ABSTRACT
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.
Subject(s)
Antibodies, Viral/immunology , Immunologic Techniques/methods , Influenza A Virus, H5N2 Subtype/physiology , Influenza, Human/prevention & control , Single-Domain Antibodies/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/administration & dosage , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Camelus/genetics , Camelus/immunology , Female , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A Virus, H5N2 Subtype/drug effects , Influenza A Virus, H5N2 Subtype/genetics , Influenza, Human/immunology , Influenza, Human/virology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Structure, Tertiary , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/geneticsABSTRACT
One effective method for the prevention and treatment of influenza infection is passive immunization. In our study, we examined the feasibility of creating an antibody-based preparation with a prolonged protective effect against influenza virus. Single-domain antibodies (sdAbs) specific for influenza virus hemagglutinin were generated. Experiments in mouse models showed 100% survivability for both intranasal sdAbs administration 24h prior to influenza challenge and 24h after infection. sdAb-gene delivery by an adenoviral vector led to gene expression for up to 14days. Protection by a recombinant adenovirus containing the sdAb gene was observed in cases of administration prior to influenza infection (14d-24h). We also demonstrated that the single administration of a combined preparation containing sdAb DNA and protein expanded the protection time window from 14d prior to 48h after influenza infection. This approach and the application of a broad-spectrum sdAbs will allow the development of efficient drugs for the prevention and treatment of viral infections produced by pandemic virus variants and other infections.
