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1
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Esman A, Salamaikina S, Kirichenko A, Vinokurov M, Fomina D, Sikamov K, Syrkina A, Pokrovskaya A, Akimkin V. Promoter Methylation of HIV Coreceptor-Related Genes CCR5 and CXCR4: Original Research. Viruses 2025; 17:465. [PMID: 40284908 PMCID: PMC12030890 DOI: 10.3390/v17040465] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2025] [Revised: 03/03/2025] [Accepted: 03/06/2025] [Indexed: 04/29/2025] Open
Abstract
The persistence of human immunodeficiency virus (HIV) within viral reservoirs poses significant challenges to eradication efforts. Epigenetic alterations, including DNA methylation, are potential factors influencing the latency and persistence of HIV. This study details the development and application of techniques to assess CpG methylation in the promoter regions of the CCR5 and CXCR4 genes, which are key HIV-1 coreceptors. Using both Sanger sequencing and pyrosequencing methods, we examined 51 biological samples from 17 people living with HIV at three time points: baseline (week 0) and post-antiretroviral therapy (ART) at weeks 24 and 48. Our results revealed that CXCR4 promoter CpG sites were largely unmethylated, while CCR5 promoter CpGs exhibited significant variability in methylation levels. Specifically, CCR5 CpG 1 showed a significant decrease in methylation from week 0 to week 48, while CXCR4 CpG 3 displayed a significant decrease between week 0 and week 24. These differences were statistically significant when compared with non-HIV-infected controls. These findings demonstrate distinct methylation patterns between CCR5 and CXCR4 promoters in people living with HIV over time, suggesting that epigenetic modifications may play a role in regulating the persistence of HIV-1. Our techniques provide a reliable framework for assessing gene promoter methylation and could be applied in further research on the epigenetics of HIV.
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Affiliation(s)
- Anna Esman
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Svetlana Salamaikina
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Alina Kirichenko
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Michael Vinokurov
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Darya Fomina
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
- State Research Center—Burnazyan Federal Medical Biophysical Center of Federal Medical Biological Agency, 123098 Moscow, Russia
| | - Kirill Sikamov
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Arina Syrkina
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
| | - Anastasia Pokrovskaya
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
- Medical Institute, Peoples’ Friendship University of Russia (RUDN University), 117198 Moscow, Russia
| | - Vasily Akimkin
- Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Central Research Institute of Epidemiology, 111123 Moscow, Russia
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2
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Ko D, McLaughlin S, Deng W, Mullins JI, Dragavon J, Harb S, Coombs RW, Frenkel LM. Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile ®. Viruses 2024; 16:510. [PMID: 38675853 PMCID: PMC11053691 DOI: 10.3390/v16040510] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Revised: 03/22/2024] [Accepted: 03/22/2024] [Indexed: 04/28/2024] Open
Abstract
HIV-1 typically infects cells via the CD4 receptor and CCR5 or CXCR4 co-receptors. Maraviroc is a CCR5-specific viral entry inhibitor; knowledge of viral co-receptor specificity is important prior to usage. We developed and validated an economical V3-env Illumina-based assay to detect and quantify the frequency of viruses utilizing each co-receptor. Plasma from 54 HIV+ participants (subtype B) was tested. The viral template cDNA was generated from plasma RNA with unique molecular identifiers (UMIs). The sequences were aligned and collapsed by the UMIs with a custom bioinformatics pipeline. Co-receptor usage, determined by codon analysis and online phenotype predictors PSSM and Geno2pheno, were compared to existing Trofile® data. The cost of V3-UMI was tallied. The sequences interpreted by Geno2pheno using the most conservative cut-off, a 2% false-positive-rate (FPR), predicted CXCR4 usage with the greatest sensitivity (76%) and specificity (100%); PSSM and codon analysis had similar sensitivity and lower specificity. Discordant Trofile® and genotypic results were more common when participants had specimens from different dates analyzed by either assay. V3-UMI reagents cost USD$62/specimen. A batch of ≤20 specimens required 5 h of technical time across 1.5 days. V3-UMI predicts HIV tropism at a sensitivity and specificity similar to those of Trofile®, is relatively inexpensive, and could be performed by most central laboratories. The adoption of V3-UMI could expand HIV drug therapeutic options in lower-resource settings that currently do not have access to phenotypic HIV tropism testing.
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Affiliation(s)
- Daisy Ko
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA 98109, USA; (D.K.)
| | - Sherry McLaughlin
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA 98109, USA; (D.K.)
| | - Wenjie Deng
- Department of Microbiology, University of Washington, Seattle, WA 98195, USA; (W.D.); (J.I.M.)
| | - James I. Mullins
- Department of Microbiology, University of Washington, Seattle, WA 98195, USA; (W.D.); (J.I.M.)
- Department of Medicine, University of Washington, Seattle, WA 98104, USA
- Department of Global Health, University of Washington, Seattle, WA 98105, USA
| | - Joan Dragavon
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA; (J.D.); (S.H.); (R.W.C.)
| | - Socorro Harb
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA; (J.D.); (S.H.); (R.W.C.)
| | - Robert W. Coombs
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA; (J.D.); (S.H.); (R.W.C.)
| | - Lisa M. Frenkel
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA 98109, USA; (D.K.)
- Department of Medicine, University of Washington, Seattle, WA 98104, USA
- Department of Global Health, University of Washington, Seattle, WA 98105, USA
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA; (J.D.); (S.H.); (R.W.C.)
- Department of Pediatrics, University of Washington, Seattle, WA 98195, USA
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3
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Ndashimye E, Reyes PS, Arts EJ. New antiretroviral inhibitors and HIV-1 drug resistance: more focus on 90% HIV-1 isolates? FEMS Microbiol Rev 2023; 47:fuac040. [PMID: 36130204 PMCID: PMC9841967 DOI: 10.1093/femsre/fuac040] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 09/13/2022] [Accepted: 09/18/2022] [Indexed: 01/21/2023] Open
Abstract
Combined HIV antiretroviral therapy (cART) has been effective except if drug resistance emerges. As cART has been rolled out in low-income countries, drug resistance has emerged at higher rates than observed in high income countries due to factors including initial use of these less tolerated cART regimens, intermittent disruptions in drug supply, and insufficient treatment monitoring. These socioeconomic factors impacting drug resistance are compounded by viral mechanistic differences by divergent HIV-1 non-B subtypes compared to HIV-1 subtype B that largely infects the high-income countries (just 10% of 37 million infected). This review compares the inhibition and resistance of diverse HIV-1 subtypes and strains to the various approved drugs as well as novel inhibitors in clinical trials. Initial sequence variations and differences in replicative fitness between HIV-1 subtypes pushes strains through different fitness landscapes to escape from drug selective pressure. The discussions here provide insight to patient care givers and policy makers on how best to use currently approved ART options and reduce the emergence of drug resistance in ∼33 million individuals infected with HIV-1 subtype A, C, D, G, and recombinants forms. Unfortunately, over 98% of the literature on cART resistance relates to HIV-1 subtype B.
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Affiliation(s)
- Emmanuel Ndashimye
- Department of Microbiology and Immunology, Western University Schulich School of Medicine & Dentistry, Western University, N6A 3K7, London, Ontario, Canada
- Joint Clinical Research Centre, -Center for AIDS Research Laboratories, 256, Kampala, Uganda
| | - Paul S Reyes
- Department of Microbiology and Immunology, Western University Schulich School of Medicine & Dentistry, Western University, N6A 3K7, London, Ontario, Canada
| | - Eric J Arts
- Department of Microbiology and Immunology, Western University Schulich School of Medicine & Dentistry, Western University, N6A 3K7, London, Ontario, Canada
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4
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Determination of HIV Tropism in Patients with Antiretroviral Therapy Failure in Arkhangelsk Region. PROBLEMS OF PARTICULARLY DANGEROUS INFECTIONS 2022. [DOI: 10.21055/0370-1069-2022-3-120-128] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The aim of the study was to determine the tropism of the human immunodeficiency virus in patients with virological failure of antiretroviral therapy (ART) from the Arkhangelsk Region based on the analysis of the env gene V3 loop nucleotide sequence.Materials and methods. We used blood plasma samples obtained from 76 HIV-infected persons from the Arkhangelsk Region with virological failure of antiretroviral therapy. The nucleotide sequences of the HIV env gene C2-V3-C3 region were studied by PCR followed by sequencing. The genotype of the studied strains was determined based on the analysis of their phylogenetic relations with reference sequences from the international GenBank database, as well as using specialized programs. To predict viral tropism, the Garrido rule and the online bioinformatic tool Geno2Pheno[coreceptor] were used. The Geno2Pheno[coreceptor] algorithm, determines the false positive rate (FPR) based on the analysis of the env gene V3 loop nucleotide sequence. Results and discussion. Significantly lower representation of R5X4/X4-tropic HIV variants in long-term infected persons with subsubtype A6 virus compared to subtype B virus has been shown. For all FPR cut-off algorithms, a significant correlation between subtype and HIV tropism was observed (p=0.0014 and p=0.013 for FPR 10 % and FPR 20 %, respectively). While among subtype B strains, at least 57 % were identified as R5X4/X4-tropic variants (for an FPR of 10 %), including two strains classified as X4-tropic; among HIV subsubtype A6 even at an FPR of 20 %, the frequency of R5X4/X4-tropic samples only slightly exceeded 22 %. It can be assumed that the dynamics of changes in HIV tropism depends on the virus subtype. Significant differences in the distribution of amino acid residues of the V3 region sequences in the examined group between R5-tropic and R5X4/X4-tropic strains of subsubtype A6 for positions 18 (χ2=7.616, p=0.0058), 21 (χ2=7.281, p=0.007), 24 (χ2=5.587, p=0.0181), and 34 (χ2=5.144, p=0.0233) have been demonstrated. Among the R5X4/X4-tropic strains of the A6 subsubtype, amino acid substitutions were registered at positions 6, 19, 21, 26, 29, 30, which were not found in the R5-tropic A6 strains. The high occurrence frequency of a number of mutations previously described as presumably associated with resistance to maraviroc and similar drugs may indicate a natural polymorphism characteristic of the A6 subsubtype, which does not correlate with resistance to CCR5 co-receptor antagonists.
