Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

Highly synergistic combinations of nanobodies that target SARS-CoV-2 reduce viral load in hACE2 transgenic mice

The ongoing emergence of SARS-CoV-2 variants threatens current vaccines and renders current therapeutic antibodies obsolete, demanding powerful new treatments that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against past and present emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind dissimilar sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies. All mouse involved experiments were performed in compliance with the Institutional Animal Care and Use Committee and mice were housed and maintained in a specific pathogen-free conditions at Seattle Children's Research Institute. Infected mice with SARSCoV- 2 were housed in a Biosafety Level 3 facility in an Animal Biohazard Containment Suite. Prophylactic intranasal application of a synergistic pair of unmodified nanobodies in 10-12 week-old female K18-hACE2 transgenic mice, a mouse model of SARS-CoV-2 infection, showed significant reduction in viral load after 3 days post-challenge with SARS-CoV-2, the first demonstration of synergy in vivo. In summary, our results show that our diverse repertoire of nanobodies can neutralize current variants of live SARS-CoV-2, pairs of nanobodies that bind distinct sites on spike show tremendous synergy in neutralizing efficacy in vitro, and the application of synergizing pair of nanobodies translates to an in vivo mouse model of SARSCoV- 2. Research funded by the Mathers Foundation, Robertson Foundation, NIH P41GM109824.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Unbinding of hACE2 and inhibitors from the receptor binding domain of SARS-CoV-2 spike protein.

The first direful biomolecular event leading to COVID-19 disease is the SARS-CoV-2 virus surface spike (S) protein-mediated interaction with the human transmembrane protein, angiotensin-converting enzyme 2 (hACE2). Prevention of this interaction presents an attractive alternative to thwart SARS-CoV-2 replications. The development of monoclonal antibodies (mAbs) in the convalescent plasma treatment, nanobody, and designer peptides, which recognizes epitopes that overlap with hACE2 binding sites in the receptor-binding domain (RBD) of S protein (S/RBD) and thereby blocking the infection has been the center stage of therapeutic research. Here we report atomistic and reliable in silico structure-energetic features of the S/RBD interactions with hACE2 and its two inhibitors (convalescent mAb, B38, and an alpaca nanobody, Ty1). The discovered potential of mean forces exhibits free energy basin and barriers along the interaction pathways, providing sufficient molecular insights to design a B38 mutant and a Ty1-based peptide with higher binding capacity. While the mutated B38 forms a 60-fold deeper free energy minimum, the designer peptide (Ty1-based) constitutes 38 amino acids and is found to form a 100-fold deeper free energy minimum in the first binding basin than their wild-type variants in complex with S/RBD. Our strategy may help to design more efficacious biologics towards therapeutic intervention against the current raging pandemic.Communicated by Ramaswamy H. Sarma.

Plant production of high affinity nanobodies that block SARS-CoV-2 spike protein binding with its receptor, human angiotensin converting enzyme.

Nanobodies® (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb) derived from camelids. Here, we demonstrate production of VHH nanobodies against the SARS-CoV-2 spike proteins in the solanaceous plant Nicotiana benthamiana through transient expression and their subsequent detection verified through western blot. We demonstrate that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2. There has been significant interest and a number of publications on the use of plants as biofactories and even some reports of producing nanobodies in plants. Our data demonstrate that functional nanobodies blocking a process necessary to initiate SARS-CoV-2 infection into mammalian cells can be produced in plants. This opens the alternative of using plants in a scheme to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture.

Preparation and characterization of nanobodies targeting SARS-CoV-2 papain-like protease.

Coronavirus Papain-like protease (PLpro) mediates the cleavage of viral polyproteins and assists the virus escaping from innate immune response. Thus, PLpro is an attractive target for the development of broad-spectrum drugs as it has a conserved structure across different coronaviruses. In this study, we purified SARS-CoV-2 PLpro as an immune antigen, constructed a nanobody phage display library, and identified a set of nanobodies with high affinity for SARS-CoV-2. In addition, enzyme activity experiments demonstrated that two nanobodies had a significant inhibitory effect on the PLpro. These nanobodies should therefore be investigated as candidates for the treatment of coronaviruses.

