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1.
Protein Sci ; 30(4): 716-727, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33586288

RESUMO

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/ml in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.


Assuntos
/metabolismo , Anticorpos Neutralizantes/metabolismo , /fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Neutralizantes/química , Sítios de Ligação , Epitopos/química , Epitopos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/química
2.
JCI Insight ; 6(6)2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33621211

RESUMO

Comorbid medical illnesses, such as obesity and diabetes, are associated with more severe COVID-19, hospitalization, and death. However, the role of the immune system in mediating these clinical outcomes has not been determined. We used multiparameter flow cytometry and systems serology to comprehensively profile the functions of T cells and antibodies targeting spike, nucleocapsid, and envelope proteins in a convalescent cohort of COVID-19 subjects who were either hospitalized (n = 20) or not hospitalized (n = 40). To avoid confounding, subjects were matched by age, sex, ethnicity, and date of symptom onset. Surprisingly, we found that the magnitude and functional breadth of virus-specific CD4+ T cell and antibody responses were consistently higher among hospitalized subjects, particularly those with medical comorbidities. However, an integrated analysis identified more coordination between polyfunctional CD4+ T cells and antibodies targeting the S1 domain of spike among subjects who were not hospitalized. These data reveal a functionally diverse and coordinated response between T cells and antibodies targeting SARS-CoV-2, which is reduced in the presence of comorbid illnesses that are known risk factors for severe COVID-19.


Assuntos
Anticorpos Antivirais/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Hospitalização , Glicoproteína da Espícula de Coronavírus , Vírion , Adulto , Idoso , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/fisiologia , Anticorpos Antivirais/metabolismo , Linfócitos T CD4-Positivos/metabolismo , /imunologia , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/imunologia , Comorbidade , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/imunologia , Feminino , Humanos , Imunidade Humoral , Masculino , Pessoa de Meia-Idade , Nucleocapsídeo , Índice de Gravidade de Doença , Proteínas Virais , Adulto Jovem
3.
Immunobiology ; 226(2): 152054, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33524881

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide causing a pandemic with millions of infected people and deaths. Currently, the scientific community is working hard to develop a specific vaccine or treatment. However, since antibody production is an important part of the adaptive immune response, to develop vaccines and therapies, we must understand the antibody response to SARS-CoV-2 infection. In this work, we summarize the most important findings of antibody-mediated immunity against SARS-CoV-2 and highlight its role in the efficient use of plasma from convalescent patients and the direct application of antibodies as treatment.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , /imunologia , /fisiologia , Formação de Anticorpos , Humanos , Imunidade Humoral , Imunização Passiva , Soroconversão
4.
mBio ; 12(1)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33593976

RESUMO

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.


Assuntos
/diagnóstico , /imunologia , /patogenicidade , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Testes de Neutralização
5.
mBio ; 12(1)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563823

RESUMO

Whether mother-to-infant SARS-CoV-2 transmission can occur during breastfeeding and, if so, whether the benefits of breastfeeding outweigh this risk during maternal COVID-19 illness remain important questions. Using RT-qPCR, we did not detect SARS-CoV-2 RNA in any milk sample (n = 37) collected from 18 women following COVID-19 diagnosis. Although we detected evidence of viral RNA on 8 out of 70 breast skin swabs, only one was considered a conclusive positive result. In contrast, 76% of the milk samples collected from women with COVID-19 contained SARS-CoV-2-specific IgA, and 80% had SARS-CoV-2-specific IgG. In addition, 62% of the milk samples were able to neutralize SARS-CoV-2 infectivity in vitro, whereas milk samples collected prior to the COVID-19 pandemic were unable to do so. Taken together, our data do not support mother-to-infant transmission of SARS-CoV-2 via milk. Importantly, milk produced by infected mothers is a beneficial source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness.IMPORTANCE Results from prior studies assaying human milk for the presence of SARS-CoV-2, the causative virus of COVID-19, have suggested milk may act as a potential vehicle for mother-to-child transmission. Most previous studies are limited because they followed only a few participants, were cross-sectional, and/or failed to report how milk was collected and/or analyzed. As such, considerable uncertainty remains regarding whether human milk is capable of transmitting SARS-CoV-2 from mother to child. Here, we report that repeated milk samples collected from 18 women following COVID-19 diagnosis did not contain SARS-CoV-2 RNA; however, risk of transmission via breast skin should be further evaluated. Importantly, we found that milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness as milk likely provides specific immunologic benefits to infants.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Leite Humano/imunologia , Complicações Infecciosas na Gravidez/imunologia , /imunologia , Adulto , Mama/virologia , Aleitamento Materno , /virologia , Feminino , Humanos , Lactente , Transmissão Vertical de Doença Infecciosa , Masculino , Leite Humano/virologia , Mães , Gravidez , Complicações Infecciosas na Gravidez/virologia , RNA Viral/isolamento & purificação , /isolamento & purificação
6.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436526

