HIV reservoir quantification using cross-subtype multiplex ddPCR

Cassidy, Noah A.J.; Fish, Carolyn S.; Levy, Claire; Roychoudhury, Pavitra; Reeves, Daniel B.; Hughes, Sean M.; Schiffer, Joshua T.; Benki-Nugent, Sarah; John‐Stewart, Grace; Wamalwa, Dalton; Jerome, Keith R.; Overbaugh, Julie; Hladik, Florian; Lehman, Dara A.

doi:10.1016/j.isci.2021.103615

Cited by 20 publications

(25 citation statements)

References 33 publications

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“…11). 140–142 ddPCR allows the detection of HIV-DNA in peripheral blood mononuclear cells (PBMCs) with minimal viral reservoirs and the total amount of HIV-DNA in them, which is an important reference for the progression of the disease in treated patients. 143…”

Section: Applicationsmentioning

confidence: 99%

“…11). [140][141][142] ddPCR allows the detection of HIV-DNA in peripheral blood mononuclear cells (PBMCs) with minimal viral reservoirs and the total amount of HIV-DNA in them, which is an important reference for the progression of the disease in treated patients. 143 SARS-CoV-2, which has caused global pandemic pneumonia since late 2019, is also an RNA virus that has been detected not only in humans but also in animals and the environment.…”

Section: Pathogen Detectionmentioning

confidence: 99%

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Advances in droplet digital polymerase chain reaction on microfluidic chips

Zhang

et al. 2023

Lab Chip

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Section: Applicationsmentioning

confidence: 99%

Section: Pathogen Detectionmentioning

confidence: 99%

Advances in droplet digital polymerase chain reaction on microfluidic chips

Zhang

et al. 2023

Lab Chip

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“…The IPDA, however, does not provide information on the capacity of the proviruses to be expressed, and is also limited by its current optimization for only HIV-1 subtype B infections ( 19 ). IPDA specific for non-subtype B infection was recently reported but needs further validation ( 21 ). The Tat/Rev Induced Limiting Dilution Assay (TILDA) is also used to quantify HIV-1 reservoir size by assessing transcriptional competence under maximal CD4+ T cell stimulation conditions for multiply-spliced HIV-1 RNA transcripts.…”

Section: Introductionmentioning

confidence: 99%

Immune activation and exhaustion marker expression on T-cell subsets in ART-treated adolescents and young adults with perinatal HIV-1 infection as correlates of viral persistence

et al. 2023

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HIV-1 infection in memory CD4+ T cells forms a latent reservoir that is a barrier to cure. Identification of immune biomarkers that correlate with HIV-1 reservoir size may aid with evaluating efficacy of HIV-1 eradication strategies, towards ART-free remission and cure. In adults living with non-perinatal HIV-1, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with measures of HIV-1 reservoir size. Immune correlates of HIV-1 are less defined in adolescents and young adults with perinatal HIV-1. With multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, TIM-3 and LAG-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and the combination (Ttem) of transitional (Ttm) and effector memory (Tem) cells, in 10 adolescents and young adults living with perinatal HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was measured with the Intact Proviral HIV-1 DNA Assay (IPDA) and an enhanced Tat/Rev limiting dilution assay (TILDA). RNA-seq was also performed on the unstimulated CD4+ T cells. The median total HIV-1 DNA concentration in memory CD4+ T cells was 211.90 copies per million CD4+ T cells. In the 7 participants with subtype B HIV-1 infection, the median intact proviral DNA load was 7.96 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on the Ttem were correlated with total HIV-1 DNA (r=0.76, p=0.015) and (r=0.72, p=0.023), respectively, but not with intact proviral load or induced reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR expression by RNA-seq. In contrast, PD-1 expression on Tcm was inversely correlated with total HIV-1 DNA (r=-0.67, p=0.039). Reservoir size by IPDA and TILDA were correlated (r=0.81, p=0.036). Thus, in this cohort of youths with long-standing treated perinatal infection, HLA-DR and TIGIT on Ttem were the key correlates of HIV-1 infected cell frequencies by total HIV-1 DNA, and not PD-1. Total HIV-1 DNA was negatively correlated with PD-1 expressing Tcm. These differences in longstanding perinatal HIV-1 infection compared with adult infection requires further study in larger cohorts.

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“…Our protocol includes genomic DNA isolation optimized to minimize DNA shearing, a reference droplet digital PCR (ddPCR) assay to quantify T cells and assess DNA shearing, and a multiplex ddPCR targeting three distinct regions across the HIV genome to quantify intact proviruses as an estimate of replication-competent proviruses in the reservoir. For complete details on the use and execution of this protocol, please refer to Cassidy et al. (2022) .…”

mentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Cassidy et al. (2022) .…”

mentioning

confidence: 99%