Improved Detection of CXCR4-Using HIV by V3 Genotyping: Application of Population-Based and “Deep” Sequencing to Plasma RNA and Proviral DNA

Swenson, Luke C.; Moores, Andrew; Low, Andrew; Thielen, Alexander; Dong, Winnie; Woods, Conan K.; Jensen, Mark A.; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P. Richard

doi:10.1097/qai.0b013e3181d0558f

Cited by 68 publications

(70 citation statements)

References 24 publications

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“…Phenotypic assays usually involve the generation of patientderived env recombinant viruses to determine their ability to infect reporter cell lines expressing HIV-1 receptors and coreceptors (16)(17)(18), while others are based in the quantification of cell-to-cell fusion events (19,20). On the other hand, genotypic tests based on population (12,14,21) or deep sequencing (22)(23)(24) take advantage of the association of certain regions in the env gene as determinants of CCR5 or CXCR4 tropism, mainly in the V3 region of the gp120, and their interpretation based on a series of algorithms and bioinformatic tools to infer the ability of HIV-1 to use any or both coreceptors to enter host cells (25)(26)(27)(28). As expected, both approaches have advantages and disadvantages, but particular emphasis has been made on their sensitivity to detect minor non-R5 variants, turnaround time, and, more important, their accuracy to determine HIV-1 coreceptor tropism (12,14,29).…”

mentioning

confidence: 99%

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

Weber

Vázquez²,

Winner³

et al. 2013

J Clin Microbiol

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mentioning

confidence: 99%

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

Weber

Vázquez²,

Winner³

et al. 2013

J Clin Microbiol

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“…A clinical proof for the validity of this exclusion is not available, and tropism changes between screening and baseline in the clinical studies may reflect the test variability or a certain instability of the viral tropism (50)(51)(52). Recent deep-sequencing analyses have demonstrated that low proportions of X4-tropic viruses can be found in almost any clinical sample, yet the clinical relevance, e.g., of X4-tropic virus minorities below 2% in a given virus population found in clinical specimens remains unclear (53).…”

Section: Resultsmentioning

confidence: 99%

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

et al. 2015

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dKey clinical studies for HIV coreceptor antagonists have used the phenotyping-based Trofile test. Meanwhile various simplerto-do genotypic tests have become available that are compatible with standard laboratory equipment and Web-based interpretation tools. However, these systems typically analyze only the most prominent virus sequence in a specimen. We present a new diagnostic HIV tropism test not needing DNA sequencing. The system, XTrack, uses physical properties of DNA duplexes after hybridization of single-stranded HIV-1 env V3 loop probes to the clinical specimen. Resulting "heteroduplexes" possess unique properties driven by sequence relatedness to the reference and resulting in a discrete electrophoretic mobility. A detailed optimization process identified diagnostic probe candidates relating best to a large number of HIV-1 sequences with known tropism. From over 500 V3 sequences representing all main HIV-1 subtypes (Los Alamos database), we obtained a small set of probes to determine the tropism in clinical samples. We found a high concordance with the commercial TrofileES test (84.9%) and the Web-based tool Geno2Pheno (83.0%). Moreover, the new system reveals mixed virus populations, and it was successful on specimens with low virus loads or on provirus from leukocytes. A replicative phenotyping system was used for validation. Our data show that the XTrack test is favorably suitable for routine diagnostics. It detects and dissects mixed virus populations and viral minorities; samples with viral loads (VL) of <200 copies/ml are successfully analyzed. We further expect that the principles of the platform can be adapted also to other sequence-divergent pathogens, such as hepatitis B and C viruses. The predominant virus variant in early stages of the clinical manifestation of disease, CCR5-tropic HIV, is found in approximately 80% of treatment-naive patients (1, 2). Although this number can vary for the different virus subtypes, the percentage of CXCR4-tropic HIV isolates is generally low and tends to rise with disease progression (3-6). Nevertheless, the fraction of CCR5-tropic viruses in clinical specimens continues to stay at Ͼ50% throughout the course of infection (7,8). As such, the molecular interactions between the viral envelope and the cellular chemokine receptor CCR5 were recognized as potentially attractive targets for drug development and have yielded compounds and drugs able to specifically block CCR5-tropic HIV (9-11). It is this selectivity of the inhibition of one (CCR5) and not the other (CXCR4) viral coreceptor that necessitates tropism testing prior to prescribing drugs of this particular class. Although the chemokine receptor binding site in the HIV envelope is constituted mainly by the V3 loop, the V1/V2 regions, and the C4 conserved region in the HIV protein gp120, coreceptor tropism is dictated predominantly by amino acid sequences of the V3 region (12, 13). But also sequences of other variable env regions can contribute as secondary sites to the viral tropism (14-17).Initially, all...

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“…Recent massive parallel sequencing also detected minor X4 populations at a higher frequency compared to ESTA. [19][20][21] Additionally, the presence of basic amino acids at codons 11, 24, or 25 does not guarantee that the variant will use CXCR4 efficiently enough to be detected using a culture-based assay. Given that HIV-1 utilization of CXCR4 evolves over time, 1,9 genotypic, but not phenotypic, assays can potentially detect viruses that have begun the evolutionary process toward use of CXCR4.…”

Section: Discussionmentioning

confidence: 99%

Development of a Novel Codon-Specific Polymerase Chain Reaction for the Detection of CXCR4-Utilizing HIV Type 1 Subtype B

McLaughlin¹,

Swenson

et al. 2013

AIDS Research and Human Retroviruses

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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. A sensitive, inexpensive genotyping assay was developed to detect viral sequences associated with CXCR4-utilizing virus (X4). Codon-specific primer pairs were used to detect X4-associated codons at five positions in the HIV-1 envelope V3 loop (11, 13, 24, 25, and 32). Sixty plasma samples from HIV-1-infected individuals were analyzed by consensus sequencing and codonspecific PCR (CS-PCR). Forty-six of these were also phenotyped by Trofile or Enhanced Sensitivity Trofile (ESTA). CS-PCR detected X4 variants 17% more often than 11/24/25 consensus sequencing alone (n = 60), 30% more often than Trofile (n = 27), and in a limited data set, 16% more often than ESTA (n = 19). CS-PCR combined with consensus sequencing had approximately 80% concordance with ESTA.

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Improved Detection of CXCR4-Using HIV by V3 Genotyping: Application of Population-Based and “Deep” Sequencing to Plasma RNA and Proviral DNA

Abstract: Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

Cited by 68 publications

References 24 publications

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

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

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

Development of a Novel Codon-Specific Polymerase Chain Reaction for the Detection of CXCR4-Utilizing HIV Type 1 Subtype B

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