RESUMO
In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.
Assuntos
Epitopos de Linfócito B/imunologia , Glutationa Transferase/imunologia , Imunogenicidade da Vacina/imunologia , Ixodidae/imunologia , Controle de Ácaros e Carrapatos , Vacinação/métodos , Sequência de Aminoácidos , Animais , Epitopos de Linfócito B/metabolismo , Glutationa Transferase/metabolismo , Ixodidae/enzimologia , Complexo de Endopeptidases do ProteassomaRESUMO
BACKGROUND: Corynebacterium pseudotuberculosis, a facultative intracellular bacterial pathogen, is the etiological agent of caseous lymphadenitis (CLA), an infectious disease that affects sheep and goats and it is responsible for significant economic losses. The disease is characterized mainly by bacteria-induced caseous necrosis in lymphatic glands. New vaccines are needed for reliable control and management of CLA. Thus, the putative virulence factors SpaC, SodC, NanH, and PknG from C. pseudotuberculosis FRC41 may represent new target proteins for vaccine development and pathogenicity studies. RESULTS: SpaC, PknG and NanH presented better vaccine potential than SodC after in silico analyses. A total of 136 B and T cell epitopes were predicted from the four putative virulence factors. A cluster analysis was performed to evaluate the redundancy degree among the sequences of the predicted epitopes; 57 clusters were formed, most of them (34) were single clusters. Two clusters from PknG and one from SpaC grouped epitopes for B and T-cell (MHC I and II). These epitopes can thus potentially stimulate a complete immune response (humoral and cellular) against C. pseudotuberculosis. Several other clusters, including two from NanH, grouped B-cell epitopes with either MHC I or II epitopes. The four target proteins were expressed in Escherichia coli. A purification protocol was developed for PknG expression. CONCLUSIONS: In silico analyses show that the putative virulence factors SpaC, PknG and NanH present good potential for CLA vaccine development. Target proteins were successfully expressed in E. coli. A protocol for PknG purification is described.
Assuntos
Vacinas Bacterianas/genética , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/patogenicidade , Expressão Gênica , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Sequência de Aminoácidos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Análise por Conglomerados , Corynebacterium pseudotuberculosis/imunologia , Corynebacterium pseudotuberculosis/metabolismo , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Escherichia coli/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fatores de Virulência/metabolismoRESUMO
The neuraminidase (NA) epitope from the Mexican AH1N1 influenza virus was identified by using sequences registered at the GenBank during the peak of a pandemic (from April 2009 to October 2010). First, NA protein sequences were submitted for multiple alignment analysis, and their three-dimensional models (3-D) were then built by using homology modeling. The most common sequence (denominated wild-type) and its mutants were submitted to linear and nonlinear epitope predictors, which included the major histocompatibility complex type II (MHC II) and B-cell peptides. The epitope prediction was in accordance with evolutionary behavior and some protein structural properties. The latter included a low NA mutation rate, NA 3-D surface exposure, and the presence of high hindrance side chain residues. After selecting the epitope, docking studies and molecular dynamics (MD) simulations were used to explore interactions between the epitope and MHC II. Afterward, several experimental assays were performed to validate the theoretical study by using antibodies from humans (infected by pandemic H1N1) and rabbits (epitope vaccination). The results show 119 complete sequences that were grouped into 28 protein sequences according to their identity (one wild-type and 27 representative mutants (1-5 mutations)). The predictors yielded several epitopes, with the best fit being the one located in the C-terminal region. Theoretical methods demonstrated that the selected epitope reached the P4, P6, P7, and P9 pockets of MHC II, whereas the experimental evidence indicates that the epitope is recognized by human antibodies and also by rabbit antibodies immunized with the peptide.
Assuntos
Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/metabolismo , Biologia Computacional , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Influenza Humana/diagnóstico , México , Modelos Animais , Dados de Sequência Molecular , Mutação/genética , Neuraminidase/genética , Neuraminidase/imunologia , Ligação Proteica , Conformação Proteica , Coelhos , VacinaçãoRESUMO
Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.
Assuntos
Flagelina/imunologia , Epitopos Imunodominantes/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Proteínas de Escherichia coli/imunologia , Flagelina/metabolismo , Epitopos Imunodominantes/metabolismo , Vacinas Antimaláricas/metabolismo , Malária Vivax/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/metabolismo , Receptor 5 Toll-Like/imunologiaRESUMO
Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue α-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 α-helix residues, whereas the B cell epitope is in the second microdomain and showed no α-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCor-overlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Infecções Estreptocócicas/imunologia , Vacinas Estreptocócicas , Streptococcus pyogenes/imunologia , Apresentação de Antígeno/imunologia , 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 , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/síntese química , Vacinas Estreptocócicas/imunologia , Vacinas Estreptocócicas/metabolismoRESUMO
The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM) to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates.