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28 Jun 2024
DOI: 10.1126/science.adm8693
DAWID S. ZYLA, ROBERTA DELLA MARCA, GELE NIEMEYER, GILLIAN ZIPURSKY, [...], AND ERICA OLLMANN SAPHIRE +17 authors
Structured Abstract
INTRODUCTION
Declining vaccination rates have resulted in a global resurgence of measles virus (MeV) and a clear need for effective treatments for those who cannot or have not yet been vaccinated or those in whom vaccines do not elicit neutralizing immunity. A promising approach involves neutralizing antibodies directed at the viral fusion protein F, but no such neutralizing antibodies are yet structurally characterized or approved.
RATIONALE
This study aims to elucidate the mechanism of action of monoclonal antibody (mAb) 77 and assess its potential as a therapeutic for MeV infection.
RESULTS
We find that mAb 77 potently neutralizes MeV infectionin vitro and significantly reduces replication and viral loads in vivo in the cotton rat model. A mechanistic assay revealed that mAb 77 effectively prevents complete membrane fusion, but it does so without blocking F activation, the initiation of fusion, or the formation of the extended intermediate.
To understand the mechanism of neutralization of mAb 77, we engineered a stabilized F ectodomain (FECTO) with two naturally occurring point mutations identified in cases of MeV encephalitis. These two point mutations allowed expression of F in the prefusion conformation without exogenous trimerization domains while still permitting later folding of this F into its postfusion state. A 2.11-Å-resolution cryo–electron microscopy (cryo-EM) structure of MeV FECTO in its prefusion state demonstrated that the two stabilizing mutations did not affect the overall structure, and a further structure of the postfusion F at 2.7-Å-resolution provided the endpoint structure needed to model intermediates in the refolding pathway.
A third cryo-EM structure, of FECTO in complex with Fab 77 (2.6-Å resolution), revealed the antibody epitope. mAb 77 simultaneously bridges domains I and II, the N-terminal part of F2, and the linker in between domain II and C-terminal heptad repeat, and on the edge of the epitope also recognizes the fusion peptide. This conformational epitope is only present in the prefusion form of F.
To reconcile this prefusion-bound structure with mechanistic information that mAb 77 neutralized at a post-triggering state, we sought to understand intermediates in refolding and the stage at which mAb 77 blocked the fusion process. A deeper analysis of the cryo-EM dataset of the FECTO–Fab 77 complex uncovered subpopulations of particles in a series of two-dimensional (2D) classes that illustrated snapshots in refolding (triggering, extended intermediate, trimer separation) and also allowed visualization of a trimeric, refolding-intermediate state arrested by bound Fab 77. A fourth structure, of this Fab 77–stabilized F fragment, was resolved to 3.6-Å resolution, revealing that the antibody still binds domains I and II and part of the F2 chain in their prefusion arrangement and that anchoring of these regions together arrests the process of refolding at this intermediate stage.
CONCLUSION
Our study reveals in vitro neutralization and in vivo protection by mAb 77, as well as the cryo-EM structure of mAb 77 in complex with MeV F and its mechanism of action, which provides information that is important for potential therapeutic use against MeV infection. We showed that mAb 77 specifically recognizes MeV F in the prefusion conformation and allows initial F activation and refolding but also that it interrupts the refolding process to prevent conformational changes required for membrane fusion and viral entry. These findings suggest a model for understanding not only antibody neutralization of MeV but also other viruses for which antibodies are thought to interrupt fusion but for which structures are not yet available.
DOI: 10.1126/science.adm8693
DAWID S. ZYLA, ROBERTA DELLA MARCA, GELE NIEMEYER, GILLIAN ZIPURSKY, [...], AND ERICA OLLMANN SAPHIRE +17 authors
Structured Abstract
INTRODUCTION
Declining vaccination rates have resulted in a global resurgence of measles virus (MeV) and a clear need for effective treatments for those who cannot or have not yet been vaccinated or those in whom vaccines do not elicit neutralizing immunity. A promising approach involves neutralizing antibodies directed at the viral fusion protein F, but no such neutralizing antibodies are yet structurally characterized or approved.
RATIONALE
This study aims to elucidate the mechanism of action of monoclonal antibody (mAb) 77 and assess its potential as a therapeutic for MeV infection.
RESULTS
We find that mAb 77 potently neutralizes MeV infectionin vitro and significantly reduces replication and viral loads in vivo in the cotton rat model. A mechanistic assay revealed that mAb 77 effectively prevents complete membrane fusion, but it does so without blocking F activation, the initiation of fusion, or the formation of the extended intermediate.
To understand the mechanism of neutralization of mAb 77, we engineered a stabilized F ectodomain (FECTO) with two naturally occurring point mutations identified in cases of MeV encephalitis. These two point mutations allowed expression of F in the prefusion conformation without exogenous trimerization domains while still permitting later folding of this F into its postfusion state. A 2.11-Å-resolution cryo–electron microscopy (cryo-EM) structure of MeV FECTO in its prefusion state demonstrated that the two stabilizing mutations did not affect the overall structure, and a further structure of the postfusion F at 2.7-Å-resolution provided the endpoint structure needed to model intermediates in the refolding pathway.
A third cryo-EM structure, of FECTO in complex with Fab 77 (2.6-Å resolution), revealed the antibody epitope. mAb 77 simultaneously bridges domains I and II, the N-terminal part of F2, and the linker in between domain II and C-terminal heptad repeat, and on the edge of the epitope also recognizes the fusion peptide. This conformational epitope is only present in the prefusion form of F.
To reconcile this prefusion-bound structure with mechanistic information that mAb 77 neutralized at a post-triggering state, we sought to understand intermediates in refolding and the stage at which mAb 77 blocked the fusion process. A deeper analysis of the cryo-EM dataset of the FECTO–Fab 77 complex uncovered subpopulations of particles in a series of two-dimensional (2D) classes that illustrated snapshots in refolding (triggering, extended intermediate, trimer separation) and also allowed visualization of a trimeric, refolding-intermediate state arrested by bound Fab 77. A fourth structure, of this Fab 77–stabilized F fragment, was resolved to 3.6-Å resolution, revealing that the antibody still binds domains I and II and part of the F2 chain in their prefusion arrangement and that anchoring of these regions together arrests the process of refolding at this intermediate stage.
CONCLUSION
Our study reveals in vitro neutralization and in vivo protection by mAb 77, as well as the cryo-EM structure of mAb 77 in complex with MeV F and its mechanism of action, which provides information that is important for potential therapeutic use against MeV infection. We showed that mAb 77 specifically recognizes MeV F in the prefusion conformation and allows initial F activation and refolding but also that it interrupts the refolding process to prevent conformational changes required for membrane fusion and viral entry. These findings suggest a model for understanding not only antibody neutralization of MeV but also other viruses for which antibodies are thought to interrupt fusion but for which structures are not yet available.
