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John Mellors
University of Pittsburgh, Pittsburg, PA, USA

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Journal article
Published: 25 June 2021 in Viruses
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Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

ACS Style

Leah Brandt; Shuang Guo; Kevin Joseph; Jana Jacobs; Asma Naqvi; John Coffin; Mary Kearney; Elias Halvas; Xiaolin Wu; Stephen Hughes; John Mellors. Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR. Viruses 2021, 13, 1235 .

AMA Style

Leah Brandt, Shuang Guo, Kevin Joseph, Jana Jacobs, Asma Naqvi, John Coffin, Mary Kearney, Elias Halvas, Xiaolin Wu, Stephen Hughes, John Mellors. Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR. Viruses. 2021; 13 (7):1235.

Chicago/Turabian Style

Leah Brandt; Shuang Guo; Kevin Joseph; Jana Jacobs; Asma Naqvi; John Coffin; Mary Kearney; Elias Halvas; Xiaolin Wu; Stephen Hughes; John Mellors. 2021. "Tracking HIV-1-Infected Cell Clones Using Integration Site-Specific qPCR." Viruses 13, no. 7: 1235.

Research article
Published: 23 June 2021 in Science Translational Medicine
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Toll-like receptor 7 (TLR7) agonists, in combination with other therapies, can induce sustained control of simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) in nonhuman primates. Here, we report the results of a randomized, double-blind, placebo-controlled phase 1b clinical trial of an oral TLR7 agonist, vesatolimod, in HIV-1–infected controllers on antiretroviral therapy (ART). We randomized participants 2:1 to receive vesatolimod (n = 17) or placebo (n = 8) once every other week for a total of 10 doses while continuing on ART. ART was then interrupted, and the time to viral rebound was analyzed using the Kaplan-Meier method. Vesatolimod was associated with induction of immune cell activation, decreases in intact proviral DNA during ART, and a modest increase in time to rebound after ART was interrupted. The delayed viral rebound was predicted by the lower intact proviral DNA at the end of vesatolimod treatment (13 days after the final dose). Inferred pathway analysis suggested increased dendritic cell and natural killer cell cross-talk and an increase in cytotoxicity potential after vesatolimod dosing. Larger clinical studies will be necessary to assess the efficacy of vesatolimod-based combination therapies aimed at long-term control of HIV infection.

ACS Style

Devi SenGupta; Cynthia Brinson; Edwin DeJesus; Anthony Mills; Peter Shalit; Susan Guo; Yanhui Cai; Jeffrey J. Wallin; Liao Zhang; Rita Humeniuk; Rebecca Begley; Romas Geleziunas; John Mellors; Terri Wrin; Norman Jones; Jeffrey Milush; April L. Ferre; Barbara L. Shacklett; Greg M. Laird; Brian Moldt; Elena Vendrame; Diana M. Brainard; Moti Ramgopal; Steven G. Deeks. The TLR7 agonist vesatolimod induced a modest delay in viral rebound in HIV controllers after cessation of antiretroviral therapy. Science Translational Medicine 2021, 13, eabg3071 .

AMA Style

Devi SenGupta, Cynthia Brinson, Edwin DeJesus, Anthony Mills, Peter Shalit, Susan Guo, Yanhui Cai, Jeffrey J. Wallin, Liao Zhang, Rita Humeniuk, Rebecca Begley, Romas Geleziunas, John Mellors, Terri Wrin, Norman Jones, Jeffrey Milush, April L. Ferre, Barbara L. Shacklett, Greg M. Laird, Brian Moldt, Elena Vendrame, Diana M. Brainard, Moti Ramgopal, Steven G. Deeks. The TLR7 agonist vesatolimod induced a modest delay in viral rebound in HIV controllers after cessation of antiretroviral therapy. Science Translational Medicine. 2021; 13 (599):eabg3071.

Chicago/Turabian Style

Devi SenGupta; Cynthia Brinson; Edwin DeJesus; Anthony Mills; Peter Shalit; Susan Guo; Yanhui Cai; Jeffrey J. Wallin; Liao Zhang; Rita Humeniuk; Rebecca Begley; Romas Geleziunas; John Mellors; Terri Wrin; Norman Jones; Jeffrey Milush; April L. Ferre; Barbara L. Shacklett; Greg M. Laird; Brian Moldt; Elena Vendrame; Diana M. Brainard; Moti Ramgopal; Steven G. Deeks. 2021. "The TLR7 agonist vesatolimod induced a modest delay in viral rebound in HIV controllers after cessation of antiretroviral therapy." Science Translational Medicine 13, no. 599: eabg3071.

Journal article
Published: 17 May 2021 in Journal of Virus Eradication
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The quantitative viral outgrowth assay (qVOA) is the gold standard for measuring inducible, replication-competent HIV-1. Using MOLT4-R5 and SupT1-R5 cell lines instead of allogeneic blasts and HIV-1 RNA detection rather than p24 enzyme-immunoassay (EIA) has been proposed to improve the sensitivity of the qVOA. It is unclear, however, how these alternative approaches affect qVOA performance. We compared three qVOAs methods across 15 persons with HIV-1 on suppressive antiretroviral therapy and found that the MOLT4-R5 method yielded a significantly higher proportion of p24-positive wells (42%) than both the allogeneic blast (29%) and SupT1-R5 (32%) assays. Additionally, 5 of 7 qVOAs that were negative by p24 EIA showed viral outgrowth by HIV-1 RNA quantification (>10-fold increase within 7 days). These findings reveal the potential for underestimation of the latent, inducible reservoir by qVOA depending on the target cells used and the measure of viral outgrowth. Use of MOLT4-R5 cells with both p24 EIA and HIV-1 RNA to detect viral outgrowth was the most sensitive method.

ACS Style

P. Nathan Enick; Joseph P. Brooker; Camille M. Tumiotto; Brittany T. Staines; Joseph J. Eron; Deborah K. McMahon; Rajesh T. Gandhi; John W. Mellors; Michele D. Sobolewski. Comparison of methods to quantify inducible HIV-1 outgrowth. Journal of Virus Eradication 2021, 7, 100043 .

AMA Style

P. Nathan Enick, Joseph P. Brooker, Camille M. Tumiotto, Brittany T. Staines, Joseph J. Eron, Deborah K. McMahon, Rajesh T. Gandhi, John W. Mellors, Michele D. Sobolewski. Comparison of methods to quantify inducible HIV-1 outgrowth. Journal of Virus Eradication. 2021; 7 (2):100043.

