RESUMO
Identifying human leukocyte antigen (HLA) haplotype-homozygous donors for the generation of induced pluripotent stem (iPS) cell lines permits the construction of biobanks immunologically compatible with significant numbers of individuals for use in therapy. However, two questions must be addressed to create such a bank: how many cell lines are necessary to match most of the recipient population and how many people should be tested to find these donors? In Japan and the UK, 50 and 100 distinct HLA-A, -B, and -DRB1 triple-homozygous haplotypes would cover 90% of those populations, respectively. Using data from the Brazilian National Registry of Bone Marrow Donors (REDOME), encompassing 4,017,239 individuals, we identified 1,906 distinct triple-homozygous HLA haplotypes. In Brazil, 559 triple-homozygous cell lines cover 95% of the population, and 3.8 million people would have to be screened. Finally, we show the contribution of the 30 most frequent triple-homozygous HLA haplotypes in Brazil to populations of different countries.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Brasil , Células-Tronco Pluripotentes Induzidas/metabolismo , Antígenos HLA/metabolismo , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Doadores de Tecidos , Antígenos de Histocompatibilidade Classe I/metabolismo , Haplótipos/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Alelos , Frequência do GeneRESUMO
Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.
Assuntos
Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunoglobulinas/metabolismo , Interferon gama/imunologia , Glicoproteínas de Membrana/metabolismo , Salmo salar/imunologia , Baço/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Biomarcadores/metabolismo , Doenças dos Peixes/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Piscirickettsia , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/veterinária , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Antígeno CD83RESUMO
Schistosomiasis remains a serious health issue nowadays for an estimated one billion people in 79 countries around the world. Great efforts have been made to identify good vaccine candidates during the last decades, but only three molecules reached clinical trials so far. The reverse vaccinology approach has become an attractive option for vaccine design, especially regarding parasites like Schistosoma spp. that present limitations for culture maintenance. This strategy also has prompted the construction of multi-epitope based vaccines, with great immunological foreseen properties as well as being less prone to contamination, autoimmunity, and allergenic responses. Therefore, in this study we applied a robust immunoinformatics approach, targeting S. mansoni transmembrane proteins, in order to construct a chimeric antigen. Initially, the search for all hypothetical transmembrane proteins in GeneDB provided a total of 584 sequences. Using the PSORT II and CCTOP servers we reduced this to 37 plasma membrane proteins, from which extracellular domains were used for epitope prediction. Nineteen common MHC-I and MHC-II binding epitopes, from eight proteins, comprised the final multi-epitope construct, along with suitable adjuvants. The final chimeric multi-epitope vaccine was predicted as prone to induce B-cell and IFN-γ based immunity, as well as presented itself as stable and non-allergenic molecule. Finally, molecular docking and molecular dynamics foresee stable interactions between the putative antigen and the immune receptor TLR 4. Our results indicate that the multi-epitope vaccine might stimulate humoral and cellular immune responses and could be a potential vaccine candidate against schistosomiasis.
Assuntos
Antígenos de Helmintos/imunologia , Linfócitos B/imunologia , Epitopos Imunodominantes/imunologia , Informática Médica/métodos , Proteínas de Membrana/imunologia , Proteínas Recombinantes de Fusão/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Vacinas/imunologia , Animais , Antígenos de Helmintos/genética , Biologia Computacional , Mapeamento de Epitopos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Celular , Imunidade Humoral , Epitopos Imunodominantes/genética , Interferon gama/metabolismo , Ativação Linfocitária , Proteínas de Membrana/genética , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Receptor 4 Toll-Like/metabolismo , Vacinas/genética , Vacinas de Subunidades Antigênicas , VacinologiaRESUMO
This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; â¼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides' 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.