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5
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Lewis ME, Simpson P, Mori J, Jubb B, Sullivan J, McFadyen L, van der Ryst E, Craig C, Robertson DL, Westby M. V3-Loop genotypes do not predict maraviroc susceptibility of CCR5-tropic virus or clinical response through week 48 in HIV-1-infected, treatment-experienced persons receiving optimized background regimens. Antivir Chem Chemother 2021; 29:20402066211030380. [PMID: 34343443 PMCID: PMC8369958 DOI: 10.1177/20402066211030380] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Viruses from 15 of 35 maraviroc-treated participants with virologic failure and CCR5-tropic (R5) virus in the MOTIVATE studies at Week 24 had reduced maraviroc susceptibility. On-treatment amino acid changes were observed in the viral envelope glycoprotein 120 third variable (V3)-loop stems and tips and differed between viruses. No amino acid change reliably predicted reduced susceptibility, indicating that resistance was genetic context-dependent. Through Week 24, poor adherence was associated with maraviroc-susceptible virologic failure, whereas reduced maraviroc susceptibility was associated with suboptimal background regimen activity, highlighting the importance of overall regimen activity and good adherence. Predictive values of pretreatment V3-loop sequences containing these Week 24 mutations or other variants present at >3% in pretreatment viruses of participants with virologic failure at Week 48 were retrospectively assessed. Week 48 clinical outcomes were evaluated for correlates with pretreatment V3-loop CCR5-tropic sequences from 704 participants (366 responders; 338 virologic failures [83 with R5 virus with maraviroc susceptibility assessment]). Seventy-five amino acid variants with >3% prevalence were identified among 23 V3-loop residues. Previously identified variants associated with resistance in individual isolates were represented, but none were associated reliably with virologic failure alone or in combination. Univariate analysis showed virologic-failure associations with variants 4L, 11R, and 19S (P < 0.05). However, 11R is a marker for CXCR4 tropism, whereas neither 4L nor 19S was reliably associated with reduced maraviroc susceptibility in R5 failure. These findings from a large study of V3-loop sequences confirm lack of correlation between V3-loop genotype and clinical outcome in participants treated with maraviroc.Clinical trial registration numbers (ClinicalTrials.gov): NCT00098306 and NCT00098722.
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Affiliation(s)
- M E Lewis
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,The Research Network Ltd, Sandwich, Kent, UK
| | - P Simpson
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,AstraZeneca, Cambridge, UK
| | - J Mori
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,hVIVO, Queen Mary BioEnterprise Innovation Centre, London, UK
| | - B Jubb
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK
| | - J Sullivan
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,Cytel, London, UK
| | - L McFadyen
- Pfizer Inc, Pharmacometrics, Sandwich, UK
| | - E van der Ryst
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,The Research Network Ltd, Sandwich, Kent, UK
| | - C Craig
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,The Research Network Ltd, Sandwich, Kent, UK
| | - D L Robertson
- MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
| | - M Westby
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, UK.,Centauri Therapeutics Limited, Discovery Park, Kent, UK
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6
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Obasa AE, Ambikan AT, Gupta S, Neogi U, Jacobs GB. Increased acquired protease inhibitor drug resistance mutations in minor HIV-1 quasispecies from infected patients suspected of failing on national second-line therapy in South Africa. BMC Infect Dis 2021; 21:214. [PMID: 33632139 PMCID: PMC7908688 DOI: 10.1186/s12879-021-05905-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Accepted: 02/16/2021] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND HIV-1C has been shown to have a greater risk of virological failure and reduced susceptibility towards boosted protease inhibitors (bPIs), a component of second-line combination antiretroviral therapy (cART) in South Africa. This study entailed an evaluation of HIV-1 drug resistance-associated mutations (RAMs) among minor viral populations through high-throughput sequencing genotypic resistance testing (HTS-GRT) in patients on the South African national second-line cART regimen receiving bPIs. METHODS During 2017 and 2018, 67 patient samples were sequenced using high-throughput sequencing (HTS), of which 56 samples were included in the final analysis because the patient's treatment regimen was available at the time of sampling. All patients were receiving bPIs as part of their cART. Viral RNA was extracted, and complete pol genes were amplified and sequenced using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV Drug Resistance Database. RESULTS Statistically significantly higher PI RAMs were observed in minor viral quasispecies (25%; 14/56) compared to non-nucleoside reverse transcriptase inhibitors (9%; 5/56; p = 0.042) and integrase inhibitor RAM (4%; 2/56; p = 0.002). The majority of the drug resistance mutations in the minor viral quasispecies were observed in the V82A mutation (n = 13) in protease and K65R (n = 5), K103N (n = 7) and M184V (n = 5) in reverse transcriptase. CONCLUSIONS HTS-GRT improved the identification of PI and reverse transcriptase inhibitor (RTI) RAMs in second-line cART patients from South Africa compared to the conventional GRT with ≥20% used in Sanger-based sequencing. Several RTI RAMs, such as K65R, M184V or K103N and PI RAM V82A, were identified in < 20% of the population. Deep sequencing could be of greater value in detecting acquired resistance mutations early.
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Affiliation(s)
- Adetayo Emmanuel Obasa
- Department of Pathology, Division of Medical Virology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, 7505, South Africa.
- Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden.
| | - Anoop T Ambikan
- Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden
| | - Soham Gupta
- Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden
| | - Ujjwal Neogi
- Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden
| | - Graeme Brendon Jacobs
- Department of Pathology, Division of Medical Virology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, Cape Town, 7505, South Africa
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7
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Melnyk A, Knyazev S, Vannberg F, Bunimovich L, Skums P, Zelikovsky A. Using earth mover's distance for viral outbreak investigations. BMC Genomics 2020; 21:582. [PMID: 33327932 PMCID: PMC7739463 DOI: 10.1186/s12864-020-06982-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Accepted: 08/11/2020] [Indexed: 11/10/2022] Open
Abstract
Background RNA viruses mutate at extremely high rates, forming an intra-host viral population of closely related variants, which allows them to evade the host’s immune system and makes them particularly dangerous. Viral outbreaks pose a significant threat for public health, and, in order to deal with it, it is critical to infer transmission clusters, i.e., decide whether two viral samples belong to the same outbreak. Next-generation sequencing (NGS) can significantly help in tackling outbreak-related problems. While NGS data is first obtained as short reads, existing methods rely on assembled sequences. This requires reconstruction of the entire viral population, which is complicated, error-prone and time-consuming. Results The experimental validation using sequencing data from HCV outbreaks shows that the proposed algorithm can successfully identify genetic relatedness between viral populations, infer transmission direction, transmission clusters and outbreak sources, as well as decide whether the source is present in the sequenced outbreak sample and identify it. Conclusions Introduced algorithm allows to cluster genetically related samples, infer transmission directions and predict sources of outbreaks. Validation on experimental data demonstrated that algorithm is able to reconstruct various transmission characteristics. Advantage of the method is the ability to bypass cumbersome read assembly, thus eliminating the chance to introduce new errors, and saving processing time by allowing to use raw NGS reads.
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Affiliation(s)
- Andrew Melnyk
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, GA, 30303, USA.
| | - Sergey Knyazev
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, GA, 30303, USA
| | - Fredrik Vannberg
- Georgia Institute of Technology, North Ave NW, Atlanta, GA, 30332, USA
| | - Leonid Bunimovich
- Georgia Institute of Technology, North Ave NW, Atlanta, GA, 30332, USA
| | - Pavel Skums
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, GA, 30303, USA
| | - Alex Zelikovsky
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, GA, 30303, USA.,I.M. Sechenov First Moscow State Medical University, Moscow, 119991, Russia
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8
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Kariuki SM, Selhorst P, Anthony C, Matten D, Abrahams MR, Martin DP, Ariën KK, Rebe K, Williamson C, Dorfman JR. Compartmentalization and Clonal Amplification of HIV-1 in the Male Genital Tract Characterized Using Next-Generation Sequencing. J Virol 2020; 94:e00229-20. [PMID: 32269124 PMCID: PMC7307092 DOI: 10.1128/jvi.00229-20] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2020] [Accepted: 03/16/2020] [Indexed: 12/17/2022] Open
Abstract
Compartmentalization of HIV-1 between the systemic circulation and the male genital tract may have a substantial impact on which viruses are available for sexual transmission to new hosts. We studied compartmentalization and clonal amplification of HIV-1 populations between the blood and the genital tract from 10 antiretroviral-naive men using Illumina MiSeq with a PrimerID approach. We found evidence of some degree of compartmentalization in every study participant, unlike previous studies, which collectively showed that only ∼50% of analyzed individuals exhibited compartmentalization of HIV-1 lineages between the male genital tract (MGT) and blood. Using down-sampling simulations, we determined that this disparity can be explained by differences in sampling depth in that had we sequenced to a lower depth, we would also have found compartmentalization in only ∼50% of the study participants. For most study participants, phylogenetic trees were rooted in blood, suggesting that the male genital tract reservoir is seeded by incoming variants from the blood. Clonal amplification was observed in all study participants and was a characteristic of both blood and semen viral populations. We also show evidence for independent viral replication in the genital tract in the individual with the most severely compartmentalized HIV-1 populations. The degree of clonal amplification was not obviously associated with the extent of compartmentalization. We were also unable to detect any association between history of sexually transmitted infections and level of HIV-1 compartmentalization. Overall, our findings contribute to a better understanding of the dynamics that affect the composition of virus populations that are available for transmission.IMPORTANCE Within an individual living with HIV-1, factors that restrict the movement of HIV-1 between different compartments-such as between the blood and the male genital tract-could strongly influence which viruses reach sites in the body from which they can be transmitted. Using deep sequencing, we found strong evidence of restricted HIV-1 movements between the blood and genital tract in all 10 men that we studied. We additionally found that neither the degree to which particular genetic variants of HIV-1 proliferate (in blood or genital tract) nor an individual's history of sexually transmitted infections detectably influenced the degree to which virus movements were restricted between the blood and genital tract. Last, we show evidence that viral replication gave rise to a large clonal amplification in semen in a donor with highly compartmentalized HIV-1 populations, raising the possibility that differential selection of HIV-1 variants in the genital tract may occur.
