ArticleLiterature Review

Nanobodies: Natural Single-Domain Antibodies

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Abstract

Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine. Expected final online publication date for the Annual Review of Biochemistry Volume 82 is June 02, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

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... Additionally, single variable domain on a heavy chain (VHH), also called nanobodies, have also been employed as the targeting domain of CARs [9]. VHH are derived from the variable domain of heavy chain-only antibodies (HcAbs), produced by the Camelidae family and sharks naturally [10,11]. Compared to conventional scFv, VHH Fig. 1 The development processes of five generations of CARs The first generation only had the intracellular CD3-ζ signal molecule, which are phosphorylated via SRC tyrosine kinase family. ...
... The activation of JAK-STAT, deriving from IL-2Rβ and incorporated between CD28/4-1BB and CD3-ζ, stimulates cell proliferation. (By Figdraw) recognize and bind target antigens with similar binding ability and specificity, as well as the solubility and stability [10]. Even if VL and constant domains are absent, VHH can still possess these characteristics [10]. ...
... (By Figdraw) recognize and bind target antigens with similar binding ability and specificity, as well as the solubility and stability [10]. Even if VL and constant domains are absent, VHH can still possess these characteristics [10]. Therefore, VHH-based targeting CAR-T cells could be as effective as traditional CAR-T cells with scFv-based targeting domains. ...
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Chimeric antigen receptor T (CAR-T) cells therapy has revolutionized the treatment paradigms for hematological malignancies, with multi-line therapy-refractory patients achieving durable complete remissions (CR) and relatively high objective response rate (ORR). So far, many CAR-T products, such as Kymriah, Yescarta and Tecartus, have been developed and got the unprecedented results. However, some patients may relapse afterwards, driving intense investigations into promoting the development of novel strategies to overcome resistance and mechanisms of relapse. Notable technical progress, such as nanobodies and CRISPR-Case9, has also taken place to ensure CAR-T cell therapy fully satisfies its medical potential. In this review, we outline the basic principles for the development and manufacturing processes of CAR-T cell therapy, summarize the similarities and differences in efficacy of different products as well as their corresponding clinical results, and discuss CAR-T immunotherapy combined with other clinical effects of drug therapy.
... There are extensive related review articles about the structural characterization, physicochemical properties and different application fields of sdAbs. Muyldermans et al. (16) and Juma et al. (17) reviewed the typical structures of HCAbsderived and IgNAR-derived sdAbs and their corresponding heavy chain antibodies detailly. Goldman et al. (18) reviewed the strategies to improve the stability of sdAbs, which showed that the excellent performance of sdAbs enables them to have development potential in many fields. ...
... The gene encoding VHH domain length is about 360 bp, which allows expanded functionality through the creation of modularity via genetic fusions to a wide array of proteins, like the creation of multispecific antibody fusions (23,24). VHH domains comprise 9 bstrands, one 4-stranded b-sheet and another 5-stranded b-sheet, connected by a conserved disulfide bond between Cys residue at position 23 (23Cys) and Cys residue at position 94 (94Cys) to stabilize the structure, packed against a conserved Trp residue (16). An additional disulfide bond connects the CDR3 loop and CDR1 in camels or CDR2 in llamas, resulting in a more constrained conformation (25). ...
... Markedly different from the paratopes of traditional antibodies, the prolate "rugby ball shaped" paratope structure of sdAbs forms a distinctly convex surface, increasing the contact frequency of epitopes, making it highly suitable to bind the rigid, concave, clefts, cavities, and restricted epitopes and can access the hydrophobic core of epitope enriched with aromatic residues (60). This may be why sdAbs showed equivalent or higher binding affinities as conventional Abs with other excellent antibody properties (16). Recently, a study reported a "best-fit ellipsoidal model" to geometrically simulate and quantify the spatial situation of the sdAbs paratope ( Figures 1D, E). ...
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Single-domain antibodies (sdAbs) showed the incredible advantages of small molecular weight, excellent affinity, specificity, and stability compared with traditional IgG antibodies, so their potential in binding hidden antigen epitopes and hazard detection in food, agricultural and veterinary fields were gradually explored. Moreover, its low immunogenicity, easy-to-carry target drugs, and penetration of the blood-brain barrier have made sdAbs remarkable achievements in medical treatment, toxin neutralization, and medical imaging. With the continuous development and maturity of modern molecular biology, protein analysis software and database with different algorithms, and next-generation sequencing technology, the unique paratope structure and different antigen binding modes of sdAbs compared with traditional IgG antibodies have aroused the broad interests of researchers with the increased related studies. However, the corresponding related summaries are lacking and needed. Different antigens, especially hapten antigens, show distinct binding modes with sdAbs. So, in this paper, the unique paratope structure of sdAbs, different antigen binding cases, and the current maturation strategy of sdAbs were classified and summarized. We hope this review lays a theoretical foundation to elucidate the antigen-binding mechanism of sdAbs and broaden the further application of sdAbs.
... In 1993, Hamers-Casterman et al. found a natural heavy chain antibody (HCAb) in camels containing only g heavy chain and missing the first domain of its constant region (CH1) ( Figure 1) (2). Subsequent studies have found that this HCAb is common in all camelids (3). The variable region of the HCAb that retains antigen-binding activity, namely the variable domain of heavy chain of heavy-chain antibody (VHH), has a molecular weight of only about 15 kDa (1/10 of that of conventional antibodies) and a size in the nanometer scale, while it retains an antigen binding affinity that is often in the low nanomolar range. ...
... In addition to immune libraries, natural libraries (12) and synthetic/semi-synthetic libraries (13) have also been proposed for generation of Nbs. These libraries are not dependent on immune responses in experimental animals, but usually, the content of the libraries needs to be much higher than that of immune libraries, especially for natural libraries (~10 9 individual clones) (3). In addition to phage display, the methods for screening the required Nbs from libraries also include cell surface display (14), ribosome display (15), mRNA/cDNA display (16), and high-throughput DNA sequencing and mass spectrometric identification (17), etc. ...
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Nanobodies are antibody fragments derived from camelids, naturally endowed with properties like low molecular weight, high affinity and low immunogenicity, which contribute to their effective use as research tools, but also as diagnostic and therapeutic agents in a wide range of diseases, including brain diseases. Also, with the success of Caplacizumab, the first approved nanobody drug which was established as a first-in-class medication to treat acquired thrombotic thrombocytopenic purpura, nanobody-based therapy has received increasing attention. In the current review, we first briefly introduce the characterization and manufacturing of nanobodies. Then, we discuss the issue of crossing of the brain-blood-barrier (BBB) by nanobodies, making use of natural methods of BBB penetration, including passive diffusion, active efflux carriers (ATP-binding cassette transporters), carrier-mediated influx via solute carriers and transcytosis (including receptor-mediated transport, and adsorptive mediated transport) as well as various physical and chemical methods or even more complicated methods such as genetic methods via viral vectors to deliver nanobodies to the brain. Next, we give an extensive overview of research, diagnostic and therapeutic applications of nanobodies in brain-related diseases, with emphasis on Alzheimer’s disease, Parkinson’s disease, and brain tumors. Thanks to the advance of nanobody engineering and modification technologies, nanobodies can be linked to toxins or conjugated with radionuclides, photosensitizers and nanoparticles, according to different requirements. Finally, we provide several perspectives that may facilitate future studies and whereby the versatile nanobodies offer promising perspectives for advancing our knowledge about brain disorders, as well as hopefully yielding diagnostic and therapeutic solutions.
... However, there are some notable structural differences. The framework 2 region of a VHH contains smaller, more hydrophilic amino acids at positions 37, 44, 45, and 47 that are thought to increase the VHH solubility [26]. Also, VHHs tend to have longer CDRH3 lengths (17 residues on average) compared to human VH domains (12 residues on average) [27]. ...
... Furthermore, VHH5 also contains a buried disulfide bond that anchors CDHR3 to CDRH2 and positions Trp98 to pack against CD8 (Fig. 2A). Although this extra pair of Cys residues in the CDR regions is rare for llama-derived VHHs, other camelid species have a higher prevalence of interloop disulfide bonds [26]. Lastly, we wondered whether our crystal structure could help inform how A99G and A100fD improve the affinity of VHH5 (Fig. 3D). ...
