RESUMEN

Factor XII (FXII) is the zymogen of the plasma protease FXIIa that activates the intrinsic coagulation pathway and the kallikrein kinin-system. The role of FXII in inflammation has been obscure. Here, we report a single-domain antibody (nanobody, Nb) fused to the Fc region of a human immunoglobulin (Nb-Fc) that recognizes FXII in a conformation-dependent manner and interferes with FXIIa formation. Nb-Fc treatment inhibited arterial thrombosis in male mice without affecting hemostasis. In a mouse model of extracorporeal membrane oxygenation (ECMO), FXII inhibition or knockout reduced thrombus deposition on oxygenator membranes and systemic microvascular thrombi. ECMO increased circulating levels of D-dimer, alkaline phosphatase, creatinine and TNF-α and triggered microvascular neutrophil adherence, platelet aggregation and their interaction, which were substantially attenuated by FXII blockade. Both Nb-Fc treatment and FXII knockout markedly ameliorated immune complex-induced local vasculitis and anti-neutrophil cytoplasmic antibody-induced systemic vasculitis, consistent with selectively suppressed neutrophil migration. In human blood microfluidic analysis, Nb-Fc treatment prevented collagen-induced fibrin deposition and neutrophil adhesion/activation. Thus, FXII is an important mediator of inflammatory responses in vasculitis and ECMO, and Nb-Fc provides a promising approach to alleviate thrombo-inflammatory disorders.

Asunto(s)

Factor XII , Inflamación , Ratones Noqueados , Neutrófilos , Anticuerpos de Dominio Único , Trombosis , Animales , Humanos , Trombosis/inmunología , Trombosis/metabolismo , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Masculino , Factor XII/metabolismo , Factor XII/antagonistas & inhibidores , Inflamación/metabolismo , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Agregación Plaquetaria/efectos de los fármacos , Factor XIIa/metabolismo , Factor XIIa/antagonistas & inhibidores , Fibrina/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo

RESUMEN

Oral delivery of proteins faces challenges due to the harsh conditions of the gastrointestinal (GI) tract, including gastric acid and intestinal enzyme degradation. Permeation enhancers are limited in their ability to deliver proteins with high molecular weight and can potentially cause toxicity by opening tight junctions. To overcome these challenges, we propose the use of montmorillonite (MMT) as an adjuvant that possesses both inflammation-oriented abilities and the ability to regulate gut microbiota. This adjuvant can be used as a universal protein oral delivery technology by fusing with advantageous binding amino acid sequences. We demonstrated that anti-TNF-α nanobody (VII) can be intercalated into the MMT interlayer space. The carboxylate groups (-COOH) of aspartic acid (D) and glutamic acid (E) interact with the MMT surface through electrostatic interactions with sodium ions (Na+). The amino groups (NH2) of asparagine (N) and glutamine (Q) are primarily attracted to the MMT layers through hydrogen bonding with oxygen atoms on the surface. This binding mechanism protects VII from degradation and ensures its release in the intestinal tract, as well as retaining biological activity, leading to significantly enhanced therapeutic effects on colitis. Furthermore, VII@MMT increases the abundance of short-chain fatty acids (SCFAs)-producing strains, including Clostridia, Prevotellaceae, Alloprevotella, Oscillospiraceae, Clostridia_vadinBB60_group, and Ruminococcaceae, therefore enhance the production of SCFAs and butyrate, inducing regulatory T cells (Tregs) production to modulate local and systemic immune homeostasis. Overall, the MMT adjuvant provides a promising universal strategy for protein oral delivery by rational designed protein.

Asunto(s)

Bentonita , Microbioma Gastrointestinal , Factor de Necrosis Tumoral alfa , Bentonita/química , Animales , Administración Oral , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Microbioma Gastrointestinal/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/inmunología , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Humanos , Inflamación/tratamiento farmacológico , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología

