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1.
Nat Commun ; 11(1): 2251, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366817

RESUMO

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Afinidade de Anticorpos/imunologia , Betacoronavirus/química , Betacoronavirus/efeitos dos fármacos , Chlorocebus aethiops , Sequência Conservada , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/imunologia , Receptores Virais/química , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
2.
Mol Immunol ; 120: 146-163, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32126449

RESUMO

Buruli ulcer is an emerging tissue-necrosis infectious disease, caused by the pathogen Mycobacterium ulcerans, leading to permanent deformity if untreated. Despite this debilitating condition, no specific disease-modifying therapeutics or vaccination is available to date. Therefore, we aimed to design an effective multi-epitope vaccine against M. ulcerans using vaccinomics approach. Briefly, the highest antigenic PE-PGRS protein was selected from which the promiscuous T- and B-cell epitopes were predicted. After rigorous assessment, 15 promising T- and B-cell epitopes were selected. The identified T-cell epitopes showed marked interactions towards their HLA-binding alleles and provided 99.8 % world population coverage. Consequently, a vaccine chimera was designed by connecting these epitopes with suitable linkers and LprG adjuvant. The vaccine construct was highly antigenic, immunogenic and non-allergenic; hence, subjected to homology modelling. The molecular docking and dynamics simulation revealed a strong and stable interaction between vaccine and toll-like receptor 2. The binding energy and dissociation constant were -15.3 kcal/mol and 5.9 × 10-12 M, respectively. The computer-simulated immune responses showed abundance of immunoglobulins, increased interferon-γ production, and macrophages activation which are crucial for immune response against M. ulcerans. Furthermore, disulfide bridging and in silico cloning were also performed. These results suggest that the vaccine, if validated experimentally, will be a promising candidate against M. ulcerans and prevent Buruli ulcer disease.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Mycobacterium ulcerans/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Úlcera de Buruli/imunologia , Úlcera de Buruli/prevenção & controle , Simulação por Computador , Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Proteínas de Membrana/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium ulcerans/química , Mycobacterium ulcerans/genética , Engenharia de Proteínas , Vacinas de Subunidades/química , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
3.
Nature ; 574(7779): 565-570, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645726

RESUMO

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.


Assuntos
Antígenos B7/química , Antígenos B7/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos B7/antagonistas & inibidores , Antígenos B7/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cristalografia por Raios X , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Linfócitos T/citologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
4.
Iran J Allergy Asthma Immunol ; 18(3): 269-280, 2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31522434

RESUMO

Dickkopf (DKK) family of proteins are known as antagonists for the Wnt-ß-catenin signaling pathway. It is suggested that the Dickkopf-1 (DKK-1) has a role in several diseases such as hepatocellular carcinomas, hepatoblastomas, Wilms' tumors, lung cancer and Myeloma bone disease. The aim of the present study was to produce a chimeric-recombinant DKK-1 protein in order to induce immune response against the antigen. The recombinant Dickkopf-1 (rDKK-1) protein was designed using bioinformatics analysis. The standard methods were used for cloning, expression and purification. The structure of recombinant protein was analyzed by spectroscopy methods. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were performed to confirm the recombinant protein using a commercial anti-DKK-1 (whole protein) polyclonal antibody. The immunogenicity of the recombinant DKK-1 was assessed by immunizing, intraperitoneally, BALB/C mice four times with the 31-kDa and 45-kDa purified rDKK-1 cloned in pET28a and pET32a vectors respectively. The antibody titer was measured in due course of time. Stronger immunogenic parts of the protein were selected based on in-silico predictions and recombinant protein was successfully designed. The chimeric gene was sub-cloned, expressed, purified and refolded. The purified protein was confirmed by Western blotting and ELISA. The three dimensional structural was confirmed by CD spectrum and predicted structures by bioinformatics tools, revealed the stability of helix structures. rDKK-1 protein was capable of inducing immune response with high titer antibody and  excessive humoral immune response. No significant difference was observed between immunization by 31-kDa and 45-kDa antigen.


