Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses.

Several ebolaviruses cause outbreaks of severe disease. Vaccines and monoclonal antibody cocktails are available to treat Ebola virus (EBOV) infections, but not Sudan virus (SUDV) or other ebolaviruses. Current cocktails contain antibodies that cross-react with the secreted soluble glycoprotein (sGP) that absorbs virus-neutralizing antibodies. By sorting memory B cells from EBOV infection survivors, we isolated two broadly reactive anti-GP monoclonal antibodies, 1C3 and 1C11, that potently neutralize, protect rodents from disease, and lack sGP cross-reactivity. Both antibodies recognize quaternary epitopes in trimeric ebolavirus GP. 1C11 bridges adjacent protomers via the fusion loop. 1C3 has a tripartite epitope in the center of the trimer apex. One 1C3 antigen-binding fragment anchors simultaneously to the three receptor-binding sites in the GP trimer, and separate 1C3 paratope regions interact differently with identical residues on the three protomers. A cocktail of both antibodies completely protected nonhuman primates from EBOV and SUDV infections, indicating their potential clinical value.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.

The experience of feeling old after a fragility fracture.

BACKGROUND: There has been little exploration of the effect of fragility fractures on patient perceptions of their age. The common assumption is that fractures "happen to old people". In individuals with a fragility fracture, our objective was to explore the experience of feeling old after sustaining a fragility fracture. METHODS: A secondary analysis of data from 145 community-dwelling women and men participating in six qualitative primary studies was conducted relying on a phenomenological approach. Participants were English-speaking, 45 years and older, who had sustained a recent fragility fracture or reported a history of previous fragility fractures. Data for the analysis included direct statements about feeling old as well any discussions relevant to age post-fracture. RESULTS: We highlight two interpretations based on how individuals with a history of fragility fracture talked about age: (1) Participants described feeling old post-fracture. Several participants made explicit statements about being "old". However, the majority of participants discussed experiences post-fracture that implied that they felt old and had resigned themselves to being old. This appeared to entail a shift in thinking and perception of self that was permanent and had become a part of their identity; and (2) Perceptions of increasing age after sustaining a fracture were reinforced by health care providers, family, and friends. CONCLUSIONS: Our findings challenge the notion that fractures "happen to old people" and suggest that fractures can make people feel old. Careful consideration of how bone health messages are communicated to patients post-fracture by health care providers is warranted. (Word Count: 248).

Patients experience a need for informal care after a fragility fracture.

In this qualitative secondary analysis, patients with a fragility fracture described needing informal care post-fracture. A significant proportion reported receiving no care or not enough care, often devising strategies to care for themselves. Requesting help from multiple individuals allowed patients to minimize the burden to family and friends. INTRODUCTION: In individuals with fragility fractures, our objectives were to examine (1) the experience of receiving informal care post-fracture; and (2) how these care experiences influenced post-fracture recovery and subsequent management of bone health. METHODS: A secondary analysis of six primary qualitative studies was conducted. Individuals in the primary studies were English-speaking women and men, 45 years and older, who were living in the community and had sustained a recent fragility fracture or reported a history of previous fragility fractures. Participants who reported at least one instance of needing informal care were categorized as receiving "enough care", "insufficient care", or "no care". RESULTS: Of 145 participants in the primary studies, 109 (75%) described needing informal care after their fracture. Of those needing care, 62 (57%) were categorized as receiving enough care while 47 (43%) were categorized as receiving insufficient or no care. The care needed affected the management of participants' fracture and bone health, including access to health care services. Participants who received insufficient or no care, especially those living alone, devised strategies to care for themselves and often requested help from multiple individuals to minimize the burden to family and friends. Compared with men, women appeared to report needing help with personal daily activities, such as bathing, and transportation to appointments related to bone health. CONCLUSION: Informal care needs are an additional burden of fragility fractures. Post-fracture interventions should consider the broader context of patients' lives and potentially support the care needs of patients as part of their services.

Management and outcomes in patients with tumor thrombus: a retrospective cohort study.

