RESUMO
Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21-99.98) and specificity (100%, 95% CI 96.31-100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
Assuntos
Testes Imunológicos/métodos , Reoviridae , Proteínas Virais , Zea mays/virologia , Animais , Camelídeos Americanos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Doenças das Plantas/virologia , Plantas , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reoviridae/imunologia , Reoviridae/isolamento & purificação , Reoviridae/metabolismo , Proteínas Virais/análise , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Snake envenoming is a globally neglected public health problem. Antivenoms produced using animal hyperimmune plasma remain the standard therapy for snakebites. Although effective against systemic effects, conventional antivenoms have limited efficacy against local tissue damage. In addition, potential hypersensitivity reactions, high costs for animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are some of the factors that have motivated the search for innovative and improved therapeutic products against such envenoming. In this study, we have developed a set of nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic components. An immune library was constructed after immunizing a Lama glama with whole venom of B. atrox, from which nanobodies were selected by phage display using partially purified hemorrhagic and myotoxic proteins. Biopanning selections retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage and myonecrosis induced by the whole venom, although the nanobody mixture failed to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and usefulness of these nanobodies to neutralize important pathologies of the venom, highlighting their potential as innovative therapeutic agents against envenoming by B. atrox, a viperid species causing many casualties in South America.
Assuntos
Antivenenos/uso terapêutico , Bothrops/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/imunologia , Hemorragia/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Miotoxicidade/tratamento farmacológico , Anticorpos de Domínio Único/uso terapêutico , Mordeduras de Serpentes/tratamento farmacológico , Animais , Camelídeos Americanos/imunologia , Imunização/métodos , Masculino , Camundongos , Resultado do TratamentoRESUMO
BACKGROUND: The diagnosis of active Toxocara canis infections in humans is challenging. Larval stages of T. canis do not replicate in human tissues and disease may result from infection with a single T. canis larva. Recently, we developed a nanobody-based electrochemical magnetosensor assay with superior sensitivity to detect T. canis excretory-secretory (TES) antigens. Here, we evaluate the performance of the assay in children from an Ecuadorian birth cohort that followed children to five years of age. METHODS: Samples were selected based on the presence of peripheral blood eosinophilia and relative eosinophil counts. The samples were analyzed by the nanobody-based electrochemical magnetosensor assay, which utilizes a bivalent biotinylated nanobody as capturing agent on the surface of streptavidin pre-coated paramagnetic beads. Detection was performed by a different nanobody chemically labelled with horseradish peroxidase. RESULTS: Of 87 samples tested, 33 (38%) scored positive for TES antigen recognition by the electrochemical magnetosensor assay. The average concentration of TES antigen in serum was 2.1 ng/ml (SD = 1.1). The positive result in the electrochemical assay was associated with eosinophilia > 19% (P = 0.001). Parasitological data were available for 57 samples. There was no significant association between positivity by the electrochemical assay and the presence of other soil-transmitted helminth infections. CONCLUSIONS: Our nanobody-based electrochemical assay provides highly sensitive quantification of TES antigens in serum and has potential as a valuable tool for the diagnosis of active human toxocariasis.
Assuntos
Antígenos de Helmintos/sangue , Técnicas Eletroquímicas/métodos , Eosinofilia/parasitologia , Proteínas de Helminto/sangue , Anticorpos de Domínio Único/imunologia , Toxocaríase/diagnóstico , Animais , Biotinilação , Camelidae , Pré-Escolar , Equador/epidemiologia , Eosinofilia/epidemiologia , Humanos , Separação Imunomagnética , Lactente , População Rural , Toxocara canis , Toxocaríase/epidemiologiaRESUMO
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. RESULTS: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113-rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. CONCLUSIONS: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.