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Parvoviruses cause dreadful enteritis in waterfowls and lead to tremendous financial losses. This study aims at developing effective way to prevent waterfowl parvoviral infection. Duck parvoviruses (DPVs) and goose parvoviruses (GPVs) were isolated from organs of infected waterfowls. The presence of virus in the specimens was identified using polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis. To reveal the genetic variation of viral capsid proteins (VPs), full length VPs gene were amplified and sequenced. The sequence data indicated the sequences diverge 4.1 to 4.4% among viral strains isolated during 1990 to 1999. The variant amino acids cluster in the common regions of VP3 at residues 203-266 and 482-534, which overlaps with the regions proposed to expose on the outer surfaces of parvoviral particles. These data implying that selective pressure from host immune system might play a part. The nucleotide sequences of VPs also reveal that DPV and GPV share 77 % similarity at the DNA, and 84.6% at the protein level. The most variable regions reside in the N-terminal of VP2 before the initiation codon of VP3 with 35% (19/54) amino acids divergence. This study also reveals the presence of conserved strain-specific residues in VPs and these residues seldom vary among different isolates of the same virus, suggesting that they might be important in maintaining viral structure or host specificity which worth further investigation. To investigate the antigenicity of VPs, the GPV genomic DNA encoding common region of VPs was fused in frame with glutathione S-transferase (GST) gene for the expression of GST-GPV (248-516) fusion protein in bacterial cells. Purified fusion protein was used as immunogen for the generation of rabbit anti-GPV (248-516) antiserum. The potential diagnostic usage was confirmed by the fact that this antiserum was able to differentiate between viral infected and uninfected primary embryonic fibroblast cells by immunocytochemical analysis. In addition, VPs in purified DPV and GPV virions were analyzed by Western blotting. This antiserum detected two prominent proteins bands with the molecule weight of 80 and 70 kilodaltons, which correspond to the sizes of VP1 and VP2 reported in the literature. The fact that VP1 of DPV reacts weakly with this antiserum suggests the existence of antigenic discrepancy between DPV and GPV. For the purpose of developing subunit vaccine for the control of Derzy's disease, recombinant full length VPs were expressed using both prokaryotic, GST and histidine-tagged fusion proteins, and eukaryotic, baculovirus and mammalian vero cell, expression systems. After large- scale production and purification, same amount of 4 recombinant VPs were individually used to immunize 1-week-old geese. The antibodies induced after immunization were then evaluated by enzyme-linked immunosorbent assay (ELISA). All four recombinant proteins stimulate approximately 7 to 8 folds increases of ELISA antibodies titers, and together with preliminary data of safety tests suggest a potential usage as subunit vaccine for the control of parvoviral infection.

Tumor immunoediting is a dynamic process in which the immune response attacks tumor cells by detecting danger signals and tumor antigens. In order to survive, tumor cells develop mechanisms to avoid detection or destruction by the immune system. To counteract this, several strategies are being developed to enhance the antitumor immune response, including the depletion of immunosuppressive cells, enhancing the activation of antitumor immune cells and increasing tumor cell immunogenicity. These therapies have seen limited success individually, however, and it is likely that combination therapy with novel targets will be necessary to see reproducible beneficial responses. Epigenetic modifications are attractive therapeutic targets because they are reversible and affect gene expression in cancer cells. Within this framework, this study aimed to elucidate the role of the chromatin remodeling complex nucleosome remodeling factor (NURF) in cancer immunoediting by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. Using two syngeneic mouse models of cancer, BPTF was found to suppress T cell antitumor activity in the tumor microenvironment. In vitro, enhanced cytolytic activity was observed for individual CD8 T cell clones only from mice bearing BPTF-silenced tumors, implicating the involvement of novel antigens. Mechanistic investigations revealed that NURF directly suppresses the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. PSMB8 inhibition reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor cell antigenicity and its depletion improves CD8 T cell antitumor immunity. In a concurrent study using different tumor lines, BPTF was also found to suppress natural killer (NK) cell antitumor immunity in vivo. Enhanced NK cell cytolytic activity toward BPTF-depleted targets in vitro was dependent on the natural cytotoxicity receptors (NCR). Molecular studies revealed that BPTF directly activates heparanase (Hpse) expression, resulting in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans. Thus, NURF represses NCR co-ligand abundance and its depletion enhances NK cell cytotoxicity. Therefore, NURF emerges as a candidate therapeutic target to enhance CD8 T or NK cell antitumor immunity.

