P. Richard Harrigan

Human leukocyte antigen-specific polymorphisms in HIV-1 Gag and their association with viral load in chronic untreated infection.

Objective:Selection of specific human leukocyte antigen (HLA)-restricted cytotoxic T-lymphocyte (CTL) escape mutations in key Gag epitopes has been associated with loss of HIV immune control on an individual basis. Here we undertake a population-based identification of HLA-associated polymorphisms in Gag and investigate their relationship with plasma viral load. Design:Cross-sectional analysis of 567 chronically HIV subtype B-infected, treatment-naive individuals. Methods:HLA class I-associated Gag substitutions were identified using phylogenetically corrected analysis methods featuring a multivariate adjustment for HLA linkage disequilibrium and a q-value correction for multiple tests. Presence of HLA-associated substitutions and markers of HIV disease status were correlated using Spearmans rank test. Results:We have created a gene-wide map of HLA class I-associated substitutions in HIV-1 subtype B Gag. This features 111 HLA-associated substitutions occurring at 51 of 500 Gag codons, more than 50% of which occur within published and/or putative HLA-restricted CTL epitopes. A modest inverse correlation was observed between the total number of HLA-associated Gag polymorphic sites within each individual and plasma viral load in chronic untreated infection (R = −0.17, P < 0.0001), supporting the hypothesis that a broad ability to target Gag in vivo contributes to viral control. A modest positive correlation was observed between the proportion of these sites exhibiting HLA-associated substitutions and plasma viral load (R = 0.09, P = 0.03), consistent with a loss of viremia control with the accumulation of CTL escape mutations. Conclusion:Results contribute to our understanding of immune-driven viral adaptation and suggest that the accumulation of CTL escape mutations in Gag results in clinically detectable consequences at the population level. These data have implications for HIV vaccines.

Changes in lipids over twelve months after initiating protease inhibitor therapy among persons treated for HIV/AIDS

BackgroundProtease inhibitors are known to alter the lipid profiles in subjects treated for HIV/AIDS. However, the magnitude of this effect on plasma lipoproteins and lipids has not been adequately quantified.ObjectiveTo estimate the changes in plasma lipoproteins and triglycerides occurring within 12 months of initiating PI-based antiretroviral therapy among HIV/AIDS afflicted subjects.MethodsWe included all antiretroviral naïve HIV-infected persons treated at St-Pauls Hospital, British Columbia, Canada, who initiated therapy with protease inhibitor antiretroviral (ARV) drugs between August 1996 and January 2002 and who had at least one plasma lipid measurement. Longitudinal associations between medication use and plasma lipids were estimated using mixed effects models that accounted for repeated measures on the same subjects and were adjusted for age, sex, time dependent CD4+ T-cell count, and time dependent cumulative use of non-nucleoside reverse transcriptase inhibitors and adherence. The cumulative number of prescriptions filled for PIs was considered time dependent. We estimated the changes in the 12 months following any initiation of a PI based regimen.ResultsA total of 679 eligible subjects were dispensed nucleoside analogues and PI at the initiation of therapy. Over a median 47 months of follow-up (interquartile range (IQR): 29–62), subjects had a median of 3 (IQR: 1–6) blood lipid measurements. Twelve months after treatment initiation of PI use, there was an estimated 20% (95% confidence interval: 17% – 24%) increase in total cholesterol and 22% (12% – 33%) increase in triglycerides.ConclusionsTwelve months after treatment initiation with PIs, statistically significant increases in total cholesterol and triglycerides levels were observed in HIV-infected patients under conditions of standard treatment. Our results contribute to the growing body of evidence implicating PIs in the development of blood lipid abnormalities. In conjunction with the predominance or men, high rates of smoking, and aging of the treated HIV-positive population, elevated lipoproteins and triglycerides may mean that patients such as these are at elevated risk for cardiovascular events in the future.

