As grandes mudanças para os pesquisadores da área de vacinas sao a optimizaçao das vacinas de DNA para uso em humanos ou animais de grande porte e criar vacinas efetivas de uma única dose usando sistemas de liberação controlada apropriados. O plasmídeo de DNA codificando a proteína de heatshock 65 (hsp65) (DNA-hsp65) foi capaz de induzir resposta imune protetora e terapêutica no modelo murino da tuberculose (TB). Apesar do sucesso da vacina baseada na DNA-hsp65 em solução para proteger camundongos contra TB, esta requer múltiplas doses de grande quantidade de DNA para imunização efetiva. No intuito de otimizar esta vacina e simplificar o protocolo de vacinação, nós co-encapsulamos DNA-hsp65 e o adjuvante dimicolato de trealose (DMT) em microesferas biodegradáveis de ácido polilático-poliglicólico (PLGA) para uma única administração. Além disso, foi também desenvolvida uma formulação prime-boost da vacina de dose única baseada na mistura de duas diferentes microesferas de PLGA, apresentando liberação rápida e lenta, de DNAhsp65 e proteína hsp65 recombinante, respectivamente. Essas formulações foram testadas em cobaias pela comparação da eficácia protetora (components efetores da imunidade protetora) e toxicidade (componentes efetores da patologia) induzida pela preparação da preparação do DNA em solução ou BCG. A formulação de dose única prime-boost nitidamente apresentou maior eficácia e reduziu a patologia nos pulmões de cobaias. / The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly(DLlactide- co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in guinea pigs by comparison with the efficacy (effector components of protective immunity) and toxicity (effector components of pathology) induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in guinea pigs.

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.

O número de jovens infectados por HIV vem aumentando nas últimas décadas, o que salienta a necessidade de estratégias vacinais que sejam imunogênicas em fase precoce de vida capazes de induzir resposta de longa duração. A vacina quimérica LAMP-1/p55Gag, associa o gene que codifica a LAMP-1 (proteína de associação da membrana lisossomal) e o gene da gag do HIV-1, direciona o tráfego da proteína viralpara os compartimentos MIIC, possibilitando a apresentação dos peptídeos virais pela classe II do Complexo Principal de Histocompatibilidade (MHC II). Esta vacina quimérica é imunogênica em camundongos BALB/c adultos e neonatos e crucial para induzir resposta T e B de longa duração, com produção de elevados níveis de anticorpos. Contudo, os mecanismos imunológicos envolvidos na indução da resposta humoral da vacina LAMP/Gag, como a geração de células T auxiliares foliculares (TFH), ainda não são conhecidos no período neonatal. O objetivo do estudo foi avaliar a geração de células TFH, T citotóxicos e B foliculares em camundongos neonatos submetidos à imunização com as vacinas LAMP/Gag (LG) e Gag (G). Inicialmente,avaliamos a imunogenicidade das vacinas gênicas na imunização neonatal aos sete dias de idade em camundongos de linhagem C57BL/6. Os resultados mostram que a imunização neonatal com a vacina LG é capaz de aumentara frequência de células secretoras de IFN-&#978 aos peptídeos imunodominantes da região gag do HIV-1 e de células T CD8&#43IFN-&#978&#43 comparadas a vacina Gag. A imunização neonatal com a vacina LG também levou a produção de títulos elevados de anticorpos IgG1 anti-p24 e aumento da porcentagem de células secretoras de IgG1 Gag-específicas. O priming neonatal com LG é capaz de promover resposta celular e humoral anti-Gag de longa duração. Além disto, a imunização neonatal (ip) com LG foi capaz de induzir células TFH (CD4&#43CXCR5&#43PD-1&#43Bcl-6&#43), linfócitos T CD8&#43 foliculares (TFC) (CD8&#43CXCR5&#43) e formação de centro germinativo (CG) nos linfonodos mesentéricos, contudo, ambas as vacinas induziram células B foliculares (CD19&#43CXCR5&#43). Apesar da menor frequência de TFH dos neonatos em relação a adultos na imunização com LG, houve frequência similar de células TFC. A imunização