Assuntos
Antígenos/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Modelos Biológicos , Conformação Proteica , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Antígenos/genética , Antígenos/imunologia , Clonagem Molecular , Biologia Computacional/métodos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Análise Espectral
5.
Iran J Allergy Asthma Immunol ; 18(4): 427-440, 2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31522451

RESUMO

Interferonbeta-1b (IFNß-1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Long-term usage of this protein can lead to the development of anti-drug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFNß-1b. The aim of this study was to predict and silence IFNß-1b T-cells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating T-cells epitopes in IFNß-1b protein. Four synthetic genes with desirable mutation constructed and PET26b+ was used as an expression vector in E. coli. The expression of this proteins confirmed by SDS-PAGE and Western blotting, consequently, IFNß-1b proteins was purified by His-tag chromatography. To determined activity of mutants' variants anti-proliferative and anti-viral activity compared to wild form was evaluated using MTT assay in A549 and Vero cells lines respectively. Also the immunogenicity of mutant proteins compared with Betaseron measured in BALB/c mice. The in vitro bioactivity analysis demonstrated that functional activities of all mutant proteins were maintained and is the same as biological activity of Betaseron. Pharmacokinetic studies suggest that, in engineered proteins that contain substitution of Histidine to Glutamic Acid at position 131 (mut 2 and mut 1+2) antibodies response reduced by about 50%, as compared to that for Betaseron. Computational analysis expedites identification and prediction of epitopes in therapeutic protein, therefore, we used immunoinformatic tools for modification of dominant T-cell epitope in IFNß-1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.


Assuntos
Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Inativação Gênica , Interferon beta-1b/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Descoberta de Drogas , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Feminino , Humanos , Imunização , Camundongos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade
6.
Mol Immunol ; 114: 643-650, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31546099

RESUMO

Peptide vaccines have many potential advantages over conventional ones including low cost, lack of need for cold-chain storage, safety and specificity. However, it is well known that approximately 90% of B-cell epitopes (BCEs) are discontinuous in nature making it difficult to mimic them for creating vaccines. In this study, the degree of discontinuity in B-cell epitopes and their conformational nature is examined. The discontinuity of B-cell epitopes is analyzed by defining 'regions' (consisting of at least three antibody-contacting residues each separated by ≤3 residues) and small fragments (antibody-contacting residues that do not satisfy the requirements for a region). Secondly, an algorithm has been developed that classifies each region's shape as straight, curved or folded on the basis that straight and folded regions are more likely to retain their native conformation as isolated peptides. We have investigated the structures of 488 B-cell epitopes from which 1282 regions and 1018 fragments have been identified. 90% of epitopes have five or fewer regions and five or fewer fragments with 14% containing only one region and 4% being truly linear (i.e. having one region and no fragments). Of the 1282 regions, 508 are straight in shape, 626 are curved and 148 are folded.


Assuntos
Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Anticorpos/química , Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Conformação Proteica
7.
Mol Biol Rep ; 46(5): 4751-4761, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31290058

RESUMO

Immunogenicity of therapeutic proteins is one of the main challenges in disease treatment. L-Asparaginase is an important enzyme in cancer treatment which sometimes leads to undesirable side effects such as immunogenic or allergic responses. Here, to decrease Erwinase (Erwinia chrysanthemiL-Asparaginase) immunogenicity, which is the main drawback of the enzyme, firstly conformational B cell epitopes of Erwinase were predicted from three-dimensional structure by three different computational methods. A few residues were defined as candidates for reducing immunogenicity of the protein by point mutation. In addition to immunogenicity and hydrophobicity, stability and binding energy of mutants were also analyzed computationally. In order to evaluate the stability of the best mutant, molecular dynamics simulation was performed. Among mutants, H240A and Q239A presented significant reduction in immunogenicity. In contrast, the immunogenicity scores of D235A slightly decreased according to two servers. Binding affinity of substrate to the active site reduced significantly in K265A and E268A. The final results of molecular dynamics simulation indicated that H240A mutation has not changed the stability, flexibility, and the total structure of desired protein. Overall, point mutation can be used for reducing immunogenicity of therapeutic proteins, in this context, in silico approaches can be used to screen suitable mutants.


