the assay cannot pick up sequestered or very small populations of X4-using HIV, the risk of forcing a population shift is evident in this small, initial study. However, in the person whose X4 phenotype persisted, no evidence of accelerated immunological or clinical deterioration has yet been reported. A more detailed presentation of this individual’s case is anticipated at an upcoming conference. Adverse events were mild to moderate and included headache and dizziness. A separate report found no impact on QT interval, the cardiac rhythmic parameter that was perturbed by Schering’s first R5 blocker. 
Source: GMHC Treatment Issues, July/September 2004
Unless otherwise stated, all references in the following conference reports are to the programme and abstracts of the XV International AIDS Conference 11-16 July 2004, Bangkok.
Abstracts are available at:
- Bermejo M, PablosJ, González N et al. Impact of CXCL12 production by dendritic cells in HIV infection. XV Intl AIDS Conference, Bangkok. Abstract MoOrA1043.
- Harrigan PR, Asselin J, Dong W et al. Prevalence, predictors and clinical impact of baseline HIV co-receptor usage in a large cohort of antiretroviral naïve individuals starting HAART. XV Intl AIDS Conference, Bangkok. Abstract MoPeB3117
- Moyle GJ, Petropoulos C, Goodrich J et al. Prevalence and predictive factors for CCR5 and CXCR4 co-receptor usage in a large cohort of HIV-1 positive individuals. XV Intl AIDS Conference, Bangkok. Abstract WePeB5725
- Ito Y, Grivel J, Chen S et al. CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue by selective induction of CC-chemokines. XV Intl AIDS Conference, Bangkok. Abstract MoPeA3057
- Bonneau KR, Diaz L, Relman D et al. Differential gene expression in primary human CD4+ T cells infected with nonsyncytia-inducing (NSI) and syncytia-inducing (SI) isolates of HIV-1 recovered from the same individual. XV Intl AIDS Conference, Bangkok. Abstract TuPeA4381
- Valentin A, Morrow M, Yarchoan R et al. Differential effects of TNF on HIV-1 expression: R5 inhibition and implications for viral evolution. XV Intl AIDS Conference, Bangkok. Abstract MoOrA1048
- Choudhary SK, Powell D, Walker R et al. HIV-1 induces apoptosis in infected thymocytes. XV Intl AIDS Conference, Bangkok. Abstract MoPeA3008
- Fätkenheuer G, Pozniak A, Johnson M et al. Evaluation of dosing frequency and food effect on viral load reduction during short-term monotherapy with UK-427,857 a novel CCR5 antagonist. XV Intl AIDS Conference, Bangkok. Abstract TuPeB4489