BACKGROUND: Tumor thrombus can be associated with an increased risk of venous thromboembolism (VTE) and poor prognosis. The risks and benefits of anticoagulation remain unclear. OBJECTIVES: To evaluate the role of anticoagulation and associated outcomes in patients with tumor thrombus. METHODS: We conducted a single-center retrospective cohort study in patients with tumor thrombus from 2019 to 2022. All patients were followed for 12 months from the diagnosis of tumor thrombus or until death if death occurred earlier. The primary outcome was the percentage of patients prescribed any dose of anticoagulation for tumor thrombus (or concurrent bland thrombus/VTE). The secondary outcomes included new thrombosis, major bleeding, clinically relevant nonmajor bleeding, and mortality. We calculated the 6- and 12-month cumulative incidence of outcomes with 95% CI and compared those given anticoagulation vs not, considering death as a competing risk. RESULTS: We included 211 patients, among whom 106 (50.2%; 95% CI, 47.9%-52.6%) were given anticoagulation for tumor thrombus or concurrent VTE (present in 21.8%). The most common type of cancer was hepatocellular carcinoma (28%). Splanchnic veins were the most commonly involved (49.3%). Anticoagulation was more likely used if tumor thrombus involved the inferior vena cava and/or the heart, with concurrent VTE, or if thrombosis service was consulted. The overall 12-month incidence of new VTE was 11.4% (95% CI, 7.3%-16.5%), that of major bleeding + clinically relevant nonmajor bleeding was 36.6% (95% CI, 29.6%-43.5%), and mortality of 52.5% (95% CI, 44.8%-59.6%), with no significant differences among groups given anticoagulation or not. CONCLUSION: Patients with tumor thrombus carry high risks of VTE, bleeding, and mortality. The impact of anticoagulation remains unclear.

Assessment of physician compliance to the CAEP 2021 Atrial Fibrillation Best Practices Checklist for rate and rhythm control in the emergency department.

OBJECTIVES: Acute atrial fibrillation and flutter (AF/AFL) are common arrhythmias treated in the emergency department (ED). The 2021 CAEP Best Practices Checklist provides clear recommendations for management of patients with acute AF/AFL. This study aimed to evaluate physician compliance to Checklist recommendations for risk assessment and ED management of AF/AFL. METHODS: This health records review assessed the management of adult patients presenting to two tertiary care EDs for management of acute AF/AFL from January to August, 2022. All ECGs demonstrating AF/AFL with a heart rate greater than 100 were compiled to capture primary and secondary causes. All visits were assessed for rate and rhythm control management, adverse events, return to ED, and safety criteria. Study physicians classified safety criteria from the Checklist into high and moderate concerns. The primary outcome was the proportion of cases with safety concerns and adverse events occurring during management in the ED. Data were analyzed using simple descriptive statistics. RESULTS: We included 429 patients with a mean age of 67.7 years and 57.1% male. ED management included rate control (20.4%), electrical (40.1%), and pharmacological (20.1%) cardioversion. Adverse events occurred in 9.5% of cases: 12.5% in rate control, 13.4% in electrical cardioversion, and 6.9% in pharmacologic cardioversion. Overall, 7.9% of cases had management safety concerns. Moderate safety concerns occurred in 4.9% of cases including failure to attain recommended heart rate at time of discharge (3.9%). Severe concerns were identified in 3.0% of cases including failure to cardiovert unstable patients (1.2%). The 30-day return-to-ED rate was 16.5% secondary to AF/AFL. CONCLUSION: ED management of AF/AFL was consistent with the CAEP Checklist and was safe overall. Opportunities for optimizing care include attaining recommended targets during rate control, avoidance of calcium channel and beta blockers in patients with systolic dysfunction, and earlier cardioversion for clinically unstable patients.

