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Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-29T11:52:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-29T19:49:36Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-29T19:49:43Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) / Made available in DSpace on 2017-08-29T19:49:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_thais_larre_oliveira.pdf: 616408 bytes, checksum: 404ac55ee19b90246334aac10658a361 (MD5) Previous issue date: 2014-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A leptospirose é uma doença infecciosa emergente causada por espiroquetas patogênicas do gênero Leptospira, com complicações humana e veterinária. Essa doença é transmitida através do contato direto com um animal reservatório ou com o ambiente contaminado com a urina destes animais. O desenvolvimento de vacinas seguras e multivalentes contra leptospirose, que substituam as bacterinas existentes, permanece um desafio. Bacterinas são reatogênicas e conferem imunidade sorovar-específica e de curta duração. Esforços para o desenvolvimento de vacinas recombinantes tem focado em proteínas de membrana externa (OMPs). A proteína de membrana externa OmpL37 é conservada entre diferentes sorovares de Leptospira e atua na adesão aos tecidos do hospedeiro. Neste estudo, nós relatamos pela primeira vez, a avaliação do potencial imunoprotetor de OmpL37 como antígeno vacinal em hamsters, sob diferentes estratégias: vacina de subunidade, vacina de DNA e prime-boost. A caracterização da resposta imune através de ELISA indireto e qRT-PCR demonstrou que os maiores níveis de IgG foram estimulados pela vacina de subunidade, a qual também induziu resposta inflamatória. A vacina de DNA falhou em induzir imunidade humoral, porém também estimulou TNF-α. Apesar da resposta induzida, nenhuma formulação protegeu significativamente os animais contra a doença. / Leptospirosis is an emerging infectious disease caused by pathogenic spirochetes of Leptospira genus, of human and veterinary concern. This infectious disease is transmitted through direct contact with an animal reservoir or an environmental contaminated with their urine. The development of safe and multivalent vaccines against leptospirosis, which replace existing bacterins, remains a challenge. Bacterins are reactogenic and afford serovar specific and short-term immunity. Efforts to develop recombinant vaccines against leptospirosis have focused on outer membrane proteins (OMPs). The outer membrane protein OmpL37 is conserved among different Leptospira serovars and plays a role in adherence to host tissues. In this study we report for the first time, the evaluation of OmpL37 immunoprotective potential as a vaccine antigen in hamsters, under different strategies: subunit vaccine, DNA vaccine and prime-boost. The characterization of the immune response by indirect ELISA and qRT-PCR showed that higher levels of IgG were stimulated by the vaccine subunit, which also induced an inflammatory response. The DNA vaccine failed to induce humoral immunity, but also stimulated TNF-α. Despite the induced response, no formulation significantly protected the animals against the disease.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-09-23T13:40:05Z No. of bitstreams: 2 Sousa, Eduardo Martins.pdf: 2216793 bytes, checksum: b28a546dbb9489fa820abe22bc2b6109 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-09-23T15:17:05Z (GMT) No. of bitstreams: 2 Sousa, Eduardo Martins.pdf: 2216793 bytes, checksum: b28a546dbb9489fa820abe22bc2b6109 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-23T15:17:05Z (GMT). No. of bitstreams: 2 Sousa, Eduardo Martins.pdf: 2216793 bytes, checksum: b28a546dbb9489fa820abe22bc2b6109 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Tuberculosis is a re-emerging infectious disease that remains a major public health problem worldwide. Although there is the BCG vaccine that is effective against severe forms of childhood TB, in adults its efficacy is variable (0-85%). In this context there is a need to develop new vaccines to control the spread of TB. This thesis proposes the development of a new recombinant fusion M. tuberculosis protein (Ag85C-MPT51-HspX) by molecular cloning, expression in E. coli with a histidine tag and purified by ion exchange chromatography. The fusion protein was constructed successfully, expressed in E. coli BL21 and purified. Tests in mice were performed to evaluate the immunogenicity of the recombinant fusion protein Ag85C-MPT51-HspX of M. tuberculosis. Mice were immunized three times with the protein Ag85C-MPT51-HspX formulated with CpG-DNA encapsulated in liposomes, CpG-DNA encapsulated in liposomes, liposome or saline as negative control and the humoral and cellular immune response was evaluated. The immunization with the vaccine formulation induced the production of high titers of specific anti-fusion