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. 2020 Aug 6:11:1971.
doi: 10.3389/fimmu.2020.01971. eCollection 2020.

Measuring the Inducible, Replication-Competent HIV Reservoir Using an Ultra-Sensitive p24 Readout, the Digital ELISA Viral Outgrowth Assay

Erin L Stuelke  1 , Katherine S James  1 , Jennifer L Kirchherr  1 , Brigitte Allard  1 , Caroline Baker  1 , Joann D Kuruc  1   2 , Cindy L Gay  1   2 , David M Margolis  1   2   3   4 , Nancie M Archin  1   2
Affiliations

Measuring the Inducible, Replication-Competent HIV Reservoir Using an Ultra-Sensitive p24 Readout, the Digital ELISA Viral Outgrowth Assay

Erin L Stuelke et al. Front Immunol. .

Abstract

Quantifying the inducible HIV reservoir provides an estimate of the frequency of quiescent HIV-infected cells in humans as well as in animal models, and can help ascertain the efficacy of latency reversing agents (LRAs). The quantitative viral outgrowth assay (QVOA) is used to measure inducible, replication competent HIV and generate estimations of reservoir size. However, traditional QVOA is time and labor intensive and requires large amounts of lymphocytes. Given the importance of reproducible and accurate assessment of both reservoir size and LRA activity in cure strategies, efforts to streamline the QVOA are of high priority. We developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay, with ultra-sensitive p24 readout, capable of femtogram detection of HIV p24 protein in contrast to the picogram limitations of traditional ELISA. For each DEVO assay, 8-12 × 106 resting CD4 + T cells from aviremic, ART-treated HIV + participants are plated in limiting dilution and maximally stimulated with PHA, IL-2 and uninfected allogeneic irradiated PBMC. CD8-depleted PHA blasts from an uninfected donor or HIV-permissive cells (e.g., Molt4/CCR5) are added to the cultures and virus allowed to amplify for 8-12 days. HIV p24 from culture supernatant is measured at day 8 by Simoa (single molecule array, ultra-sensitive p24 assay) confirmed at day 12, and infectious units per million CD4 + T cells (IUPM) are calculated using the maximum likelihood method. In all DEVO assays performed, HIV p24 was detected in the supernatant of cultures as early as 8 days post stimulation. Importantly, DEVO IUPM values at day 8 were comparable or higher than traditional QVOA IUPM values obtained at day 15. Interestingly, DEVO IUPM values were similar with or without the addition of allogeneic CD8-depleted target PHA blasts or HIV permissive cells traditionally used to expand virus. The DEVO assay uses fewer resting CD4 + T cells and provides an assessment of reservoir size in less time than standard QVOA. This assay offers a new platform to quantify replication competent HIV during limited cell availability. Other potential applications include evaluating LRA activity, and measuring clearance of infected cells during latency clearance assays.

Keywords: DEVO; HIV; IUPM; QVOA; outgrowth.