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Affiliation(s)
- Samuel Mundia Kariuki
- Division of Immunology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- International Centre for Genetic Engineering and Biotechnology, Cape Town, South Africa
- Department of Biological Sciences, School of Science, University of Eldoret, Eldoret, Kenya
| | - Philippe Selhorst
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
| | - Colin Anthony
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - David Matten
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - Melissa-Rose Abrahams
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - Darren P Martin
- Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- Insitute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - Kevin K Ariën
- Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
- Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Kevin Rebe
- Anova Health Institute, Cape Town, South Africa
- Department of Medicine, Division of Infectious Diseases and HIV Medicine, University of Cape Town, Cape Town, South Africa
| | - Carolyn Williamson
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- Insitute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - Jeffrey R Dorfman
- Division of Immunology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- Division of Medical Virology, Department of Pathology, Stellenbosch University, Cape Town, South Africa
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9
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Sidhu G, Schuster L, Liu L, Tamashiro R, Li E, Langaee T, Wagner R, Wang GP. A single variant sequencing method for sensitive and quantitative detection of HIV-1 minority variants. Sci Rep 2020; 10:8185. [PMID: 32424187 PMCID: PMC7234988 DOI: 10.1038/s41598-020-65085-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 04/20/2020] [Indexed: 10/25/2022] Open
Abstract
HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals. Drug resistant HIV variants, including minority variants, can compromise response to antiretroviral therapy. Many studies have investigated the clinical relevance of drug resistant minority variants, but the level at which minority variants become clinically relevant remains unclear. A combination of Primer-ID and deep sequencing is a promising approach that may quantify minority variants more accurately compared to standard deep sequencing. However, most studies that used the Primer-ID method have analyzed clinical samples directly. Thus, its sensitivity and quantitative accuracy have not been adequately validated using known controls. Here, we constructed defined proportions of artificial RNA and virus quasispecies and measured their relative proportions using the Primer-ID based, quantitative single-variant sequencing (qSVS) assay. Our results showed that minority variants present at 1% of quasispecies were detected reproducibly with minimal variations between technical replicates. In addition, the measured frequencies were comparable to the expected frequencies. These data validate the accuracy and reproducibility of the qSVS assay in quantifying authentic HIV minority variants, and support the use of this approach to examine the impacts of minority HIV variants on virologic response and clinical outcome.
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Affiliation(s)
- Gurjit Sidhu
- Division of Infectious Disease and Global Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA
| | - Layla Schuster
- Division of Infectious Disease and Global Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA.,Medosome Biosciences, Alachua, FL, USA
| | - Lin Liu
- Division of Infectious Disease and Global Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA.,Department of Medicine, St. Luke's Hospital, Chesterfield, MO, USA
| | - Ryan Tamashiro
- Division of Infectious Disease and Global Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA
| | - Eric Li
- Division of Infectious Disease and Global Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA
| | - Taimour Langaee
- Department of Pharmacotherapy and Translational Research, Center for Pharmacogenomics and Precision Medicine, College of Pharmacy, University of Florida, Gainesville, FL, USA
| | | | - Gary P Wang
- Division of Infectious Disease and Global Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA. .,Infectious Diseases Section, Medical Service, North Florida/South Georgia Veterans Health System, Gainesville, FL, USA.
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10
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Raymond S, Nicot F, Abravanel F, Minier L, Carcenac R, Lefebvre C, Harter A, Martin-Blondel G, Delobel P, Izopet J. Performance evaluation of the Vela Dx Sentosa next-generation sequencing system for HIV-1 DNA genotypic resistance. J Clin Virol 2019; 122:104229. [PMID: 31809945 DOI: 10.1016/j.jcv.2019.104229] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2019] [Revised: 11/15/2019] [Accepted: 11/25/2019] [Indexed: 11/20/2022]
Abstract
BACKGROUND Patients on antiretroviral therapy could benefit from HIV-1 DNA resistance genotyping for exploring virological failure with low viral load or to guide treatment simplification. Few new generation sequencing data are available. OBJECTIVE To check that the automated deep sequencing Sentosa platform (Vela DX) detected minority resistant variants well enough for HIV DNA genotyping. STUDY DESIGN We evaluated the Sentosa SQ HIV genotyping assay with automated extraction on 40 DNA longitudinal samples from treatment-experienced patients by comparison with Sanger sequencing. HIV drug resistance was interpreted using the ANRS algorithm (v29) at the threshold of 20 % and 3 %. RESULTS The Sentosa SQ HIV genotyping assay was 100 % successful to amplify and sequence PR and RT and 86 % to amplify and sequence IN when the HIV DNA load was >2.5 log copies/million cells. The Sentosa and Sanger sequencing were concordant for predicting PR-RT resistance at the threshold of 20 % in 14/18 samples successfully sequenced. A higher level of resistance was predicted by Sentosa in three samples and by Sanger in one sample. The prevalence of resistance was 7 % to PI, 59 % to NRTI, 31 % to NNRTI and 20 % to integrase inhibitors using the Sentosa SQ genotyping assay at the threshold of 3 %. Seven additional mutations <20 % were detected using the Sentosa assay. CONCLUSION Automated DNA extraction and sequencing using the Sentosa SQ HIV genotyping assay accurately predicted HIV DNA drug resistance by comparison with Sanger. Prospective studies are needed to evaluate the clinical interest of HIV DNA genotyping.
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Affiliation(s)
- Stéphanie Raymond
- INSERM U1043, CNRS UMR 5282, Toulouse University Paul Sabatier, CPTP, Toulouse, F-31300 France; CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France.
| | - Florence Nicot
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
| | - Florence Abravanel
- INSERM U1043, CNRS UMR 5282, Toulouse University Paul Sabatier, CPTP, Toulouse, F-31300 France; CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
| | - Luce Minier
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
| | - Romain Carcenac
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
| | - Caroline Lefebvre
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
| | - Agnès Harter
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
| | - Guillaume Martin-Blondel
- INSERM U1043, CNRS UMR 5282, Toulouse University Paul Sabatier, CPTP, Toulouse, F-31300 France; CHU de Toulouse, Hôpital Purpan, Service des Maladies Infectieuses et Tropicales, Toulouse, F-31300 France
| | - Pierre Delobel
- INSERM U1043, CNRS UMR 5282, Toulouse University Paul Sabatier, CPTP, Toulouse, F-31300 France; CHU de Toulouse, Hôpital Purpan, Service des Maladies Infectieuses et Tropicales, Toulouse, F-31300 France
| | - Jacques Izopet
- INSERM U1043, CNRS UMR 5282, Toulouse University Paul Sabatier, CPTP, Toulouse, F-31300 France; CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse, F-31300 France
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11
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Abstract
OBJECTIVE To study the long-term evolution of the transmitted CXCR4-using viruses. CCR5-using viruses (R5 viruses) predominate during primary HIV-1 infections (PHI) while CXCR4-using viruses are isolated in less than 10% of PHI. DESIGN Six patients infected with an R5X4 virus, detected by a sensitive phenotypic assay during PHI, were matched with six patients infected with a pure R5 virus for sex, Fiebig stage, time of antiretroviral initiation and duration of follow-up. METHODS We used MiSeq ultra-deep sequencing to determine the composition of the virus quasispecies during PHI and at the end of follow-up (median time of follow-up: 12.5 years). RESULTS X4 viruses were detected by genetic analysis in three of six samples from the R5X4 group, accounting for 1.3-100% of the virus quasispecies, during PHI, and in four of six samples (accounting for 6.7-100%) at the end of follow-up. No X4 virus was detected in the R5 group during PHI and in only one patient (accounting for 1.2%) at the end of follow-up. The complexity of the virus quasispecies at the stage of PHI was higher in the R5X4 group than in the R5 group. Complexity increased from PHI to the end of follow-up in the R5 group but remained stable in the R5X4 group. CONCLUSION CXCR4-using viruses persisted in the peripheral blood mononuclear cells of several patients on suppressive antiretroviral therapy for a median duration of 12.5 years after PHI. The genetic complexity of HIV-1 evolved differently post-PHI in patients infected with R5X4 viruses from those infected with R5 viruses.
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12
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Raymond S, Nicot F, Carcenac R, Lefebvre C, Jeanne N, Saune K, Delobel P, Izopet J. HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform. J Antimicrob Chemother 2019; 73:1152-1157. [PMID: 29444253 DOI: 10.1093/jac/dky003] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2017] [Accepted: 12/29/2017] [Indexed: 11/12/2022] Open
Abstract
Objectives To evaluate the diagnostic performance of the Vela next-generation sequencing (NGS) system in conjunction with the Sentosa SQ HIV Genotyping Assay for genotyping HIV-1. Methods Plasma RNA was extracted and templates prepared with the Sentosa SX instrument before sequencing the HIV-1 polymerase on the Sentosa SQ301 Sequencer (PGM IonTorrent). The Vela NGS System was compared with direct sequencing and the 454 GS-FLX (Roche) and MiSeq (Illumina) systems for genotypic resistance testing on clinical samples. Results The Vela NGS system detected majority resistance mutations in subtype B and CRF02-AG samples at 500 copies/mL and minority variants with a sensitivity of 5% at 100 000 copies/mL. The Vela NGS system and direct sequencing identified resistance mutations with 97% concordance in 46 clinical samples. Vela identified 1/20 of the 1%-5% mutations identified by 454, 5/12 of the 5%-20% mutations and 60/61 of the >20% mutations. Vela identified 3/14 of the 1%-5% mutations identified by MiSeq, 0/2 of the 5%-20% mutations and 47/47 of the >20% mutations. The resistance mutation quantifications by Vela and 454 were concordant (bias: 2.31%), as were those by Vela and MiSeq (bias: 1.06%). Conclusions The Vela NGS system provides automated nucleic acid extraction, PCR reagent distribution, library preparation and bioinformatics analysis. The analytical performance was very good when compared with direct sequencing, but was less sensitive than two other NGS platforms for detecting minority variants.
