133 results on '"O’Doherty, Una"'
Search Results
102. Genetic Evidence That Naive T Cells Can Contribute Significantly to the Human Immunodeficiency Virus Intact Reservoir: Time to Re-evaluate Their Role.
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Rullo, Emmanuele Venanzi, Cannon, LaMont, Pinzone, Marilia Rita, Ceccarelli, Manuela, Nunnari, Giuseppe, and O'Doherty, Una
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GENETICS ,HIV ,T cells - Published
- 2019
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103. Long HIV Type 1 Reverse Transcripts Can Accumulate Stably within Resting CD4+ T Cells While Short Ones Are Degraded.
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Swiggard, William J., O'Doherty, Una, McGain, David, Jeyakumar, Deepa, and Malim, Michael H.
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- 2004
104. Chapter 34 - HIV and HTLV
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O'Doherty, Una and Swiggard, William J.
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- 2001
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105. Long HIV Type 1 Reverse Transcripts Can Accumulate Stably within Resting CD4+ T Cells While Short Ones Are Degraded
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Swiggard, William J., O'Doherty, Una, McGain, David, Jeyakumar, Deepa, and Malim, Michael H.
- Abstract
We utilized quantitative methods to compare the efficiency of reverse transcription and stability of viral DNA within resting and activated T cells. Highly purified resting CD4+ T cells and activated T cells from healthy donors were spinoculated with HIV-1YU-2, then cultured in conditions that maintain both the viability and the quiescence of the resting cells. Spreading infection was suppressed, then kinetic PCR was used to relate the rates of synthesis of short (strong-stop, RU5) and long (gag or U3-gag second strand transfer) viral DNA to the mean number of virions initially bound to each type of cell. As shown previously, activated cells support an initial burst of high-level reverse transcription, which is then followed by a ~ 10-fold decay in cDNA levels over 4.5 days. In resting T cells, although the synthesis of late reverse transcripts was initially ~ 1000-fold less efficient than in activated T cells, the number of these cDNAs per bound input virion rose 10-fold as culture was extended to 4.5 days. The number of late reverse transcripts remained constant for 3 days after the addition of efavirinez, reflecting enhanced stability. In contrast, the short strong-step reverse transcripts were mostly degraded. Thus, late HIV-1 reverse transcripts can accumulate stably in resting T cells in the absence of detectable T cell activation. Defining the underlying basis for the stabilization of late reverse transcripts, and their associated nucleoprotein complexes, may be pertinent to the accumulation of reservoirs of latent HIV-1 in patients, and could provide a target for future therapies.
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- 2004
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106. Improved treatment for primary HIV infection by interferon-alfa therapy? Does HCV treatment in HIV/HCV coinfected patients help us to test this hypothesis? Reply to zur Wiesch and van Lunzen.
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Azzoni, Livio, Foulkes, Andrea S, Papasavvas, Emmanouil, Mexas, Angela M, Lynn, Kenneth M, Mounzer, Karam, Tebas, Pablo, Jacobson, Jeffrey M, Frank, Ian, O'Doherty, Una, Kostman, Jay, and Montaner, Luis J
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- 2013
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107. Next Generation Sequencing in a direct model of HIV infection reveals important parallels and differences to in vivo reservoir dynamics.
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Pinzone, Marilia Rita, Bertuccio, Maria Paola, VanBelzen, D. Jake, Zurakowski, Ryan, and O'Doherty, Una
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HIV infections , *RESERVOIRS , *T cells , *HUMAN genome , *NUCLEOTIDE sequencing , *HIV - Abstract
Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions we obtained HIV sequences at 2, 4 and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/mL IL-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. [ABSTRACT FROM AUTHOR]
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- 2020
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108. Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART
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Mars Stone, Jeffrey N. Martin, Mark Fitch, Abha Chopra, Simon Mallal, Mohamed Abdel-Mohsen, Marc K. Hellerstein, Vanessa A. York, Steven A. Yukl, Paul U. Cameron, Haelee Ahn, Daniel L Cameron, Frederick Hecht, Charline Bacchus-Souffan, Steven G. Deeks, Sharon R Lewin, Daniel B. Reeves, Peter W. Hunt, Joseph Hiatt, Rebecca Hoh, Jori Symons, Joshua T. Schiffer, Joseph M. McCune, Peggy Kim, and O'Doherty, Una