No description available.

<p>The delivery of therapeutic peptides and proteins is often challenged by a short circulation half-life, necessitating frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. The covalent conjugation of therapeutic peptides and proteins, and more recently oligonucleotide-based drugs, with the “stealth” polymer poly(ethylene glycol) (PEG), termed PEGylation, is one of the most commonly used approaches to increase the in vivo half-life and reduce the immunogenicity of these therapeutic biomolecules. However, after several decades of research and clinical use, the limitations of PEGylation have begun to emerge.</p><p>Conventional methods for synthesizing peptide/protein-polymer conjugates have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of control over site and stoichiometry of conjugation, which results in heterogeneous products with significantly compromised biological activity. Additionally, anti-PEG antibodies have been induced in patients treated with PEGylated drugs and have been shown to correlate with rapid clearance of these drugs. High levels of pre-existing anti-PEG antibodies have also been found in individuals naïve to PEGylated agents, which are associated with serious first-exposure allergic reactions.</p><p>To address the synthetic limitations of PEGylation, a general approach for the high-yield synthesis of site-specific (C-terminal) and stoichiometric (1:1) peptide/protein-polymer conjugates, named sortase-catalyzed polymer conjugation, was developed. Demonstrating proof-of-concept of the approach with green fluorescent protein (GFP) as a model protein, sortase A from Staphylococcus aureus was used to site-specifically attach an initiator solely at the C-terminus of GFP, followed by in situ growth of the PEG-based brush polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) from the protein macroinitiator by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeded with high specificity and near-complete (~ 95%) product conversion. Subsequent in situ ATRP in aqueous buffer produced 1:1 stoichiometric conjugates with > 90% yield, tunable MW, low dispersity, and no denaturation of the protein. The extraordinarily high yield compares favorably to order of magnitude losses typically seen in conventional PEGylation processes.</p><p>Next, the therapeutic potential of POEGMAlation, or the conjugation of POEGMA to a peptide or protein, was demonstrated by implementing the developed sortase-catalyzed polymer conjugation strategy with exendin-4 (exendin), a therapeutic peptide for treating type 2 diabetes, to synthesize exendin-C-POEGMA conjugates with a wide and tunable range of molecular weights (MWs) and low dispersity. A single subcutaneous injection of exendin-C-POEGMA conjugates lowered blood glucose for up to 120 h in a diabetic mouse model. Most intriguingly, we showed that appending PEG as oligomeric side-chains on the conjugated POEGMA and tuning the side-chain length completely eliminated the reactivity of exendin-C-POEGMA conjugates toward patient-derived anti-PEG antibodies without compromising in vivo efficacy. Clinically, the lack of anti-PEG antigenicity of POEGMA conjugates is expected to completely eliminate serious first-exposure allergic reactions and the accelerated blood clearance of POEGMA-drug conjugates due to pre-existing anti-PEG antibodies in patients.</p><p>Collectively, these results establish POEGMAlation as a next-generation PEGylation technology that is highly useful for improving the pharmacological performance of therapeutic biomolecules while providing a timely solution to the increasing levels of pre-existing anti-PEG antibodies in patients that are seriously hindering the safety and efficacy of traditional PEGylated drugs.</p> / Dissertation

Foot-and-mouth disease virus (FMDV) is an economically devastating and highly contagious livestock pathogen. It exists as seven serotypes, comprising numerous antigenically distinct subtypes. The large amount of antigenic heterogeneity has confounded attempts at developing broadly reactive vaccines. In order to overcome this issue the fundamentals of the interactions between the virus and the host humoral immune response must first be understood. Previous work in this area using monoclonal antibody (mAb) escape mutants has identified five antigenic sites for the O serotype and efforts have been made to quantify their relative importance. However, this does not represent a complete picture of serotype O antigenicity. The work conducted in this thesis demonstrates the role of a limited number of dominant substitutions in mediating the antigenic diversity of serotype O Foot-and-Mouth disease virus. Two alternative but complementary methods for identifying epitopes were developed. The first used a mathematical model to analyse newly generated serological and sequence data from 105 viruses, cultured for this purpose (and cross-reacted to 5 reference antisera), in the context of an existing crystallographic structure to identify and quantify the antigenic importance of sites on the surface of the virus. The second approach was purely structural, using existing B cell epitope prediction tools to develop a method for predicting FMDV epitopes using existing crystallographic structures of FMDV. These techniques were validated by the use of reverse genetics, which confirmed the impact on cross reactivity of two predicted novel serotype O antigenic residues, with a further four novel residues identified by looking in depth at the interactions between two genetically close, but antigenically distant viruses. This increased knowledge of the antigenic composition of serotype O FMDV contributes to our understanding of the nature of vaccine efficacy and the breadth of protection, which, in the longer term, will aid in the goal of developing vaccines to better protect livestock from such a highly antigenically variable disease.

