In a recent study published on the bioRxiv* preprint server, researchers investigated nanobody engineering to develop antivirals and diagnostic tools against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Study: Engineering of nanobodies for the neutralization and detection of SARS-CoV-2. Image credit: Juan Gaertner/Shutterstock
background
The global effort to contain the recent coronavirus disease 2019 (COVID-19) pandemic has led to the development of many antibody-based therapeutic and diagnostic technologies ranging from rapid antigen tests to monoclonal antibodies to treat severe symptoms of the COVID-19.
Many monoclonal antibodies and vaccines against COVID-19 target the SARS-CoV-2 spike protein because of its role in host cell membrane binding and viral entry. Recent studies have shown that mutations in the receptor-binding domain (RBD) of the S1 subunit of the spike protein lead to the emergence of new variants that challenge the efficacy of existing COVID-19 monoclonal antibodies and vaccines .
Current research against the immune evasion shown by some of the new variants of SARS-CoV-2 is focused on the development of new antibody-based technologies, such as single-domain antibody fragments from camelids or nanobodies. Although a few SARS-CoV-2 neutralizing nanobodies have been characterized, the use of nanobodies in diagnostic tools remains largely unexplored.
About the study
In the present study, the researchers designed multimodular nanobodies by fusing nanobody domains that bind at different binding sites. These domains were fused by flexible 20-amino acid linkers and could simultaneously bind to different epitopes, increasing binding strength and potentially reducing immune escape by emerging variants.
Combinations of four previously developed monomeric nanobodies were used to generate three trimodular nanobodies: tri-Ty1, tri-TMH, and tri-TMV. In vitro neutralization assays were performed to test the neutralization potency of the multimodular nanobodies against wild-type SARS-CoV-2 and the Alpha, Beta, Delta, and Omicron variants. An antigen microarray was used to understand how amino acid changes in the RBD influence binding of the three trimodular nanobodies.
In addition, the modular properties of the nanobodies were used to develop a diagnostic assay consisting of RBD-binding nanobodies fused to cleaved fragments of the engineered fluorescent luciferase protein NanoLuc, which acts as a signal molecule. The diagnostic assay is based on the principle that when split fragments of NanoLuc are brought into close proximity by binding of Nanobodies to SARS-CoV-2 spike trimers, fusion of the fragments will result in a fluorescent signal. The researchers believe this will help detect subnanomolar levels of SARS-CoV-2 spike proteins in a single step.
results
The results indicate an up to 100-fold increase in the neutralization efficacy of the multimodular nanobodies developed in this study compared to the mean maximal inhibitory concentration (IC50) of the individual constituent nanobodies.
The tri-TMH nanobody construct was the strongest neutralizer of wild-type SARS-CoV-2 and the Alpha variant, but showed decreased potency against the Delta variant. All three multimodular nanobodies were ineffective at neutralizing the Beta and Omicron variants. Prophylactic doses of tri-TMH administered into the nasal cavity of animal models limited lung tissue damage.
According to the authors, the E484K mutation present in the Beta and Omicron variants but absent in wild-type SARS-CoV-2 and the other variants is responsible for the reduced efficacy of the three nanobody constructs. This mutation causes changes in the amino acids, which disrupt the salt bridges and cause conformational changes in the RBD, thus affecting the binding interface of the nanobody.
The nanobody-based diagnostic assay developed in this study successfully detected the SARS-CoV-2 spike protein at concentrations as low as 200 pM. These detection levels were comparable to other antigen testing methods, such as the fluorescence resonance energy transfer (FRET)-based assay, and the results were similar to those of commercially available antigen tests.
Conclusions
Overall, the study presents a promising antiviral and diagnostic alternative to monoclonal antibodies with the development of multimodular nanobodies with increased binding avidity and the ability to bind to multiple epitopes simultaneously.
Proof-of-principle experiments indicate that the new nanobody-based diagnostic tool could detect very low concentrations of the SARS-CoV-2 spike protein. The assay requires further validation with patient samples for commercial use as a diagnostic tool. However, relatively low production costs and the absence of resource-intensive requirements, such as animal tissue cultures, make nanobodies an attractive alternative in antiviral research and testing.
With emerging SARS-CoV-2 variants challenging the efficacy of monoclonal antibodies and vaccines, relatively inexpensive and modifiable nanobodies present a feasible option for antiviral therapy and diagnostic testing.
*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.