Chicago/Turabian Style

P. Nathan Enick; Joseph P. Brooker; Camille M. Tumiotto; Brittany T. Staines; Joseph J. Eron; Deborah K. McMahon; Rajesh T. Gandhi; John W. Mellors; Michele D. Sobolewski. 2021. "Comparison of methods to quantify inducible HIV-1 outgrowth." Journal of Virus Eradication 7, no. 2: 100043.

Journal article
Published: 08 February 2021 in JCI Insight
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Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.

ACS Style

Eva M. Stevenson; Adam R. Ward; Ronald Truong; Allison S. Thomas; Szu-Han Huang; Thomas R. Dilling; Sandra Terry; John K. Bui; Talia M. Mota; Ali Danesh; Guinevere Q. Lee; Andrea Gramatica; Pragya Khadka; Winiffer D. Conce Alberto; Rajesh T. Gandhi; Deborah K. McMahon; Christina M. Lalama; Ronald J. Bosch; Bernard Macatangay; Joshua C. Cyktor; Joseph J. Eron; John W. Mellors; R. Brad Jones. HIV-specific T cell responses reflect substantive in vivo interactions with antigen despite long-term therapy. JCI Insight 2021, 6, 1 .

AMA Style

Eva M. Stevenson, Adam R. Ward, Ronald Truong, Allison S. Thomas, Szu-Han Huang, Thomas R. Dilling, Sandra Terry, John K. Bui, Talia M. Mota, Ali Danesh, Guinevere Q. Lee, Andrea Gramatica, Pragya Khadka, Winiffer D. Conce Alberto, Rajesh T. Gandhi, Deborah K. McMahon, Christina M. Lalama, Ronald J. Bosch, Bernard Macatangay, Joshua C. Cyktor, Joseph J. Eron, John W. Mellors, R. Brad Jones. HIV-specific T cell responses reflect substantive in vivo interactions with antigen despite long-term therapy. JCI Insight. 2021; 6 (3):1.

Chicago/Turabian Style

Eva M. Stevenson; Adam R. Ward; Ronald Truong; Allison S. Thomas; Szu-Han Huang; Thomas R. Dilling; Sandra Terry; John K. Bui; Talia M. Mota; Ali Danesh; Guinevere Q. Lee; Andrea Gramatica; Pragya Khadka; Winiffer D. Conce Alberto; Rajesh T. Gandhi; Deborah K. McMahon; Christina M. Lalama; Ronald J. Bosch; Bernard Macatangay; Joshua C. Cyktor; Joseph J. Eron; John W. Mellors; R. Brad Jones. 2021. "HIV-specific T cell responses reflect substantive in vivo interactions with antigen despite long-term therapy." JCI Insight 6, no. 3: 1.

Journal article
Published: 05 October 2020 in Journal of Clinical Investigation
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BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred by clinicians for nonsuppressible viremia (plasma HIV-1 RNA above 40 copies/mL) despite reported adherence to ART and the absence of drug resistance to the current ART regimen.METHODSSamples were collected from at least 2 time points from 8 donors who had nonsuppressible viremia for more than 6 months. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were amplified by PCR and sequenced for evidence of clones of cells that produced infectious viruses. Clones were confirmed by host-proviral integration site analysis.RESULTSHIV-1 genomic RNA with identical sequences were identified in plasma samples from all 8 donors. The identical viral RNA sequences did not change over time and did not evolve resistance to the ART regimen. In 4 of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication competent. Integration sites for infectious proviruses from those 4 donors were mapped to the introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were estimated to be from 50 million to 350 million cells.CONCLUSIONThese findings show that clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia. The mechanisms involved in clonal expansion and persistence need to be defined to effectively target viremia and the HIV-1 reservoir.FUNDINGNational Cancer Institute, NIH; Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute; Bill and Melinda Gates Foundation; Office of AIDS Research; American Cancer Society; National Cancer Institute through a Leidos subcontract; National Institute for Allergy and Infectious Diseases, NIH, to the I4C Martin Delaney Collaboratory; University of Rochester Center for AIDS Research and University of Rochester HIV/AIDS Clinical Trials Unit.

ACS Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Wei Shao; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus. Journal of Clinical Investigation 2020, 130, 5847 -5857.

AMA Style

Elias K. Halvas, Kevin W. Joseph, Leah D. Brandt, Shuang Guo, Michele D. Sobolewski, Jana L. Jacobs, Camille Tumiotto, John K. Bui, Joshua C. Cyktor, Brandon F. Keele, Gene D. Morse, Michael J. Bale, Wei Shao, Mary F. Kearney, John M. Coffin, Jason W. Rausch, Xiaolin Wu, Stephen H. Hughes, John W. Mellors. HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus. Journal of Clinical Investigation. 2020; 130 (11):5847-5857.

Chicago/Turabian Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Wei Shao; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. 2020. "HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus." Journal of Clinical Investigation 130, no. 11: 5847-5857.

