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1.
Nat Commun ; 12(1): 324, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436573

RESUMO

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Capsídeo/imunologia , Ligação Proteica/imunologia , /imunologia , Animais , Anticorpos Antivirais/imunologia , Feminino , Humanos , Imunogenicidade da Vacina , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Testes Sorológicos , Glicoproteína da Espícula de Coronavírus/imunologia
2.
Respir Res ; 22(1): 26, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478486

RESUMO

BACKGROUND: The epithelial-mesenchymal signaling involving SHH-FOXF1, TBX4-FGF10, and TBX2 pathways is an essential transcriptional network operating during early lung organogenesis. However, precise regulatory interactions between different genes and proteins in this pathway are incompletely understood. METHODS: To identify TBX2 and TBX4 genome-wide binding sites, we performed chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in human fetal lung fibroblasts IMR-90. RESULTS: We identified 14,322 and 1,862 sites strongly-enriched for binding of TBX2 and TBX4, respectively, 43.95% and 18.79% of which are located in the gene promoter regions. Gene Ontology, pathway enrichment, and DNA binding motif analyses revealed a number of overrepresented cues and transcription factor binding motifs relevant for lung branching that can be transcriptionally regulated by TBX2 and/or TBX4. In addition, TBX2 and TBX4 binding sites were found enriched around and within FOXF1 and its antisense long noncoding RNA FENDRR, indicating that the TBX4-FGF10 cascade may directly interact with the SHH-FOXF1 signaling. CONCLUSIONS: We highlight the complexity of transcriptional network driven by TBX2 and TBX4 and show that disruption of this crosstalk during morphogenesis can play a substantial role in etiology of lung developmental disorders.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas Hedgehog/metabolismo , Pulmão/metabolismo , Proteínas com Domínio T/metabolismo , Desenvolvimento Fetal/fisiologia , Fator 10 de Crescimento de Fibroblastos/genética , Fatores de Transcrição Forkhead/genética , Proteínas Hedgehog/genética , Humanos , Pulmão/irrigação sanguínea , Pulmão/embriologia , Ligação Proteica/imunologia , Proteínas com Domínio T/genética
3.
Mol Immunol ; 125: 43-50, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32645549

RESUMO

The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas não Estruturais Virais/imunologia , Vírus da Febre Amarela/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Humanos , Epitopos Imunodominantes/química , Modelos Moleculares , Mutação , Biblioteca de Peptídeos , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/química
4.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32522857

RESUMO

Rabbits are pivotal domestic animals for both the economy and as an animal model for human diseases. A large number of rabbits have been infected by rabbit hemorrhagic disease virus (RHDV) in natural and artificial pandemics in the past. Differences in presentation of antigenic peptides by polymorphic major histocompatibility complex (MHC) molecules to T-cell receptors (TCR) on T lymphocytes are associated with viral clearance in mammals. Here, we screened and identified a series of peptides derived from RHDV binding to the rabbit MHC class I molecule, RLA-A1. The small, hydrophobic B and F pockets of RLA-A1 capture a peptide motif analogous to that recognized by human class I molecule HLA-A*0201, with more restricted aliphatic anchors at P2 and PΩ positions. Moreover, the rabbit molecule is characterized by an uncommon residue combination of Gly53, Val55, and Glu56, making the 310 helix and the loop between the 310 and α1 helices closer to the α2 helix. A wider A pocket in RLA-A1 can induce a special conformation of the P1 anchor and may play a pivotal role in peptide assembly and TCR recognition. Our study broadens the knowledge of T-cell immunity in domestic animals and also provides useful insights for vaccine development to prevent infectious diseases in rabbits.IMPORTANCE We screened rabbit MHC class I RLA-A1-restricted peptides from the capsid protein VP60 of rabbit hemorrhagic disease virus (RHDV) and determined the structures of RLA-A1 complexed with three peptides, VP60-1, VP60-2, and VP60-10. From the structures, we found that the peptide binding motifs of RLA-A1 are extremely constraining. Thus, there is a generally restricted peptide selection for RLA-A1 compared to that for human HLA-A*0201. In addition, uncommon residues Gly53, Val55, and Glu56 of RLA-A1 are located between the 310 helix and α1 helix, which makes the steric position of the 310 helix in RLA-A1 much closer to the α2 helix than that found in other mammalian MHC class I molecules. This special conformation between the 310 helix and α1 helix plays a pivotal role in rabbit MHC class I assembly. Our results provide new insights into MHC class I molecule assembly and peptide presentation of domestic mammals. Furthermore, these data also broaden our knowledge on T-cell immunity in rabbits and may also provide useful information for vaccine development to prevent infectious diseases in rabbits.


