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1.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32893735

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
2.
Adv Exp Med Biol ; 1255: 221-230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32949403

RESUMO

Monoclonal antibodies from human sources are being increasingly recognized as valuable options in many therapeutic areas. These antibodies can show exquisite specificity and high potency while maintaining a desirable safety profile, having been matured and tolerized within human patients. However, the discovery of these antibodies presents important challenges, since the B cells encoding therapeutic antibodies can be rare in a typical blood draw and are short-lived ex vivo. Furthermore, the unique pairing of VH and VL domains in each B cell contributes to specificity and function; therefore, maintaining antibody chain pairing presents a throughput limitation. This work will review the various approaches aimed at addressing these challenges with an eye to next-generation methods for high-throughput discovery from the human B-cell repertoire.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Descoberta de Drogas , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Humanos
3.
Nat Commun ; 11(1): 4198, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826914

RESUMO

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


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Imunoglobulina A/imunologia , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Chlorocebus aethiops , Reações Cruzadas , Epitopos , Células HEK293 , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina A Secretora/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
4.
Nat Commun ; 11(1): 4303, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855401

RESUMO

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


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Chlorocebus aethiops , Mapeamento de Epitopos , Epitopos , Humanos , Biblioteca de Peptídeos , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
5.
Biosensors (Basel) ; 10(9)2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847008

RESUMO

Cytokines are a family of proteins which play a major role in the regulation of the immune system and the development of several diseases, from rheumatoid arthritis to cancer and, more recently, COVID-19. Therefore, many efforts are currently being developed to improve therapy and diagnosis, as well as to produce inhibitory drugs and biosensors for a rapid, minimally invasive, and effective detection. In this regard, even more efficient cytokine receptors are under investigation. In this paper we analyze a set of IL-6 cytokine receptors, investigating their topological features by means of a theoretical approach. Our results suggest a topological indicator that may help in the identification of those receptors having the highest complementarity with the protein, a feature expected to ensure a stable binding. Furthermore, we propose and discuss the use of these receptors in an idealized experimental setup.


Assuntos
Técnicas Biossensoriais/métodos , Interleucina-6/análise , Receptores de Interleucina-6/análise , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Aptâmeros de Nucleotídeos/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/imunologia , Limite de Detecção , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Receptores de Interleucina-6/imunologia
6.
Monoclon Antib Immunodiagn Immunother ; 39(4): 107-111, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32762609

RESUMO

In this hypothesis, we address the biological/immunological pathway leading to severe disease or death after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The underlying immune response is described with "original antigenic sin" (OAS) whereby previous infections influence the response to future virus encounters. We cite evidence for OAS-induced immunopathology in HIV-1 disease. We hypothesize that similar immune abnormalities can occur after infection with SARS-CoV-2. This hypothesis is supported by recent analysis of the antibodies in infected patients demonstrating serological and B cell abnormalities. The concept of symmetrical clonal regulation developed earlier for the immune network illustrates the pathway suggested by our hypothesis and may be helpful to develop strategies avoiding severe coronavirus disease 2019.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Evasão da Resposta Imune/imunologia , Pneumonia Viral/imunologia , Anticorpos Monoclonais/imunologia , Infecções por Coronavirus/patologia , Reações Cruzadas/imunologia , Síndrome da Liberação de Citocina/imunologia , HIV/imunologia , HIV-1/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Memória Imunológica/imunologia , Pandemias , Pneumonia Viral/patologia
7.
PLoS One ; 15(8): e0237940, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853255

RESUMO

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Comamonadaceae/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Sorogrupo , Hibridomas , Limite de Detecção
8.
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
9.
Drug Des Devel Ther ; 14: 2607-2611, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32753842

RESUMO

In March 2020, the WHO declared the COVID-19 disease as a pandemic disease. There have been studies on the COVID-19 to find a certain treatment, but yet, there is no certain cure. In this article, we present a possible way to treat severe cases of COVID-19. Based on the previous studies, there are similarities between the spike antigens of SARS-CoV and SARS-CoV-2 viruses. It is expected that these similarities (structural and affinity to the receptor of ACE2) can lead to the same pathophysiological activity of the virus by the use of ACE2 and FcγRII (the antibody-dependent enhancement mechanism). Therefore, we propose a way of washing out (by plasmapheresis) the possible antibodies against the spike protein of the virus out of patients' plasma to stop the antibody-dependent enhancement (ADE)-mediated infection of the immune system cells at the first phase of the treatment and simultaneous use of the anti-ACE2 with anti-FcγRII monoclonal antibodies at the second phase. We propose these procedures for the patients that have no significant response for typical anti-viral, ARDS and conservative therapies, and the disease persists or progresses despite sufficient therapies.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Infecções por Coronavirus/terapia , Plasmaferese/métodos , Pneumonia Viral/terapia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Pandemias , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Receptores de IgG/imunologia , Índice de Gravidade de Doença
11.
PLoS One ; 15(7): e0235546, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609743

RESUMO

Resistin and resistin-like molecules are pleiotropic cytokines that are involved in inflammatory diseases. Our previous work suggested that resistin has the potential to be used as a biomarker and therapeutic target for human pulmonary arterial hypertension. However, data are limited on the distribution of resistin in healthy human organs. In this study, we used our newly developed anti-human resistin (hResistin) antibody to immunohistochemically detect the expression, localization, and intracellular/extracellular compartmentalization of hResistin in a full human tissue panel from healthy individuals. The potential cross reactivity of this monoclonal anti-hResistin IgG1 with normal human tissues also was verified. Results showed that hResistin is broadly distributed and principally localized in the cytoplasmic granules of macrophages scattered in the interstitium of most human tissues. Bone marrow hematopoietic precursor cells also exhibited hResistin signals in their cytoplasmic granules. Additionally, hResistin labeling was observed in the cytoplasm of nervous system cells. Notably, the cytokine activity of hResistin was illustrated by positively stained extracellular material in most human tissues. These data indicate that our generated antibody binds to the secreted hResistin and support its potential use for immunotherapy to reduce circulating hResistin levels in human disease. Our findings comprehensively document the basal expression patterns of hResistin protein in normal human tissues, suggest a critical role of this cytokine in normal and pathophysiologic inflammatory processes, and offer key insights for using our antibody in future pharmacokinetic studies and immunotherapeutic strategies.


Assuntos
Anticorpos Monoclonais/imunologia , Regulação da Expressão Gênica , Resistina/imunologia , Resistina/metabolismo , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Espaço Intracelular/metabolismo , Especificidade de Órgãos , Transporte Proteico
12.
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
13.
Euro Surveill ; 25(28)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32700671

RESUMO

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Animais , Betacoronavirus/genética , Western Blotting , Células COS , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Genoma Viral , Camundongos , Pandemias , Peptidil Dipeptidase A/imunologia , Plasmídeos , Pneumonia Viral/genética , Proteínas Recombinantes/imunologia , Vírus da SARS/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção , Células Vero , Integração Viral
14.
Nat Commun ; 11(1): 3736, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719371

RESUMO

A replication-competent vesicular stomatitis virus vaccine expressing the Ebola virus (EBOV) glycoprotein (GP) (rVSV-ZEBOV) was successfully used during the 2013-16 EBOV epidemic. Additionally, chimeric and human monoclonal antibodies (mAb) against the EBOV GP have shown promise in animals and humans when administered therapeutically. Uncertainty exists regarding the efficacy of postexposure antibody treatments in the event of a known exposure of a recent rVSV-ZEBOV vaccinee. Here, we model a worst-case scenario using rhesus monkeys vaccinated or unvaccinated with the rVSV-ZEBOV vaccine. We demonstrate that animals challenged with a uniformly lethal dose of EBOV one day following vaccination, and then treated with the anti-EBOV GP mAb MIL77 starting 3 days postexposure show no evidence of clinical illness and survive challenge. In contrast, animals receiving only vaccination or only mAb-based therapy become ill, with decreased survival compared to animals vaccinated and subsequently treated with MIL77. These results suggest that rVSV-ZEBOV augments immunotherapy.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Vacinas contra Ebola/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Profilaxia Pós-Exposição , Vacinação , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Doença pelo Vírus Ebola/patologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Estimativa de Kaplan-Meier , Macaca mulatta , Resultado do Tratamento , Carga Viral/imunologia
15.
Life Sci ; 257: 118052, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634431

RESUMO

AIMS: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. MAIN METHODS: We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SIGNIFICANCE: Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.


