Resurfaced ZIKV EDIII nanoparticle immunogens elicit neutralizing and protective responses in vivo.

Zika virus (ZIKV) is a flavivirus that can cause severe disease, but there are no approved treatments or vaccines. A complication for flavivirus vaccine development is the potential of immunogens to enhance infection via antibody-dependent enhancement (ADE), a process mediated by poorly neutralizing and cross-reactive antibodies. Thus, there is a great need to develop immunogens that minimize the potential to elicit enhancing antibodies. Here we utilized structure-based protein engineering to develop "resurfaced" (rs) ZIKV immunogens based on E glycoprotein domain III (ZDIIIs), in which epitopes bound by variably neutralizing antibodies were masked by combinatorial mutagenesis. We identified one resurfaced ZDIII immunogen (rsZDIII-2.39) that elicited a protective but immune-focused response. Compared to wild type ZDIII, immunization with resurfaced rsZDIII-2.39 protein nanoparticles produced fewer numbers of ZIKV EDIII antigen-reactive B cells and elicited serum that had a lower magnitude of induced ADE against dengue virus serotype 1 (DENV1) Our findings enhance our understanding of the structural and functional determinants of antibody protection against ZIKV.

Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity.

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.

Combinatorial Resurfacing of Dengue Envelope Protein Domain III Antigens Selectively Ablates Epitopes Associated with Serotype-Specific or Infection-Enhancing Antibody Responses.

Mutagenesis of surface-exposed residues, or "resurfacing", is a protein engineering strategy that can be utilized to disrupt antibody recognition or modulate the capacity of a protein to elicit antibody responses. We apply resurfacing to engineer Dengue virus envelope protein domain III (DENV DIII) antigens with the goal of focusing humoral recognition on epitopes of interest by selective ablation of irrelevant and undesired epitopes. Cross-reactive but non-neutralizing antibodies have the potential to enhance Dengue virus (DENV) infection by a process called antibody-dependent enhancement, thought to be associated with severe secondary heterotypic infection. Thus, a focus on epitopes associated with broadly neutralizing antibodies is important both for understanding human antibody responses against DENV and for the development of a successful DENV vaccine. To engineer DENV DIII antigens focusing on the AG strand epitope associated with broadly neutralizing antibody responses, we generated yeast surface display libraries of DENV2 DIII where the AB loop (associated with cross-reactive but non-neutralizing antibody responses) and FG loop (associated with serotype-specific antibody responses) were mutagenized to allow for all possible amino acid substitutions. Loop variants that maintained the AG strand epitope and simultaneously disrupted the AB and FG loop epitopes exhibited high and diverse mutational loads that were amenable to loop exchange and transplantation into a DENV4 DIII background. Thus, several loop variants fulfill this antigenicity criteria regardless of serotype context. The resulting resurfaced DIII antigens may be utilized as AG strand epitope-focusing probes or immunogen candidates.

Peptide-Based Vaccines: Current Progress and Future Challenges.

Vaccines have had a profound impact on the management and prevention of infectious disease. In addition, the development of vaccines against chronic diseases has attracted considerable interest as an approach to prevent, rather than treat, conditions such as cancer, Alzheimer's disease, and others. Subunit vaccines consist of nongenetic components of the infectious agent or disease-related epitope. In this Review, we discuss peptide-based vaccines and their potential in three therapeutic areas: infectious disease, Alzheimer's disease, and cancer. We discuss factors that contribute to vaccine efficacy and how these parameters may potentially be modulated by design. We examine both clinically tested vaccines as well as nascent approaches and explore current challenges and potential remedies. While peptide vaccines hold substantial promise in the prevention of human disease, many obstacles remain that have hampered their clinical use; thus, continued research efforts to address these challenges are warranted.

Human monoclonal antibodies against chikungunya virus target multiple distinct epitopes in the E1 and E2 glycoproteins.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.

Isolation of Synthetic Antibodies Against BCL-2-Associated X Protein (BAX).

The BCL-2 protein family plays central roles in the mitochondrial pathway of cell apoptosis. The BCL-2-Associated X protein (BAX), along with other proapoptotic proteins, induces cell death in response to a variety of stress stimuli. Upon receipt of killing signals, cytosolic BAX is activated and translocates to mitochondria where it causes mitochondrial outer membrane permeabilization (MOMP) and initials a series of cellular events that eventually lead to cell destruction. Despite recent progress toward understanding the structure, function, and activation mechanism of BAX, detailed information about how cytosolic BAX can be inhibited is still limited. Here we describe a method of selecting synthetic antibody fragments (Fabs) against BAX using phage display. Synthetic antibodies discovered from the selection have been used as structural probes to gain novel mechanistic details on BAX inhibition. This synthetic antibody selection method could be potentially applied to other BCL-2 proteins.

Engineered Dengue Virus Domain III Proteins Elicit Cross-Neutralizing Antibody Responses in Mice.

