Screening inhibitors and monitoring the efficacy of neutralizing antibodies in convalescent and vaccinated patient samples with Lumit SARS-CoV-2 Spike RBD:ACE2 immunoassay

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was initially detected in December 2019, and became a worldwide pandemic of viral respiratory illness. SARS-CoV-2 virus enters target cells through the interaction between its surface Spike protein (S) and the Angiotensin Converting Enzyme 2 (ACE2) receptor on human cells. The importance of the Spike protein for viral internalization is highlighted by the efforts to identify molecules that would interfere with its binding to ACE2, thus preventing its cellular internalization resulting in an effective therapeutic treatment against COVID-19. Towards that goal, an assay that monitors the interaction of SARS-CoV-2 Spike RBD and human ACE2 would be a useful tool both for high throughput screening for inhibitory molecules as well as for the screening and characterization of clinical specimens that may contain virus neutralizing antibodies (NAbs). Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit (Lumit secondary antibodies). The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies from COVID-19 positive samples were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.