detection-sars-cov-2-neutralizing-antibodies-using-high-throughput

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

detection-sars-cov-2-receptor-binding-domain-antibody-using-hibit

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Ottawa Hospital Research Institute

University of Ottawa

The transformation of healthy cells to malignant often drives them to become inherently susceptible to viral infection as a trade-off to achieve uninhibited growth and immune escape. Enter oncolytic viruses (OVs), an exciting class of viruses that specifically infect cancer cells, leaving healthy tissue unharmed. Unfortunately, there is more to this story. Tumours are much more than a group of cancer cells, the surrounding tumour microenvironment (TME) comprises a collection of cells which influence and nourish the development and spread of the tumour. While initially quite promising, OV therapy has been met with a myriad of barriers due to the unwelcoming nature of the TME. Riddled with immunosuppressive factors and physical barriers, many tumours have proven impenetrable by OVs. Herein, we review the diverse array of approaches being used to target each component of the TME from enhancing entry into specific tumour types, breaking through the dense tumour stroma, eliminating cancer stem cells, and activating the immune system. We highlight the value of combination approaches which have led to complete successes in several in vivo models, some of which have entered clinical development.

Redirecting oncolytic viruses: Engineering opportunists to take control of the tumour microenvironment.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

High-throughput detection strategies for antibodies against SARS-CoV-2 in patients recovering from COVID-19, or in vaccinated individuals, are urgently required during this ongoing pandemic. Serological assays are the most widely used method to measure antibody responses in patients. However, most of the current methods lack the speed, stability, sensitivity, and specificity to be selected as a test for worldwide serosurveys. Here, we demonstrate a novel NanoBiT-based serological assay for fast and sensitive detection of SARS-CoV-2 RBD-specific antibodies in sera of COVID-19 patients. This assay can be done in high-throughput manner at 384 samples per hour and only requires a minimum of 5 μL of serum or 10 ng of antibody. The stability of our NanoBiT reporter in various temperatures (4-42 °C) and pH (4-12) settings suggests the assay will be able to withstand imperfect shipping and handling conditions for worldwide seroepidemiologic surveillance in the post-vaccination period of the pandemic. Our newly developed rapid assay is highly accessible and may facilitate a more cost-effective solution for seroconversion screening as vaccination efforts progress.

Taylor R. Jamieson

Ottawa Hospital Research Institute

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter