A nanobody-based blocking enzyme-linked immunosorbent assay for detecting antibodies against pseudorabies virus glycoprotein E

Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.

Two nanobody‑based immunoassays to differentiate antibodies against genotype 1 and 2 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus(PRRSV)infection causes significant economic loss to the global pig industry.Genotype 1 and 2 PRRSV(PRRSV-1 and-2)infections have been reported in China,Europe and America.For accurate prevention,nanobodies were first used as diagnostic reagents for PRRSV typing.In this study three nanobodies targeting both PRRSV-1 and-2,two targeting PRRSV-1 and three targeting PRRSV-2,were screened and produced.To develop two competitive ELISAs(cELISAs),the g1-2-PRRSV-Nb3-HRP nanobody was chosen for the g1-2-cELISA,to detect common antibodies against PRRSV-1 and-2,and the g1-PRRSV-Nb136-HRP nanobody was chosen for the g1-cELISA,to detect anti-PRRSV-1 antibodies.The two cELISAs were developed using PRRSV-1-N protein as coating antigen,and the amounts for both were 100 ng/well.The optimized dilution of testing pig sera was 1:20,the optimized reaction times were 30 min,and the colorimetric reaction times were 15 min.Then,the cut-off values of the g1-2-cELISA and g1-cELISA were 26.6%and 35.6%,respectively.Both of them have high sensitivity,strong specificity,good repeatability,and stability.In addition,for the 1534 clinical pig sera,an agreement rate of 99.02%(Kappa values=0.97)was determined between the g1-2-cELISA and the commercial IDEXX ELISA kit.For the g1-cELSIA,it can specifically detect anti-PRRSV-1 antibodies in the clinical pig sera.Importantly,combining two nanobody-based cELISAs can differentially detect antibodies against PRRSV-1 and-2.

Quantification of soluble epoxide hydrolase inhibitors in experimental and clinical samples using the nanobody-based ELISA

To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.

In Silico Evaluation of the Potential Interference of Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376 in the Binding of SARS-CoV-2 Spike Protein to Human Nanobody Nb20

Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (Mpro) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 Mpro: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo

Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

Nanobody-based immunosensing methods for safeguarding public health

Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public health.Taking advantage of antibody-related biotechnological advances,the utilization of an antigen-binding fragment of a heavy-chain-only antibody termed as'nanobody'holds significant biomedical potential.Compared with the conventional full-length antibody,a single-domain nanobody retaining cognate antigen specificity possesses remarkable physicochemical stability and structural adaptability,which enables a flexible and efficient molecular design of the immunosensing strategy.This minireview aims to summarize the recent progress in immunosensing methods using nanobody targeting tumor markers,environmental pollutants,and foodborne microbes.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

Nanobody cDNA Mock-Up in pHEN6c Plasmid Vector: Live Out

Whenever there is no adequate DNA replication in vitro, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, E. coli, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured E. coli sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from E. coli by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.

Tannic acid assisted anti-TNF-αnanobody assembly modulating the epithelial barrier dysregulation of allergic rhinitis

The derailed nasal epithelial barrier is associated with the disorder of tight junctions(TJ)function or expression,leading to more penetration of allergens to the barrier,accompanied by the release of cytokines,which develop allergic rhinitis(AR).Considering the increasing AR disease incidence worldwide,there is still an urgent unmet medical need to develop new therapeutics.Tumor necrosis factor-alpha(TNF-α)inhibitors have been applied in treating autoimmune diseases.However,their roles in AR remain unclear.In this study,anti-TNF-αnanobody(V)was assembled with tannic acid(V/TA)as a functional antibody drug candidate which could inhibit the release of the cytokines in ovalbumin(OVA)-induced AR murine model.Upon receiving V/TA treatment,the infiltration level of inflammatory cells,and the number of mucus-secreting cells and mast cells in the nasal mucosa recovered to a relatively normal level.Preliminary mechanism of action research revealed that the efficacy of V/TA was accompanied by the restricted level of TJ molecules:zonula occluden-1(ZO-1),occludin,claudin-1,and claudin-5.The therapeutic effect of the anti-TNF-αnanobody against AR was enhanced with the tannic acid assisted without any toxicity observed.This study supplied a promising delivery strategy of TNF-αinhibitor for the effective treatment of complicated allergic rhinitis disease,with an advantage in restoring effect on the AR-caused epithelial barrier defects than the commercial drug Infliximab.

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds.Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase(nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA(cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7%by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.