Assuntos
Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Receptores de Fator Estimulador de Colônias de Granulócitos/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos , Anticorpos Monoclonais/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Neovascularização Patológica/tratamento farmacológico , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única
16.
J Exp Med ; 217(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32692348

RESUMO

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Imunoensaio/métodos , Pneumonia Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Linhagem Celular , Quimera/genética , Quimera/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Humanos , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/virologia , Recombinação Genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
17.
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
18.
Food Chem ; 332: 127398, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32610260

RESUMO

Herein, a label-free and dual-readout immunochromatographic test strip (ITS) for the sensitive detection of Escherichia coli (E. coli) O157:H7 by taking advantages of the strong capture ability of Fe3O4@CuS nanostructures (NSs) towards bacteria and their ultrahigh photothermal effects (PTEs) was reported. Especially, without the customarily antibody (Ab)-labeled probe, Fe3O4@CuS NSs could be adsorbed onto the surfaces of bacteria to form Fe3O4@CuS-bacteria conjugates and then trapped by immobilized Abs on the test line (T-line), forming a characteristic yellow band. After direct immunoreactions, the PTEs of Fe3O4@CuS NSs endowed T-line to be irradiated by an 808 nm infrared light, obtaining satisfactory sensitivity and accuracy. Under optimal conditions, E. coli O157:H7, as low as 103 and 102 CFU/mL, could be monitored in colorimetric and photothermal modes. Additionally, E. coli O157:H7 was successfully detected in beef, chicken, milk and honey samples by this proposed platform with a recovery of 80-120%.


Assuntos
Cromatografia de Afinidade/métodos , Cobre/química , Escherichia coli O157/isolamento & purificação , Compostos Férricos/química , Microbiologia de Alimentos/métodos , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Galinhas/microbiologia , Cromatografia de Afinidade/instrumentação , Escherichia coli O157/imunologia , Microbiologia de Alimentos/instrumentação , Mel/microbiologia , Limite de Detecção , Leite/microbiologia , Fitas Reagentes/química , Carne Vermelha/microbiologia
19.
PLoS One ; 15(7): e0235815, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673351

RESUMO

Monoclonal antibodies (mAbs) for therapeutic applications should be as similar to native human antibodies as possible to minimize their immunogenicity in patients. Several transgenic animal platforms are available for the generation of fully human mAbs. Attributes such as specificity, efficacy and Chemistry, Manufacturing and Controls (CMC) developability of antibodies against a specific target are typically established for antibodies obtained from one platform only. In this study, monoclonal antibodies (mAbs) cross-reactive against human and cynomolgus LAMP1 were derived from the human immunoglobulin transgenic TRIANNI mouse and OmniChicken® platforms and assessed for their specificity, sequence diversity, ability to bind to and internalize into tumor cells, expected immunogenicity and CMC developability. Our results show that the two platforms were complementary at providing a large diversity of mAbs with respect to epitope coverage and antibody sequence diversity. Furthermore, most antibodies originating from either platform exhibited good manufacturability characteristics.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/química , Galinhas , Células HEK293 , Humanos , Imunização , Macaca fascicularis , Camundongos , Modelos Moleculares
20.
Nat Commun ; 11(1): 3418, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647286

RESUMO

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.


Assuntos
Anticorpos Antivirais/imunologia , Resistência a Múltiplos Medicamentos/imunologia , Farmacorresistência Viral/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Mutação/genética , Neuraminidase/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Afinidade de Anticorpos/imunologia , Antígenos Virais/metabolismo , Líquidos Corporais/virologia , Análise Mutacional de DNA , Cães , Epitopos/química , Epitopos/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H3N2/enzimologia , Células Madin Darby de Rim Canino , Simulação de Acoplamento Molecular , Imagem Óptica , Ligação Proteica , Análise Espectral Raman
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