protein Ag85C-MPT51-HspX IgG1 = 3.08 ± 0.04; IgG2a = 3.10 ± 0.03) and, favored the increase of specific CD4+ IFN-γ (2.14% ± 0.17), CD4+ TNF-α (2.16 ± 0.34%). The recognizing of this protein by seric IgG and IgM discriminated patients with active TB infection from healthy individuals. We conclude that CMX protein has potential to be used for the development of vaccine against M. tuberculosis as well also for TB diagnostic kits. / A tuberculose é uma doença infecciosa re-emergente que permanece como um dos maiores problemas de saúde pública mundial. Embora exista a vacina BCG que é eficiente contra formas graves de TB na infância, em adultos a eficácia é variável (0 a 85%). Nesse contexto existe a necessidade do desenvolvimento de novas vacinas para controlar a disseminação da TB. O presente trabalho teve como objetivo desenvolver uma nova proteína de fusão recombinante (Ag85C-MPT51-HspX) de M. tuberculosis a partir da clonagem molecular, expressão em E. coli com uma cauda de histidina e purificação através de cromatografia de troca iônica. A proteína de fusão foi construída com sucesso expressa em E. coli BL21 e purificada. Ensaios em camundongos foram realizados para avaliar a imunogenicidade da proteína recombinante de fusão Ag85C-MPT51-HspX de M. tuberculosis. Os camundongos foram imunizados três vezes com a proteína Ag85C-MPT51-HspX formulada com CpG-DNA encapsulada em lipossoma, CpG-DNA encapsulado em lipossoma, lipossoma ou salina como controle negativo e a resposta imune humoral e celular foi avaliada . A imunização com a formulação vacinal induziu a produção de altos títulos de anticorpos específicos anti-proteína de fusão Ag85C-MPT51-HspX (IgG1 =3,08±0,04; IgG2a=3,10±0,03), bem como, favoreceu o aumento de células T CD4+IFN-γ (2,14%±0,17), CD4+TNF-α (2,16%±0,34) específicas. A avaliação do reconhecimento desta proteína de fusão tanto por IgM quanto IgG humana sérica permitiu discriminar pacientes com tuberculose ativa de controles saudáveis, demonstrando a antigenicidade desta molécula em humanos. Conclui-se que a proteína CMX poderá ser testada tanto como vacina, assim como para o desenvolvimento de testes de diagnóstico para a tuberculose.

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. It becomes widespread among adolescent and adults as well as infants. Although availability of effective pertussis vaccines seems to decrease the incidence of the disease, B. pertussis circulation in population has not been eliminated. It is thought that the antigenic drifts in major protective antigens and continued circulation of B. pertussis strains will result in gradual loss of the efficacy of the current pertussis vaccines. Therefore, development of more effective acellular pertussis vaccines with conserved protective proteins is a convenient strategy to provide a better protection against whooping cough. In this study, immune responses against putative peptidyl-prolyl cis-trans isomerase (PPIase) which was shown to be immunogenic in B. pertussis for the first time by our immunoproteome group and FimX whose expression was found higher in our local Saadet strain were determined in mice. The genes encoding FimX and putative PPIase were amplified by PCR, cloned into pGEM&reg / -T Easy vector and sequenced. The genes were then introduced into pET-28a (+) vector and they were expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins were purified by His-tag affinity chromatography and dialyzed. After Western blot analyses, 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase were used to immunize BALB/c mice (16-18 g) at day 0 and 21. The mice were challenged intranasally with 2.5 x 109 live B. pertussis Saadet cells. Before second immunization and challenge, the sera were collected to carry out ELISA for measurement of serum-specific IgG levels. According to ELISA results, IgG levels in the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX were found significantly higher than in control groups at both first and second vaccinations (p&lt / 0.01). On the other hand, immunization with 160 &micro / g recombinant putative PPIase provided a significant increase in IgG level (p&lt / 0.05) only at second vaccination. The lungs of the mice were removed at day 2, 5, 8 after challenge and bacterial colonization was determined. No significant decrease in bacterial colonization was observed in the lungs of the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase with respect to control groups. After respiratory challenge and second immunization (at day 30) with 20 &micro / g and 80 &micro / g recombinant FimX, the spleens of the mice were removed and a spleen cell culture was obtained. Supernatants were collected after induction of the cells with the recombinant protein and cytokine ELISA was carried out to measure IFN-&gamma / level. No significant difference was observed between control and vaccinated mice in terms of IFN-&gamma / production.

