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1.
Mol Immunol ; 120: 146-163, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32126449

RESUMO

Buruli ulcer is an emerging tissue-necrosis infectious disease, caused by the pathogen Mycobacterium ulcerans, leading to permanent deformity if untreated. Despite this debilitating condition, no specific disease-modifying therapeutics or vaccination is available to date. Therefore, we aimed to design an effective multi-epitope vaccine against M. ulcerans using vaccinomics approach. Briefly, the highest antigenic PE-PGRS protein was selected from which the promiscuous T- and B-cell epitopes were predicted. After rigorous assessment, 15 promising T- and B-cell epitopes were selected. The identified T-cell epitopes showed marked interactions towards their HLA-binding alleles and provided 99.8 % world population coverage. Consequently, a vaccine chimera was designed by connecting these epitopes with suitable linkers and LprG adjuvant. The vaccine construct was highly antigenic, immunogenic and non-allergenic; hence, subjected to homology modelling. The molecular docking and dynamics simulation revealed a strong and stable interaction between vaccine and toll-like receptor 2. The binding energy and dissociation constant were -15.3 kcal/mol and 5.9 × 10-12 M, respectively. The computer-simulated immune responses showed abundance of immunoglobulins, increased interferon-γ production, and macrophages activation which are crucial for immune response against M. ulcerans. Furthermore, disulfide bridging and in silico cloning were also performed. These results suggest that the vaccine, if validated experimentally, will be a promising candidate against M. ulcerans and prevent Buruli ulcer disease.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Mycobacterium ulcerans/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Úlcera de Buruli/imunologia , Úlcera de Buruli/prevenção & controle , Simulação por Computador , Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Proteínas de Membrana/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mycobacterium ulcerans/química , Mycobacterium ulcerans/genética , Engenharia de Proteínas , Vacinas de Subunidades/química , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
2.
PLoS One ; 15(2): e0228177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040522

RESUMO

BACKGROUND: Sterile protection against malaria, most likely mediated by parasite-specific CD8+ T cells, has been achieved by attenuated sporozoite vaccination of animals as well as malaria-naïve and malaria-exposed subjects. The circumsporozoite protein (CSP)-based vaccine, RTS,S, shows low efficacy partly due to limited CD8+ T cell induction, and inclusion of such epitopes could improve RTS,S. This study assessed 8-10mer CSP peptide epitopes, present in predicted or previously positive P. falciparum 3D7 CSP 15mer overlapping peptide pools, for their ability to induce CD8+ T cell IFN-γ responses in natural malaria-exposed subjects. METHODS: Cryopreserved PBMCs from nine HLA-typed subjects were stimulated with 23 8-10mer CSP peptides from the 3D7 parasite in IFN-É£ ELISpot assays. The CD8+ T cell specificity of IFN-γ responses was confirmed in ELISpot assays using CD8+ T cell-enriched PBMC fractions after CD4+ cell depletion. RESULTS: Ten of 23 peptide epitopes elicited responses in whole PBMCs from five of the nine subjects. Four peptides tested positive in CD8+ T cell-enriched PBMCs from two previously positive responders and one new subject. All four immunodominant peptides are restricted by globally common HLA supertypes (A02, A03, B07) and mapped to regions of the CSP antigen with limited or no reported polymorphism. Association of these peptide-specific responses with anti-malarial protection remains to be confirmed. CONCLUSIONS: The relatively conserved nature of the four identified epitopes and their binding to globally common HLA supertypes makes them good candidates for inclusion in potential multi-epitope malaria vaccines.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/efeitos dos fármacos , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia
3.
Proc Natl Acad Sci U S A ; 117(6): 3063-3073, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31974305

RESUMO

The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4+ T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5-mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.


Assuntos
Doença Celíaca , Antígenos HLA-DQ , Receptores de Antígenos de Linfócitos T , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/metabolismo , Doença Celíaca/genética , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Linhagem Celular , Cristalografia por Raios X , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Glutens/química , Glutens/imunologia , Glutens/metabolismo , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
4.
BMC Genomics ; 20(Suppl 9): 921, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31874646

