The potential of nanobody engineering in the development of antivirals and diagnostic tools for SARS-CoV-2

In a recent study published in bioRxiv* Prepress server, researchers investigated the engineering of nanobodies to develop antivirals and diagnostic tools against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Stady: Engineering nanobodies for neutralization and detection of SARS-CoV-2. Image Credit: Juan Gaertner / Shutterstock


Global efforts to contain the recent coronavirus 2019 (COVID-19) pandemic have led to the development of several antibody-based therapeutic and diagnostic technologies ranging from rapid antigen tests to monoclonal antibodies in the treatment of severe COVID-19 symptoms.

Many monoclonal antibodies and COVID-19 vaccines target the SARS-CoV-2 spike protein due to its role in host cell membrane binding and virus entry. Recent studies have shown that mutations in the receptor-binding domain (RBD) of the S1 subunit of the spike protein give rise to novel variants that challenge the efficacy of existing monoclonal antibodies and COVID-19 vaccines.

Current research against immune evasion demonstrated by some novel SARS-CoV-2 variants is focused on developing new antibody-based technology such as single-domain antibody fragments or nanobodies. While a few neutral SARS-CoV-2 nanobodies have been characterized, the use of nanobodies in diagnostic tools remains largely unexplored.

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In this study, the researchers designed multi-unit nanobodies by integrating domains of nanobodies that bind to different binding sites. These domains are fused with 20 amino acid ligands and can simultaneously bind to different epitopes, increasing the strength of the association and potentially decreasing immune escape through emerging variants.

Previously developed combinations of four monomeric nanobodies were used to generate three ternary nanobodies—tri-Ty1, tri-TMH and tri-TMV. Neutralization assays were performed in the laboratory To test the neutralizing efficacy of PMUs against SARS-CoV-2 wild-type, alpha, beta, delta and omicron variants. An antigen microarray was used to understand how amino acid changes in the RBD affect the binding of the three-unit nanobodies.

Furthermore, the modulatory properties of nanobodies have been used to develop a diagnostic assay consisting of RBD-linked nanobodies fused with spliced ​​fragments of the engineered fluorescent protein NanoLuc luciferase, which serves as a signaling molecule. The diagnostic assay is based on the principle that when the split fragments of a NanoLuc are brought into proximity by binding the nanoparticles to the SARS-CoV-2 spike fragments, the fusion of the fragments will result in a fluorescent signal. The researchers believe this will help detect the near-nanoscale levels of SARS-CoV-2 spike proteins in a single step.


The results indicate an up to 100-fold increase in the neutralizing efficacy of the multi-unit nanobodies developed in this study compared to the half-maximum inhibitory concentration (IC50) of the individual component nanobodies.

The triple TMH nanobody construct was the strongest equivalent of the wild-type SARS-CoV-2 and the Alpha variant but showed lower strength versus the delta variant. The three polyunit nanobodies were ineffective in neutralizing the beta and omicron variants. Prophylactic doses of triple TMH administered into the nasal cavity of animal models have limited lung tissue damage.

According to the authors, the E484K mutation present in the Beta and Omicron variants but absent in the wild-type SARS-CoV-2 and other variants is responsible for the reduced efficacy of the three nanobody formulations. This mutation results in changes in amino acids, which disrupt salt bridges and cause conformational changes in the RBD, thus affecting the nanobody binding interface.

The nanobody-based diagnostic assay developed in this study successfully detected the SARS-CoV-2 spike protein at concentrations as low as 200 μM. These detection levels were comparable with other antigen testing methods, such as the fluorescence resonance energy transfer (FRET)-based assay, and the results were similar to commercially available antigen tests.


Overall, the study presents a promising antiviral and diagnostic alternative to monoclonal antibodies with the development of multi-subunit nanobodies with increased binding affinity and ability to bind to multiple epitopes simultaneously.

Proof-of-principle experiments indicate that the new nanobody-based diagnostic tool can detect very low concentrations of the SARS-CoV-2 protein. The assay requires further validation of patient samples for commercial use as a diagnostic tool. However, the relatively low production costs and absence of resource-intensive requirements, such as animal tissue cultures, make nanobodies an attractive alternative in antiviral research and testing.

With the emergence of SARS-CoV-2 variants that challenge the efficacy of monoclonal antibodies and vaccines, relatively inexpensive and modifiable nanobodies represent a feasible option for antiviral therapy and diagnostic testing.

*Important note

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