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RNA viruses ,CD4-Positive T-Lymphocytes ,Male ,Composite Particles ,Cellular differentiation ,HIV Infections ,Pathology and Laboratory Medicine ,Virus Replication ,Persistence (computer science) ,White Blood Cells ,0302 clinical medicine ,Immunodeficiency Viruses ,Isotopes ,Animal Cells ,Medicine and Health Sciences ,Public and Occupational Health ,030212 general & internal medicine ,Viral ,Biology (General) ,0303 health sciences ,Effector ,Physics ,Cell Differentiation ,Genomics ,Viral Load ,Middle Aged ,Vaccination and Immunization ,Infectious Diseases ,Anti-Retroviral Agents ,Medical Microbiology ,Viral Pathogens ,Viruses ,Physical Sciences ,HIV/AIDS ,Infectious diseases ,Pathogens ,Cellular Types ,Infection ,Viral load ,Research Article ,Medical conditions ,Adult ,Atoms ,QH301-705.5 ,Immune Cells ,Immunology ,Antiretroviral Therapy ,Viral diseases ,Biology ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Antiviral Therapy ,Clinical Research ,Virology ,Retroviruses ,Genetics ,Humans ,T Helper Cells ,Particle Physics ,Molecular Biology Techniques ,Microbial Pathogens ,Molecular Biology ,030304 developmental biology ,Cloning ,Blood Cells ,Lentivirus ,Organisms ,Biology and Life Sciences ,HIV ,Cell Biology ,DNA ,RC581-607 ,Deuterium ,Viral replication ,Parasitology ,Case-Control Studies ,DNA, Viral ,HIV-1 ,Preventive Medicine ,Immunologic diseases. Allergy ,Homeostasis ,Developmental Biology - Abstract
The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p, Author summary HIV infection is a life-long disease for which we do not currently have a cure. One reason for this is that the virus goes into a dormant state and hides in CD4 T cell lymphocytes. Many of the less mature infected cells slowly self-renew (i.e., replace themselves) over time, whereas other types of infected cells rapidly proliferate and expand, and are replaced more rapidly. In our study, we directly measured how rapidly different types of CD4 T cells divide in treated people with HIV. In addition to confirming that less mature CD4 T cell populations self-renew at a very slow rate, and that more mature cells proliferate and expand at a more rapid rate, we also found that cell types that divide more rapidly are more likely to contain HIV. By carefully defining how rapidly these different cell populations are replaced, we help inform studies of HIV cure interventions that specifically target these discrete populations of HIV-infected cells.
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- 2021
109. R5 HIV env and Vesicular Stomatitis Virus G Protein Cooperate To Mediate Fusion to Naïve CD4+ T Cells.
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Pace, Matthew J., Agosto, Luis, and O'Doherty, Una
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T cells , *HIV , *VESICULAR stomatitis , *VIRAL receptors , *G proteins - Abstract
Naïve CD44 T cells are resistant to both HIV R5 env and vesicular stomatitis virus G protein (VSV-G)-mediated fusion. However, viral particles carrying both HIV R5 env and VSV-G infect naïve cells by an unexplained mechanism. We show that VSV-G-pseudotyped virus cannot fuse to unstimulated cells because the viral particles cannot be endocytosed. However, virions carrying both HIV R5 env and VSV-G can fuse because CD4 binding allows viral uptake. Our findings reveal a unique mechanism by which R5 HIV env and VSV-G cooperate to allow entry to naïve CD4+ T cells, providing a tool to target naïve CD4+ T cells with R5 HIV to study HIV coreceptor signaling and latency. [ABSTRACT FROM AUTHOR]
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- 2011
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110. The CXCR4-Tropic Human Immunodeficiency Virus Envelope Promotes More-Efficient Gene Delivery to Resting CD4+ T Cells than the Vesicular Stomatitis Virus Glycoprotein G Envelope.
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Agosto, Luis M., Yu, Jianqing J., Liszewski, Megan K., Baytop, Clifford, Korokhov, Nikolay, Humeau, Laurent M., and O'Doherty, Una
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HIV , *T cells , *GENETIC transformation , *GENE therapy , *VESICULAR stomatitis , *VIRUS diseases , *GLYCOPROTEINS - Abstract
Current gene transfer protocols for resting CD4+ T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4+ T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4+ T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4+ T cells. To investigate this, we inoculated resting CD4+ T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4+ T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4+ T cells and suggest that fusion may be the critical step restricting transduction of resting CD4+ T cells by lentiviral gene therapy vectors. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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111. Human Immunodeficiency Virus Integrates Directly into Naïve Resting CD4+ T Cells but Enters Naïve Cells Less Efficiently than Memory Cells.