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Affiliation(s)
- Stéphanie Raymond
- INSERM, U1043, Toulouse F-31300, France.,CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
| | - Florence Nicot
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
| | - Romain Carcenac
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
| | - Caroline Lefebvre
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
| | - Nicolas Jeanne
- CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
| | - Karine Saune
- INSERM, U1043, Toulouse F-31300, France.,CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
| | - Pierre Delobel
- INSERM, U1043, Toulouse F-31300, France.,Université Toulouse III Paul-Sabatier, Faculté de Médecine Toulouse-Purpan, Toulouse F-31300, France.,CHU de Toulouse, Hôpital Purpan, Service des Maladies Infectieuses et Tropicales, Toulouse F-31300, France
| | - Jacques Izopet
- INSERM, U1043, Toulouse F-31300, France.,CHU de Toulouse, Hôpital Purpan, Laboratoire de Virologie, Toulouse F-31300, France
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13
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Lewis M, Mori J, Toma J, Mosley M, Huang W, Simpson P, Mansfield R, Craig C, van der Ryst E, Robertson DL, Whitcomb JM, Westby M. Clonal analysis of HIV-1 genotype and function associated with virologic failure in treatment-experienced persons receiving maraviroc: Results from the MOTIVATE phase 3 randomized, placebo-controlled trials. PLoS One 2018; 13:e0204099. [PMID: 30586365 PMCID: PMC6306210 DOI: 10.1371/journal.pone.0204099] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Accepted: 04/17/2018] [Indexed: 11/29/2022] Open
Abstract
Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1-5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key 'signature' mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain-specific mutational patterns.
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Affiliation(s)
- Marilyn Lewis
- The Research Network, Sandwich, Kent, United Kingdom
| | - Julie Mori
- hVIVO, Queen Mary BioEnterprise Innovation Centre, London, United Kingdom
| | - Jonathan Toma
- Monogram Biosciences, South San Francisco, California, United States of America
| | - Mike Mosley
- University of Oxford, Oxford, United Kingdom
| | - Wei Huang
- Monogram Biosciences, South San Francisco, California, United States of America
| | | | - Roy Mansfield
- Pfizer Global Research and Development, Sandwich Labs, Sandwich, Kent, United Kingdom
| | - Charles Craig
- The Research Network, Sandwich, Kent, United Kingdom
| | | | - David L. Robertson
- Evolution and Genomic Sciences, School of Biological Sciences, The University of Manchester, Manchester, United Kingdom
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | | | - Mike Westby
- Centauri Therapeutics Limited, Discovery Park, Kent, United Kingdom
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14
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Silver N, Paynter M, McAllister G, Atchley M, Sayir C, Short J, Winner D, Alouani DJ, Sharkey FH, Bergefall K, Templeton K, Carrington D, Quiñones-Mateu ME. Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay. AIDS Res Ther 2018; 15:18. [PMID: 30409215 PMCID: PMC6223033 DOI: 10.1186/s12981-018-0206-y] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 10/30/2018] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND The widespread global access to antiretroviral drugs has led to considerable reductions in morbidity and mortality but, unfortunately, the risk of virologic failure increases with the emergence, and potential transmission, of drug resistant viruses. Detecting and quantifying HIV-1 drug resistance has therefore become the standard of care when designing new antiretroviral regimens. The sensitivity of Sanger sequencing-based HIV-1 genotypic assays is limited by its inability to identify minority members of the quasispecies, i.e., it only detects variants present above ~ 20% of the viral population, thus, failing to detect minority variants below this threshold. It is clear that deep sequencing-based HIV-1 genotyping assays are an important step change towards accurately monitoring HIV-infected individuals. METHODS We implemented and verified a clinically validated HIV-1 genotyping assay based on deep sequencing (DEEPGEN™) in two clinical laboratories in the United Kingdom: St. George's University Hospitals Healthcare NHS Foundation Trust (London) and at NHS Lothian (Edinburgh), to characterize minority HIV-1 variants in 109 plasma samples from ART-naïve or -experienced individuals. RESULTS Although subtype B HIV-1 strains were highly prevalent (44%, 48/109), most individuals were infected with non-B subtype viruses (i.e., A1, A2, C, D, F1, G, CRF02_AG, and CRF01_AE). DEEPGEN™ was able to accurately detect drug resistance-associated mutations not identified using standard Sanger sequencing-based tests, which correlated significantly with patient's antiretroviral treatment histories. A higher proportion of minority PI-, NRTI-, and NNRTI-resistance mutations was detected in NHS Lothian patients compared to individuals from St. George's, mainly M46I/L and I50 V (associated with PIs), D67 N, K65R, L74I, M184 V/I, and K219Q (NRTIs), and L100I (NNRTIs). Interestingly, we observed an inverse correlation between intra-patient HIV-1 diversity and CD4+ T cell counts in the NHS Lothian patients. CONCLUSIONS This is the first study evaluating the transition, training, and implementation of DEEPGEN™ between three clinical laboratories in two different countries. More importantly, we were able to characterize the HIV-1 drug resistance profile (including minority variants), coreceptor tropism, subtyping, and intra-patient viral diversity in patients from the United Kingdom, providing a rigorous foundation for basing clinical decisions on highly sensitive and cost-effective deep sequencing-based HIV-1 genotyping assays in the country.
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15
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Nicot F, Jeanne N, Raymond S, Delfour O, Carcenac R, Lefebvre C, Sauné K, Delobel P, Izopet J. Performance comparison of deep sequencing platforms for detecting HIV-1 variants in the pol gene. J Med Virol 2018; 90:1486-1492. [PMID: 29750364 DOI: 10.1002/jmv.25224] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2018] [Accepted: 05/01/2018] [Indexed: 01/08/2023]
Abstract
The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (ρ = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.
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Affiliation(s)
- Florence Nicot
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France
| | - Nicolas Jeanne
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France
| | - Stéphanie Raymond
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France.,INSERM, U1043, Toulouse, France.,Faculté de Médecine Toulouse-Purpan, Université Toulouse III Paul-Sabatier, Toulouse, France
| | - Olivier Delfour
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France
| | - Romain Carcenac
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France
| | - Caroline Lefebvre
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France
| | - Karine Sauné
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France.,INSERM, U1043, Toulouse, France.,Faculté de Médecine Toulouse-Purpan, Université Toulouse III Paul-Sabatier, Toulouse, France
| | - Pierre Delobel
- INSERM, U1043, Toulouse, France.,Faculté de Médecine Toulouse-Purpan, Université Toulouse III Paul-Sabatier, Toulouse, France.,CHU de Toulouse, Hôpital Purpan, Service des Maladies Infectieuses et Tropicales, Toulouse, France
| | - Jacques Izopet
- Laboratoire de Virologie, CHU de Toulouse, Hôpital Purpan, Toulouse, France.,INSERM, U1043, Toulouse, France.,Faculté de Médecine Toulouse-Purpan, Université Toulouse III Paul-Sabatier, Toulouse, France
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16
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Stella-Ascariz N, Arribas JR, Paredes R, Li JZ. The Role of HIV-1 Drug-Resistant Minority Variants in Treatment Failure. J Infect Dis 2017; 216:S847-S850. [PMID: 29207001 DOI: 10.1093/infdis/jix430] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Human immunodeficiency virus type 1 (HIV-1) drug resistance genotyping is recommended to help in the selection of antiretroviral therapy and to prevent virologic failure. There are several ultrasensitive assays able to detect HIV-1 drug-resistance minority variants (DRMVs) not detectable by standard population sequencing-based HIV genotyping assays. Presence of these DRMVs has been shown to be clinically relevant, but its impact does not appear to be uniform across drug classes. In this review, we summarize key evidence for the clinical impact of DRMVs across drug classes for both antiretroviral treatment-naive and antiretroviral treatment-experienced patients, and highlight areas where more supporting evidence is needed.
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Affiliation(s)
| | - José Ramón Arribas
- HIV Unit, Internal Medicine Service, Hospital Universitario La Paz-IdiPAZ
| | - Roger Paredes
- HIV Unit and irsiCaixa AIDS Research Institute, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona and Universitat de Vic-UCC, Spain
| | - Jonathan Z Li
- Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School
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17
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Glebova O, Knyazev S, Melnyk A, Artyomenko A, Khudyakov Y, Zelikovsky A, Skums P. Inference of genetic relatedness between viral quasispecies from sequencing data. BMC Genomics 2017; 18:918. [PMID: 29244009 PMCID: PMC5731608 DOI: 10.1186/s12864-017-4274-5] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
BACKGROUND RNA viruses such as HCV and HIV mutate at extremely high rates, and as a result, they exist in infected hosts as populations of genetically related variants. Recent advances in sequencing technologies make possible to identify such populations at great depth. In particular, these technologies provide new opportunities for inference of relatedness between viral samples, identification of transmission clusters and sources of infection, which are crucial tasks for viral outbreaks investigations. RESULTS We present (i) an evolutionary simulation algorithm Viral Outbreak InferenCE (VOICE) inferring genetic relatedness, (ii) an algorithm MinDistB detecting possible transmission using minimal distances between intra-host viral populations and sizes of their relative borders, and (iii) a non-parametric recursive clustering algorithm Relatedness Depth (ReD) analyzing clusters' structure to infer possible transmissions and their directions. All proposed algorithms were validated using real sequencing data from HCV outbreaks. CONCLUSIONS All algorithms are applicable to the analysis of outbreaks of highly heterogeneous RNA viruses. Our experimental validation shows that they can successfully identify genetic relatedness between viral populations, as well as infer transmission clusters and outbreak sources.
