RESUMO
Thymic atrophy reduces naive T cell production and contributes to increased susceptibility to viral infection with age. Expression of tissue-restricted antigen (TRA) genes also declines with age and has been thought to increase autoimmune disease susceptibility. We find that diminished expression of a model TRA gene in aged thymic stromal cells correlates with impaired clonal deletion of cognate T cells recognizing an autoantigen involved in atherosclerosis. Clonal deletion in the polyclonal thymocyte population is also perturbed. Distinct age-associated defects in the generation of antigen-specific T cells include a conspicuous decline in generation of T cells recognizing an immunodominant influenza epitope. Increased catalase activity delays thymic atrophy, and here, we show that it mitigates declining production of influenza-specific T cells and their frequency in lung after infection, but does not reverse declines in TRA expression or efficient negative selection. These results reveal important considerations for strategies to restore thymic function.
Assuntos
Envelhecimento/imunologia , Antígenos/imunologia , Imunidade , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/imunologia , Animais , Antioxidantes/farmacologia , Apolipoproteínas B/metabolismo , Atrofia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Catalase/metabolismo , Suplementos Nutricionais , Imunidade/efeitos dos fármacos , Epitopos Imunodominantes/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/imunologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Tolerância a Antígenos Próprios/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/enzimologia , Linfócitos T/efeitos dos fármacos , Timo/patologiaRESUMO
HI, a fusion protein that consists of the alpha-toxin (Hla) and the N2 domain of iron surface determinant B (IsdB), is one of the antigens in the previously reported S. aureus vaccine rFSAV and has already entered phase II clinical trials. Previous studies revealed that HI is highly immunogenic in both mice and healthy volunteers, and the humoral immune response plays key roles in HI-mediated protection. In this study, we further investigated the protective efficacy of immunization with HI plus four different adjuvants in a mouse bacteremia model. Results showed that HI-mediated protection was altered in response to different adjuvants. Using antisera from immunized mice, we identified seven B-cell immunodominant epitopes on Hla and IsdB, including 6 novel epitopes (Hla1-18, Hla84-101, Hla186-203, IsdB342-359, IsdB366-383, and IsdB384-401). The immunodominance of B-cell epitopes, total IgG titers and the levels of IFN-γ and IL-17A from mice immunized with HI plus different adjuvants were different from each other, which may explain the difference in protective immunity observed in each immunized group. Thus, our results indicate that adjuvants largely affected the immunodominance of epitopes and the protective efficacy of HI, which may guide further adjuvant screening for vaccine development and optimization.
Assuntos
Bacteriemia/imunologia , Toxinas Bacterianas/imunologia , Proteínas de Transporte de Cátions/imunologia , Epitopos de Linfócito B/imunologia , Proteínas Hemolisinas/imunologia , Epitopos Imunodominantes/imunologia , Infecções Estafilocócicas/prevenção & controle , Animais , Bacteriemia/prevenção & controle , Modelos Animais de Doenças , Feminino , Imunização Passiva , Imunoterapia Adotiva , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/imunologiaRESUMO
BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Epitopos Imunodominantes/imunologia , Leptospira/imunologia , Leptospirose/imunologia , Adolescente , Adulto , Idoso , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/uso terapêutico , Proteínas da Membrana Bacteriana Externa/química , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Coelhos , Testes Sorológicos , Adulto JovemRESUMO
In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.
Assuntos
Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/imunologia , Antígenos Virais/imunologia , Vacinas Virais/imunologia , Febre Suína Africana/prevenção & controle , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/genética , Alphavirus/genética , Alphavirus/metabolismo , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Imunização Secundária , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Suínos , Células Vero , Vacinas Virais/administração & dosagemRESUMO
BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are defined carbohydrate portions of glycoprotein cell surface molecules common to many plant and insect species. Mammalian species recognize CCD as foreign antigens and can mount humoral immune responses against them. Approximately 20-37% of grass and venom allergic people possess circulating IgE against CCD; these antibodies are generally considered clinically irrelevant. Anti-CCD IgE is, however, recognized as a cause of false positive, clinically incongruent serum allergen test results in people; this phenomenon has not been investigated in animals. OBJECTIVE: To determine if anti-CCD IgE could be detected in sera of client-owned atopic dogs and how frequently it is found. ANIMALS: Sera from 38 dogs with a clinical diagnosis of atopic dermatitis and prior serological evidence of IgE antibodies, defined as a positive result to at least one mite and pollen (of any type). METHODS: Sera were analysed for IgE against CCD and environmental allergens with a commercially available multiplex enzyme-labelled allergen-specific IgE assay. RESULTS: Anti-CCD IgE was detected in nine of 38 (24%) of atopic dog sera. As with their human counterparts, all dogs with anti-CCD IgE had strong serological reactivity to grass pollens. CONCLUSIONS AND CLINICAL IMPORTANCE: Anti-CCD IgE can confound serological allergen testing in people; the same might be true in dogs. Further studies are warranted to investigate the clinical implications of anti-CCD IgE in dogs, including the potential for these antibodies to affect serum allergen-specific IgE assays used for clinical diagnosis, and whether they are relevant to clinical disease.
