Single domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge

Abstract

Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NAspecific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity.Fil: Cardoso, Francisco Miguel. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Ibañez, Lorena Itatí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Van Den Hoecke, Silvie. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: De Baets, Sarah. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Smet, Anouk. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Roose, Kenny. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Schepens, Bert. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Descamps, Francis J.. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Fiers, Walter. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Muyldermans, Serge. Structural Biology Research Center; Bélgica. Vrije Universiteit Brussel. Laboratory of Cellular and Molecular Immunology; BélgicaFil: Depicker, Ann. VIB. Department of Plant Systems Biology; Bélgica. Department of Biotechnology and Bioinformatics; BélgicaFil: Saelens, Xavier. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; Bélgic