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1.
Nat Commun ; 11(1): 4956, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009383

RESUMO

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Corpos Embrioides/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
3.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
4.
PLoS One ; 15(9): e0237694, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-771808

RESUMO

BACKGROUND: The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2) is responsible for the infectious respiratory disease called COVID-19 (COronaVIrus Disease 2019). In response to the growing COVID-19 pandemic, point-of-care (POC) tests have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. We conducted a prospective observational study to evaluate the performance of two POC tests, COVID-PRESTO® and COVID-DUO®, compared to the gold standard, RT-PCR (real-time reverse transcriptase polymerase chain reaction). METHODS: RT-PCR testing of SARS-Cov-2 was performed from nasopharyngeal swab specimens collected in adult patients visiting the infectious disease department at the hospital (Orléans, France). Capillary whole blood (CWB) samples from the fingertip taken at different time points after onset of the disease were tested with POC tests. The specificity and sensitivity of the rapid test kits compared to test of reference (RT-PCR) were calculated. RESULTS: Among 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 143 patients were RT-PCR negative. Results of test with POC tests were all negative for these patients, indicating a specificity of 100% for both POC tests. In the RT-PCR positive subgroup (n = 238), 133 patients were tested with COVID-PRESTO® and 129 patients were tested with COVID-DUO® (24 patients tested with both). The further the onset of symptoms was from the date of collection, the greater the sensitivity. The sensitivity of COVID-PRESTO® test ranged from 10.00% for patients having experienced their 1st symptoms from 0 to 5 days ago to 100% in patients where symptoms had occurred more than 15 days before the date of tests. For COVID-DUO® test, the sensitivity ranged from 35.71% [0-5 days] to 100% (> 15 days). CONCLUSION: COVID-PRESTO® and DUO® POC tests turned out to be very specific (none false positive) and to be sensitive enough after 15 days from onset of symptom. These easy to use IgG/IgM combined test kits are the first ones allowing a screening with CWB sample, by typing from a finger prick. These rapid tests are particularly interesting for screening in low resource settings.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pneumonia Viral/diagnóstico , Kit de Reagentes para Diagnóstico , Adulto , Idoso , Especificidade de Anticorpos , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Capilares , Infecções por Coronavirus/sangue , Dedos/irrigação sanguínea , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/sangue , Testes Imediatos , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
5.
PLoS One ; 15(9): e0237694, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941461

RESUMO

BACKGROUND: The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2) is responsible for the infectious respiratory disease called COVID-19 (COronaVIrus Disease 2019). In response to the growing COVID-19 pandemic, point-of-care (POC) tests have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. We conducted a prospective observational study to evaluate the performance of two POC tests, COVID-PRESTO® and COVID-DUO®, compared to the gold standard, RT-PCR (real-time reverse transcriptase polymerase chain reaction). METHODS: RT-PCR testing of SARS-Cov-2 was performed from nasopharyngeal swab specimens collected in adult patients visiting the infectious disease department at the hospital (Orléans, France). Capillary whole blood (CWB) samples from the fingertip taken at different time points after onset of the disease were tested with POC tests. The specificity and sensitivity of the rapid test kits compared to test of reference (RT-PCR) were calculated. RESULTS: Among 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 143 patients were RT-PCR negative. Results of test with POC tests were all negative for these patients, indicating a specificity of 100% for both POC tests. In the RT-PCR positive subgroup (n = 238), 133 patients were tested with COVID-PRESTO® and 129 patients were tested with COVID-DUO® (24 patients tested with both). The further the onset of symptoms was from the date of collection, the greater the sensitivity. The sensitivity of COVID-PRESTO® test ranged from 10.00% for patients having experienced their 1st symptoms from 0 to 5 days ago to 100% in patients where symptoms had occurred more than 15 days before the date of tests. For COVID-DUO® test, the sensitivity ranged from 35.71% [0-5 days] to 100% (> 15 days). CONCLUSION: COVID-PRESTO® and DUO® POC tests turned out to be very specific (none false positive) and to be sensitive enough after 15 days from onset of symptom. These easy to use IgG/IgM combined test kits are the first ones allowing a screening with CWB sample, by typing from a finger prick. These rapid tests are particularly interesting for screening in low resource settings.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pneumonia Viral/diagnóstico , Kit de Reagentes para Diagnóstico , Adulto , Idoso , Especificidade de Anticorpos , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Capilares , Infecções por Coronavirus/sangue , Dedos/irrigação sanguínea , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/sangue , Testes Imediatos , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
6.
BMC Res Notes ; 13(1): 372, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762746