Assuntos
Vírus da Doença Hemorrágica de Coelhos/imunologia , Vírus da Doença Hemorrágica de Coelhos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/química , Peptídeos/imunologia , Animais , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Modelos Moleculares , Peptídeos/genética , Ligação Proteica/imunologia , Conformação Proteica , Coelhos , Receptores de Antígenos de Linfócitos T/metabolismo , Alinhamento de Sequência , Linfócitos T/imunologia
5.
Proc Natl Acad Sci U S A ; 117(25): 14376-14385, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513718

RESUMO

Temporally harmonized elimination of damaged or unnecessary organelles and cells is a prerequisite of health. Under Type 2 inflammatory conditions, human airway epithelial cells (HAECs) generate proferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamines (HpETE-PEs) as proximate death signals. Production of 15-HpETE-PE depends on activation of 15-lipoxygenase-1 (15LO1) in complex with PE-binding protein-1 (PEBP1). We hypothesized that cellular membrane damage induced by these proferroptotic phospholipids triggers compensatory prosurvival pathways, and in particular autophagic pathways, to prevent cell elimination through programmed death. We discovered that PEBP1 is pivotal to driving dynamic interactions with both proferroptotic 15LO1 and the autophagic protein microtubule-associated light chain-3 (LC3). Further, the 15LO1-PEBP1-generated ferroptotic phospholipid, 15-HpETE-PE, promoted LC3-I lipidation to stimulate autophagy. This concurrent activation of autophagy protects cells from ferroptotic death and release of mitochondrial DNA. Similar findings are observed in Type 2 Hi asthma, where high levels of both 15LO1-PEBP1 and LC3-II are seen in HAECs, in association with low bronchoalveolar lavage fluid mitochondrial DNA and more severe disease. The concomitant activation of ferroptosis and autophagy by 15LO1-PEBP1 complexes and their hydroperoxy-phospholipids reveals a pathobiologic pathway relevant to asthma and amenable to therapeutic targeting.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Asma/imunologia , Autofagia/imunologia , Células Epiteliais/patologia , Ferroptose/imunologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Adulto , Animais , Asma/diagnóstico , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Sobrevivência Celular/imunologia , Células Epiteliais/imunologia , Feminino , Técnicas de Inativação de Genes , Humanos , Ácidos Hidroxieicosatetraenoicos/imunologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-13/imunologia , Interleucina-13/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Proteína de Ligação a Fosfatidiletanolamina/genética , Fosfatidiletanolaminas/imunologia , Fosfatidiletanolaminas/metabolismo , Cultura Primária de Células , Ligação Proteica/imunologia , Índice de Gravidade de Doença
6.
Immunity ; 52(5): 742-752, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433947

RESUMO

The cytotoxic activity of myeloid cells is regulated by a balance of signals that are transmitted through inhibitory and activating receptors. The Cluster of Differentiation 47 (CD47) protein, expressed on both healthy and cancer cells, plays a pivotal role in this balance by delivering a "don't eat me signal" upon binding to the Signal-regulatory protein alpha (SIRPα) receptor on myeloid cells. Here, we review the current understanding of the role of the CD47-SIRPα axis in physiological tissue homeostasis and as a promising therapeutic target in, among others, oncology, fibrotic diseases, atherosclerosis, and stem cell therapies. We discuss gaps in understanding and highlight where additional insight will be beneficial to allow optimal exploitation of this myeloid cell checkpoint as a target in human disease.


Assuntos
Antígenos de Diferenciação/imunologia , Antígeno CD47/imunologia , Homeostase/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Humanos , Imunoterapia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Ligação Proteica/imunologia , Receptores Imunológicos/metabolismo
7.
Nat Immunol ; 21(6): 626-635, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424362

RESUMO

The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in intestinal inflammation.