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Purpose Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8⁺ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8⁺ cells could provide a critical biomarker for new CIT agents. However, existing ⁸⁹Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. Methods To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an ¹⁸F radiolabel to the VHH. Results The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. Conclusion Our work reveals the potential of this anti-human CD8 VHH [¹⁸F]F-VHH5v2 to enable rapid and specific imaging of CD8⁺ cells in the clinic.
... While HCAbs have a molecular mass of 95 kDa, its variable antigen-binding domain, referred as VHH or Nanobody ® , is generally functional as a single domain despite their small size of only 15 kDa. 2,3 VHHs have typically similar structural characteristics to those of human VH domains, consisting of four framework regions (FR1/2/3/4) surrounding three complementarity determining regions (CDR1/2/3). 3,4 Compared with conventional murine or human antibodies, VHHs contain CDR3 loops 3-4 residues longer on average, which are also more divergent in both sequence and structure, and occupying a greater range of positions relative to the framework. ...
... 6,8,10,11 VHHs exhibited favorable biochemical characteristics, such as high affinity and specificity ($nM range), high solubility ($10 mg/ml), long shelf life at 4 C ($months), and high expression level in Escherichia coli. 2,12 Some VHHs display an exceptionally stable behavior, resisting temperatures above 90 C. 13 Based on these characteristics, VHHs are an attractive alternative to antigenbinding fragments from conventional antibodies such as Fabs and scFvs, in biotechnological, diagnostic, and therapeutic applications. 4,9,14,15 Indeed, several VHH-based antibody-drugs have been developed in clinical trials for human therapy recently. ...
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The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework‐2 region. Subsequently, we performed back‐mutational analyses on the chimeric VHH. Back‐mutations resulted in an increase of the thermal stability and affinity. These data suggested that back‐mutations restored the intramolecular interactions, and proper positioning and / or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.
... As a novel type of antibody fragment, nanobody has attracted extensive interest and is expected to be applied as a potential tool in the research and biomedical fields in the past two decades (Hamers-Casterman et al., 1993). Compared with conventional antibodies, nanobodies have been shown to possess many striking properties, including high affinity and specificity, small molecular size, economical and easy production, deep tissue penetration and recognition of hidden epitopes (Muyldermans, 2013;Salvador et al., 2019). Due to the beneficial properties, an increasing number of nanobodies have been explored and used in a wide range of routine and innovative applications for diagnostics and therapeutics (Salvador et al., 2019). ...
... After three rounds of bio-panning, the recombinant phage particles were enriched obviously, and all selected clones were positive to target protein. These results suggested that the panning process conducted herein is highly efficient and three rounds of panning could sufficiently enrich the recombinant clones containing specific VHHs (Muyldermans, 2013). Ultimately, nine distinct nanobodies specific for p72 protein were retrieved and identified according to their diverse amino acid sequences of CDRs, which are responsible for the antigen binding specificity. ...
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African swine fever virus (ASFV) is a large and very complex DNA virus. The major capsid protein p72 is the most predominant structural protein and constitutes the outmost icosahedral capsid of the virion. In the present study, the nanobodies against ASFV p72 protein were screened from a camelid immune VHH library by phage display technique. Nine distinct nanobodies were identified according to the amino acid sequences of the complementary determining regions (CDRs), and contain typical amino acid substitutions in the framework region 2 (FR2). Six nanobodies were successfully expressed in E . coli , and their specificity and affinity to p72 protein were further evaluated. The results showed that nanobodies Nb25 had the best affinity to both recombinant and native p72 protein of ASFV. The Nb25 possesses an extremely long CDR3 with 23 amino acids compared with other nanobodies, which may allow this nanobody to access the hidden epitopes of target antigen. Furthermore, the Nb25 can specifically recognize the virus particles captured by polyclonal antibody against ASFV in a sandwich immunoassay, and its application as a biosensor to target virus in PAM cells was verified by an immunofluorescence assay. Nanobodies have been proven to possess many favorable properties with small size, high affinity and specificity, easier to produce, low costs and deep tissue penetration that make them suitable for various biotechnological applications. These findings suggest that nanobody Nb25 identified herein could be a valuable alternative tool and has potential applications in diagnostic and basic research on ASFV.
... The use of neutralizing antibodies for the treatment of a variety of diseases is fairly common. For example, "Bezlotoxumab" is based on monoclonal antibodies against Clostridium difficile toxin B (Muyldermans, 2013;Navalkele and Chopra, 2018). However, antibody-based therapeutics are used mainly for treatment, but not for the prevention of infectious diseases, due to limited time of circulation in the body (Lu et al., 2020). ...
... Although there is evidence that VHH is non-immunogenic (Hassanzadeh-Ghassabeh et al., 2013;Muyldermans, 2013), the immunogenicity of a recombinant VHH-Fc antibody expressed via rAAV transduction had to be investigated. Our results demonstrate that anti-B11-Fc antibodies can be detected in the serum samples of rAAV-B11-Fc-transduced mice in a small quantity, with a peak concentration of 1/50 on day 60 ( Figure 5). ...
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Botulinum neurotoxin (BoNT) is one of the most dangerous bacterial toxins and a potential biological weapon component. BoNT mechanism of pathological action is based on inhibiting the release of neurotransmitters from nerve endings. To date, anti-BoNT therapy is reduced to the use of horse hyperimmune serum, which causes many side effects, as well as FDA-approved drug BabyBig which consists of human-derived anti-BoNT antibodies (IgG) for infant botulinum treatment. Therapeutics for botulism treatment based on safer monoclonal antibodies are undergoing clinical trials. In addition, agents have been developed for the specific prevention of botulism, but their effectiveness has not been proved. In this work, we have obtained a recombinant adeno-associated virus (rAAV-B11-Fc) expressing a single-domain antibody fused to the human IgG Fc-fragment (B11-Fc) and specific to botulinum toxin type A (BoNT/A). We have demonstrated that B11-Fc antibody, expressed via rAAV-B11-Fc treatment, can protect animals from lethal doses of botulinum toxin type A, starting from day 3 and at least 120 days after administration. Thus, our results showed that rAAV-B11-Fc can provide long-term expression of B11-Fc-neutralizing antibody in vivo and provide long-term protection against BoNT/A intoxication. Consequently, our study demonstrates the applicability of rAAV expressing protective antibodies for the prevention of intoxication caused by botulinum toxins.
... Further comparative studies may deepen our understanding on the regulation process of these proteins and, consequently, of correlated human diseases [86]. Finally, similarly to sharks, camelids produce small homodimeric heavy chain-only antibodies with variable binding domains that can be highly useful in medicine and biotechnology research applications because of their small size, economic production, specificity, affinity, and stability [30,87,88]. ...
Article
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Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many physiological and pathological processes not only in humans but also in all the organisms of the eukaryotic and prokaryotic kingdoms. EV shedding constitutes a fundamental universal mechanism of intra-kingdom and inter-kingdom intercellular communication. A tremendous increase of interest in EVs has therefore grown in the last decades, mainly in humans, but progressively also in animals, parasites, and bacteria. With the present review, we aim to summarize the current status of the EV research on domestic and wild animals, analyzing the content of scientific literature, including approximately 220 papers published between 1984 and 2021. Critical aspects evidenced through the veterinarian EV literature are discussed. Then, specific subsections describe details regarding EVs in physiology and pathophysiology, as biomarkers, and in therapy and vaccines. Further, the wide area of research related to animal milk-derived EVs is also presented in brief. The numerous studies on EVs related to parasites and parasitic diseases are excluded, deserving further specific attention. The literature shows that EVs are becoming increasingly addressed in veterinary studies and standardization in protocols and procedures is mandatory, as in human research, to maximize the knowledge and the possibility to exploit these naturally produced nanoparticles.
