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Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

Journal Article


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


  •   Otterstrom, Jason J. (external author)
  •   Brandenburg, Boerries (external author)
  •   Koldijk, Martin (external author)
  •   Juraszek, Jarek (external author)
  •   Tang, Chan (external author)
  •   Mashaghi, Samaneh (external author)
  •   Kwaks, Ted (external author)
  •   Goudsmit, Jaap (external author)
  •   Vogels, Ronald (external author)
  •   Friesen, Robert H. E. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2014

Citation


  • Otterstrom, J. J., Brandenburg, B., Koldijk, M. H., Juraszek, J., Tang, C., Mashaghi, S., Kwaks, T., Goudsmit, J., Vogels, R., Friesen, R. H. E. & van Oijen, A. M. (2014). Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level. Proceedings of the National Academy of Sciences of the United States of America, 111 (48), E5143-E5148.

Scopus Eid


  • 2-s2.0-84914165942

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/5113

Start Page


  • E5143

End Page


  • E5148

Volume


  • 111

Issue


  • 48

Place Of Publication


  • United States

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


  •   Otterstrom, Jason J. (external author)
  •   Brandenburg, Boerries (external author)
  •   Koldijk, Martin (external author)
  •   Juraszek, Jarek (external author)
  •   Tang, Chan (external author)
  •   Mashaghi, Samaneh (external author)
  •   Kwaks, Ted (external author)
  •   Goudsmit, Jaap (external author)
  •   Vogels, Ronald (external author)
  •   Friesen, Robert H. E. (external author)
  •   van Oijen, Antoine M.

Publication Date


  • 2014

Citation


  • Otterstrom, J. J., Brandenburg, B., Koldijk, M. H., Juraszek, J., Tang, C., Mashaghi, S., Kwaks, T., Goudsmit, J., Vogels, R., Friesen, R. H. E. & van Oijen, A. M. (2014). Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level. Proceedings of the National Academy of Sciences of the United States of America, 111 (48), E5143-E5148.

Scopus Eid


  • 2-s2.0-84914165942

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/5113

Start Page


  • E5143

End Page


  • E5148

Volume


  • 111

Issue


  • 48

Place Of Publication


  • United States