Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic.

Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a ß-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis.

Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression.

UNLABELLED: Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. IMPORTANCE: Marburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

BACKGROUND: Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. METHODS: Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. RESULTS: The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105-9.0 × 108 genomes/mL. CONCLUSIONS: The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015.

Development of Prototype Filovirus Recombinant Antigen Immunoassays.

BACKGROUND: Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. METHODS: Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. RESULTS: Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. CONCLUSIONS: The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins.

Crystal structure of Marburg virus VP24.

The VP24 protein plays an essential, albeit poorly understood role in the filovirus life cycle. VP24 is only 30% identical between Marburg virus and the ebolaviruses. Furthermore, VP24 from the ebolaviruses is immunosuppressive, while that of Marburg virus is not. The crystal structure of Marburg virus VP24, presented here, reveals that although the core is similar between the viral genera, Marburg VP24 is distinguished by a projecting ß-shelf and an alternate conformation of the N-terminal polypeptide.

The ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold.

Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.

Antibodies targeting Crimean-Congo hemorrhagic fever virus GP38 limit vascular leak and viral spread.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen transmitted by tick bites, with no vaccines or specific therapeutics approved to date. Severe disease manifestations include hemorrhage, endothelial dysfunction, and multiorgan failure. Infected cells secrete the viral glycoprotein GP38, whose extracellular function is presently unknown. GP38 is considered an important target for vaccine and therapeutic design as GP38-specific antibodies can protect against severe disease in animal models, albeit through a currently unknown mechanism of action. Here, we show that GP38 induces endothelial barrier dysfunction in vitro, and that CCHFV infection, and GP38 alone, can trigger vascular leak in a mouse model. Protective antibodies that recognize specific antigenic sites on GP38, but not a protective neutralizing antibody binding the structural protein Gc, potently inhibit endothelial hyperpermeability in vitro and vascular leak in vivo during CCHFV infection. This work uncovers a function of the secreted viral protein GP38 as a viral toxin in CCHFV pathogenesis and elucidates the mode of action of non-neutralizing GP38-specific antibodies.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Structural and mechanistic basis of neutralization by a pan-hantavirus protective antibody.

Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or therapeutics. We recently isolated a monoclonal broadly neutralizing antibody (nAb) from a Puumala virus-experienced human donor. Here, we report its structure bound to its target, the Gn/Gc glycoprotein heterodimer comprising the viral fusion complex. The structure explains the broad activity of the nAb: It recognizes conserved Gc fusion loop sequences and the main chain of variable Gn sequences, thereby straddling the Gn/Gc heterodimer and locking it in its prefusion conformation. We show that the nAb's accelerated dissociation from the divergent Andes virus Gn/Gc at endosomal acidic pH limits its potency against this highly lethal virus and correct this liability by engineering an optimized variant that sets a benchmark as a candidate pan-hantavirus therapeutic.

Reversion of Ebolavirus Disease from a Single Intramuscular Injection of a Pan-Ebolavirus Immunotherapeutic.

Intravenous (IV) administration of antiviral monoclonal antibodies (mAbs) can be challenging, particularly during an ongoing epidemic, due to the considerable resources required for performing infusions. An ebolavirus therapeutic administered via intramuscular (IM) injection would reduce the burdens associated with IV infusion and allow rapid treatment of exposed individuals during an outbreak. Here, we demonstrate how MBP134, a cocktail of two pan-ebolavirus mAbs, reverses the course of Sudan ebolavirus disease (Gulu variant) with a single IV or IM dose in non-human primates (NHPs) as late as five days post-exposure. We also investigate the utility of adding half-life extension mutations to the MBP134 mAbs, ultimately creating a half-life extended cocktail designated MBP431. When delivered as a post-exposure prophylactic or therapeutic, a single IM dose of MBP431 offered complete or significant protection in NHPs challenged with Zaire ebolavirus. In conjunction with previous studies, these results support the use of MBP431 as a rapidly deployable IM medical countermeasure against every known species of ebolavirus.

Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. Structural information is lacking in regard to the CCHFV membrane fusion glycoprotein Gc­the main target of the host neutralizing antibody response­as well as antibody­mediated neutralization mechanisms. We describe the structure of prefusion Gc bound to the antigen-binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show the two Fabs acting in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The structures also revealed the neutralization mechanism of previously reported antibodies against CCHFV, providing the molecular underpinnings essential for developing CCHFV­specific medical countermeasures for epidemic preparedness.

Combination therapy with remdesivir and monoclonal antibodies protects nonhuman primates against advanced Sudan virus disease.

A major challenge in managing acute viral infections is ameliorating disease when treatment is delayed. Previously, we reported the success of a 2-pronged mAb and antiviral remdesivir therapeutic approach to treat advanced illness in rhesus monkeys infected with Marburg virus (MARV). Here, we explored the benefit of a similar combination therapy for Sudan ebolavirus (Sudan virus; SUDV) infection. Importantly, no licensed anti-SUDV therapeutics currently exist, and infection of rhesus macaques with SUDV results in a rapid disease course similar to MARV with a mean time to death of 8.3 days. When initiation of therapy with either remdesivir or a pan-ebolavirus mAb cocktail (MBP431) was delayed until 6 days after inoculation, only 20% of macaques survived. In contrast, when remdesivir and MBP431 treatment were combined beginning 6 days after inoculation, significant protection (80%) was achieved. Our results suggest that combination therapy may be a viable treatment for patients with advanced filovirus disease that warrants further clinical testing in future outbreaks.

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Combination therapy protects macaques against advanced Marburg virus disease.

Monoclonal antibodies (mAbs) and remdesivir, a small-molecule antiviral, are promising monotherapies for many viruses, including members of the genera Marburgvirus and Ebolavirus (family Filoviridae), and more recently, SARS-CoV-2. One of the major challenges of acute viral infections is the treatment of advanced disease. Thus, extending the window of therapeutic intervention is critical. Here, we explore the benefit of combination therapy with a mAb and remdesivir in a non-human primate model of Marburg virus (MARV) disease. While rhesus monkeys are protected against lethal infection when treatment with either a human mAb (MR186-YTE; 100%), or remdesivir (80%), is initiated 5 days post-inoculation (dpi) with MARV, no animals survive when either treatment is initiated alone beginning 6 dpi. However, by combining MR186-YTE with remdesivir beginning 6 dpi, significant protection (80%) is achieved, thereby extending the therapeutic window. These results suggest value in exploring combination therapy in patients presenting with advanced filovirus disease.

Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein.

The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177-182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.