Jonathan R. Lai

Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses

ABSTRACT The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

Inhibition of Ebola Virus Entry by a C-peptide Targeted to Endosomes

Ebola virus (EboV) and Marburg virus (MarV) (filoviruses) are the causative agents of severe hemorrhagic fever. Infection begins with uptake of particles into cellular endosomes, where the viral envelope glycoprotein (GP) catalyzes fusion between the viral and host cell membranes. This fusion event is thought to involve conformational rearrangements of the transmembrane subunit (GP2) of the envelope spike that ultimately result in formation of a six-helix bundle by the N- and C-terminal heptad repeat (NHR and CHR, respectively) regions of GP2. Infection by other viruses employing similar viral entry mechanisms (such as HIV-1 and severe acute respiratory syndrome coronavirus) can be inhibited with synthetic peptides corresponding to the native CHR sequence (“C-peptides”). However, previously reported EboV C-peptides have shown weak or insignificant antiviral activity. To determine whether the activity of a C-peptide could be improved by increasing its intracellular concentration, we prepared an EboV C-peptide conjugated to the arginine-rich sequence from HIV-1 Tat, which is known to accumulate in endosomes. We found that this peptide specifically inhibited viral entry mediated by filovirus GP proteins and infection by authentic filoviruses. We determined that antiviral activity was dependent on both the Tat sequence and the native EboV CHR sequence. Mechanistic studies suggested that the peptide acts by blocking a membrane fusion intermediate.

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.

Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site

Summary Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.

A "Trojan horse" bispecific antibody strategy for broad protection against ebolaviruses.

Treating Ebola with a Trojan horse The recent major Ebola virus outbreak in West Africa high-lighted the need for effective therapeutics against this and other filoviruses. Neutralizing ebolaviruses with antibodies is a challenge because the viruses bind their entry receptor, NPC1, inside the cell within endosomes rather than on the cell surface. Furthermore, enzymes in endosomes cleave the Ebola virus surface glycoprotein (GP) to reveal its receptor binding site. Wec et al. now report a bispecific antibody strategy targeting all known ebolaviruses that overcomes this problem (see the Perspective by Labrijn and Parren). They coupled an antibody specific for a conserved, surface-exposed epitope of GP to antibodies that recognize either NPC1 or the NPC1 binding site on GP. Treating mice therapeutically with these antibodies allowed them to survive otherwise lethal ebolavirus infection. Science, this issue p. 350; see also p. 284 Bispecific antibodies show therapeutic efficacy against ebolaviruses in mice. There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor–binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such “Trojan horse” bispecific antibodies have potential as broad antifilovirus immunotherapeutics.

Role of Electrostatic Repulsion in Controlling pH-Dependent Conformational Changes of Viral Fusion Proteins

Viral fusion proteins undergo dramatic conformational transitions during membrane fusion. For viruses that enter through the endosome, these conformational rearrangements are typically pH sensitive. Here, we provide a comprehensive review of the molecular interactions that govern pH-dependent rearrangements and introduce a paradigm for electrostatic residue pairings that regulate progress through the viral fusion coordinate. Analysis of structural data demonstrates a significant role for side-chain protonation in triggering conformational change. To characterize this behavior, we identify two distinct residue pairings, which we define as Histidine-Cation (HisCat) and Anion-Anion (AniAni) interactions. These side-chain pairings destabilize a particular conformation via electrostatic repulsion through side-chain protonation. Furthermore, two energetic control mechanisms, thermodynamic and kinetic, regulate these structural transitions. This review expands on the current literature by identification of these residue clusters, discussion of data demonstrating their function, and speculation of how these residue pairings contribute to the energetic controls.

Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein

Ebola virus (EBOV) is a highly pathogenic member of the Filoviridae family of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann‐Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the “uncleaved” [GPUNCL] and “cleaved” [GPCL] forms). We identified antibodies with distinct recognition profiles: FabCL bound preferentially to GPCL (EC50=1.7 nM), whereas FabUNCL bound specifically to GPUNCL (EC50=75 nM). Neutralization assays with GP‐containing pseudotyped viruses indicated that these antibodies inhibited GPCL‐ or GPUNCL‐mediated viral entry with specificity matching their recognition profiles (IC50: 87 nM for IgGCL; 1 μM for FabUNCL). Competition ELISAs indicate that FabCL binds an epitope distinct from that of KZ52, a well‐characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of FabUNCL was also distinct from that of KZ52, suggesting that FabUNCL binds a novel neutralization epitope on GPUNCL. Furthermore, the neutralizing ability of FabCL suggests that there are targets on GPCL available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.

Synthetic Antibodies with a Human Framework That Protect Mice from Lethal Sudan Ebolavirus Challenge

The ebolaviruses cause severe and rapidly progressing hemorrhagic fever. There are five ebolavirus species; although much is known about Zaire ebolavirus (EBOV) and its neutralization by antibodies, little is known about Sudan ebolavirus (SUDV), which is emerging with increasing frequency. Here we describe monoclonal antibodies containing a human framework that potently inhibit infection by SUDV and protect mice from lethal challenge. The murine antibody 16F6, which binds the SUDV envelope glycoprotein (GP), served as the starting point for design. Sequence and structural alignment revealed similarities between 16F6 and YADS1, a synthetic antibody with a humanized scaffold. A focused phage library was constructed and screened to impart 16F6-like recognition properties onto the YADS1 scaffold. A panel of 17 antibodies were characterized and found to have a range of neutralization potentials against a pseudotype virus infection model. Neutralization correlated with GP binding as determined by ELISA. Two of these clones, E10 and F4, potently inhibited authentic SUDV and conferred protection and memory immunity in mice from lethal SUDV challenge. E10 and F4 were further shown to bind to the same epitope on GP as 16F6 with comparable affinities. These antibodies represent strong immunotherapeutic candidates for treatment of SUDV infection.

Chemical and Structural Aspects of Ebola Virus Entry Inhibitors

The Ebolaviruses are members of the family Filoviridae (“filoviruses”) and cause severe hemhorragic fever with human case fatality rates as high as 90%. Infection requires attachment of the viral particle to cells and triggering of membrane fusion between the host and viral membranes, a process that occurs in the host endosome and is facilitated by the envelope glycoprotein (GP). One potential strategy for therapeutic intervention is the development of agents (antibodies, peptides, and small molecules) that can interfere with viral entry aspects such as attachment, uptake, priming, or membrane fusion. This paper highlights recent developments in the discovery and evaluation of therapeutic entry inhibitors and identifies opportunities moving forward.

Structural Characterization of the Glycoprotein GP2 Core Domain from the CAS Virus, a Novel Arenavirus-Like Species

Abstract Fusion of the viral and host cell membranes is a necessary first step for infection by enveloped viruses and is mediated by the envelope glycoprotein. The transmembrane subunits from the structurally defined “class I” glycoproteins adopt an α-helical “trimer-of-hairpins” conformation during the fusion pathway. Here, we present our studies on the envelope glycoprotein transmembrane subunit, GP2, of the CAS virus (CASV). CASV was recently identified from annulated tree boas (Corallus annulatus) with inclusion body disease and is implicated in the disease etiology. We have generated and characterized two protein constructs consisting of the predicted CASV GP2 core domain. The crystal structure of the CASV GP2 post-fusion conformation indicates a trimeric α-helical bundle that is highly similar to those of Ebola virus and Marburg virus GP2 despite CASV genome homology to arenaviruses. Denaturation studies demonstrate that the stability of CASV GP2 is pH dependent with higher stability at lower pH; we propose that this behavior is due to a network of interactions among acidic residues that would destabilize the α-helical bundle under conditions where the side chains are deprotonated. The pH-dependent stability of the post-fusion structure has been observed in Ebola virus and Marburg virus GP2, as well as other viruses that enter via the endosome. Infection experiments with CASV and the related Golden Gate virus support a mechanism of entry that requires endosomal acidification. Our results suggest that, despite being primarily arenavirus like, the transmembrane subunit of CASV is extremely similar to the filoviruses.