Assuntos
Asparaginase/imunologia , Pectobacterium chrysanthemi/enzimologia , Pectobacterium chrysanthemi/imunologia , Engenharia de Proteínas , Asparaginase/química , Asparaginase/genética , Biologia Computacional/métodos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Pectobacterium chrysanthemi/genética , Estabilidade Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade
8.
Diagn Microbiol Infect Dis ; 95(2): 125-130, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31182246

RESUMO

Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum. Indirect immunofluorescence assay (IFA) is generally used for HGA serodiagnosis. A. phagocytophilum immunodominant P44 major outer membrane proteins are encoded by p44/msp2 multigene family, responsible for IFA reactivity. However, because multiple P44-related proteins may involve immunoreactivity in IFA, the available diagnostic antigens remain obscure. In this study, we identified 12 B-cell epitopes on triple P44-related proteins using peptide array that reacted with 4 HGA patients' sera. Then, peptide spot immunoassay using 14 synthetic peptides derived from those 12 epitopes as antigens was applied for the detection of antibody to A. phagocytophilum from patients with fever of unknown origin. The sensitivities and diagnostic efficiencies of this immunoassay were higher than those of Western blot analysis using 3 recombinant proteins previously developed. Thus, the immunoassay using our epitope-derived antigens, which has higher diagnostic performances, may have significant benefit for HGA serodiagnosis.


Assuntos
Anaplasma phagocytophilum/imunologia , Anaplasmose/diagnóstico , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/química , Epitopos de Linfócito B/imunologia , Imunoensaio/métodos , Sequência de Aminoácidos , Anaplasma phagocytophilum/isolamento & purificação , Anaplasmose/sangue , Anaplasmose/microbiologia , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Epitopos de Linfócito B/química , Humanos , Sensibilidade e Especificidade , Testes Sorológicos
9.
Appl Microbiol Biotechnol ; 103(18): 7467-7480, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31253999

RESUMO

Bovine enterovirus (BEV) VP2 protein is a structural protein that plays an important role in inducing protective immunity in the host. The function of VP2 has been characterized, but there is little information on its B cell epitopes. Three monoclonal antibodies (mAbs) directed against BEV VP2 were generated and characterized from mice immunized with the recombinant VP2 protein. Three minimal linear epitopes 152FQEAFWLEDG161, 168LIYPHQ173, and 46DATSVD51 reactive to the three mAbs were identified using western blotting analysis. Three-dimensional model of the BEV-E virion and the VP2 monomer showed that epitope 152FQEAFWLEDG161 is exposed on surface of the virion and epitopes 46DATSVD51 and 168LIYPHQ173 are located inside the virion. Alignment of the amino acid sequences corresponding to the regions containing the three minimal linear epitopes in the VP2 proteins and their cross-reactivity with the three mAbs showed that epitope 168LIYPHQ173 is completely conserved in all BEV strains. Epitope 46DATSVD51 is highly conserved among BEV-E strains and partly conserved among BEV-F strains. However, epitope 152FQEAFWLEDG161 is not conserved among BEV-F strains. Using the mAbs of 3H4 and 1E10, we found that VP2 localized in the cytoplasm during viral replication and could be used to monitor the viral antigen in infected tissues using immunohistochemistry. A preliminary 3H4-epitope-based indirect ELISA allowed us to detect anti-BEV-strain-HY12 antibodies in mice. This study indicates that the three mAbs could be useful tools for investigating the structure and function of the viral VP2 protein and the development of serological diagnostic techniques for BEV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Bovino/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Bovinos , Epitopos de Linfócito B/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Homologia de Sequência
10.
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 , 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
11.
Arch Biochem Biophys ; 664: 127-133, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30742802