Other
Published: 29 September 2020
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Background/Aims We reviewed demographic and clinical profiles, along with measures of hospital-based clinical practice to identify temporal changes in clinical practice that may have affected in-hospital outcomes of patients with COVID-19. Methods Data consisted of sociodemographic and clinical data captured in University of Pittsburgh Medical Center (UPMC) electronic medical record (EMR) systems, linked by common variables (deidentified). The analysis population included hospitalized patients (across 21 hospitals) with a primary diagnosis of COVID-19 infection during the period March 14-August 31, 2020. The primary outcome was a composite of in-hospital mechanical ventilation/mortality. We compared temporal trends in patient characteristics, clinical practice, and hospital outcomes using 4 time-defined epochs for calendar year 2020: March 14-March 31 (epoch 1); April 1-May 15, (epoch 2), May 16-June 28 (epoch 3); and June 29-August 31 (epoch 4). We report unadjusted survival estimates, followed by propensity score analyses to adjust for differences in patient characteristics, to compare in-hospital outcomes of epoch 4 patients (recently treated) to epoch 1-3 patients (earlier treated). Results Mean number of hospital admissions was 9.9 per day during epoch 4, which was ∼2-to 3-fold higher than the earlier epochs. Presenting characteristics of the 1,076 COVID-19 hospitalized patients were similar across the 4 epochs, including mean age. The crude rate of mechanical ventilation/mortality was lower in epoch 4 patients (17%) than in epoch 1-3 patients (23% to 35%). When censoring for incomplete patient follow-up, the rate of mechanical ventilation/mortality was lower in epoch 4 patients (pp=0.0002) and mortality (p=0.02). In propensity score adjusted analyses, the in-hospital relative risk (RR) of mechanical ventilation/mortality was lower in epoch 4 patients (RR=0.67, 95% CI: 0.48, 0.93). For the outcome being discharged alive within 3, 5, or 7 days of admission, adjusted odds ranged from 1.6-to 1.7-fold higher among epoch 4 patients compared to earlier treated patients. The better outcomes in epoch 4 patients were principally observed in patients under the age of 75 years. Patient level dexamethasone use was 55.6% in epoch 4 compared to 15% or less of patients in the earlier epochs. Most patients across epochs received anticoagulation drugs (principally heparin). Overall steroid (81.7% vs. 54.3%, pp=0.0001) was more frequent on the day or day after hospitalization in epoch 4 patients compared to earlier treated patients. Conclusions In our large system, recently treated hospitalized COVID-19 patients had lower rates of in-hospital mechanical ventilation/mortality and shorter length of hospital stay. Alongside of this was a change to early initiation of glucocorticoid therapy and anticoagulation. The extent to which the improvement in patient outcomes was related to changes in clinical practice remains to be established.

ACS Style

Kevin E. Kip; Graham Snyder; Donald M. Yealy; John W. Mellors; Tami Minnier; Michael P. Donahoe; Jeffrey McKibben; Kevin Collins; Oscar C. Marroquin. Temporal Changes in Clinical Practice with COVID-19 Hospitalized Patients: Potential Explanations for Better In-Hospital Outcomes. 2020, 1 .

AMA Style

Kevin E. Kip, Graham Snyder, Donald M. Yealy, John W. Mellors, Tami Minnier, Michael P. Donahoe, Jeffrey McKibben, Kevin Collins, Oscar C. Marroquin. Temporal Changes in Clinical Practice with COVID-19 Hospitalized Patients: Potential Explanations for Better In-Hospital Outcomes. . 2020; ():1.

Chicago/Turabian Style

Kevin E. Kip; Graham Snyder; Donald M. Yealy; John W. Mellors; Tami Minnier; Michael P. Donahoe; Jeffrey McKibben; Kevin Collins; Oscar C. Marroquin. 2020. "Temporal Changes in Clinical Practice with COVID-19 Hospitalized Patients: Potential Explanations for Better In-Hospital Outcomes." , no. : 1.

Preprint content
Published: 06 August 2020
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Despite the effectiveness of antiretroviral (ARV) therapy, virological failure can occur in some HIV-1 infected patients in the absence of mutations in the proteins targeted by these drugs. We previously reported that, in vitro, the lab-adapted NL4-3 strain of HIV-1 can acquire resistance to the integrase inhibitor dolutegravir (DTG) by acquiring mutations in the envelope glycoprotein (Env) that enhance the ability of HIV-1 to spread via cell-cell transmission. In this study, we investigated whether Env-mediated drug resistance extends to ARVs other than DTG and whether it occurs in other HIV-1 isolates. We demonstrate that Env mutations can broadly confer resistance to multiple classes of ARVs in the context of cell-cell but not cell-free infection and also increase resistance to ARVs when coupled with target-gene drug resistance mutations. To investigate the mechanism of Env-mediated drug resistance, we evaluated the impact of the Env mutations on Env stability and conformational dynamics. We observe that the NL4-3 Env mutants display a more stable and closed Env conformation compared to WT virus and reduced rates of gp120 shedding. We also selected for mutations in the gp41 ectodomain of clinically relevant, CCR5-tropic isolates in the presence of DTG. These Env mutants exhibit reduced susceptibility to DTG, with effects on replication kinetics and Env structure that are HIV-1 strain-dependent. Finally, to examine a possible in vivo relevance of Env-mediated drug resistance, we performed single-genome sequencing of plasma-derived virus from five patients failing an integrase inhibitor-containing regimen. This analysis revealed the presence of several mutations in the highly conserved gp120-gp41 interface despite low frequency of resistance mutations in integrase. These results suggest a “stepping-stone” model whereby mutations in Env that enhance the ability of HIV-1 to spread via a cell-cell route increase the opportunity for the virus to acquire high-level drug resistance mutations in ARV-target genes. Author summary Although combination antiretroviral (ARV) therapy has proven highly effective in controlling the progression of HIV disease, drug resistance can be a major obstacle to long-term treatment, particularly in resource-limited settings. In most cases, resistance arises from the accumulation of mutations in the ARV-target genes; however, in some cases, resistance develops without ARV target-gene mutations. We previously reported that mutations in the HIV-1 envelope glycoprotein (Env) confer resistance to an integrase inhibitor. Here we investigated the mechanism of Env-mediated drug resistance and the possible contribution of Env to virological failure in vivo. We demonstrate that Env mutations can confer broad resistance to multiple classes of ARVs and define the effect of the Env mutations on Env subunit interactions and sensitivity to neutralizing antibodies. We also selected for drug resistance mutations in Env in clinically relevant HIV-1 isolates. We observed that many Env mutations accumulated in individuals failing integrase inhibitor therapy despite a low frequency of resistance mutations in integrase. Our findings suggest that broad-based, Env-mediated drug resistance may impact current and possibly future therapeutic strategies. Our findings also provide clues towards understanding how ARV-treated patients can experience virological failure without acquiring drug resistance mutations in ARV-target genes.

ACS Style

Yuta Hikichi; Rachel Van Duyne; Phuong Pham; Jennifer L. Groebner; Ann Wiegand; John W. Mellors; Mary F. Kearney; Eric O. Freed. Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations. 2020, 1 .

AMA Style

Yuta Hikichi, Rachel Van Duyne, Phuong Pham, Jennifer L. Groebner, Ann Wiegand, John W. Mellors, Mary F. Kearney, Eric O. Freed. Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations. . 2020; ():1.

Chicago/Turabian Style

Yuta Hikichi; Rachel Van Duyne; Phuong Pham; Jennifer L. Groebner; Ann Wiegand; John W. Mellors; Mary F. Kearney; Eric O. Freed. 2020. "Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations." , no. : 1.

