title: Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level.
abstract: The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.
authors.affiliation: Harvard University Biophysics Program, Harvard Medical School, Boston, MA 02115; Centre for Synthetic Biology, Zernike Institute for Advanced Materials, University of Groningen, 9747 AG, Groningen, The Netherlands; and.
authors.forename: Jason J
authors.identifier: http://orcid.org/0000-0002-0176-9976
authors.initials: JJ
authors.lastname: Otterstrom
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Boerries
authors.initials: B
authors.lastname: Brandenburg
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Martin H
authors.initials: MH
authors.lastname: Koldijk
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Jarek
authors.initials: J
authors.lastname: Juraszek
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Chan
authors.initials: C
authors.lastname: Tang
authors.affiliation: Centre for Synthetic Biology, Zernike Institute for Advanced Materials, University of Groningen, 9747 AG, Groningen, The Netherlands; and.
authors.forename: Samaneh
authors.initials: S
authors.lastname: Mashaghi
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Ted
authors.initials: T
authors.lastname: Kwaks
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Goudsmit
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Ronald
authors.initials: R
authors.lastname: Vogels
authors.affiliation: Crucell Vaccine Institute, Janssen Center of Excellence for Immunoprophylaxis, 2333 CN, Leiden, The Netherlands.
authors.forename: Robert H E
authors.initials: RH
authors.lastname: Friesen
authors.affiliation: Centre for Synthetic Biology, Zernike Institute for Advanced Materials, University of Groningen, 9747 AG, Groningen, The Netherlands; and a.m.van.oijen@rug.nl.
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000494:pharmacology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D054884:Host-Pathogen Interactions / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000494:pharmacology; D053118:Influenza A Virus, H1N1 Subtype / Q000276:immunology / Q000502:physiology / Q000648:ultrastructure; D053122:Influenza A Virus, H3N2 Subtype / Q000276:immunology / Q000502:physiology / Q000648:ultrastructure; D009980:Influenza A virus / Q000276:immunology* / Q000502:physiology / Q000648:ultrastructure; D007251:Influenza, Human / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D007700:Kinetics; D008561:Membrane Fusion / Q000187:drug effects / Q000276:immunology*; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009010:Monte Carlo Method; D011485:Protein Binding; D014771:Virion / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D057134:Antibodies, Neutralizing; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007140:Immunoglobulin Fab Fragments
keywords: hemagglutinin; influenza; membrane fusion; neutralization stoichiometry; neutralizing antibody