"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.

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.

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.

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.

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.

Current V3 genotyping algorithms are inadequate for predicting X4 co-receptor usage in clinical isolates.

Objective: Integrating CCR5 antagonists into clinical practice would benefit from accurate assays of co-receptor usage (CCR5 versus CXCR4) with fast turnaround and low cost.

Design: Published HIV V3-loop based predictors of co-receptor usage were compared with actual phenotypic tropism results in a large cohort of antiretroviral naive individuals to determine accuracy on clinical samples and identify areas for improvement.

Methods: Aligned HIV envelope V3 loop sequences (n = 977), derived by bulk sequencing were analyzed by six methods: the 11/25 rule; a neural network (NN), two support vector machines, and two subtype-B position specific scoring matrices (PSSM).