Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2).

This work continues a series of recently published studies that employ recombinant single-domain antibody (sdAb, or nanobody®) generation technologies to battle viruses by a passive immunization approach. As a proof of principle, we describe a modified technique to efficiently generate protective molecules against a particular strain of influenza virus within a reasonably short period of time. This approach starts with the immunization of a camel (Camelus bactrianus) with the specified antigen-enriched material presented in as natural a form as possible. An avian influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2) adapted for mice was used as a model source of antigens for both the immunization and phage display-based selection procedures. To significantly increase activities of initially selected monovalent single-domain antibodies, we propose a new type of sdAb formatting that involves the addition of a special type of coiled-coil sequence, the isoleucine zipper domain (ILZ). Presumably, the ILZ-containing peptides adopt trimeric parallel conformations. After the formatting, the biological activities (virus neutralization) of the initially selected anti-influenza virus (H5N2) sdAbs were significantly increased. Intraperitoneal or intranasal administration of the formatted sdAb at 2h before or 24h after viral challenge specifically protects mice from lethal infection with influenza virus. We hope that the described approach combined with the selection focused on particular conservative epitopes will lead to the generation of sdAb-based molecules protective against a broad spectrum of influenza virus subtypes.