RESUMO
Recent advances in proteomics and mass-spectrometry have widely expanded the detectable peptide repertoire presented by major histocompatibility complex (MHC) molecules on the cell surface, collectively known as the immunopeptidome. Finely characterizing the immunopeptidome brings about important basic insights into the mechanisms of antigen presentation, but can also reveal promising targets for vaccine development and cancer immunotherapy. This report describes a number of practical and efficient approaches to analyze immunopeptidomics data, discussing the identification of meaningful sequence motifs in various scenarios and considering current limitations. Guidelines are provided for the filtering of false hits and contaminants, and to address the problem of motif deconvolution in cell lines expressing multiple MHC alleles, both for the MHC class I and class II systems. Finally, it is demonstrated how machine learning can be readily employed by non-expert users to generate accurate prediction models directly from mass-spectrometry eluted ligand data sets.
Assuntos
Motivos de Aminoácidos/imunologia , Biologia Computacional/métodos , Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/metabolismo , Epitopos/análise , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/imunologiaRESUMO
Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.
Assuntos
Anaplasma marginale/imunologia , Anaplasmose/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Imunidade Celular , Imunidade Humoral , Motivos de Aminoácidos/imunologia , Anaplasmose/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Bovinos , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Epitopos/genética , Eritrócitos/imunologia , Eritrócitos/virologia , Feminino , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Mediadores da Inflamação/imunologia , Camundongos , Peptídeos/síntese química , Peptídeos/imunologia , Baço/citologia , Baço/imunologia , Transcrição GênicaRESUMO
This study tested the hypothesis that the IFN-γ R1 287-YVSLI-91 intracellular motif regulates its endocytosis. IFN-γ exerts its biological activities by interacting with a specific cell-surface RC composed of two IFN-γ R1 and two IFN-γ R2 chains. Following IFN-γ binding and along with the initiation of signal transduction, the ligand and IFN-γ R1 are internalized. Two major types of consensus-sorting signals are described in receptors, which are rapidly internalized from the plasma membrane to intracellular compartments: tyrosine-based and dileucine-based internalization motifs. Transfection of HEK 293 cells and IFN-γ R1-deficient fibroblasts with WT and site-directed, mutagenesis-generated mutant IFN-γ R1 expression vectors helped us to identify region IFN-γ R1 287-YVSLI-291 as the critical domain required for IFN-γ-induced IFN-γ R1 internalization and Y287 and LI290-291 as part of a common structure essential for receptor endocytosis and function. This new endocytosis motif, YxxLI, shares characteristics of tyrosine-based and dileucine-based internalization motifs and is highly conserved in IFN-γ Rs across species. The IFN-γ R1 270-LI-271 dileucine motif, previously thought to be involved in this receptor endocytosis, showed to be unnecessary for receptor endocytosis.
Assuntos
Endocitose/imunologia , Leucina/química , Leucina/metabolismo , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Sequência Conservada/imunologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/fisiologia , Receptores de Interferon/genética , Tirosina/química , Tirosina/metabolismo , Receptor de Interferon gamaRESUMO
The N-terminal half or toxic fragment of Bacillus thuringiensis Cry proteins is comprised of three structural domains. In a previous paper, we showed that this region plays an important role in the immunogenicity of the B. thuringiensis Cry proteins. Due to this ability and along with their stability it is worthy of investigating whether this region has carrier potential. To approach this, an eight amino acid hydrophobic motif in alpha-helix 7 of wild-type (WT) Cry1A toxins was exchanged for a diphtheria toxin epitope (DTB). The resultant recombinant toxins were tested for their ability to induce specific anti-Cry and anti-diphtheria toxin antibodies in mice after intraperitoneal and nasal immunization. We found that recombinant Cry1A toxins retained their ability to induce serum and mucosal anti-Cry Ab as well as IgG subclasses, although with a varied magnitude. By the systemic route, the effect of the amino acid substitution in the ratio of the IgG1/IgG2a Ab, leading in some sites toward IgG1 or IgG2a is more evident. Interestingly, mice produced specific anti-DTB IgG, and IgA after intranasal immunization. Together, our results support and show the immunogenic properties of the WT Cry1A toxins as well as its carrier potential for a DTB.