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1.
Science ; 372(6546)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083463

RESUMO

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno de Histocompatibilidade H-2D/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Complexo CD3/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T , Feminino , Antígeno de Histocompatibilidade H-2D/química , Antígeno de Histocompatibilidade H-2D/imunologia , Vírus da Influenza A , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de Sinais
2.
Sci Rep ; 11(1): 5402, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686135

RESUMO

Most multiple sclerosis (MS) patients given currently available disease-modifying drugs (DMDs) experience progressive disability. Accordingly, there is a need for new treatments that can limit the generation of new waves T cell autoreactivity that drive disease progression. Notably, immune cells express GABAA-receptors (GABAA-Rs) whose activation has anti-inflammatory effects such that GABA administration can ameliorate disease in models of type 1 diabetes, rheumatoid arthritis, and COVID-19. Here, we show that oral GABA, which cannot cross the blood-brain barrier (BBB), does not affect the course of murine experimental autoimmune encephalomyelitis (EAE). In contrast, oral administration of the BBB-permeable GABAA-R-specific agonist homotaurine ameliorates monophasic EAE, as well as advanced-stage relapsing-remitting EAE (RR-EAE). Homotaurine treatment beginning after the first peak of paralysis reduced the spreading of Th17 and Th1 responses from the priming immunogen to a new myelin T cell epitope within the CNS. Antigen-presenting cells (APC) isolated from homotaurine-treated mice displayed an attenuated ability to promote autoantigen-specific T cell proliferation. The ability of homotaurine treatment to limit epitope spreading within the CNS, along with its safety record, makes it an excellent candidate to help treat MS and other inflammatory disorders of the CNS.


Assuntos
Sistema Nervoso Central/patologia , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Taurina/análogos & derivados , Animais , Apresentação do Antígeno/efeitos dos fármacos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Proliferação de Células/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Modelos Animais de Doenças , Progressão da Doença , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Recidiva , Baço/patologia , Linfócitos T/efeitos dos fármacos , Taurina/farmacologia , Ácido gama-Aminobutírico/farmacologia
3.
Medicine (Baltimore) ; 100(8): e24615, 2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33663071

RESUMO

ABSTRACT: The T-SPOT.TB assay detects cellular immune responses to 2 core Mycobacterium tuberculosis antigens, early secreted antigenic target of 6-kDa protein (ESAT-6) and culture filtrate protein-10 (CFP-10). T-SPOT.TB has been recently used for auxiliary diagnosis of active pulmonary tuberculosis (PTB). However, testing can produce inconsistent results due to differential PTB patient immune responses to these antigens, prompting us to identify factors underlying inconsistent results.Data were retrospectively analyzed from 1225 confirmed PTB patients who underwent T-SPOT.TB testing at 5 specialized tuberculosis hospitals in China between December 2012 and November 2015. Numbers of spot-forming cells (SFCs) reflecting T cell responses to ESAT-6 and CFP-10 antigens were recorded then analyzed via multivariable logistic regression to reveal factors underlying discordant T cell responses to these antigens.The agreement rate of 84.98% (82.85%-86.94%) between PTB patient ESAT-6 and CFP-10 responses demonstrated high concordance. Additionally, positivity rates were higher for ESAT-6 than for CFP-10 (84.8% vs 80.7%, P < .001), with ESAT-6 and CFP-10 microwell SFC numbers for each single positive group not differing significantly (P > .99), while spot numbers of the single positive group were lower than numbers for the double positive group (P < .001). Elderly patients (aged ≥66 years) and patients receiving retreatment were most likely to have discordance results.ESAT-6 promoted significantly more positive T-SPOT.TB results than did CFP-10 in PTB patients. Advanced age and retreatment status were correlated with discordant ESAT-6 and CFP-10 results. Assessment of factors underlying discordance may lead to improved PTB diagnosis using T-SPOT.TB.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Humanos , Imunidade Celular/imunologia , Testes Imunológicos/normas , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
4.
Int J Biol Macromol ; 171: 82-88, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33418045

RESUMO

Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 µg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Mycobacterium bovis/imunologia , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Bovina/prevenção & controle , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Bovinos , Clonagem Molecular , Códon , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Imunogenicidade da Vacina , Interferon gama/biossíntese , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Vacinação/métodos
5.
Food Chem ; 346: 128926, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33484948

