Pan-ebolavirus protective therapy by two multifunctional human antibodies.

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein.

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

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.

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.

Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

Potently neutralizing and protective human antibodies against SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

Endothelial von Willebrand factor regulates angiogenesis.

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvß3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

Development of a Kinetic ELISA and Reactive B Cell Frequency Assay to Detect Respiratory Syncytial Virus Pre-Fusion F Protein-Specific Immune Responses in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants, making vaccination an attractive preventive strategy. Due to earlier reports of vaccine-enhanced disease in RSV-naive children, assessing prior RSV infection is critical for determining eligibility for future infant vaccine trials. However, this is complicated by the presence of maternally transferred maternal antibodies. We sought to develop assays that measure immune responses to RSV pre-fusion (F) protein that discriminates between maternal and infant responses. METHODS: We measured RSV-specific responses in two groups of children <3 years of age; those with laboratory-confirmed RSV (RSV-infected) and those enrolled prior to their first RSV season (RSV-uninfected). Serial blood samples were obtained and recent infections with RSV and other respiratory viruses were assessed during follow-up. An RSV pre-F-specific kinetic enzyme-linked immunosorbent assay (kELISA) and an F-specific reactive B cell frequency (RBF) assay were developed. RESULTS: One hundred two young children were enrolled between July 2015 and April 2017; 74 were in the RSV-uninfected group and 28 were in the RSV-infected group. Participants were asked to provide sequential blood samples over time, but only 53 participants in the RSV-uninfected group and 22 participants in the RSV-infected groups provided multiple samples. In the RSV-infected group, most had positive kELISA and RBF during the study. In the RSV-uninfected group, two patterns emerged: declining kELISA values without reactive B cells, due to maternal transplacental antibody transfer, and persistently positive kELISA with reactive B cells, due to asymptomatic undiagnosed RSV infection. CONCLUSIONS: A kELISA targeting RSV pre-F epitopes and an RBF assay targeting RSV F-specific B cells generally allow discrimination between maternally and infant-derived antibodies.

Systematic analysis of human antibody response to ebolavirus glycoprotein shows high prevalence of neutralizing public clonotypes.

Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic-virus-protective antigen is poorly defined, we perform deep paired heavy- and light-chain sequencing in Ebola virus glycoprotein (EBOV-GP)-specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitates investigation of the molecular and genetic basis for the evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B cell and serum repertoire. We identify 73 public clonotypes of EBOV, 20% of which encode antibodies with neutralization activity and capacity to protect mice in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV GP, which informs the design of vaccines and improved therapeutics.

Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

Isolation of a Potently Neutralizing and Protective Human Monoclonal Antibody Targeting Yellow Fever Virus.

Yellow fever virus (YFV) causes sporadic outbreaks of infection in South America and sub-Saharan Africa. While live-attenuated yellow fever virus vaccines based on three substrains of 17D are considered some of the most effective vaccines in use, problems with production and distribution have created large populations of unvaccinated, vulnerable individuals in areas of endemicity. To date, specific antiviral therapeutics have not been licensed for human use against YFV or any other related flavivirus. Recent advances in monoclonal antibody (mAb) technology have allowed the identification of numerous candidate therapeutics targeting highly pathogenic viruses, including many flaviviruses. Here, we sought to identify a highly neutralizing antibody targeting the YFV envelope (E) protein as a therapeutic candidate. We used human B cell hybridoma technology to isolate mAbs from circulating memory B cells from human YFV vaccine recipients. These antibodies bound to recombinant YFV E protein and recognized at least five major antigenic sites on E. Two mAbs (designated YFV-136 and YFV-121) recognized a shared antigenic site and neutralized the YFV-17D vaccine strain in vitro. YFV-136 also potently inhibited infection by multiple wild-type YFV strains, in part, at a postattachment step in the virus replication cycle. YFV-136 showed therapeutic protection in two animal models of YFV challenge, including hamsters and immunocompromised mice engrafted with human hepatocytes. These studies define features of the antigenic landscape of the YFV E protein recognized by the human B cell response and identify a therapeutic antibody candidate that inhibits infection and disease caused by highly virulent strains of YFV. IMPORTANCE Yellow fever virus (YFV) is a mosquito-borne virus that occasionally causes outbreaks of severe infection and disease in South America and sub-Saharan Africa. There are very effective live-attenuated (weakened) yellow fever virus vaccines, but recent problems with their production and distribution have left many people in affected areas vulnerable. Here, we sought to isolate an antibody targeting the surface of the virus for possible use in the future as a biologic drug to prevent or treat YFV infection. We isolated naturally occurring antibodies from individuals who had received a YFV vaccine. We created antibodies and tested them. We found that the antibody with the most powerful antiviral activity was a beneficial treatment in two different small-animal models of human infection. These studies identified features of the virus that are recognized by the human immune system and generated a therapeutic antibody candidate that inhibits infection caused by highly virulent strains of YFV.

Standardized two-step testing of antibody activity in COVID-19 convalescent plasma.

