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1.
Cell Rep ; 35(1): 108950, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33794145

RESUMO

Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determine the structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three use VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy-chain N53I-enhancing binding and light-chain tyrosines recognizing F486RBD. Despite these similarities, class members bind both RBD-up and -down conformations of the spike, with a subset of antibodies using elongated CDRH3s to recognize glycan N343 on a neighboring RBD-a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2 antibody class, thus, uses modular recognition encoded by modular genetic elements to effect potent neutralization, with the VH-gene component specifying recognition of RBD and the CDRH3 component specifying quaternary interactions.


Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Região Variável de Imunoglobulina , /imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , /imunologia , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia
2.
Nat Commun ; 12(1): 1577, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707427

RESUMO

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.


Assuntos
/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /química , Animais , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Afinidade de Anticorpos , Linhagem Celular , Chlorocebus aethiops , Biblioteca Gênica , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Modelos Moleculares , Mutação , Testes de Neutralização , Pandemias , Biblioteca de Peptídeos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus/química , Células Vero
4.
PLoS Pathog ; 17(3): e1009328, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33657135

RESUMO

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.


Assuntos
/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética
5.
MAbs ; 13(1): 1893426, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33666135

RESUMO

Numerous neutralizing antibodies that target SARS-CoV-2 have been reported, and most directly block binding of the viral Spike receptor-binding domain (RBD) to angiotensin-converting enzyme II (ACE2). Here, we deliberately exploit non-neutralizing RBD antibodies, showing they can dramatically assist in neutralization when linked to neutralizing binders. We identified antigen-binding fragments (Fabs) by phage display that bind RBD, but do not block ACE2 or neutralize virus as IgGs. When these non-neutralizing Fabs were assembled into bispecific VH/Fab IgGs with a neutralizing VH domain, we observed a ~ 25-fold potency improvement in neutralizing SARS-CoV-2 compared to the mono-specific bi-valent VH-Fc alone or the cocktail of the VH-Fc and IgG. This effect was epitope-dependent, reflecting the unique geometry of the bispecific antibody toward Spike. Our results show that a bispecific antibody that combines both neutralizing and non-neutralizing epitopes on Spike-RBD is a promising and rapid engineering strategy to improve the potency of SARS-CoV-2 antibodies.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/genética , Anticorpos Antivirais/uso terapêutico , /genética , Epitopos/genética , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Glicoproteína da Espícula de Coronavírus/genética
6.
ACS Synth Biol ; 10(2): 379-390, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33534552

RESUMO

Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC50 in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.


Assuntos
/epidemiologia , /genética , Sondas Moleculares/genética , Pandemias , /imunologia , Anticorpos Antivirais/genética , Especificidade de Anticorpos/genética , Doenças Transmissíveis Emergentes/virologia , Escherichia coli/genética , Imunofluorescência , Genes Sintéticos , Genes Virais , Células HEK293 , Humanos , Sondas Moleculares/imunologia , Pandemias/prevenção & controle , Biblioteca de Peptídeos , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/genética , Biologia Sintética
7.
PLoS Pathog ; 17(2): e1009165, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33571304

RESUMO

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Convalescença , Glicoproteína da Espícula de Coronavírus , Adulto , Idoso , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , /imunologia , Chlorocebus aethiops , Clonagem Molecular , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
8.
Nat Commun ; 12(1): 944, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574228

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , /imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Feminino , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , /fisiologia , Soroconversão , Células Vero , Carga Viral
9.
Nature ; 591(7851): 639-644, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33461210

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.


Assuntos
Anticorpos Antivirais/imunologia , Imunidade Humoral/imunologia , /imunologia , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Biópsia , Estudos de Coortes , Imunofluorescência , Humanos , Imunidade Humoral/genética , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Memória Imunológica/imunologia , Intestinos/imunologia , Pessoa de Meia-Idade , Mutação , Hipermutação Somática de Imunoglobulina , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Adulto Jovem
10.
Science ; 371(6531): 823-829, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33495307

RESUMO

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Sítios de Ligação , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , /terapia , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Epitopos/imunologia , Humanos , Imunização Passiva , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos Endogâmicos BALB C , Domínios Proteicos , Engenharia de Proteínas , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/terapia , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
Nat Commun ; 12(1): 469, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473140

RESUMO

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.