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , /imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Antígenos Virais/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Microscopia Crioeletrônica , Epitopos , Humanos , Fusão de Membrana , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , /genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral
7.
EBioMedicine ; 63: 103197, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33422991

RESUMO

BACKGROUND: SARS-CoV-2 has caused a global pandemic, infecting millions of people. A safe, effective vaccine is urgently needed and remains a global health priority. Subunit vaccines are used successfully against other viruses when administered in the presence of an effective adjuvant. METHODS: We evaluated three different clinically tested adjuvant systems in combination with the SARS-CoV-2 pre-fusion stabilized (S-2P) spike protein using a one-dose regimen in mice. FINDINGS: Whilst spike protein alone was only weakly immunogenic, the addition of either Aluminum hydroxide, a squalene based oil-in-water emulsion system (SE) or a cationic liposome-based adjuvant significantly enhanced antibody responses against the spike receptor binding domain (RBD). Kinetics of antibody responses differed, with SE providing the most rapid response. Neutralizing antibodies developed after a single immunization in all adjuvanted groups with ID50 titers ranging from 86-4063. Spike-specific CD4 T helper responses were also elicited, comprising mainly of IFN-γ and IL-17 producing cells in the cationic liposome adjuvanted group, and more IL-5- and IL-10-secreting cells in the AH group. INTERPRETATION: These results demonstrate that adjuvanted spike protein subunit vaccine is a viable strategy for rapidly eliciting SARS-CoV-2 neutralizing antibodies and CD4 T cell responses of various qualities depending on the adjuvant used, which can be explored in further vaccine development against COVID-19. FUNDING: This work was supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Hidróxido de Alumínio/química , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , /virologia , Feminino , Imunização , Interferon gama/metabolismo , Interleucina-17/metabolismo , Lipossomos/química , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína da Espícula de Coronavírus/química , Esqualeno/química , Vacinas de Subunidades/imunologia
8.
Nat Commun ; 12(1): 559, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495478

RESUMO

Broadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Epitopos/química , Epitopos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia
9.
MAbs ; 12(1): 1854149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33319649

RESUMO

Monoclonal antibody (mAb) therapy has been previously exploited for viral infections, such as respiratory syncytial virus pneumonia and Ebolavirus disease. In the ongoing COVID-19 pandemic, early signals of efficacy from convalescent plasma therapy have encouraged research and development of anti-SARS-CoV-2 mAbs. While many candidates are in preclinical development, we focus here on anti-SARS-CoV-2 neutralizing mAbs (or mAb cocktails) that represent the late-stage clinical pipeline, i.e., those currently in Phase 2 or Phase 3 clinical trials. We describe the structure, mechanism of action, and ongoing trials for VIR-7831, LY-CoV555, LY-CoV016, BGB-DXP593, REGN-COV2, and CT-P59. We speculate also on the next generation of these mAbs.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , /terapia , /fisiologia , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Humanos , Imunização Passiva/métodos
10.
Trends Immunol ; 41(11): 1006-1022, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33041212

RESUMO

The 2019 coronavirus pandemic remains a major public health concern. Neutralizing antibodies (nAbs) represent a cutting-edge antiviral strategy. We focus here on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV, and discuss current progress in antibody research against rampant SARS-CoV-2 infections. We provide a perspective on the mechanisms of SARS-CoV-2-derived nAbs, comparing these with existing SARS-CoV-derived antibodies. We offer insight into how these antibodies cross-react and cross-neutralize by analyzing available structures of spike (S) glycoprotein-antibody complexes. We also propose ways of adopting antibody-based strategies - such as cocktail antibody therapeutics against SARS-CoV-2 - to overcome the possible resistance of currently identified mutants and mitigate possible antibody-dependent enhancement (ADE) pathologies. This review provides a platform for the progression of antibody and vaccine design against SARS-CoV-2, and possibly against future coronavirus pandemics.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Ligação Proteica , Vírus da SARS/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
11.
Sci Adv ; 6(42)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32948512

RESUMO

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.


Assuntos
Betacoronavirus/imunologia , Peptidil Dipeptidase A/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Visualização da Superfície Celular/métodos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Dimerização , Humanos , Cinética , Pandemias , Peptídeos/química , Peptídeos/imunologia , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Domínios Proteicos/imunologia , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Subunidades Proteicas/metabolismo , RNA Viral/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química
12.
Nat Commun ; 11(1): 4198, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826914

RESUMO

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
13.
Nat Struct Mol Biol ; 27(10): 942-949, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32753755

RESUMO

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, with 10 million infections and more than 500,000 fatalities by June 2020. To initiate infection, the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface and fusion of the viral and host membranes. Prefusion SARS-CoV-2 S is the main target of neutralizing antibodies and the focus of vaccine design. However, its limited stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a construct corresponding to the prefusion SARS-CoV-2 S ectodomain trimer, covalently stabilized by a disulfide bond in the closed conformation. Structural and antigenicity analyses show we successfully shut S in the closed state without otherwise altering its architecture. We demonstrate that this strategy is applicable to other ß-coronaviruses, such as SARS-CoV and MERS-CoV, and might become an important tool for structural biology, serology, vaccine design and immunology studies.


