The role of baseline HIV-1 RNA, drug resistance, and regimen type as determinants of response to first-line antiretroviral therapy.

The factors influencing virological response to first-line combined antiretroviral therapy (cART) in an Italian cohort of HIV-1-infected patients were examined. Eligible patients were those enrolled in a national prospective observational cohort (Antiretroviral Resistance Cohort Analysis), starting first-line cART between 2001 and 2011 and who had at least one follow-up of HIV-1 RNA. The primary endpoint was virological success, defined as the first viral load <50 copies/ml. Time to events were analyzed by Kaplan-Meier analysis and Cox proportional hazard model. One thousand three hundred five patients met the study inclusion criteria. In a multivariable model adjusting for transmission mode, presence of transmitted drug resistance, baseline CD4(+) cell count, viral subtype, and type of NRTI backbone employed, independent predictors of virological success were higher baseline viral load (≥500,000 vs. <100,000 HR 0.52; P < 0.001), a weighted genotypic susceptibility score (wGSS) <3 (HR 0.58; P = 0.003), male sex (HR 0.76 P = 0.001), and type of initial third drug employed (integrase inhibitor vs. boosted protease inhibitors HR 3.23; P < 0.001). In the subset with HIV-1 RNA >100,000 copies/ml, virologic success was only associated with the use of integrase inhibitors in the first cART regimen. Independent predictors of immunological success were baseline CD4(+) cell count and wGSS <3. High baseline HIV-1 RNA, predicted activity of the first-line regimen based on genotypic resistance testing, gender, and use of new agents were found to predict time to achieve virological success. The type of initial nucleoside analog backbone was not found to predict virological response.

Comparative determination of HIV-1 co-receptor tropism by Enhanced Sensitivity Trofile, gp120 V3-loop RNA and DNA genotyping.

BACKGROUND: Trofile is the prospectively validated HIV-1 tropism assay. Its use is limited by high costs, long turn-around time, and inability to test patients with very low or undetectable viremia. We aimed at assessing the efficiency of population genotypic assays based on gp120 V3-loop sequencing for the determination of tropism in plasma viral RNA and in whole-blood viral DNA. Contemporary and follow-up plasma and whole-blood samples from patients undergoing tropism testing via the enhanced sensitivity Trofile (ESTA) were collected. Clinical and clonal geno2pheno[coreceptor] (G2P) models at 10% and at optimised 5.7% false positive rate cutoff were evaluated using viral DNA and RNA samples, compared against each other and ESTA, using Cohen's kappa, phylogenetic analysis, and area under the receiver operating characteristic (AUROC). RESULTS: Both clinical and clonal G2P (with different false positive rates) showed good performances in predicting the ESTA outcome (for V3 RNA-based clinical G2P at 10% false positive rate AUROC = 0.83, sensitivity = 90%, specificity = 75%). The rate of agreement between DNA- and RNA-based clinical G2P was fair (kappa = 0.74, p < 0.0001), and DNA-based clinical G2P accurately predicted the plasma ESTA (AUROC = 0.86). Significant differences in the viral populations were detected when comparing inter/intra patient diversity of viral DNA with RNA sequences. CONCLUSIONS: Plasma HIV RNA or whole-blood HIV DNA V3-loop sequencing interpreted with clinical G2P is cheap and can be a good surrogate for ESTA. Although there may be differences among viral RNA and DNA populations in the same host, DNA-based G2P may be used as an indication of viral tropism in patients with undetectable plasma viremia.

Rearrangement of the JC virus regulatory region sequence in the bone marrow of a patient with rheumatoid arthritis and progressive multifocal leukoencephalopathy.

The polyomavirus JC (JCV) is the etiologic agent of progressive multifocal leukoencephalopathy (PML). JCV remains quiescent in kidneys, where it displays a stable archetypal regulatory region (RR). Conversely, rearranged JCV RR, including tandem repeat patterns found in the central nervous system (CNS) of PML patients, have been associated with neurovirulence. The precise site and mechanism of JCV RR transformation is unknown. We present herein a patient with rheumatoid arthritis treated with methotrexate, who developed PML and had a rapid fatal outcome. JCV DNA polymerase chain reaction (PCR) was positive in cerebrospinal fluid (CSF), bone marrow, blood, and urine. Double-immunohistochemical staining demonstrated that 9% of bone marrow CD138(+) plasma cells sustained productive infection by JCV, accounting for 94% of JCV-infected cells. JCV RR analysis revealed archetype and rearranged RR forms in bone marrow, whereas RR with tandem repeat was predominant in blood. These results suggest that the bone marrow may be a potential site of JCV pathogenic transformation. Further studies will be needed to determine the prevalence of JCV in bone marrow of immunosuppressed individuals at risk of PML and characterize the RR and phenotype of these JCV isolates.