Assuntos
Carboidratos/imunologia , Dermatite Atópica/veterinária , Doenças do Cão/imunologia , Imunoglobulina E/imunologia , Alérgenos/imunologia , Animais , Reações Cruzadas/imunologia , Dermatite Atópica/sangue , Dermatite Atópica/diagnóstico , Dermatite Atópica/imunologia , Doenças do Cão/sangue , Doenças do Cão/diagnóstico , Cães , Epitopos Imunodominantes/imunologia , Imunoglobulina E/sangue , Pólen/imunologiaRESUMO
Four previously identified immunodominant B-cell epitopes, located within known virulent pneumococcal proteins CbpD, PhtD, PhtE, and ZmpB, had shown promising in vivo immunological characteristics, indicating their potential to be used as vaccine antigens. In this study, we further evaluated the opsonophagocytic activity of antibodies against these epitopes and their capacity to protect mice from pneumococcal sepsis. An opsonophagocytic killing assay (OPKA) revealed that OPKA titers of human anti-peptide antibodies against pneumococcal serotypes 1, 3, and 19A were significantly higher (P < 0.001) than those of the control sera, suggesting their functional potential against virulent clinical isolates. Data obtained from mice actively immunized with any of the selected epitope analogues or with a mixture of these (G_Mix group) showed, compared to controls, enhanced survival against the highly virulent pneumococcal serotype 3 (P < 0.001). Moreover, passive transfer of hyperimmune serum from G_Mix to naive mice also conferred protection to a lethal challenge with serotype 3, which demonstrates that the observed protection was antibody mediated. All immunized murine groups elicited gradually higher antibody titers and avidity, suggesting a maturation of immune response over time. Among the tested peptides, PhD_pep19 and PhtE_pep40 peptides, which reside within the zinc-binding domains of PhtD and PhtE proteins, exhibited superior immunological characteristics. Recently it has been shown that zinc uptake is of high importance for the virulence of Streptococcus pneumoniae; thus, our findings suggest that these epitopes deserve further evaluation as novel immunoreactive components for the development of a polysaccharide-independent pneumococcal vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Proteínas de Membrana/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Avaliação Pré-Clínica de Medicamentos , Epitopos de Linfócito B/genética , Feminino , Humanos , Imunização , Epitopos Imunodominantes/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genéticaRESUMO
Human schistosomiasis is a neglected tropical disease of great importance in public health. A large number of people are infected with schistosomiasis, making vaccine development and effective diagnosis important control strategies. A rational epitope prediction workflow using Schistosoma mansoni hypothetical proteins was previously presented by our group, and an improvement to that approach is presented here. Briefly, immunodominant epitopes from parasite membrane proteins were predicted by reverse vaccinology strategy with additional in silico analysis. Furthermore, epitope recognition was evaluated using sera of individuals infected with S. mansoni. The epitope that stood out in both in silico and in vitro assays was used to compose a rational chimeric molecule to improve immune response activation. Out of 2185 transmembrane proteins, four epitopes with high binding affinities for human and mouse MHCII molecules were selected through computational screening. These epitopes were synthesized to evaluate their ability to induce TCD4+ lymphocyte proliferation in mice. Sm204830e and Sm043300e induced significant TCD4+ proliferation. Both epitopes were submitted to enzyme-linked immunosorbent assay to evaluate their recognition by IgG antibodies from the sera of infected individuals, and epitope Sm043300 was significantly recognized in most sera samples. Epitope Sm043300 also showed good affinity for human MHCII molecules in molecular docking, and its sequence is curiously highly conserved in four S. mansoni proteins, all of which are described as G-protein-coupled receptors. In addition, we have demonstrated the feasibility of incorporating this epitope, which showed low similarity to human sequences, into a chimeric molecule. The stability of the molecule was evaluated by molecular modeling aimed at future molecule production for use in diagnosis and vaccination trials.
Assuntos
Antígenos de Helmintos/imunologia , Epitopos Imunodominantes/imunologia , Schistosoma mansoni/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/genética , Linfócitos T CD4-Positivos/imunologia , Técnicas de Química Combinatória , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Cadeias HLA-DRB1/imunologia , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/metabolismo , Ativação Linfocitária , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Schistosoma haematobium/imunologia , Schistosoma mansoni/genética , Esquistossomose mansoni/sangue , Esquistossomose mansoni/imunologia , Alinhamento de Sequência , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
T lymphocytes equipped with clonotypic T cell antigen receptors (TCR) recognize immunogenic peptides only when presented in the context of their own major histocompatibility complex (MHC) molecules. Peptide loading to MHC molecules occurs in intracellular compartments (ER for class I and MIIC for class II molecules) and relies on the interaction of the respective peptides and peptide binding pockets on MHC molecules. Those peptide residues not engaged in MHC binding point towards the TCR screening for possible peptide MHC complex binding partners. Natural or intentional modification of both MHC binding registers and TCR interacting residues of peptides - leading to the formation of altered peptide ligands (APLs) - might alter the way peptides interact with TCRs and hence influence subsequent T cell activation events, and consequently T cell effector functions. This review article summarizes how APLs were detected and first described, current concepts of how APLs modify T cellular signaling, which biological mechanisms might force the generation of APLs in vivo, and how peptides and APLs might be used for the benefit of patients suffering from allergic or autoimmune diseases.
Assuntos
Imunoterapia , Ligantes , Peptídeos/imunologia , Alérgenos/química , Alérgenos/imunologia , Animais , Apresentação de Antígeno , Diferenciação Celular , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Epitopos/imunologia , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Transporte de Cátions/imunologia , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Staphylococcus aureus Resistente à Meticilina/imunologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Mapeamento de Epitopos , Feminino , Células HL-60 , Hemocianinas/imunologia , Humanos , Manganês , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Infecções Estafilocócicas/imunologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Recent research on vaccination has extended its scope from infectious diseases to chronic diseases, including Alzheimer disease, dyslipidemia, and hypertension. The aim of this study was to design DNA vaccines for high blood pressure and eventually develop human vaccine therapy to treat hypertension. Plasmid vector encoding hepatitis B core-angiotensin II (Ang II) fusion protein was injected into spontaneously hypertensive rats using needleless injection system. Anti-Ang II antibody was successfully produced in hepatitis B core-Ang II group, and antibody response against Ang II was sustained for at least 6 months. Systolic blood pressure was consistently lower in hepatitis B core-Ang II group after immunization, whereas blood pressure reduction was continued for at least 6 months. Perivascular fibrosis in heart tissue was also significantly decreased in hepatitis B core-Ang II group. Survival rate was significantly improved in hepatitis B core-Ang II group. This study demonstrated that Ang II DNA vaccine to spontaneously hypertensive rats significantly lowered high blood pressure for at least 6 months. In addition, Ang II DNA vaccines induced an adequate humoral immune response while avoiding the activation of self-reactive T cells, assessed by ELISPOT assay. Future development of DNA vaccine to treat hypertension may provide a new therapeutic option to treat hypertension.
