Comparative analysis and generation of a robust HIV-1 DNA quantification assay

Thomas, Jordan; Ruggiero, Alessandra; Procopio, Francesco A.; Pantaleo, Giuseppe; Paxton, William A.; Pollakis, Georgios

doi:10.1016/j.jviromet.2018.10.010

Cited by 11 publications

(12 citation statements)

References 41 publications

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“…Longitudinal analyses of HIV proviral dynamics will yield critical information on the timing of the shifts in the populations of HIV-infected cells that take place during cART. There are published reports that measure viral DNA load by estimating the numbers of intact proviruses [3,28] and measuring levels of proviruses that are likely to be infectious [45]; in addition, there are methods that quantify either internal portions of the HIV-1 genome or the HIV-1 long terminal repeats (LTRs), but not both simultaneously [23,[46][47][48][49][50][51]. We have developed Droplet Digital PCR (ddPCR) approaches that make it possible to simultaneously quantify both HIV-1 LTRs and internal HIV-1 DNA sequences; these methods are sensitive enough to measure small (2-to 3-fold) differences in relative abundance [52].…”

Section: Introductionmentioning

confidence: 99%

Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

Anderson

Simonetti²,

Gorelick

et al. 2020

Viruses

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Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1–2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10–15 years on therapy, there were as many as 3.5–5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.

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Section: Introductionmentioning

confidence: 99%

Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

Anderson

Simonetti²,

Gorelick

et al. 2020

Viruses

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“…Previous studies have shown that LTR has been proved to be the best target for DNA quantification [17]. In this study, real-time fluorescence based HIV detection kit was used to amplify and quantify HIV-1 DNA using LTR gene primers, which ensured the reliability and accuracy of HIV-1 DNA detection.…”

Section: Discussionmentioning

confidence: 99%

Whole blood as an alternative to peripheral blood mononuclear cell for detection of total HIV-1 DNA

Yue

Wang

et al. 2020

BMC Infect Dis

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Background A more time saving, convenient, reproducible, and scalable method is needed to assess total HIV-1 DNA levels. Methods Frozen whole blood and peripheral blood mononuclear cell (PBMC) samples both 200 μl at the same point were used to detect total HIV-1 DNA. Automatic extraction of total HIV-1 DNA was used to ensure the consistency of sample extraction efficiency. The detection reagent was HIV-1 DNA quantitative detection kit and real-time quantitative PCR was utilized. Results Of the 44 included patients, 42 were male and 2 were female, with a median age of 33 years. Thirty-three cases were collected after receiving antiviral treatment, with a median duration of treatment of 3 months, and the other 11 cases were collected before antiviral treatment. The median viral load was 1.83 log10 copies/mL, the median CD4 and CD8 count were 94 and 680 cells/μL, and the median CD4/CD8 ratio was 0.18. The results of the two samples were 3.02 ± 0.39 log10 copies/106 PBMCs in PBMC samples and 3.05 ± 0.40 log10 copies/106 PBMCs in whole blood samples. The detection results of the two methods were highly correlated and consistent by using paired t test (P = 0.370), pearson correlation (r = 0.887, P < 0.0001) and intra-group correlation coefficient (ICC = 0.887, P < 0.0001) and bland-altman [4.55% points were outside the 95% limits of agreement (− 0.340 ~ 0.390)]. Conclusions The results of the whole blood sample test for total HIV-1 DNA are consistent with those of PBMC samples. In a clinical setting it is recommended to use whole blood samples directly for the evaluation of the HIV reservoir.

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“…Cell lines such as 8E5 and ACH2 are widely used as the source of calibration DNA, though recent work has demonstrated that HIV-1 integration into these cell lines is unstable, likely due to ongoing replication, and their use may confound accurate quantification and reproducibility between labs (Sunshine et al, 2016;Wilburn et al, 2016;Busby et al, 2017;Symons et al, 2017;Rutsaert et al, 2018b;Thomas et al, 2019). Recent analysis of HIV-1 quantification methods has demonstrated the stability of HIV-1 integration into J-Lat 10.6, a Jurkat cell latently infected with full length, env deficient provirus, and suggested the use of this cell line as the gold standard for HIV-1 DNA quantification by qPCR (Sunshine et al, 2016;Thomas et al, 2019). Another key determinant of the accuracy and specificity of HIV-1 DNA quantification assays is the genomic location at which the primers and probes anneal.…”

Section: Assays To Measure the Success Of Hiv-1 Cure Viral Outgrowth mentioning

confidence: 99%

“…Amplification efficiencies between the standard and the sample must be equal to limit bias when quantifying relative to a standard curve (Rutsaert et al, 2018a). Amplification efficiency is affected by the DNA input per reaction, the presence of inhibitory contaminants and, crucially for HIV-1 quantification, recent work has shown that small mismatches between the primer and target sequence significantly impair sample quantification (Rutsaert et al, 2018b;Thomas et al, 2019). Digital droplet PCR (ddPCR) platforms mitigate these issues by facilitating absolute quantification of a sample and as such, are becoming increasingly popular in HIV-1 research and clinical trials.…”

Section: Digital Droplet Pcr Based Hiv-1 Quantificationmentioning

confidence: 99%

Measuring the Success of HIV-1 Cure Strategies

Thomas

Ruggiero

Paxton

et al. 2020

Front. Cell. Infect. Microbiol.

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HIV-1 eradication strategies aim to achieve viral remission in the absence of antiretroviral therapy (ART). The development of an HIV-1 cure remains challenging due to the latent reservoir (LR): long-lived CD4 T cells that harbor transcriptionally silent HIV-1 provirus. The LR is stable despite years of suppressive ART and is the source of rebound viremia following therapy interruption. Cure strategies such as "shock and kill" aim to eliminate or reduce the LR by reversing latency, exposing the infected cells to clearance via the immune response or the viral cytopathic effect. Alternative strategies include therapeutic vaccination, which aims to prime the immune response to facilitate control of the virus in the absence of ART. Despite promising advances, these strategies have been unable to significantly reduce the LR or increase the time to viral rebound but have provided invaluable insight in the field of HIV-1 eradication. The development and assessment of an HIV-1 cure requires robust assays that can measure the LR with sufficient sensitivity to detect changes that may occur following treatment. The viral outgrowth assay (VOA) is considered the gold standard method for LR quantification due to its ability to distinguish intact and defective provirus. However, the VOA is time consuming and resource intensive, therefore several alternative assays have been developed to bridge the gap between practicality and accuracy. Whilst a cure for HIV-1 infection remains elusive, recent advances in our understanding of the LR and methods for its eradication have offered renewed hope regarding achieving ART free viral remission.

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Comparative analysis and generation of a robust HIV-1 DNA quantification assay

Cited by 11 publications

References 41 publications

Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

Dynamic Shifts in the HIV Proviral Landscape During Long Term Combination Antiretroviral Therapy: Implications for Persistence and Control of HIV Infections

Whole blood as an alternative to peripheral blood mononuclear cell for detection of total HIV-1 DNA

Measuring the Success of HIV-1 Cure Strategies

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