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HIV tropism: diagnostic tools and implications for disease progression and treatment with entry inhibitors
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AIDS: Volume 20(10) 26 June 2006 p 1359-1367
Poveda, Evaa; Briz, Veronicaa; Quinones-Mateu, Miguelb; Soriano, Vincenta
From the aDepartment of Infectious Diseases, Hospital Carlos III, Madrid, Spain
bDepartment of Molecular Genetics, Section Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Introduction
CCR5 and CXCR4 are the major chemokine co-receptors used by HIV to enter into human cells [1-3]. Based on this co-receptor usage, a new HIV classification was established in 1998, i.e., CCR5-tropic (R5), CXCR4-tropic (X4), or dual tropic (R5/X4) HIV-1 strains [4]. Ten years earlier, Tersmette et al. identified a relationship between viral phenotype (i.e., non-syncytium-inducing, NSI or syncytium-inducing, SI) and the virulence of HIV strains [5]. We now know that, in vitro, R5 viruses usually correspond to NSI on T-cell lines and are able to replicate in monocyte-macrophages (M-tropic), all features that previously had been linked to less virulent strains. In contrast, X4 strains are SI on T-cell lines and replicate preferentially on T lymphocytes (T-tropic), all characteristics of more pathogenic virus strains [5-8]. On the basis of these findings, it is clear that HIV co-receptor usage may be associated with disease progression.
Within the last few years, the interest in HIV tropism has resurged mainly due to the appearance of promising new anti-HIV molecules that target CCR5 and CXCR4 receptors [9,10]. Given their mechanism of action, the clinical development of these compounds may require the prior knowledge of the viral tropism in a given HIV-infected individual. Moreover, a close follow up of viral tropism in patients undergoing treatment with these compounds to exclude possible phenotype shifts could be advisable [11,12]. However, clinical determination of HIV-1 co-receptor usage is difficult since only a few methods exist, which are complicated and not widely available. In this review we summarize the molecular basis of viral tropism and its impact on HIV disease progression. Furthermore, we discuss the tools currently available for determining HIV co-receptor usage in the era of HIV co-receptor antagonists.
Definition and molecular basis of HIV tropism
The definition of HIV tropism has experienced an evolution from the late 1980s, when the existence of two distinct HIV phenotypic variants was established [5,13-15]. Originally, the tropism referred to the cytopathic effects of HIV isolates on peripheral blood mononuclear cells (PBMC). Virus isolates causing syncytia (giant multinucleated cells) in cell cultures were described as SI, whereas the rest were named as NSI [5]. Thereafter, HIV isolates were classified on the basis of their replication kinetics on PBMC as slow/low and rapid/high strains [15]. More recently, a new classification has been proposed taking into account the ability of viruses to replicate in two cell lineages, monocyte-derived macrophages and CD4 T-cell lines. Accordingly, viruses are then named as M-tropic or T-tropic, respectively [16-18].
In 1996 HIV was found to require a second co-receptor to enter target cells, in addition to binding the CD4 receptor. The main co-receptors involved in HIV entry are CCR5 and CXCR4 [1-3]. As shown in Fig. 1, the CD4-gp120 attachment provokes conformational changes in the viral envelope allowing the CD4-gp120 complex interaction with CCR5 or CXCR4 [19,20].
CCR5 and CXCR4 belong to the seven-transmembrane G protein-coupled receptor family. They present an ƒ¿-helix structure composed of four transmembrane domains, three extracellular loops and one N-terminal domain [21]. The CD4-gp120 complex binds to co-receptors through the V3 variable domain of gp120, although other gp120 regions such as V1/V2 and C4 are also involved in this interaction. However, the amino acid sequence of V3 seems to be the major determinant of CCR5 or CXCR4 usage [22]. The gp120 co-receptor binding mapping suggests that for R5 viruses, the N-terminal domain and the second extracellular loop (ECL2) are essential for co-receptor recognition and functional activity [23,24], whereas for X4 strains only ECL2 is critical [25].
The close relationship between HIV co-receptor usage and some viral phenotypic features was soon recognized. Strains classified as NSI and M-tropic generally use CCR5 to enter cells, whereas those that use CXCR4 are generally SI and T-tropic [26,27]. These findings have allowed a new definition of viral tropism based on HIV co-receptor usage, which can be split into three categories: CCR5-tropic (R5), CXCR4-tropic (X4) and dual/mixed tropic R5/X4 strains [4].