RESUMO

Food allergy is on the rise, and preventive/therapeutic procedures are needed. We explored a preventive protocol for milk allergy with the oral administration of a Gly-m-Bd-30K soy-derived peptide that contains cross-reactive epitopes with bovine caseins. B/T-cross-reactive epitopes were mapped using milk-specific human sera and monoclonal antibodies on overlapping and recombinant peptides of Gly-m-Bd-30K by SPOT and cell proliferation assays. Bioinformatics tools were used to characterize epitopes on the 3D-modelled molecule, and to predict the binding to HLA alleles. The peptide was orally administrated to mice that were then IgE-sensitized to milk proteins. Immunodominant B-epitopes were mainly located on the surface of the Nt-fragment. The use of a soy-peptide-containing an immunodominant cross-reactive T-epitope, along with a single B epitope, prevents IgE-mediated milk sensitization through the induction of Th1-mediated immunity and induction of blocking IgG. The use of a safe soy-peptide may represent a promising alternative for preventing milk allergy.


Assuntos
Reações Cruzadas , Hipersensibilidade a Leite/prevenção & controle , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Proteínas de Soja/imunologia , Administração Oral , Animais , Bovinos , Epitopos/imunologia , Humanos , Camundongos , Hipersensibilidade a Leite/imunologia
6.
Clin Immunol ; 225: 108679, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33485895

RESUMO

HER2-targeted therapy has not benefited patients with low levels of HER2 expression; however, combination therapy may be effective. Primary analysis of a phase IIb trial investigating the HER2-derived vaccine nelipepimut-S (NPS) did not benefit the intention-to-treat population, but subset analysis showed a benefit in triple-negative breast cancer (TNBC) patients. The subset analysis of this multicenter, randomized, single-blind, phase IIb trial identified significant improvement in 36-month disease-free survival (DFS) between NPS (n = 55) and placebo (n = 44) in TNBC (HR 0.25, p = 0.01) and those who express HLA-A24 (HR 0.41, p = 0.05). The TNBC cohort demonstrated improved 36-month DFS in those with HER2 1+ expression (HR 0.17, p = 0.01), HLA-A24 positivity (HR 0.08, p < 0.01), or in those who received neoadjuvant chemotherapy (HR 0.21, p < 0.01). NPS vaccination with trastuzumab was associated with improved 36-month DFS among patients with TNBC. The observed benefit to this high-risk subgroup warrants confirmation in a phase III trial.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunoterapia/métodos , Fragmentos de Peptídeos/imunologia , Receptor ErbB-2/imunologia , Trastuzumab/uso terapêutico , Neoplasias de Mama Triplo Negativas/terapia , Adulto , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Antígeno HLA-A24/metabolismo , Humanos , Análise de Intenção de Tratamento , Recidiva Local de Neoplasia , Efeito Placebo , Medicina de Precisão , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Risco , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/mortalidade
7.
Int Immunopharmacol ; 90: 107241, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33321294

RESUMO

The impact of immunization with Anisakis simplex larval antigen on the occurrence and progression of experimental autoimmune encephalomyelitis (EAE) induced in mice was studied. C57BL/6J mice were immunized with the MOG35-55 peptide and one batch was treated with A. simplex total larval antigen on days 1, 8, 10 and 12 after EAE induction. Significantly higher values were obtained in the EAE clinical parameters of the antigen-treated group. Likewise, there was a significant decrease in the weights of the animals. Anisakis-treatment produced a significant decrease in anti-MOG35-55 specific IgG1 on day 21. On day 14 there was an increase in serum IL-2, IL-6, IL-10, IL-17A, and TGF-ß in the treated group. On day 21, a decrease in IL-4, IL-6, TNF-α, TGF-ß was observed. All brain determinations were made on day 21. The treatment decreased values of IL-6, IL-10, IL-17A and TNF-α. A. simplex antigen caused a significantly higher incidence of EAE and an advance in the appearance of the disease manifestations. However, treatment with the antigen was able to cause a decrease in proinflammatory cytokines (IL-6, IL-17A, and TNF-α) in nervous tissue that could establish a future preventive scenario for myelin damage.