RESUMO

BACKGROUND: The sequence diversity of dengue virus (DENV) is one of the challenges in developing an effective vaccine against the virus. Highly conserved, serotype-specific (HCSS), immune-relevant DENV sequences are attractive candidates for vaccine design, and represent an alternative to the approach of selecting pan-DENV conserved sequences. The former aims to limit the number of possible cross-reactive epitope variants in the population, while the latter aims to limit the cross-reactivity between the serotypes to favour a serotype-specific response. Herein, we performed a large-scale systematic study to map and characterise HCSS sequences in the DENV proteome. METHODS: All reported DENV protein sequence data for each serotype was retrieved from the NCBI Entrez Protein (nr) Database (txid: 12637). The downloaded sequences were then separated according to the individual serotype proteins by use of BLASTp search, and subsequently removed for duplicates and co-aligned across the serotypes. Shannon's entropy and mutual information (MI) analyses, by use of AVANA, were performed to measure the diversity within and between the serotype proteins to identify HCSS nonamers. The sequences were evaluated for the presence of promiscuous T-cell epitopes by use of NetCTLpan 1.1 and NetMHCIIpan 3.2 server for human leukocyte antigen (HLA) class I and class II supertypes, respectively. The predicted epitopes were matched to reported epitopes in the Immune Epitope Database. RESULTS: A total of 2321 nonamers met the HCSS selection criteria of entropy < 0.25 and MI > 0.8. Concatenating these resulted in a total of 337 HCSS sequences. DENV4 had the most number of HCSS nonamers; NS5, NS3 and E proteins had among the highest, with none in the C and only one in prM. The HCSS sequences were immune-relevant; 87 HCSS sequences were both reported T-cell epitopes/ligands in human and predicted epitopes, supporting the accuracy of the predictions. A number of the HCSS clustered as immunological hotspots and exhibited putative promiscuity beyond a single HLA supertype. The HCSS sequences represented, on average, ~ 40% of the proteome length for each serotype; more than double of pan-DENV sequences (conserved across the four serotypes), and thus offer a larger choice of sequences for vaccine target selection. HCSS sequences of a given serotype showed significant amino acid difference to all the variants of the other serotypes, supporting the notion of serotype-specificity. CONCLUSION: This work provides a catalogue of HCSS sequences in the DENV proteome, as candidates for vaccine target selection. The methodology described herein provides a framework for similar application to other pathogens.


Assuntos
Vírus da Dengue/imunologia , Proteínas Virais/química , Proteínas Virais/imunologia , Sequência de Aminoácidos , Sequência Conservada , Bases de Dados de Proteínas , Vacinas contra Dengue/imunologia , Epitopos de Linfócito T/química , Evolução Molecular , Proteoma , Sorogrupo
5.
Yi Chuan ; 41(11): 1041-1049, 2019 Nov 20.
Artigo em Chinês | MEDLINE | ID: mdl-31735706

RESUMO

Accurate epitope presentation prediction is a key procedure in tumour immunotherapies based on neoantigen for targeting T cell specific epitopes. Epitopes identified by mass spectrometry (MS) is valuable to train an epitope presentation prediction model. In spite of the accelerating accumulation of MS data, the number of epitopes that match most of human leukocyte antigens (HLAs) is relatively small, which makes it difficult to build a reliable prediction model. Therefore, this research attempted to use the transfer learning method to train a model to learn common features among the mixed allele specific epitopes. Then based on this pre-trained model, we used the allele-specific epitopes to train the final epitope presentation prediction model, termed Pluto. The average 0.1% positive predictive value (PPV) of Pluto outperformed the prediction model without pretraining with a margin of 0.078 on the same validation dataset. When evaluating Pluto on external HLA eluted ligand datasets, Pluto achieved an averaged 0.1% PPV of 0.4255, which is better than the prediction model without pretraining (0.3824) and other popular methods, including MixMHCpred (0.3369), NetMHCpan4.0-EL (0.4000), NetMHCpan4.0-BA (0.3188) and MHCflurry (0.3002). Moreover, when it comes to the evaluation of predicting immunogenicity, Pluto can identify more neoantigens than other tools. Pluto is publicly available at https://github.com/weipenegHU/Pluto.