Nanobody-based polyvinyl alcohol beads as antifouling adsorbents for selective removal of tumor necrosis factor- α

Highly efficient removal of tumor necrosis factor-α(TNF-α)from plasma by hemoperfusion for autoim-mune disease therapy remains a challenge in the clinical field owing to the low adsorption capacity and poor blood compatibility of adsorbents.In this work,a new class of nanobody(Nb)-coupled antifouling polyvinyl alcohol(PVA)beads was constructed as an immunosorbent for the selective removal of TNF-αfrom plasma.Notably,our immunosorbent exhibited an exceptionally high specific TNF-αadsorption ca-pacity of 416.9 ng/g in human plasma(at a plasma-to-adsorbent ratio of 300).More importantly,the ob-tained adsorbent beads showed outstanding blood compatibility.In addition,during in vivo experiments,the blood circulation device was constructed to remove TNF-αin rat models,proving that the beads had good removal performance(∼85%/60 min).Furthermore,95%of the original capacity was retained after 6-month storage,showed strong stability and prolonged storage of PVA-Nb.Above all,the results indi-cate that the novel PVA-Nb immunosorbent has possible clinical applications for treating autoimmune diseases in the clinic.

Fast screening of nanobody and AIV detection

Nanobody is the variable region of heavy chain antibody, with obvious characteristics such as small size, good stability and solubility. Many useful nanobodies have been isolated by

Generation and Characterization of a Nanobody Against SARS-CoV

The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available;thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain(RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2(ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

Selection of specific nanobodies against peanut allergen through unbiased immunization strategy and the developed immuno-assay

Peanut allergy is considered to be a major health issue with global effects.To date,no effective curative approach has been applied for the therapy of the anaphylaxis resulting from the peanut allergens.The accurate and effective detection methods for the surveillance of allergens in food are still the primary strategies to avoid allergic diseases.In this study,nanobodies(Nbs)derived from the Heavy-Chain only Antibodies(HCAbs)were selected against the general peanut protein extract through the unbiased strategy to facilitate the development of the sandwich ELISA for the detection and surveillance of peanut allergen contamination.The target antigen of the selected Nb was identified as peanut allergen Ara h 3,and a cross-reaction was observed with the member of Gly 1 from the Ara h 3 family.The applicability of the self-paired Nb P43 on the establishment of the immuno-assay was verified.A sandwich ELISA against peanut allergen was developed,which reached a linear range of 0.2-10.6μg/mL,and a limit of detection of 53.13 ng/mL.

Isolation and optimization of camelid single-domain antibodies:Dirk Saerens'work on nanobodies

It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain homodimer.These so-called heavy-chain antibodies(HCAbs)are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol.These HCAbs are easily purified from serum,and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies.The antigen binding site of the dromedary HCAb comprises one single domain,referred to as VHH or nanobody(Nb),therefore,a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens.The monoclonal Nb is produced well in bacteria,is very stable and highly soluble,and it binds the antigen with high affinity and specificity.Currently,the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors,to diagnose infections,or to treat diseases such as cancer or trypanosomiasis.

Molecular Cloning, Expression and Purification of Recombinant VHH Proteins Expressed in <i>E. coli</i>

Variable Heavy-chain Homodimer (VHH) or Nanobody is a recombinant dromedary antibody fragment which is classified as the smallest antibody fragments with the highest binding affinity and specificity of the original whole antibody. In this study the Expression of Nanobodies in E. coli WK6 cell periplasm was performed. The protein expression and purity was and analyzed by Affinity Chromatography, SDS PAGE and Western Blot. Upon elution with Imidazole, the concentrations observed using the OD280 nm of the eluted fractions EI, E2 and E3 were observed to be 0.42 μg/ml, 0.13 μg/ml and −0.46 μg/ml respectively. This gives an Antilog of 7.88 kDa which showed the calculated molecular size of our band. The SDS-PAGE gel reading was confirmed using Western blot analysis and illustrated as the specific binding of the mouse Anti-His antibody to the Histidine tag of the Nanobody. The Nanobody protein expression was then analyzed further with western blotting showed a strong signal at the region corresponding to the 15 kDa marker indicating presence of the Nanobody gene. This was taken as further confirmation of the protein expression from the bacterial cells.