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Affiliation(s)
- Olga Glebova
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, 30303, GA, USA.
| | - Sergey Knyazev
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, 30303, GA, USA
| | - Andrew Melnyk
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, 30303, GA, USA
| | - Alexander Artyomenko
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, 30303, GA, USA
| | - Yury Khudyakov
- Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, 30329, GA, USA
| | - Alex Zelikovsky
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, 30303, GA, USA
| | - Pavel Skums
- Computer Science Department, Georgia State University, 25 Park Place NE, Atlanta, 30303, GA, USA.,Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, 30329, GA, USA
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18
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Multi-drug resistant Klebsiella pneumoniae strains circulating in hospital setting: whole-genome sequencing and Bayesian phylogenetic analysis for outbreak investigations. Sci Rep 2017; 7:3534. [PMID: 28615687 PMCID: PMC5471223 DOI: 10.1038/s41598-017-03581-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Accepted: 05/09/2017] [Indexed: 01/12/2023] Open
Abstract
Carbapenems resistant Enterobacteriaceae infections are increasing worldwide representing an emerging public health problem. The application of phylogenetic and phylodynamic analyses to bacterial whole genome sequencing (WGS) data have become essential in the epidemiological surveillance of multi-drug resistant nosocomial pathogens. Between January 2012 and February 2013, twenty-one multi-drug resistant K. pneumoniae strains, were collected from patients hospitalized among different wards of the University Hospital Campus Bio-Medico. Epidemiological contact tracing of patients and Bayesian phylogenetic analysis of bacterial WGS data were used to investigate the evolution and spatial dispersion of K. pneumoniae in support of hospital infection control. The epidemic curve of incident K. pneumoniae cases showed a bimodal distribution of cases with two peaks separated by 46 days between November 2012 and January 2013. The time-scaled phylogeny suggested that K. pneumoniae strains isolated during the study period may have been introduced into the hospital setting as early as 2007. Moreover, the phylogeny showed two different epidemic introductions in 2008 and 2009. Bayesian genomic epidemiology is a powerful tool that promises to improve the surveillance and control of multi-drug resistant pathogens in an effort to develop effective infection prevention in healthcare settings or constant strains reintroduction.
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19
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Nedellec R, Herbeck JT, Hunt PW, Deeks SG, Mullins JI, Anton ED, Reeves JD, Mosier DE. High-Sequence Diversity and Rapid Virus Turnover Contribute to Higher Rates of Coreceptor Switching in Treatment-Experienced Subjects with HIV-1 Viremia. AIDS Res Hum Retroviruses 2017; 33:234-245. [PMID: 27604829 DOI: 10.1089/aid.2016.0153] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Coreceptor switching from CCR5 to CXCR4 is common during chronic HIV-1 infection, but is even more common in individuals who have failed antiretroviral therapy (ART). Prior studies have suggested rapid mutation and/or recombination of HIV-1 envelope (env) genes during coreceptor switching. We compared the functional and genotypic changes in env of viruses from viremic subjects who had failed ART just before and after coreceptor switching and compared those to viruses from matched subjects without coreceptor switching. Analysis of multiple unique functional env clones from each subject revealed extensive diversity at both sample time points and rapid diversification of sequences during the 4-month interval in viruses from both 9 subjects with coreceptor switching and 15 control subjects. Only two subjects had envs with evidence of recombination. Three findings distinguished env clones from subjects with coreceptor switching from controls: (1) lower entry efficiency via CCR5; (2) longer V1/V2 regions; and (3), lower nadir CD4 T cell counts during prior years of infection. Most of these subjects harbored virus with lower replicative capacity associated with protease (PR) and/or reverse transcriptase inhibitor resistance mutations, and the extensive diversification tended to lead either to improved entry efficiency via CCR5 or the gain of entry function via CXCR4. These results suggest that R5X4 or X4 variants emerge from a diverse, low-fitness landscape shaped by chronic infection, multiple ART resistance mutations, the availability of target cells, and reduced entry efficiency via CCR5.
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Affiliation(s)
- Rebecca Nedellec
- Department of Immunology and Microbial Science, IMM-7, The Scripps Research Institute, La Jolla, California
| | - Joshua T. Herbeck
- International Clinical Research Center, Department of Global Health, University of Washington, Seattle, Washington
| | - Peter W. Hunt
- Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of California San Francisco, San Francisco, California
| | - Steven G. Deeks
- Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of California San Francisco, San Francisco, California
| | - James I. Mullins
- Department of Microbiology, University of Washington, Seattle, Washington
| | - Elizabeth D. Anton
- Monogram Biosciences, Laboratory Corporation of America® Holding, Virology Research and Development, South San Francisco, California
| | - Jacqueline D. Reeves
- Monogram Biosciences, Laboratory Corporation of America® Holding, Virology Research and Development, South San Francisco, California
| | - Donald E. Mosier
- Department of Immunology and Microbial Science, IMM-7, The Scripps Research Institute, La Jolla, California
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20
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Performance comparison of next-generation sequencing platforms for determining HIV-1 coreceptor use. Sci Rep 2017; 7:42215. [PMID: 28186189 PMCID: PMC5301480 DOI: 10.1038/srep42215] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2016] [Accepted: 01/06/2017] [Indexed: 01/31/2023] Open
Abstract
The coreceptor used by HIV-1 must be determined before a CCR5 antagonist, part of the arsenal of antiretroviral drugs, is prescribed because viruses that enter cells using the CXCR4 coreceptor are responsible for treatment failure. HIV-1 tropism is also correlated with disease progression and so must be determined for virological studies. Tropism can be determined by next-generation sequencing (NGS), but not all of these new technologies have been fully validated for use in clinical practice. The Illumina NGS technology is used in many laboratories but its ability to predict HIV-1 tropism has not been evaluated while the 454 GS-Junior (Roche) is used for routine diagnosis. The genotypic prediction of HIV-1 tropism is based on sequencing the V3 region and interpreting the results with an appropriate algorithm. We compared the performances of the MiSeq (Illumina) and 454 GS-Junior (Roche) systems with a reference phenotypic assay. We used clinical samples for the NGS tropism predictions and assessed their ability to quantify CXCR4-using variants. The data show that the Illumina platform can be used to detect minor CXCR4-using variants in clinical practice but technical optimization are needed to improve quantification.
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Casadellà M, Paredes R. Deep sequencing for HIV-1 clinical management. Virus Res 2016; 239:69-81. [PMID: 27818211 DOI: 10.1016/j.virusres.2016.10.019] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Revised: 10/10/2016] [Accepted: 10/18/2016] [Indexed: 02/05/2023]
Abstract
The emerging HIV-1 resistance epidemic is threatening the impressive global advances in HIV-1 infection treatment and prevention achieved in the last decade. Next-generation sequencing is improving our ability to understand, diagnose and prevent HIV-1 resistance, being increasingly cost-effective and more accessible. However, NGS still faces a number of limitations that need to be addressed to enable its widespread use. Here, we will review the main NGS platforms available for HIV-1 diagnosis, the factors affecting the clinical utility of NGS testing and the evidence supporting -or not- ultrasensitive genotyping over Sanger sequencing for routine HIV-1 diagnosis. Now that global HIV-1 eradication might be within our reach, making NGS accessible also to LMICs has become a priority. Reductions in sequencing costs, particularly in library preparation, and accessibility to low-cost, robust but simplified automated bioinformatic analyses of NGS data will remain essential to end the HIV-1 pandemic.
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Affiliation(s)
- Maria Casadellà
- IrsiCaixa AIDS Research Institute, Badalona, Spain; Universitat Autònoma de Barcelona, Catalonia, Spain.
| | - Roger Paredes
- IrsiCaixa AIDS Research Institute, Badalona, Spain; Universitat Autònoma de Barcelona, Catalonia, Spain; Universitat de Vic - Central de Catalunya, Vic, Catalonia, Spain; HIV-1 Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Catalonia, Spain
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22
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Saladini F, Vicenti I. Role of phenotypic investigation in the era of routine genotypic HIV-1 drug resistance testing. Future Virol 2016. [DOI: 10.2217/fvl-2016-0080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The emergence of drug resistance can seriously compromise HIV type-1 therapy and decrease therapeutic options. Resistance testing is highly recommended to guide treatment decisions and drug activity can be accurately predicted in the clinical setting through genotypic assays. While phenotypic systems are not suitable for monitoring drug resistance in routine laboratory practice, genotyping can misclassify unusual or complex mutational patterns, particularly with recently approved antivirals. In addition, phenotypic assays remain fundamental for characterizing candidate antiretroviral compounds. This review aims to discuss how phenotypic assays contributed to and still play a role in understanding the mechanisms of resistance of both licensed and investigational HIV type-1 inhibitors.
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Affiliation(s)
- Francesco Saladini
- Department of Medical Biotechnologies, University of Siena Italy, Policlinico Le Scotte, Viale Bracci 16 53100 Siena, Italy
| | - Ilaria Vicenti
- Department of Medical Biotechnologies, University of Siena Italy, Policlinico Le Scotte, Viale Bracci 16 53100 Siena, Italy
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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. J Virol 2016; 90:7142-58. [PMID: 27226378 DOI: 10.1128/jvi.00441-16] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2016] [Accepted: 05/23/2016] [Indexed: 01/06/2023] Open
Abstract
UNLABELLED 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. IMPORTANCE Primer 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.
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No selection of CXCR4-using variants in cell reservoirs of dual-mixed HIV-infected patients on suppressive maraviroc therapy. AIDS 2016; 30:965-8. [PMID: 26752281 DOI: 10.1097/qad.0000000000001013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
We used ultradeep sequencing to investigate the evolution of the frequency of CXCR4-using viruses in the peripheral blood mononuclear cells of 22 patients infected with both CCR5 and CXCR4-using viruses treated with the CCR5 antagonist maraviroc for 24 weeks and a stable antiviral therapy. The mean CXCR4-using virus frequency in peripheral blood mononuclear cells was 59% before maraviroc intensification and 52% after 24 weeks of effective treatment, indicating no selection by maraviroc.
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25
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Gonzalez-Serna A, Genebat M, Ruiz-Mateos E, Leal M. Short-term maraviroc exposure, a clinical approach to decide on maraviroc prescription in HIV-1-infected treatment-naïve patients. Drug Des Devel Ther 2016; 10:353-4. [PMID: 26848259 PMCID: PMC4723024 DOI: 10.2147/dddt.s100639] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Affiliation(s)
- Alejandro Gonzalez-Serna
- Laboratory of Molecular Immunobiology, Hospital General Universitario Gregorio Maranon, Madrid, Spain
| | - Miguel Genebat
- Laboratory of Immunovirology, Institute of Biomedicine of Seville, Seville, Spain
| | - Ezequiel Ruiz-Mateos
- Laboratory of Immunovirology, Institute of Biomedicine of Seville, Seville, Spain
| | - Manuel Leal
- Laboratory of Immunovirology, Institute of Biomedicine of Seville, Seville, Spain
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Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism. Sci Rep 2015; 5:16944. [PMID: 26585833 PMCID: PMC4653658 DOI: 10.1038/srep16944] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2015] [Accepted: 10/22/2015] [Indexed: 11/11/2022] Open
Abstract
HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.
