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Department of Neurology, University of CaliforniaSan Francisco
Gladstone Institute of Virology and Immunology, San Francisco
ViroLogic, South San Francisco, California
Background.
Chemokine receptors serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, influence cell tropism, and may critically determine central nervous system infection pathogenesis. Using an in vitro functional entry assay, we examined utilization of 2 principal coreceptors in cerebrospinal fluid (CSF) and plasma in 46 subjects.
Methods.
Paired CSF and plasma samples were selected from subjects with a range of CD4 T cell counts. Amplified populations of env sequences were characterized as using CCR5 (R5), CXCR4 (X4), or both receptors (R5+X4). Individual clones derived from 3 subjects were analyzed for viral tropism and phylogeny.
Results.
CSF and plasma pairs were mainly concordant for R5 (36/46) or R5+X4 (5/46) viruses. However, 5 pairs were discordant, 2 of which had the R5+X4 phenotype in CSF despite having the R5 phenotype in plasma. Although R5+X4 tropism was associated with advanced immunodeficiency, all 4 subjects with acquired immunodeficiency syndrome dementia complex had R5 tropism in CSF. Clones derived from R5+X4tropic populations revealed mixtures of R5 and X4 viruses and viruses able to utilize either coreceptor, suggesting both virus exchange between compartments and autonomous CSF virus evolution.
Conclusions.
Although R5 viruses predominate in the CSF, HIV-1 populations able to utilize CXCR4 are also present. Discordant tropism in CSF and plasma may have implications for R5 inhibitor therapy.
CCR5 and CXCR4 are G-proteincoupled transmembrane receptors that serve as the obligate coreceptors for cell HIV-1 [1, 2]. After binding to CD4, the V3 loop region of the gp120 HIV envelope glycoprotein binds to one of these coreceptors before subsequent steps of membrane fusion and capsid entry [36]. Preference for one or the other of these receptors contributes to cell tropism. Most HIV strains that infect macrophages utilize CCR5 (R5 viruses) [7, 8]. CXCR4-utilizing strains (X4 viruses) mainly infect lymphocytic cell lines, although some X4 isolates have been shown to infect macrophages and microglia [9, 10]. In general, R5 viruses predominate during early infection. X4 viruses are most often detected later in the course of infection and may be associated with more-rapid CD4 T cell loss, although it is unknown whether this virus subtype is the cause or the result of a change in the course of T cell attrition [11, 12]. The role of these coreceptors is now of additional interest, since small-molecule drugs are being introduced that block these receptors and interfere with infection [2, 13].
Coreceptor utilization is likely to be important in central nervous system (CNS) HIV infection and related neurological morbidity. Most HIV strains isolated from individuals with HIV encephalitis and AIDS dementia complex (ADC) are R5 viruses [1417], consistent with the central role of macrophage infection in brain infection [1821]. Furthermore, coreceptor binding may mediate brain injury, on the basis of observations that (1) chemokine receptors expressed on neural cells can serve as targets for pathogenic signaling by HIV gp120 [22], (2) the activation of CXCR4 by its native ligand affects neuronal migration in the developing brain [2325], and (3) gp120 is toxic to neurons in culture via the CXCR4 receptor [26]. However, the influence of gp120 on neural chemokine receptors in vivo remains unclear [27].
Because most previous studies of CNS coreceptor usage have involved only case reports or small clusters of cases, we undertook this study to survey concurrent coreceptor phenotypes in cerebrospinal fluid (CSF) and plasma in a large sample of carefully selected patients. Our primary objective was to establish studies of coreceptor tropism in CSF, to explore an association between chemokine receptor tropism and the presence of neurological disease. Furthermore, since results of prior studies have suggested that coreceptor tropism might differ between the blood and CNS compartments [28, 29], we sought to directly study the relationship between coreceptor utilization in CSF and plasma in a large cohort. Finally, we analyzed cloned viruses from each compartment in 3 subjects, to investigate the mechanisms of dual versus mixed coreceptor tropism. The availability of a novel high-throughput phenotypic assay allowed us to address these issues in a relatively large sample of subjects.
SUBJECTS, MATERIALS, AND METHODS
Subjects and protocols.
Paired CSF and plasma samples were derived from participants in 2 longitudinal cohort studies approved by the University of CaliforniaSan Francisco Committee on Human Research. Specimens were selected for this study if they had minimum HIV RNA levels required to permit env gene amplification (1000 cps/mL). Informed consent was obtained from all subjects (and from the durable power of attorney of an individual with stage 2 ADC). Subjects with active CNS diseases other than ADC were excluded.
