RESUMO
Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/isolamento & purificação , Manganês/metabolismo , Proteína C/metabolismo , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Algoritmos , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Citocinas/metabolismo , Mapeamento de Epitopos , Escherichia coli/genética , Feminino , Interferon gama/metabolismo , Interleucina-17/metabolismo , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos BALB C , Proteína C/genética , Proteína C/imunologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/imunologia , Infecções Estafilocócicas/imunologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a nonspecific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was subcloned into a pSUMOMut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII2.2 server. As a result, two B cell epitopes (3545 and 8186) and four potential T cell epitopes including 215, 4550, 5561 and 6773 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptidebased vaccine designs of pollen allergy.
Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Imunoglobulina E/imunologia , Magnoliopsida/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Clonagem Molecular , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/isolamento & purificação , Escherichia coli/genética , Feminino , Humanos , Imunoglobulina E/sangue , Magnoliopsida/química , Magnoliopsida/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Pólen/química , Pólen/genética , Pólen/imunologia , Conformação Proteica , Rinite Alérgica Sazonal/sangue , Adulto JovemRESUMO
Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.
Assuntos
Alérgenos/imunologia , Bioquímica/métodos , Toxina da Cólera/metabolismo , Cryptomeria/metabolismo , Epitopos de Linfócito T/metabolismo , Escherichia coli/metabolismo , Pólen/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Alérgenos/química , Sequência de Aminoácidos , Antígenos de Plantas , Western Blotting , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitopos de Linfócito T/química , Epitopos de Linfócito T/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.
Assuntos
Vacinas contra a AIDS/imunologia , Sequência Conservada/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Epitopos de Linfócito T/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/síntese química , Adulto , Motivos de Aminoácidos/imunologia , Animais , Linhagem Celular Transformada , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/isolamento & purificação , Infecções por HIV/imunologia , HIV-1/isolamento & purificação , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A3/genética , Antígeno HLA-A3/imunologia , Antígeno HLA-B7/genética , Antígeno HLA-B7/imunologia , Teste de Histocompatibilidade , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Camundongos , Camundongos Transgênicos , Valor Preditivo dos Testes , Superantígenos/imunologia , Linfócitos T Citotóxicos/virologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/síntese químicaRESUMO
More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.
Assuntos
Alérgenos/imunologia , Imunoglobulina E/imunologia , Lolium/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Alérgenos/genética , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos , Antígenos de Plantas , Basófilos/imunologia , Sítios de Ligação , Divisão Celular , Células Clonais , Sequência Conservada , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/isolamento & purificação , Liberação de Histamina , Humanos , Hipersensibilidade/prevenção & controle , Hipersensibilidade Imediata/imunologia , Imunoterapia Ativa , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Dobramento de Proteína , Estrutura Secundária de Proteína , Recombinação Genética , Pele/imunologiaRESUMO
Although atopic allergy affects =20% of the total population, the relationship between the protein structure and immunogenic activity of the allergens is still largely unknown. We observed that group 5 grass allergens are characterized by repeated structural motifs. Using a new algorithm, TEPITOPE, we predicted promiscuous HLA-DR ligands within the repeated motifs of the Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.