Application of ultrasensitive digital ELISA for p24 enables improved evaluation of HIV-1 reservoir diversity and growth kinetics in viral outgrowth assays

Abstract

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure.

Figure 1

Digital ELISA provides a robust method of detecting HIV-1 p24. (A) Correlation of HIV-1 p24 as measured by Simoa and conventional ELISA (Pearson’s correlation coefficient, r). Two replicates were assessed for each dilution set, both for Simoa and conventional ELISA. (B) Evaluation of recovery of spiked clinical isolate HIV-1_92/BR/014 in HIV negatives samples. HIV-1_92/BR/014 was serially diluted and the assay matrix effect was evaluated by comparing measured concentration by Simoa to the expected (RMSE = root mean square error). Two replicates were assessed for each dilution paramater. (C) Frequency Distribution of HIV negative samples. Limit of Detection (LOD) for QVOA-specific digital p24 assay was determined to be 0.0515 pg/mL. To calculate LOD, a Grubb’s outlier test was performed to remove 2 significant outliers from a total of 226 HIV negative samples (Participant 6513-R). The QVOA-specific LOD was calculated by adding three standard deviations (0.005577 pg/mL) of the mean to the maximum p24 value (0.03480 pg/mL) obtained from known HIV negative samples.

Figure 2

Digital ELISA yields higher reservoir measurements from HIV-1 infected donors and detects HIV-1 p24 at earlier assay time points. (A) Correlation of HIV-1 p24 measurements by Simoa and conventional ELISA (Pearson’s correlation coefficient, r) in a set of triplicate QVOAs performed on 5 HIV-1 infected donors. (B) Digital ELISA produces significantly more positive wells over the course of the assay duration on different days as found through chi-square analysis, * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001. (C) Coupling Simoa with QVOA allowed all assays to yield a reservoir measurement while conventional ELISA did not produce a quantifiable measurement in four assays. Reservoir frequencies and associated 95% confidence intervals (error bars) are plotted for each replicate from day 20 of the QVOA culture. Regions, where confidence intervals do not overlap, are significant. BD below detection.

Figure 3

Ultrasensitive HIV-1 p24 and RNA genome detection strongly correlate and reveal growth kinetics at concentrations not detectable by conventional ELISA. Supernatants collected at days 8, 12, and 20 of QVOA that have been published previously were analyzed for the presence of HIV-1 p24 by standard ELISA, digital ELISA, and HIV-1 RNA by Panther. HIV-1 p24 concentrations measured by digital ELISA and HIV-1 RNA concentrations in QVOA supernatants directly correlate (Pearson’s correlation coefficient, r). Dotted red line indicates Lower Limit of Quantitation for ELISA (3.25 pg/mL p24 antigen) and Panther (30 RNA copies/mL) assays and Limit of Detection for Simoa, (0.0515 pg/mL p24 antigen). Solid blue line indicates the relationship equivalency of 1 pg HIV-1 p24 to 2 × 10⁴ HIV RNA copies, referred to as mature particle correlation (MPC).

Figure 4

Ultrasensitive methods for detecting HIV-1 p24 and viral RNA provide evidence of HIV-1 reactivation that does not lead to exponential outgrowth. Each panel represents a unique well from an individual QVOA displaying viral kinetics through measurements of either HIV-1 p24 molecules (red-filled circles and red solid lines) or HIV RNA copies (blue-filled squares and blue solid lines) taken at three different time points during the assay. HIV-1 p24 molecules per milliliter axis set to 1000-times the RNA genome axis. The dotted line represents 5,100 HIV RNA copies threshold (converted to 51,000 HIV RNA copies/mL). The light-blue-filled area represents area below the LOD of the digital ELISA (1 × 10⁶ p24 molecules/mL). The grey-filled area represents the area below the lower limit of quantitation (LLOQ) of the Panther Hologic HIV-1 RNA system of 30 HIV RNA copies/mL. For sample 45-A1 (marked with a pink diamond), single-genome sequencing was performed to assess viral replication (Fig. 5).

Figure 5

Low concentrations p24 requiring ultrasensitive detection approaches reveal a more diverse repertoire of reactivated HIV-1. Each circle or diamond represents a single, unique RNA genome found through P6-PR-RT single-genome sequencing performed on QVOA culture supernatants from RAVEN participant 2147-R. Black triangles represent proviral sequences found in mixed PBMC from RAVEN participant 2147-R. Asterisk denotes stop codons. Sequences were processed to visualize 1–2 nucleotide changes.