Cross-sectional characterization of HIV-1 env compartmentalization in cerebrospinal fluid over the full disease course

Abstract

Objectives: To characterize HIV-1 env compartmentalization between cerebrospinal fluid (CSF) and peripheral blood plasma over all stages of the HIV-1 disease course, and to determine the relationship between the extent of CSF HIV-1 env compartmentalization and clinical neurologic disease status.

Design: Paired blood plasma and CSF specimens were collected from 66 HIV-infected patients cross-sectionally representing all major clinical stages relating to HIV-associated neurologic disease, including primary infection, asymptomatic chronic infection, chronic infection with minor global impairment, and immune deficiency with HIV-associated dementia.

Methods: Heteroduplex tracking assays and bulk sequence analysis targeting the V1/V2, C2-V3, and V4/V5 regions of env were performed to characterize the genetic makeup of complex HIV-1 populations in the cross-sectional blood plasma and CSF specimens. The levels of blood plasma/CSF env compartmentalization were quantified and compared across the different clinical stages of HIV-1 neurologic disease.

Results: Blood plasma/CSF env compartmentalization levels varied considerably by disease stage and were generally consistent across all three regions of env characterized. Little or no compartmentalization was observed in non-impaired individuals with primary HIV-1 infection. Compartmentalization levels were elevated in chronically infected patients, but were not significantly different between mildly impaired and non-impaired patients. Patients with HIV-associated dementia showed significantly greater blood plasma/CSF env compartmentalization relative to other groups.

Conclusion: : Increased CSF compartmentalization of the HIV-1 env gene, which may reflect independent HIV-1 replication and evolution within the central nervous system, is specifically associated with HIV-associated dementia and not the less severe forms of HIV-1 neurologic disease.

Fig. 1. Analysis of HIV-1 env populations in paired blood plasma and cerebrospinal fluid

Shown are representative HTA gels for the V4/V5 region of env for six patients with varying degrees of HIV-associated neurologic disease. Two independent RT-PCR and HTA analyses were performed for each sample, and results for each replicate are shown. All bands showed run between the single-stranded probe and probe homoduplex bands, which are not shown for simplicity. The measurement of env compartmentalization (BP/CSF % difference) accounts for both the presence and absence of genetic variants in one compartment versus another, as well as differences in the relative abundance of variants present in both compartments. To monitor HTA reproducibility, two independent RT-PCR and HTA analyses were performed, and discordance between replicate HTA results was similarly quantified (replicates % difference). BP, blood plasma; CSF, cerebrospinal fluid; HTA, heteroduplex tracking assay; MCMD, minor cognitive and motor disorder; RT-PCR, reverse transcriptase-PCR.

Fig. 2. Blood plasma/cerebrospinal fluid env compartmentalization based on V1/V2 and V4/V5 heteroduplex tracking assay is associated with HIV-associated dementia

(a) V1/V2 and V4/V5 env populations in BP and CSF of each patient were compared by HTA, and the % difference for BP/CSF HTA band patterns was determined for each patient and the data compiled for all patients in each of the different disease stages. A higher % difference indicates more discordant BP/CSF viral genetic populations for the particular region of env analyzed. (b) Discordance of band patterns for RT-PCR and HTA replicates from the same RNA sample was similarly measured by % difference to monitor reproducibility of HTA results for patients in each neurologic disease category. (c) Linear regression was performed to determine whether V1/V2 and V4/V5 BP/CSF % difference values within each patient correlate (P <0.0001, r² = 0.4763) (d) The mean of V1/V2 and V4/V5 % difference results was determined for each patient to reflect global env compartmentalization between BP and CSF, and results were compiled for comparison between the different disease categories. Unadjusted P-values shown were determined by Wilcoxon rank-sum test (HAD versus other groups, P = 0.001, by univariable logistic regression). BP, blood plasma; CSF, cerebrospinal fluid; HAD, HIV-associated dementia; HTA, heteroduplex tracking assay; MCMD/I, minor cognitive and motor disorder/globally impaired; NI, not impaired; PI, primary infection; RT-PCR, reverse transcriptase-PCR.

Fig. 3. Compartmentalization of the C2-V3 env bulk sequence between blood plasma and cerebrospinal fluid

(a) Genetic distances estimated from alignments of the C2-V3 consensus sequences for blood plasma and CSF using the pairwise distance function in MEGA4 [28] and specifying the Kimura 2-parameter model of nucleotide substitution to allow transition–transversion rate differences. The resulting measure of genetic distance estimates the per-site number of nucleotide substitutions, after correcting for the probability of multiple substitutions at individual sites (*P <0.01 for HAD versus MCMD/I, NI, or PI). (b) A molecular clock was determined using consensus sequences obtained from longitudinal peripheral blood mononuclear cell samplings from eight chronically infected patients, as reported by Shankarappa et al. [27]. Genetic distances for the region of sequence overlap with this study (HXB2 nt positions 7023–7208) were estimated using the Kimura two-parameter model of nucleotide substitution and represent the per-site number of nucleotide substitutions that occurred in an individual patient as the initial sample in that patient. Each color corresponds to sequences sampled from a different patient. Lines represent the best-fit linear model (solid line) and the 95% confidence limits (dashed lines). On the basis of this molecular clock, we estimated the mean time of BP/CSF env divergence for patients in different neurologic disease categories. The indicated 95% confidence interval is based on the confidence limits of the molecular clock. BP, blood plasma; CSF, cerebrospinal fluid; HAD, HIV-associated dementia; MCMD/I, minor cognitive and motor disorder/globally impaired; MEGA, molecular evolutionary genetics analysis; NI, not impaired; PI, primary infection; RT-PCR, reverse transcriptase-PCR.

Fig. 4. Relationship between CD4⁺ cell count and blood plasma/cerebrospinal fluid env compartmentalization

(a,b) Linear regression analysis was performed to determine whether CD4⁺ cell count levels correlate with average %difference measure of BP/CSF env compartmentalization for all patients (a) or only HAD patients (b). (c) BP/CSF env compartmentalization levels in patients with poor immune status (based on CD4⁺ T cell count <250 cells/μl) were compared between the different disease groups (P values based on Wilcoxon rank-sum test). Note that separate comparisons of HAD versus MCMD/I and HAD versus NI showed comparable statistical significance (P = 0.0006 and P = 0.007, respectively). CD4⁺ T cell counts for NI/MCMD/I and HAD patients in this subgroup analysis were not significantly different (P = 0.15, Wilcoxon rank sum test), with mean ± SD of 146 ± 66 (range 17–230) and 106 ± 71 (range 9–234), respectively. BP, blood plasma; CSF, cerebrospinal fluid; HAD, HIV-associated dementia; MCMD/I, minor cognitive and motor disorder/globally impaired; NI, not impaired, PI, primary infection.