Assuntos
Algoritmos , Apresentação do Antígeno , Epitopos de Linfócito T/química , Animais , Humanos , Ligantes , Aprendizado de Máquina
6.
Iran J Allergy Asthma Immunol ; 18(4): 427-440, 2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31522451

RESUMO

Interferonbeta-1b (IFNß-1b) developed as therapeutic protein for the treatment of multiple sclerosis (MS). Studies have been shown that Long-term usage of this protein can lead to the development of anti-drug antibodies (ADAs) and this phenomenon cause total loss or reduced efficacy of IFNß-1b. The aim of this study was to predict and silence IFNß-1b T-cells epitopes by in silico methods and genetic engineering. Based on bioinformatics studies we identified optimal sets of conservative point mutations for eliminating T-cells epitopes in IFNß-1b protein. Four synthetic genes with desirable mutation constructed and PET26b+ was used as an expression vector in E. coli. The expression of this proteins confirmed by SDS-PAGE and Western blotting, consequently, IFNß-1b proteins was purified by His-tag chromatography. To determined activity of mutants' variants anti-proliferative and anti-viral activity compared to wild form was evaluated using MTT assay in A549 and Vero cells lines respectively. Also the immunogenicity of mutant proteins compared with Betaseron measured in BALB/c mice. The in vitro bioactivity analysis demonstrated that functional activities of all mutant proteins were maintained and is the same as biological activity of Betaseron. Pharmacokinetic studies suggest that, in engineered proteins that contain substitution of Histidine to Glutamic Acid at position 131 (mut 2 and mut 1+2) antibodies response reduced by about 50%, as compared to that for Betaseron. Computational analysis expedites identification and prediction of epitopes in therapeutic protein, therefore, we used immunoinformatic tools for modification of dominant T-cell epitope in IFNß-1b protein, and this strategy has capacity to create proteins which have naturally reduced immunogenicity.


Assuntos
Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Inativação Gênica , Interferon beta-1b/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Descoberta de Drogas , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Feminino , Humanos , Imunização , Camundongos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade
7.
Immunology ; 158(2): 94-103, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31323138

RESUMO

Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG-rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1- and Cry j 2-specific T-cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG-rice seeds. We verified the antigenicity of TG-rice seeds to T-cells through the analysis of the proliferative responses of T-cells in Cry j 1- or Cry j 2-immunized mice or T-cell lines to TG-rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T-cells in the immunized mice proliferated on treatment with TG-rice seed extract, but not non-transgenic wild-type rice (WT-rice) seed extract. Furthermore, T-cell lines were established from the spleen cells of the immunized mice. Each T-cell line resulted in a proliferative response to TG-rice seed extract, but not to WT-rice seed extract, suggesting that TG-rice seeds certainly express T-cell epitopes corresponding to T-cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG-rice seeds, the expression of specific T-cell epitopes suggested that TG-rice seeds express all possible T-cell epitope repertoires of Cry j 1 and Cry j 2.


Assuntos
Alérgenos/farmacologia , Antígenos de Plantas/imunologia , Epitopos de Linfócito T/imunologia , Oryza/química , Proteínas de Plantas/imunologia , Rinite Alérgica Sazonal/imunologia , Linfócitos T/efeitos dos fármacos , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Plantas/química , Antígenos de Plantas/genética , Proliferação de Células/efeitos dos fármacos , Cryptomeria/genética , Cryptomeria/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Expressão Gênica , Imunização , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Oryza/genética , Oryza/imunologia , Mapeamento de Peptídeos , Extratos Vegetais/imunologia , Extratos Vegetais/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/imunologia , Cultura Primária de Células , Rinite Alérgica Sazonal/induzido quimicamente , Rinite Alérgica Sazonal/genética , Rinite Alérgica Sazonal/patologia , Sementes/química , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Transgenes
8.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31331958

RESUMO

Infection with Coxiella burnetii, the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA class II C. burnetii epitopes, providing the basis for a novel T-cell targeted subunit vaccine. In this study, we investigated in a cohort of 22 individuals treated for persistent infection (chronic Q fever) whether they recognize the same set of epitopes or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection. In cultured enzyme-linked immunosorbent spot (ELISpot) assays, individuals with chronic Q fever showed strong class II epitope-specific responses that were largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or nonreactive in subjects with chronic Q fever. Consistent with more recent/prolonged exposure, we found, however, stronger ex vivo responses by direct ELISpot to both whole-cell C. burnetii and individual peptides in chronic patients than in convalescents. In conclusion, we have validated and expanded a previously published set of candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in treated patients with chronic Q fever and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrated that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes.


Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Coxiella burnetii/imunologia , Epitopos de Linfócito T/imunologia , Febre Q/imunologia , Idoso , Antibacterianos/uso terapêutico , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Doença Crônica , Convalescença , Coxiella burnetii/patogenicidade , ELISPOT , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Feminino , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Teste de Histocompatibilidade , Humanos , Interferon gama/genética , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Peptídeos/genética , Peptídeos/imunologia , Febre Q/tratamento farmacológico , Febre Q/genética , Febre Q/prevenção & controle , Linfócitos T/imunologia , Linfócitos T/microbiologia
9.
Infect Immun ; 87(6)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30910792

RESUMO

CD4+ T-cell mechanisms are implied in protection against pneumococcal colonization; however, their target antigens and function are not well defined. In contrast to high-throughput protein arrays for serology, basic antigen tools for CD4+ T-cell studies are lacking. Here, we evaluate the potential of a bioinformatics tool for in silico prediction of immunogenicity as a method to reveal domains of pneumococcal proteins targeted by human CD4+ T cells. For 100 pneumococcal proteins, CD4+ T-cell immunogenicity was predicted based on HLA-DRB1 binding motifs. For 20 potentially CD4+ T-cell immunogenic proteins, epitope regions were verified by testing synthetic peptides in T-cell assays using peripheral blood mononuclear cells from healthy adults. Peptide pools of 19 out of 20 proteins evoked T-cell responses. The most frequent responses (detectable in ≥20% of donors tested) were found to SP_0117 (PspA), SP_0468 (putative sortase), SP_0546 (BlpZ), SP_1650 (PsaA), SP_1923 (Ply), SP_2048 (conserved hypothetical protein), SP_2216 (PscB), and SPR_0907 (PhtD). Responding donors had diverging recognition patterns and profiles of signature cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-13 [IL-13], and/or IL-17A) against single-epitope regions. Natural HLA-DR-restricted presentation and recognition of a predicted SP_1923-derived epitope were validated through the isolation of a CD4+ T-cell clone producing IFN-γ, TNF-α, and IL-17A in response to the synthetic peptide, whole protein, and heat-inactivated pneumococcus. This proof of principle for a bioinformatics tool to identify pneumococcal protein epitopes targeted by human CD4+ T cells provides a peptide-based strategy to study cell-mediated immune mechanisms for the pneumococcal proteome, advancing the development of immunomonitoring assays and targeted vaccine approaches.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/imunologia , Proteínas de Bactérias/genética , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Leucócitos Mononucleares/imunologia , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/imunologia , Domínios Proteicos , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética
10.
BMC Bioinformatics ; 19(Suppl 13): 468, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717656

RESUMO

BACKGROUND: In the current scenario, designing of world-wide effective malaria vaccine against Plasmodium falciparum remain challenging despite the significant progress has been made in last few decades. Conventional vaccinology (isolate, inactivate and inject) approaches are time consuming, laborious and expensive; therefore, the use of computational vaccinology tools are imperative, which can facilitate the design of new and promising vaccine candidates. RESULTS: In current investigation, initially 5548 proteins of P. falciparum genome were carefully chosen for the incidence of signal peptide/ anchor using SignalP4.0 tool that resulted into 640 surface linked proteins (SLP). Out of these SLP, only 17 were predicted to contain GPI-anchors using PredGPI tool in which further 5 proteins were considered as malarial antigenic adhesins by MAAP and VaxiJen programs, respectively. In the subsequent step, T cell epitopes of 5 genome derived predicted antigenic adhesins (GDPAA) and 5 randomly selected known malarial adhesins (RSKMA) were analysed employing MHC class I and II tools of IEDB analysis resource. Finally, VaxiJen scored T cell epitopes from each antigen were considered for prediction of population coverage (PPC) analysis in the world-wide population including malaria endemic regions. The validation of the present in silico strategy was carried out by comparing the PPC of combined (MHC class I and II) predicted epitope ensemble among GDPAA (99.97%), RSKMA (99.90%) and experimentally known epitopes (EKE) of P. falciparum (97.72%) pertaining to world-wide human population. CONCLUSIONS: The present study systematically screened 5 potential protective antigens from P. falciparum genome using bioinformatics tools. Interestingly, these GDPAA, RSKMA and EKE of P. falciparum epitope ensembles forecasted to contain highly promiscuous T cell epitopes, which are potentially effective for most of the world-wide human population with malaria endemic regions. Therefore, these epitope ensembles could be considered in near future for novel and significantly effective vaccine candidate against malaria.