RESUMEN

COVID-19 has caused millions of deaths and many times more infections worldwide, emphasizing the unpreparedness of the global health system in the face of new infections and the key role for vaccines and therapeutics, including virus-neutralizing antibodies, in prevention and containment of the disease. Continuous evolution of the SARS-CoV-2 coronavirus has been causing its new variants to evade the action of the immune system, which highlighted the importance of detailed knowledge of the epitopes of already selected potent virus-neutralizing antibodies. A single-chain antibody ("nanobody") targeting the SARS-CoV-2 receptor-binding domain (RBD), clone P2C5, had exhibited robust virus-neutralizing activity against all SARS-CoV-2 variants and, being a major component of the anti-COVID-19 formulation "GamCoviMab", had successfully passed Phase I of clinical trials. However, after the emergence of the Delta and XBB variants, a decrease in the neutralizing activity of this nanobody was observed. Here we report on the successful crystal structure determination of the RBD:P2C5 complex at 3.1 Å, which revealed the intricate protein-protein interface, sterically occluding full ACE2 receptor binding by the P2C5-neutralized RBD. Moreover, the structure revealed the developed RBD:P2C5 interface centered around residues Leu452 and Phe490, thereby explaining the evasion of the Delta or Omicron XBB, but not Omicron B.1.1.529 variant, as a result of the single L452R or F490S mutations, respectively, from the action of P2C5. The structure obtained is expected to foster nanobody engineering in order to rescue neutralization activity and will facilitate epitope mapping for other neutralizing nanobodies by competition assays.

Asunto(s)

Anticuerpos Neutralizantes , SARS-CoV-2 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/química , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Dominios Proteicos , Unión Proteica , Epítopos/inmunología , Epítopos/química , Modelos Moleculares , Evasión Inmune , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Sitios de Unión

RESUMEN

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro. We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro. We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.

Asunto(s)

Neoplasias Pulmonares , Anticuerpos de Dominio Único , ATPasas de Translocación de Protón Vacuolares , Animales , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Línea Celular Tumoral , Neoplasias de la Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Ratones Endogámicos BALB C , Humanos , Invasividad Neoplásica

RESUMEN

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.

Asunto(s)

Antivenenos , Venenos Elapídicos , Naja naja , Anticuerpos de Dominio Único , Mordeduras de Serpientes , Animales , Venenos Elapídicos/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Ratones , Antivenenos/farmacología , Antivenenos/inmunología , Mordeduras de Serpientes/tratamiento farmacológico , Camelus , Cromatografía Líquida de Alta Presión , Pruebas de Neutralización

RESUMEN

Despite advancements in antiretroviral therapy (ART) suppressing HIV-1 replication, existing antiviral drugs pose limitations, including lifelong medication, frequent administration, side effects and viral resistance, necessitating novel HIV-1 treatment approaches. CD4, pivotal for HIV-1 entry, poses challenges for drug development due to neutralization and cytotoxicity concerns. Nevertheless, Ibalizumab, the sole approved CD4-specific antibody for HIV-1 treatment, reignites interest in exploring alternative anti-HIV targets, emphasizing CD4's potential value for effective drug development. Here, we explore anti-CD4 nanobodies, particularly Nb457 from a CD4-immunized alpaca. Nb457 displays high potency and broad-spectrum activity against HIV-1, surpassing Ibalizumab's efficacy. Strikingly, engineered trimeric Nb457 nanobodies achieve complete inhibition against live HIV-1, outperforming Ibalizumab and parental Nb457. Structural analysis unveils Nb457-induced CD4 conformational changes impeding viral entry. Notably, Nb457 demonstrates therapeutic efficacy in humanized female mouse models. Our findings highlight anti-CD4 nanobodies as promising HIV-1 therapeutics, with potential implications for advancing clinical treatment against this global health challenge.

Asunto(s)

Antígenos CD4 , Camélidos del Nuevo Mundo , Anticuerpos Anti-VIH , Infecciones por VIH , VIH-1 , Anticuerpos de Dominio Único , VIH-1/inmunología , VIH-1/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Humanos , Infecciones por VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Camélidos del Nuevo Mundo/inmunología , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/farmacología , Ratones , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Conformación Proteica , Femenino , Internalización del Virus/efectos de los fármacos , Células HEK293 , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Anticuerpos Monoclonales

RESUMEN

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.

Asunto(s)

Receptores CXCR4 , Anticuerpos de Dominio Único , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Receptores CXCR4/inmunología , Humanos , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Animales , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/inmunología , Células HEK293 , Afinidad de Anticuerpos

RESUMEN

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.