Assuntos
Anticorpos Monoclonais/farmacologia , /efeitos dos fármacos , /metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Ligação Proteica , /imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
12.
Viruses ; 12(12)2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348840

RESUMO

Delivering rapid protection against infectious agents to non-immune populations is a formidable public health challenge. Although passive immunotherapy is a fast and effective method of protection, large-scale production and administration of monoclonal antibodies (mAbs) is expensive and unpractical. Viral vector-mediated delivery of mAbs offers an attractive alternative to their direct injection. Integrase-defective lentiviral vectors (IDLV) are advantageous for this purpose due to the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. We engineered IDLV to produce the humanized mAb VN04-2 (IDLV-VN04-2), which is broadly neutralizing against H5 influenza A virus (IAV), and tested the vectors' ability to produce antibodies and protect from IAV in vivo. We found that IDLV-transduced cells produced functional VN04-2 mAbs in a time- and dose-dependent fashion. These mAbs specifically bind the hemagglutinin (HA), but not the nucleoprotein (NP) of IAV. VN04-2 mAbs were detected in the serum of mice at different times after intranasal (i.n.) or intramuscular (i.m.) administration of IDLV-VN04-2. Administration of IDLV-VN04-2 by the i.n. route provided rapid protection against lethal IAV challenge, although the protection did not persist at later time points. Our data suggest that administration of mAb-expressing IDLV may represent an effective strategy for rapid protection against infectious diseases.


Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Integrase de HIV/genética , Lentivirus/genética , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Ordem dos Genes , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A/imunologia , Camundongos , Infecções por Orthomyxoviridae/imunologia
13.
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
14.
PLoS One ; 15(12): e0239112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382708

RESUMO

Influenza virus A is a significant agent involved in the outbreak of worldwide epidemics, causing millions of fatalities around the world by respiratory diseases and seasonal illness. Many projects had been conducting to investigate recovered infected patients for therapeutic vaccines that have broad-spectrum activity. With the aid of the computational approach in biology, the designation for a vaccine model is more accessible. We developed an in silico protocol called iBRAB to design a broad-reactive Fab on a wide range of influenza A virus. The Fab model was constructed based on sequences and structures of available broad-spectrum Abs or Fabs against a wide range of H1N1 influenza A virus. As a result, the proposed Fab model followed iBRAB has good binding affinity over 27 selected HA of different strains of H1 influenza A virus, including wild-type and mutated ones. The examination also took by computational tools to fasten the procedure. This protocol could be applied for a fast-designed therapeutic vaccine against different types of threats.


Assuntos
Anticorpos Antivirais/química , Antígenos Virais/química , Desenho de Fármacos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Fragmentos Fab das Imunoglobulinas/química , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Anticorpos Antivirais/genética , Antígenos Virais/genética , Antígenos Virais/imunologia , Sítios de Ligação , Simulação por Computador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/biossíntese , Influenza Humana/imunologia , Influenza Humana/virologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
15.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2206-2215, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169584

RESUMO

Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.


Assuntos
Anticorpos Antivirais , Vírus da Dengue , Dengue , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/classificação , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Dengue/diagnóstico , Vírus da Dengue/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade , Sorogrupo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
16.
Arch Virol ; 165(12): 2927-2930, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33005985

RESUMO

This study describes the first isolation and genetic characterization of the newly emerging porcine circovirus type 2e (PCV2e) from Korean pigs. The PCV2e isolate did not produce a cytopathic effect in PCV-free PK-15 cells; therefore, PCV2e infection was demonstrated by immunohistochemistry with polyclonal PCV2a antibodies and polymerase chain reaction with primers specific for PCV2e. As the infected PCV-free PK-15 cells were passaged, the amount of infectious virus correlated with an increase in the amount of viral DNA (i.e., a decrease in the cycle threshold values. A full genomic analysis of the PCV2e strain SNUVR199711 was performed and showed that the genome is 1,777 nucleotides in length.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/genética , Infecções por Circoviridae/virologia , Circovirus/genética , DNA Viral/genética , Imuno-Histoquímica , Reação em Cadeia da Polimerase , República da Coreia , Sus scrofa/virologia , Suínos
17.
Sci Rep ; 10(1): 17698, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077899

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 µg/g and 130 µg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Tabaco/metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Folhas de Planta/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Domínios Proteicos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
18.
Cell Host Microbe ; 28(4): 516-525.e5, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32941787

RESUMO

B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Formação de Anticorpos , Betacoronavirus/genética , Feminino , Células HEK293 , Humanos , Imunogenética , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Pandemias , Análise de Sequência , Glicoproteína da Espícula de Coronavírus/imunologia
19.
PLoS Pathog ; 16(9): e1008857, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936838

RESUMO

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.


Assuntos
Antígenos Virais/genética , Enterovirus Humano A/genética , Variação Genética , Genoma Viral , Genótipo , Doença de Mão, Pé e Boca/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Criança , Pré-Escolar , Enterovirus Humano A/imunologia , Enterovirus Humano A/isolamento & purificação , Feminino , Doença de Mão, Pé e Boca/epidemiologia , Doença de Mão, Pé e Boca/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Taiwan
20.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895485
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