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Jihong Dai, Agosto, Luis M., Baytop, Clifford, Yu, Jianqing J., Pace, Matthew J., Liszewski, Megan K., and O'Doherty, Una
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MEDICAL research , *HIV infections , *T cells , *LYMPHOCYTES , *GENETIC transcription - Abstract
Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at or before reverse transcription, resulting in slower kinetics of reverse transcription. In a previous study, we showed that, despite this restriction at reverse transcription, HIV integration occurs in resting CD4+ T cells, albeit with slower kinetics. In that study, the resting T cells were a mixture of memory and naïve cells. Here we asked whether the more quiescent naïve cell subset could be directly infected by HIV and, if so, whether the level of integration in naïve cells was comparable to that in memory cells. We found that HIV integrates in the naïve subset of resting CD4+ T cells without prior activation of the cells. The level of integration (proviruses/cell) in naïve cells was lower than that in memory cells. This difference between naïve and memory cells was observed whether we inoculated the cells with R5 or X4 HIV and could not be explained solely by differences in coreceptor expression. The presence of endogenous dendritic cells did not change the number of proviruses/cell in memory or naïve cells, and deoxynucleoside pools were equally limiting. Our results instead indicate the existence of a novel restriction point in naïve T cells at viral fusion that results in reduced levels of fusion to naïve CD4+ T cells. We conclude that HIV can integrate into both naïve and memory cells directly. Our data further support our hypothesis that integrated proviral infection of resting T cells can be established without T-cell activation. [ABSTRACT FROM AUTHOR]
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- 2009
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112. HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration
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Agosto, Luis M., Yu, Jianqing J., Dai, Jihong, Kaletsky, Rachel, Monie, Daphne, and O'Doherty, Una
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T cells , *LYMPHOCYTES , *ANTIVIRAL agents , *VIROLOGY - Abstract
Abstract: Human Immunodeficiency Virus Type 1 (HIV-1) establishes a latent reservoir early in infection that is resistant to the host immune response and treatment with highly active antiretroviral therapy (HAART). The best understood of these reservoirs forms in resting CD4+ T cells. While it remains unclear how reservoirs form, a popular model holds that the virus can only integrate in activated CD4+ T cells. Contrary to this model, our previous results suggest that HIV-1 can integrate directly into the genomes of resting CD4+ T cells. However, a limitation of our previous studies was that they were conducted at high viral inoculum and these conditions may lead to cellular activation or saturation of restriction factors. In the present study, we tested if our previous findings were an artifact of high inoculum. To do this, we enhanced the sensitivity of our integration assay by incorporating a repetitive sampling technique that allowed us to capture rare integration events that occur near an Alu repeat. The new technique represents a significant advance as it enabled us to measure integration accurately down to 1 provirus/well in 15,000 genomes—a 40-fold enhancement over our prior assay. Using this assay, we demonstrate that HIV can integrate into resting CD4+ T cells in vitro even at low viral inoculum. These findings suggest there is no threshold number of virions required for HIV to integrate into resting CD4+ T cells. [Copyright &y& Elsevier]
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- 2007
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113. Human Immunodeficiency Virus Type 1 Can Establish Latent Infection in Resting CD4++ T Cells in the Absence of Activating Stimuli.
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Swiggard, William J., Baytop, Clifford, Jianqing J. Yu, Jihong Dai, Chuanzhao Li, Schretzenmair, Richard, Theodosopoulos, Ted, and O'Doherty, Una
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HIV , *LATENT infection , *T cells , *HIV infections , *ANTIRETROVIRAL agents , *LYMPHOCYTES , *GENOMES - Abstract
Resting CD4+ T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4+ T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4+ T cells that have not received activating stimuli and have not entered cell cycle stage G1b. A percentage of the resting CD4+ T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4+ T cells and suggest a new route for HIV-1 reservoir formation. [ABSTRACT FROM AUTHOR]
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- 2005
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114. Addition of Deoxynucleosides Enhances Human Immunodeficiency Virus Type 1 Integration and 2LTR Formation in Resting CD4+ T Cells.
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Plesa, Gabriela, Jihong Dai, Baytop, Cliff, Riley, James L., June, Carl H., and O'Doherty, Una
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NUCLEOSIDES , *HIV , *CD4 antigen , *T cells , *HIV infections - Abstract
Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4+ T cells in vitro, albeit at lower levels than in activated CD4+ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4+ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4+ T cells. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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115. Machine Learning Bolsters Evidence That D1, Nef, and Tat Influence HIV Reservoir Dynamics.