intradérmica convencional induziu aumento do número de células TFH nos linfonodos inguinais já ao terceiro dia após o reforço vacinal, embora frequência similar de células TFH, TFC e B foliculares. Neste período foi possível observar que a vacina LG também induziu a geração de células B de CG. Outra peculiaridade da vacina LG neonatal foi o aumento da expressão gênica da enzima citidina deaminase (AID). Os resultados mostram que a vacina L/AMP/Gag é imunogênica na fase neonatal de camundongos C57BL/6 quanto à geração de resposta celular e humoral antígeno-específica e resposta de longa duração, similarmente aos camundongos BALB/c. Os dados mostraram que a vacina LG é eficaz na indução de células T foliculares, na maturação de tecidos linfoides com formação de CGs e na indução de transcritos para AID. No conjunto, os achados evidenciam que a estratégia da vacina quimérica L/AMP/Gag é eficaz neste período da vida, e possui importante papel adjuvante na maturação da resposta humoral. / The number of young people infected with HIV has been increasing in recent decades, which highlights the need for vaccine strategies that are immunogenic at early phase of life able of inducing long-term response. The chimeric LAMP-1/p55Gag vaccine, associates the gene encoding LAMP-1 (lysosomal associated membrane protein) and the HIV-1 gag gene, directs the traffic of viral protein to MIIC compartments, leading to presentation of the viral peptides through class II of the Major Histocompatibility Complex (MHC II). This chimeric vaccine is immunogenic in adult and neonatal BALB/c mice and crucial to induce long-term T and B responses, producing high levels of antibodies. However, the immunological mechanisms involved in the induction of the humoral response of the LAMP/Gag vaccine, such as the generation of T follicular helper cells (TFH), are unknown in the neonatal period. The objective of this study was to evaluate the generation of TFH, and follicular cytotoxic T cells and B cells in neonates submitted to immunization with the LAMP/Gag (LG) and Gag (G) vaccines. The results show that neonatal immunization at seven days-old in C57BL/6 mice strain with the LG vaccine is able to increasing the frequency of IFN-&#978-secreting cells to the immunodominant peptides of the HIV-1 gag region and of CD8&#43IFN-&#978&#43 T cells compared to the Gag vaccine. Neonatal immunization with the LG vaccine led to the production of high titers of anti-p24 IgG1 antibodies and the increased percentage of Gag-specific IgG1 secreting cells. Neonatal priming with LG is able to promote long-lasting anti-Gag humoral and cellular response. Moreover, neonatal (ip) immunization with LG was able to induce TFH cells (CD4&#43CXCR5&#43PD-1&#43Bcl-6 &#43), follicular CD8 &#43 T cells (TFC) (CD8&#43CXCR5&#43) and germinal center (GC) formation in the mesenteric lymph nodes, whereas both vaccines induced follicular B cells (CD19&#43CXCR5&#43). Despite the lower frequency of TFH in neonates in relation to adult counterpartin the immunization with LG, a similar percentage of TFC cells was observed. Conventional immunization by intradermal immunization induced an increased number of TFH cells in inguinal lymph nodes on the third day after booster vaccination, despite the similar frequency of follicular TFH, TFC and B cells. In this period the LG vaccine also induced generation of CG B cells. Another peculiarity of the LG neonatal vaccine was the increase in the gene expression of the enzyme activation-induced cytidine deaminase (AID).The results show that the LAMP/Gag vaccine is immunogenic in the neonatal phase of C57BL/6 mice for the generation of antigen-specific humoral and cellular response and long-term response, similar to BALB/c mice. The findings showed effective induction of follicular T cells, maturation of lymphoid tissues with formation of GCs and up-regulation oftranscripts for AID. Taken together, our findings demonstrate that the LAMP/Gag chimeric vaccine strategy is effective at this time in life, and has an important adjuvant role in the maturation of the humoral response.

