Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 486
Filtrar
1.
Biochemistry (Mosc) ; 86(11): 1469-1476, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34906050

RESUMO

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.


Assuntos
Anticorpos Monoclonais Murinos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H1N1 , Simulação de Dinâmica Molecular , Neurônios/metabolismo , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Mutagênese Sítio-Dirigida , Neurônios/patologia , Domínios Proteicos
2.
Signal Transduct Target Ther ; 6(1): 315, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433803

RESUMO

The evolution of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Here we report a human angiotensin-converting enzyme 2 (ACE2)-targeting monoclonal antibody, 3E8, blocked the S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2, SARS-CoV-2 mutant variants (SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617.1, and P.1), SARS-CoV and HCoV-NL63, without markedly affecting the physiological activities of ACE2 or causing severe toxicity in ACE2 "knock-in" mice. 3E8 also blocked live SARS-CoV-2 infection in vitro and in a prophylactic mouse model of COVID-19. Cryo-EM and "alanine walk" studies revealed the key binding residues on ACE2 interacting with the CDR3 domain of 3E8 heavy chain. Although full evaluation of safety in non-human primates is necessary before clinical development of 3E8, we provided a potentially potent and "broad-spectrum" management strategy against all coronaviruses that utilize ACE2 as entry receptors and disclosed an anti-coronavirus epitope on human ACE2.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Anticorpos Monoclonais Murinos/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Vero
3.
ACS Chem Biol ; 16(8): 1344-1349, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255482

RESUMO

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.


Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Epitopos/metabolismo , Glicerofosfatos/metabolismo , Ácidos Teicoicos/metabolismo , Animais , Anticorpos Monoclonais Murinos/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Glicerofosfatos/química , Glicerofosfatos/imunologia , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia
4.
Anal Biochem ; 619: 114103, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33453163

RESUMO

Low density lipoprotein (LDL) can be oxidized in a stepwise process that leads to the production of minimally modified low density lipoprotein (mm-LDL), in which only the lipid component is oxidized, and then of fully oxidized LDL (oxLDL), in which both the lipids and the protein are oxidized. The thiobarbituric acid-reactive substances (TBARS) assay is a recognized method for determination of oxidized LDL, however this method is unable to distinguish between mm-LDL and oxLDL. In this study, seven specific monoclonal antibodies (mAbs) against human LDL were generated and selectively bound to the apolipoprotein B-100 (apoB-100) component of LDL. Oxidized LDL was produced by incubation of human LDL with 10 µM CuSO4 for various times. The TBARS assay revealed that the optimal incubation time to achieve maximal lipid oxidation was 9 h. Indirect ELISA using the newly generated mAbs was implemented to differentiate between mm-LDL and oxLDL and it was found that binding of the mAbs to oxLDL was significantly decreased after 48 h of incubation, reflecting the oxidative modification of apoB-100. Our results suggest that the optimal times for incubation of LDL with CuSO4 for generation of mm-LDL and oxLDL were 9 h and 48 h, respectively.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Lipoproteínas LDL/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C
5.
Mol Immunol ; 131: 6-12, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33450670

RESUMO

The SARS-CoV-2 virus responsible for coronavirus 2019 (COVID-19) poses a significant challenge to healthcare systems worldwide. According to the World Health Organization (WHO), the outbreak of COVID-19 has been a pandemic that infected more than 25.32 million people and caused more than 848.25 thousand deaths worldwide at the time of 1st September 2020. Despite governmental initiatives aimed to contain the spread of the disease, several countries are experiencing unmanageable increases in medical equipment and larger testing capacity. The current diagnosis based on nuclear acid requires specialized instruments, time-consuming, and laborious, the low-cost and convenient technologies were still urgently needed. Both spike and nucleocapsid are key structural proteins of COVID-19 with good immunogenicity, can serve as primary targets for immunoassay. After comparative research, we certified nucleocapsid antigen-monoclonal antibody (mAbs) system was more suitable for the COVID-19 immunodetection. Subsequently, we designed a rapid test strip based on it that can be used in large-scale screening of COVID-19 in population and more suitable for some remote and special needs areas were restricted by a medical condition or for quick and large quantities of screenings.


