Preparation and Application of Clostridium perfringens Alpha Toxin Nanobodies.

All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 µg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC50). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD50) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD50 CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD50 CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products.

Single-Domain Antibodies as Antibody-Drug Conjugates: From Promise to Practice-A Systematic Review.

BACKGROUND: Antibody-drug conjugates (ADCs) represent potent cancer therapies that deliver highly toxic drugs to tumor cells precisely, thus allowing for targeted treatment and significantly reducing off-target effects. Despite their effectiveness, ADCs can face limitations due to acquired resistance and potential side effects. OBJECTIVES: This study focuses on advances in various ADC components to improve both the efficacy and safety of these agents, and includes the analysis of several novel ADC formats. This work assesses whether the unique features of VHHs-such as their small size, enhanced tissue penetration, stability, and cost-effectiveness-make them a viable alternative to conventional antibodies for ADCs and reviews their current status in ADC development. METHODS: Following PRISMA guidelines, this study focused on VHHs as components of ADCs, examining advancements and prospects from 1 January 2014 to 30 June 2024. Searches were conducted in PubMed, Cochrane Library, ScienceDirect and LILACS using specific terms related to ADCs and single-domain antibodies. Retrieved articles were rigorously evaluated, excluding duplicates and non-qualifying studies. The selected peer-reviewed articles were analyzed for quality and synthesized to highlight advancements, methods, payloads, and future directions in ADC research. RESULTS: VHHs offer significant advantages for drug conjugation over conventional antibodies due to their smaller size and structure, which enhance tissue penetration and enable access to previously inaccessible epitopes. Their superior stability, solubility, and manufacturability facilitate cost-effective production and expand the range of targetable antigens. Additionally, some VHHs can naturally cross the blood-brain barrier or be easily modified to favor their penetration, making them promising for targeting brain tumors and metastases. Although no VHH-drug conjugates (nADC or nanoADC) are currently in the clinical arena, preclinical studies have explored various conjugation methods and linkers. CONCLUSIONS: While ADCs are transforming cancer treatment, their unique mechanisms and associated toxicities challenge traditional views on bioavailability and vary with different tumor types. Severe toxicities, often linked to compound instability, off-target effects, and nonspecific blood cell interactions, highlight the need for better understanding. Conversely, the rapid distribution, tumor penetration, and clearance of VHHs could be advantageous, potentially reducing toxicity by minimizing prolonged exposure. These attributes make single-domain antibodies strong candidates for the next generation of ADCs, potentially enhancing both efficacy and safety.

A single domain antibody-based Luminex assay for the detection of SARS-CoV-2 in clinical samples.

Within the past decade, single domain antibodies (sdAbs) have been recognized as unique affinity binding reagents that can be tailored for performance in a variety of immunoassay formats. Luminex MagPlex color-coded magnetic microspheres provide a high-throughput platform that enables multiplexed immunoassays. We developed a MagPlex bead-based assay for the detection of SARS-CoV-2, using sdAbs against SARS-CoV-2 nucleocapsid (N) protein in which we engineered the sdAb capture reagents to orient them on the beads. The oriented sdAbs provided an increase in sensitivity over randomly oriented sdAbs for samples of N diluted in buffer, which also translated into better detection of SARS-CoV-2 in clinical samples. We assessed the specificity of the assay by examining seasonal coronavirus clinical samples. In summary, we provide a proof-of-concept that a bead-based assay using sdAbs to detect SARS-CoV-2 is feasible and future research combining it with other sdAb-coated beads that can detect other viruses may provide a useful diagnostic tool.

Development and characterization of nanobody against envenomation by Naja naja oxiana.

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

Non-invasive strategy: Developing a topical IL-4Rα-specific nanobody for the treatment of allergic airway diseases.