Asunto(s)

Antiinflamatorios , Aspergillus fumigatus , Modelos Animales de Enfermedad , Queratitis , Anticuerpos de Dominio Único , beta-Glucanos , Animales , Queratitis/tratamiento farmacológico , Queratitis/microbiología , Ratones , beta-Glucanos/farmacología , Antiinflamatorios/farmacología , Humanos , Anticuerpos de Dominio Único/farmacología , Pared Celular/química , Aspergilosis/tratamiento farmacológico , Aspergilosis/microbiología , Aspergilosis/inmunología , Córnea/efectos de los fármacos , Citocinas/metabolismo , Macrófagos/efectos de los fármacos , Infecciones Fúngicas del Ojo/tratamiento farmacológico , Infecciones Fúngicas del Ojo/microbiología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología

RESUMEN

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Asunto(s)

Farmacorresistencia Bacteriana , Plásmidos , Animales , Plásmidos/genética , Ratones , Farmacorresistencia Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Antibacterianos/farmacología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/farmacología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Femenino , Salmonella/genética , Salmonella/inmunología , Salmonella/efectos de los fármacos , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Ratones Endogámicos BALB C

RESUMEN

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Asunto(s)

Infecciones por Caliciviridae , Proteínas de la Cápside , Norovirus , Anticuerpos de Dominio Único , Norovirus/genética , Norovirus/efectos de los fármacos , Norovirus/inmunología , Humanos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/terapia , Antivirales/farmacología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/química , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Cápside/metabolismo , Cápside/inmunología , Antígenos de Grupos Sanguíneos/metabolismo , Replicación Viral/efectos de los fármacos , Gastroenteritis/virología , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología

RESUMEN

Tauopathies are a group of neurodegenerative diseases characterized by the presence of tau inclusions. We have developed over fifty anti-tau single-domain antibodies (sdAbs) derived from phage display libraries of a llama immunized with recombinant and pathological tau immunogens. We examined the therapeutic potential of four of these sdAbs in a Drosophila tauopathy model following their transgenic expression either in all neurons or neuronal subtypes. Three of these sdAbs showed therapeutic potential in various assays, effectively clearing pathological tau and attenuating or preventing tau-induced phenotypes that typically manifest as defects in neuronal axonal transport, neurodegeneration, functional impairments, and shortened lifespan. Of these three, one sdAb was superior in every assay, which may at least in part be attributed to its tau-binding epitope. These findings support its development as a gene therapy for tauopathies.

Asunto(s)

Anticuerpos de Dominio Único , Tauopatías , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/inmunología , Animales , Tauopatías/inmunología , Tauopatías/patología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Humanos , Modelos Animales de Enfermedad , Drosophila , Animales Modificados Genéticamente , Neuronas/metabolismo , Neuronas/patología , Camélidos del Nuevo Mundo/inmunología , Drosophila melanogaster/inmunología

RESUMEN

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.

Asunto(s)

Anticuerpos Neutralizantes , Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Anticuerpos de Dominio Único , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Camélidos del Nuevo Mundo/inmunología , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Virus de la Diarrea Epidémica Porcina/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/inmunología , Porcinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Células Vero

RESUMEN

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

Asunto(s)

Anticuerpos Neutralizantes , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Péptidos , Virus del Síndrome Respiratorio y Reproductivo Porcino , Receptores de Superficie Celular , Anticuerpos de Dominio Único , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/efectos de los fármacos , Animales , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Porcinos , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Receptores de Superficie Celular/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Anticuerpos Neutralizantes/inmunología , Péptidos/química , Péptidos/farmacología , Péptidos/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Ratones , Replicación Viral/efectos de los fármacos , Línea Celular

RESUMEN

In planta expression of recombinant antibodies has been proposed as a strategy for herbicide resistance but is not well advanced yet. Here, an atrazine nanobody gene fused with a green fluorescent protein tag was transformed to Arabidopsis thaliana, which was confirmed with PCR, ELISA, and immunoblotting. High levels of nanobody accumulation were observed in the nucleus, cytoderm, and cytosol. The nanobody expressed in the plant had similar affinity, sensitivity, and selectivity as that expressed in Escherichia coli. The T3 homozygous line showed resistance in a dose-dependent manner up to 380 g ai/ha of atrazine, which is approximately one-third of the recommended field application rate. This is the first report of utilizing a nanobody in plants against herbicides. The results suggest that utilizing a high-affinity herbicide nanobody gene rather than increasing the expression of nanobodies in plants may be a technically viable approach to acquire commercial herbicide-resistant crops and could be a useful tool to study plant physiology.

Asunto(s)

Arabidopsis , Atrazina , Resistencia a los Herbicidas , Herbicidas , Plantas Modificadas Genéticamente , Anticuerpos de Dominio Único , Atrazina/farmacología , Herbicidas/farmacología , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Resistencia a los Herbicidas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/inmunología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/inmunología

RESUMEN

Invasive fungal disease accounts for about 3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insight about the role of ß-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.

