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1.
Med Sci (Paris) ; 36 Hors série n° 1: 56-60, 2020 Oct.
Artigo em Francês | MEDLINE | ID: mdl-33052096

RESUMO

Monoclonal antibody (mAb)-based immunotherapy is booming in oncology. In 2020, more than 40% of FDA (Food and Drug Administration)-approved antibodies (34 out of 84 antibodies, according to The Antibody Society) have an indication for cancer therapy. In contrast to standard chemotherapy, they demonstrate a much better safety profile for patients. Despite this, adverse side effects may occur due to the targeting of the antigen also expressed by healthy tissues. For this reason, emerging strategies aim at optimizing the antibody format and considering the particularities of the tumor microenvironment to confer a more specific action of the antibody at the tumor site.


Assuntos
Anticorpos Monoclonais , Antígenos de Neoplasias/isolamento & purificação , Terapia de Alvo Molecular/métodos , Neoplasias/imunologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Antígenos de Neoplasias/imunologia , Sistemas de Liberação de Medicamentos/métodos , Mapeamento de Epitopos/métodos , Humanos , Pró-Fármacos/uso terapêutico , Hipóxia Tumoral/imunologia
2.
Arch Virol ; 165(12): 2789-2798, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32970278

RESUMO

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.


Assuntos
Especificidade de Anticorpos , Cicer/virologia , Geminiviridae/isolamento & purificação , Doenças das Plantas/virologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Bacteriófagos/genética , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Geminiviridae/genética , Simulação de Acoplamento Molecular , Filogenia , Análise de Sequência de DNA , Anticorpos de Cadeia Única/isolamento & purificação
3.
J Chromatogr A ; 1629: 461465, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32827903

RESUMO

Modeling the chromatographic separations of proteins at manufacturing scale is important since downstream processing costs are often dominant. At such scales, the columns are highly overloaded heightening the challenge of predicting performance. In this work, the separation of a monoclonal antibody monomer-dimer mixture is conducted by gradient elution chromatography with ceramic hydroxyapatite (CHT) columns Type I and Type II under overloaded conditions. Phosphate gradients are shown to be preferable over sodium chloride gradients since the latter result in undesirable pH transitions generated within the column itself. Using sodium phosphate gradients separation is obtained with both CHT types, achieving approximately 90% recovery at 99% monomer purity starting with a mixture containing 30% dimer at total protein loads up to 30 mg/mL. Because of its higher binding capacity, even higher loadings can be obtained with CHT Type I without monomer breakthrough. A hybrid model is developed to describe the separation. The model, based on an empirical description of two-component, competitive isotherms at low sodium phosphate concentration coupled with the stoichiometric displacement model at higher sodium phosphate concentrations, is in good agreement with the experiments using the linear driving force (LDF) approximation to describe adsorption/desorption kinetics. The same LDF rate coefficient predicts the separation at loadings between 0.8 and 30 mg/mL. The model developed in this work can be used as a general tool to optimize operating conditions, understand what factors limit performance, and compare different operating modes.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cerâmica/química , Durapatita/química , Multimerização Proteica , Adsorção , Anticorpos Monoclonais/química , Simulação por Computador , Íons , Cinética , Modelos Químicos , Polímeros/química , Ligação Proteica , Sódio/química , Temperatura
4.
J Chromatogr A ; 1629: 461453, 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-32861093

RESUMO

We examine the use of a z2 flow distribution and collection feature for enhancing the separation efficiency of a laterally-fed membrane chromatography (or LFMC) device. The new device is designated z2LFMC. Two devices were fabricated using two different ion-exchange membranes: strong anion exchange (Q) with 0.8-micron pore size, and strong cation exchange (S) with pore size in the 3-5 µm range. The number of theoretical plates per unit membrane bed height in these z2LFMC devices were higher than those reported for other membrane chromatography devices housing similar membranes, including the older versions of LFMC devices. The enhancement in separation efficiency is explained based on computational fluid dynamic (CFD) simulations. Model protein separation experiments showed that the z2LFMC device gave similar resolution as an equivalent resin-packed column, at 40-time greater flow rate than that used with the column. At a comparable flow rate, the resolution obtained with the z2LFMC device was significantly higher than that obtained with the equivalent resin-packed column. Therefore, the z2LFMC device combines high-speed with high-resolution. We demonstrate the suitability of the z2LFMC devices for carrying out fast and efficient biopharmaceutical separations through two case studies, i.e. the fractionation of monoclonal antibody charge variants, and monoclonal antibody aggregates separation.


