Choo Beng Chew

HIV-1 compartmentalization in diverse leukocyte populations during antiretroviral therapy

CD4+ T lymphocytes are the primary target of human immunodeficiency virus type 1 (HIV‐1), but there is increasing evidence that other immune cells in the blood, including CD8+ T lymphocytes and monocytes, are also productively infected. The extent to which these additional cellular reservoirs contribute to ongoing immunodeficiency and viral persistence during therapy remains unclear. In this study, we conducted a detailed investigation of HIV‐1 diversity and genetic structure in CD4+ T cells, CD8+ T cells, and monocytes of 13 patients receiving highly active antiretroviral therapy (HAART). Analysis of molecular variance and nonparametric tests performed on HIV‐1 envelope sequences provided statistically significant evidence of viral compartmentalization in different leukocyte populations. Signature pattern analysis and predictions of coreceptor use provided no evidence that selection arising from viral tropism was responsible for the genetic structure observed. Analysis of viral genetic variation in different leukocyte populations demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. In the absence of evidence for leukocyte‐specific selection, these features were supportive of a metapopulation model of HIV‐1 replication as described previously among HIV‐1 populations in the spleen. Compartmentalization of the virus in different leukocytes may have significant implications for current models of HIV‐1 population genetics and contribute to the highly variable way in which drug resistance evolves in different individuals during HAART.

Drug Resistance and Viral Evolution in Plasma and Peripheral Blood Cells During Structured Treatment Interruption (STI) and Non-Interrupted HAART

BACKGROUND Highly active antiretroviral therapy (HAART) can successfully reduce plasma and tissue levels of HIV-1 RNA and results in reductions in HIV-related morbidity and mortality, but the slow viral evolution during therapy in cellular reservoirs is a continuing problem. In addition, little remains known how viral evolutionary process may differ between cell-free and cell-associated compartments, over time, in vivo in patients receiving HAART or STI. OBJECTIVES The main objectives of this study were to assess viral replication kinetics, drug resistance and viral evolution during HAART and STI. STUDY DESIGN We have conducted a longitudinal study of virus culture kinetics in vitro, molecular analysis of uncultured HIV-1 variants from plasma and PBMC of 6 patients on HAART, 4 patients on STI, and 6 from treatment-naïve patients. RESULTS Our data suggest that drug resistance mutations remained compartmentalized between plasma and PBMC. The divergent distribution of resistance mutations between plasma and PBMC coincided with divergent env gene evolution in these compartments. In contrast, the HIV strains from therapy-naive patients showed tight genetic and phylogenetic concordance between plasma and PBMC. Both STI and non-STI groups showed the presence of resistance mutations to both RT and protease inhibitors, which correlated with inadequate suppression of viremia and partially with the virus culture isolation in vitro. CONCLUSIONS Overall, STI for HIV patients has no added advantage over regular HAART at the virologic level and in the diminution of resistance mutations that result in therapy failure. Under both forms of anti-retroviral therapies, virus could be isolated in vitro from the PBMC showing continuing low-level viral replication under suppressive therapy. Overall, these data may be useful in predicting the late emergence of drug resistance mutations via the latent integrated provirus.

Sensitive detection of the K103N non-nucleoside reverse transcriptase inhibitor resistance mutation in treatment-naïve HIV-1 infected individuals by rolling circle amplification.

Primary or transmitted antiretroviral drug resistance mutations pose a significant obstacle for optimizing antiviral treatment. When present at low-levels, resistance mutations are less likely to be detected by standard genotyping assays. This study utilizes a novel rolling circle amplification (RCA) method using padlock probes to achieve the sensitive, specific and low-level detection of the NNRTI resistance K103N from 59 HIV+ treatment-naïve patients from Beijing, China. Using standard genotyping methods, primary drug resistance mutations to either protease or RT inhibitors were found in 25% (15/59) of patients attending hospital clinics in Beijing. Among these 15 patients with antiretroviral (ARV) resistance mutations, standard sequence-based genotyping revealed that most (10/15) had the 103N. Using a highly sensitive RCA assay, 5 more patients among the 59 treatment-naïve cohort were found to have the 103N, but at low-levels, leading to an overall rate of 103N at 25.4% (15/59) in this population. The high prevalence of the 103N suggests that baseline resistance testing should be performed before treatment in this population. Importantly, the new RCA technology allows large-scale, sensitive detection of drug resistance mutations, including detection of minority populations with minimal equipment requirement.

Varicella zoster virus quantitation in blood from symptomatic and asymptomatic individuals

Primary infection with varicella zoster virus (VZV) occurs in immunocompromised and immunocompetent individuals. Clinical and asymptomatic reactivation with shedding of infectious virus and viremia may occur. The prevalence of VZV viremia is unknown. The aim of this study was to detect VZV viremia and quantify VZV DNA using quantitative polymerase chain reaction (qPCR) in blood from different populations. A qPCR‐based method using EvaGreen® was used to quantify VZV DNA in 491 samples, including whole blood, plasma and buffy‐coat, from patients hospitalized with varicella‐associated disease (Group 1, n = 10) and three groups with no VZV disease: individuals with a first clinical diagnosis of central nervous system demyelination (Group 2, n = 213) with their age and sex‐matched controls (Group 3, n = 218); and HIV‐infected individuals (Group 4, n = 50). VZV‐specific IgG antibody titres were measured in Group 3. The proportion positive for viremia and mean detectable VZV DNA load (copies/ml) were: Group 1: 100% (10/10) and 4.6 × 106 ± 1.4 × 107; Group 2: 4% (9/213) and 1.5 × 103 ± 1.8 × 104; Group 3: 8% (17/218) and 1.1 × 103 ± 7.8 × 103; Group 4: 12% (6/50) and 7.7 × 101 ± 2.8 × 102. VZV DNA load and IgG titres were not significantly correlated (Group 3 only). VZV load in Group 1 was significantly elevated compared to Groups 2–4 (P < 0.001); the latter were not significantly different from each other (P = 0.05). VZV genotypes from clades 1–5 were identified in Group 1. VZV DNA was detected but at low frequency and viral load in both immunocompetent and immunocompromised individuals asymptomatic for VZV infection, compared to individuals with active VZV infection. J. Med. Virol. 85:1491–1497, 2013.

First Evidence for the Disease-Stage, Cell-Type, and Virus Specificity of microRNAs during Human Immunodeficiency Virus Type-1 Infection

The potential involvement of host microRNAs (miRNAs) in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+) individuals (viremic and aviremic individuals while receiving highly active antiretroviral therapy (HAART), therapy-naïve long-term non-progressors (LTNP), and HIV-negative (HIV–) healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV− samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted p-value < 0.05. The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV– controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs.