Risk of breast cancer with CXCR4-using HIV defined by V3 loop sequencing.

Objective: Evaluate the risk of female breast cancer associated with HIV-CXCR4 (X4) tropism as determined by various genotypic measures.

Methods: A breast cancer case-control study, with pairwise comparisons of tropism determination methods, was conducted. From the Women's Interagency HIV Study repository, one stored plasma specimen was selected from 25 HIV-infected cases near the breast cancer diagnosis date and 75 HIV-infected control women matched for age and calendar date.

"Deep" sequencing accuracy and reproducibility using Roche/454 technology for inferring co-receptor usage in HIV-1.

Next generation, "deep", sequencing has increasing applications both clinically and in disparate fields of research. This study investigates the accuracy and reproducibility of "deep" sequencing as applied to co-receptor prediction using the V3 loop of Human Immunodeficiency Virus-1. Despite increasing use in HIV co-receptor prediction, the accuracy and reproducibility of deep sequencing technology, and the factors which can affect it, have received only a limited level of investigation.

Theoretical and experimental assessment of degenerate primer tagging in ultra-deep applications of next-generation sequencing.

Primer IDs (pIDs) are random oligonucleotide tags used in next-generation sequencing to identify sequences that originate from the same template. These tags are produced by degenerate primers during the reverse transcription of RNA molecules into cDNA. The use of pIDs helps to track the number of RNA molecules carried through amplification and sequencing, and allows resolution of inconsistencies between reads sharing a pID.

HIV drug resistance detected during low-level viraemia is associated with subsequent virologic failure.

Background: The clinical implications of emergent HIV drug resistance on samples with low-level viraemia (LLV <1000 copies/ml) remain unclear. We undertook the present analysis to evaluate the impact of emergent HIV drug resistance at LLV on the risk of subsequent virologic failure.

Methods: One thousand, nine hundred and sixty-five patients had genotype results at LLV.

Genotypic analysis of the V3 region of HIV from virologic nonresponders to maraviroc-containing regimens reveals distinct patterns of failure.

Changes in HIV tropism from R5 to non-R5 or development of drug resistance is often associated with virologic failure in patients treated with maraviroc, a CCR5 antagonist. We sought to examine changes in HIV envelope sequences and inferred tropism in patients who did not respond to maraviroc-based regimens. We selected 181 patients who experienced early virologic failure on maraviroc-containing therapy in the MOTIVATE trials.

Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch collaborative study.

The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR).

Use of cellular HIV DNA to predict virologic response to maraviroc: performance of population-based and deep sequencing.

Background: A tropism test is required before administration of the antiretroviral drug maraviroc. However, plasma RNA testing is not possible in patients with undetectable plasma viral loads. Here we assess genotypic testing of cellular human immunodeficiency virus (HIV) DNA from peripheral blood mononuclear cells (PBMCs) to predict virologic responses in treatment-experienced patients beginning maraviroc-containing regimens.

Development of a novel codon-specific polymerase chain reaction for the detection of CXCR4-utilizing HIV type 1 subtype B.

Insight concerning the switch in HIV-1 coreceptor use will lead to a better understanding of HIV-1 pathogenesis and host-virus dynamics. Predicting CXCR4 utilization by analyzing HIV-1 envelope consensus sequences is highly specific, but minority variants in the viral population are often missed resulting in low sensitivity. Commercial phenotypic assays are costly, and the development of sensitive in-house phenotypic assays to detect CXCR4-using HIV may not be feasible for some laboratories.

Factors influencing the sensitivity and specificity of conventional sequencing in human immunodeficiency virus type 1 tropism testing.

Human immunodeficiency virus type 1 (HIV-1) V3 loop sequence can be used to infer viral coreceptor use. The effect of input copy number on population-based sequencing of the V3 loop of HIV-1 was examined through replicate deep and population-based sequencing of samples with known tropism, a heterogeneous clinical sample (624 population-based sequences and 47 deep-sequencing replicates), and a large cohort of clinical samples from phase III clinical trials of maraviroc including the MOTIVATE/A4001029 studies (n = 1,521). Proviral DNA from two independent samples from each of 101 patients from the MOTIVATE/A4001029 studies was also analyzed.

Reconstructing the dynamics of HIV evolution within hosts from serial deep sequence data.

At the early stage of infection, human immunodeficiency virus (HIV)-1 predominantly uses the CCR5 coreceptor for host cell entry. The subsequent emergence of HIV variants that use the CXCR4 coreceptor in roughly half of all infections is associated with an accelerated decline of CD4+ T-cells and rate of progression to AIDS. The presence of a 'fitness valley' separating CCR5- and CXCR4-using genotypes is postulated to be a biological determinant of whether the HIV coreceptor switch occurs.