RESUMO

In order to establish structure-function relationship for the design of a new group of oligopeptide antigen-macromolecule conjugate, multiple copies of mucin-1 B-cell epitope peptide, APDTRPAPG were conjugated with branched chain polymeric polypeptides possessing poly[L-Lys] backbone. By the synthesis, radiolabeling (125I) and in vivo treatment of BALB/c mice with epitope conjugates containing XiK/XAK type carrier, where X = Glu (EiK or EAK) or Leu (LAK), the influence of the polypeptide structure on the blood clearance profile and on tissue distribution profile concerning the epitope delivery to relevant organs (e.g. immunocompetent or involved in excretion) were investigated. We observed significant differences in the blood clearance profiles for the conjugates, the respective polypeptide carriers and free epitope peptide. All conjugates, regardless of their charge properties exhibited longer presence in the circulation than the free oligopeptide. Tissue distribution data also showed that the structural properties (e.g. amino acid composition, charge) of the carrier polypeptide have marked influence on the tissue accumulation of the epitope peptide conjugates. In contrast to conjugates with linear (K) or branched chain (LAK) polycationic polymers exhibiting rapid blood clearance and high spleen/liver uptake, amphoteric epitope peptide conjugates with different branches, but similar charge properties (EiK or EAK) had extended blood survival and generally lower tissue accumulation. The results on this systematic investigation suggest that further studies on the immune response induced by these epitope conjugates would be needed to provide correlation between biodistribution properties (presence in the blood, level of tissue accumulation) and the capacity of these conjugates to elicit antibody production.


Assuntos
Epitopos de Linfócito B/metabolismo , Mucina-1/metabolismo , Oligopeptídeos/metabolismo , Animais , Área Sob a Curva , Epitopos de Linfócito B/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/química , Distribuição Tecidual
12.
Appl Microbiol Biotechnol ; 103(6): 2689-2699, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30729288

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is leading to huge losses in the swine industry worldwide. Its nonstructural protein 2 (Nsp2), with a cysteine protease domain (PL2), is crucial for virus replication and as a trigger to host innate immune regulation. In this study, three monoclonal antibodies (mAbs) to Nsp2, designated 4A12, 4G8, and 8H11, were generated. Subsequently, a sequence of recombinant peptides with partial overlap was utilized to determine the epitopes using these mAbs. We found three novel minimal linear Nsp2 B cell epitopes, 188ELSDDSNRPV197, 42HLKRYSPPAE51, and 54CGWHCISA61, which were identified by the antibodies 4A12, 4G8, and 8H11, respectively. Structure analysis indicates that 42HLKRYSPPAE51 and 188ELSDDSNRPV197 are located separately in hypervariable region 1 and hypervariable region 2 of Nsp2. Interestingly, 54CGWHCISA61 is located in the PL2 region, which is highly conserved in all arteriviruses, particularly at the expected conserved catalytic site at Cys54. Importantly, 54CGWHCISA61 is located in the inner region of the expected 3D structure of Nsp2, which reveals that the epitope is cryptic. These findings not only provide valuable insight for vaccine design and hold diagnostic potential for the identified epitopes, but also reveal a protective mechanism against variation under selective pressure in an important epitope.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Proteínas Recombinantes/imunologia , Suínos , Replicação Viral
13.
Microb Pathog ; 128: 254-262, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30616000

RESUMO

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.


Assuntos
Antígenos Virais/imunologia , Aphthovirus/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Antígenos Virais/química , Simulação por Computador , Sequência Consenso , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/imunologia , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Alinhamento de Sequência , Sorogrupo , Proteínas Virais/química , Proteínas Virais/imunologia , Proteínas Estruturais Virais/química , Vacinas Virais/imunologia
14.
Int J Mol Med ; 43(3): 1531-1541, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664181

RESUMO

Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET­28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS­PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B­cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog­allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0­fold increase in cluster of differentiation 63 and C­C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog­allergic children compared with those of non­allergic controls. The secondary structure analysis showed that Can f 7 contains 6 ß­sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.


