RESUMO
The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.
Assuntos
Microscopia Crioeletrônica , Serina Endopeptidases , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Humanos , Serina Endopeptidases/metabolismo , Serina Endopeptidases/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Animais , Células HEK293 , Ligação Proteica , Anticorpos Neutralizantes/imunologia , Modelos Moleculares , Receptores Virais/metabolismo , Receptores Virais/químicaRESUMO
The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among É-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.
Assuntos
Coronavirus Humano 229E , Infecções por Coronavirus , Coronavirus , Animais , Antígenos CD13/química , Antígenos CD13/metabolismo , Gatos , Linhagem Celular , Coronavirus/metabolismo , Coronavirus Humano 229E/metabolismo , Cães , Humanos , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , SuínosRESUMO
The SARS-CoV-2 spike (S) glycoprotein contains an immunodominant receptor-binding domain (RBD) targeted by most neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite (designated site i) recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge, albeit selecting escape mutants in some animals. Indeed, several SARS-CoV-2 variants, including the B.1.1.7, B.1.351, and P.1 lineages, harbor frequent mutations within the NTD supersite, suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs for protective immunity and vaccine design.
Assuntos
Antígenos Virais/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , Cricetinae , Mapeamento de Epitopos , Variação Genética , Modelos Moleculares , Mutação/genética , Testes de Neutralização , Domínios Proteicos , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/ultraestruturaRESUMO
Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.
RESUMO
The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
Assuntos
Betacoronavirus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Betacoronavirus/química , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.
Assuntos
Simulação de Acoplamento Molecular , Mutação , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2 , Sítios de Ligação , Células HEK293 , Humanos , Peptidil Dipeptidase A/química , Fenótipo , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.
Assuntos
Anticorpos Neutralizantes/imunologia , Mapeamento de Epitopos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Cinética , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos/imunologia , Estrutura Quaternária de Proteína , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.
Assuntos
Coronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Coronavirus/metabolismo , Infecções por Coronavirus/imunologia , Células HEK293 , Humanos , Imunidade Humoral/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Mimetismo Molecular/imunologia , Ligação Proteica , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Glicoproteína da Espícula de Coronavírus/fisiologia , Células Vero , Internalização do VírusRESUMO
Immune imprinting describes how the first exposure to a virus shapes immunological outcomes of subsequent exposures to antigenically related strains. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron breakthrough infections and bivalent COVID-19 vaccination primarily recall cross-reactive memory B cells induced by prior Wuhan-Hu-1 spike mRNA vaccination rather than priming Omicron-specific naive B cells. These findings indicate that immune imprinting occurs after repeated Wuhan-Hu-1 spike exposures, but whether it can be overcome remains unclear. To understand the persistence of immune imprinting, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID-19 mRNA vaccine booster. We showed that the XBB.1.5 booster elicited neutralizing antibody responses against current variants that were dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. Therefore, immune imprinting persists after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 booster vaccination, which will need to be considered to guide future vaccination.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2 , Anticorpos Neutralizantes , RNA Mensageiro/genética , Vacinação , Anticorpos AntiviraisRESUMO
Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.
Assuntos
Chlamydomonas/metabolismo , Proteínas de Fusão de Membrana/química , Proteínas de Plantas/química , Plasmodium/metabolismo , Proteínas de Protozoários/química , Sequência de Aminoácidos , Evolução Biológica , Chlamydomonas/citologia , Cristalografia por Raios X , Células Germinativas/química , Células Germinativas/metabolismo , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmodium/citologia , Domínios Proteicos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.
Assuntos
Enzima de Conversão de Angiotensina 2 , Quirópteros , Coronavírus da Síndrome Respiratória do Oriente Médio , Receptores Virais , Internalização do Vírus , Animais , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Quirópteros/metabolismo , Quirópteros/virologia , Microscopia Crioeletrônica , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Ligação Proteica , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Dipeptidil Peptidase 4/metabolismo , Zoonoses ViraisRESUMO
Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant1, which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b22. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/imunologia , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , COVID-19/metabolismo , COVID-19/virologia , Feminino , Células HEK293 , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Imunização Passiva , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Testes de Neutralização , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Sintéticas/administração & dosagem , Soroterapia para COVID-19 , Vacinas de mRNARESUMO
The recent emergence of SARS-CoV-2 variants of concern1-10 and the recurrent spillovers of coronaviruses11,12 into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes/uso terapêutico , COVID-19/prevenção & controle , SARS-CoV-2/classificação , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Anticorpos Amplamente Neutralizantes/química , COVID-19/imunologia , COVID-19/virologia , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Mesocricetus/imunologia , Mesocricetus/virologia , Mutação , Testes de Neutralização , SARS-CoV-2/química , SARS-CoV-2/genética , Zoonoses Virais/imunologia , Zoonoses Virais/prevenção & controle , Zoonoses Virais/virologiaRESUMO
An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/virologia , Reações Cruzadas/imunologia , Evasão da Resposta Imune , SARS-CoV-2/classificação , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Anticorpos Amplamente Neutralizantes/química , COVID-19/imunologia , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Linhagem Celular , Cricetinae , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Masculino , Mesocricetus , Pessoa de Meia-Idade , Modelos Moleculares , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinologia , Tratamento Farmacológico da COVID-19RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 20201,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope containing a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails containing S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Reações Cruzadas/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linfócitos B/imunologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Reações Cruzadas/efeitos dos fármacos , Microscopia Crioeletrônica , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Células HEK293 , Humanos , Evasão da Resposta Imune/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Memória Imunológica/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Modelos Moleculares , Testes de Neutralização , Pandemias/prevenção & controle , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/química , Células VeroRESUMO
The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a mouse coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.
Assuntos
Microscopia Crioeletrônica , Vírus da Hepatite Murina/química , Vírus da Hepatite Murina/ultraestrutura , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Drosophila melanogaster , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Hepatite Murina/imunologia , Multimerização Proteica , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/química , Vacinas Virais/imunologia , Internalização do VírusRESUMO
The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.