A utilização de antígenos de Taenia crassiceps vem se mostrando como via alternativa no imunodiagnóstico da neurocisticercose (NC), sendo as frações de 18 e 14 kDa consideradas específicas. No presente trabalho, foram sintetizados seis peptídeos, com base nas seqüências de aminoácidos das proteínas de 10, 14 e 18 kDa de T. crassiceps e homólogas às seqüências de aminoácidos de T. solium. Os peptídeos foram divididos em \"a\" e \"b\", denominados como, P1a, P2a, P3a e P4a e P1b, P3b e P4b, sendo, as seqüências de aminoácidos dos peptídeos \"a\", uma seqüência interna dos peptídeos \"b\". Em estudo da antigenicidade dos peptídeos conduzido por teste ELISA, P1a e P4a isolados ou utilizados como múltiplos peptídeos antigênicos (MAP) contendo dois, três ou quatro peptídeos, apresentaram reatividade com soro hiperimune anti-líquido vesicular de T. crassiceps, enquanto que MAPs contendo apenas P2a e P3a não foram antigênicos. Anticorpos monoclonais (AcMos) anti-T. crassiceps (n=3) e anti-T. solium (n=19), reconheceram, em ordem decrescente, P1b (72,7%), P1a (45,5%) e P3a e P2a (9,1%), enquanto que P3b, P4a e P4b não apresentaram reatividade com os AcMos utilizados. Com soros humanos de pacientes com NC (NC) e de indivíduos supostamente saudáveis (ISS), P1b e P1a foram considerados potencialmente antigênicos, isolados ou em MAPs com três ou quatro peptídeos. A avaliação do teste ELISA com peptídeo sintético isolado ou MAP, respectivamente, P1b e MAP-a (P1a+P3a+P4a), foi realizada baseando-se em três diferentes cut off, a, b e TG-ROC. De acordo com os cut off utilizados, ELISA com P1b apresentou índices de positividade entre 59,0-79,5% com amostras de soros NC (n=39); 1,3-11,8% com ISS (n=76); 3,4-10,2% com amostras de soros de pacientes com hidatidose provindos do Rio Grande do Sul (H-R) (n=59); 0,0-50,0% com amostras de soros de pacientes com hidatidose provindos do Peru (H-P) (n=8) e 0,0-9,1% com amostras de soros de pacientes com outras parasitoses (OP) (n=33). ELISA com MAP-a apresentou índices de positividade entre 57,7-92,3% com NC (n=26); 1,4-9,6% com ISS (n=73); 0,0-13,3% com H-R (n=15) e 12,5-37,5% com H-P (n=8). Na tentativa de aumentar a positividade do teste ELISA com peptídeos sintéticos, antígeno de 18 e14 kDa de T. crassiceps foi adicionado, porém, não houve maior antigenicidade ao complexo antigênico. Os peptídeos sintéticos utilizados mostraram-se promissores para o diagnóstico sorológico da NC, porém, não foram representativos dos epítopos imunodominantes presentes em cisticercos de T. solium. Outras seqüências de aminoácidos necessitam ser ensaiadas, a fim de obter um complexo antigênico sintético equivalente ao antígeno nativo, porém, o alto custo da síntese dos peptídeos ainda é uma barreira que limita a investigação de novos MAPs. / Taenia crassiceps antigens have been showed like one alternative rote to the immunodiagnosis of neurocysticercosis (NC), where 18- and 14-kDa fractions have been considered specific. In this study, six peptides were synthesized based on 10, 14 and 18 kDa fractions of amino acids sequence of proteins of the T.crassiceps and homologous to T. solium. Peptides were divided in \"a\" and \"b\", denominated P1a, P2a, P3a and P4a and P1b, P3b and P4b, being that amino acids sequences of the \"a\" peptides are one internal sequence of the \"b\" peptides. In antigenic study of the peptides conducted by ELISA, isolated P1a and P4a peptides or used like multiple antigenic peptides (MAP) with two, three or four peptides, cross-reacted with anti-vesicular fluid of the T. crassiceps hyperimmune serum, while that, MAPs with only P2a and P3a did not showed antigenicity. Monoclonal antibodies (Mabs) anti-T. crassiceps (n=3) and anti-T.solium (n=19) Mabs recognized in decreasing order, P1b (72,7%), P1a (45,5%) and P3a and P2a (9,1%), while, P3b, P4a and P4b did not showed reactivity with Mabs used. In human serum samples of the patients with neurocysticercosis (NC) and serum healthy individuals (HI), where P1b and P1a peptides were analyzed isolated or in MAPs with three or four peptides, both of them showed antigenic potential. The evaluation of the ELISA tests with synthetic peptides isolated or in MAPs, P1b and MAP-a (P1a+P3a+P4a), respectively, was analyzed in three differents cut off, a, b and TG-Roc. Based on its, ELISA assayed with P1b showed 59,0-79,5% positivity when NC serum samples where tested (n=39); 1,3-11,8% with HI serum samples (n=76); 3,4-10,2% with human serum samples of the patients with hydatidosis from Rio Grande do Sul (H-R) (n=59); 0,0-50,0% with human serum samples of the patients with hydatidosis from Peru (H-P) (n=8) and 0,0-9,1% with human serum samples of the patients with others parasitoses (OP) (n=33). By the way, ELISA assayed with MAP-a showed 57,7-92,3% positivity with NC (n=26); 1,4-9,6% with HI (n=73); 0,0-13,3% with H-R (n=15) and 12,5-37,5% with H-P (n=8). On the attempt of increase the positivity of the ELISA test using synthetic peptides, 18- and 14-kDa fractions of the T. crassiceps was added, but they did not promoted more antigenicity. Synthetic peptides used showed promising results on neurocysticercosis serumdiagnosis, but they were no representatives of dominants epitopes present in T. solium cysticercus. Others amino acids sequences need be used to have a synthetic antigenic complex similar to native antigens, however, the high cost of the peptide synthesis still is a problem that limit the study of the news MAPs.

Submitted by Renorbio (renorbioba@ufba.br) on 2018-04-25T17:41:58Z No. of bitstreams: 1 Tese de Doutorado - Maria da Conceição Aquino de Sá.pdf: 3251110 bytes, checksum: f7580c2d7b59b8cd7c2c1409212927e0 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2018-05-04T14:16:29Z (GMT) No. of bitstreams: 1 Tese de Doutorado - Maria da Conceição Aquino de Sá.pdf: 3251110 bytes, checksum: f7580c2d7b59b8cd7c2c1409212927e0 (MD5) / Made available in DSpace on 2018-05-04T14:16:30Z (GMT). No. of bitstreams: 1 Tese de Doutorado - Maria da Conceição Aquino de Sá.pdf: 3251110 bytes, checksum: f7580c2d7b59b8cd7c2c1409212927e0 (MD5) / FUNDACAO DE AMPARO A PESQUISA DO ESTADO DA BAHIA - EDITAL 013/2014 Programa De Bolsas De Mestrado e Doutorado / A procura por produtos da caprino-ovinocultura é frequente e contribui para aumentar a economia mundial, por isso é necessário produzir em larga escala e com qualidade para atender à demanda da população. Entretanto, diversos fatores na criação desses animais ainda necessitam melhorar, principalmente na área sanitária, na qual muitas doenças acometem o rebanho, dentre elas, a Linfadenite Caseosa, doença causada por Corynebacterium pseudotuberculosis, uma bactéria que apresenta sua toxicidade através de genes de virulência, assim como pela produção de biofilme, que protege a bactéria de ambientes hostis. Dessa forma, estudos sobre esse patógeno são necessários, a exemplo do sequenciamento genômico, na busca de conhecer genes para análises de biomoléculas e com isso compreender melhor a sua antigenicidade e imunogenicidade com o objetivo de melhorar a especificidade e sensibilidade de testes sorológicos, utilizados como prevenção da doença no rebanho. Além disso, é essencial entender o mecanismo de patogenicidade desta bactéria no hospedeiro e suas formas de defesa no sistema imunológico do animal. Assim, no presente estudo, foram caracterizados os genomas de quatro cepas de C. pseudotuberculosis, comparando as cepas produtoras (OVI2C, CAPJ4) e não produtoras (OVI03, CAP3W) de biofilme. As cepas foram extraídas para produção de antígenos somáticos e de superfícies. A partir de estudos prévios com immunoblotting, foram escolhidas as cepas de origem caprina (CAPJ4 e CAP3W) para o estudo in vivo com dezoito caprinos da raça Canindé, divididos em três grupos experimentais, um grupo controle e dois grupos infectados com cepas de C. pseudotuberculosis, para avaliar a patogenia clinicamente, realizando hemograma, além de outras análises sorológicas através do ensaio de ELISA e Western Blotting. Também foi realizada a cultura de células sanguíneas para o estudo da imunidade celular com IFN- e cultivo de células mononucleares do sangue periférico para observar a modulação da expressão de proteínas do sistema GTPases, Rab 5 e Rab 7. Após esses estudos, observou-se que os genes contidos no genoma das cepas de C. pseudotuberculosis possuem similaridade, mostrando que eles são altamente conservados. A extração das bactérias