CSF analysis.
All CSF was obtained via lumbar puncture, for study purposes rather than for clinical diagnosis. CSF analysis, including determination of cell counts, cell differentials, total protein levels, and albumin levels, was performed by the San Francisco General Hospital (SFGH) Clinical Laboratories, and the remaining fluid was processed as described elsewhere [30]. CSF for virological studies was centrifuged at 1200 g for 10 min to remove cells, pooled, and stored at -80°C in individual aliquots. Blood was collected for evaluation of albumin levels and CD4 and CD8 T cell counts by flow cytometry; plasma was separated and stored for viral assays conducted in parallel with assays of CSF.
Clinical evaluation.
All subjects underwent general medical and neurological assessments. A standardized, ADC-focused neurological exam was used for ADC diagnosis and staging [31, 32]. Diagnosis of ADC conformed to criteria for the AIDS-related cognitive/motor complex outlined by the American Academy of Neurology Task Force [33]. No ADC stage was assigned for 1 subject who was excessively sedated with methadone at the time of evaluation.
General virological methods.
HIV-1 RNA levels were measured in cell-free CSF and plasma by the Roche Amplicor HIV-1 Monitor assay (versions 1.0 and 1.5; Roche Diagnostic Systems) by use of standard and ultrasensitive extraction methods, as appropriate, according to the manufacturer's guidelines. HIV-1 RNA concentrations were transformed to log10 values for all calculations.
Clonal analysis.
Clonal analysis of HIV envelope sequences was performed on the paired plasma and CSF specimens from 3 individuals, selected from our sample group on the basis of R5+X4 viruses being predominant in either CSF, plasma, or both fluids. Individual envelope-expression vector clones were derived from the pools, and their tropism was tested using the assay described above. Individual clones that were identified as utilizing both CCR5 and CXCR4 were designated as true R5X4 clones. Full-length gp160 open reading frames were also sequenced, for phylogenetic analysis and to assure clonality.
Statistical and phylogenetic analysis.
For descriptive and exploratory statistics, we used nonparametric comparisons (SSPS, version 11.0). CLUSTAL W was employed [37] to perform multiple sequence alignments of full-length gp160 nucleotide clonal sequence from 3 subjects and reference strains HXB2 and JRCSF. A neighbor-joining phylogenetic tree was generated using 100 bootstrap replicates, with branch lengths proportional to the estimated divergence along each branch.
RESULTS
Study subject demographics and background HIV-related characteristics.
A total of 46 HIV-infected subjects were included in this study (table 1). Thirty-three neurologically asymptomatic subjects were chosen from an ongoing longitudinal cohort study (Sentinel Neurological Cohort), and 13 subjects were selected from a study of the effects of antiretroviral therapy on CSF infection. Included within this second group were 4 subjects who presented with newly diagnosed, active ADC (1 with stage 1, 2 with stage 2, and 1 with stage 3). Reflecting subject selection based on viremia, the median HIV RNA levels were 4.77 log10 copies/mL in plasma and 3.84 log10 copies/mL in CSF. The median blood CD4 T cell count was 244.5 cells/mm3 (intraquartile range, 129.3326.0; range, 5612). The median CSF : plasma albumin ratios [38] and CSF white blood cell (WBC) counts were at the upper limit of normal. The median CSF red blood cell (RBC) count was 2 cells/L; none of the samples were xanthochromic, and only 4 samples had RBC counts >50 cells/L in the initial sampling tube (range, 51340 cells/L). Thus, the contribution of blood HIV contamination to the CSF results was considered negligible. Approximately 20% of subjects were receiving combination antiretroviral therapy; all of these subjects had persistent viremia. The group was representative of our stable cohort of long-term study subjects with respect to range of immunosuppression, HIV risk factors, median age (42 years), and predominance of men (94%).
X4 RLU values in plasma were inversely correlated with CD4 T cell counts (Spearman's , -0.519; P = .0002). There was no consistent relationship between X4 RLU values and either plasma or CSF HIV RNA levels. In contrast, R5 RLU values were weakly positively correlated with CSF HIV RNA levels (Spearman's , 0.345; P = .019) but were not associated with either blood CD4 T cell counts or plasma HIV RNA levels.
Differential coreceptor utilization in CSF and plasma.
Most of the sample pairs were concordant with respect to chemokine receptor utilization: 36 of 46 matched samples exhibited the R5 phenotype in both plasma and CSF, whereas 5 had the R5+X4 phenotype in both tissues. Five paired samples were discordant, 3 of which showed the R5+X4 phenotype in plasma but the R5 phenotype in CSF, whereas 2 showed the R5 phenotype in plasma but the R5+X4 phenotype in CSF. Thus, 2 of the 46 subjects had X4 viruses in CSF but not in plasma.