Assuntos
Anisakis/imunologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Antígenos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Citocinas/sangue , Encefalomielite Autoimune Experimental/imunologia , Feminino , Imunoglobulina G/imunologia , Larva/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia
8.
Methods Mol Biol ; 2225: 257-273, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108668

RESUMO

Solid tissue transplant is a growing medical need that is further complicated by a limited donor organ supply. Acute and chronic rejection occurs in nearly all transplants and reduces long-term graft survival, thus increasing the need for repeat transplantation. Viruses have evolved highly adapted responses designed to evade the host's immune defenses. Immunomodulatory proteins derived from viruses represent a novel class of potential therapeutics that are under investigation as biologics to attenuate immune-mediated rejection and damage. These immune-modulating proteins have the potential to reduce the need for traditional posttransplant immune suppressants and improve graft survival. The myxoma virus-derived protein M-T7 is a promising biologic that targets chemokine and glycosaminoglycan pathways central to kidney transplant rejection. Orthotopic transplantations in mice are prohibitively difficult and costly and require a highly trained microsurgeon to successfully perform the procedure. Here we describe a kidney-to-kidney subcapsular transplant model as a practical and simple method for studying transplant rejection, a model that requires fewer mice. One kidney can be used as a donor for transplants into six or more recipient mice. Using this model there is lower morbidity, pain, and mortality for the mice. Subcapsular kidney transplantation provides a first step approach to testing virus-derived proteins as new potential immune-modulating therapeutics to reduce transplant rejection and inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Fatores Imunológicos/farmacologia , Transplante de Rim/métodos , Myxoma virus/química , Proteínas Virais/farmacologia , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Biomarcadores/análise , Quimiocinas/biossíntese , Complemento C4b/genética , Complemento C4b/imunologia , Feminino , Expressão Gênica , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Fatores Imunológicos/biossíntese , Fatores Imunológicos/imunologia , Rim/imunologia , Rim/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptores de Interferon/biossíntese , Receptores de Interferon/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Transplante Homólogo , Proteínas Virais/biossíntese , Proteínas Virais/imunologia
9.
Food Chem ; 340: 127903, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889205

RESUMO

Microbial transglutaminase (mTG) catalyses the formation of protein crosslinks, deamidating glutamine in a side-reaction. Gluten deamidation by human tissue transglutaminase is critical to activate celiac disease pathogenesis making the addition of mTG to wheat-based products controversial. The ability of mTG (0-2000 U.kg-1) to alter gluten's structure, digestibility and the deamidation state of six immunogenic gluten peptides within bread was investigated. Gluten's structure was altered when mTG exceeded 100 U.kg-1, determined by confocal microscopy, extractability and free sulfhydryl assays. The effect of mTG on six immunogenic peptides was investigated by in vitro digestion (INFOGEST) and mass spectrometry. The addition of mTG to bread (0-2000 U.kg-1) did not alter the deamidation state or digestibility of the immunogenic peptides investigated. Overall, this investigation indicated that the addition of mTG to bread does not create activated gluten peptides. This analysis provides evidence for risk assessments of mTG as a food processing aid.


Assuntos
Pão , Glutens/química , Glutens/farmacocinética , Transglutaminases/metabolismo , Pão/análise , Doença Celíaca , Digestão , Glutens/imunologia , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologia , Proteólise , Streptomyces/enzimologia , Transglutaminases/química , Triticum/química
10.
Adv Chronic Kidney Dis ; 27(5): 404-411, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33308506

RESUMO

Hypertension emerged from early reports as a potential risk factor for worse outcomes for persons with coronavirus disease 2019 (COVID-19). Among the putative links between hypertension and COVID-19 is a key counter-regulatory component of the renin-angiotensin system (RAS): angiotensin-converting enzyme 2 (ACE2). ACE2 facilitates entry of severe acute respiratory syndrome coronavirus 2, the virus responsible for COVID-19, into host cells. Because RAS inhibitors have been suggested to increase ACE2 expression, health-care providers and patients have grappled with the decision of whether to discontinue these medications during the COVID-19 pandemic. However, experimental models of analogous viral pneumonias suggest RAS inhibitors may exert protective effects against acute lung injury. We review how RAS and ACE2 biology may affect outcomes in COVID-19 through pulmonary and other systemic effects. In addition, we briefly detail the data for and against continuation of RAS inhibitors in persons with COVID-19 and summarize the current consensus recommendations from select specialty organizations.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Antagonistas de Receptores de Angiotensina/uso terapêutico , Enzima de Conversão de Angiotensina 2/metabolismo , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , COVID-19/metabolismo , Hipertensão/tratamento farmacológico , Lesão Pulmonar Aguda/epidemiologia , Lesão Pulmonar Aguda/imunologia , Angiotensina I/imunologia , Angiotensina I/metabolismo , Angiotensina II/imunologia , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2/imunologia , COVID-19/epidemiologia , COVID-19/imunologia , Comorbidade , Humanos , Hipertensão/epidemiologia , Hipertensão/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fatores de Proteção , Receptores de Coronavírus/imunologia , Receptores de Coronavírus/metabolismo , Sistema Renina-Angiotensina , Fatores de Risco , SARS-CoV-2 , Regulação para Cima
11.
Molecules ; 25(21)2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33147821