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Figures

FIGURE 1
FIGURE 1
Limiting dilution culture of uninfected donor PBMC reveals background noise of Simoa. PBMC from an uninfected donor were stimulated in limiting dilution with PHA/IL-2 and irradiated PBMC or treated with low amount of IL-2 only. Cultures were either targeted with PHA blasts or no targets were added. Supernatant were harvested at day 8 and HIV p24 assayed by Simoa. (A) Low IL-2 treated/no targets added. (B) Low IL-2 treated/cultures targeted with PHA blasts. (C) PHA stimulated/no targets added. (D) PHA stimulated/cultures targeted with PHA blasts. Each dot represents a well at the indicated cell dilution. LLOQ, lower limit of quantitation.
FIGURE 2
FIGURE 2
The lower limit of quantitation for the DEVO assay. Freshly isolated resting CD4 + T cells from an uninfected donor were stimulated with PHA and irradiated PBMC, then co-cultured with PHA blasts over the course of 19 days. On three separate, but not consecutive days, supernatant from day 8 and 12 were spiked with HIV p24 protein at different dilutions and assayed by Simoa. Bias, total error and % total error was calculated from replicate well values using the following formula: Bias = Average−ActualValue;TotalError = |Bias| + 2×StandardDeviation;%TotalError = TotalErrorActualValue×100. The concentration with <20% total error was determined to be 49.21fg/ml**. This value was used as the DEVO lower limit of quantitation.
FIGURE 3
FIGURE 3
Comparison of IUPM values obtained in the DEVO assay at day 8 post-stimulation versus IUPM values obtained from the QVOA at day 15 post-stimulation. (A) The DEVO assay generated IUPM values that were either comparable or in some cases higher than values obtained with the traditional QVOA. However, overall, there was no significant difference in IUPM values between the two assays. P = 0.1294, Wilcoxon matched-pairs signed rank test. Twelve independent, paired assays from 12 distinct participants is shown. IUPM + 95% CI (upper and lower limits) is shown. (B) Correlation of the DEVO assay at day 8 post-stimulation with the QVOA at day 15 post-stimulation. Spearman r = 0.9282.
FIGURE 4
FIGURE 4
DEVO assay outcome using either no blasts, or PHA blasts or the HIV permissive cell line, Molt4/CCR5 to expand virus. (A) Increase in IUPM values over time in cultures receiving either PHA blasts or no exogenous targets (no blasts). Two independent assays using cells from two participants are shown (left and right panel, DV-01 and DV-02). (B) Similar IUPM values are obtained in the DEVO assay whether or not exogenous targets to expand virus are used. Each symbol represents an independent assay performed with cells from distinct participants. (C) The HIV permissible cell line Molt4/CCR5 expands virus as well as PHA blasts in the DEVO assay. Results from independent assays performed with cells from three different participants (DV-01, DV-05 and DV-06) at day 8 (green) and day 12 (purple) are shown. For each experiment, cultures either received Molt4/CCR5 or PHA blasts to expand virus. Each symbol represents a participant. Open symbols indicate values below the limit of detection. Wilcoxon matched-pairs signed rank test was used to compare statistical differences.
FIGURE 5
FIGURE 5
The DEVO assay tracked similarly overtime as the traditional QVOA. The DEVO assay and QVOA were performed with resting CD4 + T cells collected at multiple time points from 3 participants, DV-01 (first panel), DV-09 (second panel), and DV-04 (third panel). All assays were performed using cells from the same leukapheresis donation, except for VV-09 where the DEVO was performed using cells from whole blood donated within 3 months of leukapheresis donations used for the QVOA. The time elapsed between visits 1 and 2, 1 and 3, and 2 and 3 for DV-01 is 7.9, 14.1, and 6.2 months respectively; for DV-09 the time elapsed between visit 1 and 2 is 17.9 months (DEVO) and 14.9 months (QVOA); For DV-04, the time between the first and second donation is 15. 9 months. IUPM + 95% CI (upper and lower limits) is shown.
FIGURE 6
FIGURE 6
Comparison of the DEVO assay using resting CD4 + T cells isolated from whole blood vs leukapheresis product (Leuka). (A) Targets (either PHA blasts or MOLT4/CCR5) added to expand virus. (B) No targets added. Whole blood was collected 6–17 months from leukapheresis donation time. Each symbol represents an independent assay performed using cells from 4 distinct participants (DV-02, DV-03, DV-04, DV-09 and DV-11). For each experiment, cultures either received exogenous targets to expand virus or no targets. Open symbols indicate values below the limit of detection. The Mann–Whitney U test was used to compare statistical differences.
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References

    1. Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N Engl J Med. (2009) 360:692–8. 10.1056/NEJMoa0802905 - DOI - PubMed
    1. Gupta RK, Abdul-Jawad S, McCoy LE, Mok HP, Peppa D, Salgado M, et al. HIV-1 remission following CCR5Delta32/Delta32 haematopoietic stem-cell transplantation. Nature. (2019) 568:244–8. 10.1038/s41586-019-1027-4 - DOI - PMC - PubMed
    1. Eriksson S, Graf EH, Dahl V, Strain MC, Yukl SA, Lysenko ES, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog. (2013) 9:e1003174. 10.1371/journal.ppat.1003174 - DOI - PMC - PubMed
    1. Ho YC, Shan L, Hosmane NN, Wang J, Laskey SB, Rosenbloom DI, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell. (2013) 155:540–51. 10.1016/j.cell.2013.09.020 - DOI - PMC - PubMed
    1. Bruner KM, Murray AJ, Pollack RA, Soliman MG, Laskey SB, Capoferri AA, et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med. (2016) 22:1043–9. 10.1038/nm.4156 - DOI - PMC - PubMed

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