Haemophilus parasuis is a Gram-negative bacterium responsible for Glasser's Disease in pigs, though little is known regarding its antigenic or virulence factors. Our goals were to characterize the H. parasuis capsular polysaccharide (CP), determine its role in serotype-specificity and virulence, determine if CP is immunogenic, and develop diagnostic and protective products to prevent rampant H. parasuis infection within swine herds. Material from H. parasuis was purified using carbohydrate isolation techniques and compared to CPs from other Pasteurellaceae. Rabbits were immunized with CPs to generate antisera for microscopy, immunoassays, and bactericidal assays. CP antisera were conjugated to latex particles to create an agglutination assay for detection and typing of H. parasuis. CP was conjugated to Cholera Toxin B, and used to immunize mice and piglets before challenge with H. parasuis to determine its protective efficacy against Glasser's Disease. Broth-grown cells expressed CP, which reacted with antisera in microscopy and immunoassays. Broth-grown H. parasuis cells were serum-resistant unless homologous anti-CP serum was present. In contrast, agar-grown cells did not react with antisera in immunoassays, and cells were susceptible to killing by normal swine serum. CP was not expressed on the surface of agar-grown cells unless supplemented with bicarbonate. The addition of bicarbonate also contributed to the variability in CP quantity and upregulation of genes in the CP locus. Sensitized latex particles agglutinated strongest with homologous H. parasuis CPs, cells, and agar-grown cell lysates, but also reacted weakly with higher concentrations of heterologous CPs. The latex beads did not agglutinate with non-H. parasuis swine bacterial pathogens. Mice immunized with the CP-CTB conjugate produced a significantly higher IgG2/Th2 response than unimmunized mice or mice immunized with only CP, and immunized mice had fewer bacteria in their tissues that unimmunized mice. The CP conjugate produced a robust IgG antibody response to CP when used to immunized piglets, but because the control animals also survived H. parasuis challenge, the protective efficacy remains inconclusive. Therefore, the H. parasuis CP is the antigen that confers serotype identity, and can be implemented in methods and help direct future research in disease prevention and serotype tracking in H. parasuis infections. / Ph. D.

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 1 dissertacao_itaua_leston_araujo.pdf: 980896 bytes, checksum: 4007b4d62827e236b56c4fe512f94f29 (MD5) Previous issue date: 2014-02-27 / Glycoprotein D (gD) is essential for attachment and penetration of bovine herpesvirus type 5 (BoHV-5) into susceptible cells, and is a major target of host immune systems, inducing strong humoral and cellular immune responses. The immunogenicity of recombinant BoHV-5 (rgD5) expressed in Pichia pastoris was evaluated in bovines. Vaccines formulated with 100 μg rgD5 dose and adjuvants (Montanide ISA50V2 or Aluminum Hydroxide) with or without inactivated BoHV-5 or rgD5 without adjuvants were administered intramuscularly. A commercial vaccine was also used as control. The vaccine formulation rgD5 + ISA50V2 stimulated humoral immune responses after two doses, and higher titer of neutralizing antibodies were obtained in all three oil-based adjuvants formulations when compared to Hydroxide Aluminium adjuvant or the commercial vaccine. The vaccine BoHV-5 + rgD5 + ISA50V2 stimulated titers of neutralizing antibodies approximately 8 log2, which demonstrated higher correlations on the Indirect ELISA. Together, the results suggest that the recombinant gD5 conserved neutralizing epitopes and was able to stimulate a efficient humoral immune response in bovines. / A glicoproteína D (gD) é essencial para ligação e penetração de herpesvírus bovino 5 (BoHV-5) em células suscetíveis, e é um alvo importante do sistema imune do hospedeiro, induzindo respostas imunes humoral e celular. A imunogenicidade da glicoproteína D recombinante de BoHV-5 (rgD5) expressa em Pichia pastoris foi avaliada em bovinos. Vacinas formuladas com 100 μg de rgD5 por dose e adjuvante a base de óleo (Montanide ISA50V2) ou hidróxido de alumínio, com ou sem adição de BoHV-5 inativado foram administradas via intramuscular, e uma vacina comercial utilizada como controle. A vacina rgD5 + ISA50V2 estimulou resposta imune humoral após duas doses, e os mais elevados títulos de anticorpos neutralizantes foram obtidos em todas as três formulações de adjuvantes à base de óleo quando comparado com a formulação de vacinas de adjuvante de hidróxido de alumínio e vacina comercial. A vacina BoHV-5 + rgD5 + ISA50V2 estimulou títulos de anticorpos neutralizantes aproximadamente a 8 log2, demonstrando correlação significativa com o ELISA indireto. Em conjunto, os resultados sugerem que a rgD5 conservou epítopos neutralizantes e foi capaz de estimular uma resposta imune humoral eficiente em bovinos.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719

Made available in DSpace on 2014-08-20T13:32:58Z (GMT). No. of bitstreams: 1 dissertacao_vanessa_galli.pdf: 1411317 bytes, checksum: f2cb4b3ef71fd9550e04ca13f65ab510 (MD5) Previous issue date: 2011-02-28 / Mycoplasma hyopneumoniae is the causative agent of Porcine Enzootic Pneumonia (PEP), one of the most common respiratory diseases in swine industry worldwide. Commercially available vaccines are inactivated whole-cell preparations (bacterin), which provide only partial protection and do not prevent microorganism colonization. In this context, it is necessary to search new alternatives prophylaxis. Potential antigens are being tested in different vaccination strategies; however none was more efficient than commercial bacterins for PEP control. This work aimed the production and evaluation of antigenicity and immunogenicity of M. hyopneumoniae antigens delivered as naked DNA and/or recombinant subunit vaccines, aiming the development of a vaccine against PEP. Recombinant subunit vaccines were obtained by the expression of eleven M. hyopneumoniae recombinant proteins in E. coli and purification by affinity chromatography, whereas the DNA vaccines were obtained by cloning four M. hyopneumoniae genes in pcDNA3 vector. Recombinant proteins antigenicity was verified against convalescent pig serum. The humoral and cellular immune response elicited by these vaccines was evaluated in mice immunized intramuscularly. All recombinant proteins evaluated were recognized by convalescent pig serum, in ELISA and/or Western blot assay, especially MHP0418, indicating that they are expressed during disease. These recombinant proteins, as well as P37, P42, P46 and P95 showed immunogenic capacity, eliciting both Th1 and Th2 immune response. The P37, P42, P46 and P95, and the DNA vaccine pcDNa3/P46 were also able to elicit INFγ expression, the cytokine associated with cellular immune response, and decrease TNFα and IL1 expression, both associated with pig lesions, during M. hyopneumoniae infeccion, suggesting their potencial as candidate vaccines. The immunization strategy using proteins combinated potencialized the immune response, and the MHP0443 and MHP0372 proteins were the main responsable for the Mix1 and Mix2 immunogenicity, respectivally. Moreover, MHP0107, MHP0418 and MHP0372 elicited antibodies that react against proteins from M. hyopneumoniae strains 7448, 4722 and J, and did not show cross reaction with M. hyohinis and M. flocculare. Thus, these proteins could be used in imunodiagnosis assay. / Mycoplasma hyopneumoniae é o agente etiológico da Pneumonia Enzoótica Suína (PES), uma das doenças respiratórias de maior incidência na criação de suínos no mundo. As vacinas disponíveis comercialmente consistem de células inteiras inativadas (bacterina), as quais proporcionam apenas uma proteção parcial e não previnem a colonização pelo microrganismo. Neste contexto, faz-se necessária a busca de novas alternativas para a profilaxia da PES. Alguns antígenos vêm sendo testados em diferentes sistemas de vacinação, porém nenhum deles foi mais eficiente que as bacterinas comerciais no controle da PES. Este trabalho teve como objetivo a produção e avaliação da antigenicidade e imunogenicidade de antígenos de M. hyopneumoniae administrados como vacinas de DNA e/ou subunidade recombinante, visando o desenvolvimento de uma vacina contra a PES. As vacinas de subunidade recombinante foram obtidas através da expressão de onze proteínas recombinantes de M. hyopneumoniae em E. coli e purificação por cromatografia de afinidade, enquanto que as vacinas de DNA foram obtidas pela clonagem de quatro genes de M. hyopneumoniae no vetor pcDNA3. A antigenicidade das proteínas recombinantes foi verificada confrontando-as com soro de suínos convalescentes. A imunidade humoral e celular destas vacinas foi avaliada em camundongos imunizados intramuscularmente. Todas as proteínas recombinantes avaliadas foram reconhecidas pelo soro de animais convalescentes, em ensaios de ELISA e/ou Western blot, em especial a proteína MHP0418, indicando serem expressas durante o processo infeccioso. Estas proteínas recombinantes, bem como P37, P42, P46 e P95 apresentaram capacidade imunogênica, induzindo ambas as respostas imune Th1 e Th2. As proteínas P37, P42, P46 e P95, e a vacina de DNA pcDNa3/P46 também foram capazes de induzir a expressão de INFγ, citocina associada a resposta imune celular e reduzir a expressão de TNFα e IL1, relacionadas com as lesões em suínos, durante infecção por M. hyopneumoniae, sugerindo o potencial destas como candidatas vacinais. A estratégia de imunização utilizando proteínas combinadas potencializou a resposta imune, sendo que as proteínas MHP0443 e MHP0372 foram as principais responsáveis pela imunogenicidade induzida pelos Mix1 e Mix2, respectivamente. Além disso, MHP0107, MHP0418 e MHP0372 induziram anticorpos que reagiram especifiamente contra proteínas das cepas 7448, 4722 e J de M. hyopneumoniae, não apresentando reação cruzada com M. hyohinis e M. flocculare, podendo, portanto, serem utilizadas em ensaios de imunodiagnóstico.

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