Nanobodies with In Vitro Neutralizing Activity Protect Mice against H5N1 Influenza Virus Infection

LORENA ITATÍ IBAÑEZ; MARINA DE FILLETE; BERT SCHEPENS; XAVIER SAELES

GHENT, Belgium

Simposio; 1ST SYMPOSIUM ON SINGLE DOMAIN ANTIBODIES; 2010

Influenza A virus infections impose recurrent and global disease burden. Although a number of small drug antiviral therapeutics against influenza are available, their impact is limited, in particular in cases of severe complications resulting from influenza. Here we assessed the protective potential of recombinant, H5N1 virus neutralizing llama-derived VHH single domain antibodies, against H5N1 virus challenge. We first demonstrated that intranasal administration of these VHH antibodies is aneffective way to suppress challenge with a homologous influenza A virus and this route of VHH delivery was used throughout the study. Administration of bivalent VHH up to 72 hours prior to H5N1 virus challenge strongly reduced viral replication in the lungs. Interestingly, a 500 ng dose of bivalent VHH given 24 hours prior to challenge with 1 LD50 of H5N1 virus, completely abrogated viral replication. Furthermore, mice treated with 60 micrograms of bivalent VHH survived a 4 LD50 challenge with H5N1 virus and displayed no signs of morbidity while all mice in the control groups died. We demonstrated that intranasal administration of bivalent VHH up to 48 hours after challenge with H5N1 virus strongly reduced viral replication in the lungs and significantly delayed time to death compared to controls, when a severe challenge dose was applied. Finally, by selection and sequence analysis of the HA of H5N1 escape viruses, we identified two HA amino acid residues involved in binding with the  neutralizing H5N1-HA-specificVHHantibodies described here. Based on the 3-dimensional structure of H5HA, the VHH epitope maps to site B.