Deep Sequencing to Infer HIV-1 Co-Receptor Usage: Application to Three Clinical Trials of Maraviroc in Treatment-Experienced Patients

Swenson, Luke C.; Mo, Theresa; Dong, Winnie; Zhong, Xiaoyin; Woods, Conan K.; Jensen, Mark A.; Thielen, Alexander; Chapman, Douglass; Lewis, Marilyn; James, Ian; Heera, Jayvant; Valdez, Hernán; Harrigan, P. Richard

doi:10.1093/infdis/jiq030

Cited by 142 publications

(153 citation statements)

References 30 publications

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“…Most of those studies used 454 sequencing, which was limited by homopolymer errors and relative low throughput compared to the Illumina or IonTorrent platform (25). In these studies, the presence of X4 variants was defined by the detection of Ͼ2% of sequences with FPR values of Ͻ3.5% in the viral population, as seen in the MERIT and MOTIVATE cohort studies (12,13,49). However, the predetermined FPR is arbitrary, and our data show that an FPR of between 2% to 6% can indicate either X4 or R5 variants.…”

Section: Discussionmentioning

confidence: 99%

“…The position-specific scoring matrix (PSSM) analyzes the entire V3 sequence and predicts X4 variants based on a scoring of the probability of the amino acid at each position being overrepresented among X4 sequences versus R5 sequences (10). The Geno2Pheno (G2P) algorithm (11) also analyzes the entire V3 sequence and provides a quantitative score (false-positive rate [FPR]) representing the probability of falsely predicting an R5 variant as an X4 variant; the validity of this approach has been assessed in several clinical trials (12)(13)(14)(15)(16). G2P has been widely used in research and clinical settings, but it requires a preset FPR cutoff to call X4 variants.…”

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confidence: 99%

“…EPD PCR is time-consuming and labor-intensive, making it challenging to analyze more than a few dozen viral genomes per sample (20)(21)(22), which limits the depth of sampling of viral genomes when assessing the complexity of the viral population. Recently, viral population studies have started to incorporate deep-sequencing or next-generation sequencing (NGS) technologies that have the capacity to greatly extend the depth of sampling (12,(23)(24)(25)(26)(27)(28). However, conventional approaches using NGS in HIV-1 population studies have serious limitations in representing an accurate sampling of the original viral population.…”

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confidence: 99%

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Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

Zhou

Bednar

Sturdevant

et al. 2016

J Virol

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HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of <2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated. IMPORTANCEPrimer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants by using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting that X4 and R5 variants are genetically isolated and may be replicating in different cell types or that X4/R5 recombinants have reduced fitness. H uman immunodeficiency virus type 1 (HIV-1) requires the CD4 receptor and a coreceptor to infect host cells. Entry is mediated by the viral envelope protein (Env), which is processed to give two associated subunits, gp120 and gp41, that assemble into a trimeric structure embedded in the viral envelope/membrane. The binding of gp120 to CD4 triggers a structural change that exposes its variable region 3 (V3) loop, which is part of the coreceptor domain (1, 2). The majority of transmitted/founder (T/F) viruses and viruses isolated from the clinically latent stage in HIV-infected subjects use CCR5 as the coreceptor, making the virus CCR5 tropic (or an R5 virus). The virus can evolve to use CXCR4 (CXCR4 tropic [or an X4 virus]), and viruses that have switched core...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

Zhou

Bednar

Sturdevant

et al. 2016

J Virol

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“…Accurate determination of coreceptor usage is therefore necessary to optimize treatment strategies before the initiation of and during therapy with CCR5 inhibitors. Techniques such as deep V3 sequencing may be useful for identifying treatment-experienced individuals who could benefit from CCR5-antagonist-containing regimens [158]. Ongoing improvements in genotypic methodologies and algorithms may eventually render them a viable alternative to phenotypic assays, much like the genotypic resistance methods.…”

Section: In Vivo Resistance: Potential Expansion Of Pre-existing CXCmentioning

confidence: 99%

Chemokine Receptors as Therapeutic Targets in HIV Infection

Anastassopoulou¹

2012

Immunodeficiency

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“…Next-generation sequencing may prove to be the best method of detection, as it is not only sensitive, but can also give a fairly accurate estimation of the percent X4 in a population. 7,8 However, next-generation sequencing is still fairly expensive and handling the data can be onerous. Phenotyping is another option for X4 detection, but many laboratories do not have the facilities or expertise to develop a sensitive culture-based assay, and commercial assays, such as Trofile, are costly.…”

Section: Introductionmentioning

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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Deep Sequencing to Infer HIV-1 Co-Receptor Usage: Application to Three Clinical Trials of Maraviroc in Treatment-Experienced Patients

Abstract: This large study establishes deep V3 sequencing as a promising tool for identifying treatment-experienced individuals who could benefit from CCR5-antagonist-containing regimens.

Cited by 142 publications

References 30 publications

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

Chemokine Receptors as Therapeutic Targets in HIV Infection

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

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