Dynamics of HIV tropism during disease progression
The pattern and extent of CCR5 and CXCR4 expression on the surface of different human cells may influence the dynamics of viral tropism during the course of HIV infection. R5 variants are generally responsible for the establishment of infection, as they seem to be more efficiently transmitted than X4 variants [28]. X4 variants tend to emerge in later HIV stages and are associated with a more rapid CD4 cell depletion and progression to AIDS [17]. Individuals who are homozygous for the deletion of 32 base pairs within the ccr5 gene (Ģ32-ccr5) seem to be protected from HIV infection, despite multiple exposures [29-32], which indirectly suggests a strong selection for R5 strains during viral transmission episodes and early establishment of HIV infection. At this time, it remains unclear why R5 viruses predominate in the early stages of infection irrespective of whether the route of transmission is sexual, vertical or parenteral [33]. During sexual transmission of HIV, R5 strains could have a preferential advantage due to the exclusively high levels of expression of CCR5 on the genital mucosa [34-36]. However, following parenteral exposure (i.e., in hemophiliacs or injecting drug users), X4 viruses could also be efficiently transmitted but even in those populations, R5 viruses predominate in early disease stages [37].
HIV can infect CCR5-expressing dendritic cells (DC), macrophages and T cells in the underlying mucosal tissues [28,38-40]. DC express CD4, CCR5, DC-SIGN and other C-type lectin receptors that facilitate the capture and infection by HIV and SIV [41,42]. DC capture HIV through C-type lectin receptors and can productively by infected by HIV through a CCR5-dependent mechanism (cis infection). DC can also capture virus and transfer HIV without becoming infected by binding DC-SIGN to carbohydrates on gp120 (trans-transmission model). Productive infection in CD4 T cells is facilitated by DC-T-cell conjugates, which spread infection to more CD4 T lymphocytes [43,44]. The cis and trans pathways of transferring HIV are not mutually exclusive [45].
DC are responsible for the trafficking of HIV particles to the lymph nodes in an attempt to induce an immune response [28]. When viruses reach the lymphoid tissue they provoke activation of CD4 T cells which acquire a memory phenotype (CD4+RO+CCR5+) [46]. The activated memory subset, which expresses higher CCR5 than CXCR4 levels, is the main producer of virus in vivo [40,47,48]. All these facts could explain the preferential selection of R5 viruses following initial exposure, at least through sexual HIV transmission. Finally, the gut-associated lymphoid tissue is also rich in activated memory CD4 T cells, and is the main site of early HIV replication in both humans and macaques [49-51]. This observation could explain the predominant spread of R5 viruses over X4 variants in early stages of HIV infection, irrespective of the transmission route.
As described above, the recognition of X4 viruses at the beginning of HIV infection is rare. However, the progression of HIV-1 infection from asymptomatic stages to AIDS is associated in approximately half of the patients with a switch in viral co-receptor use from CCR5 to CXCR4 tropic isolates [27]. The pathogenicity of X4 HIV strains may be explained by the ability of these isolates to infect thymocytes, the precursor cells of mature CD4 T lymphocytes [52,53]. CXCR4 is highly expressed on all immature thymocytes, in contrast to CCR5 which is expressed at lower level [52]. Whereas R5 viruses may reach the thymus through infected DC and/or mature CD4 T cells without affecting thymopoiesis, X4 viruses may infect immature thymocytes, which express high levels of CXCR4, leading to a rapid depletion of thymus cells. This fact may account for the rapid CD4 T-cell depletion and faster progression to AIDS generally seen in patients with X4 variants [53-56].
For patients developing AIDS in the absence of X4 viruses, it has been proposed that different characteristics between R5 strains isolated from these individuals ('late' R5 viruses) and R5 viruses isolated from asymptomatic subjects ('early' R5 viruses) could be found. Late R5 viruses seem to have intrinsic properties distinguishing them from early R5 viruses, such as enhanced cytopathic effects due to a decreased sensitivity to inhibition by the ƒÀ-chemokine RANTES [57-59] or cause increased levels of CD4 T-cell death [60]. More recently, two different phenotypes of primary R5 viruses have been proposed; late R5 strains seem to have a reduced dependence on CD4/CCR5 levels for their entry into target cells, along with a reduced sensitivity to entry inhibitors TAK-779 and T-20 [61]. In contrast, early R5 strains require comparatively higher levels of CD4/CCR5 for cell entry and are highly sensitive to inhibition by TAK-779 and T-20 [61]. Thus, R5 viruses may evolve during the course of infection toward more efficient CCR5 usage by increasing the affinity of Env for CCR5, perhaps enhancing CD4-CCR5 interactions. By this mechanism, R5 viruses could increase their virulence and limit in some extent their susceptibility to entry inhibitors [61].