Assuntos
Eritrócitos/parasitologia , Vacinas Antimaláricas/farmacologia , Plasmodium falciparum/patogenicidade , Animais , Antígenos de Protozoários/química , Aotidae , Proteínas de Transporte/química , Epitopos , Eritrócitos/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/metabolismo , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Peptídeos/imunologia , Peptídeos/metabolismo , Proteínas de Protozoários/química , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/farmacologiaRESUMO
Bovine tuberculosis (bTB) is caused by Mycobacterium bovis and disseminated worldwide. In Argentina, the highest prevalence occurs in dairy areas. BoLA DRB3.2 is related to the adaptive immunity in mycobacterial infections. Genetic polymorphisms of this marker have been associated with resistance or susceptibility to bovine diseases. We evaluated the association between BoLA DRB3.2 polymorphisms and bTB pathology scores in dairy and beef cattle breeds of Argentina. Most bovines exhibited visible lesions compatible with tuberculosis and, furthermore, 150 (85.7%) were also positive by bacteriology. A pathology index showed a variable degree of disease, from 3 to 76 (median pathology score = 9 (IQR: 7-15)). Thirty-five BoLA DRB3.2 alleles were identified with an associated frequency from 16% to 0.3%, distributed 73% (n = 128) in heterozygosis and 27% (n = 47) in homozygosis, with 12 BoLA DRB3.2 alleles (*0101, *1101, *1501, *0201, *2707 *1001, *1002, *1201, *14011, *0501 *0902 and *0701) representing the 74.7% of the population variability. A functional analysis grouped them in 4 out of 5 clusters (A-D), suggesting a functional overlapping. Among the 90 identified genotypes, *1101/*1101, *1101/*1501 and *0101/*0101 were the most frequent (10%, 8.9% and 8.9%, respectively). No association was detected between the pathology scores and a specific DRB3.2 allele (p > .05). Animals infected with M. bovis spoligotype SB0153 showed a significantly higher pathology score than those affected by the spoligotype SB0145 (p = .018). Furthermore, the Aberdeen Angus breed exhibited highest pathological scores (p < .0001), which were associated with disseminated lesion, thus suggesting that the host component could be important to the disease progression.
Assuntos
Genótipo , Antígenos de Histocompatibilidade Classe II/genética , Polimorfismo Genético , Tuberculose Bovina/patologia , Alelos , Animais , Argentina , Bovinos , Éxons , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Masculino , Nucleotídeos , Tuberculose Bovina/genéticaRESUMO
T cell-mediated immune response plays a crucial role in controlling Trypanosoma cruzi infection and parasite burden, but it is also involved in the clinical onset and progression of chronic Chagas' disease. Therefore, the study of T cells is central to the understanding of the immune response against the parasite and its implications for the infected organism. The complexity of the parasite-host interactions hampers the identification and characterization of T cell-activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients' T cells specificity. Fifty T. cruzi peptides predicted to bind a broad range of class I and II HLA molecules were selected for in vitro screening against PBMC samples from a cohort of chronic Chagas' disease patients, using IFN-γ secretion as a readout. Seven of these peptides were shown to activate this type of T cell response, and four out of these contain class I and II epitopes that, to our knowledge, are first described in this study. The remaining three contain sequences that had been previously demonstrated to induce CD8+ T cell response in Chagas' disease patients, or bind HLA-A*02:01, but are, in this study, demonstrated to engage CD4+ T cells. We also assessed the degree of differentiation of activated T cells and looked into the HLA variants that might restrict the recognition of these peptides in the context of human T. cruzi infection.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Epitopos de Linfócito T/imunologia , Trypanosoma cruzi/imunologia , Antígenos de Protozoários/metabolismo , Argentina , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/imunologia , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/parasitologia , Simulação por Computador , ELISPOT , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Celular , Memória Imunológica , Testes de Liberação de Interferon-gama , Ativação Linfocitária , Masculino , Trypanosoma cruzi/metabolismoRESUMO