Assuntos
Anticorpos Monoclonais Murinos/química , Teste Sorológico para COVID-19 , COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Imunoensaio , Camundongos , Camundongos Endogâmicos BALB C
6.
Mikrochim Acta ; 188(1): 11, 2021 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-33389211

RESUMO

An ultrasensitive and rapid fluorescent immunoassay based on a broad-spectrum monoclonal antibody (mAb) was developed to detect pyrrolizidine alkaloids (PAs) in honey samples. First, Discovery Studio software was used to analyze and predict the target hapten, and retrorsine (RTS) was selected to react with succinic anhydride (HS) for hapten synthesization. A sensitive and broad-spectrum monoclonal antibody (mAb 13E1) was obtained for nine PAs. Then, fluorescent gold nanoclusters (AuNCs) were conjugated with mAb as a label probe and used in establishing a qualitative and quantitative lateral flow immunoassay (AuNCs-LFIA) for the determination of four PAs (retrorsine, platyphylline, senecionine, integerrimine) in honey within 14 min. The limits of detection (LOD) were 0.083 µg/kg. The recovery in spiked honey samples were 87.98-119.57%, with coefficients of variation of ≤ 11.5%. A total of 45 commercial import honey samples from nine different countries were tested through AuNCs-LFIA and UPLC-MS/MS method, and satisfactory consistency (R2 = 0.995) was obtained. The rates of positive samples were 55.56% (25/45), and the average concentrations of four PAs were 3.24-46.47 µg/kg. This ultrasensitive multi-PA method provides an alternative analytical tool for evaluating the human risk posed by the consumption of PA-contaminated honey.


Assuntos
Corantes Fluorescentes/química , Fluorimunoensaio/métodos , Nanopartículas Metálicas/química , Alcaloides de Pirrolizidina/análise , Animais , Anticorpos Monoclonais Murinos/imunologia , Feminino , Contaminação de Alimentos/análise , Ouro/química , Haptenos/imunologia , Mel/análise , Limite de Detecção , Camundongos Endogâmicos BALB C , Alcaloides de Pirrolizidina/imunologia , Software
7.
Mikrochim Acta ; 188(1): 3, 2021 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-33389215

RESUMO

A surface-enhanced Raman scattering (SERS) immunochromatographic assay (ICA) has been developed for rapid, ultrasensitive, and quantitative detection of rotavirus in feces using double Raman molecule-labeled Au-core Ag-shell nanoparticles. The Raman signals are generated by 5,5'-dithiobis-(2-nitrobenzoic acid) and the intensity of the characteristic peak at 1334-1 cm was detected as the analytical signal. The Raman signals were enhanced by the SERS-enhanced effect of both Au and Ag, the large amount of Raman molecules, and the hot-spot effect in the narrow gap between the Au core and Ag shell. The SERS ICA can quantitatively detect rotavirus in a concentration range of 8- 40,000 pg/mL, with detection limits of 80 pg/mL and 8 pg/mL based on naked eye observation and SERS signal detection, respectively. No cross-reaction was observed from other common pathogens. The standard deviation of the intra- and inter-batch repetitive tests is less than 10%, and the coincidence between SERS ICA and RT-qPCR as well as commercial colloidal gold ICA is 100%. The results indicated that this SERS ICA is able to quantitatively detect rotavirus in feces in 20 min with high sensitivity, selectivity, reproducibility, and accuracy and might be a promising method for the early detection of rotavirus in clinical analysis.


Assuntos
Cromatografia de Afinidade/métodos , Nanopartículas Metálicas/química , Rotavirus/isolamento & purificação , Análise Espectral Raman/métodos , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Ácido Ditionitrobenzoico/química , Fezes/virologia , Ouro/química , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Rotavirus/imunologia , Prata/química
8.
FEBS Lett ; 595(6): 789-798, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33159684

RESUMO

Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Epitopos/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Animais , Epitopos/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética
9.
J Cutan Pathol ; 48(4): 472-478, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32935876