Preprint content
Published: 31 January 2020
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BACKGROUND HIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred for non-suppressible viremia (plasma HIV-1 RNA above 40 copies/ml) who reported adherence to ART and did not show drug resistance to their current regimen. METHODS Samples were collected from at least two time points from eight donors who had non-suppressible viremia for more than six months on ART. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were PCR-amplified and sequenced for evidence of clones of cells that produced infectious viruses. Clones were identified by host-proviral integration site analysis. RESULTS HIV-1 genomic RNAs with identical sequences were identified in plasma samples from all eight donors. The identical viral RNA sequences did not change over time and lacked resistance to the ART regimen. In four of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication-competent. Integration sites for infectious proviruses from those four donors were mapped to introns of theMATR3,ZNF268,ZNF721/ABCA11P, andABCA11Pgenes. The sizes of the clones were from 50 million to 350 million cells. CONCLUSION Clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia despite medication adherence and absence of drug resistance. The mechanisms involved in clonal expansion and persistence need to be defined to eliminate viremia and the HIV-1 reservoir.

ACS Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus. 2020, 1 .

AMA Style

Elias K. Halvas, Kevin W. Joseph, Leah D. Brandt, Shuang Guo, Michele D. Sobolewski, Jana L. Jacobs, Camille Tumiotto, John K. Bui, Joshua C. Cyktor, Brandon F. Keele, Gene D. Morse, Michael J. Bale, Mary F. Kearney, John M. Coffin, Jason W. Rausch, Xiaolin Wu, Stephen H. Hughes, John W. Mellors. HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus. . 2020; ():1.

Chicago/Turabian Style

Elias K. Halvas; Kevin W. Joseph; Leah D. Brandt; Shuang Guo; Michele D. Sobolewski; Jana L. Jacobs; Camille Tumiotto; John K. Bui; Joshua C. Cyktor; Brandon F. Keele; Gene D. Morse; Michael J. Bale; Mary F. Kearney; John M. Coffin; Jason W. Rausch; Xiaolin Wu; Stephen H. Hughes; John W. Mellors. 2020. "HIV-1 Viremia Not Suppressible By Antiretroviral Therapy Can Originate from Large T-Cell Clones Producing Infectious Virus." , no. : 1.

Journal article
Published: 20 June 2019 in JCI Insight
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In HIV-infected individuals on long-term antiretroviral therapy (ART), more than 40% of the infected cells are in clones. Although most HIV proviruses present in individuals on long-term ART are defective, including those in clonally expanded cells, there is increasing evidence that clones carrying replication-competent proviruses are common in patients on long-term ART and form part of the HIV reservoir that makes it impossible to cure HIV infection with current ART alone. Given the importance of clonal expansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca. 1 × 105 cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III-V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6,500 independent integration sites from peripheral blood mononuclear cells before ART was initiated and after 0.5-18 years of suppressive ART. Some infected clones could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years.

ACS Style

John M. Coffin; David W. Wells; Jennifer M. Zerbato; JoAnn D. Kuruc; Shuang Guo; Brian T. Luke; Joseph J. Eron; Michael Bale; Jonathan Spindler; Francesco Roberto Simonetti; Shawn Hill; Mary F. Kearney; Frank Maldarelli; Xiaolin Wu; John W. Mellors; Stephen H. Hughes. Clones of infected cells arise early in HIV-infected individuals. JCI Insight 2019, 4, 1 .

AMA Style

John M. Coffin, David W. Wells, Jennifer M. Zerbato, JoAnn D. Kuruc, Shuang Guo, Brian T. Luke, Joseph J. Eron, Michael Bale, Jonathan Spindler, Francesco Roberto Simonetti, Shawn Hill, Mary F. Kearney, Frank Maldarelli, Xiaolin Wu, John W. Mellors, Stephen H. Hughes. Clones of infected cells arise early in HIV-infected individuals. JCI Insight. 2019; 4 (12):1.

Chicago/Turabian Style

John M. Coffin; David W. Wells; Jennifer M. Zerbato; JoAnn D. Kuruc; Shuang Guo; Brian T. Luke; Joseph J. Eron; Michael Bale; Jonathan Spindler; Francesco Roberto Simonetti; Shawn Hill; Mary F. Kearney; Frank Maldarelli; Xiaolin Wu; John W. Mellors; Stephen H. Hughes. 2019. "Clones of infected cells arise early in HIV-infected individuals." JCI Insight 4, no. 12: 1.

Research article
Published: 12 April 2019 in PLOS Computational Biology
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Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a “gold standard” for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates—as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir—rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points. The latent reservoir of resting CD4+ T cells is a major, if not the primary, obstacle to curing HIV. Quantitative viral outgrowth assays (QVOAs) are used to measure the latent reservoir in ART-suppressed HIV-infected people. Using QVOA is difficult, however, as the fraction of cells constituting the latent reservoir is typically about one in one million, far lower than other infectious disease biomarkers. To study reliability of these assays, we distributed 75 PBMC samples from five ART-suppressed HIV-infected participants among four labs, each conducting QVOA and following prespecified sample batching procedures. Using a Bayesian statistical method, we analyzed detailed assay output to understand how results varied within batches, between batches, and between labs. We found that, if batch variation can be controlled (i.e., a lab assays all samples in one batch), typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. We also found that freezing, storing, and thawing samples for later analysis caused no more than a 2-fold change in results. These outcomes, and the statistical methods developed to obtain them, should lead towards more precise and powerful assessments of HIV cure strategies.

ACS Style

Daniel I. S. Rosenbloom; Peter Bacchetti; Mars Stone; Xutao Deng; Ronald J. Bosch; Douglas D. Richman; Janet D. Siliciano; John W. Mellors; Steven Deeks; Roger G. Ptak; Rebecca Hoh; Sheila M. Keating; Melanie Dimapasoc; Marta Massanella; Jun Lai; Michele D. Sobolewski; Deanna A. Kulpa; Michael P. Busch; for the Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group. Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size. PLOS Computational Biology 2019, 15, e1006849 .

AMA Style

Daniel I. S. Rosenbloom, Peter Bacchetti, Mars Stone, Xutao Deng, Ronald J. Bosch, Douglas D. Richman, Janet D. Siliciano, John W. Mellors, Steven Deeks, Roger G. Ptak, Rebecca Hoh, Sheila M. Keating, Melanie Dimapasoc, Marta Massanella, Jun Lai, Michele D. Sobolewski, Deanna A. Kulpa, Michael P. Busch, for the Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group. Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size. PLOS Computational Biology. 2019; 15 (4):e1006849.