Assuntos
Enzima Desubiquitinante CYLD/metabolismo , Enterocolite/etiologia , Enterocolite/metabolismo , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Citrobacter rodentium , Enzima Desubiquitinante CYLD/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Enterocolite/patologia , Expressão Gênica , Humanos , Interleucina-18/antagonistas & inibidores , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Ligação Proteica/imunologia , Ubiquitinação
8.
Sci Rep ; 10(1): 6573, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313230

RESUMO

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.


Assuntos
Anticorpos/farmacologia , Antígenos CD/genética , Proteínas de Transporte/genética , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Semaforinas/genética , Anticorpos/genética , Anticorpos/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Proteínas de Transporte/imunologia , Eritrócitos/parasitologia , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica/genética , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Merozoítos/genética , Merozoítos/patogenicidade , Plasmodium falciparum/patogenicidade , Ligação Proteica/imunologia , Proteínas de Protozoários/imunologia
9.
Cancer Res ; 80(12): 2537-2549, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32265222

RESUMO

The adaptor protein TNF receptor-associated factor 6 (TRAF6) is a key mediator in inflammation. However, the molecular mechanisms controlling its activity and stability in cancer progression remain unclear. Here we show that death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) inhibits the proinflammatory signaling pathway by targeting TRAF6 for degradation, thereby suppressing inflammatory signaling-mediated tumor growth and metastasis in advanced cervical cancer cells. DRAK1 bound directly to the TRAF domain of TRAF6, preventing its autoubiquitination by interfering with homo-oligomerization, eventually leading to autophagy-mediated degradation of TRAF6. Depletion of DRAK1 in cervical cancer cells resulted in markedly increased levels of TRAF6 protein, promoting activation of the IL1ß signaling-associated pathway and proinflammatory cytokine production. DRAK1 was specifically underexpressed in metastatic cervical cancers and inversely correlated with TRAF6 expression in mouse xenograft model tumor tissues and human cervical tumor tissues. Collectively, our findings highlight DRAK1 as a novel antagonist of inflammation targeting TRAF6 for degradation that limits inflammatory signaling-mediated progression of advanced cervical cancer. SIGNIFICANCE: Serine/threonine kinase DRAK1 serves a unique role as a novel negative regulator of the inflammatory signaling mediator TRAF6 in cervical cancer progression.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Estadiamento de Neoplasias , Ligação Proteica/imunologia , Domínios Proteicos , Multimerização Proteica/imunologia , Estabilidade Proteica , Proteólise , Transdução de Sinais/imunologia , Análise Serial de Tecidos , Ubiquitinação/imunologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Commun ; 11(1): 1314, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161266

RESUMO

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.


Assuntos
Apresentação do Antígeno , Dipeptídeos/metabolismo , Antígeno HLA-A2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dipeptídeos/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/ultraestrutura , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
11.
Mol Biol Rep ; 47(3): 2137-2147, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32080807

RESUMO

The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VHH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 108/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.


Assuntos
Afinidade de Anticorpos , Antineoplásicos Imunológicos/farmacologia , Antagonistas do Receptor de Endotelina B/farmacologia , Receptor de Endotelina B/metabolismo , Anticorpos de Domínio Único/farmacologia , Afinidade de Anticorpos/imunologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Melanoma/tratamento farmacológico , Melanoma/imunologia , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Receptor de Endotelina B/imunologia , Análise de Sequência de DNA , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação
12.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32075930

