Reanalysis of Coreceptor Tropism in HIV-1-Infected Adults Using a Phenotypic Assay with Enhanced Sensitivity

Objectives:Genotypic Tropism Testing (GTT) tools are generally developed based on HIV-1 subtype B (HIV-1B) and used for HIV-1C as well but with a large discordance of prediction between different methods. We used an established phenotypic assay for comparison with GTT methods and for the determination of in vitro maraviroc sensitivity of pure R5-tropic and dual-tropic HIV-1C.Methods:Plasma was obtained from 58 HIV-1C infected Ethiopians. Envgp120 was cloned into a luciferase tagged NL4-3 plasmid. Phenotypic tropism was determined by in house method and the V3 sequences were analysed by five GTT methods. In vitro maraviroc sensitivity of R5-tropic and dual-tropic isolates were compared in the TZMbl cell-line.Results:The phenotypes were classified as R5 in 92.4% and dual tropic (R5X4) in 7.6% of 79 clones. The concordance between phenotype and genotype ranged from 64.7% to 84.3% depending on the GTT method. Only 46.9% of the R5 phenotypes were predicted as R5 by all GTT tools while R5X4 phenotypes were predicted as X4 by four methods, but not by Raymond’s method. All six tested phenotypic R5 clones, as well as five of six of dual tropic clones, showed a dose response to maraviroc.Conclusion:There is a high discordance between GTT methods, which underestimates the presence of R5 and overestimates X4 strains compared to a phenotypic assay. Currently available GTT algorithms should be further improved for tropism prediction in HIV-1C. Maraviroc has an in vitro activity against most HIV-1C viruses and could be considered as an alternative regimen in individuals infected with CCR5-tropic HIV-1C viruses.

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02710-13 ◽

2014 ◽

Vol 58 (4) ◽

pp. 2167-2185 ◽

Cited By ~ 48

Author(s):

Richard M. Gibson ◽

Ashley M. Meyer ◽

Dane Winner ◽

John Archer ◽

Felix Feyertag ◽

...

Keyword(s):

Reverse Transcriptase ◽

Deep Sequencing ◽

False Positive Rate ◽

Antiretroviral Drugs ◽

Clinical Samples ◽

Personal Genome ◽

Drug Resistant ◽

Coreceptor Tropism ◽

Enhanced Sensitivity ◽

Hiv 1

ABSTRACTWith 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derivedgag-p2/NCp7/p1/p6/pol-PR/RT/IN- andenv-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals.

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Impact of Mutations Outside the V3 Region on Coreceptor Tropism Phenotypically Assessed in Patients Infected with HIV-1 Subtype B

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00743-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5078-5084 ◽

Cited By ~ 20

Author(s):

Laura Monno ◽

Annalisa Saracino ◽

Luigia Scudeller ◽

Grazia Punzi ◽

Gaetano Brindicci ◽

...

Keyword(s):

Amino Acid ◽

Statistical Significance ◽

Net Charge ◽

Variable Regions ◽

Coreceptor Tropism ◽

Enhanced Sensitivity ◽

Subtype B ◽

End Stage ◽

The Impact ◽

Hiv 1

ABSTRACTHIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P< 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early- and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.

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Novel recombinant phenotypic assay for clonal analysis of reverse transcriptase mutations conferring drug resistance to HIV-1 variants

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkh165 ◽

2004 ◽

Vol 53 (5) ◽

pp. 766-771 ◽

Cited By ~ 5

Author(s):

S. Paolucci

Keyword(s):

Drug Resistance ◽

Reverse Transcriptase ◽

Clonal Analysis ◽

Phenotypic Assay ◽

Hiv 1

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Comparison of levels of HIV-1 resistance to protease inhibitors by recombinant versus conventional virus phenotypic assay and two genotypic interpretation procedures in treatment-naive and HAART-experienced HIV-infected patients

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkg016 ◽

2002 ◽

Vol 51 (1) ◽

pp. 135-139 ◽

Cited By ~ 9

Author(s):

S. Paolucci

Keyword(s):

Protease Inhibitors ◽

Phenotypic Assay ◽

Treatment Naïve ◽

Hiv 1

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Removal of a Single N-Linked Glycan in Human Immunodeficiency Virus Type 1 gp120 Results in an Enhanced Ability To Induce Neutralizing Antibody Responses

Journal of Virology ◽

10.1128/jvi.01691-07 ◽

2007 ◽

Vol 82 (2) ◽

pp. 638-651 ◽

Cited By ~ 127

Author(s):

Yun Li ◽

Bradley Cleveland ◽

Igor Klots ◽

Bruce Travis ◽

Barbra A. Richardson ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Pronounced Increase ◽

Enhanced Sensitivity ◽

Subtype B ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Glycans on human immunodeficiency virus (HIV) envelope protein play an important role in infection and evasion from host immune responses. To examine the role of specific glycans, we introduced single or multiple mutations into potential N-linked glycosylation sites in hypervariable regions (V1 to V3) of the env gene of HIV type 1 (HIV-1) 89.6. Three mutants tested showed enhanced sensitivity to soluble CD4. Mutant N7 (N197Q) in the carboxy-terminal stem of the V2 loop showed the most pronounced increase in sensitivity to broadly neutralizing antibodies (NtAbs), including those targeting the CD4-binding site (IgG1b12) and the V3 loop (447-52D). This mutant is also sensitive to CD4-induced NtAb 17b in the absence of CD4. Unlike the wild-type (WT) Env, mutant N7 mediates CD4-independent infection in U87-CXCR4 cells. To study the immunogenicity of mutant Env, we immunized pig-tailed macaques with recombinant vaccinia viruses, one expressing SIVmac239 Gag-Pol and the other expressing HIV-1 89.6 Env gp160 in WT or mutant forms. Animals were boosted 14 to 16 months later with simian immunodeficiency virus gag DNA and the cognate gp140 protein before intrarectal challenge with SHIV89.6P-MN. Day-of-challenge sera from animals immunized with mutant N7 Env had significantly higher and broader neutralizing activities than sera from WT Env-immunized animals. Neutralizing activity was observed against SHIV89.6, SHIV89.6P-MN, HIV-1 SF162, and a panel of subtype B primary isolates. Compared to control animals, immunized animals showed significant reduction of plasma viral load and increased survival after challenge, which correlated with prechallenge NtAb titers. These results indicate the potential advantages for glycan modification in vaccine design, although the role of specific glycans requires further examination.

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Determination of HIV-1 Chemokine Coreceptor Tropism Using Transduced Human Osteosarcoma (HOS) Cells

HIV Protocols ◽

10.1385/0-89603-369-4:29 ◽

2003 ◽

pp. 29-34

Author(s):

Mark K. Louder ◽

John R. Mascola

Keyword(s):

Coreceptor Tropism ◽

Human Osteosarcoma ◽

Hiv 1

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Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.2.495-501.2001 ◽

2001 ◽

Vol 45 (2) ◽

pp. 495-501 ◽

Cited By ~ 65

Author(s):

Atsuko Hachiya ◽

Saori Aizawa-Matsuoka ◽

Mari Tanaka ◽

Yukiko Takahashi ◽

Setsuko Ida ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Cell Clone ◽

Drug Susceptibility ◽

Phenotypic Resistance ◽

Phenotypic Assay ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.

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