Origin of Rebound Plasma HIV Includes Cells with Identical Proviruses That Are Transcriptionally Active before Stopping of Antiretroviral Therapy

ABSTRACTUnderstanding the origin of HIV variants during viral rebound may provide insight into the composition of the HIV reservoir and has implications for the design of curative interventions. HIV single-genome sequences were obtained from 10 AIDS Clinical Trials Group participants who underwent analytic antiretroviral therapy (ART) interruption (ATI). Rebounding variants were compared with those in pre-ART plasma in all 10 participants and with on-ART peripheral blood mononuclear cell (PBMC)-associated DNA and RNA (CA-RNA) in 7/10 participants. The highest viral diversities were found in the DNA and CA-RNA populations. In 3 of 7 participants, we detected multiple, identical DNA and CA-RNA sequences during suppression on ART that exactly matched plasma HIV sequences. Hypermutated DNA and CA-RNA were detected in four participants, contributing to diversities in these compartments that were higher than in the pre-ART and post-ATI plasma. Shifts in the viral rebound populations could be detected in some participants over the 2- to 3-month observation period. These findings suggest that a source of initial rebound viremia could be populations of infected cells that clonally expanded prior to and/or during ART, some of which were already expressing HIV RNA before treatment was interrupted. These clonally expanding populations of HIV-infected cells may represent an important target for strategies aimed at achieving reservoir reduction and sustained virologic remission.IMPORTANCEAntiretroviral therapy alone cannot eradicate the HIV reservoir, and viral rebound is generally rapid after treatment interruption. It has been suggested that clonal expansion of HIV-infected cells is an important mechanism of HIV reservoir persistence, but the contribution of these clonally proliferating cells to the rebounding virus is unknown. We report a study of AIDS Clinical Trials Group participants who underwent treatment interruption and compared rebounding plasma virus with that found within cells prior to treatment interruption. We found several incidences in which plasma HIV variants exactly matched that of multiple proviral DNA copies from infected blood cells sampled before treatment interruption. In addition, we found that these cells were not dormant but were generating unspliced RNA transcripts before treatment was interrupted. Identification of the HIV reservoir and determining its mechanisms for persistence may aid in the development of strategies toward a cure for HIV. (This study was presented in part at the Conference on Retroviruses and Opportunistic Infections, Seattle, WA, February 23 to 26 2015.)

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Kinetics of Plasma HIV Rebound in the Era of Modern Antiretroviral Therapy

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa270 ◽

2020 ◽

Vol 222 (10) ◽

pp. 1655-1659 ◽

Cited By ~ 1

Author(s):

Michael C Sneller ◽

Erin D Huiting ◽

Katherine E Clarridge ◽

Catherine Seamon ◽

Jana Blazkova ◽

...

Keyword(s):

Clinical Trials ◽

Antiretroviral Therapy ◽

Therapeutic Efficacy ◽

Historical Data ◽

Modern Art ◽

Treatment Interruption ◽

Viral Rebound ◽

Hiv Cure ◽

Analytical Treatment ◽

Kinetics Of

Abstract Historical data regarding time to viral rebound following analytical treatment interruption (ATI) have been used to determine therapeutic efficacy in HIV cure trials; however, such data were collected from studies conducted a decade or more ago and included participants receiving older antiretroviral therapy (ART) regimens with infrequent virologic monitoring. We conducted a study of 22 HIV-infected participants receiving modern ART to determine the kinetics of plasma viral rebound following ATI. Our data suggest that modern ART does not alter kinetics of viral rebound when compared to previous regimens and that immunologic interventions may be necessary to achieve ART-free virologic remission. Clinical Trials Registration ClinicaTrials.gov identifier: NCT03225118.

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Anti-HIV-1 ADCC Antibodies following Latency Reversal and Treatment Interruption

Journal of Virology ◽

10.1128/jvi.00603-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 7

Author(s):

Wen Shi Lee ◽

Anne B. Kristensen ◽

Thomas A. Rasmussen ◽

Martin Tolstrup ◽

Lars Østergaard ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Treatment Interruption ◽