Prolonged and substantial discordance in prevalence of raltegravir-resistant HIV-1 in plasma versus PBMC samples revealed by 454 "deep" sequencing.

The evolution of drug resistance mutations in plasma samples is relatively well-characterized. However, the viral population and diversity in other body compartments such as peripheral blood mononuclear cells (PBMC) remains poorly understood. Previous studies have mostly focused on protease and reverse transcriptase drug resistance mutations (DRMs).

Technical and regulatory shortcomings of the TaqMan version 1 HIV viral load assay.

Background: The lower limit of detection of the original Roche Amplicor HIV plasma viral load (pVL) assay (50 copies/mL) has defined HIV treatment success. The Amplicor assay, however, has been replaced by the Roche TaqMan assay(s). Changes to the limits of detection and calibration have not been validated for clinical utility.

Next-generation sequencing to assess HIV tropism.

Purpose Of Review: Deep sequencing of the V3 region of the HIV envelope gene can detect minority non-R5 variants in patients with high sensitivity and specificity. As next-generation sequencing approaches have matured, the clinical utility of deep sequencing for HIV tropism has entered the clinic. Accurate and sensitive tropism testing is essential for successful treatment with the CCR5 antagonist class of antiretrovirals.

Deep V3 sequencing for HIV type 1 tropism in treatment-naive patients: a reanalysis of the MERIT trial of maraviroc.

Background: Deep sequencing is a highly sensitive technique that can detect and quantify the proportion of non-R5 human immunodeficiency virus (HIV) variants, including small minorities, that may emerge and cause virologic failure in patients who receive maraviroc-containing regimens. We retrospectively tested the ability of deep sequencing to predict response to a maraviroc-containing regimen in the Maraviroc versus Efavirenz in Treatment-Naive Patients (MERIT) trial. Results were compared with those obtained using the Enhanced Sensitivity Trofile Assay (ESTA), which is widely used in clinical practice.

Detection of inferred CCR5- and CXCR4-using HIV-1 variants and evolutionary intermediates using ultra-deep pyrosequencing.

The emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants is associated with accelerated disease progression. CXCR4-using variants are believed to evolve from CCR5-using variants, but due to the extremely low frequency at which transitional intermediate variants are often present, the kinetics and mutational pathways involved in this process have been difficult to study and are therefore poorly understood. Here, we used ultra-deep sequencing of the V3 loop of the viral envelope in combination with the V3-based coreceptor prediction tools PSSM(NSI/SI) and geno2pheno([coreceptor]) to detect HIV-1 variants during the transition from CCR5- to CXCR4-usage.

Mutations in gp41 are correlated with coreceptor tropism but do not improve prediction methods substantially.

Background: The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations.

Methods: Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort (n=444) and from patients screened for the MOTIVATE studies (n=1,916; 859 with maraviroc outcome data).

"Dynamic range" of inferred phenotypic HIV drug resistance values in clinical practice.

Background: 'Virtual' or inferred phenotypes (vPhenotypes) are commonly used to assess resistance to antiretroviral agents in patients failing therapy. In this study, we provide a clinical context for understanding vPhenotype values.

Methods: All HIV-infected persons enrolled in the British Columbia Drug Treatment Program with a baseline plasma viral load (pVL) and follow-up genotypic resistance and pVL results were included up to October 29, 2008 (N = 5,277).

Deep sequencing to infer HIV-1 co-receptor usage: application to three clinical trials of maraviroc in treatment-experienced patients.

Background: The Maraviroc versus Optimized Therapy in Viremic Antiretroviral Treatment-Experienced Patients (MOTIVATE) studies compared maraviroc versus placebo in treatment-experienced patients with CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1), screened using the original Trofile assay. A subset with non-R5 HIV infection entered the A4001029 trial. We retrospectively examined the performance of a genotypic tropism assay based on deep sequencing of the HIV env V3 loop in predicting virologic response to maraviroc in these trials.

Genotypic inference of HIV-1 tropism using population-based sequencing of V3.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.

Methods: Viral RNA is extracted from blood plasma.

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

Background: Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods.

Methods: Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA.

Phylogenetic analysis of population-based and deep sequencing data to identify coevolving sites in the nef gene of HIV-1.

Rapidly evolving viruses such as HIV-1 display extensive sequence variation in response to host-specific selection, while simultaneously maintaining functions that are critical to replication and infectivity. This apparent conflict between diversifying and purifying selection may be resolved by an abundance of epistatic interactions such that the same functional requirements can be met by highly divergent sequences. We investigate this hypothesis by conducting an extensive characterization of sequence variation in the HIV-1 nef gene that encodes a highly variable multifunctional protein.

HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins.

Background: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA)-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies.