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1.
J Agric Food Chem ; 67(45): 12547-12557, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31613616

RESUMO

This study aims at understanding the changes in the structural properties of Gly m 4 soy allergen as a result of the influence of the temperature and pressure deviations. The primary emphasis was placed on analyzing the surface properties of suspected linear and conformational epitopes present in the Gly m 4 allergen. All three epitopes of Gly m 4 were studied, and the results showed that the molecule has significant structural changes in terms of solvent-accessible surface area (SASA) and radius of gyration, which showed that the increased pressures resulted in compaction. However, at lower temperatures and higher pressures (300 K and 6 kbar), swelling in the molecule was observed with a significant increase in the surface area. The study also tracked the changes in surface areas of individual residues that are part of the selected epitopes. Residues, such as D-27 and T-51, were found to have significant changes in their SASA as a result of temperature and pressure deviations.


Assuntos
Alérgenos/química , Antígenos de Plantas/química , Soja/imunologia , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Mapeamento de Epitopos , Modelos Moleculares , Pressão , Soja/química , Temperatura Ambiente
2.
Chemistry ; 25(61): 13945-13955, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31404475

RESUMO

The human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N-acetylgalactosamine (GalNAc) containing structures present on pathogens, self-glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc-containing structures derived from the blood group A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of methyl α-GalNAc (α-MeGalNAc), a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood group A antigen, the Forssman antigen, or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.


Assuntos
Acetilgalactosamina/química , Lectinas Tipo C/metabolismo , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Mapeamento de Epitopos , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/genética , Ligantes , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
3.
Adv Exp Med Biol ; 1140: 377-388, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347059

RESUMO

Identifying antigen-antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen-antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen-antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.


Assuntos
Complexo Antígeno-Anticorpo/análise , Mapeamento de Epitopos , Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Antígenos de Neoplasias/análise , Epitopos , Humanos
4.
Nat Commun ; 10(1): 3068, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296843

RESUMO

Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/sangue , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Linhagem Celular Tumoral , Cercopithecus aethiops , Infecções por Coronavirus/sangue , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Testes de Neutralização , Ligação Proteica/imunologia , Domínios Proteicos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Células Vero , Internalização do Vírus
5.
Molecules ; 24(11)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159327

RESUMO

Food allergies originate from adverse immune reactions to some food components. Ingestion of food allergens can cause effects of varying severity, from mild itching to severe anaphylaxis reactions. Currently there are no clues to predict the allergenic potency of a molecule, nor are cures for food allergies available. Cutting-edge research on allergens is aimed at increasing information on their diffusion and understanding structure-allergenicity relationships. In this context, purified recombinant allergens are valuable tools for advances in the diagnostic and immunotherapeutic fields. Chitinases are a group of allergens often found in plant fruits, but also identified in edible insects. They are classified into different families and classes for which structural analyses and identification of epitopes have been only partially carried out. Moreover, also their presence in common allergen databases is not complete. In this review we provide a summary of the identified food allergenic chitinases, their main structural characteristics, and a clear division in the different classes.


Assuntos
Alérgenos/imunologia , Quitinases/imunologia , Hipersensibilidade Alimentar/imunologia , Alérgenos/química , Alérgenos/classificação , Animais , Quitinases/química , Reações Cruzadas/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Humanos , Imunoglobulina E/imunologia , Insetos/química , Insetos/enzimologia , Insetos/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Relação Estrutura-Atividade
6.
Nat Commun ; 10(1): 2727, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227708

RESUMO

A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.


Assuntos
Anticorpos Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Cromatografia de Afinidade , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Mapeamento de Epitopos , Voluntários Saudáveis , Humanos , Espectrometria de Massas , Proteínas Opsonizantes/imunologia , Proteínas Opsonizantes/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/imunologia , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo
7.
Arch Virol ; 164(9): 2285-2295, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250104

RESUMO

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.


Assuntos
Doença Nodular Cutânea/virologia , Vírus da Doença Nodular Cutânea/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Mapeamento de Epitopos , Dosagem de Genes , Doença Nodular Cutânea/diagnóstico , Vírus da Doença Nodular Cutânea/química , Vírus da Doença Nodular Cutânea/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética
8.
Virol J ; 16(1): 75, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159841

RESUMO

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Parvovirus Suíno/imunologia , Animais , Antígenos Virais/química , Mapeamento de Epitopos , Epitopos/química , Masculino , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologia , Suínos
9.
PLoS Pathog ; 15(5): e1007772, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31100098

RESUMO

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.


Assuntos
Anticorpos Neutralizantes/imunologia , Desenho de Drogas , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Genótipo , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Masculino , Testes de Neutralização , Estudos Prospectivos , Homologia de Sequência , Adulto Jovem
10.
Mol Immunol ; 111: 128-135, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31054406

RESUMO

The main challenge in the development of antibody is to select the appropriate antigen particularly when a truncated protein is used for immunization or as vaccine antigen. In previous studies, fragment selection was mainly based on epitopes and less often on the structure. Fewer studies have paid attention to the prediction of the truncated protein 3D structure and retained its similarity in the native and truncated proteins. Here we used in silico analysis to select two fragments of Pyruvate Kinase M2 (PKM2), as a tumor marker. One fragment, M-tPKM2, had a shorter sequence with one epitope although the predicted 3D structure was similar to the native PKM2. The other fragment, R-tPKM2, had a longer sequence and thus more epitopes, but had a different structure from the native PKM2. Recombinant truncated proteins were expressed in E. coli and purified via affinity chromatography. Secondary structure elements in purified proteins were determined by Circular Dichroism, then they were utilized to develop antibodies in mice. Both antigens could elicit high immune response against themselves (OD450 = 3.326 ± 0.562 for M-tPKM2; OD450 = 3.562 ± 0.110 for R-tPKM2). However, significantly higher response against PKM2 was observed among the mice immunized with M-tPKM2 (p < 0.0001 by One way ANOVA followed by Tukey's post hoc comparison). Also, the monoclonal antibody produced against the M-tPKM2 could recognize the native PKM2 in the MCF7 cells. Our finding suggested that for the purpose of designing an antigen with the ability to produce a potent antibody against the target protein, it is better to select sequences which have a similar structure in truncated and native proteins, even at the cost of having shorter sequences and fewer epitopes.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Epitopos/imunologia , Animais , Linhagem Celular Tumoral , Mapeamento de Epitopos/métodos , Escherichia coli/imunologia , Feminino , Humanos , Imunização/métodos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Piruvato Quinase/imunologia , Proteínas Recombinantes/imunologia
11.
Emerg Microbes Infect ; 8(1): 749-759, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130109

RESUMO

The Zika virus (ZIKV) outbreak and its link to microcephaly triggered a public health concern. To examine antibody response in a patient infected with ZIKV, we used single-cell PCR to clone 31 heavy and light chain-paired monoclonal antibodies (mAbs) that bind to ZIKV envelope (E) proteins isolated from memory B cells of a ZIKV-infected patient. Three mAbs (7B3, 1C11, and 6A6) that showed the most potent and broad neutralization activities against the African, Asian, and American strains were selected for further analysis. mAb 7B3 showed an IC50 value of 11.6 ng/mL against the circulating American strain GZ02. Epitope mapping revealed that mAbs 7B3 and 1C11 targeted residue K394 of the lateral ridge (LR) epitope of the EDIII domain, but 7B3 has a broader LR epitope footprint and recognizes residues T335, G337, E370, and N371 as well. mAb 6A6 recognized residues D67, K118, and K251 of the EDII domain. Interestingly, although the patient was seronegative for DENV infection, mAb 1C11, originating from the VH3-23 and VK1-5 germline pair, neutralized both ZIKV and DENV1. Administration of the mAbs 7B3, 1C11, and 6A6 protected neonatal SCID mice infected with a lethal dose of ZIKV. This study provides potential therapeutic antibody candidates and insights into the antibody response after ZIKV infection.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Imunização Passiva , Proteínas do Envelope Viral/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Adulto , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , China , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/isolamento & purificação , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos SCID , Testes de Neutralização , Análise de Sobrevida , Resultado do Tratamento , Infecção por Zika virus/imunologia
12.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083520

RESUMO

Alpha-synuclein is considered the major pathological protein associated with Parkinson's disease, but there is still no effective immunotherapy which targets alpha-synuclein. In order to create a safer and more effective therapy against PD, we are targeting an epitope of alpha-synuclein rather than full-length alpha-synuclein. We have selected several antigenic domains (B-cell epitope) through antigenicity prediction, and also made several recombinant protein fragments from alpha-synuclein upon antigenicity prediction in an E. coli system. We then tested the function of each of the peptides and recombinant fragments in aggregation, their toxicity and antigenicity. We have discovered that the full-length recombinant (aa1-140) can aggregate into oligomers or even fibrils, and fragment aa15-65 can promote the aggregation of aa1-140. It is worth noting that it not only promotes whole protein aggregation, but also self-aggregates as seen by western blotting and silver staining assays. We have tested all candidates on primary neurons for their toxicity and discovered that aa15-65 is the most toxic domain compared to all other fragments. The antibody targeting this domain also showed both anti-aggregation activity and some therapeutic effect. Therefore, we believe that we have identified the most potent therapeutic domain of alpha synuclein as a therapeutic target.