Viral Rebound ◽

Analytical Treatment ◽

Antigenic Stimulus ◽

Latently Infected ◽

Infected Cells ◽

Functional Antibodies ◽

Hiv 1 ◽

Reversing Agents

ABSTRACT There is growing interest in utilizing antibody-dependent cellular cytotoxicity (ADCC) to eliminate infected cells following reactivation from HIV-1 latency. A potential barrier is that HIV-1-specific ADCC antibodies decline in patients on long-term antiretroviral therapy (ART) and may not be sufficient to eliminate reactivated latently infected cells. It is not known whether reactivation from latency with latency-reversing agents (LRAs) could provide sufficient antigenic stimulus to boost HIV-1-specific ADCC. We found that treatment with the LRA panobinostat or a short analytical treatment interruption (ATI), 21 to 59 days, was not sufficient to stimulate an increase in ADCC-competent antibodies, despite viral rebound in all subjects who underwent the short ATI. In contrast, a longer ATI, 2 to 12 months, among subjects enrolled in the Strategies for Management of Antiretroviral Therapy (SMART) trial robustly boosted HIV-1 gp120-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro. These results show that there is a lag between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies toward eliminating latently infected cells. IMPORTANCE The “shock and kill” HIV-1 cure strategy aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. Several latency reversing agents (LRAs) have been examined in vivo, but LRAs alone have not been able to achieve HIV-1 remission and prevent viral rebound following analytical treatment interruption (ATI). In this study, we examined whether LRA treatment or ATI can provide sufficient antigenic stimulus to boost HIV-1-specific functional antibodies that can eliminate HIV-1-infected cells. Our study has implications for the antigenic stimulus required for antilatency strategies and/or therapeutic vaccines to boost functional antibodies and assist in eliminating the latent reservoir.

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Effect of Short-Term Antiretroviral Therapy Interruption on Levels of Integrated HIV DNA

Journal of Virology ◽

10.1128/jvi.00285-18 ◽

2018 ◽

Vol 92 (12) ◽

Cited By ~ 12

Author(s):

Zachary Strongin ◽

Radwa Sharaf ◽

D. Jake VanBelzen ◽

Jeffrey M. Jacobson ◽

Elizabeth Connick ◽

...

Keyword(s):

Clinical Trials ◽

Antiretroviral Therapy ◽

Peripheral Blood ◽

Treatment Interruption ◽

Viral Reservoir ◽

Short Term ◽

Size Selection ◽

Hiv Dna ◽

Aids Clinical Trials ◽

Dna Size

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.

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Impact of HLA Class I Alleles on Timing of HIV Rebound After Antiretroviral Treatment Interruption

Pathogens and Immunity ◽

10.20411/pai.v2i3.222 ◽

2017 ◽

Vol 2 (3) ◽

pp. 431 ◽

Cited By ~ 5

Author(s):

You Jeong Park ◽

Behzad Etemad ◽

Hayat Ahmed ◽

Vivek Naranbhai ◽

Evgenia Aga ◽

...

Keyword(s):

Hla Class I ◽

Treatment Interruption ◽

Single Copy ◽

Pooled Analysis ◽

Vital Role ◽

Class I ◽

Viral Rebound ◽

Peripheral Blood Mononuclear ◽

Hiv Reservoir ◽

Hiv Dna

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.

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HIV-1 DNA predicts disease progression and post-treatment virological control

eLife ◽

10.7554/elife.03821 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 183

Author(s):

James P Williams ◽

Jacob Hurst ◽

Wolfgang Stöhr ◽

Nicola Robinson ◽

Helen Brown ◽

...

Keyword(s):

Disease Progression ◽

Treatment Interruption ◽

Viral Rebound ◽

Clinical Progression ◽

Clinical Trial Registration ◽

Plasma Virus ◽

Latently Infected ◽

Infected Cells ◽

Multivariable Analyses ◽

Hiv 1

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20

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Infectious Virus Persists in CD4+T Cells and Macrophages in Antiretroviral Therapy-Suppressed Simian Immunodeficiency Virus-Infected Macaques

Journal of Virology ◽

10.1128/jvi.00065-19 ◽

2019 ◽

Vol 93 (15) ◽

Cited By ~ 16

Author(s):

Celina M. Abreu ◽

Rebecca T. Veenhuis ◽

Claudia R. Avalos ◽

Shelby Graham ◽

Suzanne E. Queen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antiretroviral Therapy ◽

Infectious Virus ◽

Treatment Interruption ◽

Viral Rebound ◽

Hiv Cure ◽

Macaque Model ◽

Immunodeficiency Virus ◽

Latently Infected

ABSTRACTUnderstanding the cellular and anatomical sites of latent virus that contribute to human immunodeficiency virus (HIV) rebound is essential for eradication. In HIV-positive patients, CD4+T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+T cells in compartments other than blood and lymph nodes is unclear. Macrophages (Mϕ) are infected by HIV/simian immunodeficiency virus (SIV) and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication-competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+T cell and Mϕ functional latent reservoirs were measured in various tissues using cell-specific QVOAs. Our results showed that blood, spleen, and lung in the majority of suppressed animals contain latently infected Mϕs. Surprisingly, the numbers of CD4+T cells, monocytes, and Mϕs carrying infectious genomes in blood and spleen were at comparable frequencies (∼1 infected cell per million). We also demonstrate thatex vivoviruses produced in the Mϕ QVOA are capable of infecting activated CD4+T cells. These results strongly suggest that latently infected tissue Mϕs can reestablish productive infection upon treatment interruption. This study provides the first comparison of CD4+T cell and Mϕ functional reservoirs in a macaque model. It is the first confirmation of the persistence of latent genomes in monocytes in blood and Mϕs in the spleen and lung of SIV-infected ART-suppressed macaques. Our results demonstrate that transcriptionally silent genomes in Mϕs can contribute to viral rebound after ART interruption and should be considered in future HIV cure strategies.IMPORTANCEThis study suggests that CD4+T cells found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. In addition, this study demonstrates that macrophages in tissues are another cellular reservoir for SIV and may contribute to viral rebound after treatment interruption. This new insight into the size and location of the SIV reservoir could have great implications for HIV-infected individuals and should be taken into consideration for the development of future HIV cure strategies.