resultou em antígenos de superfície F3, que apresentaram reatividade no immunoblotting frente aos soros de ovinos experimentalmente infectados. De todos os antígenos, os de origem caprina, apresentaram melhor antigenicidade. No experimento in vivo, após a infecção dos animais, foram realizadas outras colheitas de sangue e foi possível observar que a cepa produtora de biofilme é mais resistente, causando danos maiores aos animais. Para as condições de infecção experimental, os valores do hemograma estão dentro dos parâmetros. Na análise sorológica os caprinos infectados experimentalmente responderam à exposição primária, o que contribui para o aumento da imunoglobulina IgG. A produção de IFN-  também foi observada. Foi verificado que as cepas de C. pseudotuberculosis produtora e não produtora de biofilme podem alterar a expressão de proteínas Rab 5 e Rab 7 em macrófagos derivados de células mononucleares do sangue periférico cultivados in vitro. Esses resultados forneceram informações importantes para a produção de testes diagnósticos, com maior acurácia. / The consumption of sheep and goat products is frequent and increases the values of world economy, so it is necessary to produce large scale and quality to meet the demand of population. However, several factors in the breeding of these animals still have to improve, especially in sanitary area, where many diseases affect the cattle, among them, Caseous Lymphadenitis, caused by Corynebacterium pseudotuberculosis, a bacterium that presents its toxicity through virulence genes, also by production of biofilm that protects the bacteria from hostile environments. Thus, studies of this pathogen are necessary, such as genomic sequencing, in search to know antigenic genes for biomolecule analysis and with this to understand better its antigenicity and immunogenicity, to be possible improve the specificity and sensitivity of serological tests, that are used as prevention of disease in the herd. Besides, it is essential to understand the mechanism of pathogenicity of this bacterium in host and its forms of defense against the animals immune system. At the present study, the genomes of four strains of C. pseudotuberculosis were characterized, comparing the biofilm producing (OVI2C, CAPJ4) and non-producing (OVI03, CAP3W) strains. These strains were extracted for production of somatic and surfaces antigens. From previous studies with immunoblottingting, strains of goat origin (CAPJ4 and CAP3W) were chosen for in vivo study with 18 Canindé goats, divided into three experimental groups, one control group and two groups infected with strains of C. pseudotuberculosis, to evaluate clinically the pathogenesis and through hemogram, in addition to other serological tests as ELISA assay and Western Blotting. Also was performed blood cell culture for the study of cellular immunity with IFN-, as culture of peripheral blood mononuclear cells to observe the modulation of expression of GTPases, Rab 5 and Rab 7 system proteins. After these studies, it was observed that the genes contained in the genome of the strains of C. pseudotuberculosis are genetically homogeneous, showing that they are highly conserved. The extraction of bacteria resulted in F3 surface antigens, that showed reactivity in immunoblottingting against sera from experimentally infected sheep. Of all antigens, those of goat origin had better antigenicity. At the in vivo experiment, after the infection of animals, other blood samples were collected and it was possible to observe that the biofilm producing strain is more resistant, causing greater damages to animals. For the conditions of experimental infection, blood count values are between normal limits. In the serological analysis, the experimentally infected goats responded to primary exposure, which contributes to increase of immunoglobulin IgG. The production of IFN- was also observed. In conclusion, it was observed that strains of C. pseudotuberculosis producing and not producing biofilm can modify the expression of Rab proteins in macrophages derived from mononuclear cells of peripheral blood cultivated in vitro. These results provided important information for production of diagnostic tests, with greater accuracy.