The chemokine receptors CCR5 and CXCR4 are the principal coreceptors for entry of HIV-1 into target cells.1,2 Viruses have the ability to use CCR5 (R5) or CXCR4 (X4) coreceptors either solely or in combination (R5X4).3 Most HIV strains isolated from individuals with HIV encephalitis and AIDS dementia are R5-tropic viruses, which is consistent with the central role of macrophage in brain infection.4-6 Most HIV strains infecting macrophages use R5 coreceptors.7,8 X4-using viruses mainly infect T lymphocytes, although some X4-tropic isolates could also infect macrophages and microglia.9,10
HIV encephalitis in patients infected with viruses resistant to nucleoside reverse transcriptase inhibitor, nonnucleoside reverse transcriptase inhibitor, and/or protease inhibitor could be a complication of HIV infection11-13 and required a novel therapeutic class of treatment. The CCR5 antagonists are specifically active against HIV-1 R5-tropic strains,2,14-16 such as maraviroc and vicriviroc, block HIV entry into the cell, and may be considered as an eventual therapeutic option in these cases.
The objectives of this study were to investigate the predicted tropism of viruses present in cerebrospinal fluid (CSF) and plasma in a group of 22 HIV-1-infected patients with neurological disorders and to search for eventual discordance of virus predicted tropism between both compartments.
The neurological disorders of the 22 patients studied were as follows: 16 HIV encephalitis, 4 bacterial meningitis, 1 neurosyphilis, and 1 cerebral toxoplasmosis (Table 1). The HIV-1-associated encephalitis was considered as a diagnosis of exclusion based on clinical symptoms (cognitive behavioral troubles and minor psychomotor slowing), CSF analysis (mild lymphocytic pleocytosis, elevated protein, and detectable HIV-1 viral load), and computed tomography and/or magnetic resonance imaging. Six patients (27.2%) were not treated by antiretroviral treatment at the time of neurological disorders. The median age was 45 years and men were predominant (63.6%). At the time of neurological disorders, blood and CSF samples were collected to explore etiology of these disorders.
In this context, HIV-1 viral load, bulk genotypic resistance tests, and predicted tropism were determined on paired CSF and plasma samples. V3 env was amplified and bulk sequenced, and HIV-1 coreceptor usage was determined from the V3 env region sequence by Geno2Pheno (http://coreceptor.bioinf.mpi-sb.mpg.de/cgi-bin/coreceptor.pl) and PSSM (http://ubik.microbiol.washington.edu/computing/pssm/) algorithms for B and non-B subtypes as previously described.17-19 Viruses were classified in 2 categories: lack (R5) or presence of X4-tropic viruses (R5X4/X4). Then, mixed/dual-tropic or X4-tropic viruses were grouped together. HIV-1 plasma and CSF viral load was performed using the Roche Amplicor test kit version 1.5 (Roche Diagnostics, Meylan, France). The reverse transcriptase (RT) and protease genes were analyzed by population sequencing according to the Agence Nationale de Recherche sur le SIDA (ANRS) consensus protocol (http://www.hivfrenchresistance.org). The genotypic susceptibility score (GSS) was calculated according to the 2006 ANRS AC-11 genotype interpretation algorithm (http://www.hivfrenchresistance.org): 0 or 1 if resistant or susceptible to the drugs. HIV-1 subtyping was performed by phylogenetic analysis of RT and protease region sequences. The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers for the RT, protease, and gp120 sequences are FJ549947-FJ550075. Comparisons between groups were performed by using the nonparametric Mann-Whitney test or χ2 test. Statview software v5.0 was used.
Concordant predicted tropism was mainly observed in CSF and plasma compartments, corresponding to 17 of 22 cases for R5 tropism and 2 of 22 cases for R5X4/X4 tropism. A discordant predicted tropism was observed for 3 pairs: viruses had R5 tropism in CSF despite having R5X4/X4 tropism in plasma (Table 1). R5X4/X4-tropic viruses in CSF were never observed with R5-tropic viruses in plasma. The prevalence of R5X4/X4-tropic viruses was 22% in this group of 22 patients.
The HIV RNA levels were similar in CSF and plasma (median 4.1 log10 copies/mL in both compartments). The median CD4 T-cell count was 239 cells per cubic millimeter (range, 13-969), and the median CD4 nadir was 102 cells per cubic millimeter (range, 1-969) (Table 1). No correlation was observed between CD4 cell count and virus predicted tropism in CSF or plasma. There was also no correlation between virus predicted tropism and HIV-1 viral load in plasma and CSF. The CD4 nadir and virus predicted tropism in plasma tended to be linked (P = 0.07).