The oxidative damage to DNA, protein and lipid, may be accumulated and play a role in the process of human cell aging. Oxidative stress may be due to the aerobic respiration and ozone-induced radiation which result in reactive oxygen species known as free radicals. Therefore, the antioxidant and free radical scanvenger which may reduce the oxidative damage, are of great interest, both in academic research and in the business world. The present study aims to evaluate the antioxidative potential of a very unique vegetable-fruit extract (Tsan-Ron-Bau-Yuan). The extract consists of over forty domestic vegetables and fruits, without using chemical fertilizers and pesticides during their entire growth period, is produced through a sophisticated freeze-dry technology. The anti-hydroxyl radical antioxidative potential were evaluated using the following four methods: (1) the plasmid DNA (2) the protein (3) the cell line and (4) the red blood cell . The results clearly demonstrat that the aqueous fraction of this extract can remarkably reduce the oxidative damage as evidenced by the DNA and protein model. In addition, the susceptibility of human red cells to oxidative stress can also be alleviated to some extents based on the RBC deformability studies. The efficacy of protection of the oxidative damage mediated by .OH generated by the Fenton¡¦s reaction was in the order of : aquaeous extract >100% ethanol extract > ethanol/ethylacetatee extract > 50% ethanol extract. Conversely, no significant protection to the action of hydrogen peroxide was observed in the cell line. Lysozyme which is ubiquitous among various natural products has negligible contribution as an antioxidant as revealed in the RBC deformability tests. Swine enzootic pneumoniae ¡]SEP¡^, is a disease caused by Mycoplasma hyopneumoniae infection, and usually lead to considerable economic loss. Though extensive research during the past years, the molecular mechanism about the infective pathway of M. hyopneumoniae was still elusive. The membrane proteins of this microorganism were considered as critical adhesion molecules and therefore are potential candidates for vaccine development. Through immunoscreening, we had isolated five recombinant phage clones expressing 10 kDa, 32 kDa, 36 kDa, 42 kDa and 60 kDa antigen proteins from the lEMBL3 library of Mycoplasma hyopneumoniae. The clone carrying the P42 gene was subcloned and further characterized as a heat shock protein gene. In the present study, the heat-shock protein gene encoding a 42 kDa/ 65 kDa protein¡]P42/P65¡^was cloned into the mammalian expression vector pcDNA3 and obtained plasmid pcDNA42. The immune response induced by pcDNA42 was evaluated in mice. The IgG titer was obviously elevated during the first eight weeks with the IgG1 titer slightly higher than IgG2a. However, the IgM titer was not changed signifcantly. Studies on the macrophage activity and T cell cytotoxity were still undergoning.

This thesis is focused on the development of a gene delivery system, especially for the purpose of DNA vaccination. DNA expression vectors have the potential to be useful therapeutics for a wide variety of applications. A carrier system was designed to realize the delivery of genes to cells and the promotion of controlled adequate expression in the target cells. The low gene delivery efficiency observed with systems composed of polyplexes is mainly due to low stability of polycation e.g polyethylenimine-DNA complexes and inability of most of the complexes to the reach nucleus after entering the cells. The encapsulation of polyethylenimine-DNA complexes inside the alginate microspheres was expected to provide protection from nuclease-based attack, thereby, increasing the stability of the complex and also to achieve controlled release of the complex at the target tissue. In this study, controlled release of complexes from alginate microspheres was studied with DNA staining. In Tris-HCl buffer, the release of PEI-DNA complexes were completed in 48 h, however in cell culture medium (DMEM) 18 % of complexes were released in 48 h because of presence of Ca+2 ions in DMEM. Also, in order to provide mucosal gene delivery for mucosal immunization polyethylene glycol (PEG) was introduced into the composition of microspheres and the two systems were compared in terms of release kinetics of the complexes. In the presence of PEG, release of PEI-DNA complexes from alginate microspheres in the cell culture medium (DMEM) were enhanced and 50 % of PEI-DNA were released from the microspheres in 48 h. To understand the effect of the PEG on the surface of microspheres zeta potential analysis and microscopic examination were carried out. By increasing percentage of PEG (0, 15, 30, 50) in microspheres, less negative zeta potential value were measured. Mucoadhesion of alginate and PEG-alginate microspheres were evaluated by using modified microbalance method, and in the presence of PEG enhancement of mucoadhesion was observed. In this way a gene delivery system with a possible route through mucosa of tissues was prepared.