Impact of antiretroviral therapy on HIV tropism
The use of HAART has dramatically modified the natural course of HIV infection [62], in such a way that life expectancy for HIV-infected individuals in Western countries could approach that of HIV-negative persons. However, concerns about a potential influence of antiretroviral therapy on the selection of X4 viruses have recently alerted that this hypothetical benefit of HAART could not be indefinite. In a cross-sectional study performed on 28 patients infected with HIV-1 subtype C, the emergence of X4 viruses was associated with the use of antiretroviral treatment [63]. Moreover, in a longitudinal follow up of 32 patients with complete suppression of plasma viremia for longer than 5 years on HAART, a switch from R5 to X4 variants was found in 11 of 23 patients, while X4 variants remained as predominant in the nine patients with R4 viruses at baseline [64]. Interestingly, patients with X4 strains showed lower CD4 cell gains or net CD4 cell declines despite complete suppression of plasma HIV RNA [64]. These results suggest that HAART might facilitate the necessary conditions for the gradual emergence of X4 variants.
X4 variants seem to show higher replication capacity compared to R5 virus strains [65]. The preferential replication of X4 viruses in the microenvironment of sites where residual virus production takes place and their selection in thymocytes or naive CD4 T cells could explain the emergence of X4 variants in cellular reservoirs in patients on HAART. Another reason why a shift from R5 to X4 might occur during HAART could be related to peripheral expansion of long-lived (naive CD45RA+) T cells in which archival X4 variants were present [64]. Subsequent spreading of X4 viruses and infection of thymocytes or naive CD4 T cells could then compromise the extent of immune reconstitution while taking HAART.
It is important to note that, contrary to reports of a preferential shift from R5 to X4 viruses following the use of antiretroviral therapy, others studies have shown that HAART might delay selection of X4 viruses. In a study conducted in 32 patients, 24 of whom harboured exclusively R5 viruses before the initiation of antiretroviral therapy and eight had dual/mixed (R5/X4) virus populations, no change in viral tropism was noted in subjects with R5 viruses but in half of those with R5/X4 strains, in whom the X4 component of the virus population became undetectable [66]. However, it should be noted that suppression of the X4 component was only transient and all patients subsequently showed a reversion to a dual/mixed X4/R5 HIV population. In another study conducted in 20 antiretroviral-experienced HIV-infected children who initiated a salvage regimen based on lopinavir-ritonavir, virological responses were accompanied by a shift in virus co-receptor use. X4 viruses were preferentially suppressed, suggesting that a switch in co-receptor use could thereby contribute to the clinical efficacy of some anti-HIV drugs [67]. Hypothetically, R5 viruses might have an advantage to replicate when plasma viral load is completely suppressed under HAART. In this situation, residual viral replication taken place in some tissue compartments (such as the central nervous system) having low permeability to some drugs (such as protease inhibitors), and latent reservoirs such as CD4 memory T cells, could be favoured as these type of cells express CCR5 at high levels [66].
Implications of viral tropism in the era CCR5/CXCR4 co-receptor antagonists
In spite of a well-established relationship between co-receptor use and HIV disease progression, viral tropism is not generally determined in the clinical follow up of HIV-infected patients. Plasma viremia and CD4 cell counts continue to be the main parameters on the basis of which prognosis and therapeutic decisions are taken [68]. However, the current development of CCR5 and CXCR4 antagonists might force modification of this paradigm. The inclusion of these compounds into the therapeutic armamentarium may require prior knowledge of the HIV co-receptor use for each patient, and periodic monitoring of co-receptor usage could be similarly required in patients treated with these drugs. Thus, knowledge of the HIV co-receptor usage will soon be available for an increasing number of patients and therefore could be considered for prognostic purposes in larger populations. This information might eventually influence therapeutic decisions.