Chicago/Turabian Style

Daniel I. S. Rosenbloom; Peter Bacchetti; Mars Stone; Xutao Deng; Ronald J. Bosch; Douglas D. Richman; Janet D. Siliciano; John W. Mellors; Steven Deeks; Roger G. Ptak; Rebecca Hoh; Sheila M. Keating; Melanie Dimapasoc; Marta Massanella; Jun Lai; Michele D. Sobolewski; Deanna A. Kulpa; Michael P. Busch; for the Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group. 2019. "Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size." PLOS Computational Biology 15, no. 4: e1006849.

Evaluation study
Published: 01 March 2019 in Journal of Clinical Microbiology
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A real-time quantitative reverse transcriptase PCR assay with single-copy sensitivity targeting the integrase region of HIV-1 (integrase single-copy assay [iSCA] v1.0) has been widely used to quantify plasma viremia in individuals on antiretroviral therapy (ART). iSCA v1.0 requires the use of an ultracentrifuge, and only about half of the nucleic acid extracted from plasma is assayed for HIV-1 RNA.

ACS Style

Melissa A. Tosiano; Jana L. Jacobs; Kathleen A. Shutt; Joshua C. Cyktor; John W. Mellors. A Simpler and More Sensitive Single-Copy HIV-1 RNA Assay for Quantification of Persistent HIV-1 Viremia in Individuals on Suppressive Antiretroviral Therapy. Journal of Clinical Microbiology 2019, 57, 1 .

AMA Style

Melissa A. Tosiano, Jana L. Jacobs, Kathleen A. Shutt, Joshua C. Cyktor, John W. Mellors. A Simpler and More Sensitive Single-Copy HIV-1 RNA Assay for Quantification of Persistent HIV-1 Viremia in Individuals on Suppressive Antiretroviral Therapy. Journal of Clinical Microbiology. 2019; 57 (3):1.

Chicago/Turabian Style

Melissa A. Tosiano; Jana L. Jacobs; Kathleen A. Shutt; Joshua C. Cyktor; John W. Mellors. 2019. "A Simpler and More Sensitive Single-Copy HIV-1 RNA Assay for Quantification of Persistent HIV-1 Viremia in Individuals on Suppressive Antiretroviral Therapy." Journal of Clinical Microbiology 57, no. 3: 1.

Research article
Published: 25 January 2019 in PLOS ONE
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Blockade of the programmed cell death protein/ligand 1 (PD-1/PD-L1) pathway with monoclonal antibodies (mAb) is now commonly used for cancer immunotherapy and has therapeutic potential in chronic viral infections including HIV-1. PD-1/PD-L1 blockade could augment HIV-1-specific immune responses and reverse HIV-1 latency, but the latter effect has not been clearly shown. We tested the ability of the human anti-PD-L1 mAb BMS-936559 and the human anti-PD-1 mAb nivolumab to increase HIV-1 virion production ex vivo from different peripheral blood mononuclear cell populations obtained from donors on suppressive antiretroviral therapy (ART). Fresh peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, total CD4+ T cells, and resting CD4+ T cells were purified from whole blood of HIV-1-infected donors and cultured in varying concentrations of BMS-936559 (20, 5, or 1.25μg/mL) or nivolumab (5 or 1.25μg/mL), with or without anti-CD3/CD28 stimulatory antibodies. Culture supernatants were assayed for virion HIV-1 RNA by qRT-PCR. Ex vivo exposure to BMS-936559 or nivolumab, with or without anti-CD3/CD28 stimulation, did not consistently increase HIV-1 virion production from blood mononuclear cell populations. Modest (2-fold) increases in virus production were observed in a subset of donors and in some cell types but were not reproducible in longitudinal samples. Cell surface expression of PD-1 and PD-L1 were not associated with changes in virus production. Ex vivo blockade of the PD-1 axis alone has limited effects on HIV-1 latency.

ACS Style

John K. Bui; Joshua C. Cyktor; Elizabeth Fyne; Shalyn Campellone; Stephen W. Mason; John W. Mellors. Blockade of the PD-1 axis alone is not sufficient to activate HIV-1 virion production from CD4+ T cells of individuals on suppressive ART. PLOS ONE 2019, 14, e0211112 .

AMA Style

John K. Bui, Joshua C. Cyktor, Elizabeth Fyne, Shalyn Campellone, Stephen W. Mason, John W. Mellors. Blockade of the PD-1 axis alone is not sufficient to activate HIV-1 virion production from CD4+ T cells of individuals on suppressive ART. PLOS ONE. 2019; 14 (1):e0211112.

Chicago/Turabian Style

John K. Bui; Joshua C. Cyktor; Elizabeth Fyne; Shalyn Campellone; Stephen W. Mason; John W. Mellors. 2019. "Blockade of the PD-1 axis alone is not sufficient to activate HIV-1 virion production from CD4+ T cells of individuals on suppressive ART." PLOS ONE 14, no. 1: e0211112.

Journal article
Published: 01 August 2018 in Virology
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The relationships between HIV-1 DNA copy number, proviral transcriptional activity, and residual plasma viremia in individuals off and on ART are not well defined. To address this, we performed a cross-sectional study of 12 viremic donors and 23 ART-treated virologically suppressed (plasma HIV-1 RNA<20 copies/ml) donors. We report a strong association between HIV-1 DNA copy number and HIV-1 transcriptional activity in blood that persists on suppressive ART, but not between transcriptional activity and the levels of persistent viremia on ART. The latter finding contrasts with that in viremic donors and suggests that most HIV transcription in donors on suppressive ART does not result in virion production. This uncoupling of proviral transcription and viremia warrants closer investigation.

ACS Style

Feiyu Hong; Jana L. Jacobs; Evgenia Aga; Anthony R. Cillo; Elizabeth Fyne; Dianna L. Koontz; Lu Zheng; John W. Mellors. Associations between HIV-1 DNA copy number, proviral transcriptional activity, and plasma viremia in individuals off or on suppressive antiretroviral therapy. Virology 2018, 521, 51 -57.