Inhibiting IL-4 and IL-13 are critical cytokines that induce the pathogenic responses of allergic airway diseases. Currently, monoclonal antibodies targeting IL-4Rα are administered subcutaneously to treat eosinophilic rhinosinusitis and allergic asthma. However, these treatments have several drawbacks. To address these issues, we have developed a novel IL-4Rα-targeting nanobody designed for non-invasive delivery to local inflammatory sites in allergic airway diseases. H5, selected via the ribosomal display applied screening from synthetic nanobody library, underwent dimerization and in-silico affinity maturation using AlphaFold2 and GROMACS resulting in a substantial/dramatic enhancement of its binding affinity. H5 effectively controlled inflammatory markers such as MUC5AC, CCL26, and FOXJ1 in human nasal epithelial cells (HNECs) by inhibiting IL-4 and IL-13 signaling. The bivalent form of H5 showed efficacy in easily accessible cells, such as multi-ciliated cells, while the monovalent variant targeted hard-to-reach cells, such as basal cells of HNECs. In summary, we developed a nanobody that could effectively inhibit inflammatory signaling in HNECs via intranasal administration, showing promise as a non-invasive rhinitis treatment.

Development of a synthetic library of humanized nanobodies for targeted IL-6 inhibition.

Interleukin-6 (IL-6) is a cytokine that can bind to IL-6 receptor and induce pleiotropic effects. It serves as a critical biomarker, involved in inflammation amplification, tumor progression, and many other disease developments. Nanobodies, featuring small structure and high affinity, are a powerful and versatile tool in medical diagnostics and therapeutics. Here, based on a scaffold optimized for humanization and stability, we developed a synthetic phage display library that rapidly generated high-affinity and humanized nanobodies, negating the need for animal immunization. Using enhanced green fluorescent protein (eGFP) as a benchmark, we demonstrated that the library produced humanized nanobodies with high function and great intracellular stability. The library was then subjected to screening against IL-6. We identified a standout nanobody, NbL3, which exhibited high affinity (22.16 nM) and stability and significantly inhibited IL-6-enhanced migration on the human breast cancer cell MCF-7 at a relatively low concentration. NbL3's strong blocking activity provides a promising therapeutic alternative for the IL-6-targeted intervention strategy, underscoring the broader potential of our synthetic library as a versatile platform for the development of humanized nanobodies against multiple antigens.

RadioLabeling of the Mutant form of Anti-PlGF Nanobody by 99mTc-Tricarbonyl.

Background: Molecular imaging is a highly effective method for diagnosing cancer and evaluating treatment. A molecular tracer often consists of two segments: A targeting segment, which can be antibodies, antibody fragments, or VHH (nanobody), and a detection segment, such as radioisotopes. The small size of VHH allows for excellent tissue penetration and fast clearance, resulting in minimal nonspecific background, which makes them appealing for use as imaging agents. 99m-technetium (99mTc), one of the well-known radioisotopes, is particularly useful in routine clinical imaging. Objectives: This study aims to construct 99mTc-anti-placenta growth factor (PlGF) nanobody and assess its radiochemical purity (RCP). Methods: The mutant form of anti-PlGF nanobody was expressed in E. coli TG1 and purified using Ni-NTA column affinity chromatography. The purified nanobodies were confirmed by SDS-PAGE and western blotting. A 99mTc-tricarbonyl solution was added to phosphate-buffered saline (PBS) containing the mutant nanobody for labeling, and the mixture was purified using a PD-10 column. Results: The RCP of 99mTc-tricarbonyl is > 98%. After the addition of radioisotopes to the mixture of nanobodies, purity reached 70% in 2 hours and remained constant during incubation. After purifying the labeled nanobody, activity was measured, and the highest amount of labeled nanobodies was collected in the second part. The stability of the labeled nanobody in PBS and in competition with histidine for 4 hours was checked by thin-layer chromatography (TLC). Conclusions: The findings of this study reveal that the RCP of the labeled nanobody was above 95% after 4 hours, indicating the labeled antibody's stability. These results are promising and could be utilized in future in vitro and in vivo studies.

Single domain antibody: Development and application in biotechnology and biopharma.