RESUMO

The major histocompatibility complex (MHC) class I region of humans, chimpanzees (Pan troglodytes), and bonobos (Pan paniscus) is highly similar, and orthologues of HLA-A, -B, and -C are present in both Pan species. Based on functional characteristics, the different HLA-A allotypes are classified into different supertypes. One of them, the HLA A03 supertype, is widely distributed among different human populations. All contemporary known chimpanzee and bonobo MHC class I A allotypes cluster genetically into one of the six HLA-A families, the HLA-A1/A3/A11/A30 family. We report here that the peptide-binding motif of the Patr-A*05:01 allotype, which is commonly present in a cohort of western African chimpanzees, has a strong preference for binding peptides with basic amino acids at the carboxyl terminus. This phenomenon is shared with the family members of the HLA A03 supertype. Based on the chemical similarities in the peptide-binding pocket, we inferred that the preference for binding peptides with basic amino acids at the carboxyl terminus is widely present among the human, chimpanzee, and bonobo MHC-A allotypes. Subsequent in silico peptide-binding predictions illustrated that these allotypes have the capacity to target conserved parts of the proteome of human immunodeficiency virus type 1 (HIV-1) and the simian immunodeficiency virus SIVcpz.IMPORTANCE Most experimentally infected chimpanzees seem to control an HIV-1 infection and are therefore considered to be relatively resistant to developing AIDS. Contemporary free-ranging chimpanzees may carry SIVcpz, and there is evidence for AIDS-like symptoms in these free-ranging animals, whereas SIV infections in bonobos appear to be absent. In humans, the natural control of an HIV-1 infection is strongly associated with the presence of particular HLA class I allotypes. The ancestor of the contemporary living chimpanzees and bonobos survived a selective sweep targeting the MHC class I repertoire. We have put forward a hypothesis that this may have been caused by an ancestral retroviral infection similar to SIVcpz. Characterization of the relevant MHC allotypes may contribute to understanding the shaping of their immune repertoire. The abundant presence of MHC-A allotypes that prefer peptides with basic amino acids at the C termini suggests that these molecules may contribute to the control of retroviral infections in humans, chimpanzees, and bonobos.


Assuntos
Genes MHC Classe I/imunologia , Antígeno HLA-A3/imunologia , Primatas/imunologia , Alelos , Sequência de Aminoácidos , Animais , HIV-1/imunologia , Antígeno HLA-A3/metabolismo , Antígenos de Histocompatibilidade , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Pan paniscus/imunologia , Pan troglodytes/imunologia , Peptídeos/metabolismo , Filogenia , Ligação Proteica/imunologia , Infecções por Retroviridae/imunologia , Vírus da Imunodeficiência Símia/imunologia
13.
Int J Med Sci ; 17(2): 161-169, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32038099

RESUMO

Rationale: Placental-like chondroitin sulfate A (pl-CSA) is known to be exclusively synthesized in multiple cancer tissues and associated with disease severity. Here, we aimed to assess whether pl-CSA is released into bio-fluids and can serve as a cancer biomarker. Methods: A novel ELISA was developed to analyse pl-CSA content in bio-fluids using pl-CSA binding protein and an anti-pl-CSA antibody. Immunohistochemical staining of tissue chips was used as the gold standard control. Results: The developed ELISA method was specific and sensitive (1.22 µg/ml). The pl-CSA content was significantly higher in lysates and supernatants of cancer cell lines than in those of normal cell lines, in plasma from mouse cancer models than in that from control mice, and in plasma from patients with oesophageal, cervical, ovarian, or lung cancer than in that from healthy controls. Similar to the tissue chip analysis, which showed a significant difference in pl-CSA positivity between cancer tissues and normal adjacent tissues, the plasma pl-CSA analysis had 100% sensitivity and specificity for differentiating oesophageal and lung cancer patients from healthy controls. Importantly, in oesophageal and lung cancer patients, the pl-CSA content was significantly higher in late-stage disease than in early-stage disease, and it dramatically decreased after surgical resection of the tumour. Conclusion: These data indicate a direct link between plasma pl-CSA content and tumour presence, indicating that plasma pl-CSA may be a non-invasive biomarker with clinical applicability for the screening and surveillance of patients with multiple types of solid tumours.


Assuntos
Sulfatos de Condroitina/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Neoplasias/genética , Animais , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/imunologia , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/imunologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/patologia , Placenta/metabolismo , Gravidez , Ligação Proteica/imunologia
14.
Cell ; 180(3): 471-489.e22, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004464

RESUMO

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.


Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Antígenos CD4/metabolismo , Células CHO , Estudos de Coortes , Cricetulus , Epitopos/imunologia , Feminino , Células HEK293 , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mutação , Ligação Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
15.
Immunology ; 160(1): 78-89, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32107769

RESUMO

Annexins are well-known Ca2+ phospholipid-binding proteins, which have a wide variety of cellular functions. The role of annexin A1 (AnxA1) in the innate immune system has focused mainly on the anti-inflammatory and proresolving properties through its binding to the formyl-peptide receptor 2 (FPR2)/ALX receptor. However, studies suggesting an intracellular role of AnxA1 are emerging. In this study, we aimed to understand the role of AnxA1 for interleukin (IL)-1ß release in response to activators of the nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3) inflammasome. Using AnxA1 knockout mice, we observed that AnxA1 is required for IL-1ß release in vivo and in vitro. These effects were due to reduction of transcriptional levels of IL-1ß, NLRP3 and caspase-1, a step called NLRP3 priming. Moreover, we demonstrate that AnxA1 co-localize and directly bind to NLRP3, suggesting the role of AnxA1 in inflammasome activation is independent of its anti-inflammatory role via FPR2. Therefore, AnxA1 regulates NLRP3 inflammasome priming and activation in a FPR2-independent manner.


