Genome-wide analysis identifies novel loci influencing plasma apolipoprotein E concentration and Alzheimer's disease risk.

The APOE 2/3/4 polymorphism is the greatest genetic risk factor for Alzheimer's disease (AD). This polymorphism is also associated with variation in plasma ApoE level; while APOE*4 lowers, APOE*2 increases ApoE level. Lower plasma ApoE level has also been suggested to be a risk factor for incident dementia. To our knowledge, no large genome-wide association study (GWAS) has been reported on plasma ApoE level. This study aimed to identify new genetic variants affecting plasma ApoE level as well as to test if baseline ApoE level is associated with cognitive function and incident dementia in a longitudinally followed cohort of the Ginkgo Evaluation of Memory (GEM) study. Baseline plasma ApoE concentration was measured in 3031 participants (95.4% European Americans (EAs)). GWAS analysis was performed on 2580 self-identified EAs where both genotype and plasma ApoE data were available. Lower ApoE concentration was associated with worse cognitive function, but not with incident dementia. As expected, the risk for AD increased from E2/2 through to E4/4 genotypes (P for trend = 4.8E-75). In addition to confirming the expected and opposite associations of APOE*2 (P = 4.73E-79) and APOE*4 (P = 8.73E-12) with ApoE level, GWAS analysis revealed nine additional independent signals in the APOE region, and together they explained about 22% of the variance in plasma ApoE level. We also identified seven new loci on chromosomes 1, 4, 5, 7, 11, 12 and 20 (P range = 5.49E-08 to 5.36E-10) that explained about 9% of the variance in ApoE level. Plasma ApoE level-associated independent variants, especially in the APOE region, were also associated with AD risk and amyloid deposition in the brain, indicating that genetically determined ApoE level variation may be a risk factor for developing AD. These results improve our understanding of the genetic determinants of plasma ApoE level and their potential value in affecting AD risk.

Continued Slow Decay of the Residual Plasma Viremia Level in HIV-1-Infected Adults Receiving Long-term Antiretroviral Therapy.

UNLABELLED: We measured plasma human immunodeficiency virus type 1 (HIV-1) RNA levels by means of single-copy assay in 334 participants receiving virologically suppressive antiretroviral therapy (ART). A residual viremia load of ≥1 copy/mL after 4 years of ART was predicted by a higher pre-ART HIV-1 RNA level, higher CD8(+) T-cell count during treatment, and a lower ratio of CD4+ T cells to CD8+ T cells during treatment but not by initial ART regimen. In a longitudinal subset of 64 individuals, continued decay of the plasma HIV-1 RNA level was observed, with an average annual decrease of 6% and an estimated half-life of 11.5 years. In contrast to prior reports, the persistent viremia level continues to slowly decline during years 4-12 of suppressive ART. CLINICAL TRIALS REGISTRATION: NCT00001137.

Early Antiretroviral Therapy in South African Children Reduces HIV-1-Infected Cells and Cell-Associated HIV-1 RNA in Blood Mononuclear Cells.

We measured cell-associated human immunodeficiency virus (HIV)-1 DNA (CAD) and RNA (CAR) and plasma HIV-1 RNA in blood samples from 20 children in the Children with HIV Early Antiretroviral (CHER) cohort after 7-8 years of suppressive combination antiretroviral therapy (cART). Children who initiated cART early (<2 months; n = 12) had lower HIV-1 CAD (median, 48 vs 216; P < .01) and CAR (median, 5 vs 436; P < .01) per million peripheral blood mononuclear cells than children who started later (≥ 2 months; n = 8). Plasma HIV-1 RNA levels were not significantly lower in early-treated children (0.5 vs 1.2 copies/mL; P = .16). Early treatment at <2 months of age reduces the number of HIV-infected cells and HIV CAR.