Assuntos
Produtos Biológicos/isolamento & purificação , Cromatografia/métodos , Membranas Artificiais , Anticorpos Monoclonais/isolamento & purificação , Simulação por Computador , Hidrodinâmica , Pressão , Reologia
5.
J Chromatogr A ; 1627: 461420, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823115

RESUMO

Monitoring preparative protein chromatographic steps by in-line spectroscopic tools or fraction analytics results in medium or large sized data matrices. Multivariate Curve Resolution (MCR) serve to compute or to estimate the concentration values of the pure components only from these data matrices. However, MCR methods often suffer from an inherent solution ambiguity which underlies the factorization problem. The typical unimodality of the chromatographic profiles of pure components can support the chemometric analysis. Here we present the pure components estimation process within the framework of the area of feasible solutions, which is a systematic approach to represent the range of all possible solutions. The unimodality constraint in combination with Pareto optimization is shown to be an effective method for the pure component calculation. Applications are presented for chromatograms on a model protein mixture containing ribonuclease A, cytochrome c and lysozyme and on a two-dimensional chromatographic separation of a monoclonal antibody from its aggregate species. The root mean squared errors of the first case study are 0.0373, 0.0529 and 0.0380 g/L compared to traditional off-line analytics. The second case study illustrates the potential of recovering hidden components with MCR from off-line reference analytics.


Assuntos
Produtos Biológicos/análise , Cromatografia/métodos , Preparações Farmacêuticas/análise , Anticorpos Monoclonais/isolamento & purificação , Estudos de Viabilidade , Análise dos Mínimos Quadrados , Análise Multivariada , Proteínas/isolamento & purificação , Reprodutibilidade dos Testes
6.
Nat Med ; 26(9): 1422-1427, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32651581

RESUMO

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia
7.
J Chromatogr A ; 1625: 461237, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709313

RESUMO

The quest for ligands alternative to Protein A for the purification of monoclonal antibodies (mAbs) has been pursued for almost three decades. Yet, the IgG-binding peptides known to date still fall short of the host cell protein (HCP) logarithmic removal value (LRV) set by Protein A media (2.5-3.1). In this study, we present an integrated computational-experimental approach leading to the discovery of peptide ligands that provide HCP LRVs on par with Protein A. First, the screening of 60,000 peptide variants was performed using a high-throughput search algorithm to identify sequences that ensure IgG affinity binding. Select sequences WQRHGI, MWRGWQ, RHLGWF, and GWLHQR were then negatively screened in silico against a panel of model HCPs to ensure the selection of peptides with high binding selectivity. Candidate ligands WQRHGI and MWRGWQ were conjugated to chromatographic resins and characterized by isothermal binding and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC10%), respectively. The resulting Qmax were 52.6 mg of IgG per mL of adsorbent for WQRHGI and 57.48 mg/mL for MWRGWQ, while the DBC10% (2 minutes residence time) were 30.1 mg/mL for WQRHGI and 36.4 mg/mL for MWRGWQ. Evaluation of the peptides by isothermal titration calorimetry (ITC) confirmed the binding energy predicted in silico, and an amino acid scanning study corroborated the affinity-like binding activity of the peptides. WQRHGI-WorkBeads resin was finally characterized by purification of a monoclonal antibody from a Chinese Hamster Ovary (CHO) cell culture harvest, affording a remarkable HCP LRV of 2.7, and consistent product yield and purity over 100 chromatographic cycles. These results demonstrate the potential of WQRHGI as an effective alternative to Protein A for antibody purification.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Peptídeos/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo
8.
J Chromatogr A ; 1625: 461117, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709364

RESUMO

To obtain consistent chromatographic behavior, it is important to develop resin packing methods in accordance with the characteristics of each resin. Resins, particularly those with a significant level of compressibility, require proper knowledge of the packing methodology to ensure scalable performance. The study demonstrates the applicability of pressure-flow modeling based on the Blake-Kozeny equation for cellulose based resins, using the MEP HyperCel (Pall) resin as a case study. This approach enabled the understanding of the appropriate bed compressibility and the determination of the minimum column diameter that can predict bed integrity during commercial manufacturing scale operation. Studies suggested that scale-dependent wall effects become negligible for column diameters exceeding 20 cm. Pressure-flow modeling produced a minimum compression recommendation of 0.206 for the MEP HyperCel resin. Columns with diameters up to 80 cm packed with this bed compression yielded incompressible beds with pressure-flow curves consistent with model predictions. Model parameter (particle diameter, viscosity, porosity) values were then varied to demonstrate how changing operating conditions influence model predictions. This analysis supported the successful troubleshooting of unexpected high pressures at the commercial manufacturing scale using MEP HyperCel resin, further supporting the applicability of this approach.