Heavy-chain antibodies (HCAbs) are a unique type of antibodies devoid of light chains, and comprised of two heavy chains-only that recognize their cognate antigen by virtue of a single variable domain also referred to as VHH, single domain antibody (sdAb), or nanobody (Nb). These functional HCAbs, serendipitous discovered about three decades ago, are exclusively found in camelids, comprising dromedaries, camels, llamas, and vicugnas. Nanobodies have become an essential tool in biomedical research and medicine, both in diagnostics and therapeutics due to their beneficial properties: small size, high stability, strong antigen-binding affinity, low immunogenicity, low production cost, and straightforward engineering into more potent affinity reagents. The occurrence of HCAbs in camelids remains intriguing. It is believed to be an evolutionary adaptation, equipping camelids with a robust adaptive immune defense suitable to respond to the pressure from a pathogenic invasion necessitating a more profound antigen recognition and neutralization. This evolutionary innovation led to a simplified HCAb structure, possibly supported by genetic mutations and drift, allowing adaptive mutation and diversification in the heavy chain variable gene and constant gene regions. Beyond understanding their origins, the application of nanobodies has significantly advanced over the past 30 years. Alongside expanding laboratory research, there has been a rapid increase in patent application for nanobodies. The introduction of commercial nanobody drugs such as Cablivi, Nanozora, Envafolimab, and Carvykti has boosted confidence among in their potential. This review explores the evolutionary history of HCAbs, their ontogeny, and applications in biotechnology and pharmaceuticals, focusing on approved and ongoing medical research pipelines.

Versatility and stability of melamine foam-based biosensors (f-ELISA) using antibodies, nanobodies, and peptides as sensing probes.

Macroporous three-dimensional (3D) framework structured melamine foam-based Enzyme-Linked Immunosorbent Assay (f-ELISA) biosensors were developed for rapid, reliable, sensitive, and on-site detection of trace amount of biomolecules and chemicals. Various ligands can be chemically immobilized onto the melamine foam, which brings in the possibility of working with antibodies, nanobodies, and peptides, respectively, as affinity probes for f-ELISA biosensors with improved stability. Different chemical reagents can be used to modify the foam materials, resulting in varied reactivities with antibodies, nanobodies, and peptides. As a result, the f-ELISA sensors produced from these modified foams exhibit varying levels of sensitivity and performance. This study demonstrated that the chemical reagents used for immobilizing antibodies, nanobodies, and peptides could affect the sensitivities of the f-ELISA sensors, and their storage stabilities under different temperatures varied depending on the sensing probes used, with f-ELISA sensors employing nanobodies as probes exhibiting the highest stability. This study not only showcases the versatility of the f-ELISA system but also opens new avenues for developing cost-effective, portable, and user-friendly diagnostic tools with optimized sensitivity and stability.

Chemo-enzymatic Functionalization of Bovine Milk Exosomes with an EGFR Nanobody for Target-specific Drug Delivery.

Bovine milk exosomes (BmExo) have been identified as versatile nanovesicles for anti-cancer drugs delivery due to their natural availability and biocompatibility. However, tumor-specific delivery based on BmExo often requires post-isolation modifications of the membrane surface with active-targeting ligands. In this study, we report an alternative approach to functionalize BmExo with nanobody using Sortase A-mediated site-specific ligation for drug delivery. The BmExo membrane was first coated with a diglycine-containing amphiphile molecule, NH2-GG-PEG2000-DSPE, through hydrophobic insertion, following by ligation with EGFR nanobody (7D12) by Sortase A (SrtA). The successful construction of BmExo-7D12 was confirmed by Western blotting analysis, electron microscopy, and dynamic light scattering (DLS). As a demonstration model, BmExo-7D12 loaded with the chemotherapeutic drug doxorubicin (Dox) was shown to be able to deliver Dox to cancer cells in response to the expression of EGFR as manifested by immunocytochemistry and flow cytometry analysis. Finally, the cytotoxicity assay showed that BmExo-7D12-Dox was more effective in killing tumor cells with high EGFR expression while significantly reduced the non-specific toxicity to EGFR negative cells. This study developed an effective approach to functionalize BmExo with nanobody for target-specific drug delivery. This approach should prove to be versatile and efficient for the generation of protein-ligands modified BmExo.

Alpaca-derived nanobody targeting the hydrophobic pocket of the HIV-1 gp41 NHR broadly neutralizes HIV-1 by blocking six-helix bundle formation.

