English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/204670
Share/Impact:
Statistics
logo share SHARE   Add this article to your Mendeley library MendeleyBASE
Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:

Title

Identification of MERS-CoV neutralizing nanobodies by direct cloning from a bone marrow cDNA library

AuthorsStalin Raj, V.; Okba, Nisreen M. A.; Gutierrez-Alvarez, Francisco J. ; Drabek, Dubravka; Widagdo, W.; Lamers, Mart M.; Solá Gurpegui, Isabel ; Bensaid, A. M.; Segalés, Joaquim; Berend Jan; Koopmans, M.; Sutter, Gerd; Osterhaus, Albert D. M. E.; Enjuanes Sánchez, Luis ; Haagmans, Bart L.
Issue Date4-Jun-2017
CitationXIVth International Nidovirus Symposium (2017)
AbstractMiddle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal respiratory disease in humans. Dromedary camels have been identified as a reservoir for this virus. Due to the continuous emergence of new zoonotic viruses, there is a need to fast approach for identification and development of prophylactic treatment. We identified and characterized MERS-CoV specific camelid nanobodies (nbs) that block MERS- CoV entry in vitro and in vivo. To develop MERS-CoV specific nbs, two camels were immunized with MVA encoding the MERS-CoV S gene and challenged with live MERS-CoV. High levels of MERS-CoV neutralizing antibodies were detected in the sera of the dromedary camels at 14 days post challenge. Bone marrow was used to directly clone the nb repertoire in an E-coli expression vector, and individual clones were screened using MERS-CoV infected cells. MERS-CoV positive clones were further characterized using ELISAs, in vitro blocking assay, affinitive measurement, epitope prediction, sequencing, virus neutralization, and finally tested in hDPP4 transgenic mice. As many as 204/560 clones (36%) were able to specifically bind to MERS-CoV infected cells. To further characterize the antigen specificity of the nbs we tested their reactivity against the S protein and 188 (92.15%) nbs were directed against the MERS-CoV S protein of which 46 (22.5%) bound to the receptor binding domain. Sequencing revealed that the nbs clustered into 14 different CDR-3 groups. Four nbs had a relatively high affinity (1.0E-09 to 1.0E-10 M) and neutralized MERS-CoV with very high efficiency, at a nanogram concentration. Cross-competition assays reveled that all 4 nbs compete for binding to the same unique epitope. Moreover, epitope prediction showed that the RBD contact residues extensively overlap with the receptor binding site, indicating that nb83 neutralizes MERS-CoV most likely by blocking the binding to its cellular receptor. Due to the smaller size, nbs may not be used for therapeutic purposes. Therefore, we expressed the 4 nbs as human IgG-Fc fused dimer. These dimers are 3 times more potent than monomer in vitro. In addition, prophylactically treated transgenic mice with Fc-fused nb were protected against MERS-CoV infection. In conclusion, our rapid nb identification method allows identification of neutralizing nbs against MERS-CoV, which may be used as a prophylactic treatment for MERS.
DescriptionTrabajo presentado en el XIVth International Nidovirus Symposium, celebrado en Kansas City, Missouri (Estados Unidos), del 4 al 9 de junio de 2017
URIhttp://hdl.handle.net/10261/204670
Appears in Collections:(VICYT) Colección Especial COVID-19
(CNB) Comunicaciones congresos
Files in This Item:
File Description SizeFormat 
accesoRestringido.pdf15,38 kBAdobe PDFThumbnail
View/Open
Show full item record
Review this work
 


WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.