Mitochondrial DNA haplogroups and incidence of lipodystrophy in HIV-infected patients on long-term antiretroviral therapy.

We investigated the rates of lipodystrophy events, according to mitochondrial DNA haplogroup, in 187 patients starting combination antiretroviral therapy and following it. Incidence rates of lipoatrophy and fat accumulation were 8.2 and 4.8 per 100 person-years of follow-up, respectively. In multivariable models, patients with haplogroup K were at higher risk of any lipodystrophy [adjusted relative risk (aRR) 4.02, P = 0.0009], lipoatrophy (competing-risk aRR 2.42, P = 0.09; cause-specific aRR 2.99, P = 0.031), and fat accumulation (competing-risk aRR, 2.63, P = 0.11; cause-specific aRR 5.27, P = 0.019) than those with haplogroup H. Mitochondrial haplogroups may explain part of the genetic predisposition to lipodystrophy during combination antiretroviral therapy.

The effect of polymorphisms in candidate genes on the long-term risk of lipodystrophy and dyslipidemia in HIV-infected white patients starting antiretroviral therapy.

We investigated whether polymorphisms in human candidate genes could be associated with a different risk of developing lipodystrophy and dyslipidemia in HIV-infected patients starting combination antiretroviral therapy (cART). Genomic DNA samples from white HIV-1-infected patients were analyzed for seven polymorphisms located in the MDR1, TNF-α, APM1, APOE, and LPL genes. Lipid data were retrospectively collected beginning with the initiation of cART. Lipodystrophy was assessed cross-sectionally and then prospectively. The association with lipodystrophy and National Cholesterol Evaluation Program Adult Treatment Panel III-defined lipid thresholds was analyzed using survival analysis and logistic regression. One-hundred and seventy-four patients were genotyped. In 151 patients assessed for lipodystrophy, MDR1 3435 T homozygosis was associated with a higher hazard (adjusted hazard ratio, aHR, versus CT 0.25; p=0.02) and tumor necrosis factor (TNF)-α 308 G homozygosis with a lower hazard (vs. AA aHR 2.14; p=0.04) of developing trunk fat accumulation after adjusting for gender and initial cART type. The TNF 238 GG genotype was associated with a higher risk of developing low HDL-cholesterol levels (adjusted odd ratio, aOR, 5.91; p=0.01) while patients carrying the LPL S477X mutation were at lower risk of reaching high non-HDL-cholesterol levels (aOR 0.39; p=0.05). The APOEe3/3 genotype patients were at lower risk (aOR 0.26, p=0.015), whereas the adiponectin 276 GT carriers were at higher risk of developing hypertriglyceremia (vs. GG aOR 3.10; p=0.04). Knowledge of the effect of genetic determinants on dyslipidemia and lipodystrophy may prompt the investigation of potential pathogenetic mechanisms and might eventually be used for guiding individualized treatment decisions.

Efficient in vitro expansion of JC virus-specific CD8(+) T-cell responses by JCV peptide-stimulated dendritic cells from patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the brain caused by JC virus (JCV) for which there is no cure. PML patients who have JCV-specific CD8(+) cytotoxic T lymphocytes (CTL) in their blood have a better clinical outcome. We compared JCV-specific CTL responses in vitro elicited either by JCV peptide-loaded dendritic cells (DC) or by direct peptide stimulation of lymphocytes from 20 HLA-A0201(+) healthy controls, HIV(+) and PML patients. JCV peptide-loaded DC elicited a stronger CTL expansion in 13/15 responders. DC can induce a potent JCV-specific CTL response in vitro, and may constitute a promising approach for PML immunotherapy.

Characterization of JC virus in cerebrospinal fluid from HIV-1 infected patients with progressive multifocal leukoencephalopathy: insights into viral pathogenesis and disease prognosis.

OBJECTIVES: To analyze virological and immunological features of AIDS-related progressive multifocal leukoencepalophathy (PML) and their association to disease prognosis. METHODS: In HIV-infected patients with virologically confirmed PML, JC virus (JCV) DNA load and levels of Macrophage Chemoattractant Protein (MCP)-1 were determined in cerebrospinal fluid. JCV genotypes, rearrangements and JCV DNA binding sites for cellular transcription factors were analyzed by sequencing the viral VP1 region and regulatory region (RR). RESULTS: 45 patients were analyzed: 60% were exposed to highly active antiretroviral therapy (HAART) after PML and 24% before the disease onset. JCV DNA load in cerebrospinal fluid was a strong predictor of patients survival. Lower levels of JCV DNA in cerebrospinal fluid were associated with the following virologic factors: viral genotype 4 (p = 0.043), more rearrangements in the RR (p = 0.046), duplication of RR block B (p = 0.028), and duplication of binding sites for cellular transcription factor NF-1 (p = 0.060). In patients with prior antiretroviral exposure there was a trend towards a higher number of binding sites for cellular transcription factors (p = 0.068). Lower JCV load was also predicted by exposure to HAART (p = 0.010), higher baseline CD4 counts (p = 0.009) and higher cerebrospinal fluid MCP-1 levels (p = 0.036). In a multiple regression model, MCP-1 levels were independently associated with JCV load. CONCLUSION: HAART leads to a partial immune-mediated control of JCV replication; the virus may tend to escape through the selection of rearrangements in the RR, some associated with enhanced viral replication efficiency, other resulting in multiplication of binding sites for cellular transcription factors.