Assuntos
Angiotensina II/imunologia , Hipertensão/prevenção & controle , Imunoterapia Ativa , Vacinas de DNA/uso terapêutico , Angiotensina II/genética , Angiotensina II/fisiologia , Animais , Apresentação de Antígeno , Aorta/patologia , Avaliação Pré-Clínica de Medicamentos , Genes Sintéticos , Células HeLa , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Humanos , Hipertensão/genética , Hipertensão/patologia , Imunização , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Isoanticorpos/biossíntese , Rim/patologia , Fígado/patologia , Ativação Linfocitária , Masculino , Miocárdio/patologia , Ratos , Ratos Endogâmicos SHR , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologiaAssuntos
Antígenos de Plantas/imunologia , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Juniperus/imunologia , Proteínas de Plantas/imunologia , Rinite Alérgica Sazonal/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Plantas/química , Humanos , Hibridomas , Epitopos Imunodominantes/química , Conformação Molecular , Proteínas de Plantas/química , Pólen/imunologiaAssuntos
Antialérgicos/uso terapêutico , Hipersensibilidade Imediata/prevenção & controle , Imunoglobulina E/imunologia , Indóis/uso terapêutico , Nitrofuranos/uso terapêutico , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antialérgicos/síntese química , Antialérgicos/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo/efeitos dos fármacos , Sítios de Ligação de Anticorpos , Dessensibilização Imunológica , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Haptenos/imunologia , Liberação de Histamina/efeitos dos fármacos , Humanos , Hipersensibilidade Imediata/tratamento farmacológico , Hipersensibilidade Imediata/imunologia , Epitopos Imunodominantes/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Indóis/síntese química , Ligantes , Mastócitos/metabolismo , Nitrofuranos/síntese químicaAssuntos
Dessensibilização Imunológica/métodos , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/terapia , Adolescente , Alérgenos/imunologia , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Dessensibilização Imunológica/tendências , Seguimentos , Humanos , Epitopos Imunodominantes/imunologia , Terapia de Alvo Molecular , Poaceae , Pólen/imunologiaRESUMO
The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Epitopos Imunodominantes/imunologia , Vacinas contra a AIDS/química , Sequência de Aminoácidos , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Proteína gp41 do Envelope de HIV/química , Humanos , Epitopos Imunodominantes/química , Micelas , Dados de Sequência Molecular , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrofotometria Infravermelho , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/metabolismoRESUMO
BACKGROUND: T-cell targeted peptide epitope tolerogens from grass pollen allergens may be useful in treating seasonal allergic rhinitis, but there is urgent need for optimisation of approaches from improved understanding of mechanism. OBJECTIVE: We sought to identify human leukocyte antigen (HLA)-DR1-restricted epitopes from the Timothy grass pollen allergen, Phleum pratense, and characterise T-cell immune regulation following intranasal administration of a single, immunodominant epitope. METHODS: T-cell epitopes within P pratense were identified using HLA-DR1 transgenic mice and tetramer-guided epitope mapping (TGEM) in HLA-DR1-positive individuals with grass allergy. An immunodominant epitope was tested in HLA-DR1 transgenics for impact on responses to whole Phl p5 b or peptide. Microarrays and quantitative PCR were used to characterise T-cell immunity. RESULTS: Peptide 26 (p26) was identified in HLA-DR1 transgenic mice and by TGEM analysis of HLA-DR1-positive individuals with grass allergy. p26 shows promiscuous binding to a wide range of HLA class II alleles, making it of relevance across immunogenetically diverse patients. The epitope is conserved in rye and velvet grass, making it applicable across a spectrum of grass pollen allergy. Intranasal pretreatment of mice with p26 results in significantly reduced T-cell responses. Transcriptomic array analysis in mice showed T-cell regulation in the intranasal treatment group associated with increased expression of members of the Cbl-b and Itch E3 ubiquitin ligase pathway. CONCLUSIONS: We defined an immunodominant P pratense epitope, p26, with broad binding across multiple HLA class II alleles. Intranasal treatment of mice with p26 results in T-cell regulation to whole allergen, involving the Cbl-b and Itch regulatory pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Alérgenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígeno HLA-DR1/imunologia , Epitopos Imunodominantes/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Proteínas Proto-Oncogênicas c-cbl/fisiologia , Rinite Alérgica Sazonal/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Adulto , Animais , Feminino , Humanos , Imunidade Celular , Masculino , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Pessoa de Meia-Idade , Phleum/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reino Unido , Adulto JovemRESUMO