Assuntos
Biologia Computacional/métodos , Genoma , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Vacinologia , Sequência de Aminoácidos , Antígenos de Protozoários/imunologia , Análise por Conglomerados , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Humanos , Malária Falciparum/genética , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular , Proteínas de Protozoários/imunologia
11.
Cancer Sci ; 110(4): 1156-1168, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30767336

RESUMO

Osteosarcoma is the most common malignancy of bone that affects young people. Neoadjuvant chemotherapy and surgery have significantly improved the prognosis. However, the prognosis of non-responders to chemotherapy is still poor. To develop peptide-based immunotherapy for osteosarcoma, we previously identified CTL epitopes derived from papillomavirus binding factor (PBF) in the context of human leukocyte antigen (HLA)-A2, HLA-A24 and HLA-B55. In the present study, we identified two novel CTL epitopes, QVT (QVTVWLLEQK) and LSA (LSALPPPLHK), in the context of HLA-A11 using a sequence of screenings based on the predicted affinity of peptides, in vitro folding ability of peptide/HLA-A11 complex, reactivity of peptide/HLA-A11 tetramer and interferon (IFN)-γ production of T cells that was induced by mixed lymphocyte peptide culture under a limiting dilution condition. CTL clones directed to QVT and LSA peptides showed specific cytotoxicity against HLA-A11+ PBF+ osteosarcoma (HOS-A11) cells. In contrast, another epitope, ASV (ASVLSRRLGK), could highly induce cognate tetramer-positive CTL. This might be because the ASV peptide mimics the peptide ASV (R6Q) (ASVLSQRLGK) derived from bacterial polypeptides, ROK family proteins. However, ASV-induced CTL did not show cytokine production against the cognate peptide. In conclusion, the CTL epitopes QVT and LSA peptides might be useful for the development of immunotherapy targeting PBF for patients with osteosarcoma.


Assuntos
Epitopos de Linfócito T/imunologia , Antígeno HLA-A11/genética , Proteínas de Membrana/imunologia , Osteossarcoma/genética , Osteossarcoma/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Sequência de Aminoácidos , Reações Cruzadas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Epitopos de Linfócito T/química , Antígeno HLA-A11/química , Antígeno HLA-A11/imunologia , Humanos , Proteínas de Membrana/química , Osteossarcoma/metabolismo , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica
12.
Microb Pathog ; 128: 254-262, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30616000

RESUMO

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.


Assuntos
Antígenos Virais/imunologia , Aphthovirus/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Antígenos Virais/química , Simulação por Computador , Sequência Consenso , Epitopos/química , Epitopos/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/imunologia , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Alinhamento de Sequência , Sorogrupo , Proteínas Virais/química , Proteínas Virais/imunologia , Proteínas Estruturais Virais/química , Vacinas Virais/imunologia
13.
Arch Virol ; 164(2): 483-495, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30415392

RESUMO

Restoring antiviral immunity is a promising immunotherapeutic approach to the treatment of chronic hepatitis B virus (HBV) infection. Dendritic cells play a crucial role in triggering antiviral immunity. In this study, we identified immunodominant epitopes prevalent in CD8+ T cell responses. We characterized the hierarchy of HBV epitopes targeted by CD8+ T cells following autologous monocyte-derived dendritic cell (moDC) expansion in HBV-infected subjects with distinct disease stages: treatment-naïve (TN group, n = 168), treatment with complete virological response (TR group, n = 72), and resolved HBV infection (RS group, n = 28). T cell responses against 32 HBV epitopes were measured upon moDC expansion. Several subdominant epitopes that triggered HBV-specific CD8+ T cell responses were identified. These epitopes' responses varied in individuals with different disease stages. Moreover, the most immunodominant and immunoprevalent epitope included the envelope residues 256-270 (Env256-270), corresponding to amino acid residues 93-107 in the small HBV surface protein (SHBs) across three patient groups. The frequency of Env256-270-specific interferon-γ-producing T cells was the highest in the RS group and the lowest in the TN group. In addition, individuals with HLA-A*02:03/02:06/02:07 were capable of responding to Env256-270. Env256-270-specific CD8+ T cells tolerated amino acid variations within the epitope detected in HBV genotypes B and C. This suggests that Env256-270 in SHBs is crucial in HBV-specific T cell immunity following autologous moDC expansion. It might be a potential target epitope for dendritic-cell-based immunotherapy for CHB patients with complete viral suppression by long-term NAs treatment.