Assuntos
Betacoronavirus/química , Betacoronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Microscopia Crioeletrônica , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas , Multimerização Proteica , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
14.
Biomed Pharmacother ; 130: 110559, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32768882

RESUMO

As the number of people infected with the newly identified 2019 novel coronavirus (SARS-CoV2) is continuously increasing every day, development of potential therapeutic platforms is vital. Based on the comparatively high similarity of receptor-binding domain (RBD) in SARS-CoV2 and SARS-CoV, it seems crucial to assay the cross-reactivity of anti-SARS-CoV monoclonal antibodies (mAbs) with SARS-CoV2 spike (S)-protein. Indeed, developing mAbs targeting SARS-CoV2 S-protein RBD could show novel applications for rapid and sensitive development of potential epitope-specific vaccines (ESV). Herein, we present an overview on the discovery of new CoV followed by some explanation on the SARS-CoV2 S-protein RBD site. Furthermore, we surveyed the novel therapeutic mAbs for targeting S-protein RBD such as S230, 80R, F26G18, F26G19, CR3014, CR3022, M396, and S230.15. Afterwards, the mechanism of interaction of RBD and different mAbs were explained and it was suggested that one of the SARS-CoV-specific human mAbs, namely CR3022, could show the highest binding affinity with SARS-CoV2 S-protein RBD. Finally, some ongoing challenges and future prospects for rapid and sensitive advancement of therapeutic mAbs targeting S-protein RBD were discussed. In conclusion, it may be proposed that this review may pave the way for recognition of RBD and different mAbs to develop potential therapeutic ESV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pandemias , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos Virais/metabolismo , Sítios de Ligação de Anticorpos , Coronavirus/química , Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos/imunologia , Humanos , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais/imunologia
16.
Nat Commun ; 11(1): 4303, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855401

RESUMO

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Chlorocebus aethiops , Mapeamento de Epitopos , Epitopos , Humanos , Biblioteca de Peptídeos , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
17.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641474

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.


Assuntos
Anticorpos Neutralizantes/farmacologia , Citomegalovirus/genética , Proteínas Mutantes Quiméricas/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Fusão Celular , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Células Gigantes/ultraestrutura , Células Gigantes/virologia , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/virologia , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo
18.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
19.
Proc Natl Acad Sci U S A ; 117(25): 13967-13974, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32503918

RESUMO

Molecular dynamics and free energy simulations have been carried out to elucidate the structural origin of differential protein-protein interactions between the common receptor protein angiotensin converting enzyme 2 (ACE2) and the receptor binding domains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [A. E. Gorbalenya et al., Nat. Microbiol. 5, 536-544 (2020)] that causes coronavirus disease 2019 (COVID-19) [P. Zhou et al., Nature 579, 270-273 (2020)] and the SARS coronavirus in the 2002-2003 (SARS-CoV) [T. Kuiken et al., Lancet 362, 263-270 (2003)] outbreak. Analysis of the dynamic trajectories reveals that the binding interface consists of a primarily hydrophobic region and a delicate hydrogen-bonding network in the 2019 novel coronavirus. A key mutation from a hydrophobic residue in the SARS-CoV sequence to Lys417 in SARS-CoV-2 creates a salt bridge across the central hydrophobic contact region, which along with polar residue mutations results in greater electrostatic complementarity than that of the SARS-CoV complex. Furthermore, both electrostatic effects and enhanced hydrophobic packing due to removal of four out of five proline residues in a short 12-residue loop lead to conformation shift toward a more tilted binding groove in the complex in comparison with the SARS-CoV complex. On the other hand, hydrophobic contacts in the complex of the SARS-CoV-neutralizing antibody 80R are disrupted in the SARS-CoV-2 homology complex model, which is attributed to failure of recognition of SARS-CoV-2 by 80R.


Assuntos
Betacoronavirus/fisiologia , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Receptores Virais/metabolismo , Aminoácidos/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Infecções por Coronavirus , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Pandemias , Pneumonia Viral , Domínios Proteicos , Eletricidade Estática
20.
Science ; 369(6504): 650-655, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571838

RESUMO

Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by determining with cryo-eletron microscopy its structure in complex with the S protein to an overall resolution of 3.1 angstroms and local resolution of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Antígenos Virais/imunologia , Linfócitos B/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/terapia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Genes de Cadeia Pesada de Imunoglobulina , Humanos , Memória Imunológica , Pessoa de Meia-Idade , Mutação , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Fosfoproteínas , Pneumonia Viral/terapia , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Adulto Jovem
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