Assuntos
Alérgenos/genética , Alérgenos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Expressão Gênica , Lipocalinas/genética , Lipocalinas/imunologia , Adolescente , Alérgenos/química , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Códon , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Lactente , Lipocalinas/química , Lipocalinas/isolamento & purificação , Masculino , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes
15.
Viral Immunol ; 32(2): 84-88, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30523737

RESUMO

Enterovirus 71 (EV-A71) has attracted widespread attention in the past decade because of its higher neurotropicity in addition to causing hand-foot-and-mouth disease (HFMD). Mapping epitopes of viral proteins may promote our understanding of antiviral humoral immunity, and is useful for clinical application. In this study, the linear B cell epitopes within nonstructural proteins of EV-A71 were identified using bioinformatics methods combined with peptide enzyme-linked immunosorbent assay (ELISA). Twenty epitopes were predicted and located at 2ABC (7), 3ABC (5), and 3D (8) protein, respectively. Of 20 epitope-containing peptides, 14 were verified by ELISA (S/CO >2.1), 9 of which had higher reactivity (S/CO >5.0). Furthermore, synthetic peptide SP09 (EV-A71-3ABC26-41) can react with healthy children sera, and its immunoreactivity was closest to that of EV-A71-VP1 protein. The protein BLAST analysis revealed that SP09 may contain a common epitope due to the high homology of amino acid sequences with other members of human Enterovirus species A. These findings may be useful in the development serological tests for the diagnosis of HFMD caused by a broad range of human Enterovirus species A.


Assuntos
Enterovirus Humano A/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Proteínas não Estruturais Virais/imunologia , Animais , Proteínas do Capsídeo/imunologia , Biologia Computacional , Enterovirus Humano A/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/genética , Doença de Mão, Pé e Boca , Humanos , Peptídeos/síntese química , Peptídeos/imunologia , Coelhos , Proteínas não Estruturais Virais/química
16.
Mol Immunol ; 105: 181-189, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30550980

RESUMO

Aggregation of therapeutic proteins is a key factor in the generation of unwanted immunogenicity, and can result in reduced serum half-life, neutralization of function and adverse health effects. There is currently little information regarding how aggregates interact with B-cell receptors or cognate antibodies at the protein sequence level, or whether non-native, aggregate-induced epitopes predominate in these interactions. Using an antibody fragment (single chain antibody variable fragment; scFv) that forms aggregates readily at low temperature, anti-scFv IgG antibody responses were generated by intraperitoneal injection of BALB/c strain mice with monomer or aggregate preparations. Aggregate-specific immunosignatures were identified by oligo-peptide microarray fine epitope mapping, using overlapping 15mer peptides based on the linear sequence of scFv, printed onto glass slides. IgG antibodies from mice immunized with aggregated scFv preferentially recognized a patch of overlapping peptides. This region mapped to a ß-strand located at the interface between the VH and VL domains. Molecular dynamics simulations indicated that the VL domain is less stable than the VH domain, suggesting the interface region between the two domains becomes exposed during partial unfolding of the scFv during aggregate formation. These data are consistent with the hypothesis that epitopes from partially unfolded states are revealed, or are more fully exposed, in the aggregated state, and that this can augment the IgG antibody response. This observation offers the theoretical possibility that epitopes preferentially associated with aggregates can be identified from the anti-drug antibody serum IgG response which may, in turn, lead to better methods for detection of anti-drug antibody responses, and improved design of therapeutic proteins to control immunogenicity.


Assuntos
Epitopos de Linfócito B/imunologia , Imunoglobulina G/imunologia , Agregados Proteicos/imunologia , Desdobramento de Proteína , Anticorpos de Cadeia Única/imunologia , Animais , Epitopos de Linfócito B/química , Feminino , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos BALB C , Anticorpos de Cadeia Única/química
17.
Nat Commun ; 9(1): 4809, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442944

RESUMO

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Receptores ErbB/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Neoplasias Cutâneas/terapia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Imunidade Adaptativa , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Receptores ErbB/agonistas , Receptores ErbB/genética , Feminino , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/biossíntese , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Anticorpos de Cadeia Única/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Mol Immunol ; 104: 20-26, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30399490