Dengue virus is the most globally prevalent mosquito-transmitted virus. Primary infection with one of four cocirculating serotypes (DENV-1 to -4) causes a febrile illness, but secondary infection with a heterologous serotype can result in severe disease, due in part to antibody-dependent enhancement of infection (ADE). In ADE, cross-reactive but nonneutralizing antibodies, or subprotective levels of neutralizing antibodies, promote uptake of antibody-opsonized virus in Fc-γ receptor-positive cells. Thus, elicitation of broadly neutralizing antibodies (bNAbs), but not nonneutralizing antibodies, is desirable for dengue vaccine development. Domain III of the envelope glycoprotein (EDIII) is targeted by bNAbs and thus is an attractive immunogen. However, immunization with EDIII results in sera with limited neutralization breadth. We developed "resurfaced" EDIII immunogens (rsDIIIs) in which the A/G strand epitope that is targeted by bNAb 4E11 is maintained but less desirable epitopes are masked. RsDIIIs bound 4E11, but not serotype-specific or nonneutralizing antibodies. One rsDIII and, unexpectedly, wild-type (WT) DENV-2 EDIII elicited cross-neutralizing antibody responses against DENV-1 to -3 in mice. While these sera were cross-neutralizing, they were not sufficiently potent to protect AG129 immunocompromised mice at a dose of 200 µl (50% focus reduction neutralization titer [FRNT50], ∼1:60 to 1:130) against mouse-adapted DENV-2. Our results provide insight into immunogen design strategies based on EDIII.IMPORTANCE Dengue virus causes approximately 390 million infections per year. Primary infection by one serotype causes a self-limiting febrile illness, but secondary infection by a heterologous serotype can result in severe dengue syndrome, which is characterized by hemorrhagic fever and shock syndrome. This severe disease is thought to arise because of cross-reactive, non- or poorly neutralizing antibodies from the primary infection that are present in serum at the time of secondary infection. These cross-reactive antibodies enhance the infection rather than controlling it. Therefore, induction of a broadly and potently neutralizing antibody response is desirable for dengue vaccine development. Here, we explore a novel strategy for developing immunogens based on domain III of the E glycoprotein, where undesirable epitopes (nonneutralizing or nonconserved) are masked by mutation. This work provides fundamental insight into the immune response to domain III that can be leveraged for future immunogen design.

Mechanistic and Fc requirements for inhibition of Sudan virus entry and in vivo protection by a synthetic antibody.

The Sudan virus (SUDV), an ebolavirus, causes severe hemorrhagic fever with human case fatality rates of ∼50%. Previous work from our lab demonstrated the synthetic antibody F4 potently inhibits viral entry and protects against lethal virus challenge in mice [Chen et al., ACS Chem. Biol., 2014, 9, 2263-2273]. Here, we explore mechanistic requirements as well as contribution of the Fc region and function on neutralization and in vivo protection. Live cell imaging demonstrates that the antibody colocalizes with vesicular stomatitis virus particles containing the Sudan virus glycoprotein (VSV-GPSUDV) and that the antibody is rapidly degraded within cellular endosomes. A viral escape mutant contained substitutions on the N-heptad repeat (NHR) segment of GP2, the fusion subunit. Truncation studies indicated that the size of the Fc impacts virus neutralization potential. Finally, we examined the protective efficacy of Fc-null mutants in mice, and found that Fc function was not required for high levels of protection. Altogether, these results indicate that neutralization of SUDV GP-mediated cell entry likely involves blockade of viral membrane fusion within endosomes, and that inhibition of viral entry is the likely mechanism of in vivo protection.

Bispecific antibodies for viral immunotherapy.

Bispecific antibody engineering, in which binding specificities toward 2 distinct epitopes are combined into a single molecule, can greatly enhance immunotherapeutic properties of monoclonal antibodies. While the bispecific antibody approach has been applied widely to targets for indications such as cancer and inflammation, the development of such agents for viral immunotherapy is only now emerging. Here, we review recent advances in the development of bispecific antibodies for viral immunotherapy, highlighting promising in vitro and in vivo results.

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

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.

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site.

The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.

Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry.

A switch from parallel to antiparallel strand orientation in a coiled-coil X-ray structure via two core hydrophobic mutations.

The coiled-coil is one of the most ubiquitous and well studied protein structural motifs. Significant effort has been devoted to dissecting subtle variations of the typical heptad repeat sequence pattern that can designate larger topological features such as relative α-helical orientation and oligomer size. Here we report the X-ray structure of a model coiled-coil peptide, HA2-Del-L2seM, which forms an unanticipated core antiparallel dimer with potential sites for discrete higher-order multimerization (trimer or tetramer). In the X-ray structure, a third, partially-ordered α-helix is weakly associated with the antiparallel dimer and analytical ultracentrifugation experiments indicate the peptide forms a well-defined tetramer in solution. The HA2-Del-L2seM sequence is closely related to a parent model peptide, HA2-Del, which we previously reported adopts a parallel trimer; HA2-Del-L2seM differs by only hydrophobic leucine to selenomethione mutations and thus this subtle difference is sufficient to switch both relative α-helical topology and number of α-helices participating in the coiled-coil. Comparison of the X-ray structures of HA2-Del-L2seM (reported here) with the HA2-Del parent (reported previously) reveals novel interactions involving the selenomethionine residues that promote antiparallel coiled-coil configuration and preclude parallel trimer formation. These novel atomic insights are instructive for understanding subtle features that can affect coiled-coil topology and provide additional information for design of antiparallel coiled-coils.