Assuntos
Produtos Biológicos/isolamento & purificação , Cromatografia Líquida/métodos , Anticorpos Monoclonais/isolamento & purificação , Géis/química , Porosidade , Pressão , Viscosidade
9.
Science ; 369(6506): 956-963, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32540903

RESUMO

Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Adulto , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/uso terapêutico , Afinidade de Anticorpos , Especificidade de Anticorpos , Betacoronavirus/fisiologia , Sítios de Ligação , Linhagem Celular , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Epitopos , Feminino , Humanos , Imunização Passiva , Pulmão/virologia , Masculino , Mesocricetus , Pessoa de Meia-Idade , Testes de Neutralização , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Carga Viral , Replicação Viral
10.
PLoS Negl Trop Dis ; 14(6): e0008203, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579555

RESUMO

BACKGROUND: Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1-4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiating DENV serotypes and none are currently commercially available. METHODOLOGY/PRINCIPLE FINDINGS: We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitivity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 serotyping were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07-100%. SIGNIFICANCE: Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Dengue/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Sorogrupo , Proteínas não Estruturais Virais/análise , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Brasil , Estudos de Coortes , Honduras , Humanos , Índia , América Latina , Camundongos Endogâmicos C57BL , Sensibilidade e Especificidade
11.
Science ; 368(6496): 1274-1278, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: covidwho-260594

RESUMO

Neutralizing antibodies could potentially be used as antivirals against the coronavirus disease 2019 (COVID-19) pandemic. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. The antibodies B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin-converting enzyme 2 (ACE2). A competition assay indicated different epitopes on the RBD for these two antibodies, making them a potentially promising virus-targeting monoclonal antibody pair for avoiding immune escape in future clinical applications. Moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide a structural basis for rational vaccine design.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Infecções por Coronavirus/terapia , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/terapia , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Modelos Animais de Doenças , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Pulmão/imunologia , Pulmão/virologia , Camundongos , Testes de Neutralização , Pandemias , Domínios Proteicos , Carga Viral/imunologia
12.
Cell ; 182(1): 73-84.e16, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32425270

RESUMO

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Linfócitos B/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Análise de Célula Única , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Convalescença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Pandemias , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Éxons VDJ
13.
Antiviral Res ; 179: 104820, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32405117

RESUMO

SARS-CoV-2-caused COVID-19 cases are growing globally, calling for developing effective therapeutics to control the current pandemic. SARS-CoV-2 and SARS-CoV recognize angiotensin-converting enzyme 2 (ACE2) receptor via the receptor-binding domain (RBD). Here, we identified six SARS-CoV RBD-specific neutralizing monoclonal antibodies (nAbs) that cross-reacted with SARS-CoV-2 RBD, two of which, 18F3 and 7B11, neutralized SARS-CoV-2 infection. 18F3 recognized conserved epitopes on SARS-CoV and SARS-CoV-2 RBDs, whereas 7B11 recognized epitopes on SARS-CoV RBD not fully conserved in SARS-CoV-2 RBD. The 18F3-recognizing epitopes on RBD did not overlap with the ACE2-binding sites, whereas those recognized by 7B11 were close to the ACE2-binding sites, explaining why 7B11 could, but 18F3 could not, block SARS-CoV or SARS-CoV-2 RBD binding to ACE2 receptor. Our study provides an alternative approach to prevent SARS-CoV-2 infection using anti-SARS-CoV nAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Betacoronavirus/genética , Sítios de Ligação , Reações Cruzadas , Epitopos/imunologia , Células HEK293 , Humanos , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/imunologia , Vírus da SARS/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
14.
Science ; 368(6496): 1274-1278, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32404477

RESUMO

Neutralizing antibodies could potentially be used as antivirals against the coronavirus disease 2019 (COVID-19) pandemic. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. The antibodies B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin-converting enzyme 2 (ACE2). A competition assay indicated different epitopes on the RBD for these two antibodies, making them a potentially promising virus-targeting monoclonal antibody pair for avoiding immune escape in future clinical applications. Moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide a structural basis for rational vaccine design.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Infecções por Coronavirus/terapia , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/terapia , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Modelos Animais de Doenças , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Pulmão/imunologia , Pulmão/virologia , Camundongos , Testes de Neutralização , Pandemias , Domínios Proteicos , Carga Viral/imunologia
20.
Biotechnol Bioeng ; 117(7): 1990-2007, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32297972

RESUMO

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/química , Infecções por Coronavirus/diagnóstico , Hibridomas/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Chaperonas Moleculares , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Coronavirus/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vacinas contra Influenza , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Testes Sorológicos , Solubilidade
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