Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.

Assembly and Entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2): Evaluation Using Virus-Like Particles.

Research on infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is currently restricted to BSL-3 laboratories. SARS-CoV2 virus-like particles (VLPs) offer a BSL-1, replication-incompetent system that can be used to evaluate virus assembly and virus-cell entry processes in tractable cell culture conditions. Here, we describe a SARS-CoV2 VLP system that utilizes nanoluciferase (Nluc) fragment complementation to track assembly and entry. We utilized the system in two ways. Firstly, we investigated the requirements for VLP assembly. VLPs were produced by concomitant synthesis of three viral membrane proteins, spike (S), envelope (E), and matrix (M), along with the cytoplasmic nucleocapsid (N). We discovered that VLP production and secretion were highly dependent on N proteins. N proteins from related betacoronaviruses variably substituted for the homologous SARS-CoV2 N, and chimeric betacoronavirus N proteins effectively supported VLP production if they contained SARS-CoV2 N carboxy-terminal domains (CTD). This established the CTDs as critical features of virus particle assembly. Secondly, we utilized the system by investigating virus-cell entry. VLPs were produced with Nluc peptide fragments appended to E, M, or N proteins, with each subsequently inoculated into target cells expressing complementary Nluc fragments. Complementation into functional Nluc was used to assess virus-cell entry. We discovered that each of the VLPs were effective at monitoring virus-cell entry, to various extents, in ways that depended on host cell susceptibility factors. Overall, we have developed and utilized a VLP system that has proven useful in identifying SARS-CoV2 assembly and entry features.

Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2.

To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2.