Assuntos
Anexina A1/metabolismo , Inflamassomos/imunologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Administração Intranasal , Animais , Cartilagem Articular , Caspase 1/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Gota/induzido quimicamente , Gota/imunologia , Gota/patologia , Humanos , Inflamassomos/metabolismo , Injeções Intra-Articulares , Pulmão/imunologia , Pulmão/patologia , Macrófagos , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Ligação Proteica/imunologia , Dióxido de Silício/administração & dosagem , Dióxido de Silício/toxicidade , Silicose/imunologia , Silicose/patologia , Transcrição Genética/imunologia , Ácido Úrico/administração & dosagem , Ácido Úrico/toxicidade
16.
Immunogenetics ; 72(3): 143-153, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31970435

RESUMO

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos HLA-A/imunologia , Antígeno HLA-A1/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Antígenos HLA-A/química , Antígeno HLA-A1/química , Humanos , Modelos Moleculares , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica
17.
EBioMedicine ; 52: 102632, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31981983

RESUMO

BACKGROUND: Small cell lung cancer (SCLC) has a poor prognosis, and its treatment options are limited. Delta-like protein 3 (DLL3) is expressed specifically in SCLC and is considered a promising therapeutic target for patients with this disease. Rovalpituzumab tesirine (Rova-T) was the first antibody-drug conjugate targeting DLL3. Although Rova-T development was unfortunately terminated, DLL3 remains an ideal target for SCLC. Near infrared photoimmunotherapy (NIR-PIT) is a new form of cancer treatment that employs an antibody-photosensitiser conjugate followed by NIR light exposure and damage target cells specifically. In this study, we demonstrate DLL3-targeted NIR-PIT to develop a novel molecularly targeted treatment for SCLC. METHODS: The anti-DLL3 monoclonal antibody rovalpituzumab was conjugated to an IR700 photosensitiser (termed 'rova-IR700'). SCLC cells overexpressing DLL3 as well as non-DLL3-expressing controls were incubated with rova-IR700 and then exposed to NIR-light. Next, mice with SCLC xenografts were injected with rova-IR700 and irradiated with NIR-light. FINDINGS: DLL3-overexpressing cells underwent immediate destruction upon NIR-light exposure, whereas the control cells remained intact. The xenograft in mice treated with rova-IR700 and NIR-light shrank markedly, whereas neither rova-IR700 injection nor NIR-light irradiation alone affected tumour size. INTERPRETATION: Our data suggest that targeting of DLL3 using NIR-PIT could be a novel and promising treatment for SCLC. FUNDING: Research supported by grants from the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, JSPS), Medical Research Encouragement Prize of The Japan Medical Association, The Nitto Foundation, Kanae Foundation for the Promotion of Medical Science.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luz , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fototerapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Humanos , Imunoconjugados/farmacologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Fototerapia/métodos , Ligação Proteica/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Int Immunopharmacol ; 80: 106141, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31982825

