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Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo, and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates., Competing Interests: Declaration of interests L.E.W. serves as a consultant for BigHat Biosciences. The content of this article is solely the responsibility of the authors and does not represent the official views of BigHat Biosciences. C.B.C. serves as a consultant to GlaxoSmithKline, Sanofi, TDCowen Investments, Guidepoint Global, Debiopharm, and CommenseBio and receives royalties from UpToDate. The laboratory of C.B.C. receives funding for unrelated work from Moderna. J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, GlaxoSmithKline, and BTG International Inc; is a member of the Scientific Advisory Board of Meissa Vaccines; a former member of the Scientific Advisory Board of Gigagen (Grifols); and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study. The opinions, interpretations, conclusions, and recommendations contained herein are those of the authors and are not necessarily endorsed by the US Department of Defense. E.D., T.E.E., and B.J.D. are employees of Integral Molecular, B.J.D. is a shareholder of Integral Molecular. Vanderbilt University has applied for a patent pertinent to some of the materials in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Novel therapeutic monoclonal antibodies (MAbs) must accommodate comprehensive breadth of activity against diverse sarbecoviruses and high neutralization potency to overcome emerging variants. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) in complex with MAb WRAIR-2063, a moderate-potency neutralizing antibody with exceptional sarbecovirus breadth, that targets the highly conserved cryptic class V epitope. This epitope overlaps substantially with the spike protein N-terminal domain (NTD) -interacting region and is exposed only when the spike is in the open conformation, with one or more RBDs accessible. WRAIR-2063 binds the RBD of SARS-CoV-2 WA-1, all variants of concern (VoCs), and clade 1 to 4 sarbecoviruses with high affinity, demonstrating the conservation of this epitope and potential resiliency against variation. We compare structural features of additional class V antibodies with their reported neutralization capacity to further explore the utility of the class V epitope as a pan-sarbecovirus vaccine and therapeutic target. IMPORTANCE Characterization of MAbs against SARS-CoV-2, elicited through vaccination or natural infection, has provided vital immunotherapeutic options for curbing the COVID-19 pandemic and has supplied critical insights into SARS-CoV-2 escape, transmissibility, and mechanisms of viral inactivation. Neutralizing MAbs that target the RBD but do not block ACE2 binding are of particular interest because the epitopes are well conserved within sarbecoviruses and MAbs targeting this area demonstrate cross-reactivity. The class V RBD-targeted MAbs localize to an invariant site of vulnerability, provide a range of neutralization potency, and exhibit considerable breadth against divergent sarbecoviruses, with implications for vaccine and therapeutic development., Competing Interests: The authors declare a conflict of interest. Patent application number PCT/US 63/140,763, PCT WO 2022/159839 A1 was filed containing the mAbs described in this publication for authors S.J.K., K.M., V.D., and N.L.M. M.G.J. and K.M. are named as inventors on international patent application WO/2021/178971 A1 entitled “Vaccines against SARS-CoV-2 and other coronaviruses.” M.G.J. is named as an inventor on international patent application WO/2018/081318 and U.S. patent 10,960,070 entitled “Prefusion coronavirus spike proteins and their use.” The other authors declare no competing interests.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro , and a capacity to limit disease in vivo .