Given their mechanism of action, the benefit of co-receptor antagonists will be limited to a subset of HIV-infected patients, those harbouring virus populations using either CCR5 or CXCR4 co-receptors. At this time it is unclear whether indirect effects may be produced following the inhibition of such co-receptors. For example, the CCR5 chemokine receptor seems to play a role in hepatitis C virus (HCV) infection. CCR5 density in the T-cell surface might determine the intensity of T-cell recruitment in the HCV-infected liver, which could influence the extent of liver damage. While some authors have found a relationship between low CCR5 levels and better outcome of HCV infection (higher viral clearance and reduced liver inflammation) [69,70] others have not confirmed these findings [71]. Thus, it is warranted to assess carefully hepatic disease in HCV/HIV-coinfected patients treated with CCR5 antagonists.
There are several co-receptor antagonists currently in clinical development, some of them at the latest stages awaiting the results of phase III clinical trials [72]. Two CCR5 antagonists, Maraviroc and Vicriviroc (SCH-D) will probable be the first drugs of this new family to be approved for clinical use. Even though a phase II study with Vicriviroc in treatment-naive HIV-infected patients was recently discontinued due to the relatively poor antiviral activity of the drug with respect to a comparative arm, studies in treatment-experienced patients are ongoing. Furthermore, a promising pharmacokinetic interaction of Vicriviroc with ritonavir may increase its overall performance [73]. Aplaviroc, which also belongs to this family of HIV entry inhibitors, was recently dismissed following the report of several cases of liver toxicity in clinical trials [74]. Finally, among CXCR4 antagonists, AMD070 is currently the unique molecule in clinical development as treatment of HIV infection. Phase Ib/IIa trials are ongoing. Preliminary data with this oral drug show that it is well tolerated [72].
For other antiretroviral drugs side effects and resistance profile are the key elements determining further clinical development. Two main resistance pathways have been proposed to explain HIV escape from CCR5 and CXCR4 antagonists. Firstly, a shift in co-receptor usage. Secondly, changes in the HIV gp120 protein due to mutations mainly in the V3 loop may permit the interaction of HIV gp120 with its co-receptor despite the presence of the inhibitor. A switch in co-receptor usage does not seem to be the main mechanism of resistance to evade the antiviral activity of CCR5 antagonists. In contrast, changes in different regions of gp120 (V3, C2, V2, C4) seem to be the major factor responsible for the selection of resistance to co-receptor antagonists [75-77]. However, care is required in the clinical management of individuals harbouring mixed R5/X4 virus populations. In a phase II clinical study with Maraviroc, one patient infected with an R5/X4 mixed virus population was inadvertently enrolled, and selection of X4 strains occurred during treatment, whereas R5 strains resurged following Maraviroc discontinuation [11]. Likewise, another patient enrolled in a clinical trial with Aplaviroc experienced a shift from R5 to R5/X4 on day 10 of treatment. Pre-existence of a minor population of X4 strains at baseline could be demonstrated, ruling out a possible de novo selection of this variant during treatment with the CCR5 antagonist [12].
It is a general belief that a shift from viruses using CCR5 to CXCR4 while being treated with CCR5 antagonists could have dreadful consequences on HIV disease progression. An expansion of X4 viruses could be favoured, leading to a rapid depletion of immature thymocytes, followed by a drop in CD4 cell counts and faster progression to AIDS. On the contrary, using CXCR4 antagonists, any shift from X4 to R5 viruses could be hypothetically beneficial, given that R5 viruses tend to be associated with lower virulence. Although all these issues need to be solved and hopefully will soon be answered upon completion of the currently ongoing clinical trials, it is clear that if co-receptor antagonists finally become a part of the anti-HIV armamentarium, it will be critical to have at one's disposal laboratory methods to easily determine co-receptor HIV tropism.
Tools for determining HIV tropism
Several assays have been developed to determine HIV tropism (see Table 1). At this time it remains unclear which are the most convenient and/or whether new methods are still needed. The MT-2 assay was widely used during the late 1980s to test the cytopathic effect of HIV isolates and served to establish the classification of HIV strains into SI and NSI viruses. The MT-2 cell assay is based on the unique expression of CXCR4 but not CCR5 on the surface of those cells. Thus, any evidence of HIV replication on culture of MT-2 cells indirectly reflects the presence of X4 viruses. The main disadvantage of the MT-2 assay is the need for viral stocks from stimulated patient PBMC [78]. The adequate collection of virus isolates is very laborious and requires highly qualified personnel, which limits the introduction of this assay in clinical facilities complementing the routine clinical practice. On the other hand, HIV isolates derived from stimulated patient PBMC might differ somewhat from plasma circulating viruses even in the same person. Thus the MT-2 assay may not be the most appropriate for use in patients being treated with co-receptor antagonists. This method may be more useful for obtaining prior knowledge of preferential co-receptor usage in viruses from patients before starting therapy with these compounds, especially with CCR5 antagonists, in order to exclude the presence of X4 viruses. These variants could be present at cellular reservoirs and being missed by testing plasma, a limitation that probably also applies to other methods based on viral isolates derived from PBMC.
