Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

“…DeepGen HIV was designed to use the same two overlapping amplicons covering the 3= end of Gag and the entire pol gene used in the phenotypic HIV-1 assay ViralARTS HIV (13), with the addition of the V3 region of the gp120 in the env gene, which allows the possibility to reflex from the ultrasensitive genotypic test (DeepGen HIV) to the comprehensive cell-based ViralARTS HIV assay using the same PCR products. The 97% amplification success of the two overlapping fragments covering the gag-p2/NCp7/p1/p6/ pol-PR/RT/IN region from plasma samples with Ն1,000 copies/ml of HIV RNA (13) was matched here by the successful amplification (92%) of the env-V3 fragment from plasma samples with a similar viral load, which is comparable to the success rates observed in other studies (23,62,109). Moreover, we have been able to PCR amplify intermittently these long fragments from plasma samples with viral loads between 50 and 1,000 copies/ml, depending on the quality of the clinical specimen (data not shown).…”

“…Written informed consent was obtained from the patients before participation in the study, as previously described (40,70). HIV-1 coreceptor tropism was determined at baseline using two phenotypic assays, i.e., the original version of the Trofile (19) and VeriTrop (23), and by population sequencing analyzed with Geno2Pheno (27), with false-positive rates (FPR) (predicted frequency of classifying an R5 sequence as a non-R5 virus) based on optimized cutoffs associated with the analysis of clinical data from MOTIVATE (2.5% and 5.75%) (63). Finally, plasma samples were obtained from HIV-infected individuals at the Infectious Diseases Unit Virgen del Rocio University Hospital (Seville, Spain) participating in a study to evaluate the use of an 8-day maraviroc monotherapy clinical test (MCT) (71,72).…”

“…Similarly, since treatment with CCR5 antagonists requires prior knowledge of the HIV-1 coreceptor tropism in the patient, i.e., CCR5-or CXCR4-tropic viruses (R5 and X4, respectively), dual tropism (R5/X4), or a mixture of both R5 and X4 viruses (7,15), a multitude of phenotypic and genotypic approaches to determine HIV-1 coreceptor tropism have been developed (8,16,17). Phenotypic assays to deter-mine HIV-1 drug resistance or tropism usually involve the generation of patient-derived pol (11)(12)(13) or env recombinant (18)(19)(20) viruses, respectively, to quantify their ability to infect susceptible cell lines expressing the appropriate HIV-1 receptors and coreceptors; in the case of HIV-1 tropism, these measures may also be based on the quantification of cell-to-cell fusion events (21)(22)(23). Conversely, genotypic HIV-1 tropism tests (8,15,16,(24)(25)(26) take advantage of the properties of specific regions in the env gene as determinants of CCR5 or CXCR4 tropism, mainly the V3 region of gp120, and their interpretations are based on a series of bioinformatics methods to infer the ability of HIV-1 to use either or both coreceptors to enter host cells (27)(28)(29)(30).…”

HIV-1 Group O Genotypes and Phenotypes: Relationship to Fitness and Susceptibility to Antiretroviral Drugs

“…Because group O and M share <40% sequence similarity in the V3 loop, various algorithms might not predict coreceptor usage of HIV-1 group O. [16][17][18] Previous studies indicate that most HIV-1 group O isolates may show limited susceptibility to protease inhibitors due to the presence of secondary PI resistance mutations (10I, 15V, 36I, 41K, 62V, 64V, 71V, and 93L) in most strains and might also be difficult to manage. 13,19 In fact, two case studies reported rapid resistance upon treatment of group O-infected individuals with PI-based regimens.…”

An in vitro Model to Mimic Selection of Replication-Competent HIV-1 Intersubtype Recombination in Dual or Superinfected Patients

“…One million (1×10 6 ) U87.CD4.CCR5 cells were cultured overnight in 100 mm petri dishes (Day 1) and were transfected 24 hours later (Day 2) with pDM128fLuc (containing an HIV-1 LTR-driven luciferase gene) using FuGENE 6 transfection reagent (Roche) as previously described [46]. Also on day 1, 6.5×10 4 HEK-293T cells were plated in 24-well plates, and were transfected 24 hours later with recombinant env -containing plasmids pREC_nfl_HIV_envA/D.…”