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design., Competing Interests: Declaration of interests M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation and on the scientific advisory boards of Moderna and Immunome. The Diamond and Fremont laboratories received support from Emergent BioSolutions under a sponsored research agreement. The Diamond laboratory has unrelated research agreements from Moderna and Vir Biotechnology. D.H.F. is a founder of Courier Therapeutics. J.R.L. is a consultant for Celdara Medical. DC2.112 and DC2.315 are the subject of a US patent application with R.J.M., J.A.Q., and J.R.L. as co-inventors. R.H.F., E.D., and B.J.D. are employees of Integral Molecular, and B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory has received unrelated sponsored research agreements from Astra Zeneca and IDBiologics. M.L.G. is an unpaid member of the scientific advisory boards of Protein Metrics and GenNext. G.A. is a founder of SeromYx Systems., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins., Competing Interests: Declaration of interests J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines, and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from IDBiologics and AstraZeneca. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory at Washington University School of Medicine has received sponsored research agreements from Emergent BioSolutions, Moderna, and Vir Biotechnology. All other authors report no conflicts. M.E.F., E.D., and B.J.D. are employees of Integral Molecular and B.J.D. is a shareholder. G.A. is a founder and the Chief Scientific Officer (Consulting) of Seromyx., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Three clinically relevant ebolaviruses - Ebola (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) viruses, are responsible for severe disease and occasional deadly outbreaks in Africa. The largest Ebola virus disease (EVD) epidemic to date in 2013-2016 in West Africa highlighted the urgent need for countermeasures, leading to the development and FDA approval of the Ebola virus vaccine rVSV-ZEBOV (Ervebo ® ) in 2020 and two monoclonal antibody (mAb)-based therapeutics (Inmazeb ® [atoltivimab, maftivimab, and odesivimab-ebgn] and Ebanga ® (ansuvimab-zykl) in 2020. The humoral response plays an indispensable role in ebolavirus immunity, based on studies of mAbs isolated from the antibody genes in peripheral blood circulating ebolavirus-specific human memory B cells. However, antibodies in the body are not secreted by circulating memory B cells in the blood but rather principally by plasma cells in the bone marrow. Little is known about the protective polyclonal antibody responses in convalescent plasma. Here we exploited both single-cell antibody gene sequencing and proteomic sequencing approaches to assess the composition of the ebolavirus glycoprotein (GP)-reactive antibody repertoire in the plasma of an EVD survivor. We first identified 1,512 GP-specific mAb variable gene sequences from single cells in the memory B cell compartment. Using mass spectrometric analysis of the corresponding GP-specific plasma IgG, we found that only a portion of the large B cell antibody repertoire was represented in the plasma. Molecular and functional analysis of proteomics-identified mAbs revealed recognition of epitopes in three major antigenic sites - the GP head domain, the glycan cap, and the base region, with a high prevalence of neutralizing and protective mAb specificities that targeted the base and glycan cap regions on the GP. Polyclonal plasma antibodies from the survivor reacted broadly to EBOV, BDBV, and SUDV GP, while reactivity of the potently neutralizing mAbs we identified was limited mostly to the homologous EBOV GP. Together these results reveal a restricted diversity of neutralizing humoral response in which mAbs targeting two antigenic sites on GP - glycan cap and base - play a principal role in plasma-antibody-mediated protective immunity against EVD., Competing Interests: SB and NC are employees of Abterra Biosciences. JL, ArG, ED, and BD are employees of Integral Molecular. GA is a founder of Seromyx Systems Inc. LZ is employer and co-owner of Mapp Biopharmaceutical. BD is a shareholder of Integral Molecular. AdG was an employee of Mapp Biopharmaceutical. JC has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gilchuk, Guthals, Bonissone, Shaw, Ilinykh, Huang, Bombardi, Liang, Grinyo, Davidson, Chen, Gunn, Alter, Saphire, Doranz, Bukreyev, Zeitlin, Castellana and Crowe.)

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies., Competing Interests: Declaration of interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from and IDBiologics and AstraZeneca. Vanderbilt University has applied for a patent that is related to antibodies discussed in this work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Zika virus (ZIKV), a mosquito-transmitted flavivirus, caused a large epidemic in Latin America between 2015 and 2017. Effective ZIKV vaccines and treatments are urgently needed to prevent future epidemics and severe disease sequelae. People infected with ZIKV develop strongly neutralizing antibodies linked to viral clearance and durable protective immunity. To understand the mechanisms of protective immunity and to support the development of ZIKV vaccines, we characterize here a strongly neutralizing antibody, B11F, isolated from a patient who recovered from ZIKV. Our results indicate that B11F targets a complex epitope on the virus that spans domains I and III of the envelope glycoprotein. While previous studies point to quaternary epitopes centered on domain II of the ZIKV E glycoprotein as targets of strongly neutralizing and protective human antibodies, we uncover a new site spanning domains I and III as a target of strongly neutralizing human antibodies. IMPORTANCE People infected with Zika virus develop durable neutralizing antibodies that prevent repeat infections. In the current study, we characterize a ZIKV-neutralizing human monoclonal antibody isolated from a patient after recovery. Our studies establish a novel site on the viral envelope that is targeted by human neutralizing antibodies. Our results are relevant to understanding how antibodies block infection and to guiding the design and evaluation of candidate vaccines., (Copyright © 2021 Graham et al.)