... A variety of recombinant antibody fragments have been isolated against SARS-CoV-2 to date, using cloning approaches from antibodies from COVID-19 patients (Ebihara et al., 2021), antibody gene libraries derived from convalescent COVID-19 patients (Bertoglio et al., 2021;Mendoza-Salazar et al., 2022;Minenkova et al., 2022), or by immunising animals such as alpacas (Güttler et al., 2021). Phage display has also been utilised to isolate nanobodies (single-domain antibodies derived from camelids; Muyldermans et al., 2013) with SARS-CoV-2 neutralising ability , as well as scFv-Fc fusion proteins against nucleocapsid protein for use in diagnostics (Kim et al., 2021). Numerous studies have also reported the use of phage display to isolate scFvs against RBD but without investigating their virus neutralisation ability (Salem et al., 2022;Parray et al., 2020). ...
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COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2. From its initial appearance in Wuhan, China in 2019, it developed rapidly into a global pandemic. In addition to vaccines, therapeutic antibodies play an important role in immediately treating susceptible individuals to lessen severity of the disease. In this study, phage display technology was utilised to isolate human scFv antibody fragments that bind the receptor-binding domain (RBD) of SARS-CoV-2 Wuhan-Hu-1 spike protein. Of eight RBD-binding scFvs isolated, two inhibited interaction of RBD with ACE2 protein on VeroE6 cells. Both scFvs also exhibited binding to SARS-CoV-2 Delta variant spike protein but not to Omicron variant spike protein in a Raman spectroscopy immunotest. The study demonstrates the potential of recombinant antibody approaches to rapidly isolate antibody moieties with virus neutralisation potential.
... Nanobody is a recombinant single variable domain of a heavy chain fragment that was first isolated from the Camelidae family (Hamers-Casterman et al., 1993). While maintaining the ability for antigen binding, nanobody has useful biochemical properties such as tiny size, excellent solubility, and high stability (Muyldermans, 2013). The representative example of the Nb-based GPCR sensor is Nb80-GFP developed by the Zastrow group, which specifically binds to the active conformation of the β2AR (Rasmussen et al., 2011a). ...
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G protein-coupled receptors (GPCRs) regulate a wide range of physiological and pathophysiological cellular processes, thus it is important to understand how GPCRs are activated and function in various cellular contexts. In particular, the activation process of GPCRs is dynamically regulated upon various extracellular stimuli, and emerging evidence suggests the subcellular functions of GPCRs at endosomes and other organelles. Therefore, precise monitoring of the GPCR activation process with high spatiotemporal resolution is required to investigate the underlying molecular mechanisms of GPCR functions. In this review, we will introduce genetically encoded fluorescent biosensors that can precisely monitor the real-time GPCR activation process in live cells. The process includes the binding of extracellular GPCR ligands, conformational change of GPCR, recruitment of G proteins or β-arrestin, GPCR internalization and trafficking, and the GPCR-related downstream signaling events. We will introduce fluorescent GPCR biosensors based on a variety of strategies such as fluorescent resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), circular permuted fluorescent protein (cpFP), and nanobody. We will discuss the pros and cons of these GPCR biosensors as well as their applications in GPCR research.
... Fumonisins (FBs) are a group of hydrophilic mycotoxins produced largely by the fungi Fusarium (verticillioides, proliferatum, moniliforme, anthophilum, dlamini, globosum, fujikuroi, napiforme, nygamai, oxysporum) and Aspergillus (awamori, niger) [2,4,5]. They frequently contaminate corn (maize), corn-based foodstuffs, tion of recombinant nanobodies by microorganisms is highly cost-effective, and nanobodies can be easily utilized as building blocks for multi-domain constructs [16,17]. Nbs have high stability to organic solvents, which is useful for mycotoxin immunoassays. ...
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Background: Fumonisin B1 (FB1) is a secondary metabolite produced mainly by Fusarium verticillioides or Fusarium proliferatum. It poses a huge threat to the sustainable animal industry and human health as well via food chains (egg, meat and milk). Although E. coli-expressed nanobodies are documented for diagnostic applications, nanobodies remain elusive as FB1 detoxifiers in feed and food. Results: In the present study, the E. coli-expressed nanobody was assessed to remove FB1 in fresh milk, embryonated eggs and broilers. Firstly, 2 alpacas received intramuscularly FB1-adjuvanted BSA 6 times, and then the variable domain of the heavy-chain antibody (VHH) of fb1 genes were amplified to clone into the pCANTAB 5 E vector in order to generate a VHH-FB1 phage antibody display library, yielding 3.4 × 1010 capacity with 96.7% positivity. Afterwards, 5 anti-FB1 nanobodies were expressed and identified. Furthermore, maximal 43.2% FB1 was removed from milk by 1:2000 concentration of nanobody 5 (Nb5). Furthermore, SPF-embryonated eggs were inoculated into albumens with nanobody-treated FB1. The Nb5 group yielded an 83.3% hatching rate, higher body weight, lower gizzard ulceration and fewer FB1 residuals. In order to warrant the above results, 50 broilers aged 10 days were received orally with 20 ppm of FB1 for 20 days. At the same time, birds were fed orally with 50 μg of Nb5 or bivalent nanobody 11 (BiNb11). Finally, the Nb5 group showed a higher relative body weight gain and lower gastric ulcerations and fewer inflammations in the thymus and bursa. Conclusions: Based on the above evidence, the Nb5 nanobody may be considered as an additional FB1 detoxifier, contributing to FB1 decontamination.
... Therefore, the acquisition of optimal PET images is usually carried out 4-7 days after tracer injection. To solve the long circulation time in the body, Zhang et al., screened and identified a specific high-affinity PD-L1 nanobody (single domain antibody) (Muyldermans, 2013), designated KN035. It has received Investigational New Drug (IND) approval for treatment of advanced or metastatic solid tumors (ClinicalTrials.gov ...
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Recently, immune checkpoint therapy (ICT) represented by programmed cell death1 (PD-1) and its major li-gands, programmed death ligand 1 (PD-L1), has achieved significant success. Detection of PD-L1 by immuno-histochemistry (IHC) is a classic method to guide the treatment of ICT patients. However, PD-L1 expression in the tumor microenvironment is highly complex. Thus, PD-L1 IHC is inadequate to fully understand the relevance of PD-L1 levels in the whole body and their dynamics to improve therapeutic outcomes. Intriguingly, numerous studies have revealed that molecular imaging technologies could potentially meet this need. Therefore, the purpose of this narrative review is to summarize the preclinical and clinical application of ICT guided by molecular imaging technology, and to explore the future opportunities and practical difficulties of these innovations.
... The novel single-domain antibodies, also named nanobodies (Nbs) or heavy-chain variable domains (VHH), which originate from Camelidae and sharks lacking light chains and the first constant domain of the heavy chain (CH1), are increasingly used in detection in these years [17,18]. Compared to monoclonal antibodies (mAbs), Nbs are the smallest antigen-binding fragments with the molecular weight size of about 15 kDa and exhibit several advantages, such as highly soluble and stable, good affinity and specificity [19]. Importantly, Nbs could be expressed in different expression systems [20], such as prokaryotic or eukaryotic expression system, and are suitable to bioengineer into various forms, like bivalent or multivalent forms. ...
Article
African swine fever (ASF) is a highly contagious and lethal hemorrhagic disease, which has brought great distress and economic losses to the world pig industry. For lack of approved vaccines and effective treatment, accurate early detection, both ASF virus (ASFV) antigen and antibody detection in the field, is one of the most important aspects for preventing the outbreak and spread of ASFV. Here we screened the specific nanobodies against the ASFV-P30 protein from the library of immunized Bactrian camel and fused them with horseradish peroxidase (HRP) for expression in HEK293T cells. Moreover, the P30 protein was designed and displayed on the surface of ferritin, the fusion nanocages. Then we developed a novel competitive ELISA (cELISA) based on ferritin-displayed P30 nanoparticles protein (P30-Fn) and nanobody-horseradish peroxidase fusions (P30–Nb71-vHRP) for rapid detection of ASFV antibody in serum sample with high sensitivity and specificity. The cELISA can detect as far as 1:200 diluted clinically positive serums and show no cross-reactivity with positive serums against others porcine viruses, such as PRRSV, PPV, CSFV, TGEV, PEDV, PCV and SIV. The agreement rate is 98.1% with the test results of the commercial ELISA kit for the detection of a total of 267 clinical samples, and the kappa value is 0.96. Furthermore, the cELISA exhibits a good repeatability and the intra- and inter-assay coefficient of variation are less than 10%. In summary, the newly developed cELISA is a simple, rapid, sensitive and low-cost immunoassay, which has a great potential for ASFV antibody detection in clinical samples.
... NBs are a subset of light chain missing heavychain antibodies found in the blood of camelid (camel, alpaca, etc.) animals that contain only one heavy chain variable region (VHH). Interestingly, VHHs, the key crystals in which NBs function, are approximately 2.5 nm in size and retain the ability to bind antigens, which are the smallest known antigen-binding crystals (Muyldermans, 2013). ...
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Acute respiratory distress syndrome (ARDS) is a common critical illness in respiratory care units with a huge public health burden. Despite tremendous advances in the prevention and treatment of ARDS, it remains the main cause of intensive care unit (ICU) management, and the mortality rate of ARDS remains unacceptably high. The poor performance of ARDS is closely related to its heterogeneous clinical syndrome caused by complicated pathophysiology. Based on the different pathophysiology phases, drugs, protective mechanical ventilation, conservative fluid therapy, and other treatment have been developed to serve as the ARDS therapeutic methods. In recent years, there has been a rapid development in nanomedicine, in which nanoparticles as drug delivery vehicles have been extensively studied in the treatment of ARDS. This study provides an overview of pharmacologic therapies for ARDS, including conventional drugs, natural medicine therapy, and nanomedicine. Particularly, we discuss the unique mechanism and strength of nanomedicine which may provide great promises in treating ARDS in the future.
... In addition, the humanization of sdAbs provides a safe option for long-term treatment (55,56). 6) SdAbs can be efficiently, easily and economically produced recombinantly in bacteria, mammalian cell lines, yeast and plants at an affordable cost (11,57), while the production of canonical monoclonal antibodies requires mammalian expression system, which is complex in technology and expensive to maintain. Apart from these outstanding characteristics, sdAbs also have some limitations. ...
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Increasing antibiotic resistance to bacterial infections causes a serious threat to human health. Efficient detection and treatment strategies are the keys to preventing and reducing bacterial infections. Due to the high affinity and antigen specificity, antibodies have become an important tool for diagnosis and treatment of various human diseases. In addition to conventional antibodies, a unique class of “heavy-chain-only” antibodies (HCAbs) were found in the serum of camelids and sharks. HCAbs binds to the antigen through only one variable domain Referred to as VHH (variable domain of the heavy chain of HCAbs). The recombinant format of the VHH is also called single domain antibody (sdAb) or nanobody (Nb). Sharks might also have an ancestor HCAb from where SdAbs or V-NAR might be engineered. Compared with traditional Abs, Nbs have several outstanding properties such as small size, high stability, strong antigen-binding affinity, high solubility and low immunogenicity. Furthermore, they are expressed at low cost in microorganisms and amenable to engineering. These superior properties make Nbs a highly desired alternative to conventional antibodies, which are extensively employed in structural biology, unravelling biochemical mechanisms, molecular imaging, diagnosis and treatment of diseases. In this review, we summarized recent progress of nanobody-based approaches in diagnosis and neutralization of bacterial infection and further discussed the challenges of Nbs in these fields.
... Llamas produce functional Abs that lack light chains. The variable domain of llama heavy chain Ab molecules (VHH) contains three CDR regions that are fully capable of antigen recognition (30,45). A naïve Llama VHH phage library (Abcore, Ramona, CA) was used to generate an AID-Pol mutagenized f3TR1-VHH library. ...
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We describe a purified biochemical system to produce monoclonal antibodies (Abs) in vitro using activation-induced deoxycytidine deaminase (AID) and DNA polymerase η (Polη) to diversify immunoglobulin variable gene (IgV) libraries within a phage display format. AID and Polη function during B-cell affinity maturation by catalyzing somatic hypermutation (SHM) of immunoglobulin variable genes (IgV) to generate high-affinity Abs. The IgV mutational motif specificities observed in vivo are conserved in vitro. IgV mutations occurred in antibody complementary determining regions (CDRs) and less frequently in framework (FW) regions. A unique feature of our system is the use of AID and Polη to perform repetitive affinity maturation on libraries reconstructed from a preceding selection step. We have obtained scFv Abs against human glucagon-like peptide-1 receptor (GLP-1R), a target in the treatment of type 2 diabetes, and VHH nanobodies targeting Fatty Acid Amide Hydrolase (FAAH), involved in chronic pain, and artemin, a neurotropic factor that regulates cold pain. A round of in vitro affinity maturation typically resulted in a 2- to 4-fold enhancement in Ab-Ag binding, demonstrating the utility of the system. We tested one of the affinity matured nanobodies and found that it reduced injury-induced cold pain in a mouse model.
... However, the traditional ELISA procedure requires several repeated cycles of reagent addition, incubation and microplate washing to add new reagents or to remove unbound reagents, resulting in a longer detection time [18]. With the development of gene engineering and antibody engineering, various miniaturized recombinant antibodies have received extensive attention [19,20]. Moreover, the recombinant antibody-enzyme fusion protein, as a novel bifunctional tracer, has shown significant advantages in terms of simplifying the operation process, improving result stability and reducing the detection time in immunoassays [21][22][23][24][25]. ...
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Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase–nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples. On the basis of Nb28-Nluc, the BLEIA can be completed with a one-step incubation and detection, with only a substrate replacement from 3,3′,5,5′-tetramethylbenzidine (TMB) to a Nluc assay reagent (Furimazine). Under the optimal experimental conditions, the proposed one-step BLEIA achieved a detection limit of 3.7 ng/mL (IC10) within 3 h. Moreover, the BLEIA method showed good repeatability and accuracy in the spike recovery experiments with recoveries of 83.88% to 120.23% and relative standard deviations (RSDs) of 5.2% to 24.7%, respectively. Particularly, the BLEIA displayed superior performances, such as fewer operations and more rapid and sensitive detection as compared with Nb28-based enzyme-linked immunosorbent assay. Therefore, the proposed one-step BLEIA has great potential for the sensitive and accurate screening of OTA in food samples.
... Because of their small size (12-15 kDa), superior biophysical stability, and antigen-binding properties, Nbs are considered as the next generation of antibodies, with great potential for medical applications, including therapy in humans [24][25][26]. Furthermore, Nbs are usually well expressed in bacteria, are stable monomeric fragments with good thermostability (up to 90 °C), and have high solubility, resistance to pH changes, and are encoded by a gene fragment of around 360-380 bp, with the latter being a property that allows them to be engineered easily [23,[27][28][29][30]. In addition, Nbs offer great potential for a variety of applications such as biosensors as well as contrast probes for fluorescent or MRI imaging owing to their strong binding affinity and specificity towards their cognate antigens [31][32][33][34]. ...
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Ovarian cancer ranks fifth in cancer-related deaths among women. Since ovarian cancer patients are often asymptomatic, most patients are diagnosed only at an advanced stage of disease. This results in a 5-year survival rate below 50%, which is in strong contrast to a survival rate as high as 94% if detected and treated at an early stage. Monitoring serum biomarkers offers new possibilities to diagnose ovarian cancer at an early stage. In this study, nanobodies targeting the ovarian cancer biomarkers human epididymis protein 4 (HE4), secretory leukocyte protease inhibitor (SLPI), and progranulin (PGRN) were evaluated regarding their expression levels in bacterial systems, epitope binning, and antigen-binding affinity by enzyme-linked immunosorbent assay and surface plasmon resonance. The selected nanobodies possess strong binding affinities for their cognate antigens (KD~0.1–10 nM) and therefore have a pronounced potential to detect ovarian cancer at an early stage. Moreover, it is of utmost importance that the limits of detection (LOD) for these biomarkers are in the pM range, implying high specificity and sensitivity, as demonstrated by values in human serum of 37 pM for HE4, 163 pM for SLPI, and 195 pM for PGRN. These nanobody candidates could thus pave the way towards multiplexed biosensors.
... Accordingly, nanobodies (V HH domains) derived from heavy chain-only antibodies of camelids were shown to be versatile tools for diagnosis and treatment of various diseases and, more recently, for immunoassay development in the field of type I allergy (24-30). In addition, nanobodies have similar antigen affinities as monoclonal antibodies, are chemically and physically very stable, and have a simple structure (i.e., single domain antibody) (31,32). These facts suggest that nanobodies might be promising tools for antibody-based allergy treatment. ...
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The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10 ⁻¹⁰ M) and had slow off-rates (10 ⁻⁴ s ⁻¹ ). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.
... To validate our design strategy, we tested it experimentally on singledomain antibodies, because of their monomeric nature, ease of production in prokaryotic systems, and small size (16). Nonethe less, the computational design pipeline described here can readily be applied to other antibody fragments, including whole Fv regions, on which designed CDR motifs can be structurally matched and grafted in the same way on either heavy or lightchain CDRs. ...
Article
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De novo design methods hold the promise of reducing the time and cost of antibody discovery while enabling the facile and precise targeting of predetermined epitopes. Here, we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterization showed that all designs are stable and bind their intended targets with affinities in the nanomolar range without in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as similar predictions are obtained when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to preselected epitopes.
... Compared with conventional antibodies, they are also easier and cheaper to produce [258]. The recombinant antigen-specific, single-domain VHH with dimensions in the nanometre range is also known as a nanobody or single-domain antibody [259]. ...
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In many countries, medical levels of zinc (typically as zinc oxide) are added to piglet diets in the first two weeks post-weaning to prevent the development of post-weaning diarrhoea (PWD). However, high levels of zinc constitute an environmental polluting agent, and may contribute to the development and/or maintenance of antimicrobial resistance (AMR) among bacteria. Consequently, the EU banned administering medical levels of zinc in pig diets as of June 2022. However, this may result in an increased use of antibiotic therapeutics to combat PWD and thereby an increased risk of further AMR development. The search for alternative measures against PWD with a minimum use of antibiotics and in the absence of medical levels of zinc has therefore been intensified over recent years, and feed-related measures, including feed ingredients, feed additives, and feeding strategies, are being intensively investigated. Furthermore, management strategies have been developed and are undoubtedly relevant; however, these will not be addressed in this review. Here, feed measures (and vaccines) are addressed, these being probiotics, prebiotics, synbiotics, postbiotics, proteobiotics, plants and plant extracts (in particular essential oils and tannins), macroalgae (particularly macroalgae-derived polysaccharides), dietary fibre, antimicrobial peptides, specific amino acids, dietary fatty acids, milk replacers, milk components, creep feed, vaccines, bacteriophages, and single-domain antibodies (nanobodies). The list covers measures with a rather long history and others that require significant development before their eventual use can be extended. To assess the potential of feed-related measures in combating PWD, the literature reviewed here has focused on studies reporting parameters of PWD (i.e., faeces score and/or faeces dry matter content during the first two weeks post-weaning). Although the impact on PWD (or related parameters) of the investigated measures may often be inconsistent, many studies do report positive effects. However, several studies have shown that control pigs do not suffer from diarrhoea, making it difficult to evaluate the biological and practical relevance of these improvements. From the reviewed literature, it is not possible to rank the efficacy of the various measures, and the efficacy most probably depends on a range of factors related to animal genetics and health status, additive doses used, composition of the feed, etc. We conclude that a combination of various measures is probably most recommendable in most situations. However, in this respect, it should be considered that combining strategies may lead to additive (e.g., synbiotics), synergistic (e.g., plant materials), or antagonistic (e.g., algae compounds) effects, requiring detailed knowledge on the modes of action in order to design effective strategies.
... Heavy chain antibodies (HCAb) or variable heavy antibodies (VHH) or nanobodies [19] Dairy Processing -From Basics to Advances in colostrum, it is responsible for conferring immunity on newborns within the first few weeks of life [26]. Lactoferrin is involved in various physiological functions such as regulating homeostasis and cell proliferation, besides being a very potent antimicrobial agent. ...
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Camel milk is a rich source of protein with well-recognized medicinal properties to treat various diseases. The objective of this work is to understand the role of camel milk lactoferrin in immunomodulation and in disease treatment. It has been found that camel milk lactoferrin is a very suitable nutraceutical agent by virtue of its bioactivity, immuno-compatibility, and safety. It can be used for the treatment of infectious, metabolic, and neurodegenerative diseases, besides cancer. It is a cost-effective biomolecule that also has high relative abundance and bioavailability.
... In addition to the conventional mAbs, great efforts have been invested to develop camelid heavy chain only antibodies (VHHs) as antitoxins against TcdA and TcdB because of their small sizes, high affinity, specificity, and stability (34,35). For example, in earlier studies we found that VHH 5D binds to the pore-forming region in TcdB DRBD and prevents the pHinduced pore formation of the toxin, and VHH E3 binds to the N-terminal four-helix bundle in TcdB GTD that may interfere with membrane association of the GTD (10). ...
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Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile, which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.
... In addition to conventional antibodies, a nanobody is a small (12)(13)(14)(15) single domain antibody fragment that exposes a convex paratope that forms a prolate shape, allowing it to efficiently blocks the essential epitopes on pathogens that are inaccessible to conventional antibodies [8,9]. In contrast, RNA viruses, such as SARS-CoV-2, typically have higher mutation rates that allow escape from neutralizing antibodies or nanobodies [10]. ...
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Purpose To inhibit the transmission of SARS-CoV-2, we developed engineered exosomes that were conjugated with anti-spike nanobodies and type I interferon β (IFN-β). We evaluated the efficacy and potency of nanobody-IFN-β 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-β conjugated exosomes by fusing an anti-spike nanobody and IFN-β 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/β cells in the presence of nanobody-IFN-β 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-β conjugated exosomes. Results We confirmed the anti-spike nanobody and IFN-β expressions on the exosomes. Exosomes conjugated with nanobody-hIFN-β 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-β was selectively delivered to SARS-CoV-2 infected cells, resulting in the upregulation of ISGs expression. Conclusion Exosomes conjugated with nanobody-IFN-β 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-β.
... Single-domain antibody, with a molecular weight of ~12 to 15 kDa, is approximately one-tenth of the size of a conventional IgG in ~150 kDa [90,91]. As small biomolecules, sdAbs exhibit efficient tissue penetration [92,93]. ...
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With severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an emergent human virus since December 2019, the world population is susceptible to coronavirus disease 2019 (COVID-19). SARS-CoV-2 has higher transmissibility than the previous coronaviruses, associated by the ribonucleic acid (RNA) virus nature with high mutation rate, caused SARS-CoV-2 variants to arise while circulating worldwide. Neutralizing antibodies are identified as immediate and direct-acting therapeutic against COVID-19. Single-domain antibodies (sdAbs), as small biomolecules with non-complex structure and intrinsic stability, can acquire antigen-binding capabilities comparable to conventional antibodies, which serve as an attractive neutralizing solution. SARS-CoV-2 spike protein attaches to human angiotensin-converting enzyme 2 (ACE2) receptor on lung epithelial cells to initiate viral infection, serves as potential therapeutic target. sdAbs have shown broad neutralization towards SARS-CoV-2 with various mutations, effectively stop and prevent infection while efficiently block mutational escape. In addition, sdAbs can be developed into multivalent antibodies or inhaled biotherapeutics against COVID-19.
... Nanobodies are at the forefront of antiviral drug research because of their unique advantages (Detalle et al., 2016). Nanobodies, also called single-domain antibodies (sdAbs), consist of one variable domain including 130 amino acids (Muyldermans, 2013). With a small molecule and single-domain nature, nanobodies can recognize special epitopes located in protein crevices (Ma et al., 2021;Zhou et al., 2022). ...
