Abstract
Greater than 90% of HIV-1 proviruses are thought to be defective and incapable of viral replication. While replication competent proviruses are of primary concern with respect to disease progression or transmission, studies have shown that even defective proviruses are not silent and can produce viral proteins, which may contribute to inflammation and immune responses. Viral protein expression also has implications for immune-based HIV-1 clearance strategies, which rely on antigen recognition. Thus, sensitive assays aimed at quantifying both replication-competent proviruses and defective, yet translationally competent proviruses are needed to understand the contribution of viral protein to HIV-1 pathogenesis and determine the effectiveness of HIV-1 cure interventions. Previously, we reported a modified HIV-1 gag p24 digital enzyme-linked immunosorbent assay with single molecule array (Simoa) detection of cell-associated viral protein. Here we report a novel p24 protein enrichment method coupled with the digital immunoassay to further extend the sensitivity and specificity of viral protein detection. Immunocapture of HIV gag p24 followed by elution in a Simoa-compatible format resulted in higher protein recovery and lower background from various biological matrices and sample volumes. Quantification of as little as 1 fg of p24 protein from cell lysates from cells isolated from peripheral blood or tissues from ART-suppressed HIV participants, as well as simian–human immunodeficiency virus–infected non-human primates (NHPs), with high recovery and reproducibility is demonstrated here. The application of these enhanced methods to patient-derived samples has potential to further the study of the persistent HIV state and examine in vitro response to therapies, as well as ex vivo study of translationally competent cells from a variety of donors.
Highlights
Antiretroviral therapy (ART) has dramatically improved life quality and survival for people living with HIV (PLWHs)
We evaluated the time needed for immunocapture by studying various incubation times of antibody conjugated beads incubated with 1 ng/mL recombinant p24 spiked into single molecule array (Simoa) lysis buffer
We evaluated anti-CD3/antiCD28 bead stimulation of CD4+ T cells isolated from blood of five additional HIV+, ART-suppressed individuals and observed a similar pattern of volume-proportional increases following IPSimoa vs. without IP Simoa (p < 0.01) (Figure 3B). p24 values were below assay limits in the flow-through following protein capture on beads, indicating the IP method efficiently captured the expressed p24
Summary
Antiretroviral therapy (ART) has dramatically improved life quality and survival for people living with HIV (PLWHs). Biomarkers that can robustly measure viral persistence in PLWHs on ART in a way that reports on proviruses capable of driving rebound off therapy or producing viral protein that influence HIV pathogenesis and immune functions are important tools for more in-depth understanding of HIV persistence and assessment of therapeutic interventions. The quantitative viral outgrowth assay (QVOA) measures the replication-competent HIV reservoir but likely underestimates reservoir size as some genetically intact viruses may not grow in culture after a single round of ex vivo stimulation. Protein-based assays overestimate replication competent reservoir size, measuring this translationally competent reservoir provides mechanistic insight into HIV persistence, immune response, and evaluation of interventional strategies aimed at clearing these infected cells
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