The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015-2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests. Integral Molecular offers ZIKV RVPs as a commercial reagent. J. C. Whitbeck, A. Thomas, K. Kadash-Edmondson, A. Grinyo-Escuer, Lewis J. Stafford, E. Davidson, B. J. Doranz are all current or former members of Integral Molecular. B. J. Doranz is a shareholder of Integral Molecular.

The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.

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., Competing Interests: Declaration of Interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Sanofi and is on the Scientific Advisory Boards of CompuVax and Meissa Vaccines, is a recipient of previous unrelated research grants from Moderna and Sanofi, and is founder of IDBiologics. Vanderbilt University has applied for a patent that is related to this work. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design., Competing Interests: ND, AA, OS, EC, JP, MR, AS, LA, FR No competing interests declared, EW, DC, FZ, SE, SQ, KM, LG Inventor of the following patent application, which is co-owned by the Chan Zuckerberg Biohub and Stanford University: PCT patent application entitled ANTIBODIES AGAINST DENGUE VIRUS AND RELATED METHODS, Serial no. PCT/US2019/045427, filed August 7, 2019, MF, ED Employee of Integral Molecular, BD Employee and shareholder of Integral Molecular, (© 2019, Durham et al.)

There is still no safe and effective vaccine against dengue virus infection. Epidemics of dengue virus infection are increasingly a threat to human health around the world. Antibodies generated in response to dengue infection have been shown to impact disease development and effectiveness of dengue vaccine. In this study, we investigated monoclonal antibody responses to an experimental dengue vaccine in rhesus macaques. Variable regions of both heavy chain (VH) and light chain (VL) were cloned from single antibody-secreting B cells. A total of 780 monoclonal antibodies (mAbs) composed of paired VH and VL were characterized. Results show that the vaccination induces mAbs with diverse germline sequences and a wide range of binding affinities. Six potent neutralizing mAbs were identified among 130 dengue envelope protein binders. Critical amino acids for each neutralizing antibody binding to the dengue envelope protein were identified by alanine scanning of mutant libraries. Diverse epitopes were identified, including epitopes on the lateral ridge of DIII, the I-III hinge, the bc loop adjacent to the fusion loop of DII, and the β-strands and loops of DI. Significantly, one of the neutralizing mAbs has a previously unknown epitope in DII at the interface of the envelope and membrane protein and is capable of neutralizing all four dengue serotypes. Taken together, the results of this study not only provide preclinical validation for the tested experimental vaccine, but also shed light on a potential application of the rhesus macaque model for better dengue vaccine evaluation and design of vaccines and immunization strategies., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Some of the coauthors are employees of Merck Co. Inc.; JMP, TB, and BJD are employees of Integral Molecular; and BJD is a shareholder of Integral Molecular.

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type-specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections.

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. In vivo 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. IMPORTANCE 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.)

Chikungunya virus (CHIKV) is an emerging mosquito-borne virus that has caused explosive outbreaks worldwide. Although neutralizing monoclonal antibodies (mAbs) against CHIKV inhibit infection in animals, the contribution of Fc effector functions to protection remains unknown. Here, we evaluated the activity of therapeutic mAbs that had or lacked the ability to engage complement and Fcγ receptors (FcγR). When administered as post-exposure therapy in mice, the Fc effector functions of mAbs promoted virus clearance from infected cells and reduced joint swelling-results that were corroborated in antibody-treated transgenic animals lacking activating FcγR. The control of CHIKV infection by antibody-FcγR engagement was associated with an accelerated influx of monocytes. A series of immune cell depletions revealed that therapeutic mAbs required monocytes for efficient clearance of CHIKV infection. Overall, our study suggests that in mice, FcγR expression on monocytes is required for optimal therapeutic activity of antibodies against CHIKV and likely other related viruses., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.)