Das West-Nil-Virus (WNV) ist eine Zoonose mit weltweit zunehmender Verbreitung. Natürliches Reservoir dieses Flavivirus sind Vögel, aber auch Säugetiere wie z.B. Menschen können infiziert werden. In einigen Fällen führt eine WNV-Infektion zu schweren neurologischen Erkrankungen. Infolgedessen werden effektive und biologisch sichere Impfstrategien gegen dieses Virus benötigt. Eine Alternative zu herkömmlichen Impfmethoden beschreibt die DNA-Immunisierung. In dieser Arbeit wurde ein potentieller DNA-Impfstoff gegen das WNV hergestellt. Die Immunisierung des DNA-Vektors induzierte starke zelluläre und humorale Immunantworten in Mäusen. Zudem waren die Tiere gegen eine WNV-Infektion geschützt. Zusätzliche Impfungen mit rekombinantem WNV-Protein führten zu einer weiteren Steigerung der Immunogenität des DNA-Impfstoffkandidaten. Des Weiteren sollte der nicht-virale Gentransfer im Allgemeinen optimiert werden. Ein neu entwickeltes Transportsystem für Plasmid-DNA, bestehend aus natürlichen Histonextrakten und Polyethylenimin, resultierte in einer verbesserten Proteinexpression in in vitro transfizierten Zellen und wurde von diesen sehr gut toleriert. Daher wäre diese Strategie auch für zukünftige DNA-Impftechniken denkbar. Der Einfluss von WNV auf die Expression zellulärer miRNAs in Wirtszellen wurde bisher noch nicht untersucht. Dennoch könnten auf diese Weise potentielle molekulare Biomarker für eine frühe WNV-Diagnose identifiziert werden. Mittels Microarray-Technik wurde die Expression zellulärer miRNAs analysiert. Verschiedene miRNA-Spezies waren infolge einer WNV-Infektion leicht herunter- bzw. hochreguliert und stellen mögliche diagnostische Biomarker für das Virus dar.

As grandes mudanças para os pesquisadores da área de vacinas sao a optimizaçao das vacinas de DNA para uso em humanos ou animais de grande porte e criar vacinas efetivas de uma única dose usando sistemas de liberação controlada apropriados. O plasmídeo de DNA codificando a proteína de heatshock 65 (hsp65) (DNA-hsp65) foi capaz de induzir resposta imune protetora e terapêutica no modelo murino da tuberculose (TB). Apesar do sucesso da vacina baseada na DNA-hsp65 em solução para proteger camundongos contra TB, esta requer múltiplas doses de grande quantidade de DNA para imunização efetiva. No intuito de otimizar esta vacina e simplificar o protocolo de vacinação, nós co-encapsulamos DNA-hsp65 e o adjuvante dimicolato de trealose (DMT) em microesferas biodegradáveis de ácido polilático-poliglicólico (PLGA) para uma única administração. Além disso, foi também desenvolvida uma formulação prime-boost da vacina de dose única baseada na mistura de duas diferentes microesferas de PLGA, apresentando liberação rápida e lenta, de DNAhsp65 e proteína hsp65 recombinante, respectivamente. Essas formulações foram testadas em cobaias pela comparação da eficácia protetora (components efetores da imunidade protetora) e toxicidade (componentes efetores da patologia) induzida pela preparação da preparação do DNA em solução ou BCG. A formulação de dose única prime-boost nitidamente apresentou maior eficácia e reduziu a patologia nos pulmões de cobaias. / The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly(DLlactide- co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in guinea pigs by comparison with the efficacy (effector components of protective immunity) and toxicity (effector components of pathology) induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in guinea pigs.

