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Abstract Antibodies to Ebola virus glycoprotein (EBOV GP) represent an important correlate of the vaccine efficiency and infection survival. Both neutralization and some of the Fc-mediated effects are known to contribute the protection conferred by antibodies of various epitope specificities. At the same time, the role of the complement system remains unclear. Here, we compare complement activation by two groups of representative monoclonal antibodies (mAbs) interacting with the glycan cap (GC) or the membrane-proximal external region (MPER) of GP. Binding of GC-specific mAbs to GP induces complement-dependent cytotoxicity (CDC) in the GP-expressing cell line via C3 deposition on GP in contrast to MPER-specific mAbs. In the mouse model of EBOV infection, depletion of the complement system leads to an impairment of protection exerted by one of the GC-specific, but not MPER-specific mAbs. Our data suggest that activation of the complement system represents an important mechanism of antiviral protection by GC antibodies.

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-21-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.

Summary: Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic-virus-protective antigen is poorly defined, we perform deep paired heavy- and light-chain sequencing in Ebola virus glycoprotein (EBOV-GP)-specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitates investigation of the molecular and genetic basis for the evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B cell and serum repertoire. We identify 73 public clonotypes of EBOV, 20% of which encode antibodies with neutralization activity and capacity to protect mice in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV GP, which informs the design of vaccines and improved therapeutics.

One year after a Zaire ebolavirus (EBOV) outbreak occurred in the Boende Health Zone of the Democratic Republic of the Congo during 2014, we sought to determine the breadth of immune response against diverse filoviruses including EBOV, Bundibugyo (BDBV), Sudan (SUDV), and Marburg (MARV) viruses. After assessing the 15 survivors, 5 individuals demonstrated some degree of reactivity to multiple ebolavirus species and, in some instances, Marburg virus. All 5 of these survivors had immunoreactivity to EBOV glycoprotein (GP) and EBOV VP40, and 4 had reactivity to EBOV nucleoprotein (NP). Three of these survivors showed serologic responses to the 3 species of ebolavirus GPs tested (EBOV, BDBV, SUDV). All 5 samples also exhibited ability to neutralize EBOV using live virus, in a plaque reduction neutralization test. Remarkably, 3 of these EBOV survivors had plasma antibody responses to MARV GP. In pseudovirus neutralization assays, serum antibodies from a subset of these survivors also neutralized EBOV, BDBV, SUDV, and Taï Forest virus as well as MARV. Collectively, these findings suggest that some survivors of naturally acquired ebolavirus infection mount not only a pan-ebolavirus response, but also in less frequent cases, a pan-filovirus neutralizing response.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

Summary: Real-time cell analysis (RTCA) enables high-throughput, quantitative kinetic measurements of cytopathic effect (CPE) in virus-infected cells. Here, we detail a RTCA approach for assessing antibody neutralization. We describe how to evaluate the neutralizing potency of monoclonal antibodies (mAbs) and identify viral escape mutants to antibody neutralization for severe respiratory syndrome coronavirus 2 (SARS-CoV-2).For complete details on the use and execution of this protocol, please refer to Zost et al. (2020) and Suryadevara et al. (2021).

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses.

Summary: The COVID-19 pandemic revealed an urgent need for rapid profiling of neutralizing antibody responses and development of antibody therapeutics. The current Food and Drug Administration-approved serological tests do not measure antibody-mediated viral neutralization, and there is a need for standardized quantitative neutralization assays. We report a high-throughput two-step profiling approach for identifying neutralizing convalescent plasma. Screening and downselection for serum antibody binding to the receptor-binding domain are followed by quantitative neutralization testing using a chimeric vesicular stomatitis virus expressing spike protein of SARS-CoV-2 in a real-time cell analysis assay. This approach enables a predictive screening process for identifying plasma units that neutralize SARS-CoV-2. To calibrate antibody neutralizing activity in serum from convalescent plasma donors, we introduce a neutralizing antibody standard reagent composed of two human antibodies that neutralize SARS-CoV strains, including SARS-CoV-2 variants of concern. Our results provide a framework for establishing a standardized assessment of antibody-based interventions against COVID-19.