RESUMO

Bothropasin is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca venom, the snake responsible for most bites in Southeastern Brazil. SVMPs, such as bothropasin, are involved in the main bothropic envenoming symptoms, which include hemorrhage, inflammation, necrosis and blood coagulation deficiency. B-cell epitope mapping of SVMPs can lead to the identification of peptides capable of inducing neutralizing antibodies without causing toxic effects, therefore improving anti-venom production. Here, using the SPOT synthesis technique, we have identified an epitope located in the catalytic domain of bothropasin (202KARMYELANIVNEILRYLYMH222) which was synthesized and named BotEp1. The peptide was used to immunize Swiss mice and Anti-BotEp1 serum cross-reacted with bothropasin and crude venoms from B. jararaca and B. atrox venoms. Furthermore, Anti-BotEp1 antibodies were able to completely neutralize the hemorrhagic activity of a chromatographic fraction from B. jararaca venom, which contains hemorrhagic SVMPs. In addition, the coagulation activity of the hemorrhagic fraction showed to be diminished when tested in serum from rabbit immunized with BotEp1 (compared to serum from non-immunized animal). Our results show the identification of neutralizing epitopes in bothropasin and provide basis for the use of synthetic peptides to improve the production of immunotherapeutics.


Assuntos
Bothrops/imunologia , Venenos de Crotalídeos/imunologia , Epitopos de Linfócito B/imunologia , Metaloendopeptidases/imunologia , Peptídeos/imunologia , Animais , Venenos de Crotalídeos/síntese química , Venenos de Crotalídeos/química , Epitopos de Linfócito B/química , Metaloendopeptidases/síntese química , Metaloendopeptidases/química , Camundongos , Peptídeos/síntese química , Peptídeos/química , Domínios Proteicos
19.
Sci Rep ; 8(1): 14904, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30297733

RESUMO

Epitope identification is essential for developing effective antibodies that can detect and neutralize bioactive proteins. Computational prediction is a valuable and time-saving alternative for experimental identification. Current computational methods for epitope prediction are underused and undervalued due to their high false positive rate. In this work, we targeted common properties of linear B-cell epitopes identified in an individual protein class (metalloendopeptidases) and introduced an alternative method to reduce the false positive rate and increase accuracy, proposing to restrict predictive models to a single specific protein class. For this purpose, curated epitope sequences from metalloendopeptidases were transformed into frame-shifted Kmers (3 to 15 amino acid residues long). These Kmers were decomposed into a matrix of biochemical attributes and used to train a decision tree classifier. The resulting prediction model showed a lower false positive rate and greater area under the curve when compared to state-of-the-art methods. Our predictions were used for synthesizing peptides mimicking the predicted epitopes for immunization of mice. A predicted linear epitope that was previously undetected by an experimental immunoassay was able to induce neutralizing-antibody production in mice. Therefore, we present an improved prediction alternative and show that computationally identified epitopes can go undetected during experimental mapping.


Assuntos
Anticorpos Neutralizantes/biossíntese , Biologia Computacional/métodos , Epitopos de Linfócito B/imunologia , Venenos de Serpentes/imunologia , Algoritmos , Sequência de Aminoácidos , Aminoácidos/química , Animais , Árvores de Decisões , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Feminino , Imunização , Metaloproteases/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeos/química , Curva ROC , Reprodutibilidade dos Testes
20.
J Med Chem ; 61(21): 9568-9582, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30351939

RESUMO

We present here for the first time the synthesis and immunological evaluation of a fully synthetic three-component anticancer vaccine candidate that consists of a ß-glycotripeptoid core mimicking a cluster of Tn at the surface of tumor cells (B epitope), conjugated to the OVA 323-339 peptide (T-cell epitope) and a Toll-like receptor 7 (TLR7) agonist for potent adjuvanticity. The immunological evaluation of this construct and of precursor components demonstrated the synergistic activity of the components within the conjugate to stimulate innate and adaptive immune cells (DCs, T-helper, and B-cells). Surprisingly, immunization of mice with the tricomponent GalNAc-based construct elicited a low level of anti-Tn IgG but elicited a very high level of antibodies that recognize the TLR7 agonist. This finding could represent a potential vaccine therapeutic approach for the treatment of some autoimmune diseases such as lupus.


Assuntos
Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Peptidomiméticos/síntese química , Peptidomiméticos/farmacologia , Receptor 7 Toll-Like/agonistas , Sequência de Aminoácidos , Animais , Técnicas de Química Sintética , Camundongos , Camundongos Endogâmicos C57BL , Peptidomiméticos/química , Peptidomiméticos/imunologia
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