A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529)

Abstract

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have risen to dominance, which contains far more mutations in the spike protein in comparison to previously reported variants, compromising the efficacy of most existing vaccines or therapeutic monoclonal antibodies. Nanobody screened from high-throughput naïve libraries is a potential candidate for developing preventive and therapeutic antibodies. Methods Four nanobodies specific to the SARS-CoV-2 wild-type receptor-binding domain (RBD) were screened from a naïve phage display library. Their affinity and neutralizing activity were evaluated by surface plasmon resonance assays, surrogate virus neutralization tests, and pseudovirus neutralization assays. Preliminary identification of the binding epitopes of nanobodies by peptide-based ELISA and competition assay. Then four multivalent nanobodies were engineered by attaching the monovalent nanobodies to an antibody-binding nanoplatform constructed based on the lumazine synthase protein cage nanoparticles isolated from the Aquifex aeolicus (AaLS). Finally, the differences in potency between the monovalent and multivalent nanobodies were compared using the same methods. Results Three of the four specific nanobodies could maintain substantial inhibitory activity against the Omicron (B.1.1.529), of them, B-B2 had the best neutralizing activity against the Omicron (B.1.1.529) pseudovirus (IC 50 = 1.658 μg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC 50 = 0.653 μg/mL). The results of peptide-based ELISA indicated that LS-B-B2 might react with the linear epitopes in the SARS-CoV-2 RBD conserved regions, which would clarify the mechanisms for the maintenance of potent neutralization of Omicron (B.1.1.529) preliminary. Conclusion Our study indicated that the AaLS could be used as an antibody-binding nanoplatform to present nanobodies on its surface and improve the potency of nanobodies. The multivalent nanobody LS-B-B2 may serve as a potential agent for the neutralization of SARS-CoV-2 variants.

Asymmetric Assembly and Self-Adjuvanted Antigen Delivery Platform for Improved Antigen Uptake and Antitumor Effect

“…In this study, the BLS asymmetric assembly was used to equally display antigen peptides and CPPs on both sides of the BLS scaffold. The 60-mer LS is also a carrier protein of interest and could transport more “passenger” molecules . However, predictably, the random arrangement of large-sized proteins and small peptides on their surfaces may interfere with the function of the peptides.…”

“…The 60-mer LS is also a carrier protein of interest and could transport more "passenger" molecules. 29 However, predictably, the random arrangement of large-sized proteins and small peptides on their surfaces may interfere with the function of the peptides. The asymmetric assembly of BLS allows for flexible selection of the size or function of the displayed peptides/proteins, enabling rapid functional modification of the scaffold.…”

Plant virus‐derived nanoparticles decorated with genetically encoded SARS‐CoV‐2 nanobodies display enhanced neutralizing activity

“…Fourth, we analysed the capacity of our VNPs for virus neutralization and compared the multivalent nanoparticles with the benchmark dimeric format. Due to obvious restrictions inherent to working with infectious biosafety level 3 live SARS-CoV-2, we opted for a pseudovirus assay based on VSV pseudotyped with the SARS-CoV-2 glycoprotein S (Lu et al, 2021(Lu et al, , 2023. Purified VHH-expressing VNPs exhibited efficient pseudovirus neutralizing activity, with an IC 50 of approximately 0.3 lg/mL for both PVX-derived VNPs, and IC 50 of 0.4 and 1.5 lg/mL for the TEV-derived VNPs displaying VHH1 or VHH2, respectively.…”

Anti-Idiotypic Nanobody Alkaline Phosphatase Fusion Protein-Triggered On–Off–On Fluorescence Immunosensor for Aflatoxin in Cereals

“…12,13 Most importantly, the small size and single-domain conformation of Nbs make them more suitable for genetic engineering, 14 allowing direct fusion with signal probes and reducing the use of secondary antibodies with increasing sensitivity. Currently, various bifunctional nanobody fusions have been reported, including nanobodyalkaline phosphatase, 15,16 green fluorescent protein (GFP)nanobodies, 17 multivalent nanobodies 18,19 and biotinylated nanobodies. 20−22 In our previous study, anti-idiotypic nanobody Nb2−5 was developed from an immune phage-displayed nanobody library and used as an aflatoxin surrogate in immunoassays.…”

“…A number of anti-idiotypic antibodies have been developed to establish nontoxic immunoassays for mycotoxins. − Nanobodies (Nbs) are single-domain antibodies composed only of heavy chain variable regions, which can be easily produced by bacterial propagation with high yield, avoiding batch-to-batch variations. , Most importantly, the small size and single-domain conformation of Nbs make them more suitable for genetic engineering, allowing direct fusion with signal probes and reducing the use of secondary antibodies with increasing sensitivity. Currently, various bifunctional nanobody fusions have been reported, including nanobody-alkaline phosphatase, , green fluorescent protein (GFP)-nanobodies, multivalent nanobodies , and biotinylated nanobodies. − …”