Broadly neutralizing antibodies are potential therapeutics and can aid rational vaccine development. Here, the authors show that the human monoclonal antibody H3v-47 recognizes a highly conserved epitope in HA of H3N2 viruses, inhibits virus replication by blocking egress and other mechanisms, and protects mice from disease.

Avian H7N9 influenza viruses cause sporadic outbreaks of human infections and threaten to cause a major pandemic. The breadth of B cell responses to natural infection and the dominant antigenic sites recognized during first exposure to H7 HA following infection are incompletely understood. Here, we studied the B cell response to H7 HA of 2 individuals who had recovered from natural H7N9 virus infection. We used competition binding, hydrogen-deuterium mass spectrometry, and single-particle negative stain electron microscopy to identify the patterns of molecular recognition of the antibody responses to H7 HA. We found that circulating H7-reactive B cells recognized a diverse antigenic landscape on the HA molecule, including HA head domain epitopes in antigenic sites A and B and in the trimer interface-II region and epitopes in the stem region. Most H7 antibodies exhibited little heterosubtypic breadth, but many recognized a wide diversity of unrelated H7 strains. We tested the antibodies for functional activity and identified clones with diverse patterns of inhibition, including neutralizing, hemagglutination- or egress-inhibiting, or HA trimer–disrupting activities. Thus, the human B cell response to primary H7 natural infection is diverse, highly functional, and broad for recognition of diverse H7 strains.

Summary: Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

Human antibodies cross-reactive for several viruses within the Ebolavirus genus have been identified. Here the authors present the crystal structure of such a neutralizing monoclonal antibody (mAb) targeting the stalk of Bundibugyo virus glycoprotein and show that mAb binding may interfere with trimeric bundle assembly and/or the viral membrane.

Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Three clinically relevant ebolaviruses – Ebola (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) viruses, are responsible for severe disease and occasional deadly outbreaks in Africa. The largest Ebola virus disease (EVD) epidemic to date in 2013-2016 in West Africa highlighted the urgent need for countermeasures, leading to the development and FDA approval of the Ebola virus vaccine rVSV-ZEBOV (Ervebo®) in 2020 and two monoclonal antibody (mAb)-based therapeutics (Inmazeb® [atoltivimab, maftivimab, and odesivimab-ebgn] and Ebanga® (ansuvimab-zykl) in 2020. The humoral response plays an indispensable role in ebolavirus immunity, based on studies of mAbs isolated from the antibody genes in peripheral blood circulating ebolavirus-specific human memory B cells. However, antibodies in the body are not secreted by circulating memory B cells in the blood but rather principally by plasma cells in the bone marrow. Little is known about the protective polyclonal antibody responses in convalescent plasma. Here we exploited both single-cell antibody gene sequencing and proteomic sequencing approaches to assess the composition of the ebolavirus glycoprotein (GP)-reactive antibody repertoire in the plasma of an EVD survivor. We first identified 1,512 GP-specific mAb variable gene sequences from single cells in the memory B cell compartment. Using mass spectrometric analysis of the corresponding GP-specific plasma IgG, we found that only a portion of the large B cell antibody repertoire was represented in the plasma. Molecular and functional analysis of proteomics-identified mAbs revealed recognition of epitopes in three major antigenic sites - the GP head domain, the glycan cap, and the base region, with a high prevalence of neutralizing and protective mAb specificities that targeted the base and glycan cap regions on the GP. Polyclonal plasma antibodies from the survivor reacted broadly to EBOV, BDBV, and SUDV GP, while reactivity of the potently neutralizing mAbs we identified was limited mostly to the homologous EBOV GP. Together these results reveal a restricted diversity of neutralizing humoral response in which mAbs targeting two antigenic sites on GP – glycan cap and base – play a principal role in plasma-antibody-mediated protective immunity against EVD.

Summary: Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.

Abstract Fine ZnS:Cu, obtained by method of self-propagating high-temperature synthesis was investigated. As flux in the mixture NaCl was used, Zn and S were taken in stoichiometric ratio; Cu concentration in charge consisted ~1.5 wt.%. Using SEM data, it was established that obtained ZnS:Cu consists from two fractions—first with particles sizes ~10 μm and more, and other with sizes 50–500 nm. It was established that composition of ZnS:Cu fractions was essentially different. According to EDS data, Cu concentration in particles of fraction with 50–500 nm sizes consists ~2 wt.%, and in particles with sizes ~10 μm and more the presence of Cu was not detected. The reasons that lead to the selective doping of particles in dependence on their size and also the role of NaCl in processes undergoing during synthesis of material are discussed.