AMA Style

Feiyu Hong, Jana L. Jacobs, Evgenia Aga, Anthony R. Cillo, Elizabeth Fyne, Dianna L. Koontz, Lu Zheng, John W. Mellors. Associations between HIV-1 DNA copy number, proviral transcriptional activity, and plasma viremia in individuals off or on suppressive antiretroviral therapy. Virology. 2018; 521 ():51-57.

Chicago/Turabian Style

Feiyu Hong; Jana L. Jacobs; Evgenia Aga; Anthony R. Cillo; Elizabeth Fyne; Dianna L. Koontz; Lu Zheng; John W. Mellors. 2018. "Associations between HIV-1 DNA copy number, proviral transcriptional activity, and plasma viremia in individuals off or on suppressive antiretroviral therapy." Virology 521, no. : 51-57.

Basic science
Published: 27 March 2018 in AIDS
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To define the relationships between molecular measures of viral persistence in blood (i.e., plasma viremia, cellular HIV-1 DNA, and mRNA) and expressed or inducible virus from resting CD4 + T-cells of individuals on suppressive ART.We compared molecular measurements of HIV-1 in plasma and in uncultured peripheral blood mononuclear cells (PBMC) to the levels of virions produced by either unstimulated or phorbol myristate acetate and ionomycin (PMA/iono)-stimulated PBMC or resting CD4 + T-cells from 21 donors on suppressive ART.We found that unstimulated virion release from cultured resting CD4 + T-cells was positively correlated with the levels of plasma viremia in vivo (Spearman rho=0.67, p = 0.0017). We also found that levels of both cellular HIV-1 DNA and unspliced HIV-1 mRNA per million uncultured PBMC were positively correlated with the levels of inducible virion release from both PMA/iono-stimulated PBMC (total HIV-1 DNA: rho=0.64, p = 0.0017; unspliced HIV-1 RNA: rho=0.77, p < 0.001) and PMA/iono-stimulated resting CD4 + T-cells (total HIV-1 DNA: rho=0.75, p < 0.001; unspliced HIV-1 RNA: rho=0.75, p < 0.001).These results show for the first time that there are strong associations between in vivo measures of HIV-1 persistence and ex vivo measures of spontaneous and inducible virus production from cultured PBMC and resting CD4 + T-cells. Findings from this study provide insight into the biology of HIV-1 persistence and suggest methods to guide the evaluation of clinical strategies to reduce the size of the viral reservoir.

ACS Style

Anthony R. Cillo; Francis Hong; Angela Tsai; Alivelu Irrinki; Jasmine Kaur; Derek D. Sloan; Mattie Follen; Romas Geleziunas; Tomas Cihlar; Sandra S. Win; Jeffrey P. Murry; John W. Mellors. Blood biomarkers of expressed and inducible HIV-1. AIDS 2018, 32, 699 -708.

AMA Style

Anthony R. Cillo, Francis Hong, Angela Tsai, Alivelu Irrinki, Jasmine Kaur, Derek D. Sloan, Mattie Follen, Romas Geleziunas, Tomas Cihlar, Sandra S. Win, Jeffrey P. Murry, John W. Mellors. Blood biomarkers of expressed and inducible HIV-1. AIDS. 2018; 32 (6):699-708.

Chicago/Turabian Style

Anthony R. Cillo; Francis Hong; Angela Tsai; Alivelu Irrinki; Jasmine Kaur; Derek D. Sloan; Mattie Follen; Romas Geleziunas; Tomas Cihlar; Sandra S. Win; Jeffrey P. Murry; John W. Mellors. 2018. "Blood biomarkers of expressed and inducible HIV-1." AIDS 32, no. 6: 699-708.

Case reports
Published: 07 November 2017 in PLOS Medicine
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It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.

ACS Style

Timothy J. Henrich; Hiroyu Hatano; Oliver Bacon; Louise E. Hogan; Rachel Rutishauser; Alison Hill; Mary Kearney; Elizabeth M. Anderson; Susan P. Buchbinder; Stephanie E. Cohen; Mohamed Abdel-Mohsen; Christopher W. Pohlmeyer; Remi Fromentin; Rebecca Hoh; Albert Y. Liu; Joseph M. McCune; Jonathan Spindler; Kelly Metcalf-Pate; Kristen S. Hobbs; Cassandra Thanh; Erica A. Gibson; Daniel R. Kuritzkes; Robert F. Siliciano; Richard W. Price; Douglas D. Richman; Nicolas Chomont; Janet D. Siliciano; John W. Mellors; Steven A. Yukl; Joel N. Blankson; Teri Liegler; Steven G. Deeks. HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study. PLOS Medicine 2017, 14, e1002417 .

AMA Style

Timothy J. Henrich, Hiroyu Hatano, Oliver Bacon, Louise E. Hogan, Rachel Rutishauser, Alison Hill, Mary Kearney, Elizabeth M. Anderson, Susan P. Buchbinder, Stephanie E. Cohen, Mohamed Abdel-Mohsen, Christopher W. Pohlmeyer, Remi Fromentin, Rebecca Hoh, Albert Y. Liu, Joseph M. McCune, Jonathan Spindler, Kelly Metcalf-Pate, Kristen S. Hobbs, Cassandra Thanh, Erica A. Gibson, Daniel R. Kuritzkes, Robert F. Siliciano, Richard W. Price, Douglas D. Richman, Nicolas Chomont, Janet D. Siliciano, John W. Mellors, Steven A. Yukl, Joel N. Blankson, Teri Liegler, Steven G. Deeks. HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study. PLOS Medicine. 2017; 14 (11):e1002417.

Chicago/Turabian Style

Timothy J. Henrich; Hiroyu Hatano; Oliver Bacon; Louise E. Hogan; Rachel Rutishauser; Alison Hill; Mary Kearney; Elizabeth M. Anderson; Susan P. Buchbinder; Stephanie E. Cohen; Mohamed Abdel-Mohsen; Christopher W. Pohlmeyer; Remi Fromentin; Rebecca Hoh; Albert Y. Liu; Joseph M. McCune; Jonathan Spindler; Kelly Metcalf-Pate; Kristen S. Hobbs; Cassandra Thanh; Erica A. Gibson; Daniel R. Kuritzkes; Robert F. Siliciano; Richard W. Price; Douglas D. Richman; Nicolas Chomont; Janet D. Siliciano; John W. Mellors; Steven A. Yukl; Joel N. Blankson; Teri Liegler; Steven G. Deeks. 2017. "HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study." PLOS Medicine 14, no. 11: e1002417.