Asunto(s)

Aspergillus fumigatus , Dominio Catalítico , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Aspergillus fumigatus/inmunología , Aspergillus fumigatus/enzimología , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/inmunología , Antifúngicos/química , Antifúngicos/farmacología , Animales , Ratones , Glucano Endo-1,3-beta-D-Glucosidasa

RESUMEN

OBJECTIVE: To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells. METHODS: Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjµgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 µg/mL. CONCLUSION: The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.

Asunto(s)

Proteína del Gen 3 de Activación de Linfocitos , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/farmacología , Humanos , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos CD/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/química , Animales , Biblioteca de Péptidos , Camelus/inmunología , Camelus/genética , Línea Celular Tumoral , Escherichia coli/genética , Linfocitos T/inmunología , Expresión Génica

RESUMEN

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and ß-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and ß-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Asunto(s)

Antibacterianos , Carbapenémicos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Anticuerpos de Dominio Único , Pseudomonas aeruginosa/efectos de los fármacos , Animales , Carbapenémicos/farmacología , Antibacterianos/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Ratones , Anticuerpos de Dominio Único/farmacología , Pruebas de Sensibilidad Microbiana , Camélidos del Nuevo Mundo , Farmacorresistencia Bacteriana

RESUMEN

Ulcerative colitis (UC) manifests clinically with chronic intestinal inflammation and microflora dysbiosis. Although biologics can effectively control inflammation, efficient delivery to the colon and colon epithelial cells remains challenging. Milk-derived extracellular vesicles (EV) show promise as an oral delivery tool, however, the ability to load biologics into EV presents challenges to therapeutic applications. Here, we demonstrate that fusing cell-penetrating peptide (TAT) to green fluorescent protein (GFP) enabled biologics loading into EV and protected against degradation in the gastrointestinal environment in vitro and in vivo after oral delivery. Oral administration of EV loaded with anti-tumour necrosis factor-α (TNF-α) nanobody (VHHm3F) (EVVHH) via TAT significantly reduced tissue TNF-α levels and alleviated pathologies in mice with acute UC, compared to VHH alone. In mice with chronic UC, simultaneously introducing VHH and an antimicrobial peptide LL37 into EV (EVLV), then administering orally improved intestinal barrier, inflammation and microbiota balance, resulted in relief of UC-induced depression and anxiety. Collectively, we demonstrated that oral delivery of EVLV effectively alleviated UC in mice and TAT efficiently loaded biologics into EV to confer protection from degradation in the gastrointestinal tract. This therapeutic strategy is promising for UC and is a simple and generalizable approach towards drug-loaded orally-administrable EV treatment for other diseases.

Asunto(s)

Colitis Ulcerosa , Vesículas Extracelulares , Leche , Anticuerpos de Dominio Único , Factor de Necrosis Tumoral alfa , Animales , Colitis Ulcerosa/tratamiento farmacológico , Vesículas Extracelulares/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/uso terapéutico , Péptidos Antimicrobianos/farmacología , Catelicidinas , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Péptidos de Penetración Celular/farmacología , Humanos , Administración Oral , Masculino , Femenino

RESUMEN

Pentameric ligand-gated ion channels provide rapid chemical-electrical signal transmission between cells in the central and peripheral nervous system. Their dysfunction is associated with many nervous system disorders. They are composed of five identical (homomeric receptors) or homologous (heteromeric receptors) subunits. VHH nanobodies, or single-chain antibodies, are the variable domain, VHH, of antibodies that are composed of the heavy chain only from camelids. Their unique structure results in many specific biochemical and biophysical properties that make them an excellent alternative to conventional antibodies. This Perspective explores the published VHH nanobodies which have been isolated against pentameric ligand-gated ion channel subfamilies. It outlines the genetic and chemical modifications available to alter nanobody function. An assessment of the available functional and structural data indicate that it is feasible to create therapeutic agents and impart, through their modification, a given desired modulatory effect of its target receptor for current stoichiometric-specific VHH nanobodies.

Asunto(s)

Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología , Humanos , Animales , Canales Iónicos Activados por Ligandos/metabolismo , Canales Iónicos Activados por Ligandos/inmunología , Canales Iónicos Activados por Ligandos/química , Modelos Moleculares

RESUMEN

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

Asunto(s)

Amiloide , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/antagonistas & inhibidores , Humanos , Amiloide/metabolismo , Amiloide/antagonistas & inhibidores , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Unión Proteica