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1.
Nat Commun ; 12(1): 1715, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731724

RESUMO

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Betacoronavirus/imunologia , Epitopos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Betacoronavirus/classificação , Camelus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/genética , Humanos , Camundongos , Conformação Proteica , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
2.
Emerg Microbes Infect ; 9(1): 1080-1091, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32471334

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.


Assuntos
Formação de Anticorpos , Apresentação de Antígeno , Infecções por Coronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Sítios de Ligação , Infecções por Coronavirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Células HEK293 , Humanos , Imunogenicidade da Vacina , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Coelhos , Glicoproteína da Espícula de Coronavírus/biossíntese , Replicação Viral
3.
Emerg Microbes Infect ; 8(1): 1593-1603, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31711379

RESUMO

The ongoing Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks pose a worldwide public health threat. Blocking MERS-CoV zoonotic transmission from dromedary camels, the animal reservoir, could potentially reduce the number of primary human cases. Here we report MERS-CoV transmission from experimentally infected llamas to naïve animals. Directly inoculated llamas shed virus for at least 6 days and could infect all in-contact naïve animals 4-5 days after exposure. With the aim to block virus transmission, we examined the efficacy of a recombinant spike S1-protein vaccine. In contrast to naïve animals, in-contact vaccinated llamas did not shed infectious virus upon exposure to directly inoculated llamas, consistent with the induction of strong virus neutralizing antibody responses. Our data provide further evidence that vaccination of the reservoir host may impede MERS-CoV zoonotic transmission to humans.


Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Camelídeos Americanos , Camelus/virologia , Infecções por Coronavirus/virologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Vacinas Virais/genética , Vacinas Virais/imunologia , Zoonoses/imunologia , Zoonoses/prevenção & controle , Zoonoses/transmissão , Zoonoses/virologia
4.
Emerg Microbes Infect ; 8(1): 516-530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30938227

RESUMO

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Domínios Proteicos , Receptores Virais/genética , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/genética
5.
Sci Adv ; 4(8): eaas9667, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30101189

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.


Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Camelus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Camundongos , Testes de Neutralização , Ligação Proteica , Anticorpos de Domínio Único
6.
Proc Natl Acad Sci U S A ; 115(22): E5135-E5143, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29760102

RESUMO

Porcine deltacoronavirus (PDCoV), identified in 2012, is a common enteropathogen of swine with worldwide distribution. The source and evolutionary history of this virus is, however, unknown. PDCoV belongs to the Deltacoronavirus genus that comprises predominantly avian CoV. Phylogenetic analysis suggests that PDCoV originated relatively recently from a host-switching event between birds and mammals. Insight into receptor engagement by PDCoV may shed light into such an exceptional phenomenon. Here we report that PDCoV employs host aminopeptidase N (APN) as an entry receptor and interacts with APN via domain B of its spike (S) protein. Infection of porcine cells with PDCoV was drastically reduced by APN knockout and rescued after reconstitution of APN expression. In addition, we observed that PDCoV efficiently infects cells of unusual broad species range, including human and chicken. Accordingly, PDCoV S was found to target the phylogenetically conserved catalytic domain of APN. Moreover, transient expression of porcine, feline, human, and chicken APN renders cells susceptible to PDCoV infection. Binding of PDCoV to an interspecies conserved site on APN may facilitate direct transmission of PDCoV to nonreservoir species, including humans, potentially reflecting the mechanism that enabled a virus, ancestral to PDCoV, to breach the species barrier between birds and mammals. The APN cell surface protein is also used by several members of the Alphacoronavirus genus. Hence, our data constitute the second identification of CoVs from different genera that use the same receptor, implying that CoV receptor selection is subjected to specific restrictions that are still poorly understood.


Assuntos
Doenças Transmissíveis Emergentes , Infecções por Coronavirus , Coronavirus/fisiologia , Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Animais , Anticorpos Antivirais/imunologia , Antígenos CD13/metabolismo , Gatos , Linhagem Celular , Galinhas , Chlorocebus aethiops , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/veterinária , Doenças Transmissíveis Emergentes/virologia , Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Cães , Especificidade de Hospedeiro , Humanos , Células Madin Darby de Rim Canino , Camundongos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos , Células Vero , Zoonoses/transmissão , Zoonoses/virologia
7.
Proc Natl Acad Sci U S A ; 114(40): E8508-E8517, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28923942

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Camelus , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Humanos , Mucinas , Glicoproteína da Espícula de Coronavírus/genética , Ligação Viral
8.
J Allergy Clin Immunol ; 134(3): 613-621.e7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24958565

RESUMO

BACKGROUND: Thymic stromal lymphopoietin (TSLP) controls allergic TH2 inflammatory responses through induction of distinct activation programs in dendritic cells (DCs). However, knowledge about TSLP receptor expression and functional consequences of receptor activation by DCs residing in the human respiratory tract is limited. OBJECTIVE: We wanted to identify TSLP-responding DC populations in the human upper airway mucosa and assess the TSLP-mediated effects on such DCs in allergic airway responses. RESULTS: We found that the TSLP receptor was constitutively and preferentially expressed by myeloid CD1c(+) DCs in the human airway mucosa and that the density of this DC subset in nasal mucosa increased significantly after in vivo allergen challenge of patients with allergic rhinitis. In vitro, TSLP strongly enhanced the capacity of CD1c(+) DCs to activate allergen-specific memory CD4(+) T cells. Moreover, TSLP rapidly induced CCR7 expression on CD1c(+) DCs. However, TH2 cytokines attenuated TSLP-mediated CCR7 induction, thus inhibiting the TSLP-induced DC migration potential to the draining lymph nodes. CONCLUSION: Our results suggest that TSLP-mediated activation of human nasal mucosal CD1c(+) DCs triggers CCR7-dependent migration to the draining lymph nodes and enhances their capacity to initiate TH2 responses. However, the observation that TH2 cytokines abrogate the induction of CCR7 implies that during a TH2-mediated inflammatory reaction, TLSP-activated CD1c(+) DCs are retained in the inflamed tissue to further exacerbate local inflammation by activating local antigen-specific memory TH2 cells.


Assuntos
Citocinas/imunologia , Células Dendríticas/imunologia , Células Mieloides/imunologia , Mucosa Respiratória/imunologia , Rinite Alérgica/imunologia , Células Th2/imunologia , Adolescente , Adulto , Idoso , Alérgenos/imunologia , Antígenos CD1/metabolismo , Diferenciação Celular/imunologia , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Memória Imunológica , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores de Citocinas/metabolismo , Regulação para Cima , Adulto Jovem , Linfopoietina do Estroma do Timo
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