Estimating peptide-major histocompatibility complex (pMHC) binding using structural computational methods has an impact on understanding overall immune function triggering adaptive immune responses in MHC class II molecules. We developed a strategy for optimizing pMHC structure interacting with water molecules and for calculating the binding energy of receptor + ligand systems, such as HLA-DR1 + HA, HLA-DR1 + CLIP, HLA-DR2 + MBP, and HLA-DR3 + CLIP, as well as a monosubstitution panel. Taking pMHC's structural properties, we assumed that ΔH â« -TΔS would generate a linear model for estimating relative free energy change, using three semiempirical quantum methods (PM6, PM7, and FMO-SCC-DFTB3) along with the implicit solvent models, and considering proteins in neutral and charged states. Likewise, we confirmed our approach's effectiveness in calculating binding energies having high correlation with experimental data and low root-mean-square error (<2 kcal/mol). All in all, our pipeline differentiates weak from strong peptide binders as a reliable method for studying pMHC interactions.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Modelos Moleculares , Peptídeos/metabolismo , Teoria Quântica , Antígenos de Histocompatibilidade Classe II/química , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
The set of peptides presented on a cell's surface by MHC molecules is known as the immunopeptidome. Current mass spectrometry technologies allow for identification of large peptidomes, and studies have proven these data to be a rich source of information for learning the rules of MHC-mediated antigen presentation. Immunopeptidomes are usually poly-specific, containing multiple sequence motifs matching the MHC molecules expressed in the system under investigation. Motif deconvolution -the process of associating each ligand to its presenting MHC molecule(s)- is therefore a critical and challenging step in the analysis of MS-eluted MHC ligand data. Here, we describe NNAlign_MA, a computational method designed to address this challenge and fully benefit from large, poly-specific data sets of MS-eluted ligands. NNAlign_MA simultaneously performs the tasks of (1) clustering peptides into individual specificities; (2) automatic annotation of each cluster to an MHC molecule; and (3) training of a prediction model covering all MHCs present in the training set. NNAlign_MA was benchmarked on large and diverse data sets, covering class I and class II data. In all cases, the method was demonstrated to outperform state-of-the-art methods, effectively expanding the coverage of alleles for which accurate predictions can be made, resulting in improved identification of both eluted ligands and T-cell epitopes. Given its high flexibility and ease of use, we expect NNAlign_MA to serve as an effective tool to increase our understanding of the rules of MHC antigen presentation and guide the development of novel T-cell-based therapeutics.
Assuntos
Algoritmos , Biologia Computacional/métodos , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Motivos de Aminoácidos , Animais , Benchmarking , Bovinos , Linhagem Celular , Bases de Dados de Proteínas , Conjuntos de Dados como Assunto , Humanos , Ligantes , Aprendizado de Máquina , Espectrometria de Massas , Peptídeos/metabolismo , Ligação ProteicaRESUMO
Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Monócitos/imunologia , RNA Bacteriano/imunologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella abortus/genética , Brucella abortus/imunologia , Brucella abortus/fisiologia , Brucelose/imunologia , Brucelose/microbiologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação para Baixo/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , RNA Bacteriano/genética , Células THP-1RESUMO
Predicting the binding affinity of peptides able to interact with major histocompatibility complex (MHC) molecules is a priority for researchers working in the identification of novel vaccines candidates. Most available approaches are based on the analysis of the sequence of peptides of known experimental affinity. However, for MHC class II receptors, these approaches are not very accurate, due to the intrinsic flexibility of the complex. To overcome these limitations, we propose to estimate the binding affinity of peptides bound to an MHC class II by averaging the score of the configurations from finite-temperature molecular dynamics simulations. The score is estimated for 18 different scoring functions, and we explored the optimal manner for combining them. To test the predictions, we considered eight peptides of known binding affinity. We found that six scoring functions correlate with the experimental ranking of the peptides significantly better than the others. We then assessed a set of techniques for combining the scoring functions by linear regression and logistic regression. We obtained a maximum accuracy of 82% for the predicted sign of the binding affinity using a logistic regression with optimized weights. These results are potentially useful to improve the reliability of in silico protocols to design high-affinity binding peptides for MHC class II receptors.