An Antibody against a Novel and Conserved Epitope in the Hemagglutinin 1 Subunit Neutralizes Numerous H5N1 Influenza Viruses

Abstract

The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.

A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

“…1C). These key H5 residues do not overlap any known epitopes described above or detected with other human and mouse antibodies (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). The four H5M9 epitope key residues do not overlap known H1 antigenic sites (36), but positions 53, 274, and 276 correspond to site C of H3 viruses (35).…”

Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies

“…The conservation of neutralizing epitopes is mostly restricted to the membrane-distal subdomain of the influenza HA protein, the HA1 region (45)(46)(47). Effective nonneutralizing Abs may not be limited by the same epitope constraints placed on neutralizing Abs because they do not need to stop virus binding or entry.…”

Entry of Influenza A Virus with a α2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin

“…In this experiment, acid-induced fusion of WSN HA-expressing HeLa cells can be inhibited by anti-FN antibody (data not shown). A previously described postattachment assay was used to confirm whether the anti-FN antibody is able to block viral entry even after virus attachment (41). The number of M2-expressing cells was examined at the 8-h postoperative time point.…”

“…The viral entry assay was a modified version of a previously described protocol (41). In brief, MDCK cells cultured in a 24-well plate were incubated with WSN virus at an MOI of 2 at 4°C for 6 h. The cells were washed with ice-chilled PBS three times and then incubated with 0.2 to 2 g of anti-FN antibody/ml at 4°C for 2 h. The antibody was removed, and fresh MEM with TPCK-trypsin was added to the monolayer.…”