Clinical trial
Published: 11 September 2017 in Journal of Clinical Investigation
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It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10-4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.

ACS Style

Gert U. Van Zyl; Mary Grace Katusiime; Ann Wiegand; William McManus; Michael Bale; Elias K. Halvas; Brian Luke; Valerie F. Boltz; Jonathan Spindler; Barbara Laughton; Susan Engelbrecht; John M. Coffin; Mark F. Cotton; Wei Shao; John W. Mellors; Mary F. Kearney. No evidence of HIV replication in children on antiretroviral therapy. Journal of Clinical Investigation 2017, 127, 3827 -3834.

AMA Style

Gert U. Van Zyl, Mary Grace Katusiime, Ann Wiegand, William McManus, Michael Bale, Elias K. Halvas, Brian Luke, Valerie F. Boltz, Jonathan Spindler, Barbara Laughton, Susan Engelbrecht, John M. Coffin, Mark F. Cotton, Wei Shao, John W. Mellors, Mary F. Kearney. No evidence of HIV replication in children on antiretroviral therapy. Journal of Clinical Investigation. 2017; 127 (10):3827-3834.

Chicago/Turabian Style

Gert U. Van Zyl; Mary Grace Katusiime; Ann Wiegand; William McManus; Michael Bale; Elias K. Halvas; Brian Luke; Valerie F. Boltz; Jonathan Spindler; Barbara Laughton; Susan Engelbrecht; John M. Coffin; Mark F. Cotton; Wei Shao; John W. Mellors; Mary F. Kearney. 2017. "No evidence of HIV replication in children on antiretroviral therapy." Journal of Clinical Investigation 127, no. 10: 3827-3834.

Journal article
Published: 11 July 2017 in Pathogens and Immunity
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Identifying host determinants associated with HIV reservoir size and viral rebound timing after an analytic treatment interruption (ATI) is an important step in the search for an HIV functional cure. A pooled analysis of 103 participants from four AIDS Clinical Trials Group ATI studies to identify the association between HLA class I alleles with HIV reservoir size and viral rebound timing. Total HIV DNA and cell-associated HIV RNA (CA-RNA) were quantified in pre-ATI peripheral blood mononuclear cell samples, and residual plasma viremia was measured using the single-copy assay. HLA class I typing was performed and we generated an odds ratio (OR) of predicted HLA effect on HIV viremia control for each individual and compared this with time to viral rebound, and levels of HIV DNA and CA-RNA. There was no significant association between the HLA ORs and levels of HIV DNA or CA-RNA, but carriage of protective HLA-B alleles (lower OR scores) was associated with delayed viral rebound (P=0.02). Higher OR scores at the HLA-C locus were associated with longer duration of ART treatment (P=0.01) and this trend was also seen with the combined OR score (P=0.007). Individuals with protective HLA-B alleles had delayed viral rebound after treatment interruption that was not explained by differences in baseline reservoir size. The results indicate the vital role of cellular host immunity in preventing HIV rebound and the importance of taking into account the HLA status of study participants being evaluated in HIV cure trials.

ACS Style

You Jeong Park; Behzad Etemad; Hayat Ahmed; Vivek Naranbhai; Evgenia Aga; Ronald J. Bosch; John W. Mellors; Daniel R. Kuritzkes; Michael Para; Rajesh T. Gandhi; Mary Carrington; Jonathan Z. Li. Impact of HLA Class I Alleles on Timing of HIV Rebound After Antiretroviral Treatment Interruption. Pathogens and Immunity 2017, 2, 431 -445.

AMA Style

You Jeong Park, Behzad Etemad, Hayat Ahmed, Vivek Naranbhai, Evgenia Aga, Ronald J. Bosch, John W. Mellors, Daniel R. Kuritzkes, Michael Para, Rajesh T. Gandhi, Mary Carrington, Jonathan Z. Li. Impact of HLA Class I Alleles on Timing of HIV Rebound After Antiretroviral Treatment Interruption. Pathogens and Immunity. 2017; 2 (3):431-445.

Chicago/Turabian Style

You Jeong Park; Behzad Etemad; Hayat Ahmed; Vivek Naranbhai; Evgenia Aga; Ronald J. Bosch; John W. Mellors; Daniel R. Kuritzkes; Michael Para; Rajesh T. Gandhi; Mary Carrington; Jonathan Z. Li. 2017. "Impact of HLA Class I Alleles on Timing of HIV Rebound After Antiretroviral Treatment Interruption." Pathogens and Immunity 2, no. 3: 431-445.

Journal article
Published: 20 December 2016 in Retrovirology
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Although next generation sequencing (NGS) offers the potential for studying virus populations in unprecedented depth, PCR error, amplification bias and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. Here we report a method, termed ultrasensitive Single-Genome Sequencing (uSGS), for NGS library construction and analysis that eliminates PCR errors and recombinants, and generates single-genome sequences of the same quality as the “gold-standard” of HIV-1 single-genome sequencing assay but with more than 100-fold greater depth. Primer ID tagged cDNA was synthesized from mixtures of cloned BH10 wild-type and mutant HIV-1 transcripts containing ten drug resistance mutations. First, the resultant cDNA was divided and NGS libraries were generated in parallel using two methods: uSGS and a method applying long PCR primers to attach the NGS adaptors (LP-PCR-1). Second, cDNA was divided and NGS libraries were generated in parallel comparing 3 methods: uSGS and 2 methods adapted from more recent reports using variations of the long PCR primers to attach the adaptors (LP-PCR-2 and LP-PCR-3). Consistently, the uSGS method amplified a greater proportion of cDNAs, averaging 30% compared to 13% for LP-PCR-1, 21% for LP-PCR-2 and 14% for LP-PCR-3. Most importantly, when the uSGS sequences were binned according to their primer IDs, 94% of the bins did not contain PCR recombinant sequences versus only 55, 75 and 65% for LP-PCR-1, 2 and 3, respectively. Finally, when uSGS was applied to plasma samples from HIV-1 infected donors, both frequent and rare variants were detected in each sample and neighbor-joining trees revealed clusters of genomes driven by the linkage of these mutations, showing the lack of PCR recombinants in the datasets. The uSGS assay can be used for accurate detection of rare variants and for identifying linkage of rare alleles associated with HIV-1 drug resistance. In addition, the method allows accurate in-depth analyses of the complex genetic relationships of viral populations in vivo.