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Hütter G, Bodor J, Ledger S, Boyd M, Millington M, Tsie M, Symonds G. CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape. Viruses 2015. [PMID: 26225991 PMCID: PMC4576177 DOI: 10.3390/v7082816] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.
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Affiliation(s)
- Gero Hütter
- Cellex GmbH, Fiedlerstr. 36, 01307 Dresden, Germany.
| | - Josef Bodor
- Department of Cell Therapy, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague 2, Czech Republic.
| | - Scott Ledger
- Faculty of Medicine, University of New South Wales, Sydney 2052 NSW, Australia.
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Raymond S, Maillard A, Amiel C, Peytavin G, Trabaud MA, Desbois D, Bellecave P, Delaugerre C, Soulie C, Marcelin AG, Descamps D, Izopet J, the ANRS ACll Resistance Study Group, Reigadas S, Bellecave P, Pinson-Recordon P, Fleury H, Masquelier B, Signori-Schmuck A, Morand P, Bocket L, Mouna L, Andre P, Tardy JC, Trabaud MA, Descamps D, Charpentier C, Peytavin G, Brun-Vezinet F, Haim-Boukobza S, Roques AM, Soulie C, Lambert-Niclot S, Malet I, Wirden M, Fourati S, Marcelin AG, Calvez V, Flandre P, Assoumou L, Costagliola D, Morand-Joubert L, Delaugerre C, Schneider V, Amiel C, Giraudeau G, Maillard A, Nicot F, Izopet J. Virological failure of patients on maraviroc-based antiretroviral therapy. J Antimicrob Chemother 2015; 70:1858-64. [DOI: 10.1093/jac/dkv026] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2014] [Accepted: 01/25/2015] [Indexed: 11/14/2022] Open
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29
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Torres AJL, Brígido LFDM, Abrahão MHN, Angelo ALD, de Jesus Ferreira G, Coelho LP, Ferreira JL, Jorge CRMP, Netto EM, Brites C. High degree of concordance between flow cytometry and geno2pheno methods for HIV-1 tropism determination in proviral DNA. Braz J Infect Dis 2015; 19:163-9. [PMID: 25701547 PMCID: PMC9425389 DOI: 10.1016/j.bjid.2014.11.007] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2014] [Revised: 10/05/2014] [Accepted: 11/16/2014] [Indexed: 11/30/2022] Open
Abstract
Use of CCR5 antagonists requires previous viral tropism determination. The available methods have high cost, are time-consuming, or require highly trained personnel, and sophisticated equipment. We compared a flow cytometry-based tropism assay with geno2pheno method to determine HIV-1 tropism in AIDS patients, in Bahia, Brazil. We tested peripheral blood mononuclear cells of 102 AIDS patients under antiretroviral therapy by using a cytometry-based tropism assay and geno2pheno assay. Cellular membrane receptors were identified by using CXCR4, CCR5 and CD4 monoclonal antibodies, while detection of cytoplasmic mRNAs for gag and pol HIV regions was achieved by using a labeled probe. Genotypic identification of X4 and R5 tropic viruses was attempted by geno2pheno algorithm. There was a high degree of concordance between cytometry-based tropism assay and geno2pheno algorithm in determination of HIV-1 tropism. Cytometry-based tropism assay demonstrated higher sensitivity and specificity in comparison to geno2pheno, which was used as a gold-standard. One sample could not be amplified by geno2pheno method, but was classified as duotropic by cytometry-based tropism assay. We did not find any association between CD4+ count or plasma HIV-1 RNA viral load and tropism results. The overall performances of cytometry-based tropism assay and geno2pheno assay were almost identical in determination of HIV-1 tropism.
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Affiliation(s)
- Alex José Leite Torres
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil.
| | | | - Marcos Herculano Nunes Abrahão
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil
| | - Ana Luiza Dias Angelo
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil
| | - Gilcivaldo de Jesus Ferreira
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil
| | - Luana Portes Coelho
- Grupo de pesquisa para caracterização de Retrovírus em humanos do Instituto Adolfo Lutz, São Paulo, SP, Brazil
| | - João Leandro Ferreira
- Grupo de pesquisa para caracterização de Retrovírus em humanos do Instituto Adolfo Lutz, São Paulo, SP, Brazil
| | - Célia Regina Mayoral Pedroso Jorge
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil
| | - Eduardo Martins Netto
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil
| | - Carlos Brites
- Laboratório de Pesquisa em Infectologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, BA, Brazil
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30
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Mbondji-Wonje C, Ragupathy V, Zhao J, Nanfack A, Lee S, Torimiro J, Nyambi P, Hewlett IK. Genotypic prediction of tropism of highly diverse HIV-1 strains from Cameroon. PLoS One 2014; 9:e112434. [PMID: 25379669 PMCID: PMC4224497 DOI: 10.1371/journal.pone.0112434] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2014] [Accepted: 10/07/2014] [Indexed: 01/04/2023] Open
Abstract
BACKGROUND The use of CCR5 antagonists involves determination of HIV-1 tropism prior to initiation of treatment. HIV-1 tropism can be assessed either by phenotypic or genotypic methods. Genotypic methods are extensively used for tropism prediction. However, their validation in predicting tropism of viral isolates belonging to group M non-B subtypes remains challenging. In Cameroon, the genetic diversity of HIV-1 strains is the broadest reported worldwide. To facilitate the integration of CCR5 antagonists into clinical practice in this region, there is a need to evaluate the performance of genotypic methods for predicting tropism of highly diverse group M HIV-1 strains. METHODS Tropism of diverse HIV-1 strains isolated from PBMCs from Cameroon was determined using the GHOST cell assay. Prediction, based on V3 sequences from matched plasma samples, was determined using bioinformatics algorithms and rules based on position 11/25 and net charge applied independently or combined according to Delobel's and Garrido's rules. Performance of genotypic methods was evaluated by comparing prediction generated with tropism assigned by the phenotypic assay. RESULTS Specificity for predicting R5-tropic virus was high, ranging from 83.7% to 97.7% depending on the genotypic methods used. Sensitivity for X4-tropic viruses was fairly low, ranging from 33.3% to 50%. In our study, overall, genotypic methods were less able to accurately predict X4-tropic virus belonging to subtype CRF02_AG. In addition, it was found that of the methods we used the Garrido rule has the highest sensitivity rate of over 50% with a specificity of 93%. CONCLUSION Our study demonstrated that overall, genotypic methods were less sensitive for accurate prediction of HIV-1 tropism in settings where diverse HIV-1 strains co-circulate. Our data suggest that further optimization of genotypic methods is needed and that larger studies to determine their utility for tropism prediction of diverse HIV-1 strains may be warranted.
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Affiliation(s)
- Christelle Mbondji-Wonje
- Laboratory of Molecular Virology, Division of Emerging and Transmission Transmitted Diseases, Office of Blood Review and Research, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, United States of America
- Faculty of Medicine, Pharmacy and Biomedical sciences, University of Douala, Douala, Cameroon
| | - Viswanath Ragupathy
- Laboratory of Molecular Virology, Division of Emerging and Transmission Transmitted Diseases, Office of Blood Review and Research, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, United States of America
| | - Jiangqin Zhao
- Laboratory of Molecular Virology, Division of Emerging and Transmission Transmitted Diseases, Office of Blood Review and Research, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, United States of America
| | - Aubin Nanfack
- Chantal Biya International Reference Centre, Yaoundé, Cameroon
- Department of Pathology, New York University School of Medicine, New York, New York, United States of America
| | - Sherwin Lee
- Laboratory of Molecular Virology, Division of Emerging and Transmission Transmitted Diseases, Office of Blood Review and Research, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, United States of America
| | - Judith Torimiro
- Chantal Biya International Reference Centre, Yaoundé, Cameroon
| | - Phillipe Nyambi
- Department of Pathology, New York University School of Medicine, New York, New York, United States of America
| | - Indira K. Hewlett
- Laboratory of Molecular Virology, Division of Emerging and Transmission Transmitted Diseases, Office of Blood Review and Research, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD, 20993, United States of America
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31
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Sede MM, Moretti FA, Laufer NL, Jones LR, Quarleri JF. HIV-1 tropism dynamics and phylogenetic analysis from longitudinal ultra-deep sequencing data of CCR5- and CXCR4-using variants. PLoS One 2014; 9:e102857. [PMID: 25032817 PMCID: PMC4102574 DOI: 10.1371/journal.pone.0102857] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Accepted: 06/25/2014] [Indexed: 11/25/2022] Open
Abstract
OBJECTIVE Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships. METHODS Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained. RESULTS Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence. CONCLUSIONS CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.
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Affiliation(s)
- Mariano M. Sede
- Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS), Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina
- Consejo de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Franco A. Moretti
- Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS), Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina
- Consejo de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Natalia L. Laufer
- Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS), Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina
- Consejo de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - Leandro R. Jones
- Consejo de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
- Laboratorio de Virología y Genética Molecular, Facultad de Ciencias Naturales, sede Trelew, Universidad Nacional de la Patagonia San Juan Bosco, Chubut, Argentina
| | - Jorge F. Quarleri
- Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS), Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina
- Consejo de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
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Knapp DJHF, McGovern RA, Poon AFY, Zhong X, Chan D, Swenson LC, Dong W, Harrigan PR. "Deep" sequencing accuracy and reproducibility using Roche/454 technology for inferring co-receptor usage in HIV-1. PLoS One 2014; 9:e99508. [PMID: 24959876 PMCID: PMC4069016 DOI: 10.1371/journal.pone.0099508] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Accepted: 05/15/2014] [Indexed: 11/19/2022] Open
Abstract
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. To accomplish this, repeated deep sequencing results were generated using the Roche GS-FLX (454) from a number of sources including a non-homogeneous clinical sample (N = 47 replicates over 18 deep sequencing runs), and a large clinical cohort from the MOTIVATE and A400129 studies (N = 1521). For repeated measurements of a non-homogeneous clinical sample, increasing input copy number both decreased variance in the measured proportion of non-R5 using virus (p<<0.001 and 0.02 for single replicates and triplicates respectively) and increased measured viral diversity (p<0.001; multiple measures). Detection of sequences with a mean abundance less than 1% abundance showed a 2 fold increase in median coefficient of variation (CV) in repeated measurements of a non-homogeneous clinical sample, and a 2.7 fold increase in CV in the MOTIVATE/A400129 dataset compared to sequences with ≥1% abundance. An unexpected source of error included read position, with low accuracy reads occurring more frequently towards the edge of sequencing regions (p<<0.001). Overall, the primary source of variability was sampling error caused by low input copy number/minority species prevalence, though other sources of error including sequence intrinsic, temporal, and read-position related errors were detected.
