A broadly-neutralizing antibody against Ebolavirus glycoprotein that potentiates the breadth and neutralization potency of other antibodies.

Ebolavirus disease (EVD) is caused by multiple species of Ebolavirus. Monoclonal antibodies (mAbs) against the virus glycoprotein (GP) are the only class of therapeutic approved for treatment of EVD caused by Zaire ebolavirus (EBOV). Therefore, mAbs targeting multiple Ebolavirus species may represent the next generation of EVD therapeutics. Broadly reactive anti-GP mAbs were produced; among these, mAbs 11886 and 11883 were broadly neutralizing in vitro. A 3.0 Å cryo-electron microscopy structure of EBOV GP bound to both mAbs shows that 11886 binds a novel epitope bridging the glycan cap (GC), 310 pocket and GP2 N-terminus, whereas 11883 binds the receptor binding region (RBR) and GC. In vitro, 11886 synergized with a range of mAbs with epitope specificities spanning the RBR/GC, including 11883. Notably, 11886 increased the breadth of neutralization by partner mAbs against different Ebolavirus species. These data provide a strategic route to design improved mAb-based next-generation EVD therapeutics.

Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.

Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories.

Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.

The evolution and determinants of neutralization of potent head-binding antibodies against Ebola virus.

Monoclonal antibodies against the Ebola virus (EBOV) surface glycoprotein are effective treatments for EBOV disease. Antibodies targeting the EBOV glycoprotein (GP) head epitope have potent neutralization and Fc effector function activity and thus are of high interest as therapeutics and for vaccine design. Here we focus on the head-binding antibodies 1A2 and 1D5, which have been identified previously in a longitudinal study of survivors of EBOV infection. 1A2 and 1D5 have the same heavy- and light-chain germlines despite being isolated from different individuals and at different time points after recovery from infection. Cryoelectron microscopy analysis of each antibody in complex with the EBOV surface GP reveals key amino acid substitutions in 1A2 that contribute to greater affinity, improved neutralization potency, and enhanced breadth as well as two strategies for antibody evolution from a common site.

Potent Omicron-neutralizing antibodies isolated from a patient vaccinated 6 months before Omicron emergence.

Therapeutic antibodies are an important tool in the arsenal against coronavirus infection. However, most antibodies developed early in the pandemic have lost most or all efficacy against newly emergent strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly those of the Omicron lineage. Here, we report the identification of a panel of vaccinee-derived antibodies that have broad-spectrum neutralization activity. Structural and biochemical characterization of the three broadest-spectrum antibodies reveal complementary footprints and differing requirements for avidity to overcome variant-associated mutations in their binding footprints. In the K18 mouse model of infection, these three antibodies exhibit protective efficacy against BA.1 and BA.2 infection. This study highlights the resilience and vulnerabilities of SARS-CoV-2 antibodies and provides road maps for further development of broad-spectrum therapeutics.

Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 reliably immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

Structure of the Inmazeb cocktail and resistance to Ebola virus escape.

Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, lessened disease and improved survival in a controlled trial. Here, we present the cryo-EM structure at 3.1 Å of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with the antibodies in the Inmazeb cocktail. This structure allows the modeling of previously disordered portions of the glycoprotein glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities and residues critical for resistance to escape by these and other clinically relevant antibodies. We further provide direct evidence that Inmazeb protects against the rapid emergence of escape mutants, whereas monotherapies even against conserved epitopes do not, supporting the benefit of a cocktail versus a monotherapy approach.

Anti-Ebola virus mAb 3A6 with unprecedented potency protects highly viremic animals from fatal outcome and physically lifts its glycoprotein target from the virion membrane.

Monoclonal antibodies (mAbs) against Ebola virus (EBOV) glycoprotein (GP1,2) are the standard of care for Ebola virus disease (EVD). Anti-GP1,2 mAbs targeting the stalk and membrane proximal external region (MPER) potently neutralize EBOV in vitro. However, their neutralization mechanism is poorly understood because they target a GP1,2 epitope that has evaded structural characterization. Moreover, their in vivo efficacy has only been evaluated in the mouse model of EVD. Using x-ray crystallography and cryo-electron tomography of 3A6 complexed with its stalk- GP1,2 MPER epitope we reveal a novel mechanism in which 3A6 elevates the stalk or stabilizes a conformation of GP1,2 that is lifted from the virion membrane. In domestic guinea pig and rhesus monkey EVD models, 3A6 provides therapeutic benefit at high viremia levels, advanced disease stages, and at the lowest dose yet demonstrated for any anti-EBOV mAb-based monotherapy. These findings can guide design of next-generation, highly potent anti-EBOV mAbs.

Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)­directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.