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Cannon L, Fehrman S, Pinzone M, Weissman S, and O'Doherty U
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Background: The primary hurdle to curing HIV is due to the establishment of a reservoir early in infection. In an effort to find new treatment strategies, we and others have focused on understanding the selection pressures exerted on the reservoir by studying how proviral sequences change over time., Methods: To gain insights into the dynamics of the HIV reservoir we analyzed longitudinal near full-length sequences from 7 people living with HIV between 1 and 20 years following the initiation of antiretroviral treatment. We used this data to employ Bayesian mixed effects models to characterize the decay of the reservoir using single-phase and multiphasic decay models based on near full-length sequencing. In addition, we developed a machine-learning approach utilizing logistic regression to identify elements within the HIV genome most associated with proviral decay and persistence. By systematically analyzing proviruses that are deleted for a specific element, we gain insights into their role in reservoir contraction and expansion., Results: Our analyses indicate that biphasic decay models of intact reservoir dynamics were better than single-phase models with a stronger statistical fit. Based on the biphasic decay pattern of the intact reservoir, we estimated the half-lives of the first and second phases of decay to be 18.2 (17.3 to 19.2, 95%CI) and 433 (227 to 6400, 95%CI) months, respectively.In contrast, the dynamics of defective proviruses differed favoring neither model definitively, with an estimated half-life of 87.3 (78.1 to 98.8, 95% CI) months during the first phase of the biphasic model. Machine-learning analysis of HIV genomes at the nucleotide level revealed that the presence of the splice donor site D1 was the principal genomic element associated with contraction. This role of D1 was then validated in an in vitro system. Using the same approach, we additionally found supporting evidence that HIV nef may confer a protective advantage for latently infected T cells while tat was associated with clonal expansion., Conclusions: The nature of intact reservoir decay suggests that the long-lived HIV reservoir contains at least 2 distinct compartments. The first compartment decays faster than the second compartment. Our machine-learning analysis of HIV proviral sequences reveals specific genomic elements are associated with contraction while others are associated with persistence and expansion. Together, these opposing forces shape the reservoir over time., Competing Interests: The authors report no competing financial interests., (Copyright © 2024 Pathogens and Immunity.)
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- 2024
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116. Highlights from the Inaugural HIV Reservoirs and Immune Control Conference, October 1 st -4 th 2023, Malahide Ireland.
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O'Doherty U, Martinez-Picado J, and Sáez-Cirión A
- Abstract
The inaugural FASEB HIV Reservoirs and Immune Control Conference brought researchers together from across the globe to discuss reservoir dynamics in clinical cohorts. It extended over 4 days in the seaside town of Malahide, Ireland. The scientific sessions covered a broad range of topics, including: 1) HIV pathogenesis and control, 2) reservoirs and viral expression, 3) pediatric reservoirs, 4) innate immunity and B cell responses, 5) environmental factors affecting pathogenesis, 6) loss of virologic control, and 7) HIV-2. The following article provides a brief summary of the meeting proceedings and includes a supplementary document with the meeting abstracts., Competing Interests: The authors report no competing financial interests., (Copyright © 2023 Pathogens and Immunity.)
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- 2023
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117. Serotonin reduction in post-acute sequelae of viral infection.
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Wong AC, Devason AS, Umana IC, Cox TO, Dohnalová L, Litichevskiy L, Perla J, Lundgren P, Etwebi Z, Izzo LT, Kim J, Tetlak M, Descamps HC, Park SL, Wisser S, McKnight AD, Pardy RD, Kim J, Blank N, Patel S, Thum K, Mason S, Beltra JC, Michieletto MF, Ngiow SF, Miller BM, Liou MJ, Madhu B, Dmitrieva-Posocco O, Huber AS, Hewins P, Petucci C, Chu CP, Baraniecki-Zwil G, Giron LB, Baxter AE, Greenplate AR, Kearns C, Montone K, Litzky LA, Feldman M, Henao-Mejia J, Striepen B, Ramage H, Jurado KA, Wellen KE, O'Doherty U, Abdel-Mohsen M, Landay AL, Keshavarzian A, Henrich TJ, Deeks SG, Peluso MJ, Meyer NJ, Wherry EJ, Abramoff BA, Cherry S, Thaiss CA, and Levy M
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- Humans, COVID-19 complications, Disease Progression, Inflammation, Virus Diseases, Post-Acute COVID-19 Syndrome blood, Post-Acute COVID-19 Syndrome pathology, Serotonin blood
- Abstract
Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes., Competing Interests: Declaration of interests E.J.W. is an advisor for Danger Bio, Janssen, New Limit, Marengo, Pluto Immunotherapeutics Related Sciences, Rubius Therapeutics, Santa Ana Bio, Synthekine, and Surface Oncology. E.J.W. is a founder of and holds stock in Surface Oncology, Danger Bio, and Arsenal Biosciences. N.J.M. reports consulting fees from Endpoint Health Inc and AstraZeneca and receives funding from Quantum Leap Healthcare Collaborative outside of the published work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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118. Rapid manufacturing of non-activated potent CAR T cells.
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Ghassemi S, Durgin JS, Nunez-Cruz S, Patel J, Leferovich J, Pinzone M, Shen F, Cummins KD, Plesa G, Cantu VA, Reddy S, Bushman FD, Gill SI, O'Doherty U, O'Connor RS, and Milone MC
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- Animals, Humans, Immunotherapy, Adoptive methods, Mice, T-Lymphocytes, Leukemia, Receptors, Chimeric Antigen
- Abstract
Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2022
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119. Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Dynamics Tracking.
