scholarly journals Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

2016 ◽  
Vol 113 (31) ◽  
pp. 8783-8788 ◽  
Author(s):  
Hiromi Imamichi ◽  
Robin L. Dewar ◽  
Joseph W. Adelsberger ◽  
Catherine A. Rehm ◽  
Una O’Doherty ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Amplicon Sequencing ◽  
Combination Antiretroviral Therapy ◽  
Peripheral Blood Mononuclear ◽  
Protein Coding ◽  
Hiv Rna ◽  
Single Genome ◽  
Hiv 1 ◽  
Rna Levels

Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5′LTR-to-3′LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of “defective” proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements ofgag,pol,env,rev, andnefto encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not “silent,” but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

scholarly journals Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types

2017 ◽  
Vol 55 (8) ◽  
pp. 2544-2553 ◽  
Author(s):  
Christina Yek ◽  
Marta Massanella ◽  
Tashi Peling ◽  
Kristen Lednovich ◽  
Sangeetha V. Nair ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Mononuclear Cells ◽  
Dried Blood Spots ◽  
Proteinase K ◽  
Peripheral Blood Mononuclear ◽  
Detection Rates ◽  
Hiv Rna ◽  
Resource Limited ◽  
Hiv 1 ◽  
Rna Levels

ABSTRACTThe search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs;n= 72), seminal plasma (n= 20), cerebrospinal fluid (CSF;n= 36), dried blood spots (DBS;n= 104), and dried plasma spots (DPS;n= 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings.

scholarly journals Defective HIV-1 proviruses produce viral proteins

2020 ◽  
Vol 117 (7) ◽  
pp. 3704-3710 ◽  
Author(s):  
Hiromi Imamichi ◽  
Mindy Smith ◽  
Joseph W. Adelsberger ◽  
Taisuke Izumi ◽  
Francesca Scrimieri ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Viral Proteins ◽  
Open Reading Frames ◽  
Peripheral Blood Mononuclear ◽  
Hiv Rna ◽  
Rna Transcripts ◽  
Extracellular Virus ◽  
Hiv 1

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as “defective” by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant “graveyard” of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell–cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.

Differences in HIV RNA levels before the initiation of antiretroviral therapy among 1864 individuals with known HIV-1 seroconversion dates

AIDS ◽  
2004 ◽  
Vol 18 (12) ◽  
pp. 1697-1705 ◽  
Author(s):  
Giota Touloumi ◽  
Nikos Pantazis ◽  
Abdel G Babiker ◽  
Sarah A Walker ◽  
Olga Katsarou ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Hiv Rna ◽  
Hiv 1 ◽  
Rna Levels

scholarly journals Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients

2016 ◽  
Vol 54 (6) ◽  
pp. 1641-1643 ◽  
Author(s):  
Sylvie Zida ◽  
Edouard Tuaillon ◽  
Makoura Barro ◽  
Arnaud Kwimatouo Lekpa Franchard ◽  
Thérèse Kagoné ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Dried Blood Spots ◽  
Peripheral Blood Mononuclear ◽  
Rna Detection ◽  
Blood Spots ◽  
Hiv Rna ◽  
Rna Quantification ◽  
Blood Mononuclear Cells ◽  
The Impact ◽  
Hiv 1

The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/106peripheral blood mononuclear cells.

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 Phylogenetic analysis of HIV-1 archived DNA in blood and gut-associated lymphoid tissue in two patients under antiretroviral therapy

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Patricia Recordon-Pinson ◽  
Annie Gosselin ◽  
Petronela Ancuta ◽  
Jean-Pierre Routy ◽  
Hervé Fleury
Keyword(s):  
Antiretroviral Therapy ◽  
Genome Sequencing ◽  
Dna Sequences ◽  
Lymphoid Tissue ◽  
Mononuclear Cells ◽  
Therapeutic Vaccine ◽  
Sequencing Analysis ◽  
Proviral Dna ◽  
Single Genome ◽  
Hiv 1

AbstractOne of the approaches to cure human immunodeficiency virus (HIV) is the use of therapeutic vaccination. We have launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles in aviremic patients under antiretroviral therapy (ART). A HIV-1 polypeptidic therapeutic vaccine based on viral sequence data obtained from circulating blood was proposed; here, our aim was to compare the proviral DNA in blood and gut-associated lymphoid tissue (GALT). Peripheral blood mononuclear cells and gut biopsies were obtained from two HIV-1 infected patients under successful antiretroviral therapy. Total DNA was extracted including the proviral DNA. The HIV-1 reverse transcriptase was sequenced in both compartments using next generation sequencing followed by single genome sequencing; phylogenetic trees were established and compared. The proviral sequences of both compartments intra-patient exhibited a very low genetic divergence while it was possible to differentiate the sequences inter-patients; single genome sequencing analysis of two couples of samples confirmed that there was no compartmentalization of the sequences intra-patient. We conclude that, considering these two cases, the proviral DNA sequences in blood and GALT are similar and that the epitope analysis of HIV-1 provirus in blood should be considered as relevant to that observed in the GALT, a hard-to-reach major compartment, and can therefore be used for therapeutic 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.

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