Single-Domain Antibodies with In Vitro and In Vivo Neutralizing Activity Protect Mice Against H5N1 Influenza Virus Infection

IBAÑEZ L. I.; DE FILETTE M.; VANDENABEELE P.; SAELENS X.

Hong Kong

Congreso; Options for the control of Influenza VII; 2010

 Influenza A virus infections cause a recurrent and global disease burden. A few small drug antiviral therapeutics against influenza are available, but their effect is limited, particularly in the presence of severe complications resulting from influenza. Here, we assessed the protective potential of recombinant, H5N1 virus neutralizing, llama-derived single domain antibodies (VHHs) against H5N1 virus challenge in vivo. BALB/c mice housed in SPF conditions were treated intranasally with recombinant H5N1 HA-specific or with control VHH antibodies and challenged with mouse-adapted NIBRG-14ma virus. NIBRG-14 is an H5N1 virus obtained by reverse genetics. VHHs were monovalent or bivalent. H5N1-HA-specific VHH antibodies [Infl-C8 and Infl-C8(15GS)2] or irrelevant control VHH antibodies [RSV-D3 and RSV-D3(15GS)2] were administered as a single dose ranging from 0.5-100 micrograms per mouse, depending on the experiment. VHHs were administered up to 72 hours before or after NIBRG-14ma challenge. In all experiments, mice were weighed daily after challenge and loss of 30% of body weight was used as the endpoint for euthanizing moribund mice. Lung virus titers were determined by end-point dilution in MDCK cells. Influenza virus RNA levels were determined by quantitative RT-PCR. H5N1-HA-specific VHH escape mutant viruses were selected and isolated by growth and plaque purification of NIBRG-14ma virus in the presence of H5N1-HA-specific VHH antibodies in MDCK cells. The nucleotide sequence of independently isolated VHH-escape viruses was determined and used to deduce the site of VHH-HA interaction based on H5-HA crystal structures. Administration of bivalent VHH up to 72 hours before H5N1 virus challenge strongly reduced viral replication in the lungs. A 500-ng dose of bivalent VHH, given 24 hours before challenge with 1 LD50 of H5N1 virus, completely abrogated viral replication. Furthermore, mice treated with 60 micrograms of bivalent VHH survived a challenge with 4 LD50 of H5N1 virus and displayed no signs of morbidity, while all mice in the control groups died. We also demonstrated that intranasal administration of bivalent VHH up to 48 hours after challenge with a high dose of H5N1 virus strongly reduced viral replication in the lungs and significantly delayed time to death compared to controls. Finally, by in vitro selection and sequence analysis of the HA of H5N1 escape viruses, we identified two HA amino acid residues involved in binding to the neutralizing H5N1-HA-specific VHH antibodies described here. Based on the three-dimensional structure of H5 HA, we mapped the VHH epitope to site B. We report the in vivo prophylactic and therapeutic potential of novel H5N1-neutralizing VHH molecules. In addition, we mapped the VHH-epitope binding site in H5 HA by selecting escape mutants. VHH molecules represent a valuable class of novel antivirals for treatment of influenza A virus infection. VHHs are easy to produce in recombinant prokaryotes or yeast and they can easily be delivered intranasally. They are smaller than conventional antibodies, very stable, and can easily be used as building blocks to assemble multivalent anti-influenza drugs.