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Hepatitis E virus (HEV) is thought to be a zoonotic pathogen that causes serious economic loss and threatens human health. However, there is a lack of efficient antiviral strategies. As a more promising tool for antiviral therapy, nanobodies (also named single-domain antibodies, sdAbs) exhibit higher specificity and affinity than traditional antibodies. In this study, nanobody anti-genotype four HEV open reading frame 2 (ORF2) was screened using phage display technology, and two nanobodies (nb14 and nb53) with high affinity were prokaryotically expressed. They were identified to block HEV ORF2 virus like particle (VLP) sp239 (aa 368–606) absorbing HepG2 cells in vitro . With the previously built animal model, the detection indicators of fecal shedding, viremia, seroconversion, alanine aminotransferase (ALT) levels, and liver lesions showed that nb14 could completely protect rabbits from swine HEV infection, and nb53 partially blocked swine HEV infection in rabbits. Collectively, these results revealed that nb14, with its anti-HEV neutralizing activity, may be developed as an antiviral drug for HEV.
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The management and treatment of infectious bacterial diseases in wound healing have both become significant research areas in the biomedical field. While current treatments show limitations related to toxicity and exposure time, nanotechnology has become a potential alternative to overcome such challenges. The application of different nanomaterials, with a wide range of elemental compositions, morphologies, and features, has become an essential tool in managing wound healing infections. This book chapter shows an updated view of the newest trends in the control and treatment of bacterial proliferation in the wound bed by utilizing various metal- and nonmetal-based nanostructures.
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Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non‐globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross‐linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes. PDB Code(s): 7NOZ;
Article
Aflatoxingenetic fungi producing highly toxic and carcinogenic mycotoxins and threatening human health widely exist in our living environment and food. Taking aflatoxingenetic fungi as an analyte example, here we report a way to improve the sensitivity of paper-based sandwich immunosensor for macromolecule. With a biomarker-specific nanobody and polyclonal antibody (IgG), two modes of paper-based immunosensors were constructed, immobilization nanobody mode and immobilization IgG mode. It was found that the sensitivity of the immobilization nanobody mode was at least 500 times higher than that of the immobilization IgG mode. The reasons for the large difference in sensitivity were also theoretically. Under the optimized conditions, the immobilization nanobody mode sensitivity reached 0.035 μg/mL aflatoxingenetic mycelia, each assay was finished within 15 min, and the spiked recoveries of aflatoxingenetic mycelium reference in peanuts were 77.0%~91.6%. And then, two important application cases were successfully demonstrated to identify the abundance of aflatoxingenetic fungi in market peanuts and peanut rhizosphere soil for environmental safety evaluation. This is the first report of time-resolved paper-based sandwich immunosensor for aflatoxingenetic fungi with nanobody immobilization, and the first sensitivity comparison between nanobody immobilization and IgG immobilization, which provides a new approach to develop highly sensitive immunosensor for macromolecular hazard materials.
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The bioconjugation of proteins to small molecules serves as an invaluable tool for probing biological mechanisms and creating biomaterials. The most powerful bioconjugation stratagems are those which have rapid kinetics,...
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Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins produced by the pathogen Villosiclava virens of rice false smut, which has recently become one of the most devastating diseases in rice-growing regions worldwide. In this research, the nanobody phage display library was established after an alpaca was immunized with the hemiustilaginoidin F-hapten coupled with bovine serum albumin (BSA). Heterologous antigen selection and combing trypsin with competition alternant elution methods were performed for nanobody screening. Two nanobodies, namely, Nb-B15 and Nb–C21, were selected for the establishment of indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs). For Nb–B15 and Nb-C21, their IC50 values were 11.86 μg/mL and 11.22 μg/mL, and the detection ranges were at 3.41–19.98 μg/mL and 1.17–32.13 μg/mL, respectively. Two nanobodies had a broad spectrum to quantify the contents of total ustilaginoidins in rice samples according to cross-reactivity. The recognition mechanisms of Nb-B15 and Nb-C21 against ustilaginoidin A were elucidated by molecular modeling and docking. The key amino acid sites for the binding of Nb–B15 or Nb–C21 to ustilaginoidin A were mainly located in the FR1 and CDR1 regions. As Nb-B15 was superior to Nb–C21 in the aspects of protein expression, ELISA titer, and tolerance to organic solvents, it was selected for application in the detection of actual contaminated rice samples. The total ustilaginoidin contents of rice samples were analyzed by Nb–B15-based ic–ELISA and HPLC-DAD, between which the results were found to be consistent. The developed immunoassay based on the nanobody from the alpaca can be employed as a rapid and effective method for detection of total utilaginoidins in contaminated rice samples.
Chapter
With the potential to organize as homodimers, heterodimers, or higher‐order species, G protein‐coupled receptors (GPCRs) represent increasingly complex therapeutic targets. Even if cell surface expression enhances their pharmacological tractability, they still comprise difficult candidates since GPCR dimers or oligomers can engage in distinct signaling transduction pathways and biological outcomes compared to their monomeric components. Researchers are thus taking advantage of various techniques in an attempt to better understand GPCR organization and function. In the last several years, a large array of biochemical and biophysical techniques has been developed to assess the organization and functional consequences associated with the formation of GPCR oligomers. We are also beginning to grasp that these macromolecular complexes may be associated with their own respective complements of effector proteins or proteomes. This may in part provide an explanation for the differences observed in biologically relevant signaling outcomes. Proteomic approaches allow us to better appreciate GPCR dimers as unique species. Here, we provide a short review of the diverse approaches used to study and understand signaling of GPCR dimers and oligomers.
Article
Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, we demonstrated that SFTSV surface glycoprotein (Glycoprotein N, Gn) was a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti‐Gn neutralizing antibodies played a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages, including small molecular weight, high affinity, low immunogenicity, convenient production by gene engineering, etc. In this study, we combined next generation sequencing (NGS) with proteomics technology based on affinity purification‐mass spectrometry (AP‐MS) and bioinformatics analysis to high‐throughput screen monoclonal anti‐Gn nanobodies from camel immunized with Gn protein. We identified 19 anti‐Gn monoclonal nanobody sequences, of which 6 sequences were selected selected for recombinant protein expression and purification. Among these 6 anti‐Gn nanobodies, nanobody 57493 was validated to be highly specific for Gn. The innovative high‐throughput technical route developed in this study could also be expanded to the production of nanobodies specific for other viruses like SARS‐CoV‐2. This article is protected by copyright. All rights reserved.
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Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli . These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.
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As already indicated in Chapter 2, Nanomaterials to aid wound healing and infection control, various types of metallic- and nonmetallic nanomaterials can be used to aid the wound healing process and to keep bacterial infections in check. While serving as powerful tools on their own, various types of nanomaterials can unravel their true potential by being embedded inside a variety of scaffolds and coatings, differing both in chemistry and morphologies. Being embedded inside the material or by covering its surface, NMs alter its physicochemical properties and serve as a means for localized controlled release of various bioactive compounds. By employing such strategies, one can obtain highly functional materials, having great potential to enter clinical applications. This chapter is aimed to summarize the current advances in the field of nanocomposite wound dressings, scaffolds, and coatings. These are used as efficient tools to improve the wound healing efficiency as well as inhibit and/or eradicate microbial infections.
Chapter
The antiviral activity of nanoparticles is facilitated by their unique properties that make them attractive tools for viral treatment. The nanoscale size of particles can facilitate cellular uptake and allow reaching anatomically privileged sites. The large surface area-to-volume ratios can ensure decoration with large payloads of functional molecules that can increase the interaction with pathogens, leading to optimized drug dosing and delivery and increasing stability. The surface charge can facilitate binding to infectious agents and subsequently inhibit the initial steps of infection. In addition, nanoparticles can contain biomimetic characteristics that enhance their intrinsic antiviral properties as in the case of silver and gold nanoparticles and dendrimers.
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Num1 is a multifunctional protein that both tethers mitochondria to the plasma membrane and anchors dynein to the cell cortex during nuclear inheritance. Previous work has examined the impact loss of Num1-based mitochondrial tethering has on dynein function in Saccharomyces cerevisiae; here, we elucidate its impact on mitochondrial function. We find that like mitochondria, Num1 is regulated by changes in metabolic state, with the protein levels and cortical distribution of Num1 differing between fermentative and respiratory growth conditions. In cells lacking Num1, we observe a reproducible respiratory growth defect, suggesting a role for Num1 in not only maintaining mitochondrial morphology, but also function. A structure-function approach revealed that, unexpectedly, Num1-mediated cortical dynein anchoring is important for normal growth under respiratory conditions. The severe respiratory growth defect in Δnum1 cells is not specifically due to dynein's canonical function in nuclear migration but is dependent on the presence of dynein, as deletion of DYN1 in Δnum1 cells partially rescues respiratory growth. We hypothesize that misregulated dynein present in cells that lack Num1 negatively impacts mitochondrial function resulting in defects in respiratory growth.