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Chen H, Li Z, Feng S, Wang A, Richard-Greenblatt M, Hutson E, Andrianus S, Glaser LJ, Rodino KG, Qian J, Jayaraman D, Collman RG, Glascock A, Bushman FD, Lee JS, Cherry S, Fausto A, Weiss SR, Koo H, Corby PM, O'Doherty U, Garfall AL, Vogl DT, Stadtmauer EA, and Wang P
- Abstract
Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples., Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients., Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections., Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.
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- 2021
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120. Heavy metal protease takes a tiki torch to HIV assembly.
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O'Doherty U and Freed EO
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- CD4-Positive T-Lymphocytes, Gene Products, gag, Metalloproteases, HIV-1, Metals, Heavy
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- 2019
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121. Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR.
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Lada SM, Huang K, VanBelzen DJ, Montaner LJ, O'Doherty U, and Richman DD
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- DNA, Viral genetics, DNA, Viral isolation & purification, Gene Products, gag genetics, HIV Long Terminal Repeat genetics, Humans, Leukocytes, Mononuclear virology, Reproducibility of Results, Electrophoresis, Gel, Pulsed-Field, HIV Infections virology, HIV-1 genetics, Proviruses genetics, Real-Time Polymerase Chain Reaction standards, Virus Integration physiology
- Abstract
We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results ( r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA., (Copyright © 2018 American Society for Microbiology.)
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- 2018
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122. HLA-C downregulation by HIV-1 adapts to host HLA genotype.
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Bachtel ND, Umviligihozo G, Pickering S, Mota TM, Liang H, Del Prete GQ, Chatterjee P, Lee GQ, Thomas R, Brockman MA, Neil S, Carrington M, Bwana B, Bangsberg DR, Martin JN, Kallas EG, Donini CS, Cerqueira NB, O'Doherty UT, Hahn BH, Jones RB, Brumme ZL, Nixon DF, and Apps R
- Subjects
- Amino Acid Sequence, Disease Reservoirs virology, Down-Regulation, Genetic Variation, Genotype, HIV Infections virology, HIV-1 immunology, Host-Pathogen Interactions genetics, Host-Pathogen Interactions immunology, Human Immunodeficiency Virus Proteins chemistry, Human Immunodeficiency Virus Proteins genetics, Human Immunodeficiency Virus Proteins immunology, Humans, Immune Evasion, Viral Regulatory and Accessory Proteins chemistry, Viral Regulatory and Accessory Proteins genetics, Viral Regulatory and Accessory Proteins immunology, HIV Infections genetics, HIV Infections immunology, HIV-1 pathogenicity, HLA-C Antigens genetics
- Abstract
HIV-1 can downregulate HLA-C on infected cells, using the viral protein Vpu, and the magnitude of this downregulation varies widely between primary HIV-1 variants. The selection pressures that result in viral downregulation of HLA-C in some individuals, but preservation of surface HLA-C in others are not clear. To better understand viral immune evasion targeting HLA-C, we have characterized HLA-C downregulation by a range of primary HIV-1 viruses. 128 replication competent viral isolates from 19 individuals with effective anti-retroviral therapy, show that a substantial minority of individuals harbor latent reservoir virus which strongly downregulates HLA-C. Untreated infections display no change in HLA-C downregulation during the first 6 months of infection, but variation between viral quasispecies can be detected in chronic infection. Vpu molecules cloned from plasma of 195 treatment naïve individuals in chronic infection demonstrate that downregulation of HLA-C adapts to host HLA genotype. HLA-C alleles differ in the pressure they exert for downregulation, and individuals with higher levels of HLA-C expression favor greater viral downregulation of HLA-C. Studies of primary and mutant molecules identify 5 residues in the transmembrane region of Vpu, and 4 residues in the transmembrane domain of HLA-C, which determine interactions between Vpu and HLA. The observed adaptation of Vpu-mediated downregulation to host genotype indicates that HLA-C alleles differ in likelihood of mediating a CTL response that is subverted by viral downregulation, and that preservation of HLA-C expression is favored in the absence of these responses. Finding that latent reservoir viruses can downregulate HLA-C could have implications for HIV-1 cure therapy approaches in some individuals., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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123. Effect of Short-Term Antiretroviral Therapy Interruption on Levels of Integrated HIV DNA.