Of the 22 cases, non-B and B subtypes were represented in 7 (31.8%) and 13 (59.1%), respectively, and were concordant in CSF and plasma for all patients with successful amplification. Two viruses are complex mosaic, and the determination of subtypes was not possible (9.1%).
The medians of GSS were similar between CSF and plasma for nucleoside reverse transcriptase inhibitor, nonnucleoside reverse transcriptase inhibitor, and protease inhibitor (median 6, 3, and 8, respectively) (Table 2). Overall, the GSS was similar in plasma and CSF, but minor differences have been found for 3 patients. In addition, the genotypic resistance profiles have been found different for several patients (including the 3 last patients) according to the antiretroviral classes (Table 2).
The tropism of viruses present in CSF and plasma in a group of 22 HIV-1-infected patients with neurological disorders was investigated by genotypic approach to search for eventual discordance of virus predicted tropism between both compartments. The majority of subjects (19 of 22) had then concordant virus predicted tropism in both compartments. All patients having R5-tropic viruses in plasma had R5-tropic viruses in CSF. Patients having R5X4/X4-using viruses in plasma could have R5X4/X4-tropic (2 of 22) or R5-using viruses (3 of 22) in CSF. The case of R5-tropic viruses in plasma and R5X4/X4-using viruses in CSF was never observed in this study. Discordant predicted coreceptor use (3 of 22 R5X4 in plasma and R5 in CSF) could indicate a compartmentalization of CSF infection, as previously suggested by a study showing distinct env sequences in CSF and plasma.20 Others studies have also shown distinct resistance patterns in CSF and plasma, arguing for the possibility of compartmentalization of HIV infection.21-23
In the present study, the R5-tropic viruses (17 plasma samples and 20 CSF samples) were more common than R5X4/X4-using viruses (5 plasma samples and 2 CSF samples), as previously described by Spudich et al who conducted a similar study in a different population of patients mainly asymptomatic, but with similar median CD4 T-cell count (244.5 cells/mm3). The high prevalence of R5-tropic predicted strains in CSF reflected more efficient replication in target cells, such as macrophages and microglial cells, and argued in favor of the important role of R5 receptor in HIV neuropathogenesis as previously demonstrated.7,8,24 The prevalence of R5X4/X4-using viruses was relatively low (22%), but it seems to be related to the nadir of CD4 (P = 0.07). The possible link between tropism of viruses and nadir CD4+ T-cell count was previously demonstrated. Indeed, enrichment for dual/mixed/X4-tropic viruses among treated patients was largely but incompletely explained by lower pretreatment nadir CD4+ T-cell counts in 2 cohorts.25 Furthermore, patients with R5X4/X4-tropic viruses have significantly lower median baseline CD4+ cell count than patients having R5 virus in ACTG5211.26
One explanation of the relatively low prevalence of R5X4/X4-tropic viruses in this population could be related to the genotypic method used. However, a good correlation between genotypes and phenotypes in a set of 118 V1-V3 molecular clones was demonstrated27 and was confirmed in an independent cohort of 103 patients where V1-V3 gp120 region was bulk sequenced.19 These studies argue that V3 sequencing could represent an alternative to phenotypic assays for assessing HIV-1 tropism. This approach could be used for predicting coreceptor use from B and non-B env genotypes; however, this type of application to non-B subtype virus needs further validation because of little available data on prediction algorithms for non-B subtype virus at present time.
It will be important to determine tropism in the perspective of use of CCR5 antagonists in case of HIV encephalitis to limit the viral replication in central nervous system as it was shown that vicriviroc and maraviroc penetrated in central nervous system.28,29 This CCR5 antagonist use could be envisaged in an optimized regimen or in addition to the therapy where neurological dysfunction remains, although the virus is controlled in plasma. Then, the tropism could be determined in CSF or DNA from peripheral blood mononuclear cells.
In conclusion, the majority of these 22 patients have R5-tropic viruses in CSF (20 of 22) that is concordant with the predicted tropism in plasma. However, R5X4/X4 tropism in plasma does not necessarily mean the same predicted tropism in CSF compartment. Then, clinical therapeutic trials testing the clinical response to CCR5 antagonists in patients with neurological disorders could be envisaged to analyze the effects of this therapeutic class in case of R5 tropism in CSF whatever the tropism in plasma.
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