In Silico Optimization of SARS-CoV-2 Spike Specific Nanobodies.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide, caused a global pandemic, and killed millions of people. The spike protein embedded in the viral membrane is essential for recognizing human receptors and invading host cells. Many nanobodies have been designed to block the interaction between spike and other proteins. However, the constantly emerging viral variants limit the effectiveness of these therapeutic nanobodies. Therefore, it is necessary to find a prospective antibody designing and optimization approach to deal with existing or future viral variants. METHODS: We attempted to optimize nanobody sequences based on the understanding of molecular details by using computational approaches. First, we employed a coarse-grained (CG) model to learn the energetic mechanism of the spike protein activation. Next, we analyzed the binding modes of several representative nanobodies with the spike protein and identified the key residues on their interfaces. Then, we performed saturated mutagenesis of these key residue sites and employed the CG model to calculate the binding energies. RESULTS: Based on analysis of the folding energy of the angiotensin-converting enzyme 2 (ACE2) -spike complex, we constructed a detailed free energy profile of the activation process of the spike protein which provided a clear mechanistic explanation. In addition, by analyzing the results of binding free energy changes following mutations, we determined how the mutations can improve the complementarity with the nanobodies on spike protein. Then we chose 7KSG nanobody as a template for further optimization and designed four potent nanobodies. Finally, based on the results of the single-site saturated mutagenesis in complementarity determining regions (CDRs), combinations of mutations were performed. We designed four novel, potent nanobodies, all exhibiting higher binding affinity to the spike protein than the original ones. CONCLUSIONS: These results provide a molecular basis for the interactions between spike protein and antibodies and promote the development of new specific neutralizing nanobodies.

Preventing SARS-CoV-2 Infection Using Anti-spike Nanobody-IFN-ß Conjugated Exosomes.

PURPOSE: To inhibit the transmission of SARS-CoV-2, we developed engineered exosomes that were conjugated with anti-spike nanobodies and type I interferon ß (IFN-ß). We evaluated the efficacy and potency of nanobody-IFN-ß conjugated exosomes to treatment of SARS-CoV-2 infection. METHODS: Milk fat globule epidermal growth factor 8 (MFG-E8) is a glycoprotein that binds to phosphatidylserine (PS) exposed on the exosomes. We generated nanobody-IFN-ß conjugated exosomes by fusing an anti-spike nanobody and IFN-ß with MFG-E8. We used the SARS-CoV-2 pseudovirus with the spike of the D614G mutant that encodes ZsGreen to mimic the infection process of the SARS-CoV-2. The SARS-CoV-2 pseudovirus was infected with angiotensin-converting enzyme-2 (ACE2) expressing adenocarcinomic human alveolar basal epithelial cells (A549) or ACE2 expressing HEK-blue IFNα/ß cells in the presence of nanobody-IFN-ß conjugated exosomes. By assessing the expression of ZsGreen in target cells and the upregulation of interferon-stimulated genes (ISGs) in infected cells, we evaluated the anti-viral effects of nanobody-IFN-ß conjugated exosomes. RESULTS: We confirmed the anti-spike nanobody and IFN-ß expressions on the exosomes. Exosomes conjugated with nanobody-hIFN-ß inhibited the interaction between the spike protein and ACE2, thereby inhibiting the infection of host cells with SARS-CoV-2 pseudovirus. At the same time, IFN-ß was selectively delivered to SARS-CoV-2 infected cells, resulting in the upregulation of ISGs expression. CONCLUSION: Exosomes conjugated with nanobody-IFN-ß may provide potential benefits in the treatment of COVID-19 because of the cooperative anti-viral effects of the anti-spike nanobody and the IFN-ß.