Assuntos
Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Adulto , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Dendríticas/química , Células Dendríticas/citologia , Mapeamento de Epitopos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Feminino , Hepatite B/genética , Hepatite B/fisiopatologia , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Interferon gama/genética , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/imunologia , Adulto Jovem
14.
Appl Biochem Biotechnol ; 187(1): 90-100, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29948995

RESUMO

One of the most dangerous human pathogens with high prevalence worldwide is Streptococcus pyogenes, which has major impacts on global morbidity and mortality. A major challenge for S. pyogenes vaccine development is the detection of epitopes that confer protection from infection by multiple S. pyogenes types. Our aim was to identify the most conserved and immunogenic antigens of S. pyogenes, which can be a potential candidate for vaccine design in the future. Eight important surface proteins were analyzed. Using different prediction servers, strongest epitopes were selected. They had the ability to stimulate the humoral and cell-mediated immune system. Molecular docking was performed for measuring free-binding energy of selected epitopes. Seven epitopes from three surface proteins were selected as potential candidates for vaccine development. Conservation of selected epitopes among different Streptococcus types was checked. Further in vitro and in vivo tests are required to validate the suitability of the epitopes for vaccine design.


Assuntos
Linfócitos B/imunologia , Vacinas Bacterianas/imunologia , Epitopos de Linfócito T/imunologia , Streptococcus pyogenes/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Simulação de Acoplamento Molecular , Receptores de Antígenos de Linfócitos T/imunologia
15.
PLoS Comput Biol ; 14(11): e1006457, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30408041

RESUMO

A number of machine learning-based predictors have been developed for identifying immunogenic T-cell epitopes based on major histocompatibility complex (MHC) class I and II binding affinities. Rationally selecting the most appropriate tool has been complicated by the evolving training data and machine learning methods. Despite the recent advances made in generating high-quality MHC-eluted, naturally processed ligandome, the reliability of new predictors on these epitopes has yet to be evaluated. This study reports the latest benchmarking on an extensive set of MHC-binding predictors by using newly available, untested data of both synthetic and naturally processed epitopes. 32 human leukocyte antigen (HLA) class I and 24 HLA class II alleles are included in the blind test set. Artificial neural network (ANN)-based approaches demonstrated better performance than regression-based machine learning and structural modeling. Among the 18 predictors benchmarked, ANN-based mhcflurry and nn_align perform the best for MHC class I 9-mer and class II 15-mer predictions, respectively, on binding/non-binding classification (Area Under Curves = 0.911). NetMHCpan4 also demonstrated comparable predictive power. Our customization of mhcflurry to a pan-HLA predictor has achieved similar accuracy to NetMHCpan. The overall accuracy of these methods are comparable between 9-mer and 10-mer testing data. However, the top methods deliver low correlations between the predicted versus the experimental affinities for strong MHC binders. When used on naturally processed MHC-ligands, tools that have been trained on elution data (NetMHCpan4 and MixMHCpred) shows better accuracy than pure binding affinity predictor. The variability of false prediction rate is considerable among HLA types and datasets. Finally, structure-based predictor of Rosetta FlexPepDock is less optimal compared to the machine learning approaches. With our benchmarking of MHC-binding and MHC-elution predictors using a comprehensive metrics, a unbiased view for establishing best practice of T-cell epitope predictions is presented, facilitating future development of methods in immunogenomics.


Assuntos
Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/metabolismo , Algoritmos , Alelos , Vacinas Anticâncer/imunologia , Conjuntos de Dados como Assunto , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunogenicidade da Vacina , Ligantes , Aprendizado de Máquina , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Reprodutibilidade dos Testes , Linfócitos T/imunologia
16.
J Med Chem ; 61(21): 9568-9582, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30351939

RESUMO

We present here for the first time the synthesis and immunological evaluation of a fully synthetic three-component anticancer vaccine candidate that consists of a ß-glycotripeptoid core mimicking a cluster of Tn at the surface of tumor cells (B epitope), conjugated to the OVA 323-339 peptide (T-cell epitope) and a Toll-like receptor 7 (TLR7) agonist for potent adjuvanticity. The immunological evaluation of this construct and of precursor components demonstrated the synergistic activity of the components within the conjugate to stimulate innate and adaptive immune cells (DCs, T-helper, and B-cells). Surprisingly, immunization of mice with the tricomponent GalNAc-based construct elicited a low level of anti-Tn IgG but elicited a very high level of antibodies that recognize the TLR7 agonist. This finding could represent a potential vaccine therapeutic approach for the treatment of some autoimmune diseases such as lupus.


