NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

• Published in: bioRxiv 2020-12
• Main Authors: Wagner, Teresa R, Kaiser, Philipp D, Gramlich, Marius, Ostertag, Elena, Ruetalo, Natalia, Junker, Daniel, Haering, Julia, Traenkle, Bjoern, Becker, Matthias, Dulovic, Alex, Schweizer, Helen, Nueske, Stefan, Scholz, Armin, Zeck, Anne, Schenke-Layland, Katja, Nelde, Annika, Strengert, Monika, Walz, Juliane S, Zocher, Georg, Stehle, Thilo, Schindler, Michael, Schneiderhan-Marra, Nicole, Rothbauer, Ulrich
• Format: Article
• Language: English
• Subjects: ACE2; Angiotensin; Angiotensin-converting enzyme 2; COVID-19; Crystal structure; Crystallization; Epitopes; High-throughput screening; Immune response; Immune status; Immunoglobulin G; Nanobodies; Pandemics; Peptidyl-dipeptidase A; Severe acute respiratory syndrome coronavirus 2; Vaccination
• DOI: 10.1101/2020.09.22.308338

Abstract As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide. Competing Interest Statement The authors have declared no competing interest. Footnotes * In the substantially revised version we added novel data including the crystal structure of Nanobody NM1230 in complex with RBD in the results section. We additionally generated and described a novel bivalent Nanobody (NM1267) as the most potent IgG surrogate which was applied in the described NeutrobodyPlex assay. Finally the NeutrobodyPlex was used to analysed a large patient cohort of 120 individuals (all result section) With Elena Ostertag, Georg Zocher and Thilo Stehle we added three additional authors which performed the RBD:NM1230 crystallization analysis. In the revised version Supplementary Information are added to the main text. All parts were adapted for clarification