Single-Domain Antibody for Binding the Conserved Epitope in the Sars-Cov-2 Spike Protein Receptor-Binding Domain

Several COVID-19 vaccines have been developed in the last three years using various tecnhiques. Multiple virus-neutralizing antibodies against SARS-CoV-2 have been also obtained to combat the pandemic. However, the use of these medications for prevention and potential treatment faces significant challenges due to the emergence of new mutant virus variants, both more contagious and escaping neutralization by the immune system, that is why it is necessary to continuously renew the vaccines and develop new therapeutic antibodies. The study was aimed to use the technology of generating single-domain antibodies (nanobodies) to target the surface spike (S) protein RBD conserved epitope of the broad spectrum of SARS-CoV-2 variants. Recombinant proteins that corresponded to RBDs of three important SARS-SoV-2 strains and the full-length S protein (Wuhan) were used as antigens for immunization of a camel in order to induce production of appropriate antibodies and/or as immobilized proteins for further cross selection of the nanobody clones with pre-set specificity by the phage display. A nanobody capable of effectively recognizing the conservative region in the S protein RBDs of the broad spectrum of pandemic SARS-CoV-2 variants, including Omicron, was selected from the generated immune library. Along with conventional use in immunoassays and diagnosis, the generated nanobody can be potentially used as a module for target-specific binding used to trap coronavirus in human upper airways during the development of novel combination antiviral drugs.Copyright © 2023 Pirogov Russian National Research Medical University. All rights reserved.

A Competitive Panning Method Reveals an Anti-SARS-CoV-2 Nanobody Specific for an RBD-ACE2 Binding Site.

Most neutralizing antibodies neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by directly blocking the interactions between the spike glycoprotein receptor-binding domain (RBD) and its receptor, human angiotensin-converting enzyme 2 (ACE2). Here, we report a novel nanobody (Nb) identified by an RBD-ACE2 competitive panning method that could specifically bind to the RBD of SARS-CoV-2 with a high affinity (EC50 = 0.03 nM) and successfully block the binding between the RBD and ACE2 recombinant protein. A structural simulation of the RBD-VHH complex also supports a mechanism of the Nb to block the interaction between the RBD and ACE2. A pseudovirus assay of the Nb showed it could neutralize the WT pseudovirus with high potency (IC50 = 0.026 µg/mL). Furthermore, we measured its binding to phages displaying RBDs of different SARS-CoV-2 variants and found that it could bind to recombinant phages displaying the RBD of beta and delta variants. This study also provides a method of phage library competitive panning, which could be useful for directly screening high-affinity antibodies targeting important functional regions.

Efficient virus detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis.

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to pandemic threats in the form of vaccines and antigen tests. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive, biogenic materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. Recent work has demonstrated that nanobodies are suitable target proteins. These proteins represent a very versatile alternative antibody format and can quickly be adapted to detect novel antigens by camelidae immunization or synthetic libraries. In this study, we exemplarily produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the spike protein receptor binding domain (RBD) as Cts1 fusions and screened their antigen binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of inexpensive antigen tests utilizing unconventionally secreted nanobodies as antigen trap and a matching ubiquitous and biogenic surface for immobilization.

General Trends of the Camelidae Antibody VHHs Domain Dynamics.

Conformational flexibility plays an essential role in antibodies' functional and structural stability. They facilitate and determine the strength of antigen-antibody interactions. Camelidae express an interesting subtype of single-chain antibody, named Heavy Chain only Antibody. They have only one N-terminal Variable domain (VHH) per chain, composed of Frameworks (FRs) and Complementarity Determining regions (CDRs) like their VH and VL counterparts in IgG. Even when expressed independently, VHH domains display excellent solubility and (thermo)stability, which helps them to retain their impressive interaction capabilities. Sequence and structural features of VHH domains contributing to these abilities have already been studied compared to classical antibodies. To have the broadest view and understand the changes in dynamics of these macromolecules, large-scale molecular dynamics simulations for a large number of non-redundant VHH structures have been performed for the first time. This analysis reveals the most prevalent movements in these domains. It reveals the four main classes of VHHs dynamics. Diverse local changes were observed in CDRs with various intensities. Similarly, different types of constraints were observed in CDRs, while FRs close to CDRs were sometimes primarily impacted. This study sheds light on the changes in flexibility in different regions of VHH that may impact their in silico design.