Several cell lines that express defined sets of receptors on their surface are currently been used to determine HIV co-receptor use. GHOST cells derived from a human osteosarcoma cell line express CD4 and various chemokine receptors on their surface [79]. Likewise, U87, a glioma malignant cell line that has been engineered to express CD4 and various chemokine co-receptors on the cell surface, is currently one of the most used to determine HIV tropism [80]. U87 cells have several advantages over other target cell types (as GHOST cells), such as their ability to growth over solid plates, which is preferable to suspension cultures for most detection methods. Furthermore, U87 cells support robust replication of HIV while maintaining stable high levels of expression of distinct surface receptors, a circumstance that tends to vanish using other cell types [81]. Among the disadvantages of the use of cell lines to determine viral tropism one is of particular relevance. The levels of receptor expression may by far exceed those found on natural target cells for HIV, which may preclude obtaining results that may directly reflect what happens under real conditions [81].
As described above, HIV entry inhibitors are one of the latest generations of antiretroviral drugs explored for the treatment of HIV infection. Their clinical development has pushed the design of tests able to assess viral tropism and/or measure resistance. At this time, there are at least two commercially available recombinant assays that allow determining at the same time viral susceptibility to HIV entry inhibitors and viral tropism: Phenoscript (VIRalliance, Paris, France) [82-84] and PhenoSense (Monogram Biosciences, San Francisco, California, USA) [85-87]. Both assays amplify the HIV-1 envelope glycoprotein gene sequence from patient's plasma samples to produce either replication-competent or replication-defective recombinant viruses, respectively. These viruses are then used to infect cell lines that express CD4 in combination with either CCR5 or CXCR4 co-receptors, which permits determination of viral tropism. When viral production is recognized in both CCR5- and CXCR4-expressing cells, it generally means that a mixture of R5 and X4 viruses is present in a given plasma specimen, although eventually a unique dual tropic R5/X4 virus, as already demonstrated by clonal analyses, may also account for this result [36].
The use of recombinant virus tropism assays, in the way described earlier, has made large scale studies possible, a goal that would not be affordable using classical tropism cell culture methods based on viral isolates derived from patient PBMC. However, a major limitation of recombinant virus assays is the threshold for detection of minority quasispecies in the presence of mixed viral populations. For instance, the limit of detection for minor populations is 10% for R5 viruses and 20% for X4 viruses using the Phenoscript assay [82]. This limitation might have important implications in patients undergoing treatment with CCR5 antagonists, in whom emergence of X4 viruses present as a minor population at baseline could be favoured. This was the case in at least two patients enrolled in studies with Aplaviroc and Maraviroc, as reported previously [11,12].
The molecular basis of HIV tropism is still under investigation. However, current evidence suggests that is determined mainly by the amino acid sequence of the V3 loop of the HIV gp120 envelope protein. Over the last few years, efforts have been made to identify which residues within the V3 domain are particularly involved in determining viral co-receptor usage [88]. Although no single changes seem to be responsible for tropism behaviour, several clusters of genotypes largely determine viral tropism. Several algorithms have been produced in an attempt to predict HIV co-receptor usage based on the V3 genetic sequence. Some of them are available in websites of public access [89-91] (Table 2). Although the predictive value of these algorithms is still limited, it is improving continuously based on the information derived from paired genotypes/phenotypes. It is expected that with further improvements, the information on viral tropism derived from genotype-based assays will soon be available for more comprehensive clinical studies. In that way, HIV tropism could be part of the clinical assessment of HIV-infected patients and help to tailor treatment decisions at an individual level.
Conclusions
Viral tropism is an important characteristic of HIV variants as it seems to independently influence disease progression. Moreover, the use of current antiretroviral therapy might impact on viral tropism in a way that is still poorly understood. With the eventual introduction of co-receptor antagonists in the HIV armamentarium, there is an urgent need for rapid and reliable tools for determining viral tropism. Only in this way, will viral tropism information be introduced into routine clinical practice.
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