RESUMO

OBJECTIVE: COVID19 has caused a global and ongoing pandemic. The need for population seroconversion data is apparent to monitor and respond to the pandemic. Using a lateral flow assay (LFA) testing platform, the seropositivity in 63 New York Blood Center (NYBC) Convelescent Plasma (CP) donor samples were evaluated for the presence of COVID19 specific IgG and IgM. RESULTS: CP donors showed diverse antibody result. Convalescent donor plasma contains SARS-CoV-2 specific antibodies. Weak antibody bands may identify low titer CP donors. LFA tests can identify antibody positive individuals that have recovered from COVID19. Confirming suspected cases using antibody detection could help inform the patient and the community as to the relative risk to future exposure and a better understanding of disease exposure.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Doadores de Sangue , Técnicas de Laboratório Clínico/métodos , Convalescença , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/diagnóstico , Testes Imediatos , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos , Infecções por Coronavirus/terapia , Coloide de Ouro , Humanos , Imunização Passiva , Plasma , Domínios Proteicos , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soroconversão
7.
Int J Nanomedicine ; 15: 4919-4932, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764925

RESUMO

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Diagnosing AD before symptoms arise will facilitate earlier intervention. The early diagnostic approaches are thus urgently needed. Methods: The multifunctional nanoparticles W20/XD4-SPIONs were constructed by the conjugation of oligomer-specific scFv antibody W20 and class A scavenger receptor (SR-A) activator XD4 onto superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs' stability and uniformity in size were measured by dynamic light scattering and transmission electron microscopy. The ability of W20/XD4-SPIONs for recognizing Aß oligomers (AßOs) and promoting AßOs phagocytosis was assessed by immunocytochemistry and flow cytometry analysis. The blood-brain barrier permeability of W20/XD4-SPIONs was determined by a co-culture transwell model. The in vivo probe distribution of W20/XD4-SPIONs in AD mouse brains was detected by magnetic resonance imaging (MRI). Results: W20/XD4-SPIONs, as an AßOs-targeted molecular MRI contrast probe, readily reached pathological AßOs regions in brains and distinguished AD transgenic mice from WT controls. W20/XD4-SPIONs retained the property of XD4 for SR-A activation and significantly promoted microglial phagocytosis of AßOs. Moreover, W20/XD4-SPIONs exhibited the properties of good biocompatibility, high stability and low cytotoxicity. Conclusion: Compared with W20-SPIONs or XD4-SPIONs, W20/XD4-SPIONs show the highest efficiency for AßOs-targeting and significantly enhance AßOs uptake by microglia. As a molecular probe, W20/XD4-SPIONs also specifically and sensitively bind to AßOs in AD brains to provide an MRI signal, demonstrating that W20/XD4-SPIONs are promising diagnostic agents for early-stage AD. Due to the beneficial effect of W20 and XD4 on neuropathology, W20/XD4-SPIONs may also have therapeutic potential for AD .


Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/imunologia , Imunoconjugados/química , Nanopartículas de Magnetita/química , Receptores Depuradores/metabolismo , Anticorpos de Cadeia Única/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Especificidade de Anticorpos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Diagnóstico Precoce , Imunoconjugados/farmacologia , Imagem por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Nanopartículas Multifuncionais/química , Fagocitose/efeitos dos fármacos , Anticorpos de Cadeia Única/imunologia
8.
PLoS One ; 15(8): e0234396, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756556

RESUMO

INTRODUCTION: Early conversion to a CNI-free immunosuppression with SRL was associated with an improved 1- and 3- yr renal function as compared with a CsA-based regimen in the SMART-Trial. Mixed results were reported on the occurrence of donor specific antibodies under mTOR-Is. Here, we present long-term results of the SMART-Trial. METHODS AND MATERIALS: N = 71 from 6 centers (n = 38 SRL and n = 33 CsA) of the original SMART-Trial (ITT n = 140) were enrolled in this observational, non-interventional extension study to collect retrospectively and prospectively follow-up data for the interval since baseline. Primary objective was the development of dnDSA. Blood samples were collected on average 8.7 years after transplantation. RESULTS: Development of dnDSA was not different (SRL 5/38, 13.2% vs. CsA 9/33, 27.3%; P = 0.097). GFR remained improved under SRL with 64.37 ml/min/1.73m2 vs. 53.19 ml/min/1.73m2 (p = 0.044). Patient survival did not differ between groups at 10 years. There was a trend towards a reduced graft failure rate (11.6% SRL vs. 23.9% CsA, p = 0.064) and less tumors under SRL (2.6% SRL vs. 15.2% CsA, p = 0.09). CONCLUSIONS: An early conversion to SRL did not result in an increased incidence of dnDSA nor increased long-term risk for the recipient. Transplant function remains improved with benefits for the graft survival.


Assuntos
Inibidores de Calcineurina/administração & dosagem , Imunossupressão/métodos , Imunossupressores/administração & dosagem , Transplante de Rim , Sirolimo/administração & dosagem , Adulto , Especificidade de Anticorpos , Esquema de Medicação , Feminino , Seguimentos , Taxa de Filtração Glomerular , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Estimativa de Kaplan-Meier , Transplante de Rim/efeitos adversos , Transplante de Rim/mortalidade , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Serina-Treonina Quinases TOR/antagonistas & inibidores , Fatores de Tempo , Doadores de Tecidos
9.
Nat Commun ; 11(1): 3971, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769993

RESUMO

Efficacy evaluation through human trials is crucial for advancing a vaccine candidate to clinics. Next-generation sequencing (NGS) can be used to quantify B cell repertoire response and trace antibody lineages during vaccination. Here, we demonstrate this application with a case study of Hecolin®, the licensed vaccine for hepatitis E virus (HEV). Four subjects are administered the vaccine following a standard three-dose schedule. Vaccine-induced antibodies exhibit a high degree of clonal diversity, recognize five conformational antigenic sites of the genotype 1 HEV p239 antigen, and cross-react with other genotypes. Unbiased repertoire sequencing is performed for seven time points over six months of vaccination, with maturation pathways characterize for a set of vaccine-induced antibodies. In addition to dynamic repertoire profiles, NGS analysis reveals differential patterns of HEV-specific antibody lineages and highlights the necessity of the long vaccine boost. Together, our study presents a quantitative strategy for vaccine evaluation in small-scale human studies.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Vírus da Hepatite E/imunologia , Vacinação , Vacinas contra Hepatite Viral/imunologia , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , Epitopos/imunologia , Genótipo , Vírus da Hepatite E/genética , Humanos , Fatores de Tempo , Doadores de Tecidos , Adulto Jovem
10.
BMC Res Notes ; 13(1): 372, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: covidwho-696522

RESUMO

OBJECTIVE: COVID19 has caused a global and ongoing pandemic. The need for population seroconversion data is apparent to monitor and respond to the pandemic. Using a lateral flow assay (LFA) testing platform, the seropositivity in 63 New York Blood Center (NYBC) Convelescent Plasma (CP) donor samples were evaluated for the presence of COVID19 specific IgG and IgM. RESULTS: CP donors showed diverse antibody result. Convalescent donor plasma contains SARS-CoV-2 specific antibodies. Weak antibody bands may identify low titer CP donors. LFA tests can identify antibody positive individuals that have recovered from COVID19. Confirming suspected cases using antibody detection could help inform the patient and the community as to the relative risk to future exposure and a better understanding of disease exposure.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Doadores de Sangue , Técnicas de Laboratório Clínico/métodos , Convalescença , Infecções por Coronavirus/diagnóstico , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/diagnóstico , Testes Imediatos , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos , Infecções por Coronavirus/terapia , Coloide de Ouro , Humanos , Imunização Passiva , Plasma , Domínios Proteicos , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soroconversão
11.
Virology ; 548: 182-191, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838941

