In early 1996, the underlying biological basis for this distinction became clear when researchers discovered that, in addition to latching onto the CD4 molecule in order to gain entry into T cells, HIV isolates utilized one of two different coreceptors: either CCR5 or CXCR4. It quickly became apparent that CCR5 use correlated with the NSI/M-tropic classification while viruses that used CXCR4 were SI/T-tropic. As it turns out, the MT-2 cell line only expresses CXCR4. The older nomenclature has been replaced by the simpler designations of R5-tropic or X4-tropic. Rare "dual tropic" viruses that can use either co-receptor have been reported, but this designation is more commonly (and perhaps misleadingly) applied to mixed populations of R5-and X4-tropic HIV.
One salutary result of this progress is the development of pharmaceutical compounds designed to inhibit the interaction between HIV and its various coreceptors. However, the larger questions of why HIV bifurcates into two variants with differing tropisms and how these variants relate to disease pathogenesis remain unanswered, leaving a cloud of uncertainty looming over the clinical development of both R5 and X4 inhibitors. Similarly, now that large human studies of coreceptor inhibitors are underway, outstanding questions regarding the biological functions of CCR5 and CXCR4 receptors in humans have inspired research and regulatory interest in the possibility of unpredictable toxicities. The recent termination of the development of GSK's CCR5 inhibitor, aplaviroc, due to several cases of severe liver toxicity further underscores the need for vigilance in this regard.
Notably, chemokine receptors can be rather promiscuous in their ability to bind different chemokines. CCR5 is known to interact with CCL3, CCL4, and CCL8 in addition to CCL5. The reverse is also true; certain chemokines can interact with more than one receptor. CCL5, for example, can bind CCR1 and CCR3 in addition to CCR5. The functions of all the known chemokine receptors and chemokines are not fully characterized, but broadly speaking, they seem to be involved in cell migration and/or inflammation.
In terms of which cells possess the two HIV coreceptors, CCR5 is primarily found on activated T cells but CCR5 expression has also been reported (primarily from mouse studies) on multiple other cell types including macrophages, dendritic cells, neutrophils and hepatic stellate cells. CXCR4 is broadly expressed in cells of both the immune and the central nervous systems. A critical question facing developers of co-receptor inhibitors is whether CCR5 and CXCR4 inhibitor compounds interfere with chemokine/receptor interactions, and if so, whether such interference has harmful consequences.
The second study discussed by Swain was conducted by a group of Belgian researchers led by Christophe Moreno and involved the same mouse model of Con A-induced hepatitis. The researchers reported similar findings to Swain's group, namely, increased mortality in CCR5 knockout mice. They also reported that, in normal mice, serum levels of the CCR5 ligands CCL3, CCL4, and CCL5 were significantly increased following Con A injection and that CCR5- expressing liver mononuclear cells (comprising T cells, macrophages, natural killer cells and NKT cells) were recruited to the liver. The CCR5 knockout mice also exhibited increased production of interleukin 4, tumor necrosis factor, CCL3, CCL4, and CCL5, and a notable infiltration of T cells, macrophages, natural killer cells and NKT cells into the liver, among which were cells expressing the chemokine receptor CCR1 (which can also bind to CCL3 and CCL5). The researchers tried blocking CCR5 ligands with antibodies to see whether the hepatitis would improve. Blocking CCL5 significantly reduced serum ALT levels and hepatic mononuclear cell infiltration, whereas blocking CCL3 and CCL4 had no effect. Thus, it appears that the absence of CCR5-expressing cells can result in increased levels of circulating CCL5, potentially exacerbating immune-mediated liver damage. This study was published in the journal Hepatology (Hepatology 42:854-862, 2005).
