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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.
Copyright © 2020 Elsevier Inc. All rights reserved.

BACKGROUND AND PURPOSE - G protein-gated inwardly rectifying K (K 3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal K 3 channels consist of K 3.1 and K 3.2 subtypes, while cardiac K 3 channels consist of K 3.1 and K 3.4 subtypes. Previously, we identified a family of urea-containing K 3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for K 3.1/3.2 relative to K 3.1/3.4 channels. Here, we characterize a non-urea activator, VU0810464, which displays nanomolar potency as a K 3.1/3.2 activator, improved selectivity for neuronal K 3 channels, and improved brain penetration.
EXPERIMENTAL APPROACH - We used whole-cell electrophysiology to measure the efficacy and potency of VU0810464 in neurons and the selectivity of VU0810464 for neuronal and cardiac K 3 channel subtypes. We tested VU0810464 in vivo in stress-induced hyperthermia and elevated plus maze paradigms. Parallel studies with ML297, the prototypical activator of K 3.1-containing K 3 channels, were performed to permit direct comparisons.
KEY RESULTS - VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal K 3 channel activators, but VU0810464 was more selective for neuronal K 3 channels. VU0810464, like ML297, reduced stress-induced hyperthermia in a K 3-dependent manner in mice. ML297, but not VU0810464, decreased anxiety-related behaviour as assessed with the elevated plus maze test.
CONCLUSION AND IMPLICATIONS - VU0810464 represents a new class of K 3 channel activator with enhanced selectivity for K 3.1/3.2 channels. VU0810464 may be useful for examining K 3.1/3.2 channel contributions to complex behaviours and for probing the potential of K 3 channel-dependent manipulations to treat neurological disorders.
© 2019 The British Pharmacological Society.

The human B cell response to natural filovirus infections early after recovery is poorly understood. Previous serologic studies suggest that some Ebola virus survivors exhibit delayed antibody responses with low magnitude and quality. Here, we sought to study the population of individual memory B cells induced early in convalescence. We isolated monoclonal antibodies (MAbs) from memory B cells from four survivors treated for Ebola virus disease (EVD) 1 or 3 months after discharge from the hospital. At the early time points postrecovery, the frequency of Ebola-specific B cells was low and dominated by clones that were cross-reactive with both Ebola glycoprotein (GP) and with the secreted GP (sGP) form. Of 25 MAbs isolated from four donors, only one exhibited neutralization activity. This neutralizing MAb, designated MAb EBOV237, recognizes an epitope in the glycan cap of the surface glycoprotein. murine lethal challenge studies showed that EBOV237 conferred protection when given prophylactically at a level similar to that of the ZMapp component MAb 13C6. The results suggest that the human B cell response to EVD 1 to 3 months postdischarge is characterized by a paucity of broad or potent neutralizing clones. However, the neutralizing epitope in the glycan cap recognized by EBOV237 may play a role in the early human antibody response to EVD and should be considered in rational design strategies for new Ebola virus vaccine candidates. The pathogenesis of Ebola virus disease (EVD) in humans is complex, and the mechanisms contributing to immunity are poorly understood. In particular, it appears that the quality and magnitude of the human B cell response early after recovery from EVD may be reduced compared to most viral infections. Here, we isolated human monoclonal antibodies from B cells of four survivors of EVD at 1 or 3 months after hospital discharge. Ebola-specific memory B cells early in convalescence were low in frequency, and the antibodies they encoded demonstrated poor neutralizing potencies. One neutralizing antibody that protected mice from lethal infection, EBOV237, was identified in the panel of 25 human antibodies isolated. Recognition of the glycan cap epitope recognized by EBOV237 suggests that this antigenic site should be considered in vaccine design and treatment strategies for EVD.
Copyright © 2019 American Society for Microbiology.

Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits, including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent vaccine immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV, and BDBV. As immunization with a cocktail of antigenically related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to sera from guinea pigs receiving the monovalent vaccines, sera from guinea pigs receiving the trivalent vaccine bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only a slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389 to 403, 397 to 415, and 477 to 493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus infection survivors demonstrated that the immune sera block the binding of antibodies specific for the GP glycan cap, the GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, administration of a cocktail of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus. The symptoms of the disease caused by the ebolaviruses Ebola, Bundibugyo, and Sudan are similar, and their areas of endemicity overlap. However, because of the limited antigenic relatedness of the ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous and not against heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, and this might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here, an in-depth analysis of the antibody responses to administration of a cocktail of human parainfluenza virus type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses compared to that induced by individual vaccines.
Copyright © 2019 American Society for Microbiology.

West Nile virus (WNV), a member of the Flavivirus genus, is a leading cause of viral encephalitis in the United States. The development of neutralizing antibodies against the flavivirus envelope (E) protein is critical for immunity and vaccine protection. Previously identified candidate therapeutic mouse and human neutralizing monoclonal antibodies (mAbs) target epitopes within the E domain III lateral ridge and the domain I-II hinge region, respectively. To explore the neutralizing antibody repertoire elicited by WNV infection for potential therapeutic application, we isolated ten mAbs from WNV-infected individuals. mAb WNV-86 neutralized WNV with a 50% inhibitory concentration of 2 ng ml, one of the most potently neutralizing flavivirus-specific antibodies ever isolated. WNV-86 targets an epitope in E domain II, and preferentially recognizes mature virions lacking an uncleaved form of the chaperone protein prM, unlike most flavivirus-specific antibodies. In vitro selection experiments revealed a neutralization escape mechanism involving a glycan addition to E domain II. Finally, a single dose of WNV-86 administered two days post-infection protected mice from lethal WNV challenge. This study identifies a highly potent human neutralizing mAb with therapeutic potential that targets an epitope preferentially displayed on mature virions.

Synthetically engineered DNA-encoded monoclonal antibodies (DMAbs) are an in vivo platform for evaluation and delivery of human mAb to control against infectious disease. Here, we engineer DMAbs encoding potent anti-Zaire ebolavirus (EBOV) glycoprotein (GP) mAbs isolated from Ebola virus disease survivors. We demonstrate the development of a human IgG1 DMAb platform for in vivo EBOV-GP mAb delivery and evaluation in a mouse model. Using this approach, we show that DMAb-11 and DMAb-34 exhibit functional and molecular profiles comparable to recombinant mAb, have a wide window of expression, and provide rapid protection against lethal mouse-adapted EBOV challenge. The DMAb platform represents a simple, rapid, and reproducible approach for evaluating the activity of mAb during clinical development. DMAbs have the potential to be a mAb delivery system, which may be advantageous for protection against highly pathogenic infectious diseases, like EBOV, in resource-limited and other challenging settings.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections.

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage-gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic-clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain-dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T-type calcium channel family, as an epilepsy modifier in the Scn2a transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a Dravet phenotype. Scn1a mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia-induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.
Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.