ABSTRAIT: OBJECTIFS: La fibrillation auriculaire aiguë et le flutter (FA/FAT) sont des arythmies courantes traitées aux urgences (SU). La liste de vérification des pratiques exemplaires 2021 du CAEP fournit des recommandations claires pour la prise en charge des patients atteints de FA/FAT aiguës. Cette étude visait à évaluer la conformité des médecins aux recommandations de la liste de contrôle pour l'évaluation des risques et la gestion de la FA/FAT. MéTHODES: Cet examen des dossiers de santé a évalué la prise en charge des patients adultes qui se sont présentés à deux urgences de soins tertiaires pour la prise en charge des FA/FAT aiguës de janvier à août 2022. Tous les ECG démontrant une FA/FAT avec une fréquence cardiaque supérieure à 100 ont été compilés pour capturer les causes primaires et secondaires. Toutes les visites ont été évaluées pour la gestion du contrôle des taux et du rythme, les événements indésirables, le retour à l'urgence et les critères de sécurité. Les médecins de l'étude ont classé les critères d'innocuité de la liste de contrôle en préoccupations élevées et modérées. Le résultat principal était la proportion de cas présentant des préoccupations en matière de sécurité et des événements indésirables survenant pendant la prise en charge à l'urgence. Les données ont été analysées à l'aide de statistiques descriptives simples. RéSULTATS: Nous avons inclus 429 patients avec un âge moyen de 67.7 ans et 57.1% d'hommes. La prise en charge de l'urgence comprenait une cardioversion contrôlée (20.4 %), électrique (40.1 %) et pharmacologique (20.1 %). Des événements indésirables se sont produits dans 9.5% des cas : 12.5% dans le contrôle du taux, 13.4% dans la cardioversion électrique et 6.9% dans la cardioversion pharmacologique. Dans l'ensemble, 7.9 % des cas concernaient la sécurité de la direction. Des problèmes de sécurité modérés sont survenus dans 4.9 % des cas, y compris l'incapacité d'atteindre la fréquence cardiaque recommandée au moment du congé (3.9 %). Des préoccupations graves ont été identifiées dans 3.0 % des cas, y compris l'échec à cardiovert patients instables (1.2 %). Le taux de retour aux urgences après 30 jours était de 16,5 % secondaire aux FA/FAT. CONCLUSION: La gestion de l'AF/AFL par le DG était conforme à la liste de vérification du CAEP et était sécuritaire dans l'ensemble. Les possibilités d'optimisation des soins comprennent l'atteinte des cibles recommandées lors du contrôle des taux, l'évitement du canal calcique et des bêtabloquants chez les patients présentant une dysfonction systolique et une cardioversion antérieure pour les patients cliniquement instables.

How well do ED physician practices align with the CAEP acute atrial fibrillation checklist for stroke prevention and disposition?

OBJECTIVES: Acute atrial fibrillation (AF)/flutter (AFL) is a common emergency department (ED) presentation. In 2021, an updated version of CAEP's Acute AF/AFL Best Practices Checklist was published, seeking to guide management. We assessed the alignment with and safety of application of the Checklist, regarding stroke prevention and disposition. METHODS: This health records review included adults presenting to two tertiary care academic EDs between January and August 2022 with a diagnosis of acute AF/AFL. Patients were excluded if their initial heart rate was < 100 or if they were hospitalized. Data extracted included: demographics, CHADS-65 score, clinical characteristics, ED treatment and disposition, and outpatient prescriptions and referrals. Our primary outcome was the proportion of patient encounters with one or more identified safety issues. Each case was assessed according to seven predetermined criteria from elements of the CAEP Checklist and either deemed "safe" or to contain one or more safety issues. We used descriptive statistics with 95% confidence intervals. RESULTS: 358 patients met inclusion criteria. The mean age was 66.9 years, 59.2% were male and 77.4% patients had at least one of the CHADS-65 criteria. 169 (47.2%) were not already on anticoagulation and 99 (27.6%) were discharged home with a new prescription for anticoagulation. The primary outcome was identified in 6.4% (95% CI 4.3-9.5) of encounters, representing 28 safety issues in 23 individuals. The safety concerns included: failure to prescribe anticoagulation when indicated (n = 6), inappropriate dosing of a direct oral anticoagulant (DOAC) (n = 2), inappropriate prescription of rate or rhythm control medication (n = 9), and failure to recommend appropriately timed follow-up for new rate control medication (n = 11). CONCLUSIONS: There was a very high level of ED physician alignment with CAEP's Best Practices Checklist regarding disposition and stroke prevention. There are opportunities to further improve care with respect to recommendation of anticoagulation and reducing inappropriate prescriptions of rate or rhythm medications.