To date, most studies have not revealed obvious, serious health problems among delta32 homozygotes or heterozygotes, but the literature on delta32 homozygotes remains relatively sparse. There has been one report that delta32 homozygotes infected with hepatitis C experience less inflammation but more fibrosis (scarring) of the liver, compared to infected individuals who lack the delta32 mutation. Data published previously suggested that delta32 homozygotes are more susceptible to hepatitis C infection and have a diminished response to treatment, but recent studies have contradicted these assertions. The delta32 mutation has also been strongly associated with a disease called primary sclerosing cholangitis (PSC). PSC is a disease involving inflammation and scarring of the bile ducts, which can cause bile to accumulate in the liver, damaging liver cells and leading to cirrhosis.
Perhaps the most dramatic human data come from a very recent study of West Nile Virus (WNV) suggesting that delta32 homozygotes may be more susceptible to symptomatic infection with this mosquito-borne pathogen (J Exp Med 203;1:35-40, 2006). The investigation was conducted based on results of a prior study in CCR5 knockout mice demonstrating that the mice experienced exacerbated symptoms as a consequence of reduced T cell trafficking to the brain (J Exp Med 202;8:1087-1098, 2005). The human study analyzed two different cohorts of individuals with laboratory confirmed symptomatic WNV infection. The results found that delta32 homozygotes were significantly overrepresented in both cohorts relative to the expected frequency of the mutation in the population. In one of the two cohorts, delta32 homozygote genotype was also associated with an increased risk of death. The study authors went so far as to conclude: "Our results have important implications regarding the potential safety of CCR5- blocking agents now under development for the treatment of HIV/AIDS. Clinical care of individuals taking these medicines while residing in WNV-endemic areas may mandate strict measures to limit mosquito exposure and a high index of suspicion for symptoms consistent with WNV."
The extent to which any or all of these problems might occur in the setting of pharmacological CCR5 inhibition cannot be known until a larger amount of safety data accumulates on CCR5 inhibitors. It is possible that the redundancy present in the chemokine/chemokine receptor system may allow other receptors to assume the function of CCR5 in delta32 homozygotes, and that something similar may occur in people receiving CCR5 inhibitors. But the safety and toxicity issues associated with CCR5 receptor blocking are significant enough that regulatory authorities are requiring extensive long term follow up -- up to 5 years -- of individuals participating in clinical trials of CCR5 inhibitors.
Recently, however, the supposition that X4 virus causes rapid progression has been questioned. The countervailing hypothesis is that X4 virus emerges as a consequence of the severe T cell depletion seen in advanced disease, perhaps due to the loss of appropriate cellular targets for R5-using HIV (this argument is rehearsed in excruciating mathematical detail in a new paper in the Journal of Virology, see J. Virol 80;2:802-9, 2006). This line of reasoning is further based on evidence that R5 HIV has a competitive advantage over X4 HIV. The precise nature of this advantage remains uncertain, but a number of possible explanations have emerged:
There are data that support each of these notions, but as of yet, nothing conclusive. It is fair to say, however, that some of the heightened early concern about the potential danger of tropism shifts has waned. A shift to X4 virus has been reported in several recipients of CCR5 inhibitors, but it does not appear to have harmful clinical consequences. CCR5 inhibitor studies are currently using an assay that attempts to quantify proportions of R5- and X4-using HIV as a screening tool. When applied to recipients of normal HAART regimens, the assay has found that roughly 40-50% of HIV+ people show evidence of X4 virus, though there is no association between X4 presence and disease stage.
At least one such drug (AMD070) is undergoing clinical evaluation. AMD070 has been tested in a very small group of HIV-infected individuals, and the ACTG is currently sponsoring a larger Phase II trial that has enrolled just four people after more than a year of accruing. Recruitment was recently temporarily stopped due to hepatotoxicity seen in a parallel dog study. Upon announcement of the halting of the ACTG study, Anormed opened a similar study. The FDA is likely to keep a close eye on these studies should they move forward.
For more information about coreceptor inhibitors and other drugs in development, see Rob Camp's 2006 Clinical Pipeline report with links to data.
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