scholarly journals CpG Methylation Profiles of HIV-1 Pro-Viral DNA in Individuals on ART

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 799
Author(s):  
Valerie F. Boltz ◽  
Cristina Ceriani ◽  
Jason W. Rausch ◽  
Wei Shao ◽  
Michael J. Bale ◽  
...  
Keyword(s):  
Cytosine Methylation ◽  
Mononuclear Cells ◽  
Cell Clone ◽  
Cpg Methylation ◽  
Viral Dna ◽  
Peripheral Blood Mononuclear ◽  
T Cell Clone ◽  
Latent Hiv ◽  
Ltr Promoter ◽  
Hiv 1

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

scholarly journals Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors

2019 ◽  
Vol 116 (51) ◽  
pp. 25891-25899 ◽  
Author(s):  
Sean C. Patro ◽  
Leah D. Brandt ◽  
Michael J. Bale ◽  
Elias K. Halvas ◽  
Kevin W. Joseph ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Cell Clone ◽  
Integration Site ◽  
Multiple Displacement Amplification ◽  
Drug Resistant ◽  
Peripheral Blood Mononuclear ◽  
Site Analysis ◽  
T Cell Clone ◽  
Integration Site Analysis ◽  
Hiv 1

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

scholarly journals Epigenetic Compound Screening Uncovers Small Molecules for Reactivation of Latent HIV-1

2020 ◽  
Vol 65 (1) ◽  
pp. e01815-20
Author(s):  
Ariane Zutz ◽  
Lin Chen ◽  
Franziska Sippl ◽  
Andreas Humpe ◽  
Christian Schölz
Keyword(s):  
High Throughput Screening ◽  
Mononuclear Cells ◽  
Model Systems ◽  
Reporter System ◽  
Peripheral Blood Mononuclear ◽  
Compound Screening ◽  
Immunological Destruction ◽  
New Treatment ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACTDuring infection with the human immunodeficiency virus type 1 (HIV-1), latent reservoirs are established that circumvent full eradication of the virus by antiretroviral therapy (ART) and are the source for viral rebound after cessation of therapy. As these reservoirs are phenotypically indistinguishable from infected cells, current strategies aim to reactivate these reservoirs, followed by pharmaceutical and immunological destruction of the cells. Here, we employed a simple and convenient cell-based reporter system, which enables sample handling under biosafety level (BSL)-1 conditions, to screen for compounds that were able to reactivate latent HIV-1. The assay showed a high dynamic signal range and reproducibility with an average Z-factor of 0.77, classifying the system as robust. The assay was used for high-throughput screening (HTS) of an epigenetic compound library in combination with titration and cell-toxicity studies and revealed several potential new latency-reversing agents (LRAs). Further validation in well-known latency model systems verified earlier studies and identified two novel compounds with very high reactivation efficiencies and low toxicity. Both drugs, namely, N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) and 2′,3′-difluoro-[1,1′-biphenyl]-4-carboxylic acid, 2-butylhydrazide (SR-4370), showed comparable performances to other already known LRAs, did not activate CD4+ T cells, and did not cause changes in the composition of peripheral blood mononuclear cells (PBMCs), as shown by flow cytometry analyses. Both compounds may represent effective new treatment possibilities for reversal of latency in HIV-1-infected individuals.

scholarly journals Early ART-initiation Reduces HIV-1 Proviral DNA Levels in Children from the CHER Trial

2021 ◽  
Author(s):  
Helen Payne ◽  
Man Chan ◽  
Sarah Watters ◽  
Kennedy Otwombe ◽  
Yuan Hsiao ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Factors Associated ◽  
Proviral Dna ◽  
Art Initiation ◽  
Latent Hiv ◽  
Dna Reduction ◽  
Living With Hiv ◽  
Hiv 1