The highly conserved hydrophobic pocket region of HIV-1 gp41 NHR triple-stranded coiled coil is crucial for the binding of CHR to NHR to form a six-helix bundle (6-HB). This pocket is only exposed instantaneously during fusion, making it an ideal target for antibody drug design. However, IgG molecule is too big to enter the pocket during the fusion process. Therefore, to overcome the steric hindrance and kinetic obstacles caused by the formation of gp41 pre-hairpin fusion intermediate, we obtained nanobodies (Nbs) targeting NHR by immunizing alpaca with an NHR-trimer mimic. Specifically, we identified a Nb, Nb-172, that exhibited potent and broadly neutralizing activity against HIV-1 pseudoviruses, HIV-1 primary isolates, and T20-resistant HIV-1 strains. In addition, the combinatorial use of mD1.22 and Nb-172 exhibited synergism in inhibiting HIV-1 infection inactivating cell-free virions. Nb-172 can competitively bind to the hydrophobic pocket of gp41 NHR to inhibit 6-HB formation. These findings suggest that Nb-172 merits further investigation as a potential therapeutic for HIV-1 infection.

Artificial targeting of autophagy components to mitochondria reveals both conventional and unconventional mitophagy pathways.

Macroautophagy/autophagy enables lysosomal degradation of a diverse array of intracellular material. This process is essential for normal cellular function and its dysregulation is implicated in many diseases. Given this, there is much interest in understanding autophagic mechanisms of action in order to determine how it can be best targeted therapeutically. In mitophagy, the selective degradation of mitochondria via autophagy, mitochondria first need to be primed with signals that allow the recruitment of the core autophagy machinery to drive the local formation of an autophagosome around the target mitochondrion. To determine how the recruitment of different core autophagy components can drive mitophagy, we took advantage of the mito-QC mitophagy assay (an outer mitochondrial membrane-localized tandem mCherry-GFP tag). By tagging autophagy proteins with an anti-mCherry (or anti-GFP) nanobody, we could recruit them to mitochondria and simultaneously monitor levels of mitophagy. We found that targeting ULK1, ATG16L1 and the different Atg8-family proteins was sufficient to induce mitophagy. Mitochondrial recruitment of ULK1 and the Atg8-family proteins induced a conventional mitophagy pathway, requiring RB1CC1/FIP200, PIK3C3/VPS34 activity and ATG5. Surprisingly, the mitophagy pathway upon recruitment of ATG16L1 proceeded independently of ATG5, although it still required RB1CC1 and PIK3C3/VPS34 activity. In this latter pathway, mitochondria were alternatively delivered to lysosomes via uptake into early endosomes.

Incompatibility in cell adhesion constitutes a barrier to interspecies chimerism.

Interspecies blastocyst complementation holds great potential to address the global shortage of transplantable organs by growing human organs in animals. However, a major challenge in this approach is the limited chimerism of human cells in evolutionarily distant animal hosts due to various xenogeneic barriers. Here, we reveal that human pluripotent stem cells (PSCs) struggle to adhere to animal PSCs. To overcome this barrier, we developed a synthetic biology strategy that leverages nanobody-antigen interactions to enhance interspecies cell adhesion. We engineered cells to express nanobodies and their corresponding antigens on their outer membranes, significantly improving adhesion between different species' PSCs during in vitro assays and increasing the chimerism of human PSCs in mouse embryos. Studying and manipulating interspecies pluripotent cell adhesion will provide valuable insights into cell interaction dynamics during chimera formation and early embryogenesis.

Streamlined on-column refolding and purification of nanobodies from inclusion bodies expressed as fusion proteins.

This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrapTM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli. This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli. Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.

Construction and preclinical evaluation of a 124I-labelled bispecific antibody targeting PD-L1 and PD-L2.