The COVID-19 pandemic revealed an urgent need for rapid profiling of neutralizing antibody responses and development of antibody therapeutics. The current Food and Drug Administration-approved serological tests do not measure antibody-mediated viral neutralization, and there is a need for standardized quantitative neutralization assays. We report a high-throughput two-step profiling approach for identifying neutralizing convalescent plasma. Screening and downselection for serum antibody binding to the receptor-binding domain are followed by quantitative neutralization testing using a chimeric vesicular stomatitis virus expressing spike protein of SARS-CoV-2 in a real-time cell analysis assay. This approach enables a predictive screening process for identifying plasma units that neutralize SARS-CoV-2. To calibrate antibody neutralizing activity in serum from convalescent plasma donors, we introduce a neutralizing antibody standard reagent composed of two human antibodies that neutralize SARS-CoV strains, including SARS-CoV-2 variants of concern. Our results provide a framework for establishing a standardized assessment of antibody-based interventions against COVID-19.

Neutralizing COVID-19 Convalescent Plasma in Adults Hospitalized With COVID-19: A Blinded, Randomized, Placebo-Controlled Trial.

BACKGROUND: Convalescent plasma has been one of the most common treatments for COVID-19, but most clinical trial data to date have not supported its efficacy. RESEARCH QUESTION: Is rigorously selected COVID-19 convalescent plasma with neutralizing anti-SARS-CoV-2 antibodies an efficacious treatment for adults hospitalized with COVID-19? STUDY DESIGN AND METHODS: This was a multicenter, blinded, placebo-controlled randomized clinical trial among adults hospitalized with SARS-CoV-2 infection and acute respiratory symptoms for < 14 days. Enrolled patients were randomly assigned to receive one unit of COVID-19 convalescent plasma (n = 487) or placebo (n = 473). The primary outcome was clinical status (disease severity) 14 days following study infusion measured with a seven-category ordinal scale ranging from discharged from the hospital with resumption of normal activities (lowest score) to death (highest score). The primary outcome was analyzed with a multivariable ordinal regression model, with an adjusted odds ratio (aOR) < 1.0 indicating more favorable outcomes with convalescent plasma than with placebo. In secondary analyses, trial participants were stratified according to the presence of endogenous anti-SARS-CoV-2 antibodies ("serostatus") at randomization. The trial included 13 secondary efficacy outcomes, including 28-day mortality. RESULTS: Among 974 randomized patients, 960 were included in the primary analysis. Clinical status on the ordinal outcome scale at 14 days did not differ between the convalescent plasma and placebo groups in the overall population (aOR, 1.04; one-seventh support interval [1/7 SI], 0.82-1.33), in patients without endogenous antibodies (aOR, 1.15; 1/7 SI, 0.74-1.80), or in patients with endogenous antibodies (aOR, 0.96; 1/7 SI, 0.72-1.30). None of the 13 secondary efficacy outcomes were different between groups. At 28 days, 89 of 482 (18.5%) patients in the convalescent plasma group and 80 of 465 (17.2%) patients in the placebo group had died (aOR, 1.04; 1/7 SI, 0.69-1.58). INTERPRETATION: Among adults hospitalized with COVID-19, including those seronegative for anti-SARS-CoV-2 antibodies, treatment with convalescent plasma did not improve clinical outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov; No.: NCT04362176; URL: www. CLINICALTRIALS: gov.

Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking.

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

Broad and potently neutralizing monoclonal antibodies isolated from human survivors of New World hantavirus infection.

New World hantaviruses (NWHs) are endemic in North and South America and cause hantavirus cardiopulmonary syndrome (HCPS), with a case fatality rate of up to 40%. Knowledge of the natural humoral immune response to NWH infection is limited. Here, we describe human monoclonal antibodies (mAbs) isolated from individuals previously infected with Sin Nombre virus (SNV) or Andes virus (ANDV). Most SNV-reactive antibodies show broad recognition and cross-neutralization of both New and Old World hantaviruses, while many ANDV-reactive antibodies show activity for ANDV only. mAbs ANDV-44 and SNV-53 compete for binding to a distinct site on the ANDV surface glycoprotein and show potently neutralizing activity to New and Old World hantaviruses. Four mAbs show therapeutic efficacy at clinically relevant doses in hamsters. These studies reveal a convergent and potently neutralizing human antibody response to NWHs and suggest therapeutic potential for human mAbs against HCPS.

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein.

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

Canonical features of human antibodies recognizing the influenza hemagglutinin trimer interface.

Broadly reactive antibodies targeting the influenza A virus hemagglutinin (HA) head domain are thought to be rare and to require extensive somatic mutations or unusual structural features to achieve breadth against divergent HA subtypes. Here we describe common genetic and structural features of protective human antibodies from several individuals recognizing the trimer interface (TI) of the influenza A HA head, a recently identified site of vulnerability. We examined the sequence of TI-reactive antibodies, determined crystal structures for TI antibody-antigen complexes, and analyzed the contact residues of the antibodies on HA to discover common genetic and structural features of TI antibodies. Our data reveal that many TI antibodies are encoded by a light chain variable gene segment incorporating a shared somatic mutation. In addition, these antibodies have a shared acidic residue in the heavy chain despite originating from diverse heavy chain variable gene segments. These studies show that the TI region of influenza A HA is a major antigenic site with conserved structural features that are recognized by a common human B cell public clonotype. The canonical nature of this antibody-antigen interaction suggests that the TI epitope might serve as an important target for structure-based vaccine design.

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals.

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals.

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo ), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.