Abstract BACKGROUND: Reduction of the reservoir of latent HIV-infected cells might increase the possibility of long-term remission in individuals living with HIV. We investigated factors associated with HIV-1 proviral DNA levels in children receiving different antiretroviral therapy (ART) strategies in the Children with HIV Early Antiretroviral Therapy (CHER) trial. METHODS: Infants with HIV <12 weeks old with CD4% ≥25% were randomized in the CHER trial to early limited ART for 40 or 96 weeks (ART-40W, ART-96W), or deferred ART (ART-Def). For ART-Def infants or following ART interruption in ART-40W/ART-96W, ART was started/re-started for clinical progression or CD4% <25%. In 229 participants, HIV-1 proviral DNA was quantified by PCR from stored peripheral blood mononuclear cells from children who had received ≥24 weeks ART and two consecutive undetectable HIV-1 RNA 12-24 weeks apart. HIV-1 proviral DNA was compared between ART-Def and ART-96W at week 96, and in all arms at week 248. Factors associated with HIV-1 proviral DNA levels were evaluated using linear regression.FINDINGS: Longer duration of ART was significantly associated with lower HIV-1 proviral DNA at both 96 (p=0.0003) and 248 weeks (p=0.0011). Higher total CD8 count at ART initiation was associated with lower HIV-1 proviral DNA at both 96 (p=0.0225) and 248 weeks (p=0.0398). Week 248 HIV-1 proviral DNA was significantly higher in those with positive HIV-1 serology at week 84 than those with negative serology (p=0.0042).INTEPRETATION: Longer ART duration is key to HIV-1 proviral DNA reduction. Further understanding is needed of the effects of “immune-attenuation” through early HIV-1 exposure.FUNDING: Wellcome Trust, National Institutes of Health, Medical Research Council.

scholarly journals Prediction and Validation of Immunogenic Domains of Pneumococcal Proteins Recognized by Human CD4+T Cells

2019 ◽  
Vol 87 (6) ◽  
Author(s):  
Martijn D. B. van de Garde ◽  
Els van Westen ◽  
Martien C. M. Poelen ◽  
Nynke Y. Rots ◽  
Cécile A. C. M. van Els
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Clone ◽  
Bioinformatics Tool ◽  
Peripheral Blood Mononuclear ◽  
Necrosis Factor Alpha ◽  
T Cell Clone ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACTCD4+T-cell mechanisms are implied in protection against pneumococcal colonization; however, their target antigens and function are not well defined. In contrast to high-throughput protein arrays for serology, basic antigen tools for CD4+T-cell studies are lacking. Here, we evaluate the potential of a bioinformatics tool forin silicoprediction of immunogenicity as a method to reveal domains of pneumococcal proteins targeted by human CD4+T cells. For 100 pneumococcal proteins, CD4+T-cell immunogenicity was predicted based on HLA-DRB1 binding motifs. For 20 potentially CD4+T-cell immunogenic proteins, epitope regions were verified by testing synthetic peptides in T-cell assays using peripheral blood mononuclear cells from healthy adults. Peptide pools of 19 out of 20 proteins evoked T-cell responses. The most frequent responses (detectable in ≥20% of donors tested) were found to SP_0117 (PspA), SP_0468 (putative sortase), SP_0546 (BlpZ), SP_1650 (PsaA), SP_1923 (Ply), SP_2048 (conserved hypothetical protein), SP_2216 (PscB), and SPR_0907 (PhtD). Responding donors had diverging recognition patterns and profiles of signature cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-13 [IL-13], and/or IL-17A) against single-epitope regions. Natural HLA-DR-restricted presentation and recognition of a predicted SP_1923-derived epitope were validated through the isolation of a CD4+T-cell clone producing IFN-γ, TNF-α, and IL-17A in response to the synthetic peptide, whole protein, and heat-inactivated pneumococcus. This proof of principle for a bioinformatics tool to identify pneumococcal protein epitopes targeted by human CD4+T cells provides a peptide-based strategy to study cell-mediated immune mechanisms for the pneumococcal proteome, advancing the development of immunomonitoring assays and targeted vaccine approaches.

scholarly journals Early ART-initiation and longer ART duration reduces HIV-1 proviral DNA levels in children from the CHER trial

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Helen Payne ◽  
Man K. Chan ◽  
Sarah A. Watters ◽  
Kennedy Otwombe ◽  
Nei-Yuan Hsiao ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Factors Associated ◽  
Proviral Dna ◽  
Art Initiation ◽  
Latent Hiv ◽  
Dna Reduction ◽  
Living With Hiv ◽  
Hiv 1