RESUMO

BACKGROUND: Lymphovascular invasion (LVI) is believed to be the mechanism by which melanoma cells can disseminate to regional lymph nodes and distant sites and may be predictive of adverse outcome. Lymphovascular invasion often difficult to detect on hematoxylin-eosin (HE) stained sections, are readily identified with dual immunohistochemistry (IHC) for melanocytic and vascular markers. METHODS: A total of 100 primary cutaneous malignant melanoma cases that had a Breslow thickness of 1-4 mm and lacked LVI by conventional HE assessment were included. We compared the LVI detection rates of double staining for CD31/S100 and CD34/S100, and D2-40/S100, and examined the association of LVI with clinical outcomes. RESULTS: The dual immunohistochemical positivity for CD31/S100, CD34/S100, and D2-40/S100 were 40(40%), 17(17%) and 35(35%), respectively. On multivariate analysis, LVI was an independent predictor of SLN status. Multivariate analysis revealed that LVI and male gender were independent risk factors for overall survival. CONCLUSIONS: The recognition of LVI is improved by dual IHC and predicts SLN metastasis. The detection of LVI using dual IHC, especially by a combination of CD31/S100 and D2-40/S100 is a useful step that inclusion should be recommended in basic evaluation parameters for cutaneous melanoma.


Assuntos
Melanócitos/patologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/imunologia , Antígenos CD34/imunologia , Biomarcadores Tumorais/metabolismo , Criança , Endotélio Vascular/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Metástase Linfática/patologia , Vasos Linfáticos/patologia , Masculino , Melanoma/metabolismo , Melanoma/mortalidade , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Estudos Retrospectivos , Fatores de Risco , Proteínas S100/imunologia , Linfonodo Sentinela/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/mortalidade , Adulto Jovem
10.
J Exp Med ; 218(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33211088

RESUMO

SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.


Assuntos
Anticorpos Monoclonais Murinos , Anticorpos Neutralizantes , Anticorpos Antivirais , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , COVID-19 , Linhagem Celular , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Feminino , Humanos , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , SARS-CoV-2
11.
J Mol Cell Biol ; 12(12): 980-986, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33377928

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide severe coronavirus disease 2019 (COVID-19) pandemic since December 2019. There is a great demand for effective therapies for the prevention and treatment of COVID-19. Developing therapeutic neutralizing antibodies (NAbs), which could block viral infection, is such a promising approach, as NAbs have been successfully applied to the treatment of other viral infections. The recent advances of antibody technology have greatly accelerated the discovery of SARS-CoV-2 NAbs, and many of which are now actively tested in clinical trials. Here, we review the approaches applied for SARS-CoV-2 NAb development, and discuss the emerging technologies underlining the antibody discovery. We further summarize the common features of these antibodies including the shared neutralizing epitopes and sequence features.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/isolamento & purificação , Anticorpos Monoclonais Murinos/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Diversidade de Anticorpos , COVID-19/virologia , Descoberta de Drogas , Epitopos/química , Epitopos/imunologia , Humanos , Camundongos , Modelos Moleculares , Pandemias , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
12.
MAbs ; 12(1): 1850394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33323006

RESUMO

Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular traps (NETs), are involved in multiple pathological processes, and NET formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity toward intact NS but not to individual NS components, indicating that 2C5 could potentially target NS in NETs. In this study, NETs were generated in vitro using neutrophils and HL-60 cells differentiated into granulocyte-like cells. The specificity of 2C5 toward NETs was evaluated by ELISA, which showed that it binds to NETs with the specificity similar to that for purified nucleohistone substrate. Immunofluorescence showed that 2C5 stains NETs in both static and perfused microfluidic cell cultures, even after NET compaction. Modification of liposomes with 2C5 dramatically enhanced liposome association with NETs. Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies.


Assuntos
Anticorpos Monoclonais Murinos , Especificidade de Anticorpos , Armadilhas Extracelulares , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Armadilhas Extracelulares/química , Armadilhas Extracelulares/imunologia , Células HL-60 , Humanos , Camundongos , Camundongos Endogâmicos BALB C
13.
J Immunol ; 205(12): 3348-3357, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33177161

RESUMO

Relative control of HIV-1 infection has been linked to genetic and immune host factors. In this study, we analyzed 96 plasma proteome arrays from chronic untreated HIV-1-infected individuals using the classificatory random forest approach to discriminate between uncontrolled disease (plasma viral load [pVL] >50,000 RNA copies/ml; CD4 counts 283 cells/mm3, n = 47) and relatively controlled disease (pVL <10,000 RNA copies/ml; CD4 counts 657 cells/mm3, n = 49). Our analysis highlighted the TNF molecule's relevance, in particular, TL1A (TNFSF15) and its cognate DR3 (TNFSRF25), both of which increased in the relative virus control phenotype. DR3 levels (in plasma and PBMCs) were validated in unrelated cohorts (including long-term nonprogressors), thus confirming their independence from CD4 counts and pVL. Further analysis in combined antiretroviral treatment (cART)-treated individuals with a wide range of CD4 counts (137-1835 cells/mm3) indicated that neither TL1A nor DR3 levels reflected recovery of CD4 counts with cART. Interestingly, in cART-treated individuals, plasma TL1A levels correlated with regulatory T cell frequencies, whereas soluble DR3 was strongly associated with the abundance of effector HLA-DR+CD8+ T cells. A positive correlation was also observed between plasma DR3 levels and the HIV-1-specific T cell responses. In vitro, costimulation of PBMC with DR3-specific mAb increased the magnitude of HIV-1-specific responses. Finally, in splenocytes of DNA.HTI-vaccinated mice, costimulation of HTI peptides and a DR3 agonist (4C12) intensified the magnitude of T cell responses by 27%. These data describe the role of the TL1A-DR3 axis in the natural control of HIV-1 infection and point to the use of DR3 agonists in HIV-1 vaccine regimens.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Feminino , Infecções por HIV/sangue , HIV-1/metabolismo , Humanos , Masculino , Camundongos , Membro 25 de Receptores de Fatores de Necrose Tumoral/sangue , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue
14.
PLoS Pathog ; 16(8): e1008753, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866207

RESUMO

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.


Assuntos
Vacinas contra a AIDS/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Monoclonais Murinos/imunologia , Epitopos/genética , Anticorpos Anti-HIV/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Macaca mulatta , Multimerização Proteica/genética , Multimerização Proteica/imunologia
15.
MAbs ; 12(1): 1802187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32835602

RESUMO

SERINC5 is a multi-pass transmembrane protein that is thought to play a role in serine incorporation during cellular membrane biosynthesis. This protein has also been identified as a human immunodeficiency virus Type 1 (HIV-1) restriction factor. The paucity of monoclonal antibodies (mAbs) against SERINC5 has posed a challenge for the study of the endogenous protein. Here we report the development of novel anti-SERINC5 mAbs that target three distinct loops on the protein. We demonstrate that these SERINC5 mAbs can be used to detect endogenously expressed SERINC5 protein in various cell lines using Western blot, whole-cell ELISA, flow cytometry, and immunocytochemistry. We further show that some of these antibodies can detect SERINC5 that is present in HIV-1 viral stocks. These antibodies will aid in the characterization of the functions and mechanisms of action of SERINC5 in different cell types.


Assuntos
Anticorpos Monoclonais Murinos/química , HIV-1/imunologia , Proteínas de Membrana/imunologia , Vírion/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C
16.
Biochem J ; 477(17): 3219-3235, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32789497

RESUMO

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.


Assuntos
Anticorpos Monoclonais Murinos , Antineoplásicos Imunológicos , Fragmentos Fab das Imunoglobulinas , Antígenos do Grupo Sanguíneo de Lewis , Antígenos CD15 , Simulação de Acoplamento Molecular , Neoplasias , Oligossacarídeos , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Antígenos CD15/química , Antígenos CD15/imunologia , Camundongos , Neoplasias/química , Neoplasias/imunologia , Oligossacarídeos/química , Oligossacarídeos/imunologia
17.
Protein Expr Purif ; 174: 105682, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32502709

RESUMO

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.


