NeutrobodyPlex nanobodies help monitor immune responses to SARS-CoV-2

As the world faces unprecedented challenges posed by the COVID-19 pandemic caused by the severe acute respiratory syndrome virus coronavirus 2 (SARS-CoV-2), it is clear that there is an unmet need for diagnostic methods, therapeutic intervention and effective vaccination. .

Neutralizing molecules such as antibodies or their derivatives have become crucial tools for the treatment of COVID-19. These binding molecules also offer a unique way to monitor the neutralizing immune response in people infected or vaccinated with SARS-CoV-2.

Since the global COVID-19 outbreak, an increasing number of neutralizing antibodies targeting SARS-CoV-2 RBD have been detected in COVID-19 patients, underscoring the importance of RBD-specific antibodies that can block the RBD: ACE2 Interaction Site and thus helps develop a protective immune response.

A promising alternative to traditional antibodies or IgGs are single domain antibodies (nanobodies, Nbs) that are derived from heavy chain antibodies in camelids. Thanks to their compact folding and small size, Nbs show good chemical stability, fast tissue penetration and solubility. Nbs can be produced rapidly with high yields in bacteria and, in their monovalent form, bind to their target with very high affinities.

Researchers from the University of Tuebingen, Eberhard-Karls University, Center for Clinical and Experimental Research on Infections, Tuebingen University Hospital, Helmholtz Center for Infection Research, Germany, recently described a set of 11 Nbs derived from an immunized alpaca that showed high affinity binding. to the peak receptor domain of glycosylated SARS-CoV-2 (RBD) in a study published on the preprint server bioRxiv. *

Schematic representation of the generation of Nbs that block the SARS-CoV-2 RBD: ACE2 interaction site Nanobodies (Nbs) are engineered genetically from heavy chain alpaca antibodies only. The interaction between the homotrimeric spike protein of SARS-CoV-2 and ACE2 can be blocked by RBD-specific Nb. Protein structures adapted from PDB 3OGO (Nb) and 6CS2 (ACE2).

Nanobodies block the interaction between virus proteins and human receptors

With the help of an in vitro multiplex binding assay, the team demonstrated that 8 of the Nbs were effective in blocking the interaction of the S1 domain, RBD, and homotrimeric peak protein with angiotensin converting enzyme 2 (ACE2), which it is the docking site of the virus in human cells.

Using detailed epitope mapping and competitive binding analysis, the researchers grouped all the Nbs that block the RBD: ACE2 interaction into three distinct sets. They demonstrated the neutralizing effect of Nb with low nanomolar range IC50 values ​​in a cell-based SARS-CoV-2 neutralization assay.

When tested, combinations of Nbs from different pools had significantly lower IC50 values ​​in both functional assays, indicating a strong synergistic effect of Nbs targeting different epitopes in the RBD simultaneously.

“Interestingly, the IC50 values ​​obtained for inhibitory Nbs in RBD and homotrimeric peak show a higher correlation compared to the IC50 values ​​obtained for the S1 domain”

The team applied the most potent combination of Nbs (NeutrobodyPlex) in a competitive multiplex binding assay and were able to detect a neutralizing immune response in plasma samples from COVID-19 infected individuals.

NeutrobodyPlex can help monitor the immune status of SARS-CoV-2 patients

Based on the study’s findings, the team designed a new diagnostic test called NeutrobodyPlex to monitor the appearance and presence of neutralizing antibodies in the serum of individuals infected with SARS-CoV-2. With the help of combinations of high affinity Nbs that cover the RBD: ACE2 interface, the team was able to directly and precisely displace the IgG in the serum samples of these RBD epitopes.

“To our knowledge, the NeutrobodyPlex using Nbs that blocks the RBD: ACE2 interaction site shows for the first time an antigen-resolved analysis of the presence of human IgG in convalescent individuals suffering from SARS-CoV-2 infection.”

All the identified monovalent Nb (except NM1225) showed high affinities in the low nanomolar range. Thus, there is no need to reformat these Nbs into bivalent forms by fusing them to an Fc domain or combining binding sites. Employing better screening strategies, Nbs can be developed that can bind to predefined domains on larger antigens.

According to the team, the NeutrobodyPlex Nbs that blocks the RBD: ACE2 interaction site is the first to show antigen-resolved analysis of the presence of human IgG in convalescent individuals with SARS-CoV-2 infection. The research team believes that these Nbs may hold great promise in both prophylactic and therapeutic approaches in the fight against COVID-19.

Nbs also offer a novel approach to the detection of neutralizing immune responses in people infected or vaccinated with the virus, helping to monitor the immune status of patients and to develop effective vaccines. Compared to other neutralizing antibody detection tests, this new assay enables high-throughput, automatable analysis. It can be done with non-live, non-infectious viral material, improving safety and reducing costs.

*Important news

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.