EGFR-targeted and gemcitabine-incorporated chemogene for combinatorial pancreatic cancer treatment

Pancreatic cancer stands out as a recognized intractable tumor due to its high malignancy and mortality rates,which are largely attributed to the insensitivity of current clinical chemotherapies or multidrug-resistance.Combinatorial chemo and gene therapy that integrates different therapeutic targets,may increase the chemosensitivity of pancreatic cancer and synergistically enhance the antitumor efficacy.However,conventional co-delivery of gene and chemo drugs is intensively dependent on complex nanoparticle delivery systems,thus would be limited by unstable drug packaging,nonspecific biodistribution,and biosafety problem.Herein,we rationally designed an epidermal growth factor-receptor(EGFR)-targeted and gemcitabine-incorporated oligonucleotide(termed as chemogene)with anti-Bcl-2 sequence,which achieves simple and precise integration of gemcitabine into a gene regulative agent,as well as the EGFR-targeted delivery for pancreatic cancer therapy.Through solid-phase synthesis,gemcitabine,as the first-line chemodrug for pancreatic cancer,is introduced to the antisense oligonucleotide to replace all cytosine nucleosides to obtain the gemcitabine-integrated chemogene(Ge-ASO^(Bcl-2)).Thereafter,Ge-ASO^(Bcl-2)is covalently coupled with EGFR nanobody to construct the final targeted chemogene without any exogenous carriers.Notably,this nanobody-conjugated chemogene exhibits remarkable tumor targeting capability and antitumor effects both in vitro and in vivo,which initiates a first step toward the application of combinatorial chemo and gene therapy for future pancreatic cancer treatment.

Single-domain antibodies as therapeutics for solid tumor treatment

Single-domain antibodies(sdAbs),initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs,have emerged as a promising alternative to conventional therapeutic antibodies.These sdAbs have many superior physicochemical and pharmacological properties,including small size,good solubility and thermostability,easier accessible epitopes,and strong tissue penetration.However,the inherent challenges associated with the animal origin of sdAbs limit their clinical use.In recent years,various innovative humanization technologies,including complementarity-determining region(CDR)grafting or complete engineering of fully human sdAbs,have been developed to mitigate potential immunogenicity issues and enhance their compatibility.This review provides a comprehensive exploration of sdAbs,emphasizing their distinctive features and the progress in humanization methodologies.In addition,we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment,such as sdAbedrug conjugates,multispecific sdAbs,sdAb-based delivery systems,and sdAb-based cell therapy.

Characterization of heavy-chain antibody gene repertoires in Bactrian camels

Camelids are the only mammals that can produce functional heavy-chain antibodies(HCAbs).Although HCAbs were discovered over 30 years ago,the antibody gene repertoire of Bactrian camels remains largely underexplored.To characterize the diversity of variable genes of HCAbs(VHHs),germline and rearranged VHH repertoires are constructed.Phylogenetics analysis shows that all camelid VHH genes are derived from a common ancestor and the nucleotide diversity of VHHs is similar across all camelid species.While species-specific hallmark sites are identified,the non-canonical cysteines specific to VHHs are distinct in Bactrian camels and dromedaries compared with alpacas.Though low divergence at the germline repertoire between wild and domestic Bactrian camels,higher expression of VHHs is observed in some wild Bactrian camels than that of domestic ones.This study not only adds our understanding of VHH repertoire diversity across camelids,but also provides useful resources for HCAb engineering.

Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models

The weakened protective efficacy of COVID-19 vaccines and antibodies caused by SARS-CoV-2 variants presents a global health emergency,which underscores the urgent need for universal therapeutic antibody intervention for clinical patients.Here,we screened three alpacas-derived nanobodies(Nbs)with neutralizing activity from twenty RBD-specific Nbs.The three Nbs were fused with the Fc domain of human IgG,namely aVHH-11-Fc,aVHH-13-Fc and aVHH-14-Fc,which could specifically bind RBD protein and competitively inhibit the binding of ACE2 receptor to RBD.They effectively neutralized SARS-CoV-2 pseudoviruses D614G,Alpha,Beta,Gamma,Delta,and Omicron sub-lineages BA.1,BA.2,BA.4,and BA.5 and authentic SARS-CoV-2 prototype,Delta,and Omicron BA.1,BA.2 strains.In mice-adapted COVID-19 severe model,intranasal administration of aVHH-11-Fc,aVHH-13-Fc and aVHH-14-Fc effectively protected mice from lethal challenges and reduced viral loads in both the upper and lower respiratory tracts.In the COVID-19 mild model,aVHH-13-Fc,which represents the optimal neutralizing activity among the above three Nbs,effectively protected hamsters from the challenge of SARS-CoV-2 prototype,Delta,Omicron BA.1 and BA.2 by significantly reducing viral replication and pathological alterations in the lungs.In structural modeling of aVHH-13 and RBD,aVHH-13 binds to the receptor-binding motif region of RBD and interacts with some highly conserved epitopes.Taken together,our study illustrated that alpaca-derived Nbs offered a therapeutic countermeasure against SARS-CoV-2,including those Delta and Omicron variants which have evolved into global pandemic strains.