The influence of magnesium impurities on luminescent properties of ZnS:Cu,Mg using obtained by self-propagating high-temperature synthesis (SHS) has been investigated. Special attention was paid to changes of photoluminescence spectra caused by relaxation processes in ZnS:Cu,Mg-SHS. It was shown that introduction of magnesium into ZnS:Cu-SHS leads to a change in symmetry of ZnS crystal lattice. It leads to relaxation quenching the photoluminescence bands caused by presence of copper impurities in ZnS.

The nature and anatomic location of the protective memory CD8+ T cell subset induced by intranasal vaccination remain poorly understood. We developed a vaccination model to assess the anatomic location of protective memory CD8+ T cells and their role in lower airway infections. Memory CD8+ T cells elicited by local intranasal, but not systemic, vaccination with an engineered non-replicative CD8+ T cell-targeted antigen confer enhanced protection to a lethal respiratory viral challenge. This protection depends on a distinct CXCR3LO resident memory CD8+ T (Trm) cell population that preferentially localizes to the pulmonary interstitium. Because they are positioned close to the mucosa, where infection occurs, interstitial Trm cells act before inflammation can recruit circulating memory CD8+ T cells into the lung tissue. This results in a local protective immune response as early as 1 day post-infection. Hence, vaccine strategies that induce lung interstitial Trm cells may confer better protection against respiratory pathogens.

Recent studies suggest that some monoclonal antibodies (mAbs) specific for ebolavirus glycoprotein (GP) can protect experimental animals against infections. Most mAbs isolated from ebolavirus survivors appeared to target the glycan cap or the stalk region of the viral GP, which is the envelope protein and the only antigen inducing virus-neutralizing antibody response. Some of the mAbs were demonstrated to be protective in vivo. Here, a panel of mAbs from four individual survivors of ebolavirus infection that target the glycan cap or stem region were selected for investigation of the mechanisms of their antiviral effect. Comparative characterization of the inhibiting effects on multiple steps of viral replication was performed, including attachment, post-attachment, entry, binding at low pH, post-cleavage neutralization of virions, viral trafficking to endosomes, cell-to-cell transmission, viral egress, and inhibition when added early at various time points post-infection. In addition, Fc-domain related properties were characterized, including activation and degranulation of NK cells, antibody-dependent cellular phagocytosis and glycan content. The two groups of mAbs (glycan cap versus stem) demonstrated very different profiles of activities suggesting usage of mAbs with different epitope specificity could coordinate inhibition of multiple steps of filovirus infection through Fab- and Fc-mediated mechanisms, and provide a reliable therapeutic approach.

Summary: Some monoclonal antibodies (mAbs) recovered from survivors of filovirus infections can protect against infection. It is currently unknown whether natural infection also induces some antibodies with the capacity for antibody-dependent enhancement (ADE). A panel of mAbs obtained from human survivors of filovirus infection caused by Ebola, Bundibugyo, or Marburg viruses was evaluated for their ability to facilitate ADE. ADE was observed readily with all mAbs examined at sub-neutralizing concentrations, and this effect was not restricted to mAbs with a particular epitope specificity, neutralizing capacity, or subclass. Blocking of specific Fcγ receptors reduced but did not abolish ADE that was associated with high-affinity binding antibodies, suggesting that lower-affinity interactions still cause ADE. Mutations of Fc fragments of an mAb that altered its interaction with Fc receptors rendered the antibody partially protective in vivo at a low dose, suggesting that ADE counteracts antibody-mediated protection and facilitates dissemination of filovirus infections. : In this paper, Kuzmina et al. demonstrate that filovirus antibodies from human survivors present at low concentrations are capable of enhancement of infection, suggesting that low levels of antibodies in humans may facilitate virus spread. The enhancement can be caused by antibodies of various epitope specificities, neutralizing capacities, and subclasses. Keywords: Ebola virus, filovirus, antibody, enhancement of infection, FC receptor, epitope

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia--because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice.