A rapid and efficient method to identify neutralizing nanobodies against SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic, caused by the rapid outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is having a major impact on healthcare systems and economies around the world. . Several COVID-19 vaccines have obtained Emergency Use Authorizations (EUAs) from global regulatory bodies such as the U.S. Food and Drug Administration (FDA). With the continued emergence of SARS-CoV-2 variants due to genomic mutation, efficacy of Available vaccines against some strains of the virus are declining.

Background

There is an urgent need for new cost-effective, high-throughput, and adaptable pipelines to identify effective treatments against COVID-19 and other similar viruses with the potential to cause pandemics It has been with.

Mechanistically, spike protein Involved in host entry of SARS-CoV-2. This protein detects and binds to host angiotensin-converting enzyme 2 (ACE2), promoting membrane fusion and leading to host infection. SARS-CoV-2 genomic variants such as N501 are associated with increased transmissibility and virulence compared to the original SARS-CoV-2 strain that emerged in Wuhan. Moreover, some variants can evade immune responses induced after COVID-19 vaccination or natural infection.

Camelidae Some shark species also contain nanobodies, which are known to be a unique class of heavy chain-only antibodies. Nanobodies (VHH) and shark variables new antigen The receptor (VNAR) is an order of magnitude smaller than the corresponding full-length IgG. A major advantage of using these nanobodies as antiviral biologics is their facile mass production at multi-kilogram scales in prokaryotic systems. In addition, nanobodies have a longer shelf life and are more tissue-permeable.

Nanobodies are administered via different routes, eg orally (eg V565) or inhaled (eg ALX-0171).Recent pro swan In research, nanobody phage display libraries and in vitro When ex vivo high-throughput screening.

Main findings

A synthetic humanized nanobody library was developed by combining randomized complementarity determining region (CDR) frameworks to diversify antigen recognition. Among the 10 concentrated RBD binders, RBD-1-2G (NCATS-BL8125) and RBD-2-1F were the most potent showing IC.₅₀ 490 and 470 nM for SARS-CoV-2 pseudotyping particles, respectively.

Development of a bivalent or trivalent format of the RBD-1-2G domain has enhanced the affinity and neutralization capacity against both pseudotyped particles and the SARS-CoV-2 virus. In addition, two distinct binding modes were noted for the four nanobodies in combination with the stable SARS-CoV-2 S protein ectodomain. The epitope of the ‘group 1’ nanobodies was found to overlap with the receptor binding motif (RBM), whereas her N-terminal domain of the adjacent monomer was targeted by the ‘group 2’ binders. The latter failed to inhibit her ACE binding and did not overlap with RBM.

We also observed that three copies of RBD-1-2G could bind to the same spike trimer. Pseudotyped particles containing the N501Y mutation were neutralized by the RBD-1-2G nanobody. Viral load in WA1 and B.1.1.7 infections was reduced by RBD-1-2G-Fc treatment, and molecular dynamics simulations revealed nanobodies binding epitopes focused exclusively on the spike-ACE2 interface. rice field. B.1.1.7 Note that one of the reasons for the affinity of RBD-1-2G for RBD may be the presence of a tyrosine at position 501.

B.1.1.7/Alpha (N501Y) was used to determine the effects of mutations on nanobodies. This enhanced her RBD binding to the ACE2 receptor. RBD-1-2G was able to bind both Alpha (N501Y) and WT RBD, but not the new mutant lineage that could be caused by the E484 mutation. No binding was also seen with Delta and Lambda variants without the E484 mutation.

future outlook

The efficacy of nanobodies as therapeutic agents is primarily governed by factors such as protein solubility, potential immunogenicity and binding affinity. Several new approaches have been reported for the development of effective therapeutics against the SARS-CoV-2 spike domain. Previous studies have shown that direct immunization of llamas produces effective nanobodies that neutralize pseudotyped particles in the low nanomolar range. In the future, it will be necessary to develop techniques for producing llama hybridomas.

A recent study generated anti-SARS-CoV-2 binders using ribosome display of a synthetic llama library by performing an affinity maturation step. A mutation library was then created by combining the sequences. Similarly, such an affinity maturation process could serve to help RBD-1-2G binders restore binding to novel SARS-CoV-2 variants.

Rapid screening of engineered humanized nanobody libraries to detect compatible binders may be beneficial in countering the immunogenic defenses possessed by viral pathogens. The discovery method described in this study was able to generate high-affinity llama binders in a short span of less than two weeks. In the future, this library could also be used to treat other diseases besides COVID-19 and serve as a reserve for rapid neutralizing nanobody discovery.