Vaccination with autologous cancer cells aims to enhance adaptive immune responses to tumour-associated antigens. The incorporation of Fms-like tyrosine kinase 3-ligand (FLT3L) treatment to the vaccination scheme has been shown previously to increase the immunogenicity of cancer vaccines, thereby enhancing their therapeutic potential. While evidence has been provided that FLT3L confers its effect through the increase of absolute dendritic cell (DC) numbers, it is currently unknown which DC populations are responsive to FLT3L and which effect FLT3L treatment has on DC functions. Here we show that the beneficial effects of FLT3L treatment resulted predominantly from a marked increase of two specific DC populations, the CD8 DCs and the recently identified merocytic DC (mcDC). These two DC populations (cross)-present cell-associated antigens to T cells in a natural killer (NK)-independent fashion. FLT3L treatment augmented the absolute numbers of these DCs, but did not change their activation status nor their capacity to prime antigen-specific T cells. While both DC populations effectively primed CD8(+) T cell responses to cell-associated antigens, only mcDC were capable to prime CD4(+) T cells to cell-associated antigens. Consequentially, the transfer of tumour vaccine-pulsed mcDC, but not of CD8 DCs, protected mice from subsequent tumour challenge in a vaccination model and resulted in eradication of established tumours in a therapeutic approach. These results show that the beneficial effect of FLT3L is associated with the induction of mcDC and suggests that selective targeting to mcDC or instilling mcDC 'characteristics' into conventional DC populations could significantly enhance the efficacy of tumour vaccines.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antígenos CD8/metabolismo , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Proteínas de Membrana/uso terapêutico , Neoplasias/terapia , Adjuvantes Imunológicos/farmacologia , Transferência Adotiva , Animais , Antígenos de Superfície/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Epitopos Imunodominantes/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/prevenção & controle , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Baço/citologia , Baço/imunologia , Análise de Sobrevida , VacinaçãoRESUMO
Few common carbohydrate epitopes consisting of terminal beta-(1,2)-xylose and/or alpha-(1,3)-fucose residues are shared by a variety of glycoproteins from plants, insects and parasitic worms, termed cross-reactive carbohydrate determinant (CCD), and frequently recognized by IgE antibodies of patients with food and/or respiratory allergy, though clinical relevancy of such CCD-specific IgE is still controversial. Attention has also been focused on CCDs from the undesired post-translational modification of recombinant therapeutic proteins produced by transgenic plants and insects. In the present study, to clarify immunogenic potentials of CCD-bearing glycoproteins, the antibody response to a model plant glycoprotein, horseradish peroxidase (HRP) was investigated in a mouse model. C3H/He mice were immunized with HRP plus Al(OH)(3) or Freund's adjuvant, and IgG and IgE responses to CCDs in addition to HRP were analyzed by ELISA using some distinct glycoproteins with known N-glycan structures. IgE response to HRP was induced remarkably, whereas that to CCD was weaker and delayed. Moreover, apparent ratio of the CCD-specific antibodies to HRP-specific ones tended to be higher in IgG2a and IgG2b isotypes than IgG1, IgG3 and IgE. In contrast to rabbit antibodies, the CCD-specific antibodies from the mice gave poor reactivity with bromelain and honeybee phospholipase A2, suggesting the critical role of both beta-(1,2)-xylose and alpha-(1,3)-mannose in the CCD-recognition by the mouse antibodies. Moreover, the mouse antibodies showed weaker cross-reactivity to pollen- and insect-derived glycoproteins than the rabbit ones. Thus, in this mouse model, not only IgE but also IgG2 antibody responses to CCDs were induced by immunizing with a CCD-bearing glycoprotein, suggesting that CCDs affected not only Th2-type but also Th1-type antibody response at least in C3H/He mice.
Assuntos
Reações Cruzadas/imunologia , Peroxidase do Rábano Silvestre/imunologia , Epitopos Imunodominantes/metabolismo , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Animais , Células Cultivadas , Mapeamento de Epitopos , Feminino , Fucose/análogos & derivados , Fucose/química , Peroxidase do Rábano Silvestre/metabolismo , Imunidade Humoral , Imunização Secundária , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Proteínas de Insetos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Coelhos , Especificidade da Espécie , Xilose/análogos & derivados , Xilose/químicaRESUMO
Triptolide is a biologically active component purified from Chinese herbal plant Tripterygium wilfordii Hook F. It is widely used in East Asia for treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, Bechect's disease, psoriasis, atopic dermatitis, and asthma. However, its immunological mechanisms are poorly understood. IL-12 and IL-23 are closely related heterodimeric cytokines that share the common subunit p40. They are produced by APCs and are key factors in the generation and effector functions of Th1 and Th17 cells, respectively. They have been strongly implicated in the pathogenesis of several autoimmune disorders. In this study, we investigated the molecular mechanism whereby triptolide inhibits the expression of the p40 gene in APCs. We demonstrate that triptolide does so at the transcriptional level in part through targeting CCAAT/enhancer-binding protein-alpha (C/EBPalpha), which directly interacts with the p40 promoter and inhibits its transcription in inflammatory macrophages. Triptolide can activate the transcription of C/EBPalpha, and phosphorylation of Ser21 and Thr222/226 critical for C/EBPalpha inhibition of p40. Further, activation of C/EBPalpha by triptolide is dependent on upstream kinases ERK1/2 and Akt-GSK3beta. This study provides mechanistic insights into the immunomodulatory capacity of triptolide and has strong implications for its therapeutic applications in autoimmune diseases.