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Drug combinations are synergistic when their combined efficacy exceeds the sum of the individual actions, but they rarely include ineffective drugs that become effective only in combination. We identified several "enabling pairs" of neutralizing and non-neutralizing anti-ebolavirus monoclonal antibodies, whose combination exhibited new functional profiles, including transforming a non-neutralizing antibody to a neutralizer. Sub-neutralizing concentrations of antibodies 2G4 or m8C4 enabled non-neutralizing antibody FVM09 (IC 50 >1 μM) to exhibit potent neutralization (IC 50 1-10 nM). While FVM09 or m8C4 alone failed to protect Ebola-virus-infected mice, a combination of the two antibodies provided 100% protection. Furthermore, non-neutralizers FVM09 and FVM02 exponentially enhanced the potency of two neutralizing antibodies against both Ebola and Sudan viruses. We identified a hotspot for the binding of these enabling antibody pairs near the interface of the glycan cap and GP2. Enabling cooperativity may be an underappreciated phenomenon for viruses, with implications for the design and development of immunotherapeutics and vaccines., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination. IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines., (Copyright © 2017 American Society for Microbiology.)

Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo IMPORTANCE: Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive "recall" or "anamnestic" response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism., (Copyright © 2016 Xu et al.)

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies., Competing Interests: W.R.S. is a founder of Compuvax, Inc. J.E.C. is a member of the Scientific Advisory Boards of Compuvax and Meissa Vaccines.

Unlabelled: The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms., Importance: Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.

We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells., Importance: Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/ml), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer., Importance: CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Following natural dengue virus (DENV) infection, humans produce some antibodies that recognize only the serotype of infection (type specific) and others that cross-react with all four serotypes (cross-reactive). Recent studies with human antibodies indicate that type-specific antibodies at high concentrations are often strongly neutralizing in vitro and protective in animal models. In general, cross-reactive antibodies are poorly neutralizing and can enhance the ability of DENV to infect Fc receptor-bearing cells under some conditions. Type-specific antibodies at low concentrations also may enhance infection. There is an urgent need to determine whether there are conserved antigenic sites that can be recognized by cross-reactive potently neutralizing antibodies. Here, we describe the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the DENV domain II fusion loop (FL) envelope protein region from subjects following vaccination or natural infection. Most of the FL-specific antibodies exhibited a conventional phenotype, characterized by low-potency neutralizing function and antibody-dependent enhancing activity. One clone, however, recognized the bc loop of domain II adjacent to the FL and exhibited a unique phenotype of ultrahigh potency, neutralizing all four serotypes better than any other previously described MAb recognizing this region. This antibody not only neutralized DENV effectively but also competed for binding against the more prevalent poor-quality antibodies whose binding was focused on the FL. The 1C19 human antibody could be a promising component of a preventative or therapeutic intervention. Furthermore, the unique epitope revealed by 1C19 suggests a focus for rational vaccine design based on novel immunogens presenting cross-reactive neutralizing determinants., Importance: With no effective vaccine available, the incidence of dengue virus (DENV) infections worldwide continues to rise, with more than 390 million infections estimated to occur each year. Due to the unique roles that antibodies are postulated to play in the pathogenesis of DENV infection and disease, there is consensus that a successful DENV vaccine must protect against all four serotypes. If conserved epitopes recognized by naturally occurring potently cross-neutralizing human antibodies could be identified, monovalent subunit vaccine preparations might be developed. We characterized 30 DENV cross-neutralizing human monoclonal antibodies (MAbs) and identified one (1C19) that recognized a novel conserved site, known as the bc loop. This antibody has several desirable features, as it neutralizes DENV effectively and competes for binding against the more common low-potency fusion loop (FL) antibodies, which are believed to contribute to antibody-mediated disease. To our knowledge, this is the first description of a potent serotype cross-neutralizing human antibody to DENV.

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.