- Author
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Strongin Z, Sharaf R, VanBelzen DJ, Jacobson JM, Connick E, Volberding P, Skiest DJ, Gandhi RT, Kuritzkes DR, O'Doherty U, and Li JZ
- Subjects
- HIV-1 genetics, Humans, Leukocytes, Mononuclear virology, Proviruses genetics, Viral Load drug effects, Withholding Treatment, Anti-HIV Agents therapeutic use, DNA, Viral genetics, HIV Infections virology, Viral Load genetics, Virus Integration genetics
- Abstract
Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group ATI studies were assayed for integrated HIV DNA levels. Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV integrated DNA size-exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu- gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in the levels of integrated HIV DNA between the pre- and post-ATI time points, with a median ratio of post- to pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI, followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on the levels of integrated proviral DNA in the peripheral blood. IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated HIV DNA and is less sample- and labor-intensive than current assays. Using stored samples from AIDS Clinical Trials Group studies, we found that short-term ART discontinuation had minimal impact on integrated HIV DNA levels after ART resumption, providing reassurance about the reservoir effects of short-term treatment interruption trials., (Copyright © 2018 American Society for Microbiology.)
- Published
- 2018
- Full Text
- View/download PDF
124. Monitoring Integration over Time Supports a Role for Cytotoxic T Lymphocytes and Ongoing Replication as Determinants of Reservoir Size.
- Author
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Pinzone MR, Graf E, Lynch L, McLaughlin B, Hecht FM, Connors M, Migueles SA, Hwang WT, Nunnari G, and O'Doherty U
- Subjects
- Acute Disease, Anti-HIV Agents therapeutic use, Chronic Disease, DNA, Viral blood, DNA, Viral genetics, Disease Reservoirs virology, HIV genetics, HIV Infections drug therapy, Humans, Longitudinal Studies, Viral Load immunology, Virus Replication immunology, HIV immunology, HIV physiology, HIV Infections immunology, HIV Infections virology, T-Lymphocytes, Cytotoxic immunology, T-Lymphocytes, Cytotoxic virology, Virus Integration immunology
- Abstract
The dynamics of HIV reservoir accumulation off antiretroviral therapy (ART) is underexplored. Levels of integrated HIV DNA in peripheral blood mononuclear cells (PBMCs) were longitudinally monitored before and after antiviral therapy. HIV integration increased over time in both elite controllers (ECs; n = 8) and noncontrollers (NCs; n = 6) before ART, whereas integration remained stable in patients on ART (n = 4). The median annual fold change was higher in NCs than in ECs and negatively correlated with CD4/CD8 T-cell ratio. Cytotoxic T lymphocyte (CTL) function as assessed by infected CD4 T-cell elimination (ICE) and granzyme B activity did not significantly change over time in ECs, suggesting that the gradual increase in integrated HIV DNA observed in ECs was not a result of progressive loss of immune-mediated control. Also, acutely infected (n = 7) but not chronically infected (n = 6) patients exhibited a significant drop in integrated HIV DNA 12 months after ART initiation. In conclusion, in the absence of ART, integrated HIV accumulates over time both in NCs and in ECs, at variable individual rates. Starting ART early in infection leads to a greater drop in integrated HIV DNA than does initiating treatment after years of infection. The increase in integrated HIV DNA over time suggests that early treatment may be of benefit in limiting HIV reservoirs., Importance: The establishment of a latent reservoir represents a barrier to cure among HIV-infected individuals. The dynamics of HIV reservoir accumulation over time in patients before antiviral therapy is underexplored, in large part because it is difficult to accurately and reproducibly measure the size of HIV reservoir in this setting. In our study, we compared the dynamics of integrated HIV DNA over time in ECs and NCs before and after ART was initiated. We found that integrated HIV DNA levels progressively increase over time in the absence of ART, but with a higher, albeit variable, rate in NCs compared to ECs. In addition, integrated HIV DNA declines more dramatically when ART is initiated in acute rather than chronic HIV infection, suggesting important differences between acute and chronic infection. Our study highlights the role of HIV replication and CTL control in reservoir accumulation in sanctuary sites and why ART appears to be more effective in acute infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2016
- Full Text
- View/download PDF
125. Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy.
- Author
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Lee SA, Bacchetti P, Chomont N, Fromentin R, Lewin SR, O'Doherty U, Palmer S, Richman DD, Siliciano JD, Yukl SA, Deeks SG, and Burbelo PD
- Subjects
- Antiretroviral Therapy, Highly Active, CD4 Lymphocyte Count, CD4-Positive T-Lymphocytes drug effects, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes virology, DNA, Viral drug effects, DNA, Viral genetics, Female, HIV Antibodies biosynthesis, HIV Infections immunology, HIV Infections virology, HIV Integrase genetics, HIV Integrase immunology, HIV-1 genetics, HIV-1 growth & development, Humans, Intestines drug effects, Intestines immunology, Intestines virology, Lymph Nodes drug effects, Lymph Nodes immunology, Lymph Nodes virology, Male, Middle Aged, RNA Splicing, RNA, Viral drug effects, RNA, Viral genetics, Viral Load immunology, Anti-HIV Agents therapeutic use, HIV Antibodies drug effects, HIV Infections drug therapy, HIV-1 drug effects, Viral Load drug effects
- Abstract
Background: A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir"). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART)., Methods: We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables., Results: Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results., Conclusions: Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.