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Toll-Like Receptor 7 Agonist GS-9620 Induces HIV Expression and HIV-Specific Immunity in Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy

Journal of Virology ◽

10.1128/jvi.02166-16 ◽

2017 ◽

Vol 91 (8) ◽

Cited By ~ 60

Author(s):

Angela Tsai ◽

Alivelu Irrinki ◽

Jasmine Kaur ◽

Tomas Cihlar ◽

George Kukolj ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Mononuclear Cells ◽

Antiviral Immunity ◽

Type I ◽

Toll Like Receptor ◽

Peripheral Blood Mononuclear ◽

Effector Functions ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv

ABSTRACT Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs. IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies that specifically target and eliminate the latent HIV reservoir are needed. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on the compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.

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Continuous Prophylactic Antiretrovirals/Antiretroviral Therapy Since Birth Reduces Seeding and Persistence of the Viral Reservoir in Children Vertically Infected With Human Immunodeficiency Virus

Clinical Infectious Diseases ◽

10.1093/cid/ciaa718 ◽

2020 ◽

Author(s):

Marta Massanella ◽

Thanyawee Puthanakit ◽

Louise Leyre ◽

Thidarat Jupimai ◽

Panadda Sawangsinth ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiretroviral Therapy ◽

Cross Sectional Study ◽

Viral Reservoir ◽

Cross Sectional ◽

Hiv Reservoir ◽

Hiv Dna ◽

Art Initiation ◽

Immunodeficiency Virus ◽

Infected Cells

Abstract Background Early antiretroviral therapy (ART) restricts the size of the human immunodeficiency virus (HIV) reservoir in infants. However, whether antiretroviral (ARV) prophylaxis given to exposed vertically infected children exerts similar effects remains unknown. Methods We measured total and integrated HIV DNA, as well as the frequency of CD4 T cells producing multiply spliced RNA (msRNA) after stimulation (inducible reservoir) in vertically infected Thai infants. Eighty-five infants were followed longitudinally for up to 3 years. We compared the size of the reservoir in children who received continuous ARV prophylaxis since birth vs those who never received or discontinued prophylaxis before initiating ART. We used samples from a cross-sectional cohort of 37 Thai children who had initiated ART within 6 months of life to validate our findings. Results Before ART, levels of HIV DNA and the frequencies of cells producing msRNA were significantly lower in infants who received continuous ARV prophylaxis since birth compared to those in whom ARV prophylaxis was discontinued or never initiated (P < .020 and P < .001, respectively). Upon ART initiation, total and integrated HIV DNA levels decayed significantly in both groups (P < .01 in all cases). Interestingly, the initial differences in the frequencies of infected cells persisted during 3 years on ART. The beneficial effect of prophylaxis on the size of the HIV reservoir was confirmed in the cross-sectional study. Importantly, no differences were observed between children who discontinued prophylactic ARVs before starting ART and those who delayed ART initiation without receiving prior prophylaxis. Conclusions Neonatal ARV prophylaxis with direct transition to ART durably limits the size of the HIV reservoir.

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Antibody Responses After Analytic Treatment Interruption in Human Immunodeficiency Virus-1-Infected Individuals on Early Initiated Antiretroviral Therapy

Open Forum Infectious Diseases ◽

10.1093/ofid/ofw100 ◽

2016 ◽

Vol 3 (2) ◽

Cited By ~ 5

Author(s):

Kathryn E. Stephenson ◽

George H. Neubauer ◽

Christine A. Bricault ◽

Jennifer Shields ◽

Madeleine Bayne ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Antiretroviral Therapy ◽

Treatment Interruption ◽

Antibody Responses ◽

Viral Rebound ◽

Analytic Treatment ◽

Antibody Maturation ◽

Neutralizing Capacity ◽

Immunodeficiency Virus ◽

Hiv 1

Abstract The examination of antibody responses in human immunodeficiency virus (HIV)-1-infected individuals in the setting of antiretroviral treatment (ART) interruption can provide insight into the evolution of antibody responses during viral rebound. In this study, we assessed antibody responses in 20 subjects in AIDS Clinical Trials Group A5187, wherein subjects were treated with antiretroviral therapy during acute/early HIV-1 infection, underwent analytic treatment interruption, and subsequently demonstrated viral rebound. Our data suggest that early initiation of ART arrests the maturation of HIV-1-specific antibody responses, preventing epitope diversification of antibody binding and the development of functional neutralizing capacity. Antibody responses do not appear permanently blunted, however, because viral rebound triggered the resumption of antibody maturation in our study. We also found that antibody responses measured by these assays did not predict imminent viral rebound. These data have important implications for the HIV-1 vaccine and eradication fields.

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