RéSUMé: OBJECTIFS: La fibrillation auriculaire aiguë (FA)/flutter (FAT) est une présentation courante aux urgences (SU). En 2021, une version mise à jour de la liste de vérification des pratiques exemplaires en matière de FA/FAT aiguë du CAEP a été publiée, dans le but de guider la direction. Nous avons évalué l'harmonisation et la sécurité de l'application de la liste de contrôle en ce qui concerne la prévention et la disposition des AVC. MéTHODES: Cet examen des dossiers de santé comprenait des adultes qui se sont présentés à deux urgences universitaires de soins tertiaires entre janvier et août 2022 avec un diagnostic d'AF/AFL aigu. Les patients étaient exclus si leur fréquence cardiaque initiale était inférieure à 100 ou s'ils étaient hospitalisés. Les données extraites comprenaient les données démographiques, le score CHADS-65, les caractéristiques cliniques, le traitement et la disposition des urgences, ainsi que les prescriptions et les références ambulatoires. Notre résultat principal était la proportion de patients qui rencontraient un ou plusieurs problèmes de sécurité identifiés. Chaque cas a été évalué selon sept critères prédéterminés à partir des éléments de la liste de vérification du PPVE et jugé « sécuritaire ¼ ou comportant un ou plusieurs problèmes de sécurité. Nous avons utilisé des statistiques descriptives avec des intervalles de confiance de 95 %. RéSULTATS: 358 patients répondaient aux critères d'inclusion. L'âge moyen était de 66.9 ans, 59.2% étaient des hommes et 77.4% des patients avaient au moins un des critères CHADS-65. 169 (47.2%) n'étaient pas déjà sous anticoagulation et 99 (27.6%) ont été renvoyés à la maison avec une nouvelle prescription d'anticoagulation. Le critère de jugement principal a été identifié dans 6.4 % (IC à 95 % 4.3­9.5) des rencontres, ce qui représente 28 problèmes d'innocuité chez 23 personnes. Parmi les préoccupations en matière d'innocuité, mentionnons l'omission de prescrire un anticoagulant lorsque cela est indiqué (n = 6), l'administration inappropriée d'un anticoagulant oral direct (n = 2), la prescription inappropriée d'un médicament pour contrôler le rythme ou le rythme (n = 9), et l'omission de recommander un suivi bien chronométré vers le haut pour le nouveau médicament de contrôle de taux (n = 11). CONCLUSIONS: Il y avait un très haut niveau d'harmonisation des médecins de l'urgence avec la liste de vérification des pratiques exemplaires de l'ACMU en ce qui concerne la disposition et la prévention des accidents vasculaires cérébraux. Il est possible d'améliorer davantage les soins en ce qui concerne la recommandation d'anticoagulation et de réduire les prescriptions inappropriées de médicaments à taux ou à rythme.

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.

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.

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.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Glycan shield of the ebolavirus envelope glycoprotein GP.

The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions.

Evaluation of anthoxanthins and their actions on digestive enzyme inhibition when used independently and in combination.

Carbohydrate digestibility is a key determinant for elevated postprandial hyperglycemia (PPHG). Apart from dietary restrictions, one of the strategies to reduce PPHG is to limit the activity of carbohydrate digestive enzymes within the gastrointestinal tract in order to reduce monosaccharide absorption rates. The present work aimed to assess the inhibitory capabilities of digestive enzymes (e.g., α-glucosidase and α-amylase) by anthoxanthins when used independently, in combination with acarbose, or with a different anthoxanthin. Our results showed that quercetin, myricetin, and luteolin presented lower IC50 values than acarbose and inhibited α-glucosidase through mixed-type inhibition. On the other hand, acarbose when compared with these anthoxanthins, remained the most potent inhibitor of α-amylase. Combinatorial treatment (i) acarbose-quercetin and (ii) myricetin-luteolin showed synergistic activity (CI value less than 0.9) in α-glucosidase inhibition. An additive effect (CI value between 0.9 and 1.1) in α-glucosidase inhibition was observed when acarbose-myricetin, acarbose-luteolin or when a combination of two different anthoxanthins (quercetin-myricetin and quercetin-luteolin) was used. This study suggests the potential use of anthoxanthins as functional food ingredients to mitigate PPHG towards the management of T2DM.

Pan-ebolavirus serology study of healthcare workers in the Mbandaka Health Region, Democratic Republic of the Congo.