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Affiliation(s)
| | | | - Art F. Y. Poon
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada
| | - Xiaoyin Zhong
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada
| | - Dennison Chan
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada
| | | | - Winnie Dong
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada
| | - P. Richard Harrigan
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC, Canada
- Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada
- * E-mail:
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Quiñones-Mateu ME, Avila S, Reyes-Teran G, Martinez MA. Deep sequencing: becoming a critical tool in clinical virology. J Clin Virol 2014; 61:9-19. [PMID: 24998424 DOI: 10.1016/j.jcv.2014.06.013] [Citation(s) in RCA: 90] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2014] [Revised: 06/12/2014] [Accepted: 06/14/2014] [Indexed: 02/07/2023]
Abstract
Population (Sanger) sequencing has been the standard method in basic and clinical DNA sequencing for almost 40 years; however, next-generation (deep) sequencing methodologies are now revolutionizing the field of genomics, and clinical virology is no exception. Deep sequencing is highly efficient, producing an enormous amount of information at low cost in a relatively short period of time. High-throughput sequencing techniques have enabled significant contributions to multiples areas in virology, including virus discovery and metagenomics (viromes), molecular epidemiology, pathogenesis, and studies of how viruses to escape the host immune system and antiviral pressures. In addition, new and more affordable deep sequencing-based assays are now being implemented in clinical laboratories. Here, we review the use of the current deep sequencing platforms in virology, focusing on three of the most studied viruses: human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus.
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Affiliation(s)
- Miguel E Quiñones-Mateu
- University Hospital Translational Laboratory, University Hospitals Case Medical Center, Cleveland, OH, USA; Department of Pathology, Case Western Reserve University, Cleveland, OH, USA
| | - Santiago Avila
- Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico; Centro de Investigaciones en Enfermedades Infecciosas, Mexico City, Mexico
| | - Gustavo Reyes-Teran
- Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico; Centro de Investigaciones en Enfermedades Infecciosas, Mexico City, Mexico
| | - Miguel A Martinez
- Fundació irsicaixa, Universitat Autònoma de Barcelona, Hospital Universitari Germans Trias i Pujol, Badalona, Spain
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Sollerkvist LP, Gaseitsiwe S, Mine M, Sebetso G, Mphoyakgosi T, Diphoko T, Essex M, Ehrnst A. Increased CXCR4 use of HIV-1 subtype C identified by population sequencing in patients failing antiretroviral treatment compared with treatment-naive patients in Botswana. AIDS Res Hum Retroviruses 2014; 30:436-45. [PMID: 24205895 DOI: 10.1089/aid.2013.0203] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
HIV-1 uses the coreceptors CCR5 and/or CXCR4 for cell entry. Monotropic CCR5-using variants are found early in the infection while CXCR4-using variants may appear after progression to AIDS. CXCR4 use may consist of both monotropic and dualtropic viruses. The viral phenotype is important in evaluating the response to CCR5 inhibitors, a new class of antiviral drugs. The coreceptor use of HIV-1 was investigated using population sequencing in 24 patients from Botswana, carrying HIV-1 subtype C and failing antiretroviral treatment, while 26 treatment-naive patients acted as controls. Single genome sequencing was used to discern minor HIV-1 populations in the treatment-experienced group. The Geno2Pheno method was employed to predict the coreceptor use phenotype from HIV-1 env gp120 V3 DNA sequences. The glycan-charge model adjusted for subtype C was also used for phenotype prediction. The viral phenotype of population sequences was predicted using Geno2Pheno in 24/24 treatment-experienced patients, of whom eight (33%) were predicted to harbor CXCR4-using strains as compared to 2/26 in the treatment-naive group (p=0.03). Single genome sequencing generated 4-23 clones/patient in the treatment-experienced group. Altogether, 90/295 (31%) putative CXCR4-using clones were identified. In 10/24 (42%) treated patients at least one clone was predicted to be CXCR4-using, further increasing the amount of identified treatment-experienced patients with CXCR4 use. Although subtype C is usually associated with comparatively little CXCR4 use, the frequency of CXCR4 use in treatment-experienced patients with subtype C can be higher, which may have implications for the administration of CCR5 inhibitors in this patient group.
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Affiliation(s)
| | - Simani Gaseitsiwe
- Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden
- Botswana-Harvard School of Public Health AIDS Initiative Partnership for HIV Research and Education, Gaborone, Botswana
| | - Madisa Mine
- Ministry of Health, Botswana Harvard HIV Reference Laboratory, Gaborone, Botswana
| | - Gaseene Sebetso
- Ministry of Health, Botswana Harvard HIV Reference Laboratory, Gaborone, Botswana
| | | | - Thabo Diphoko
- Botswana-Harvard School of Public Health AIDS Initiative Partnership for HIV Research and Education, Gaborone, Botswana
| | - Max Essex
- Botswana-Harvard School of Public Health AIDS Initiative Partnership for HIV Research and Education, Gaborone, Botswana
- Department of Immunology and Infectious Diseases, and the Harvard School of Public Health AIDS Initiative, Harvard School of Public Health, Boston, Massachusetts
| | - Anneka Ehrnst
- Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden
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Asin-Milan O, Wei Y, Sylla M, Vaisheva F, Chamberland A, Tremblay CL. Performance of a clonal-based HIV-1 tropism phenotypic assay. J Virol Methods 2014; 204:53-61. [PMID: 24731927 DOI: 10.1016/j.jviromet.2014.04.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Revised: 03/31/2014] [Accepted: 04/04/2014] [Indexed: 11/29/2022]
Abstract
Adequate determination of HIV-1 tropism is important in clinical and research settings. Genotypic and phenotypic approaches to evaluate tropism have been described. Phenotypic assays are widely used to determine HIV-1 tropism because of their sensitivity to detect minor CXCR4-using variants (X4). However they cannot differentiate mixed quasi-species of R5 and X4 viruses from dual-tropic viruses. We describe here a clonal-based HIV-1 tropism phenotypic assay. Env-pseudo-typed viruses were produced by co-transfection of the env expression plasmid pcDNA3.1/V5HisTOPO and a backbone vector pNL4-3.Luc.E-R- that expresses the entire HIV-1 genome except for env and vpr in 293T cell cultures. Co-receptor use was tested by infecting U87.CD4.CCR5+ and U87.CD4.CXCR4+ cells in the presence or absence of co-receptor inhibitors, using 10 clones from each sample. The ability of the assay to detect minor variants in a viral population was assessed by mixing X4 and R5 clones using different ratios. Both R5 and X4 minority variants were detected when present at greater than 0.4% in a mixture of envelope populations. This assay can be useful in both clinical and research laboratories.
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Affiliation(s)
- Odalis Asin-Milan
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada
| | - Yi Wei
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada
| | - Mohamed Sylla
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada
| | | | - Annie Chamberland
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada
| | - Cécile L Tremblay
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada; Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Canada.
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Next-Generation Sequencing to Help Monitor Patients Infected with HIV: Ready for Clinical Use? Curr Infect Dis Rep 2014; 16:401. [DOI: 10.1007/s11908-014-0401-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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Kagan RM, Johnson EP, Siaw MF, Van Baelen B, Ogden R, Platt JL, Pesano RL, Lefebvre E. Comparison of genotypic and phenotypic HIV type 1 tropism assay: results from the screening samples of Cenicriviroc Study 202, a randomized phase II trial in treatment-naive subjects. AIDS Res Hum Retroviruses 2014; 30:151-9. [PMID: 23875707 DOI: 10.1089/aid.2013.0123] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Cenicriviroc is a once-daily oral CCR5/CCR2 antagonist in development for treatment of HIV infection. CVC Study 202 (652-2-202; NCT01338883) excluded treatment-naive subjects demonstrated to harbor non-R5 (CXCR4-tropic or dual-mixed) tropic HIV-1 by either genotypic or phenotypic tropism testing. Here we compare the results of genotypic and phenotypic tropism testing in Study 202. A total of 304 subjects screened had paired genotypic and phenotypic results. Genotypic tropism testing (GTT) incorporated triplicate population sequencing using the geno2pheno algorithm and the PSSM algorithm, followed by ultradeep sequencing (UDS) for samples with R5 results. All samples were further evaluated with a phenotypic test, the enhanced-sensitivity Trofile assay (ESTA). Concordance between GTT and ESTA was 80% and increased to 84% when only geno2pheno was used for triplicate population sequencing. GTT (geno2pheno) classified 18% of the samples as non-R5 compared to 16% by ESTA. Only one-third of samples with non-R5 results by either test were classified as non-R5 by both tests. Median CD4((+)) cell counts were lower in patients with concordant non-R5 results by UDS and ESTA than in subjects with an R5 result by either assay (p=0.0004). UDS detected non-R5 virus in an additional 27/304 subjects (median 15% non-R5, interquartile range: 3.7-62%) with R5 results by ESTA. In conclusion, the geno2pheno algorithm improves concordance of GTT with a clinically validated phenotypic tropism assay as does the use of UDS. These findings provide support for recent guidelines indicating that genotypic tropism testing may be considered as an alternative to phenotypic testing.
