| Abstract: | ![]()
H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (6–9) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion. |