Although multiple antigenically distinct ebolavirus species can cause human disease, previous serosurveys focused on only Zaire ebolavirus (EBOV). Thus, the extent of reactivity or exposure to other ebolaviruses, and which sociodemographic factors are linked to this seroreactivity, are unclear. We conducted a serosurvey of 539 healthcare workers (HCW) in Mbandaka, Democratic Republic of the Congo, using ELISA-based analysis of serum IgG against EBOV, Sudan ebolavirus (SUDV) and Bundibugyo ebolavirus (BDBV) glycoproteins (GP). We compared seroreactivity to risk factors for viral exposure using univariate and multivariable logistic regression. Seroreactivity against different GPs ranged from 2.2-4.6%. Samples from six individuals reacted to all three species of ebolavirus and 27 samples showed a species-specific IgG response. We find that community health volunteers are more likely to be seroreactive against each antigen than nurses, and in general, that HCWs with indirect patient contact have higher anti-EBOV GP IgG levels than those with direct contact. Seroreactivity against ebolavirus GP may be associated with positions that offer less occupational training and access to PPE. Those individuals with broadly reactive responses may have had multiple ebolavirus exposures or developed cross-reactive antibodies. In contrast, those individuals with species-specific BDBV or SUDV GP seroreactivity may have been exposed to an ebolavirus not previously known to circulate in the region.

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.

Cross-reactive neutralizing human survivor monoclonal antibody BDBV223 targets the ebolavirus stalk.

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.

Structural basis of broad ebolavirus neutralization by a human survivor antibody.

The structural features that govern broad-spectrum activity of broadly neutralizing anti-ebolavirus antibodies (Abs) outside of the internal fusion loop epitope are currently unknown. Here we describe the structure of a broadly neutralizing human monoclonal Ab (mAb), ADI-15946, which was identified in a human survivor of the 2013-2016 outbreak. The crystal structure of ADI-15946 in complex with cleaved Ebola virus glycoprotein (EBOV GPCL) reveals that binding of the mAb structurally mimics the conserved interaction between the EBOV GP core and its glycan cap ß17-ß18 loop to inhibit infection. Both endosomal proteolysis of EBOV GP and binding of mAb FVM09 displace this loop, thereby increasing exposure of ADI-15946's conserved epitope and enhancing neutralization. Our work also mapped the paratope of ADI-15946, thereby explaining reduced activity against Sudan virus, which enabled rational, structure-guided engineering to enhance binding and neutralization of Sudan virus while retaining the parental activity against EBOV and Bundibugyo virus.

Structural Basis of Pan-Ebolavirus Neutralization by a Human Antibody against a Conserved, yet Cryptic Epitope.

Only one naturally occurring human antibody has been described thus far that is capable of potently neutralizing all five ebolaviruses. Here we present two crystal structures of this rare, pan-ebolavirus neutralizing human antibody in complex with Ebola virus and Bundibugyo virus glycoproteins (GPs), respectively. The structures delineate the key protein and glycan contacts for binding that are conserved across the ebolaviruses, explain the antibody's unique broad specificity and neutralization activity, and reveal the likely mechanism behind a known escape mutation in the fusion loop region of GP2. We found that the epitope of this antibody, ADI-15878, extends along the hydrophobic paddle of the fusion loop and then dips down into a highly conserved pocket beneath the N-terminal tail of GP2, a mode of recognition unlike any other antibody elicited against Ebola virus, and likely critical for its broad activity. The fold of Bundibugyo virus glycoprotein, not previously visualized, is similar to the fold of Ebola virus GP, and ADI-15878 binds to each virus's GP with a similar strategy and angle of attack. These findings will be useful in deployment of this antibody as a broad-spectrum therapeutic and in the design of immunogens that elicit the desired broadly neutralizing immune response against all members of the ebolavirus genus and filovirus family.IMPORTANCE There are five different members of the Ebolavirus genus. Provision of vaccines and treatments able to protect against any of the five ebolaviruses is an important goal of public health. Antibodies are a desired result of vaccines and can be delivered directly as therapeutics. Most antibodies, however, are effective against only one or two, not all, of these pathogens. Only one human antibody has been thus far described to neutralize all five ebolaviruses, antibody ADI-15878. Here we describe the molecular structure of ADI-15878 bound to the relevant target proteins of Ebola virus and Bundibugyo virus. We explain how it achieves its rare breadth of activity and propose strategies to design improved vaccines capable of eliciting more antibodies like ADI-15878.