HIV-1 DNA decay dynamics in blood during more than a decade of suppressive antiretroviral therapy.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) DNA dynamics during long-term antiretroviral therapy (ART) are not defined. METHODS: Blood mononuclear cells obtained during 7-12 years of effective ART were assayed for total HIV-1 DNA and 2-long terminal repeat (LTR) circles by quantitative polymerase chain reaction (qPCR). Slopes of HIV-1 DNA were estimated by participant-specific linear regressions. Plasma was assayed for residual viremia (HIV-1 RNA) by qPCR. RESULTS: Thirty participants were studied. HIV-1 DNA decreased significantly from years 0-1 and 1-4 of ART with median decay slopes of -0.86 (interquartile range, -1.05, -0.59) and -0.11 (-0.17, -0.06) log10(copies/10(6) CD4+ T-cells)/year, respectively (P < .001). Decay was not significant for years 4-7 (-0.02 [-0.06, 0.02]; P = .09) or after year 7 of ART (-0.006 [-0.030, 0.015]; P = .17). All participants had detectable HIV-1 DNA after 10 years (median 439 copies/10(6) CD4+ T-cells; range: 7-2074). Pre-ART HIV-1 DNA levels were positively associated with pre-ART HIV-1 RNA levels (Spearman = 0.71, P < .001) and with HIV-1 DNA at years 4, 7, and 10 on ART (Spearman ≥ 0.75, P < .001). No associations were found (P ≥ .25) between HIV-1 DNA slopes or levels and % activated CD8+ T-cells (average during years 1-4) or residual viremia (n = 18). 2-LTR circles were detected pre-ART in 20/29 and in 8/30 participants at last follow-up. CONCLUSIONS: Decay of HIV-1 DNA in blood is rapid in the first year after ART initiation (86% decline), slows during years 1-4 (23% decline/year), and subsequently plateaus. HIV-1 DNA decay is not associated with the levels of CD8+ T-cell activation or persistent viremia. The determinants of stable HIV-1 DNA persistence require further elucidation. Clinical Trials Registration. NCT00001137.

Improved single-copy assays for quantification of persistent HIV-1 viremia in patients on suppressive antiretroviral therapy.

A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.

New tools for quantifying HIV-1 reservoirs: plasma RNA single copy assays and beyond.

Quantification of plasma HIV-1 RNA below the limit of FDA-approved assays by a single copy quantitative PCR assays (SCA) has provided significant insights into HIV-1 persistence despite potent antiretroviral therapy as well as a means to assess the impact of therapeutic strategies, such as treatment intensification, on residual viremia. In this review, we discuss insights gained from plasma HIV-1 RNA SCA and highlight the need for additional assays to characterize better the cellular and tissue reservoirs of HIV-1. Accurate, reproducible, and sensitive assays to quantify HIV-1 reservoirs, before and after therapeutic interventions, are essential tools in the quest for a cure of HIV-1 infection.

Residual plasma viraemia and infectious HIV-1 recovery from resting memory CD4 cells in patients on antiretroviral therapy: results from ACTG A5173.

BACKGROUND: In HIV-1-infected patients receiving antiretroviral therapy (ART), the relationship between residual viraemia and ex vivo recovery of infectious virus from latently infected CD4 cells is uncertain. METHODS: We measured residual viraemia (HIV-1 RNA copies/ml) by single-copy assay (SCA) and the latent reservoir by infectious virus recovery from resting memory CD4 cells (infectious units per million cells [IUPM]) in patients who initiated ART. We assessed immune activation by measuring CD38 expression on T-cells. RESULTS: Ten patients who initiated ART and maintained a plasma HIV-1 RNA level < 200 copies/ml had residual viraemia and IUPM measured every 24 weeks. Five of 10 patients had longitudinal IUPM measured at weeks 24-96; the remainder had IUPM measured 1-3 times over 24-72 weeks. Analyses of 29 paired measurements revealed a positive association between level of residual viraemia and IUPM (0.56 higher log10 HIV-1 RNA copies/ml per 1 log10 higher IUPM; P = 0.005). Residual viraemia level was positively associated with CD38 density and percentage on CD8+ T-cells in concurrent samples and with pre-ART HIV-1 RNA levels. CONCLUSIONS: In patients with HIV-1 RNA levels < 200 copies/ml 24-96 weeks after initiating ART, the level of viraemia is positively associated with infectious virus recovery from resting memory CD4 cells. Whether this association persists after longer-term suppressive ART needs to be determined. If additional studies show that residual viraemia measured by SCA reflects the size of the latent reservoir in patients who have had virological suppression for longer periods of time, this could facilitate testing of potentially curative strategies to reduce this important reservoir.