- Published
- 2016
- Full Text
- View/download PDF
126. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA.
- Author
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Pasternak AO, DeMaster LK, Kootstra NA, Reiss P, O'Doherty U, and Berkhout B
- Subjects
- Gene Expression Profiling, HIV genetics, Humans, HIV physiology, Proviruses genetics, RNA, Viral biosynthesis, Transcription, Genetic, Virus Activation
- Abstract
Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
127. A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Therapy.
- Author
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DeMaster LK, Liu X, VanBelzen DJ, Trinité B, Zheng L, Agosto LM, Migueles SA, Connors M, Sambucetti L, Levy DN, Pasternak AO, and O'Doherty U
- Subjects
- Flow Cytometry, Humans, Optical Imaging, T-Lymphocyte Subsets chemistry, Anti-Retroviral Agents therapeutic use, CD4 Antigens analysis, CD8 Antigens analysis, HIV Infections drug therapy, HIV Infections virology, T-Lymphocyte Subsets virology, gag Gene Products, Human Immunodeficiency Virus biosynthesis
- Abstract
Unlabelled: A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4(+) T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4(+) and CD4(-) cells. In this study, we directly infected resting CD4(+) T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag(+) cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/β T cells that express HIV protein may be a component of the long-lived reservoir., Importance: A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag(+) CD4(-) cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs., (Copyright © 2016 DeMaster et al.)
- Published
- 2015
- Full Text
- View/download PDF
128. Towards an HIV cure: a global scientific strategy.
- Author
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Deeks SG, Autran B, Berkhout B, Benkirane M, Cairns S, Chomont N, Chun TW, Churchill M, Di Mascio M, Katlama C, Lafeuillade A, Landay A, Lederman M, Lewin SR, Maldarelli F, Margolis D, Markowitz M, Martinez-Picado J, Mullins JI, Mellors J, Moreno S, O'Doherty U, Palmer S, Penicaud MC, Peterlin M, Poli G, Routy JP, Rouzioux C, Silvestri G, Stevenson M, Telenti A, Van Lint C, Verdin E, Woolfrey A, Zaia J, and Barré-Sinoussi F
- Subjects
- HIV Infections virology, Humans, Virus Latency drug effects, Virus Replication drug effects, Antiretroviral Therapy, Highly Active methods, HIV physiology, HIV Infections drug therapy
- Abstract
Given the limitations of antiretroviral therapy and recent advances in our understanding of HIV persistence during effective treatment, there is a growing recognition that a cure for HIV infection is both needed and feasible. The International AIDS Society convened a group of international experts to develop a scientific strategy for research towards an HIV cure. Several priorities for basic, translational and clinical research were identified. This Opinion article summarizes the group's recommended key goals for the international community.
- Published
- 2012
- Full Text
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129. R5 HIV env and vesicular stomatitis virus G protein cooperate to mediate fusion to naive CD4+ T Cells.
- Author
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Pace MJ, Agosto L, and O'Doherty U
- Subjects
- HIV-1 genetics, HIV-1 metabolism, Humans, Membrane Glycoproteins genetics, Receptors, CCR5 metabolism, Vesicular stomatitis Indiana virus genetics, Vesicular stomatitis Indiana virus metabolism, Vesicular stomatitis Indiana virus pathogenicity, Viral Envelope Proteins genetics, Virion genetics, env Gene Products, Human Immunodeficiency Virus genetics, CD4-Positive T-Lymphocytes virology, Membrane Fusion, Membrane Glycoproteins metabolism, Viral Envelope Proteins metabolism, Virion metabolism, Virus Internalization, env Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Naïve CD4(4) T cells are resistant to both HIV R5 env and vesicular stomatitis virus G protein (VSV-G)-mediated fusion. However, viral particles carrying both HIV R5 env and VSV-G infect naïve cells by an unexplained mechanism. We show that VSV-G-pseudotyped virus cannot fuse to unstimulated cells because the viral particles cannot be endocytosed. However, virions carrying both HIV R5 env and VSV-G can fuse because CD4 binding allows viral uptake. Our findings reveal a unique mechanism by which R5 HIV env and VSV-G cooperate to allow entry to naïve CD4(+) T cells, providing a tool to target naïve CD4(+) T cells with R5 HIV to study HIV coreceptor signaling and latency.
- Published
- 2011
- Full Text
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130. HIV integration site distributions in resting and activated CD4+ T cells infected in culture.