RESUMO

With an increasing fatality rate, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a promising threat to human health worldwide. Recently, the World Health Organization (WHO) has announced the infectious disease caused by SARS-CoV-2, which is known as coronavirus disease-2019 (COVID-2019), as a global pandemic. Additionally, the positive cases are still following an upward trend worldwide and as a corollary, there is a need for a potential vaccine to impede the progression of the disease. Lately, it has been documented that the nucleocapsid (N) protein of SARS-CoV-2 is responsible for viral replication and interferes with host immune responses. We comparatively analyzed the sequences of N protein of SARS-CoV-2 for the identification of core attributes and analyzed the ancestry through phylogenetic analysis. Subsequently, we predicted the most immunogenic epitope for the T-cell and B-cell. Importantly, our investigation mainly focused on major histocompatibility complex (MHC) class I potential peptides and NTASWFTAL interacted with most human leukocyte antigen (HLA) that are encoded by MHC class I molecules. Further, molecular docking analysis unveiled that NTASWFTAL possessed a greater affinity towards HLA and also available in a greater range of the population. Our study provides a consolidated base for vaccine design and we hope that this computational analysis will pave the way for designing novel vaccine candidates.


Assuntos
Betacoronavirus/imunologia , Proteínas do Nucleocapsídeo/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Hipersensibilidade a Drogas/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/química , Fragmentos de Peptídeos/imunologia , Fosfoproteínas , Estrutura Secundária de Proteína , Vacinas Virais/imunologia
12.
PLoS One ; 15(10): e0238609, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112881

RESUMO

INTRODUCTION: Although immune responses to the Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) have a potential to modulate the immune responses to each other, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV specific, DENV non cross-reactive antibody responses by identifying JEV specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV specific antibodies associate with dengue disease severity. METHODOLOGY AND RESULTS: 22 JEV specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed in individuals who were non-immune to JEV and DENV (JEV-DENV-, N = 30), those who were only immune to the JEV and not DENV (JEV+DENV-, N = 30), those who were only immune to DENV(JEV-DENV+, N = 30) and in those who were immune to both viruses (JEV+DENV+, N = 30). 7/22 peptides were found to be highly immunogenic and specific and these 7 peptides were used as a pool to further evaluate JEV-specific responses. All 30/30 JEV+DENV- and 30/30 JEV+DENV+ individuals, and only 3/30 (10%) JEV-DENV+ individuals responded to this pool. We further evaluated this pool of 7 peptides in patients following primary and secondary dengue infection during the convalescent period and found that the JEV-specific peptides, were unlikely to cross react with DENV IgG antibodies. We further compared this in-house ELISA developed with the peptide pool with an existing commercial JEV IgG assay to identify JEV-specific IgG following vaccination, and our in-house ELISA was found to be more sensitive. We then proceeded to investigate if the presence of JEV-specific antibodies were associated with dengue disease severity, and we found that those who had past severe dengue (n = 175) were significantly more likely (p<0.0001) to have JEV-specific antibodies than those with past non-severe dengue (n = 175) (OR 5.3, 95% CI 3.3 to 8.3). CONCLUSIONS: As our data show that this assay is highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Encefalite Japonesa (Espécie)/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Especificidade de Anticorpos , Criança , Sequência Conservada , Reações Cruzadas , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/epidemiologia , Encefalite Japonesa/imunologia , Encefalite Japonesa/virologia , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Sorogrupo , Sri Lanka/epidemiologia , Vacinação , Proteínas Virais/genética , Proteínas Virais/imunologia , Adulto Jovem
14.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
15.
Proc Natl Acad Sci U S A ; 117(39): 24384-24391, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32913053

RESUMO

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269 +CD8+ and A2/Orf1ab3183 +CD8+ populations indicated that A2/S269 +CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269 +CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269 +CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269 +CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Antígeno HLA-A2/imunologia , Pneumonia Viral/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Poliproteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas Virais/química , Proteínas Virais/imunologia
16.
Front Immunol ; 11: 1714, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32793244