Absolute Leukocyte Telomere Length in HIV-Infected and Uninfected Individuals: Evidence of Accelerated Cell Senescence in HIV-Associated Chronic Obstructive Pulmonary Disease

Combination antiretroviral therapy (cART) has extended the longevity of human immunodeficiency virus (HIV)-infected individuals. However, this has resulted in greater awareness of age-associated diseases such as chronic obstructive pulmonary disease (COPD). Accelerated cellular senescence may be responsible, but its magnitude as measured by leukocyte telomere length is unknown and its relationship to HIV-associated COPD has not yet been established. We measured absolute telomere length (aTL) in peripheral leukocytes from 231 HIV-infected adults. Comparisons were made to 691 HIV-uninfected individuals from a population-based sample. Subject quartiles of aTL were assessed for relationships with measures of HIV disease severity, airflow obstruction, and emphysema severity on computed tomographic (CT) imaging. Multivariable regression models identified factors associated with shortened aTL. Compared to HIV-uninfected subjects, the mean aTL in HIV-infected patients was markedly shorter by 27 kbp/genome (p<0.001); however, the slopes of aTL vs. age were not different (p=0.469). Patients with longer known durations of HIV infection (p=0.019) and lower nadir CD4 cell counts (p=0.023) had shorter aTL. Shorter aTL were also associated with older age (p=0.026), smoking (p=0.005), reduced forced expiratory volume in one second (p=0.030), and worse CT emphysema severity score (p=0.049). HIV-infected subjects demonstrate advanced cellular aging, yet in a cART-treated cohort, the relationship between aTL and age appears no different from that of HIV-uninfected subjects.

A molecular phylogenetics-based approach for identifying recent hepatitis C virus transmission events.

UNLABELLEDnImproved surveillance methods are needed to better understand the current hepatitis C virus (HCV) disease burden and to monitor the impact of prevention and treatment interventions on HCV transmission dynamics. Sanger sequencing (HCV NS5B, HVR1 and Core-E1-HVR1) and phylogenetics were applied to samples from individuals diagnosed with HCV in British Columbia, Canada in 2011. This included individuals with two or three sequential samples collected <1 year apart. Patristic distances between sequential samples were used to set cutoffs to identify recent transmission clusters. Factors associated with transmission clustering were analyzed using logistic regression. From 618 individuals, 646 sequences were obtained. Depending on the cutoff used, 63 (10%) to 92 (15%) unique individuals were identified within transmission clusters of predicted recent origin. Clustered individuals were more likely to be <40 years old (Adjusted Odds Ratio (AOR) 2.12, 95% CI 1.21-3.73), infected with genotype 1a (AOR 6.60, 95% CI 1.98-41.0), and to be seroconverters with estimated infection duration of <1 year (AOR 3.13, 95% CI 1.29-7.36) or >1 year (AOR 2.19, 95% CI 1.22-3.97).nnnCONCLUSIONnSystematic application of molecular phylogenetics may be used to enhance traditional surveillance methods through identification of recent transmission clusters.

Methamphetamine injecting is associated with phylogenetic clustering of hepatitis C virus infection among street-involved youth in Vancouver, Canada