- Author
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Brady T, Agosto LM, Malani N, Berry CC, O'Doherty U, and Bushman F
- Subjects
- Base Sequence, Cells, Cultured, DNA, Viral genetics, Genome, HIV-1 genetics, Humans, Lymphocyte Activation, Molecular Sequence Data, Virus Latency, CD4-Positive T-Lymphocytes virology, HIV-1 physiology, Virus Integration genetics
- Abstract
Objective: The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting., Design: Primary resting or activated CD4 T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined., Methods: We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions., Results: HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes., Conclusion: These data indicate that integration site distributions show modest but significant differences between resting and activated CD4 T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression.
- Published
- 2009
- Full Text
- View/download PDF
131. Human immunodeficiency virus integrates directly into naive resting CD4+ T cells but enters naive cells less efficiently than memory cells.
- Author
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Dai J, Agosto LM, Baytop C, Yu JJ, Pace MJ, Liszewski MK, and O'Doherty U
- Subjects
- Cell Line, Cell Separation, Genome genetics, HIV genetics, Transcription, Genetic genetics, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes virology, HIV immunology, Immunity, Innate immunology, Immunologic Memory immunology, Virus Integration immunology
- Abstract
Resting CD4(+) T cells restrict human immunodeficiency virus (HIV) infection at or before reverse transcription, resulting in slower kinetics of reverse transcription. In a previous study, we showed that, despite this restriction at reverse transcription, HIV integration occurs in resting CD4(+) T cells, albeit with slower kinetics. In that study, the resting T cells were a mixture of memory and naïve cells. Here we asked whether the more quiescent naïve cell subset could be directly infected by HIV and, if so, whether the level of integration in naïve cells was comparable to that in memory cells. We found that HIV integrates in the naïve subset of resting CD4(+) T cells without prior activation of the cells. The level of integration (proviruses/cell) in naïve cells was lower than that in memory cells. This difference between naïve and memory cells was observed whether we inoculated the cells with R5 or X4 HIV and could not be explained solely by differences in coreceptor expression. The presence of endogenous dendritic cells did not change the number of proviruses/cell in memory or naïve cells, and deoxynucleoside pools were equally limiting. Our results instead indicate the existence of a novel restriction point in naïve T cells at viral fusion that results in reduced levels of fusion to naïve CD4(+) T cells. We conclude that HIV can integrate into both naïve and memory cells directly. Our data further support our hypothesis that integrated proviral infection of resting T cells can be established without T-cell activation.
- Published
- 2009
- Full Text
- View/download PDF
132. Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells.
- Author
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Plesa G, Dai J, Baytop C, Riley JL, June CH, and O'Doherty U
- Subjects
- CD4-Positive T-Lymphocytes cytology, Disease Reservoirs, HIV Long Terminal Repeat genetics, HIV Long Terminal Repeat physiology, HIV-1 genetics, Humans, Nucleosides chemistry, Virus Integration, Virus Latency, CD4-Positive T-Lymphocytes virology, HIV-1 drug effects, Nucleosides metabolism
- Abstract
Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4+ T cells in vitro, albeit at lower levels than in activated CD4+ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4+ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4+ T cells.
- Published
- 2007
- Full Text
- View/download PDF
133. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli.
- Author
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Swiggard WJ, Baytop C, Yu JJ, Dai J, Li C, Schretzenmair R, Theodosopoulos T, and O'Doherty U
- Subjects
- Acquired Immunodeficiency Syndrome pathology, Acquired Immunodeficiency Syndrome virology, Antigens, CD immunology, CD4-Positive T-Lymphocytes pathology, CD4-Positive T-Lymphocytes virology, Cell Cycle, Cell Line, Disease Reservoirs, Gene Expression Regulation, Viral, Genome, Viral, HIV-1 genetics, HIV-1 pathogenicity, Humans, Lymphocyte Activation, Proviruses genetics, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Virus Integration, Acquired Immunodeficiency Syndrome immunology, CD4-Positive T-Lymphocytes immunology, HIV-1 physiology, Virus Latency
- Abstract
Resting CD4(+) T cells are the best-defined reservoir of latent human immunodeficiency virus type 1 (HIV-1) infection, but how the reservoir is formed is unclear. Understanding how the reservoir of latently infected cells forms is critical because it is a major barrier to curing HIV infection. The system described here may provide an in vitro model of latent HIV-1 infection in resting CD4(+) T cells. We demonstrated that HIV-1 integrates into the genomes of in vitro-inoculated resting CD4(+) T cells that have not received activating stimuli and have not entered cell cycle stage G(1b). A percentage of the resting CD4(+) T cells that contain integrated DNA produce virus upon stimulation, i.e., are latently infected. Our results show that latent HIV-1 infection occurs in unstimulated resting CD4(+) T cells and suggest a new route for HIV-1 reservoir formation.
- Published
- 2005
- Full Text
- View/download PDF
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