The phylogenetic analysis indicated both segregation and mixture between CSF and plasma clones. In subject 7047, with overall concordant R5+X4 activity, distantly related clusters of R5 clones in CSF and R5X4 clones in plasma were found that were not present in the other fluid. In subject 7069, the discordance between plasma and CSF resulted chiefly from a related group of R5X4 and X4 clones (14, 35, and 11) in CSF with relatively high X4 activity, which suggested evolution independent of the plasma compartment. However, there was also intermixing of R5 clones from CSF and plasma. In subject 7120, the 1 R5X4 plasma clone 23 was most closely related to a group of CSF X4 clones, showing that this minority plasma virus shared a source with the group of CSF X4 viruses. Similarly, the R5 plasma clone 07 was closely related to the R5 CSF clone 15. Thus, in each of these 3 cases, there was the suggestion of both exchange with closely related, functionally similar clones in both fluids as well as elements of segregation with grouped clones clustering in 1 or the other compartment.
DISCUSSION
In this cross-sectional study of CSF and blood from 46 HIV-infected subjects, most CSF HIV populations utilized CCR5 as the principal coreceptor, and the majority of subjects had concordant tropism in the 2 compartments. However, approximately one-fifth of subjects displayed the R5+X4 phenotype in 1 or both fluids, and one-tenth of subjects had discordant plasma and CSF tropism. Our detailed analysis of tropism and phylogenetic relationships among clones picked from paired specimens of plasma and CSF provides evidence of extensive, subtle discordance between and variation within compartments. The present study provides a more detailed view of relationships in this important functional property between HIV species in the CSF and plasma than that previously available. Our results have implications not only for better understanding of viral compartmentalization and neuropathogenesis but also for optimizing future therapy with chemokine-receptor entry inhibitors.
A principal rationale for undertaking this study was to provide a profile of chemokine receptor utilization in the CSF as a background for further inquiry into CSF HIV infection and, perhaps, into the neurotoxicity of HIV. Although the CSF cannot be considered a compartment identical to brain tissue, it can be more readily sampled and is directly related to the brain parenchyma via the choroid plexus and perivascular spaces [39]. Most HIV isolates from patients with ADC have been reported to utilize CCR5, including the "classic" isolates JRFL, isolated from brain [14]; YU-2, directly cloned from brain [40]; and others [41]. The 4 subjects with ADC in our study, including 1 discordant subject with the R5+X4 plasma virus phenotype and greater X4 than R5 activity in the tropism assay, all had R5 tropism in the CSF. This finding may argue against the potential importance of the CXCR4 receptor in HIV neuropathogenesis. However, our clonal analysis indicates that, despite the tropism phenotype in a pooled sample, minority subspecies may be present. It is possible that a limited amount of X4 or R5X4 virus may stimulate the CXCR4 receptor and influence neuronal function and survival, even when R5 viruses predominate. Evidence that some brain-derived CXCR4-utilizing HIV strains in subjects with ADC are highly macrophage tropic [9] supports the importance of macrophages and, perhaps, of microglia in sustaining HIV encephalitis, the common pathological substrate of ADC [42]. Further studies of coreceptor phenotypes are necessary to determine whether macrophage tropism or coreceptor utilization is a more critical determinant of neurological injury.
One of the central questions regarding HIV infection of the CNS is the extent to which it is compartmentalized or isolated from the infection detected in blood samples [43]. HIV in the CSF may derive from both blood (via the choroid plexus and meninges) and the brain. The term "transitory" infection refers to blood-derived HIV of recent origin and with rapid exchange, presumably carried within trafficking T lymphocytes, whereas "autonomous" infection refers to infection supported in brain or meningeal macrophages [44]. In the 46 subjects in our study, infection was either concordant between CSF and blood or discordant with respect to coreceptor utilization. Concordant pooled specimens do not distinguish between transitory infection, with identical CSF and blood viruses, and autonomous infection, with common phenotypic selection. Discordant coreceptor phenotypes indicate compartmentalization of CSF infection.