BACKGROUNDnAmong prospective cohorts of people who inject drugs (PWID), phylogenetic clustering of HCV infection has been observed. However, the majority of studies have included older PWID, representing distant transmission events. The aim of this study was to investigate phylogenetic clustering of HCV infection among a cohort of street-involved youth.nnnMETHODSnData were derived from a prospective cohort of street-involved youth aged 14-26 recruited between 2005 and 2012 in Vancouver, Canada (At Risk Youth Study, ARYS). HCV RNA testing and sequencing (Core-E2) were performed on HCV positive participants. Phylogenetic trees were inferred using maximum likelihood methods and clusters were identified using ClusterPicker (Core-E2 without HVR1, 90% bootstrap threshold, 0.05 genetic distance threshold).nnnRESULTSnAmong 945 individuals enrolled in ARYS, 16% (n=149, 100% recent injectors) were HCV antibody positive at baseline interview (n=86) or seroconverted during follow-up (n=63). Among HCV antibody positive participants with available samples (n=131), 75% (n=98) had detectable HCV RNA and 66% (n=65, mean age 23, 58% with recent methamphetamine injection, 31% female, 3% HIV+) had available Core-E2 sequences. Of those with Core-E2 sequence, 14% (n=9) were in a cluster (one cluster of three) or pair (two pairs), with all reporting recent methamphetamine injection. Recent methamphetamine injection was associated with membership in a cluster or pair (P=0.009).nnnCONCLUSIONnIn this study of street-involved youth with HCV infection and recent injecting, 14% demonstrated phylogenetic clustering. Phylogenetic clustering was associated with recent methamphetamine injection, suggesting that methamphetamine drug injection may play an important role in networks of HCV transmission.

Differentiation of acute from chronic hepatitis C virus infection by nonstructural 5B deep sequencing: A population‐level tool for incidence estimation

The ability to classify acute versus chronic hepatitis C virus (HCV) infections at the time of diagnosis is desirable to improve the quality of surveillance information. The aim of this study was to differentiate acute from chronic HCV infections utilizing deep sequencing. HCV nonstructural 5B (NS5B) amplicons (nu2009=u200994) were generated from 77 individuals (13 acute and 64 chronic HCV infections) in British Columbia, Canada, with documented seroconversion time frames. Amplicons were deep sequenced and HCV genomic diversity was measured by Shannon entropy (SE) and a single nucleotide variant (SNV) analysis. The relationship between each diversity measure and the estimated days since infection was assessed using linear mixed models, and the ability of each diversity measure to differentiate acute from chronic infections was assessed using generalized estimating equations. Both SE and the SNV diversity measures were significantly different for acute versus chronic infections (Pu2009<u20090.009). NS5B nucleotide diversity continued to increase for at least 3 years postinfection. Among individuals with the least uncertainty with regard to duration of infection (nu2009=u200939), the area under the receiver operating characteristic curve (AUROC) was high (0.96 for SE; 0.98 for SNV). Although the AUROCs were lower (0.86 for SE; 0.80 for SNV) when data for all individuals were included, they remain sufficiently high for epidemiological purposes. Synonymous mutations were the primary discriminatory variable accounting for over 78% of the measured genetic diversity. Conclusions: NS5B sequence diversity assessed by deep sequencing can differentiate acute from chronic HCV infections and, with further validation, could become a powerful population‐level surveillance tool for incidence estimation. (Hepatology 2015;61:1842–1850)

Compounds producing an effective combinatorial regimen for disruption of HIV‐1 latency

Highly active antiretroviral therapy (HAART) has improved the outlook for the HIV epidemic, but does not provide a cure. The proposed “shock‐and‐kill” strategy is directed at inducing latent HIV reservoirs, which may then be purged via boosted immune response or targeting infected cells. We describe five novel compounds that are capable of reversing HIV latency without affecting the general T‐cell activation state. The new compounds exhibit synergy for reactivation of latent provirus with other latency‐reversing agents (LRAs), in particular ingenol‐3‐angelate/PEP005. One compound, designated PH02, was efficient at reactivating viral transcription in several cell lines bearing reporter HIV‐1 at different integration sites. Furthermore, it was capable of reversing latency in resting CD4+ T lymphocytes from latently infected aviremic patient cells on HAART, while producing minimal cellular toxicity. The combination of PH02 and PEP005 produces a strong synergistic effect for reactivation, as demonstrated through a quantitative viral outgrowth assay (qVOA), on CD4+ T lymphocytes from HIV‐1‐infected individuals. We propose that the PH02/PEP005 combination may represent an effective novel treatment for abrogating persistent HIV‐1 infection.