Assuntos
Células Apresentadoras de Antígenos/efeitos dos fármacos , Proteína alfa Estimuladora de Ligação a CCAAT/efeitos dos fármacos , Diterpenos/farmacologia , Imunossupressores/farmacologia , Interleucina-12/biossíntese , Interleucina-23/biossíntese , Fenantrenos/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/imunologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Compostos de Epóxi/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Epitopos Imunodominantes/biossíntese , Epitopos Imunodominantes/efeitos dos fármacos , Epitopos Imunodominantes/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/imunologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Effective protective immunity against respiratory infections with intracellular pathogens requires pathogen-specific cytotoxic T cells (CTL) in the lung. However, vaccines that induce pathogen-specific CTL in the lung are poorly explored. Dendritic cells (DC) have increasingly been exploited as vaccines against infections. However, few studies have investigated the ability of mucosal DC vaccines to elicit protective CTL responses in the lung. Our objective was to develop an efficacious mucosal DC vaccine to generate protective CTL against respiratory infections with intracellular pathogens. Bone marrow-derived DC (BM-DC) pulsed with a single immunodominant CTL epitope, listeriolysin O (LLO) 91-99, of Listeria monocytogenes (LM) were intratracheally administered into mice. The frequency and function of epitope-specific CTL in mediastinal lymph nodes (MLN) and spleen were assessed for their ability to protect against LM infection. After intratracheal administration, lipopolysaccharide (LPS)-treated LLO 91-99-loaded BM-DC (LPS-LLO DC) more frequently migrated to MLN than LPS-untreated LLO 91-99-loaded BM-DC (LLO DC). Using tetrameric H2-K(d)/LLO 91-99 peptide complex, specific CD8(+) T cells were found in MLN as well as the spleen in LPS-LLO DC-immunized mice, but not in LLO-DC-immunized mice. Both MLN and spleen cells obtained from LPS-LLO DC-immunized mice produced large amounts of IFN-gamma in response to LLO 91-99 with high epitope-specific CTL activities. Vaccination with LPS-LLO DC, but not LLO DC, protected mice against lethal respiratory infection with LM. These data suggest that mucosal vaccination with LPS-treated immunodominant CTL epitope-loaded DC is a promising strategy for generating protective CTL against respiratory infections with intracellular pathogens.
Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Células Dendríticas/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Epitopos Imunodominantes/imunologia , Listeria monocytogenes/imunologia , Linfócitos T Citotóxicos/imunologia , Administração por Inalação , Animais , Vacinas Bacterianas/administração & dosagem , Movimento Celular , Avaliação Pré-Clínica de Medicamentos , Imunização , Interferon gama/biossíntese , Listeriose/prevenção & controle , Pulmão/patologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Fragmentos de Peptídeos/imunologia , Pneumonia Bacteriana/prevenção & controle , Receptores CCR7/biossíntese , Baço/imunologia , Traqueia/imunologiaRESUMO
Mitochondrial uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis. We explored the neuroprotective role of UCP4 with its stable overexpression in SH-SY5Y cells, after exposure to either MPP(+) or dopamine to induce ATP deficiency and oxidative stress. Cells overexpressing UCP4 proliferated faster in normal cultures and after exposure to MPP(+) and dopamine. Differentiated UCP4-overexpressing cells survived better when exposed to MPP(+) with decreased LDH release. Contrary to the mild uncoupling hypothesis, UCP4 overexpression resulted in increased absolute ATP levels (with ADP/ATP ratios similar to those of controls under normal conditions and ADP supplementation) associated with increased respiration rate. Under MPP(+) toxicity, UCP4 overexpression preserved ATP levels and mitochondrial membrane potential (MMP) and reduced oxidative stress; the preserved ATP level was not due to increased glycolysis. Under MPP(+) toxicity, the induction of UCP2 expression in vector controls was absent in UCP4-overexpressing cells, suggesting that UCP4 may compensate for UCP2 expression. UCP4 function does not seem to adhere to the mild uncoupling hypothesis in its neuroprotective mechanisms under oxidative stress and ATP deficiency. UCP4 overexpression increases cell survival by inducing oxidative phosphorylation, preserving ATP synthesis and MMP, and reducing oxidative stress.