Earlier studies by Di Stefano et al. used cytopathological characterization of MT-2 cells as an index of tropism to evaluate blood and CSF isolates [29]. Before chemokine receptor utilization had been clearly characterized, HIV isolates that induced fusion of this T cell line in culture were referred to as "syncytia-inducing" (SI), and this cultural phenotype was noted most commonly in advanced systemic HIV infection. Those strains not causing this pathologythat is, non-SI (NSI) strainswere detected in early infection. The cytopathological studies thus described the switch from R5 to X4 viruses that is now more clearly understood [45]. Using this MT-2 assay, Di Stefano et al. found 68% SI strains in blood and 77% NSI strains in CSF. They also noted viral phenotypic discordance between the compartments, with NSI strains in CSF and SI strains in blood simultaneously. Our findings complement but are distinct from theirs, in terms of (1) study populations (theirs had a low median CD4 T cell count of 30 cells/L and many neurologically ill subjects); (2) the fact that the MT-2 cytopathologic assay may overemphasize minor populations of X4 virus, which might expand during culture; and (3) the more general selection of strains by culture amplification in their study, compared with direct gene amplification in ours. The current assay likely provides a more representative view of the predominating virus populations in the samples. Brew et al. reported results similar to those of Di Stefano et al., by assessment of cytopathological effect and tropism [28]. Observations of phenotypic compartmentalization are also consistent with the CSF : plasma discordance noted in more-extensive analysis of env sequences [46, 47] or pol resistance mutations [4853] to define virus genotypes in the 2 compartments. We observed discordance in both directions, including 2 examples of the R5 phenotype in plasma and the R5+X4 phenotype in CSF. This latter finding is striking, given the overall frequency of the R5 phenotype in CSF and the concept that "autonomous" infection in the CSF is more likely sustained in macrophages rather than lymphocytes [54, 55].
The detailed clonal analysis of paired plasma and CSF from 3 subjects adds additional depth to the understanding of compartmentalization of this functional characteristic. HIV infection involves a variable and evolving quasispecies displaying diversity in coreceptor utilization, and there is both exchange between blood and CSF and local segregation of this characteristic. Clonal analysis demonstrated the representative value of the population assay as performed on the CSF and plasma specimens. However, an R5+X4 phenotype in the native specimen pool could indicate the presence of true R5X4 viruses or a mixture of pure R5 and X4 viruses. The analysis also showed that a small minority population might not be appreciated from the pooled specimen. Closely related sequences with R5, R5X4, or X4 activity should also provide useful material for assessing structure-activity relationships of binding to the 2 receptors. Although limited to 3 subjects, our phylogenetic analysis supports and expands upon the phenotypic heterogeneity of viral clones. Since the CSF strains did not entirely cluster together, our findings suggest multiple exchanges or trafficking episodes into the CNS, rather than a single viral trafficking event leading to a founder effect in viral evolution. Extension of this approach to longitudinal sampling should more fully address the extent to which some viral genotypes replicate independently while others exchange between compartments. In addition, future clonal analyses in subjects with ADC will explore the possibility that these individuals harbor more compartmentalizedand, thus, likely autonomousCSF infection.
Finally, the segregation of coreceptor tropism across the blood-brain barrier may have implications for treatment of HIV by use of chemokine entry inhibitors [2, 5658]. Screening of viral tropism in plasma alone may be inadequate for determination of optimal therapy with entry inhibitors. For example, 2 subjects in whom we detected R5 virus in plasma but R5+X4 virus in CSF might develop viral escape in CSF in response to R5 inhibitor therapy. Alternatively, 1 of our subjects with ADC might neurologically benefit from R5 inhibitor therapy despite R5+X4 tropism in his plasma, given R5 tropism in his CSF. Other factors may be important in the success of these inhibitors [12]. Assessment of coreceptor utilization in CSF and other tissue compartments will contribute to understanding the uses and shortcomings of this mode of therapy during clinical trials.
CONCLUSIONS
The coreceptor tropism assay provides a powerful, high-throughput assay for assessing HIV tropism. Further use of the tropism assay and clonal analysis of CSF and plasma from subjects with ADC will help define the role of coreceptor utilization in the development of neurological morbidity. Longitudinal studies employing these methods, by defining changing populations over time, should provide information on viral evolution and on the balance between viral exchange and autonomous replication in CSF and blood. The additional functional information provided by the tropism assay in pooled and cloned specimens enhances the picture provided by sequence data alone. This approach should facilitate further understanding of the evolution, selection, and clinical importance of changes in chemokine coreceptor utilization by HIV in the nervous system.
Acknowledgments
We thank Steven Deeks for helpful critique of this article. We also thank the subjects who volunteered for these studies, as well as the staffs of the San Francisco General Hospital (SFGH)/University of CaliforniaSan Francisco (UCSF) General Clinical Research Center and UCSF/SFGH Core Virology Laboratory, for their invaluable help.
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