Assuntos
Anticorpos Monoclonais Murinos , Anticorpos Antivirais , Epitopos , Infecções por Parvoviridae , Parvovirus Canino , Proteínas não Estruturais Virais , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linhagem Celular , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/metabolismo , Parvovirus Canino/química , Parvovirus Canino/genética , Parvovirus Canino/imunologia , Parvovirus Canino/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo
18.
Int J Biol Macromol ; 162: 490-500, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32574737

RESUMO

Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas de Artrópodes , Epitopos , Metaloendopeptidases , Diester Fosfórico Hidrolases , Venenos de Aranha , Aranhas , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Engenharia de Proteínas , Venenos de Aranha/química , Venenos de Aranha/genética , Venenos de Aranha/imunologia
19.
Immunohorizons ; 4(4): 153-164, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32276922

RESUMO

SLAM-associated protein (SAP) is an adaptor molecule that facilitates critical effector functions in immune cells, and its deficiency causes X-linked lymphoproliferative disease type 1 in which effector responses directed against EBV are severely compromised. The primary objective of this study was to phenotypically and functionally characterize a rare, CD8 T cell-restricted bimodal SAP expression pattern observed in healthy, human donors with the widely used 1C9-SAP mAb clone. We initially observed this pattern during the clinical validation of our flow cytometry-based assay to diagnose X-linked lymphoproliferative disease type 1 in our laboratory. For this validation study, we used multiparameter flow cytometry to identify cytosolic SAP expression in lymphocyte subsets, and CD8 T cells from the donors displaying the rare SAP expression pattern mentioned above were separately further evaluated by intracellular cytokine and CD107a staining to examine polyfunctionality following PMA/ionomycin and HLA class I allele-restricted EBV peptide epitope-induced T cell activation. Our data revealed that SAP 1C9-hi CD8 T cells clearly displayed higher polyfunctional responses versus SAP 1C9-lo CD8 T cells following PMA/ionomycin stimulation. Furthermore, polyfunctional EBV-specific CD8 T cell responses segregated with the SAP 1C9-hi CD8 T cells and not the SAP 1C9-lo CD8 T cells. Additionally, and rather intriguingly, short- and long-term T cell stimulation selectively diminished the signal for the 1C9-hi subset. Overall, our data suggest that although rare, this unique SAP expression pattern merits further evaluation as it has the potential to provide some insight into fundamental processes as they might relate to host-pathogen dynamics.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fenótipo , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/imunologia , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/metabolismo , Adulto , Doadores de Sangue , Células Cultivadas , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/farmacologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Citometria de Fluxo , Herpesvirus Humano 4/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina G/imunologia , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Acetato de Tetradecanoilforbol/farmacologia
20.
Clin Cancer Res ; 26(14): 3694-3706, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32273277

RESUMO

PURPOSE: The murine Lym-1 mAb targets a discontinuous epitope (Lym-1 epitope) on several subtypes of HLA-DR, which is upregulated in a majority of human B-cell lymphomas and leukemias. Unlike CD19, the Lym-1 epitope does not downregulate upon crosslinking, which may provide an advantage as a target for CAR T-cell therapy. Lym-1 CAR T cells with a conventional 4-1BB and CD3ζ (BB3z) signaling domain exhibited impaired ex vivo expansion. This study aimed to identify the underlying mechanisms and develop strategies to overcome this effect. EXPERIMENTAL DESIGN: A functional humanized Lym-1 antibody (huLym-1-B) was identified and its scFv form was used for CAR design. To overcome observed impaired expansion in vitro, a huLym-1-B CAR using DAP10 and DAP12 (DAP) signaling domains was evaluated for ex vivo expansion and in vivo function. RESULTS: Impaired expansion in huLym-1-B-BB3z CAR T cells was shown to be due to ligand-dependent suboptimal CAR signaling caused by interaction of the CAR binding domain and the surface of human T cells. Using the novel DAP signaling domain construct, the effects of suboptimal CAR signaling were overcome to produce huLym-1-B CAR T cells with improved expansion ex vivo and function in vivo. In addition, the Lym-1 epitope does not significantly downregulate in response to huLym-1-B-DAP CAR T cells both ex vivo and in vivo. CONCLUSIONS: DAP intracellular domains can serve as signaling motifs for CAR, and this new construct enables nonimpaired production of huLym-1-B CAR T cells with potent in vivo antitumor efficacy.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Imunoterapia Adotiva/métodos , Linfoma de Células B/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Murinos/genética , Linhagem Celular Tumoral , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Domínios Proteicos/genética , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...