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third coronavirus leading to a global health outbreak. Despite the high mortality rates from SARS-CoV-1 and Middle-East respiratory syndrome (MERS)-CoV infections, which both sparked the interest of the scientific community, the underlying physiopathology of the SARS-CoV-2 infection, remains partially unclear. SARS-CoV-2 shares similar features with SARS-CoV-1, notably the use of the angiotensin conversion enzyme 2 (ACE2) as a receptor to enter the host cells. However, some features of the SARS-CoV-2 pandemic are unique. In this work, we focus on the association between obesity, metabolic syndrome, and type 2 diabetes on the one hand, and the severity of COVID-19 infection on the other, as it seems greater in these patients. We discuss how adipocyte dysfunction leads to a specific immune environment that predisposes obese patients to respiratory failure during COVID-19. We also hypothesize that an ACE2-cleaved protein, angiotensin 1-7, has a beneficial action on immune deregulation and that its low expression during the SARS-CoV-2 infection could explain the severity of infection. This introduces angiotensin 1-7 as a potential candidate of interest in therapeutic research on CoV infections.


Assuntos
Adipocinas/imunologia , Angiotensina I/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/patologia , Fragmentos de Peptídeos/imunologia , Pneumonia Viral/patologia , Síndrome Respiratória Aguda Grave/patologia , Adipocinas/sangue , Diabetes Mellitus Tipo 2/imunologia , Humanos , Síndrome Metabólica/imunologia , Obesidade/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo
17.
Lancet Neurol ; 19(7): 591-600, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32562684

RESUMO

BACKGROUND: Robust evidence supports the role of α-synuclein pathology as a driver of neuronal dysfunction in Parkinson's disease. PD01A is a specific active immunotherapy with a short peptide formulation targeted against oligomeric α-synuclein. This phase 1 study assessed the safety and tolerability of the PD01A immunotherapeutic in patients with Parkinson's disease. METHODS: We did a first-in-human, randomised, phase 1 study of immunisations with PD01A, followed by three consecutive study extensions. Patients aged 45-65 years with a clinical diagnosis of Parkinson's disease (≤4 years since diagnosis and Hoehn and Yahr Stage 1 to 2), imaging results (dopamine transporter single photon emission CT and MRI) consistent with their Parkinson's disease diagnosis, and on stable doses of Parkinson's disease medications for at least 3 months were recruited at a single private clinic in Vienna, Austria. Patients were randomly assigned (1:1), using a computer-generated sequence with varying block size, to receive four subcutaneous immunisations with either 15 µg or 75 µg PD01A injected into the upper arms and followed up initially for 52 weeks, followed by a further 39 weeks' follow-up. Patients were then randomly assigned (1:1) again to receive the first booster immunisation at 15 µg or 75 µg and were followed up for 24 weeks. All patients received a second booster immunisation of 75 µg and were followed up for an additional 52 weeks. Patients were masked to dose allocation. Primary (safety) analyses included all treated patients. These four studies were registered with EU Clinical Trials Register, EudraCT numbers 2011-002650-31, 2013-001774-20, 2014-002489-54, and 2015-004854-16. FINDINGS: 32 patients were recruited between Feb 14, 2012, and Feb 6, 2013, and 24 were deemed eligible and randomly assigned to receive four PD01A priming immunisations. One patient had a diagnosis change to multiple system atrophy and was withdrawn and two patients withdrew consent during the studies. 21 (87%) of 24 patients received all six immunisations and completed 221-259 weeks in-study (two patients in the 15 µg dose group and one patient in the 75 µg dose group discontinued). All patients experienced at least one adverse event, but most of them were considered unrelated to study treatment (except for transient local injection site reactions, which affected all but one patient). Serial MRI assessments also ruled out inflammatory processes. Systemic treatment-related adverse events were fatigue (n=4), headache (n=3), myalgia (n=3), muscle rigidity (n=2), and tremor (n=2). The geometric group mean titre of antibodies against the immunising peptide PD01 increased from 1:46 at baseline to 1:3580 at week 12 in the 15 µg dose group, and from 1:76 to 1:2462 at week 12 in the 75 µg dose group. Antibody titres returned to baseline over 2 years, but could be rapidly reactivated after booster immunisation from week 116 onwards, reaching geometric group mean titres up to 1:20218. INTERPRETATION: Repeated administrations of PD01A were safe and well tolerated over an extended period. Specific active immunotherapy resulted in a substantial humoral immune response with target engagement. Phase 2 studies are needed to further assess the safety and efficacy of PD01A for the treatment of Parkinson's disease. FUNDING: AFFiRiS, Michael J Fox Foundation.