RESUMO

Human cytomegalovirus (HCMV) is the most common congenital infection. A glycoprotein B (gB) subunit vaccine (gB/MF59) is the most efficacious clinically tested to date, having achieved 50% protection against primary infection of HCMV-seronegative women. We previously identified that gB/MF59 vaccination primarily elicits non-neutralizing antibody responses, with variable binding to gB genotypes, and protection associated with binding to membrane-associated gB. We hypothesized that gB-specific non-neutralizing antibody binding breadth and function are dependent on epitope and genotype specificity, and ability to interact with membrane-associated gB. We mapped twenty-four gB-specific monoclonal antibodies (mAbs) from naturally HCMV-infected individuals for gB domain specificity, genotype preference, and ability to mediate phagocytosis or NK cell activation. gB-specific mAbs were primarily specific for Domain II and demonstrated variable binding to gB genotypes. Two mAbs facilitated phagocytosis with binding specificities of Domain II and AD2. This investigation provides novel understanding on the relationship between gB domain specificity and antigenic variability on gB-specific antibody effector functions.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Proteínas do Envelope Viral/imunologia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Citomegalovirus/genética , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Masculino , Proteínas do Envelope Viral/genética , Adulto Jovem
12.
EBioMedicine ; 58: 102890, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32707445

RESUMO

BACKGROUND: The novel coronavirus (SARS-CoV-2) shares approximately 80% whole genome sequence identity and 66% spike (S) protein identity with that of SARS-CoV. The cross-neutralization between these viruses is currently not well-defined. METHODS: Here, by using the live SARS-CoV-2 virus infection assay as well as HIV-1 based pseudotyped-virus carrying the spike (S) gene of the SARS-CoV-2 (ppSARS-2) and SARS-CoV (ppSARS), we examined whether infections with SARS-CoV and SARS-CoV-2 can induce cross-neutralizing antibodies. FINDINGS: We confirmed that SARS-CoV-2 infects cells via angiotensin converting enzyme 2 (ACE2), the functional receptor for SARS-CoV, and we also found that the recombinant receptor binding domain (RBD) of the S protein of SARS-CoV effectively inhibits ppSARS-2 entry in Huh7.5 cells. However, convalescent sera from SARS-CoV and SARS-CoV-2 patients showed high neutralizing activity only against the homologous virus, with no or limited cross-neutralization activity against the other pseudotyped virus. Similar results were also observed in vaccination studies in mice. INTERPRETATION: Our study demonstrates that although both SARS-CoV and SARS-CoV-2 use ACE2 as a cellular receptor, the neutralization epitopes are not shared by these two closely-related viruses, highlighting challenges towards developing a universal vaccine against SARS-CoV related viruses. FUNDING: This work was supported by the National Key Research and Development Program of China, the National Major Project for Control and Prevention of Infectious Disease in China, and the One Belt and One Road Major Project for infectious diseases.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Reações Cruzadas , Vírus da SARS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Betacoronavirus/genética , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Vírus da SARS/genética , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
13.
PLoS One ; 15(7): e0234792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614850

RESUMO

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.


Assuntos
Proteínas Angiogênicas/biossíntese , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Substituição de Aminoácidos , Proteínas Angiogênicas/química , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Encéfalo/citologia , Proteínas de Transporte/análise , Linhagem da Célula , Epitopos/imunologia , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Desenvolvimento Muscular , Proteína MyoD/análise , Especificidade de Órgãos , Conformação Proteica , Domínios Proteicos , Coelhos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia , Sequências Repetitivas de Aminoácidos , Pele/citologia , Especificidade da Espécie , Tatuagem , Adulto Jovem
14.
PLoS One ; 15(7): e0236172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726321

RESUMO

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.