PURPOSE: NB12 is a bispecific antibody that consists of two anti-programmed cell death-ligand 1 (PD-L1) nanobodies and two anti-programmed cell death-ligand 2 (PD-L2) nanobodies. The aim of this study was to design a novel tracer, [124I]I-NB12, targeting PD-L1/2 and perform preclinical evaluations to dynamically monitor PD-L1/2 expression for determining cancer patient responsiveness to ICI therapy. METHODS: NB12 was labelled with the radionuclide 124I at room temperature (RT). An in vitro binding assay was performed to assess the affinity of [124I]I-NB12 for PD-L1 and PD-L2. Cellular uptake, pharmacokinetic, and biodistribution experiments were performed to evaluate the biological properties. Micro-PET/CT imaging with [124I]I-NB12 was conducted at different time points. Immunohistochemical and haematoxylin and eosin (HE) staining experiments were carried out using tumour tissues. Routine blood, biochemical indices and major organ pathology were used to evaluate the biosafety of the tracers. RESULTS: The radiochemical yield of [124I]I-NB12 was 84.62 ± 3.90%, and the radiochemical purity (RCP) was greater than 99%. [124I]I-NB12 had a high affinity for the PD-L1 (Kd = 19.82 nM) and PD-L2 (Kd = 2.93 nM). Cellular uptake experiments confirmed that the uptake of [124I]I-NB12 by A549-PDL1/2 cells was greater than that by A549 cells. The half-lives of the distribution phase and elimination phase were 0.26 h and 4.08 h, respectively. Micro-PET/CT showed significant [124I]I-NB12 uptake in the tumour region of A549-PDL1/2 tumour-bearing mice compared with A549 tumour-bearing mice 24 h postinjection. Immunohistochemical and HE staining experiments confirmed that tumour-bearing mice was successfully constructed. CONCLUSION: We constructed a bispecific antibody that targets PD-L1 and PD-L2, namely, [124I]I-NB12. Biological evaluation revealed its specificity and affinity for PD-L1/2, and micro-PET/CT confirmed the feasibility of visualizing tumour PD-L1/2 in vivo. Using [124I]I-NB12 may be a promising strategy for identifying cancer patients that can potentially benefit from ICI therapy.

Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism.

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or ß-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

A single-domain antibody library based on a stability-engineered human VH3 scaffold.

Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.

Anti-Inflammatory Efficacy of Nanobody Specific to ß-Glucan on a Fungal Cell Wall in a Murine Model of Fungal Keratitis.

Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to ß-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin-eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.

Monovalent, bivalent and biparatopic nanobodies targeting S1 protein of porcine epidemic diarrhea virus efficiently neutralized the virus infectivity.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets, which has brought huge economic losses to the pork industry worldwide since its first discovery in the early 1970s in Europe. Passive immunization with neutralizing antibodies against PEDV is an effective prevention measure. To date, there are no effective therapeutic drugs to treat the PEDV infection. RESULTS: We conducted a screening of specific nanobodies against the S1 protein from a phage display library obtained from immunized alpacas. Through competitive binding to antigenic epitopes, we selected instead of chose nanobodies with high affinity and constructed a multivalent tandem. These nanobodies were shown to inhibit PEDV infectivity by the neutralization assay. The antiviral capacity of nanobody was found to display a dose-dependent pattern, as demonstrated by IFA, TCID50, and qRT-PCR analyses. Notably, biparatopic nanobody SF-B exhibited superior antiviral activity. Nanobodies exhibited low cytotoxicity and high stability even under harsh temperature and pH conditions, demonstrating their potential practical applicability to animals. CONCLUSIONS: Nanobodies exhibit remarkable biological properties and antiviral effects, rendering them a promising candidate for the development of anti-PEDV drugs.

Shark anti-idiotypic variable new antigen receptor specific for an alpaca nanobody: Exploration of a nontoxic substitute to ochratoxin A in immunoassay.

Nontoxic substitutes to mycotoxins can facilitate the development of eco-friendly immunoassays. To explore a novel nontoxic substitute to ochratoxin A (OTA), this study screened shark anti-idiotypic variable new antigen receptors (VNARs) against the alpaca anti-OTA nanobody Nb28 through phage display. After four rounds of biopanning of a naïve VNAR phage display library derived from six adult Chiloscyllium plagiosum sharks, one positive clone, namely, P-3, was validated through a phage enzyme-linked immunosorbent assay (phage ELISA). The recombinant anti-idiotypic VNAR AId-V3 was obtained by prokaryotic expression, and the interactions between Nb28 and AId-V3 were investigated via computer-assisted simulation. The affinity of AId-V3 for Nb28 and its heptamer Nb28-C4bpα was measured using Biacore assay. Combining Nb28-C4bpα with AId-V3, a novel direct competitive ELISA (dcELISA) was developed for OTA analysis, with a limit of detection of 0.44 ng/mL and a linear range of 1.77-32.25 ng/mL. The good selectivity, reliability, and precision of dcELISA were confirmed via cross-reaction analysis and recovery experiments. Seven commercial pepper powder samples were tested using dcELISA and validated using high-performance liquid chromatography. Overall, the shark anti-idiotypic VNAR was demonstrated as a promising nontoxic substitute to OTA, and the proposed method was confirmed as a reliable tool for detecting OTA in food.