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy.

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.

Reduced rate of diagnostic positive detection of JC virus DNA in cerebrospinal fluid in cases of suspected progressive multifocal leukoencephalopathy in the era of potent antiretroviral therapy.

Fifty-nine human immunodeficiency virus (HIV)-infected patients with suspected progressive multifocal leukoencephalopathy and 224 controls were tested for JC virus (JCV) DNA in cerebrospinal fluid by PCR. The diagnostic positive detection rate dropped from 89.5% (95% confidence intervals of 75.5 to 103.5%) in the pre-highly active antiretroviral therapy (HAART) era to 57.5% (95% confidence intervals of 42.1 to 72.9%) in the HAART era; the specificity remained unchanged. Predictors of failure to detect JCV DNA were exposure to HAART at disease onset and higher CD4 counts.

Macrophage chemoattractant protein-1 levels in cerebrospinal fluid correlate with containment of JC virus and prognosis of acquired immunodeficiency syndrome--associated progressive multifocal leukoencephalopathy.

In the highly active antiretroviral therapy (HAART) era, the role of the inflammatory response in acquired immunodeficiency syndrome (AIDS)-related progressive multifocal leukoencephalopathy (PML) remains controversial. In this study, JC virus DNA load and levels of cytokines were determined in cerebrospinal fluid (CSF) from 32 human immunodeficiency virus (HIV)-1-infected patients with confirmed PML who underwent HAART; cytokines were also measured in 12 HIV-positive controls. Predictors of survival were analyzed by Cox's models. Macrophage chemoattractant protein (MCP)-1 levels were significantly higher in PML patients than in controls (mean +/- SD, 2.45 +/- 0.64 versus 1.32 +/- 0.64 log(10) pg/ml, P<.0001). In PML patients, the higher concentration of MCP-1 correlated with lower JC viral load (r=-.405, P=.036). Higher concentrations of MCP-1 in CSF were associated with longer survival on HAART after adjusting for CD4 counts (for each log(10) pg/ml higher, hazard ratio for death 0.28, 95% confidence interval 0.08--1.00). Predictors of shorter survival were lower baseline CD4 counts, higher JCV DNA concentrations, lower Karnofsky, and no prior HAART exposure. These results showed that higher CSF levels of MCP-1, an inflammatory cytokine, were correlated with better prognosis in HAART-treated patients with PML.

Analysis of JC virus genotype distribution and transcriptional control region rearrangements in human immunodeficiency virus-positive progressive multifocal leukoencephalopathy patients with and without highly active antiretroviral treatment.

After the introduction of highly active antiretroviral therapy (HAART), the incidence of many acquired immunodeficiency syndrome (AIDS)-related opportunistic infections, but not of progressive multifocal leukoencephalopathy (PML), has been dramatically decreased. However, it has been shown that about 50% of the HAART-treated PML patients had a significantly prolonged (>6 months) survival time, in comparison to the short (<6 months) survival time of the classical form of PML. In order to verify if a particular genotype or genomic rearrangements of JC virus (JCV) could affect the clinical course of PML, the authors performed nucleotide sequencing of 25 virion protein (VP1) and 18 transcriptional control region (TCR) DNA amplified in the cerebrospinal fluid (CSF) of HAART-untreated PML patients, of 17 HAART-treated PML patients, and in the urine of 23 healthy individuals. In nontreated PML patients, 52% and 44% of amplified JCV were respectively type 1 and type 2, whereas in HAART-treated PML patients, 59% of the amplified JCV were type 1, 23% type 2, and 18% type 4, without differences between long and short survivors. In both groups, the amplified TCR had unique and extensive rearrangements, whereas archetype TCR without rearrangements was detected in all the healthy subjects and in the CSF of two long-survivor PML patients. The data obtained indicate that the introduction of HAART has induced changes in JCV genotype distribution and probably reduced the rate of rearrangements of TCR region among PML patients.