Assuntos
Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Peptidomiméticos/síntese química , Peptidomiméticos/farmacologia , Receptor 7 Toll-Like/agonistas , Sequência de Aminoácidos , Animais , Técnicas de Química Sintética , Camundongos , Camundongos Endogâmicos C57BL , Peptidomiméticos/química , Peptidomiméticos/imunologia
17.
Sci Rep ; 8(1): 15696, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30356071

RESUMO

Epitope-specific CD4+ T lymphocytes were magnetically enriched using ferromagnetic Ni and Fe-Au nanowires coated with a monomer containing a major histocompatibility complex class II-bound peptide epitope (pMHCII). The enriched lymphocytes were subsequently quantified using fluorescence-activated cell sorting (FACS). This was the first use of magnetic nanowires for cell sorting using FACS, and improvements in both specificity and fluorescent signal strength were predicted due to higher particle moments and lengths than conventional paramagnetic beads. Three different types of nanowires (Ni, Fe with Au tip and Fe-Au multilayers) were made by electrodeposition. Ni nanowires separated fewer T cells than Au tipped Fe nanowires, likely because Ni has a lower magnetic moment than Fe. Fe-Au multilayer nanowires separated more T cells than Au-tipped Fe nanowires because there was more monomer per nanowire. Also, increasing the amount of monomer increased the number of CD4+ cells separated. Compared to conventional paramagnetic beads, the nanowires had lower specificity for CD4+ T cells, but had stronger fluorescent signals due to more fluorophores per particle. This results in broader FACS baseline separation between the positive and negative cells, which is useful to detect T cells, even those with lower binding affinity for pMHCII ligands.


Assuntos
Linfócitos T CD4-Positivos/química , Epitopos de Linfócito T/química , Citometria de Fluxo/métodos , Ouro/química , Ferro/química , Imãs/química , Nanofios/química , Níquel/química , Animais , Galvanoplastia/métodos , Antígenos de Histocompatibilidade Classe II/imunologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Sensibilidade e Especificidade , Baço/citologia
18.
J Transl Med ; 16(1): 298, 2018 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-30368237

RESUMO

BACKGROUND: Chikungunya virus (CHIKV), causes massive outbreaks of chikungunya infection in several regions of Asia, Africa and Central/South America. Being positive sense RNA virus, CHIKV replication within the host resulting in its genome mutation and led to difficulties in creation of vaccine, drugs and treatment strategies. Vector control strategy has been a gold standard to combat spreading of CHIKV infection, but to eradicate a species from the face of earth is not an easy task. Therefore, alongside vector control, there is a dire need to prevent the infection through vaccine as well as through antiviral strategies. METHODS: This study was designed to find out conserved B cell and T cell epitopes of CHIKV structural proteins through immuno-informatics and computational approaches, which may play an important role in evoking the immune responses against CHIKV. RESULTS: Several conserved cytotoxic T-lymphocyte epitopes, linear and conformational B cell epitopes were predicted for CHIKV structural polyprotein and their antigenicity was calculated. Among B-cell epitopes "PPFGAGRPGQFGDI" showed a high antigenicity score and it may be highly immunogenic. In case of T cell epitopes, MHC class I peptides 'TAECKDKNL' and MHC class II peptides 'VRYKCNCGG' were found extremely antigenic. CONCLUSION: The study led to the discovery of various epitopes, conserved among various strains belonging to different countries. The potential antigenic epitopes can be successfully utilized in designing novel vaccines for combating and eradication of CHIKV disease.


Assuntos
Vírus Chikungunya/imunologia , Simulação de Acoplamento Molecular , Vacinas de Subunidades/imunologia , Alelos , Alérgenos/imunologia , Sequência de Aminoácidos , Sequência Conservada , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Filogenia , Vacinas de Subunidades/química
19.
Front Immunol ; 9: 2213, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30374343