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.

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking.

We previously described potent inhibition of Ebola virus entry by a 'C-peptide' based on the GP2 C-heptad repeat region (CHR) targeted to endosomes ('Tat-Ebo'). Here, we report the synthesis and evaluation of C-peptides conjugated to cholesterol, and Tat-Ebo analogs containing covalent side chain-side chain crosslinks to promote α-helical conformation. We found that the cholesterol-conjugated C-peptides were potent inhibitors of Ebola virus glycoprotein (GP)-mediated cell entry (~10(3)-fold reduction in infection at 40 µM). However, this mechanism of inhibition is somewhat non-specific because the cholesterol-conjugated peptides also inhibited cell entry mediated by vesicular stomatitis virus glycoprotein G. One side chain-side chain crosslinked peptide had moderately higher activity than the parent compound Tat-Ebo. Circular dichroism revealed that the cholesterol-conjugated peptides unexpectedly formed a strong α-helical conformation that was independent of concentration. Side chain-side chain crosslinking enhanced α-helical stability of the Tat-Ebo variants, but only at neutral pH. These result provide insight into mechanisms of C-peptide inhibiton of Ebola virus GP-mediated cell entry.

Peptides that bind specifically to an antibody from a chronic lymphocytic leukemia clone expressing unmutated immunoglobulin variable region genes.

Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the disease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) engage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined the binding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable domains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected peptide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in families based on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb revealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (micromolar KD), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations (nanomolar KD) and with immunoglobulin heavy chain variable (IGHV)-mutated antibodies selected by environmental antigens. This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthy subjects with monoclonal B lymphocytosis.

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.

Conformational properties of peptides corresponding to the ebolavirus GP2 membrane-proximal external region in the presence of micelle-forming surfactants and lipids.

Ebola virus and Sudan virus are members of the family Filoviridae of nonsegmented negative-strand RNA viruses ("filoviruses") that cause severe hemorrhagic fever with fatality rates as high as 90%. Infection by filoviruses requires membrane fusion between the host and the virus; this process is facilitated by the two subunits of the envelope glycoprotein, GP1 (the surface subunit) and GP2 (the transmembrane subunit). The membrane-proximal external region (MPER) is a Trp-rich segment that immediately precedes the transmembrane domain of GP2. In the analogous glycoprotein for HIV-1 (gp41), the MPER is critical for membrane fusion and is the target of several neutralizing antibodies. However, the role of the MPER in filovirus GP2 and its importance in membrane fusion have not been established. Here, we characterize the conformational properties of peptides representing the GP MPER segments of Ebola virus and Sudan virus in the presence of micelle-forming surfactants and lipids, at pH 7 and 4.6. Circular dichroism spectroscopy and tryptophan fluorescence indicate that the GP2 MPER peptides bind to micelles of sodium dodecyl sulfate and dodecylphosphocholine (DPC). Nuclear magnetic resonance spectroscopy of the Sudan virus MPER peptide revealed that residues 644-651 interact directly with DPC, and that this interaction enhances the helical conformation of the peptide. The Sudan virus MPER peptide was found to moderately inhibit cell entry by a GP-pseudotyped vesicular stomatitis virus but did not induce leakage of a fluorescent molecule from a large unilammellar vesicle comprised of 1-palmitoyl-2-oleoylphosphatidylcholine or cause hemolysis. Taken together, this analysis suggests the filovirus GP2 MPER binds and inserts shallowly into lipid membranes.

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" [GP(UNCL)] and "cleaved" [GP(CL)] forms). We identified antibodies with distinct recognition profiles: Fab(CL) bound preferentially to GP(CL) (EC(50)=1.7 nM), whereas Fab(UNCL) bound specifically to GP(UNCL) (EC(50)=75 nM). Neutralization assays with GP-containing pseudotyped viruses indicated that these antibodies inhibited GP(CL)- or GP(UNCL)-mediated viral entry with specificity matching their recognition profiles (IC(50): 87 nM for IgG(CL); 1 µM for Fab(UNCL)). Competition ELISAs indicate that Fab(CL) 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 Fab(UNCL) was also distinct from that of KZ52, suggesting that Fab(UNCL) binds a novel neutralization epitope on GP(UNCL). Furthermore, the neutralizing ability of Fab(CL) suggests that there are targets on GP(CL) 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.