Assuntos
Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Hemaglutinação/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Testes de Neutralização , Vacinação
15.
Euro Surveill ; 25(28)2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32700670

RESUMO

Serological reactivity was analysed in plasma from 436 individuals with a history of disease compatible with COVID-19, including 256 who had been laboratory-confirmed with SARS-CoV-2 infection. Over 99% of laboratory-confirmed cases developed a measurable antibody response (254/256) and 88% harboured neutralising antibodies (226/256). Antibody levels declined over 3 months following diagnosis, emphasising the importance of the timing of convalescent plasma collections. Binding antibody measurements can inform selection of convalescent plasma donors with high neutralising antibody levels.


Assuntos
Anticorpos Neutralizantes/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Pneumonia Viral/sangue , Pneumonia Viral/terapia , Adolescente , Adulto , Idoso , Anticorpos Neutralizantes/uso terapêutico , Especificidade de Anticorpos , Doadores de Sangue/estatística & dados numéricos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Inglaterra , Humanos , Imunização Passiva/estatística & dados numéricos , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Estatísticas não Paramétricas , Fatores de Tempo , Adulto Jovem
16.
Proc Natl Acad Sci U S A ; 117(30): 17957-17964, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661157

RESUMO

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.


Assuntos
Adjuvantes Imunológicos , Linfócitos B/imunologia , Reações Cruzadas/imunologia , Memória Imunológica , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/metabolismo , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Imunização Secundária , Masculino , Plasmócitos/imunologia , Plasmócitos/metabolismo
17.
J Clin Invest ; 130(10): 5235-5244, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: covidwho-635481

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Carga Viral , Eliminação de Partículas Virais , Adulto , Idoso , Especificidade de Anticorpos , Reações Cruzadas , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Pandemias , Índice de Gravidade de Doença
18.
J Clin Invest ; 130(10): 5235-5244, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32634129

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Carga Viral , Eliminação de Partículas Virais , Adulto , Idoso , Especificidade de Anticorpos , Reações Cruzadas , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Pandemias , Índice de Gravidade de Doença
20.
EBioMedicine ; 58: 102890, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: covidwho-666030

RESUMO

BACKGROUND: The novel coronavirus (SARS-CoV-2) shares approximately 80% whole genome sequence identity and 66% spike (S) protein identity with that of SARS-CoV. The cross-neutralization between these viruses is currently not well-defined. METHODS: Here, by using the live SARS-CoV-2 virus infection assay as well as HIV-1 based pseudotyped-virus carrying the spike (S) gene of the SARS-CoV-2 (ppSARS-2) and SARS-CoV (ppSARS), we examined whether infections with SARS-CoV and SARS-CoV-2 can induce cross-neutralizing antibodies. FINDINGS: We confirmed that SARS-CoV-2 infects cells via angiotensin converting enzyme 2 (ACE2), the functional receptor for SARS-CoV, and we also found that the recombinant receptor binding domain (RBD) of the S protein of SARS-CoV effectively inhibits ppSARS-2 entry in Huh7.5 cells. However, convalescent sera from SARS-CoV and SARS-CoV-2 patients showed high neutralizing activity only against the homologous virus, with no or limited cross-neutralization activity against the other pseudotyped virus. Similar results were also observed in vaccination studies in mice. INTERPRETATION: Our study demonstrates that although both SARS-CoV and SARS-CoV-2 use ACE2 as a cellular receptor, the neutralization epitopes are not shared by these two closely-related viruses, highlighting challenges towards developing a universal vaccine against SARS-CoV related viruses. FUNDING: This work was supported by the National Key Research and Development Program of China, the National Major Project for Control and Prevention of Infectious Disease in China, and the One Belt and One Road Major Project for infectious diseases.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Reações Cruzadas , Vírus da SARS/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Betacoronavirus/genética , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Vírus da SARS/genética , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
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