RESUMO

Theileriosis poses a serious threat to ruminants in tropical and subtropical countries. It is a tick-borne disease, caused by an apicomplexan parasite, Theileria. The high disease burden in animals causes huge economic losses to marginal farmers. Further, with increasing cases of resistance to commonly used drugs, it is highly desirable to develop better and cost-effective vaccines against theileriosis. The only available vaccine, live attenuated parasite vaccine, has many drawbacks and hence is unsuitable for controlling this disease. Immuno-informatics has emerged as a useful tool in down selection of potential molecules for vaccine development. In this study, we have used an immuno-informatics driven genome-wide screening strategy to identify potential vaccine targets containing important and effective dominant immunogens against Theileria. The proteome of Theileria annulata was screened for proteins with probability of plasma membrane localization or GPI anchor. The proteins non-homologous to the host (bovine) were selected and their antigenicity was analyzed. The B-cell epitopes were identified in the selected proteins and mapped in the modeled structure of the proteins. A total of 19 linear epitopes in 12 proteins, exposed in the extracellular space and having the potential to induce protective antibodies were obtained. Additionally, CTL epitopes which are peptides with 9-mer core sequence, were also identified, modeled and docked with bovine MHC-I structures. The CTL epitopes showing high binding energy with the bovine MHC-I were further engineered in silico to design a putative multi-epitope vaccine candidate against Theileria parasites. The docking studies and molecular dynamics studies with the predicted multi-epitope vaccine candidate and modeled bovine TLR4 exhibited strong binding energy, suggesting that the complex is stable and the putative multi-epitope vaccine candidate can be a potentially good candidate for vaccine development.


Assuntos
Biologia Computacional/métodos , Epitopos/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Theileria annulata/imunologia , Theileriose/imunologia , Animais , Bovinos , Desenho de Fármacos , Epitopos/química , Epitopos/metabolismo , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Sistema Imunitário/imunologia , Simulação de Acoplamento Molecular , Proteoma/imunologia , Proteoma/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Vacinas Protozoárias/administração & dosagem , Theileria annulata/metabolismo , Theileria annulata/fisiologia , Theileriose/parasitologia , Theileriose/prevenção & controle , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
20.
Front Immunol ; 9: 2236, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30344521

RESUMO

HLA expression levels have been suggested to be genetically controlled by single nucleotide polymorphisms (SNP) in the untranslated regions (UTR), and expression variants have been associated with the outcome of chronic viral infection and hematopoietic stem cell transplantation (HSCT). In particular, the 3'UTR rs9277534-G/A SNP in HLA-DPB1 has been associated with graft-versus-host-disease after HSCT (Expression model); however its relevance in different immune cells and its mode of action have not been systematically addressed. In addition, there is a strong though not complete overlap between the rs9277534-G/A SNP and structural HLA-DPB1 T cell epitope (TCE) groups which have also been associated with HSCT outcome (TCE Structural model). Here we confirm and extend previous findings of significantly higher HLA-DPB1 expression in B cell lines, unstimulated primary B cells, and monocytes homozygous for rs9277534-G compared to those homozygous for rs9277534-A. However, these differences were abrogated by interferon-γ stimulation or differentiation into dendritic cells. We identify at least seven 3'UTR rs9277534-G/A haplotypes differing by a total of 37 SNP, also characterized by linkage to length variants of a short tandem repeat (STR) in intron 2 and TCE group assignment. 3'UTR mapping did not show any significant differences in post-transcriptional regulation assessed by luciferase assays between two representative rs9277534-G/A haplotypes for any of eight overlapping fragments. Moreover, no evidence for alternative splicing associated with the intron 2 STR was obtained by RT-PCR. In an exemplary cohort of 379 HLA-DPB1 mismatched donor-recipient pairs, risk prediction by the Expression model and the Structural TCE model was 36.7% concordant, with the majority of discordances due to non-applicability of the Expression model. HLA-DPB1 from different TCE groups expressed in the absence of the 3'UTR at similar levels by transfected HeLa cells elicited significantly different mean alloreactive CD4+ T-cell responses, as assessed by CD137 upregulation assays in 178 independent cultures. Taken together, our data provide new insights into the cell type-specific and mechanistic basis of the association between the rs9277534-G/A SNP and HLA-DPB1 expression, and show that, despite partial overlap between both models in HSCT risk-prediction, differential alloreactivity determined by the TCE structural model occurs independently from HLA-DPB1 differential expression.


Assuntos
Regiões 3' não Traduzidas/imunologia , Regulação da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/imunologia , Cadeias beta de HLA-DP/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Polimorfismo de Nucleotídeo Único/imunologia , Regiões 3' não Traduzidas/genética , Alelos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Frequência do Gene , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/genética , Cadeias beta de HLA-DP/genética , Haplótipos , Células HeLa , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Linfócitos T/imunologia , Linfócitos T/metabolismo
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