ACS Style

Valerie F. Boltz; Jason Rausch; Wei Shao; Junko Hattori; Brian Luke; Frank Maldarelli; John W. Mellors; Mary F. Kearney; John M. Coffin. Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA. Retrovirology 2016, 13, 1 -17.

AMA Style

Valerie F. Boltz, Jason Rausch, Wei Shao, Junko Hattori, Brian Luke, Frank Maldarelli, John W. Mellors, Mary F. Kearney, John M. Coffin. Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA. Retrovirology. 2016; 13 (1):1-17.

Chicago/Turabian Style

Valerie F. Boltz; Jason Rausch; Wei Shao; Junko Hattori; Brian Luke; Frank Maldarelli; John W. Mellors; Mary F. Kearney; John M. Coffin. 2016. "Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA." Retrovirology 13, no. 1: 1-17.

Validation study
Published: 16 December 2016 in Journal of Virological Methods
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HIV-1 sequence variation is a major obstacle to developing molecular based assays for multiple subtypes. This study sought to independently assess performance characteristics of the ViroSeq™ HIV-1 Integrase RUO Genotyping Kit (Celera, US) for samples of multiple different HIV-1 subtypes. 264 samples were tested in the validation, 106 from integrase inhibitor naïve patients' sent for routine HIV-1 drug resistance testing after failing a 1st- or 2nd-line regimen, and 158 samples from an external virology quality assurance program (VQA). For the latter, 53 unique VQA samples were tested in two to five different laboratories to assess assay reproducibility. For all assays, viral RNA was extracted using the ViroSeq extraction module, reverse transcribed, and amplified in a one-step reaction. Four sequencing primers were used to span codons 1-288 of integrase. The Rega subtyping tool was used for subtype assignment. Integrase polymorphisms and mutations were determined as differences from the HXB2 sequence and by the Stanford database, respectively. Sequences obtained from the different laboratories were aligned and sequence homology determined. HIV-1 RNA in the 264 samples ranged from 3.15 to 6.74logcopies/ml. Successful amplification was obtained for 97% of samples (n=256). The 8 samples that failed to amplify were subtype D (n=3), subtype C (n=1), CRF01_AE (n=1), subtype A1 (n=2), and an unassigned subtype (n=1). Of the 256 that successfully amplified samples, 203 (79%) were successfully sequenced with bidirectional coverage. Of the 53 unsuccessful samples, 13 (5%) failed sequencing and 40 (16%) did not have full bidirectional sequence, as a result of failure of sequencing primers: Primer A (n=1); Primer B (n=18); Primer C (n=1); Primer D (n=7) or short sequences (n=16). For the 135 VQA samples (30 unique samples) that were assayed by different laboratories, homology of the sequences obtained ranged from 92.1% to 100%. However, Laboratory 2 detected more mixtures (74%) compared to the other four laboratories, whereas Laboratory 1 detected the least number of mixtures (35%), likely due to differences between the labs in the methods of sequence analysis. Mutations associated with integrase resistance were observed in seven of the 106 (7%) clinical samples [one sample: Q148K; E138K; G140A; two samples: T97A and four samples: L74I]. Of the four samples with L74I, 3 were subtype G. Of the total 264 samples tested, 243 (92%) of samples were able to be amplified and sequenced to generate an integrase genotype. Sequencing results were similar between the testing laboratories with the exception of mixture detection. Mutations associated with integrase inhibitor resistance were observed in only 7% of integrase inhibitor naive samples, and some of these mutations are likely to be due to subtype-specific polymorphisms rather than selection by an integrase inhibitor.

ACS Style

Carole Wallis; Raquel V Viana; Shanmugam Saravanan; Carlos Silva de Jesus; Clement Zeh; Elias K Halvas; John W. Mellors. Performance of Celera RUO integrase resistance assay across multiple HIV-1 subtypes. Journal of Virological Methods 2016, 241, 41 -45.

AMA Style

Carole Wallis, Raquel V Viana, Shanmugam Saravanan, Carlos Silva de Jesus, Clement Zeh, Elias K Halvas, John W. Mellors. Performance of Celera RUO integrase resistance assay across multiple HIV-1 subtypes. Journal of Virological Methods. 2016; 241 ():41-45.

Chicago/Turabian Style

Carole Wallis; Raquel V Viana; Shanmugam Saravanan; Carlos Silva de Jesus; Clement Zeh; Elias K Halvas; John W. Mellors. 2016. "Performance of Celera RUO integrase resistance assay across multiple HIV-1 subtypes." Journal of Virological Methods 241, no. : 41-45.

Short report
Published: 04 July 2016 in Retrovirology
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The NCI Retrovirus Integration Database is a MySql-based relational database created for storing and retrieving comprehensive information about retroviral integration sites, primarily, but not exclusively, HIV-1. The database is accessible to the public for submission or extraction of data originating from experiments aimed at collecting information related to retroviral integration sites including: the site of integration into the host genome, the virus family and subtype, the origin of the sample, gene exons/introns associated with integration, and proviral orientation. Information about the references from which the data were collected is also stored in the database. Tools are built into the website that can be used to map the integration sites to UCSC genome browser, to plot the integration site patterns on a chromosome, and to display provirus LTRs in their inserted genome sequence. The website is robust, user friendly, and allows users to query the database and analyze the data dynamically. Availability: https://rid.ncifcrf.gov; or http://home.ncifcrf.gov/hivdrp/resources.htm.

ACS Style

Wei Shao; Jigui Shan; Mary F. Kearney; Xiaolin Wu; Frank Maldarelli; John W. Mellors; Brian Luke; John M. Coffin; Stephen H. Hughes. Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes. Retrovirology 2016, 13, 47 .

AMA Style

Wei Shao, Jigui Shan, Mary F. Kearney, Xiaolin Wu, Frank Maldarelli, John W. Mellors, Brian Luke, John M. Coffin, Stephen H. Hughes. Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes. Retrovirology. 2016; 13 (1):47.

Chicago/Turabian Style

Wei Shao; Jigui Shan; Mary F. Kearney; Xiaolin Wu; Frank Maldarelli; John W. Mellors; Brian Luke; John M. Coffin; Stephen H. Hughes. 2016. "Retrovirus Integration Database (RID): a public database for retroviral insertion sites into host genomes." Retrovirology 13, no. 1: 47.