Nanobodies With In Vitro Neutralizing Activity Protect Mice Against H5N1 Influenza Virus Infection

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

Buenos Aires

Workshop; 3rd ICGEB WORKSHOP; 2012

Institute Leloir, Buenos Aires

Influenza A virus infections cause recurrent and global disease burden. Although small drug antiviral therapeutics against influenza are available, their impact is limited in cases of severe complications. Here we assessed the protective potential of recombinant, H5N1 virus neutralizing llama-derived single domain antibodies, VHHs, against H5N1 virus challenge in vivo.Mice were treated intranasally with H5N1-HA-specific VHH antibodies H5-VHHm/b or irrelevant control VHH antibodies RSV-VHHm/b and challenged with mouse-adapted NIBRG-14ma virus. Single dose ranging from 100-0.5 micrograms per mouse, depending on the experiment, were administered. Mouse body weight was determined daily after challenge and loss of 25 % 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. Selection and isolation of H5N1-HA-specific VHH escape mutant viruses was performed by growth and plaque purification of NIBRG-14ma virus in the presence of H5N1-HA-specific VHHs 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 structure.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. 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 also 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 in vitro selection and sequence analysis of the HA of H5N1 escape viruses, we identified two amino acid residues involved in binding with the neutralizing H5N1-HA-specific VHH antibodies that maps to site B of HA.We here report the in vivo prophylactic and therapeutic potential of novel H5N1-neutralizing VHH molecules. In addition, we have mapped the VHH-epitope binding site in H5 HA by selecting escape mutants. VHH molecules represent a valuable class of novel antivirals to threat influenza A virus infection, that can easily be delivered intranasally. Importantly, VHHs are easy to produce; they are smaller than conventional antibodies, are very stable and can easily be used as building blocks to assemble multivalent anti-influenza drugs.