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Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting (FACS) to enrich for a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic E. coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high throughput discovery of bacterial surface-binding molecules. This article is protected by copyright. All rights reserved.
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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 0.2-10.6 μg/mL, and a limit of detection of 53.13 ng/mL.
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Rationale Recent studies have demonstrated the feasibility of CD38-specific antibody constructs for in vivo imaging of multiple myeloma. However, detecting multiple myeloma in daratumumab-pretreated patients remains difficult due to overlapping binding epitopes of the CD38-specific imaging antibody constructs and daratumumab. Therefore, the development of an alternative antibody construct targeting an epitope of CD38 distinct from that of daratumumab is needed. We report the generation of a fluorochrome-conjugated nanobody recognizing such an epitope of CD38 to detect myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo. Methods We conjugated the CD38-specific nanobody JK36 to the near-infrared fluorescent dye Alexa Fluor 680. The capacity of JK36 AF680 to bind and detect CD38-expressing cells pretreated with daratumumab was evaluated on CD38-expressing tumor cell lines in vitro, on primary myeloma cells from human bone marrow biopsies ex vivo, and in a mouse tumor model in vivo. Results Fluorochrome-labeled nanobody JK36 AF680 showed specific binding to CD38-expressing myeloma cells pretreated with daratumumab in vitro and ex vivo and allowed for specific imaging of CD38-expressing xenografts in daratumumab-pretreated mice in vivo. Conclusions Our study demonstrates that a nanobody recognizing a distinct, non-overlapping epitope of CD38 allows the specific detection of myeloma cells under daratumumab therapy in vitro, ex vivo, and in vivo.
Article
Chimeric antigen receptor (CAR) T-cell immunotherapy has become one of the research hotspots in the treatment of malignant tumors nowadays. However, the available tumor surface antigens are limited in number. Most tumor-associated antigens are intracellular molecules that can’t be targeted by conventional CAR T cells. As the major histocompatibility complex (MHC)/peptide complex is a presentation form of intracellular proteins on the surface of tumor cells, here, we chose the Glypican-3 (GPC3) oncoprotein and Wilms tumor 1 (WT1) oncoprotein as examples to explore whether nanobody (Nb)-based T cell receptor (TCR)-like CAR T cells could kill tumor cells by targeting the MHC/peptide complexes. Using the immune nanobody phage display library, we developed human leukocyte antigen (HLA)-A2/GPC3- and HLA-A2/WT1-specific nanobodies for the first time and then incorporated these nanobodies in two TCR-like CARs, targeting HLA-A2/GPC3 and HLA-A2/WT1 respectively. These TCR-like Nb CAR-redirected T cells could selectively recognize and lyse MHC/peptide complex-expressing tumor cells in vitro assays and subcutaneous mouse tumor models. This study offers a possible strategy for targeting intracellular antigens and widening the application of CAR T-cell therapy.
Article
In this issue of Structure, Maso et al. (2022) discover nanobodies that inhibit the SOS response of Escherichia coli by targeting the LexA repressor-protease. High-resolution structures of the novel LexA-nanobody complexes reveal they function by stabilizing LexA in its inactive conformation and preventing co-proteolysis by RecA∗.
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Point-of-care (POC) assays have emerged as powerful analytical tools combining several attractive characteristics such as simplicity, cost-effectiveness, and rapidness. Among them, assay portability can be further enhanced by coupling POC devices to smartphones as analytical detectors. Therefore, in this chapter we critically review and discuss interesting and important advances of smartphone-based sensing analytical platforms in the medical and food analysis fields. To monitor research trends, a scientometric study was performed using structured keywords in the Scopus database. The bibliometric analysis revealed that both fields are relatively young and especially in the case of food analysis, smartphone-based diagnostics remains predominantly in a proof-of-concept stage. In addition, certain POC assays such as lateral flow assays or paper-based assays are the most common assays hyphenated to smartphones with the aim to improve quantification capabilities, online result processing, and data communication. Nevertheless, certain bottlenecks currently inhibit smartphone diagnostics to fully express their analytical potential due to absence of clinical trials in the medical field or insufficient validation in food analysis. In addition, interphone result variation, especially in the case of optical detection, is another significant challenge that needs to be dealt with. In conclusion, a comprehensive overview on smartphone sensing is provided aiming to emphasize the merits and drawbacks of this constantly evolving technology.
Chapter
The integration of microfluidic technology together with biosensors is an attractive choice for the fabrication of miniaturized components in a single device and opens new opportunities for future biosensing applications, including portability, ease of use, and high-throughput analysis. Pairing microfluidic devices with biosensors has lead to the development of a wide range of new functional features for improving biosensing performance. Various conventional materials such as silicon, glass, ceramic, polydimethylsiloxane, and paper are the most common materials used in microfluidic biosensors; meanwhile, advancements in polymer material science have driven the emerging three-dimensional-printed microfluidics to enable facile design and fabrication of complexed microfluidic architectures coupled with sensing components for novel microfluidic biosensors with new features. On the other hand, advanced materials have also contributed to the improvement of signal transduction in microfluidic biosensors. In particular, graphene-based two-dimensional (2D) nanomaterials, 2D transition metal nanomaterials (such as transition metal oxides, transition metal dichalcogenides), MXenes, and black phosphorus were used for the development of high-performance electrochemical transducers in microfluidic biosensors. Here, we discuss and summarize various advanced functional materials for the fabrication of microfluidic platforms for biosensing, as well as the use of nanomaterials to improve signal transduction in microfluidic biosensors.
Chapter
In the last few decades, printed microfluidics exhibited attractive unique advantages, such as fast turnaround time and high flexibility in customization and fabrication, showing potentials in on-site production and downstream biosensing. The advancement in technical methods of fabrication, assembly, and integration has simplified the construction of these biosensors without sacrificing their biosensing performance. Since their development in the 1990s, microfluidic technologies have enabled the processing of fluid on micron scale, hence reducing the need for a large volume of the sample, from milliliter in conventional biosensing methods to pico-or micro-liter. It also supports automation in fluidic actuation to realize sample-to-answer biosensing, an essential feature for its use as a point-of-care and point-of-use test. This chapter first gives a brief overview of printed microfluidic biosensors, an introduction to the origins of microfluidics, and types of printed microfluidics compared to traditional microfluidics. It then covers each type of printed microfluidics (2D, pseudo-3D, 3D, and 4D) and its corresponding fabrication and assembly methods. Next, it discusses different approaches to conjugating the recognition elements, such as nucleic acid probes and antibodies, into the printed microfluidics and the integration with actuators and signal detecting units to make functional biosensors. Optical and electrochemical detection techniques are our main focus. The chapter then highlights in-depth researches on using these biosensors for two biomedical applications, i.e., disease screening and food safety. Lastly, we discuss the outlook on the development of printed microfluidics. This chapter aims to provide the reader with the updated knowledge on the advanced technologies for constructing printed microfluidic biosensors and increasing awareness of the importance of these advanced techniques and their practical real-life applications.
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The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to a pandemic threat in the form of vaccines and antigen tests. The causative agent of COVID-19 is SARS-CoV-2. The spike protein on the virus surface interacts with the human angiotensin-converting enzyme (ACE2) via the so-called receptor binding domain (RBD), facilitating virus entry. The RBD thus represents a prime target for vaccines, therapeutic antibodies, and antigen test systems. 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 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. In this study, we produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the viral RBD as Cts1 fusions and screened their RBD 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 an inexpensive antigen test utilizing unconventionally secreted nanobodies as RBD trap and a matching ubiquitous and biogenic surface for immobilization.