Assuntos
Doença de Parkinson/tratamento farmacológico , alfa-Sinucleína/química , alfa-Sinucleína/uso terapêutico , Animais , Anticorpos/metabolismo , Mapeamento de Epitopos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/toxicidade , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade
13.
Nat Commun ; 10(1): 2073, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061402

RESUMO

Isolation of broadly neutralizing human monoclonal antibodies (HmAbs) targeting the E2 glycoprotein of Hepatitis C virus (HCV) has sparked hope for effective vaccine development. Nonetheless, escape mutations have been reported. Ideally, a potent vaccine should elicit HmAbs that target regions of E2 that are most difficult to escape. Here, aimed at addressing this challenge, we develop a predictive in-silico evolutionary model for E2 that identifies one such region, a specific antigenic domain, making it an attractive target for a robust antibody response. Specific broadly neutralizing HmAbs that appear difficult to escape from are also identified. By providing a framework for identifying vulnerable regions of E2 and for assessing the potency of specific antibodies, our results can aid the rational design of an effective prophylactic HCV vaccine.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Proteínas do Envelope Viral/imunologia , Simulação por Computador , Desenho de Drogas , Mapeamento de Epitopos/métodos , Epitopos/genética , Epitopos/imunologia , Evolução Molecular , Hepacivirus/genética , Hepatite C/prevenção & controle , Hepatite C/virologia , Humanos , Modelos Biológicos , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/imunologia
14.
Parasitol Res ; 118(7): 2263-2270, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089811

RESUMO

Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA's and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox.


Assuntos
Antígenos de Helmintos/imunologia , Epitopos/imunologia , Filariose/diagnóstico , Onchocerca volvulus/imunologia , Oncocercose/diagnóstico , Peptídeos/imunologia , Adulto , Idoso , Animais , Brugia Malayi/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Feminino , Filariose/parasitologia , Humanos , Masculino , Pessoa de Meia-Idade , Oncocercose/parasitologia , Proteoma , Sensibilidade e Especificidade , Testes Sorológicos , Adulto Jovem
15.
J Immunol Res ; 2019: 9804584, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019982

RESUMO

The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.


Assuntos
Anticorpos Neutralizantes/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Animais , Anticorpos Monoclonais/imunologia , Bacteriófagos/imunologia , HIV-1 , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Simulação de Acoplamento Molecular , Testes de Neutralização , Peptídeos/administração & dosagem , Peptídeos/imunologia , Ligação Proteica/efeitos dos fármacos
16.
Virol Sin ; 34(3): 306-314, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31020574

RESUMO

Previous studies have indicated that two monoclonal antibodies (mAbs; A1-10 and H1-84) of the hemagglutinin (HA) antigen on the H1N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue (Guo et al. in Immunobiology 220:941-946, 2015). However, characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2 (LVLWGIHHP191-199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.


Assuntos
Antígenos Heterófilos/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Peptídeos/imunologia , Software
17.
Virus Res ; 266: 34-42, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30965063

RESUMO

The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499-638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV.


Assuntos
Epitopos de Linfócito B/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Cercopithecus aethiops , Sequência Conservada , Infecções por Coronavirus/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Feminino , Células HEK293 , Humanos , Imunização , Camundongos Endogâmicos BALB C , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
18.
Monoclon Antib Immunodiagn Immunother ; 38(2): 79-84, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30939066

RESUMO

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Cavalos , Camundongos
19.
Int J Mol Sci ; 20(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003530

RESUMO

The mosquito-borne viral disease caused by the Dengue virus is an expanding global threat. Diagnosis in low-resource-settings and epidemiological surveillance urgently requires new immunoprobes for serological tests. Structure-based epitope prediction is an efficient method to design diagnostic peptidic probes able to reveal specific antibodies elicited in response to infections in patients' sera. In this study, we focused on the Dengue viral envelope protein (E); computational analyses ranging from extensive Molecular Dynamics (MD) simulations and energy-decomposition-based prediction of potentially immunoreactive regions identified putative epitope sequences. Interestingly, one such epitope showed internal dynamic and energetic properties markedly different from those of other predicted sequences. The epitope was thus synthesized as a linear peptide, modified for chemoselective immobilization on microarrays and used in a serological assay to discriminate Dengue-infected individuals from healthy controls. The synthetic epitope probe showed a diagnostic performance comparable to that of the full antigen in terms of specificity and sensitivity. Given the high level of sequence identity among different flaviviruses, the epitope was immune-reactive towards Zika-infected sera as well. The results are discussed in the context of the quest for new possible structure-dynamics-based rules for the prediction of the immunoreactivity of selected antigenic regions with potential pan-flavivirus immunodiagnostic capacity.


Assuntos
Vírus da Dengue/imunologia , Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais , Biologia Computacional , Reações Cruzadas/imunologia , Dengue/sangue , Dengue/virologia , Vírus da Dengue/patogenicidade , Mapeamento de Epitopos , Humanos , Simulação de Dinâmica Molecular , Peptídeos/imunologia , Zika virus/imunologia , Zika virus/patogenicidade , Infecção por Zika virus/sangue , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
20.
Nat Commun ; 10(1): 1842, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015529

RESUMO

The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteína 9 Associada à CRISPR/imunologia , Epitopos de Linfócito T/genética , Mutagênese/imunologia , Streptococcus pyogenes/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Células Apresentadoras de Antígenos/imunologia , Proteína 9 Associada à CRISPR/genética , Engenharia Celular/métodos , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/imunologia , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Células HEK293 , Antígenos HLA-A/imunologia , Voluntários Saudáveis , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Medicina de Precisão/efeitos adversos , Medicina de Precisão/métodos , Streptococcus pyogenes/genética
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