RESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Linhagem Celular , Microscopia Crioeletrônica , Ebolavirus/ultraestrutura , Epitopos/imunologia , Feminino , Glicoproteínas/química , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos BALB C , Modelos Moleculares , Primatas , Receptores Fc/metabolismo , Proteínas Recombinantes/imunologia , Viremia/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Substâncias Protetoras/farmacologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Ligação Competitiva , COVID-19/imunologia , COVID-19/virologia , Quimiocinas/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese/genética , Testes de Neutralização , Domínios Proteicos , Células VeroRESUMO
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.
Assuntos
Alphavirus/imunologia , Anticorpos Antivirais/imunologia , Reações Cruzadas/imunologia , Proteínas Virais/imunologia , Liberação de Vírus/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Linhagem Celular , Vírus Chikungunya/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Encefalomielite Equina/imunologia , Encefalomielite Equina/virologia , Mapeamento de Epitopos , Feminino , Cavalos , Humanos , Concentração de Íons de Hidrogênio , Articulações/patologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica , RNA Viral/metabolismo , Receptores Fc/metabolismo , Temperatura , Vírion/metabolismo , Internalização do VírusRESUMO
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.
Assuntos
Aerossóis/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina do Leste/imunologia , Encefalomielite Equina/imunologia , Encefalomielite Equina/prevenção & controle , Adulto , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Microscopia Crioeletrônica , Modelos Animais de Doenças , Vírus da Encefalite Equina do Leste/ultraestrutura , Encefalomielite Equina/virologia , Epitopos/química , Feminino , Glicoproteínas/imunologia , Humanos , Camundongos , Modelos Moleculares , Mutagênese/genética , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/imunologia , Sindbis virus/imunologia , Vírion/imunologia , Vírion/ultraestrutura , Internalização do VírusRESUMO
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.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Betacoronavirus/química , Ligação Competitiva , COVID-19 , Linhagem Celular , Reações Cruzadas , Modelos Animais de Doenças , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Pessoa de Meia-Idade , Testes de Neutralização , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Profilaxia Pré-Exposição , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.
Assuntos
Anticorpos Antivirais/genética , Regiões Determinantes de Complementaridade/genética , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Regiões Determinantes de Complementaridade/imunologia , Epitopos/genética , Epitopos/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Doença do Vírus de Marburg/tratamento farmacológico , Doença do Vírus de Marburg/genética , Doença do Vírus de Marburg/virologia , Marburgvirus/patogenicidade , Mutação/genética , Mutação/imunologia , Proteínas do Envelope Viral , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
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.
RESUMO
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.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Criança , Lactente , Humanos , Pré-Escolar , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteínas Virais de Fusão , Imunidade , Ensaio de Imunoadsorção EnzimáticaRESUMO
The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/genética , Humanos , Camundongos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
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.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H1N1/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/química , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologiaRESUMO
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.
RESUMO
Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.
Assuntos
SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Epitopos/imunologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Domínios Proteicos , SARS-CoV-2/genética , Saccharomyces cerevisiae/genética , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis-and in some cases trogocytosis-but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Reações Antígeno-Anticorpo , Linfócitos B/citologia , Linfócitos B/metabolismo , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Subunidades Proteicas/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , SARS-CoV-2/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Variação Antigênica , Sítios de Ligação , COVID-19/imunologia , COVID-19/virologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , Mutação , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.
Assuntos
Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , COVID-19/genética , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica , Mapeamento de Epitopos/métodos , Epitopos/química , Epitopos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Células VeroRESUMO
Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution.
RESUMO
The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo . The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.
RESUMO
The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity 1,2 . From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC 50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type ( wt ) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (S RBD ) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the S RBD . Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the S RBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.
RESUMO
Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date 1,2 . In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.
RESUMO
The rational design of dengue virus (DENV) vaccines requires a detailed understanding of the molecular basis for antibody-mediated immunity. The durably protective antibody response to DENV after primary infection is serotype specific. However, there is an incomplete understanding of the antigenic determinants for DENV type-specific (TS) antibodies, especially for DENV serotype 3, which has only one well-studied, strongly neutralizing human monoclonal antibody (mAb). Here, we investigated the human B cell response in children after natural DENV infection in the endemic area of Nicaragua and isolated 15 DENV3 TS mAbs recognizing the envelope (E) glycoprotein. Functional epitope mapping of these mAbs and small animal prophylaxis studies revealed a complex landscape with protective epitopes clustering in at least 6-7 antigenic sites. Potently neutralizing TS mAbs recognized sites principally in E glycoprotein domains I and II, and patterns suggest frequent recognition of quaternary structures on the surface of viral particles.