RESUMEN
Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD â¼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
Asunto(s)
Anticuerpos de Dominio Único , Canales de Sodio Activados por Voltaje , Animales , Células Cultivadas , Escherichia coli/genética , Humanos , Síndrome de QT Prolongado/metabolismo , Mamíferos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.
La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.
Asunto(s)
Anticuerpos Antivirales , Infecciones por Coronavirus/terapia , Sueros Inmunes/inmunología , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Pandemias , Neumonía Viral , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Argentina , Betacoronavirus , COVID-19 , Caballos , Humanos , Inmunización Pasiva , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Pruebas de Neutralización , SARS-CoV-2 , Sueroterapia para COVID-19RESUMEN
The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.
La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.
Asunto(s)
Humanos , Animales , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Infecciones por Coronavirus/terapia , Sueros Inmunes/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Argentina , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/química , Fragmentos Fab de Inmunoglobulinas/química , Pruebas de Neutralización , Pandemias , Betacoronavirus , SARS-CoV-2 , COVID-19 , CaballosRESUMEN
Vaccination is as one of the most beneficial biopharmaceutical interventions against pathogens due to its ability to induce adaptive immunity through targeted activation of the immune system. Each vaccine needs a tailor-made set of tests in order to monitor its quality throughout the development and manufacturing. The analysis of the conformational state of protein nanoparticles is one of the key steps in vaccine quality control. The enzyme lumazine synthase from Brucella spp. (BLS) acts as a potent oral and systemic immunogen. BLS has been used as a carrier of foreign peptides, protein domains and whole proteins, serving as a versatile platform for vaccine engineering purposes. Here, we show the generation and characterization of four families of nanobodies (Nbs) which only recognize BLS in its native conformational state and that bind to its active site. The present results support the use of conformation-sensitive Nbs as molecular probes during the development and production of vaccines based on the BLS platform. Finally, we propose Nbs as useful molecular tools targeting other protein scaffolds with potential applications in nano-and biotechnology.
Asunto(s)
Complejos Multienzimáticos , Anticuerpos de Dominio Único , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Brucella/enzimología , Escherichia coli/genética , Células HEK293 , Humanos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/fisiología , Conformación Proteica , Pliegue de Proteína , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/fisiología , Vacunas de SubunidadRESUMEN
The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Anticuerpos de Dominio Único/inmunología , ADP Ribosa Transferasas/inmunología , ADP Ribosa Transferasas/toxicidad , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/toxicidad , Supervivencia Celular/efectos de los fármacos , Clostridioides difficile/patogenicidad , Perros , Mapeo Epitopo , Epítopos/inmunología , Células HT29 , Humanos , Células de Riñón Canino Madin Darby , Microscopía de Interferencia , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/toxicidad , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genéticaRESUMEN
Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Complejos Multienzimáticos/inmunología , Receptor Toll-Like 4/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Biopolímeros , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Colorantes Fluorescentes , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
The polymeric display of proteins is a method that could be used to increase the immunogenicity of antigens and to enhance the interaction strength of binding domains for their target ligands through an avidity effect. However, the coupling of proteins to oligomeric scaffolds is challenging. The chemical conjugation and recombinant fusion techniques have limitations that prevent their general use. In this work we describe a simple and effective method for coupling proteins to the decameric structure of Brucella abortus Lumazine Synthase based on the use of a pair of high affinity heterodimeric coiled coil peptides complementary fused to the scaffold and the target protein. Results obtained with a series of proteins demonstrate the capability of this approach to generate polyvalent particles. Furthermore, we show that the method is able to increase the immunogenicity of antigens and produce polyfunctional particles with promising biomedical and nanotechnological applications.
Asunto(s)
Biopolímeros/química , Leucina Zippers , Péptidos/química , Proteínas/química , Brucella abortus/enzimología , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Desnaturalización Proteica , TemperaturaRESUMEN
ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Anticuerpos Antibacterianos/inmunología , Toxinas Bacterianas/metabolismo , Camélidos del Nuevo Mundo/inmunología , Salmonella typhimurium/metabolismo , Factores de Virulencia/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/inmunología , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Regulación de la Expresión Génica , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Salmonella typhimurium/inmunología , Células VeroRESUMEN
Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.
Asunto(s)
Anticuerpos/química , Enfermedad Celíaca/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Gliadina/sangre , Secuencia de Aminoácidos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Modelos Animales de Enfermedad , Gliadina/administración & dosificación , Gliadina/química , Humanos , Masculino , Datos de Secuencia Molecular , Unión Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.
Asunto(s)
Anticuerpos/química , Cadenas Pesadas de Inmunoglobulina/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Camélidos del Nuevo Mundo/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Conformación Molecular , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Tiburones/inmunologíaRESUMEN
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 P2x7 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.