RESUMO

Neuroinflammation significantly contributes to brain injury and neurological deterioration following intracerebral hemorrhage (ICH). MicroRNA-152(miR-152) was reported to be downregulated in ICH patients and to possess anti-inflammatory properties in other diseases. In this study, we aimed to explore the role of miR-152 in ICH, and the underlying mechanisms, using a collagenase-induced rat ICH model and hemin-exposure as a cell model. We first confirmed that miR-152 was consistently downregulated in both models. Overexpression of miR-152 in microglial BV2 cells reduced hemin-induced inflammatory response and reactive oxygen species (ROS) generation, thus protecting co-cultured neuronal HT22 cells. Moreover, overexpression of miR-152 by intracerebroventricular lentivirus injection in ICH rats significantly alleviated neurodecifits, brain edema, and hematoma. These changes were associated with a marked reduction in ICH-induced neuronal death, as detected by co-staining of NeuN and TUNEL, and ICH-induced neuroinflammation, as revealed by inflammatory cytokine levels as well as by the number of Iba1 positive-stained cells in the perihematomal region. Mechanistically, miR-152 significantly inhibited ICH-induced TXNIP expression, and its overexpression blocked the interaction between TXNIP and NOD-like receptor pyrin domain containing 3(NLRP3), thus inhibiting NLRP3-driven inflammasome activation to attenuate neuroinflammation in vivo and in vitro. Moreover, the results of si-TXNIP transfection further confirmed that TXNIP inhibition was involved in the reduction of NLRP3 inflammasome activation by the overexpression of miR-152. Collectively, the present study demonstrates that miR-152 confers protection against ICH-induced neuroinflammation and brain injury by inhibiting TXNIP-mediated NLRP3 inflammasome activation, indicating a potential strategy for ICH treatment.


Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Hemorragia Cerebral Intraventricular/genética , Inflamassomos/imunologia , MicroRNAs/metabolismo , Tiorredoxinas/genética , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Hemorragia Cerebral Intraventricular/induzido quimicamente , Hemorragia Cerebral Intraventricular/imunologia , Hemorragia Cerebral Intraventricular/patologia , Ventrículos Cerebrais/imunologia , Ventrículos Cerebrais/patologia , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Técnicas de Silenciamento de Genes , Hemina/imunologia , Humanos , Inflamassomos/metabolismo , Injeções Intraventriculares , Masculino , Camundongos , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Colagenase Microbiana/administração & dosagem , Colagenase Microbiana/toxicidade , Microglia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neurônios , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Interferente Pequeno/metabolismo , Ratos , Tiorredoxinas/metabolismo
19.
J Exp Med ; 217(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31757867

RESUMO

We previously generated a panel of human monoclonal antibodies (mAbs) against Zika virus (ZIKV) and identified one, ZIKV-116, that shares germline usage with mAbs identified in multiple donors. Here we show that ZIKV-116 interferes with ZIKV infection at a post-cellular attachment step by blocking viral fusion with host membranes. ZIKV-116 recognizes the lateral ridge of envelope protein domain III, with one critical residue varying between the Asian and African strains responsible for differential binding affinity and neutralization potency (E393D). ZIKV-116 also binds to and cross-neutralizes some dengue virus serotype 1 (DENV1) strains, with genotype-dependent inhibition explained by variation in a domain II residue (R204K) that potentially modulates exposure of the distally located, partially cryptic epitope. The V-J reverted germline configuration of ZIKV-116 preferentially binds to and neutralizes an Asian ZIKV strain, suggesting that this epitope may optimally induce related B cell clonotypes. Overall, these studies provide a structural and molecular mechanism for a cross-reactive mAb that uniquely neutralizes ZIKV and DENV1.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Domínios Proteicos/imunologia , Proteínas do Envelope Viral/química , Zika virus/imunologia , Aedes , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Linhagem Celular Tumoral , Chlorocebus aethiops , Reações Cruzadas , Cristalografia por Raios X , Dengue/imunologia , Dengue/virologia , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química , Ligação Proteica/imunologia , Conformação Proteica , Células Vero , Proteínas do Envelope Viral/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
20.
Mol Immunol ; 118: 110-116, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31869742

RESUMO

The accurate transmission of signals by the canonical ERK1/2 kinase pathway critically relies on the proper assembly of an intricate multiprotein complex by the scaffold protein Shoc2. However, the details of the mechanism by which Shoc2 guides ERK1/2 signals are not clear, in part, due to the lack of research tools targeting specific protein binding moieties of Shoc2. We report generation and characterization of single domain antibodies against human Shoc2 using a universal synthetic library of humanized nanobodies. Our results identify eight synthetic single-domain antibodies and show that two evaluated antibodies have binding affinities to Shoc2 in the nanomolar range. High affinity antibodies were uniquely suited for the analysis of the Shoc2 complex assembly. Selected single-domain antibodies were also functional in intracellular assays. This study illustrates that Shoc2 single-domain antibodies can be used to understand functional mechanisms governing complex multiprotein signaling modules and have promise in application for therapies that require modulation of the ERK1/2-associated diseases.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Transdução de Sinais/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Ligação Proteica/imunologia
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