Structural Studies to Guide the Development of Broadly Reactive Antibodies Against Ebolaviruses and Lassa Virus

Identification: Saphire, Erica


Structural Studies to Guide the Development of Broadly Reactive Antibodies Against Ebolaviruses and Lassa Virus
Erica Ollmann Saphire1, Kate M. Hastie1, Jacob C. Milligan1, Brandyn R. West1, Sharon Schendel1, Ayato Takada2, James E. Crowe, Jr.3, Alexander Bukreyev4, Robert F. Garry5, Luis M. Branco6
1Dept. of Immunology and Microbiology, Scripps Research, La Jolla, CA, USA; 2Hokkaido University, Sapporo, Japan; 3Vanderbilt University School of Medicine, Nashville TN, USA
4University of Texas Medical Branch, Galveston TX, USA; 5Tulane University School of Medicine, New Orleans, LA, USA; 6Zalgen Labs, Germantown, MD, USA
The Ebolavirus genus includes several viruses (Bundibugyo (BDBV), Ebola (EBOV), Sudan (SUDV) and Tai Forest (TAFV) that are highly pathogenic in humans and cause periodic outbreaks of lethal disease. Lassa virus (LASV) is endemic in West Africa and causes up to 300,00 infections annually. Both Lassa and Ebolaviruses display a single trimeric glycoprotein (termed GPC and GP, respectively) composed of three monomers that include GP1 and GP2 domains, which come together to form a trimer of heterodimers. GPC and GP are primary targets for protective antibody responses following Lassa and ebolavirus infection. Structural analyses of GPC and GP in complex with protective antibodies using X-ray crystallography and single particle electron microscopy provide critical information for understanding the basis of protective potency that can be used to design improved therapeutics that act against of a range of virus variants.
The Fab domains of two monoclonal antibodies (mAbs), one isolated from mice immunized with EBOV and SUDV virus-like particles, and the other isolated from an Ebola virus disease survivor, in complex with GP were analyzed by negative stain electron microscopy and X-ray crystallography, respectively. These mAbs are unique in that they have pan-ebolavirus neutralizing activity, and thus could have therapeutic applications in outbreaks for which the causative ebolavirus variant is unknown. The mouse- and survivor-derived ebolavirus mAbs each recognized the GP fusion loop region that lies in the “waist” of the GP molecule. The mouse-derived mAb bridges the GP1-GP2 of adjacent monomers and interacts with the GP via electrostatic and hydrophobic interactions. The negative stain structures of the mouse-derived mAb in complex with EBOV, BDBV and SUDV GP revealed that this mAb binds to the fusion loop using an identical angle of attack. The x-ray crystallography structure of the survivor mAb Fab domain in complex with GP showed that its footprint overlaps with that of the mouse-derived mAb. However, this mAb has a binding site that dips into a highly conserved pocket under the N-terminal tail of GP2.   
For antibodies against LASV GPC, neutralization potency is associated with higher affinity and H-bonding, but decreased trimer stability, suggesting that tight binding of the GP1-GP2 monomer, over stabilization of the trimer is key for neutralization at this site. Antibodies like these, against the primary neutralization site, Competition Group B (GPC-B) target only prefusion GPC that encompasses the fully assembled GP1-GP2 complex. We compared crystal structures of three different GPC-B mAbs having varying neutralization potency, each in complex with LASV GPC. Each mAb bound two GP monomers simultaneously to lock the trimer in the prefusion conformation, and the three mAbs targeted an overlapping epitope. Structural differences allowed us to pinpoint those contacts that enhance neutralization. We then used this information to make rational substitutions to increase both the potency and breadth of neutralization against different Lassa virus lineages.  
Together these structural studies can guide the development of improved, broadly reactive mAbs for post-exposure therapy and inform key design considerations for vaccine development.


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