Assuntos
Imunoterapia/métodos , Doença de Parkinson/tratamento farmacológico , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/uso terapêutico , Peptídeos/imunologia , Peptídeos/uso terapêutico , alfa-Sinucleína/antagonistas & inibidores , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Método Simples-Cego
18.
Surgery ; 168(3): 478-485, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439208

RESUMO

BACKGROUND: Intestinal ischemia-reperfusion injury results in morbidity and mortality from both local injury and systemic inflammation and acute lung injury. Extracellular cold-inducible RNA-binding protein is a damage associated molecular pattern that fuels systemic inflammation and potentiates acute lung injury. We recently discovered a triggering receptor expressed on myeloid cells-1 serves as a novel receptor for extracellular cold-inducible RNA-binding protein. We developed a 7-aa peptide, named M3, derived from the cold-inducible RNA-binding protein, which interferes with cold-inducible RNA-binding protein's binding to a triggering receptor expressed on myeloid cells-1. Here, we hypothesized that M3 protects mice against intestinal ischemia-reperfusion injury. METHODS: Intestinal ischemia was induced in C57BL/6 mice via clamping of the superior mesenteric artery for 60 minutes. At reperfusion, mice were treated intraperitoneally with M3 (10 mg/kg body weight) or normal saline vehicle. Mice were killed 4 hours after reperfusion and blood and lungs were collected for various analysis. A 24-hours survival after intestinal ischemia-reperfusion was assessed. RESULTS: Serum levels of organ injury markers aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and lactate were increased with intestinal ischemia-reperfusion, while treatment with M3 significantly decreased their levels. Serum, intestinal, and lung levels of proinflammatory cytokines and chemokines were also increased by intestinal ischemia-reperfusion, and treatment with M3 significantly reduced these values. Intestinal ischemia-reperfusion caused significant histological intestinal and lung injuries, which were mitigated by M3. Treatment with M3 improved the survival from 40% to 80% after intestinal ischemia-reperfusion. CONCLUSION: Inhibition of triggering receptor expressed on myeloid cells-1 by an extracellular cold-inducible RNA-binding protein-derived small peptide (M3) decreased inflammation, reduced lung injury, and improved survival in intestinal ischemia-reperfusion injury. Thus, blocking the extracellular cold-inducible RNA-binding protein-triggering receptor expressed on myeloid cells-1 interaction is a promising therapeutic avenue for mitigating intestinal ischemia-reperfusion injury.


Assuntos
Intestinos/irrigação sanguínea , Fragmentos de Peptídeos/uso terapêutico , Proteínas de Ligação a RNA/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/farmacologia , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo
20.
J Biotechnol ; 318: 51-56, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32387449

RESUMO

Multiple sclerosis (MS) affects 2.3 million patients worldwide with no effective treatments available thus far. Depletion of autoreactive T-cells is considered the basis for immunotherapeutic approaches. For this purpose the peptides BV5S2, BV6S5, and BV13S1 have been identified as candidates for the development of a MS vaccine. Herein, the plant-based simultaneous production of these peptides is described as an effort to generate a new model of MS immunotherapy. A polyprotein comprising the sequence of the target peptides was designed having the picornaviral 2A sequence in between to mediate the release of the individual peptides upon translation. A codon optimized gene was cloned in vectors mediating constitutive (CaMV35S promoter) or inducible (AlcA promoter) expression. No transgenic tobacco plants were recovered from the constitutive vector suggesting toxicity of the target peptides. In contrast, several transformed lines were obtained with the inducible vector. The individual BV5S2, BV6S5, and BV13S1 peptides were detected in transformed lines upon ethanol-mediated induction and a quantitative analysis based on a OVA conjugate carrying the three peptides revealed accumulation levels up to 0.5 µg g-1 FW leaves. The plant-made peptides were able to induce humoral responses in orally immunized mice. This platform will be useful in the development of alternative immunotherapies against MS having low cost and safety as main attributes. Moreover the platform represents an attractive alternative for the expression of antigens having detrimental effects in plants.


Assuntos
Imunoterapia , Esclerose Múltipla/terapia , Fragmentos de Peptídeos/genética , Plantas Geneticamente Modificadas/genética , Receptores de Antígenos de Linfócitos T/genética , Animais , Cisteína Endopeptidases/genética , Expressão Gênica , Vetores Genéticos , Humanos , Imunização , Camundongos , Esclerose Múltipla/imunologia , Fragmentos de Peptídeos/imunologia , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Antígenos de Linfócitos T/imunologia , Tabaco/genética , Tabaco/metabolismo , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Proteínas Virais/genética
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