Abstract Background Reduction of the reservoir of latent HIV-infected cells might increase the possibility of long-term remission in individuals living with HIV. We investigated factors associated with HIV-1 proviral DNA levels in children receiving different antiretroviral therapy (ART) strategies in the children with HIV early antiretroviral therapy (CHER) trial. Methods Infants with HIV  <  12 weeks old with CD4%  ≥  25% were randomized in the CHER trial to early limited ART for 40 or 96 weeks (ART-40 W, ART-96 W), or deferred ART (ART-Def). For ART-Def infants or following ART interruption in ART-40 W/ART-96 W, ART was started/re-started for clinical progression or CD4%  <  25%. In 229 participants, HIV-1 proviral DNA was quantified by PCR from stored peripheral blood mononuclear cells from children who had received  ≥  24 weeks ART and two consecutive undetectable HIV-1 RNA 12–24 weeks apart. HIV-1 proviral DNA was compared between ART-Def and ART-96 W at week 96, and in all arms at week 248. Factors associated with HIV-1 proviral DNA levels were evaluated using linear regression. Findings Longer duration of ART was significantly associated with lower HIV-1 proviral DNA at both 96 (p  =  0.0003) and 248 weeks (p  =  0.0011). Higher total CD8 count at ART initiation was associated with lower HIV-1 proviral DNA at both 96 (p  =  0.0225) and 248 weeks (p  =  0.0398). Week 248 HIV-1 proviral DNA was significantly higher in those with positive HIV-1 serology at week 84 than those with negative serology (p  =  0.0042). Intepretation Longer ART duration is key to HIV-1 proviral DNA reduction. Further understanding is needed of the effects of “immune-attenuation” through early HIV-1 exposure. Funding Wellcome Trust, National Institutes of Health, Medical Research Council.

Novel Phorbol Esters Exert Dichotomous Effects on Inhibition of HIV-1 Infection and Activation of Latent HIV-1 Expression

2005 ◽  
Vol 16 (5) ◽  
pp. 303-313
Author(s):  
Yu Zhong ◽  
Yuji Matsuya ◽  
Hideo Nemoto ◽  
Masao Mori ◽  
Haruo Saito ◽  
...  
Keyword(s):  
Mtt Assay ◽  
Mononuclear Cells ◽  
Phorbol Esters ◽  
High Concentration ◽  
Viral Reservoirs ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected ◽  
Low Cytotoxicity ◽  
Latent Hiv ◽  
Hiv 1

Two new phorbol esters, NPB-11 (12- O-methoxymethylphorbol-13-decanoate) and NPB-15 (12- O-benzyloxymethylphorbol-13-decanoate) were synthesized. The compounds exhibited potent anti-HIV-1 activity and low cytotoxicity in MT-4 cells by MTT assay even at a high concentration [50% cytotoxic concentrations (CC50) were 8.32 and 4.39 μg/ml, respectively]. Two inhibitors strongly suppressed HIV-1 (IIIB strain) replication in MT-4 cells with a 50% effective concentration (EC50) of 1.3 and 0.27 ng/ml, respectively. NPB-11 efficiently blocked replication of both X4 and R5 HIV-1 in PHA-activated peripheral blood mononuclear cells and MT-4 cells as revealed by p24 assay. The antiviral activity appeared to be mediated, at least partially, by the down-regulation of the expression of CD4 and the HIV-1 co-receptors, CXCR4 and CCR5. The compounds were also capable of selectively up-regulating HIV-1 expression in a variety of latently infected cell lines and inducing cell death in HIV-1 infected cells. The effect of NPBs on the induction of HIV-1 was specifically blocked by nontoxic doses of a protein kinase C blocker, staurosporine. NPB-11 blocked the spread of HIV-1 released from latently infected ACH-2 cells to MT-4 cells in a co-culture system. When combined with AZT, NPB-11 synergistically inhibited HIV-1 replication in MTT assay using MT-4 cells. These data suggest that these agents might be useful in reducing persistent viral reservoirs in patients and as adjuvant therapy in patients treated with HAART.

Sign in / Sign up

Export Citation Format

Share Document