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Affiliation(s)
- Ron M. Kagan
- Quest Diagnostics Nichols Institute, San Juan Capistrano, California
| | - Erik P. Johnson
- Quest Diagnostics Nichols Institute, San Juan Capistrano, California
| | - Martin F. Siaw
- Quest Diagnostics Nichols Institute, San Juan Capistrano, California
| | | | - Richard Ogden
- Tobira Therapeutics Inc., South San Francisco, California
| | - Jamie L. Platt
- Quest Diagnostics Nichols Institute, San Juan Capistrano, California
| | - Rick L. Pesano
- Quest Diagnostics Nichols Institute, San Juan Capistrano, California
| | - Eric Lefebvre
- Tobira Therapeutics Inc., South San Francisco, California
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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. Antimicrob Agents Chemother 2014; 58:2167-85. [PMID: 24468782 DOI: 10.1128/aac.02710-13] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
With 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-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-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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Pérez-Olmeda M, Alcami J. Determination of HIV tropism and its use in the clinical practice. Expert Rev Anti Infect Ther 2014; 11:1291-302. [DOI: 10.1586/14787210.2013.852469] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Prosperi MCF, Yin L, Nolan DJ, Lowe AD, Goodenow MM, Salemi M. Empirical validation of viral quasispecies assembly algorithms: state-of-the-art and challenges. Sci Rep 2013; 3:2837. [PMID: 24089188 PMCID: PMC3789152 DOI: 10.1038/srep02837] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2013] [Accepted: 09/13/2013] [Indexed: 11/22/2022] Open
Abstract
Next generation sequencing (NGS) is superseding Sanger technology for analysing intra-host viral populations, in terms of genome length and resolution. We introduce two new empirical validation data sets and test the available viral population assembly software. Two intra-host viral population 'quasispecies' samples (type-1 human immunodeficiency and hepatitis C virus) were Sanger-sequenced, and plasmid clone mixtures at controlled proportions were shotgun-sequenced using Roche's 454 sequencing platform. The performance of different assemblers was compared in terms of phylogenetic clustering and recombination with the Sanger clones. Phylogenetic clustering showed that all assemblers captured a proportion of the most divergent lineages, but none were able to provide a high precision/recall tradeoff. Estimated variant frequencies mildly correlated with the original. Given the limitations of currently available algorithms identified by our empirical validation, the development and exploitation of additional data sets is needed, in order to establish an efficient framework for viral population reconstruction using NGS.
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Affiliation(s)
- Mattia C. F. Prosperi
- University of Manchester, Faculty of Medical and Human Sciences, Northwest Institute of Bio-Health Informatics, Centre for Health Informatics, Institute of Population Health, Manchester, UK
- University of Florida, College of Medicine, Department of Pathology, Immunology and Laboratory Medicine, Gainesville, Florida, USA
| | - Li Yin
- University of Florida, College of Medicine, Department of Pathology, Immunology and Laboratory Medicine, Gainesville, Florida, USA
- Florida Center for AIDS Research, Gainesville, Florida, USA
| | - David J. Nolan
- University of Florida, College of Medicine, Department of Pathology, Immunology and Laboratory Medicine, Gainesville, Florida, USA
| | - Amanda D. Lowe
- University of Florida, College of Medicine, Department of Pathology, Immunology and Laboratory Medicine, Gainesville, Florida, USA
- Florida Center for AIDS Research, Gainesville, Florida, USA
| | - Maureen M. Goodenow
- University of Florida, College of Medicine, Department of Pathology, Immunology and Laboratory Medicine, Gainesville, Florida, USA
- Florida Center for AIDS Research, Gainesville, Florida, USA
| | - Marco Salemi
- University of Florida, College of Medicine, Department of Pathology, Immunology and Laboratory Medicine, Gainesville, Florida, USA
- Florida Center for AIDS Research, Gainesville, Florida, USA
- Emerging Pathogens Institute, Gainesville, Florida, USA
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Genotypic analysis of the V3 region of HIV from virologic nonresponders to maraviroc-containing regimens reveals distinct patterns of failure. Antimicrob Agents Chemother 2013; 57:6122-30. [PMID: 24080655 DOI: 10.1128/aac.01534-13] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
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. All patients had R5 HIV by the original Trofile assay before entry. We used population-based sequencing methods and the geno2pheno algorithm to examine changes in tropism and V3 sequences at the time of failure. Using deep sequencing, we assessed whether V3 sequences observed at failure emerged from preexisting subpopulations. From population genotyping data at failure, 90 patients had R5 results, and 91 had non-R5 results. Of the latter group, the geno2pheno false-positive rate (FPR) value fell from a median of 20 at screening to 1.1 at failure. By deep sequencing, the median percentage of non-R5 variants in these patients rose from 1.4% to 99.5% after a median of 4 weeks on maraviroc. In 70% of cases, deep sequencing could detect a pretreatment CXCR4-using subpopulation, which emerged at failure. Overall, there were two distinct patterns of failure of maraviroc. Patients failing with R5 generally had few V3 substitutions and low non-R5 prevalence by deep sequencing. Patients with non-R5 HIV who were failing developed very-high-prevalence non-R5 HIV (median, 99%) and had very low geno2pheno values.
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Capobianchi MR, Giombini E, Rozera G. Next-generation sequencing technology in clinical virology. Clin Microbiol Infect 2013; 19:15-22. [PMID: 23279287 DOI: 10.1111/1469-0691.12056] [Citation(s) in RCA: 117] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2012] [Revised: 09/17/2012] [Accepted: 09/22/2012] [Indexed: 12/18/2022]
Abstract
Recent advances in nucleic acid sequencing technologies, referred to as 'next-generation' sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represents the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a dataset of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner.
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Affiliation(s)
- M R Capobianchi
- National Institute for Infectious Diseases 'L. Spallanzani', Rome, Italy.
| | - E Giombini
- National Institute for Infectious Diseases 'L. Spallanzani', Rome, Italy
| | - G Rozera
- National Institute for Infectious Diseases 'L. Spallanzani', Rome, Italy
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Raymond S, Saliou A, Nicot F, Delobel P, Dubois M, Carcenac R, Saune K, Marchou B, Massip P, Izopet J. Characterization of CXCR4-using HIV-1 during primary infection by ultra-deep pyrosequencing. J Antimicrob Chemother 2013; 68:2875-81. [DOI: 10.1093/jac/dkt290] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
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Delobel P, Cazabat M, Saliou A, Loiseau C, Coassin L, Raymond S, Requena M, Marchou B, Massip P, Izopet J. Primary resistance of CCR5-tropic HIV-1 to maraviroc cannot be predicted by the V3 sequence. J Antimicrob Chemother 2013; 68:2506-14. [DOI: 10.1093/jac/dkt249] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
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Huang A, Kantor R, DeLong A, Schreier L, Istrail S. QColors: an algorithm for conservative viral quasispecies reconstruction from short and non-contiguous next generation sequencing reads. In Silico Biol 2013; 11:193-201. [PMID: 23202421 PMCID: PMC5530257 DOI: 10.3233/isb-2012-0454] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Next generation sequencing technologies have recently been applied to characterize mutational spectra of the heterogeneous population of viral genotypes (known as a quasispecies) within HIV-infected patients. Such information is clinically relevant because minority genetic subpopulations of HIV within patients enable viral escape from selection pressures such as the immune response and antiretroviral therapy. However, methods for quasispecies sequence reconstruction from next generation sequencing reads are not yet widely used and remains an emerging area of research. Furthermore, the majority of research methodology in HIV has focused on 454 sequencing, while many next-generation sequencing platforms used in practice are limited to shorter read lengths relative to 454 sequencing. Little work has been done in determining how best to address the read length limitations of other platforms. The approach described here incorporates graph representations of both read differences and read overlap to conservatively determine the regions of the sequence with sufficient variability to separate quasispecies sequences. Within these tractable regions of quasispecies inference, we use constraint programming to solve for an optimal quasispecies subsequence determination via vertex coloring of the conflict graph, a representation which also lends itself to data with non-contiguous reads such as paired-end sequencing. We demonstrate the utility of the method by applying it to simulations based on actual intra-patient clonal HIV-1 sequencing data.
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Affiliation(s)
- Austin Huang
- Division of Infectious Disease, Computer Science Department, Brown University, Box 1910, Providence, RI 02912, USA.
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McLaughlin S, Swenson LC, Hu S, Hughes P, Harrigan PR, Coombs RW, Frenkel LM. Development of a novel codon-specific polymerase chain reaction for the detection of CXCR4-utilizing HIV type 1 subtype B. AIDS Res Hum Retroviruses 2013; 29:814-25. [PMID: 23343425 PMCID: PMC3636599 DOI: 10.1089/aid.2012.0024] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
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 codon-specific 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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Sensitive cell-based assay for determination of human immunodeficiency virus type 1 coreceptor tropism. J Clin Microbiol 2013; 51:1517-27. [PMID: 23486708 DOI: 10.1128/jcm.00092-13] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.
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Abstract
Technologic advances in human immunodeficiency virus type 1 (HIV-1) sequencing have revolutionized the study of antiretroviral drug resistance and are increasingly moving from the laboratory to clinical practice. These techniques are able to detect HIV-1 drug resistance mutations present at low frequencies not detectable by current HIV-1 genotyping assays. For a number of commonly used antiretroviral medications, such as nonnucleoside reverse transcriptase inhibitors, the detection of these drug-resistant minority variants significantly increases the risk of treatment failure. The level of evidence, however, is insufficient to determine the impact of HIV-1 minority variants for several other classes of antiretroviral medications. Clinicians should be aware of the novel technologies that are moving into routine clinical use and the clinical implications of HIV-1 minority variants. Additional studies are needed to determine the optimal platform for clinical application of these new technologies and to provide guidance to clinicians on the type and frequency of clinically important HIV-1 minority variants.
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Affiliation(s)
- Jonathan Z Li
- Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
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Watson SJ, Welkers MRA, Depledge DP, Coulter E, Breuer JM, de Jong MD, Kellam P. Viral population analysis and minority-variant detection using short read next-generation sequencing. Philos Trans R Soc Lond B Biol Sci 2013; 368:20120205. [PMID: 23382427 PMCID: PMC3678329 DOI: 10.1098/rstb.2012.0205] [Citation(s) in RCA: 142] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.
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Affiliation(s)
- Simon J Watson
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
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Gall A, Kaye S, Hué S, Bonsall D, Rance R, Baillie GJ, Fidler SJ, Weber JN, McClure MO, Kellam P. Restriction of V3 region sequence divergence in the HIV-1 envelope gene during antiretroviral treatment in a cohort of recent seroconverters. Retrovirology 2013; 10:8. [PMID: 23331949 PMCID: PMC3605130 DOI: 10.1186/1742-4690-10-8] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2011] [Accepted: 04/27/2012] [Indexed: 12/21/2022] Open
Abstract
Background Dynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods. Results Here we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect. Conclusions Deep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.
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Affiliation(s)
- Astrid Gall
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
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