Author:
Jimenez-Campos Anahi G.,Maestas Lucas I.,Velappan Nileena,Beck Brian,Ye Chunyan,Wernsing Keith,Mata-Solis Yaniksa,Bruno William J.,Bussmann Silas C.,Bradfute Steven,Baca Justin T.,Rininsland Frauke H.
Abstract
AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life and prosperity. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in asymptomatic and presymptomatic carriers. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro). The test system consists of a peptide that fluoresces when cleaved by SARS PLpro that is active in crude, unprocessed lysates from human tongue scrapes and saliva. Test results are obtained in 30 minutes or less using widely available fluorescence plate readers, or a battery-operated portable instrument for testing at point of care (PoC) sites and in low-resource and hard to reach populations. Proof-of-concept was obtained in a clinical study on clinical specimens collected from patients with COVID-19 like symptoms who tested positive (n=10) or negative (n=10) with diagnostic LIAT RT-PCR using nasal mid turbinate swabs. When saliva from these patients was tested with in-house endpoint RT-PCR only 3 specimens were negative and 17 were positive. PLpro activity correlated in 17 of these cases (3 out of 3 negatives and 14 out of 16 positives, with one invalid specimen. Despite the small number of samples, the agreement was significant (p value = 0.01). In one presumptive false positive patient viral RNA was detected in saliva 5 days later by endpoint RT-PCR. Two false negatives were detected, one from a sample with a late Ct value of 35 in diagnostic RT-PCR, indicating that an active infection was no longer present. The PLpro activity assay is easily scalable and expected to detect all viable SARS-CoV-2 variants and recombinants, making it attractive as a screening and surveillance tool. Additionally, we show feasibility of the platform as a new homogeneous phenotypic assay for rapid screening of SARS-CoV-2 antiviral drugs and neutralizing antibodies.
Publisher
Cold Spring Harbor Laboratory