A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System.

Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an Escherichia coli protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a Kd of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.

Sensitivity-enhanced nanoplasmonic biosensor using direct immobilization of two engineered nanobodies for SARS-CoV-2 spike receptor-binding domain detection.

Sensitive, rapid, and easy-to-implement biosensors are critical in responding to highly contagious and fast-spreading severe acute respiratory syndrome coronavirus (SARS-CoV-2) mutations, enabling early infection screening for appropriate isolation and treatment measures to prevent the spread of the virus. Based on the sensing principle of localized surface plasmon resonance (LSPR) and nanobody immunological techniques, an enhanced sensitivity nanoplasmonic biosensor was developed to quantify the SARS-CoV-2 spike receptor-binding domain (RBD) in serum within 30 min. The lowest concentration in the linear range can be detected down to 0.01 ng/mL by direct immobilization of two engineered nanobodies. Both the sensor fabrication process and immune strategy are facile and inexpensive, with the potential for large-scale application. The designed nanoplasmonic biosensor achieved excellent specificity and sensitivity for SARS-CoV-2 spike RBD, providing a potential option for accurate early screening of the novel coronavirus disease 2019 (COVID-19).

Synthetic nanobodies as tools to distinguish IgG Fc glycoforms.

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

[SARS-CoV-2 neutralizing monoclonal antibodies and nanobodies: a review].

Coronavirus disease (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), with strong contagiousness, high susceptibility and long incubation period. cell entry by SARS-CoV-2 requires the binding between the receptor-binding domain of the viral spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). Here, we briefly reviewed the mechanisms underlying the interaction between SARS-CoV-2 and ACE2, and summarized the latest research progress on SARS-CoV-2 neutralizing monoclonal antibodies and nanobodies, so as to better understand the development process and drug research direction of COVID-19. This review may facilitate understanding the development of neutralizing antibody drugs for emerging infectious diseases, especially for COVID-19.

Applications of nanobodies in the prevention, detection, and treatment of the evolving SARS-CoV-2.

Global health and economy are deeply influenced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its newly emerging variants. Nanobodies with nanometer-scale size are promising for the detection and treatment of SARS-CoV-2 and its variants because they are superior to conventional antibodies in terms of cryptic epitope accessibility, tissue penetration, cost, formatting adaptability, and especially protein stability, which enables their aerosolized specific delivery to lung tissues. This review summarizes the progress in the prevention, detection, and treatment of SARS-CoV-2 using nanobodies, as well as strategies to combat the evolving SARS-CoV-2 variants. Generally, highly efficient generation of potent broad-spectrum nanobodies targeting conserved epitopes or further construction of multivalent formats targeting non-overlapping epitopes can promote neutralizing activity against SARS-CoV-2 variants and suppress immune escape.

Simple Affinity-Based Method for Concentrating Viruses from Wastewater Using Engineered Curli Fibers.

Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.

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

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 (IC50 = 1.658 µg/mL). The antiviral ability of multivalent nanobody LS-B-B2 was improved in the Omicron (B.1.1.529) pseudovirus assays (IC50 = 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.

Nanobody derived using a peptide epitope from the Spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants.

The emergence of new escape mutants of the SARS-CoV-2 virus has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS-CoV variants and other viruses in a short period of time becomes essential. Blocking SARS-CoV-2 entry into human host cells by disrupting the Spike glycoprotein-angiotensin-converting enzyme 2 (ACE2) interaction has already been exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a nine-amino acid peptide from the receptor-binding motif (RBM) of the Spike (S) protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus-containing SARS-CoV-2 Spike as the surface glycoprotein. Moreover, using mCherry fluorescence based reporter assay we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped Spike virus with a bivalent version of the N1.2 nanobody. In summary, our study presents a rapid, and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. We propose that this approach can also be widely extended to target other viruses and pathogens in the future.