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Cell-based Surface Plasmon Resonance imaging (SPRi) with anti-endoglin VHH antibodies microarray on a gold-covered biochip was used to characterize the binding to endoglin expressed on the surface membrane of SC cells. SPRi showed the specific capture of SC cells by the immobilized anti-endoglin VHHs, but not of THP-1 cells that do not express endoglin on their plasmatic membrane. The SPRi reflectivity shifts with SC cells bound by anti-endoglin VHHs ranged from 3.59 to 5.54% (average 4.66 ±1.18%) and with THP-1 cells from 2.23 to 3.05% (average 2.62±1.32% (p<0.001)) and the SC cell densities with anti-endoglin VHHs ranged from 417 to 747.7, (average 548 cells/2.28 × 10⁵ μm² ± 132). Anti-endoglin VHHs bind epitopes located in the extracellular domains of endoglin with different avidity as suggested by the differences in SC cell densities. The cell-based SPRi of anti-endoglin VHH microarrays can be used both in studies aimed at a better understanding of endoglin function in living cells and in VHH applications as nanoprobes.
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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.
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Milk proteins are widely used for food supplementation, despite the potential risk of food allergy, especially against β-lactoglobulin (BLG), which makes BLG surveillance critical. Possible interaction of detecting antibodies with BLG-derived peptides will result in unprecise inspection. Thus, in this study, it was proposed to generate nanobodies (Nbs) and validate the immunological detection of intact BLG rather than hydrolytic peptides. Nbs were successfully retrieved and characterized with high stability and target specificity. A competitive enzyme-linked immunosorbent assay (cELISA) was developed with a linear range from 39 to 10,000 ng/mL and a detection limit (LOD) of 4.55 ng/mL, with a recovery of 86.30%-95.09% revealed by analysis of spiked samples. Meanwhile, a sandwich ELISA (sELISA) was established with Nb82 and BLG polyclonal antibody (pAb-BLG) providing a linear range from 29.7 to 1250 ng/mL and an LOD of 13.82 ng/mL with a recovery of 87.82%-103.97%. The interaction of selected Nbs with BLG-derived peptides was investigated by Nb structure modeling and BLG docking. No binding on hydrolytic peptides was revealed, confirming the precision of Nb-mediated immunoassays. In summary, this study successfully identified BLG-specific Nbs for immunoassay development and guaranteed the monitoring of intact BLG without interference of hydrolytic peptides, providing experimental evidence that our Nbs recognize intact food allergen.
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Enteric microbial pathogens, including Escherichia coli , Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic E . coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E . coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro . The probiotic strain E . coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E . coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen ( Shigella flexneri ), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eukaryotic pathogen ( Cryptosporidium parvum ) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.
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The purpose of our study was to develop a tool for blocking the function of a specific leukocyte ecto-enzyme in vivo. ART2.2 is a toxin-related ecto-enzyme that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. ART2.2 induces T cell death by activating the cytolytic P2؋7 purinoceptor via ADP-ribosylation. Here, we report the generation of ART2.2-blocking single domain antibodies from an immunized llama. The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses. Their long CDR3 endows VHH domains with the extraordinary capacity to extend into and block molecular clefts. Following intravenous injection , the ART2.2-specific VHH domains effectively shut off the enzymatic and cytotoxic activities of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but not the related enzymes ART1 or ART2.1), rapid (within 15 min after injection), and reversible (24 h after injection). Our findings constitute a proof of principle that opens up a new avenue for targeting leukocyte ecto-enzymes in vivo and that can serve as a model also for developing new antidotes against ADP-ribosylating toxins.-Koch-Nolte, F., Reyelt, J., Schö␤ow, B., Schwarz, N., Scheu-plein, F., Rothenburg, S., Haag, F., Alzogaray, V., Cauerhff, A., and Goldbaum, F. A. Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo. FASEB J. 21, 3490-3498 (2007)
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The eukaryotic ubiquitin-proteasome system is responsible for most aspects of regulatory and quality-control protein degradation in cells. Its substrates, which are usually modified by polymers of ubiquitin, are ultimately degraded by the 26S proteasome. This 2.6-MDa protein complex is separated into a barrel-shaped proteolytic 20S core particle (CP) of 28 subunits capped on one or both ends by a 19S regulatory particle (RP) comprising at least 19 subunits. The RP coordinates substrate recognition, removal of substrate polyubiquitin chains, and substrate unfolding and translocation into the CP for degradation. Although many atomic structures of the CP have been determined, the RP has resisted high-resolution analysis. Recently, however, a combination of cryo-electron microscopy, biochemical analysis, and crystal structure determination of several RP subunits has yielded a near-atomic-resolution view of much of the complex. Major new insights into chaperone-assisted proteasome assembly have also recently emerged. Here we review these novel findings. Expected final online publication date for the Annual Review of Biochemistry Volume 82 is June 02, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention. Li, T., Bourgeois, J.-P., Celli, S., Glacial, F., Le Sourd, A.-M., Mecheri, S., Weksler, B., Romero, I., Couraud, P.-O., Rougeon, F., and Lafaye, P. Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.
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S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a φ-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca(2+) ion coordination. A Ca(2+)-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology.
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We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.
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Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.
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Information about protein interactions is crucial for the understanding of cellular processes. Current methods for the investigation of protein-protein interactions (PPIs) require either removal of the proteins from their normal cellular environment, perturbation of the cells or costly instrumentation and advanced technical expertise (Fields and Song, Nature 340:245-246, 1989; Deane et al., Mol Cell Proteomics 1:349-356, 2002; Kerppola, Nat Rev Mol Cell Biol 7:449-456, 2006; Blanchard et al., Mol Cell Proteomics 5:2175-2184, 2006; Miller et al., Mol Cell Proteomics 6:1027-1038, 2007; Miyawaki, Dev Cell 4:295-305, 2003; Parrish et al., Curr Opin Biotechnol 17:387-393, 2006; Sekar and Periasamy, J Cell Biol 160:629-633, 2003). Here, we describe a simple assay to directly visualize and analyze PPIs in single living cells. By adapting a lac operator/repressor system, we generated a stable nuclear interaction platform. A fluorescent bait protein is tethered to the interaction platform and assayed for co-localization of fluorescent prey fusion proteins. This fluorescent two-hybrid (F2H) assay allows the investigation of cell cycle dependent PPIs. With this cell based assay protein interactions even from different subcellular compartments can be visualized in real time (Zolghadr et al., Mol Cell Proteomics 7:2279-2287, 2008). The simple optical readout enables automated imaging systems to segment and analyze the acquired data for high-throughput screening of PPIs in living cells in response to external stimuli and chemical compounds.
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The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio.
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The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (V(H)Hs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant V(H)Hs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant V(H)H pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the V(H)Hs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant V(H)H trypsin resistance was similar to that of wild-type V(H)Hs, although the trypsin resistance of one V(H)H mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(H)H thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(H)H stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.
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The polymeric immunoglobulin receptor (pIgR) ensures the transport of dimeric immunoglobulin A (dIgA) and pentameric immunoglobulin M (pIgM) across epithelia to the mucosal layer of for example the intestines and the lungs via transcytosis. Per day the human pIgR mediates the excretion of 2 to 5 grams of dIgA into the mucosa of luminal organs. This system could prove useful for therapies aiming at excretion of compounds into the mucosa. Here we investigated the use of the variable domain of camelid derived heavy chain only antibodies, also known as VHHs or Nanobodies®, targeting the human pIgR, as a transport system across epithelial cells. We show that VHHs directed against the human pIgR are able to bind the receptor with high affinity (∼1 nM) and that they compete with the natural ligand, dIgA. In a transcytosis assay both native and phage-bound VHH were only able to get across polarized MDCK cells that express the human pIgR gene in a basolateral to apical fashion. Indicating that the VHHs are able to translocate across epithelia and to take along large particles of cargo. Furthermore, by making multivalent VHHs we were able to enhance the transport of the compounds both in a MDCK-hpIgR and Caco-2 cell system, probably by inducing receptor clustering. These results show that VHHs can be used as a carrier system to exploit the human pIgR transcytotic system and that multivalent compounds are able to significantly enhance the transport across epithelial monolayers.