Made available in DSpace on 2014-06-11T19:24:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-06Bitstream added on 2014-06-13T19:51:42Z : No. of bitstreams: 1 pezavento_sfgz_me_botfm.pdf: 924376 bytes, checksum: e516f8ff865bbc2e7b75d6fc6e8d7450 (MD5) / A única vacina disponível contra a tuberculose (TB) é o bacilo de Calmette- Guérin (BCG), que é constituída por uma cepa de Mycobacterium bovis vivo atenuado, mas que possui variabilidade de proteção de 0 a 80%. Portanto é necessário o desenvolvimento de novas vacinas para o controle dessa infecção. Dentre as novas formulações profiláticas em teste para TB destacam-se as vacinas gênicas, sendo a DNAhsp65 a vacina investigada por nosso grupo. A DNAhsp65 é uma construção que contém o gene que codifica a proteína de choque térmico de 65KDa do M. leprae. Em virtude da elevada homologia entre a hsp65 micobacteriana e a hsp60 dos mamíferos, pode ocorrer mimetismo antigênico, ou seja, reatividade cruzada entre anticorpos e células T específicas para hsp65 micobacteriana e hsp60 humana. Nesse contexto, nosso grupo tem estudado o possível efeito colateral da DNAhsp65 no desenvolvimento de doenças autoimunes. Neste projeto foi investigado o efeito (proteção, exacerbação ou inocuidade) de diferentes formulações vacinais para TB na encefalite autoimune experimental (EAE). Os objetivos foram caracterizar a resposta imune específica induzida pela DNAhsp65 em ratos Lewis e avaliar o efeito imunomodulador tanto da estratégia clássica de imunização (3 doses de DNAhsp65 por via im) quanto da estratégia prime-boost BCG / DNAhsp65, nas características clínicas, imunológicas e histológicas da EAE. Quanto à imunogenicidade da DNAhsp65, foram testadas duas doses da vacina administradas via intramuscular (100 e 300 μg), sendo que somente a maior dose induziu IgG2b específica para hsp65 e produção de IFN- em cultura de células esplênicas estimuladas in vitro com rhsp65. Para avaliar o efeito da imunização com DNAhsp65 ou do prime-boost BCG /DNAhsp65 no desenvolvimento da EAE, os animais foram previamente imunizados com 3 doses de 300 μg de DNAhsp65 ou... / BCG is the only accepted vaccine against tuberculosis (TB) but its protective ability is very limited. Many new vaccines are therefore being evaluated. Our group has been working with DNAhsp65 that is a genetic construction containing the gene of hsp65 from Mycobacterium leprae. In previous experimental work we demonstrated that both, DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with 3 doses of DNAhsp65 or primed with BCG followed by 2 DNAhsp65 boosters. The animals were then immunized with myelin associated with Complete Freund Adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, inflammation at the central nervous system (CNS) and immune response against myelin. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and inflammation at the CNS did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN- and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate MS, being therefore... (Complete abstract click electronic access below)

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-05T16:49:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-ElianeCoimbra.pdf: 2000368 bytes, checksum: 2ec0dc2be6907bc65cd246b36e95e551 (MD5) / Made available in DSpace on 2017-04-05T16:49:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-ElianeCoimbra.pdf: 2000368 bytes, checksum: 2ec0dc2be6907bc65cd246b36e95e551 (MD5) Previous issue date: 2012-09-04 / O câncer cervical é considerado o segundo tipo de tumor a ocasionar maior número de mortes entre as mulheres no mundo,e ainfecção pelopapilomavirus humano (HPV) éa principalcausarelacionada com o desenvolvimento desta neoplasia. No Brasil, o câncer cervicalproduz uma média de 2,5 óbitos/100.000 indivíduos.Atualmente existem duas vacinas no mercado, baseadas em “Vírus-Like Particles”(VLPs), mas o alto custo inviabiliza seu uso pelos países em desenvolvimento. As VLPs de HPV são produzidasa partir daproteína capsidial L1, contra aqual é conferidaforte imunidade humoral.Uma estratégia vacinalorientada para a prevenção do câncer cervical em humanosé muito importante para a saúde pública, no entanto, como hámuitas pessoas já infectadas peloHPV, uma abordagem terapêutica se torna necessária.Neste trabalho foi proposta a expressão do gene L1 de HPV-16 em células da levedura Pichia pastoris, como base de uma estratégia preventiva e a construção de uma vacina de DNA baseada na proteína E5 do HPV-16, identificadacomo potencialantígeno tumoral para terapia docâncer. O gene L1 otimizado de HPV-16 foi clonado em vetor de expressão pPGK para integração no genoma de P. pastoris, sua transcrição foi confirmada por RT-PCR e a expressão da proteína detectada por dot blot, colony blot e western blot.A vacina de DNA foi construída pela inserçãodo gene E5 de HPV-16 otimizado e adicionado doepítopo AU1, novetor pCIneo.Para estudo inicial da funcionalidadeda construção vacinalpCI-E5sintH16, foi realizadaumaanálise in vitroem célulasHEK-293, através de RT-PCRe western blot.O estudo do gene E5 de HPVcomoantígeno terapêutico,apesar depouco exploradoainda, é bastante relevante. / Cervical cancer is the second type of tumor to cause deaths among women worldwide. The infection of human papillomavirus (HPV) is the leading cause related to the development of this neoplasia.In Brazil, cervical cancer produces an average of 2.5 deaths per 100.000 individuals. There are currently two vaccines on the market, based on Virus-Like Particles, but the high cost (U.S. $ 350.00) prevents its use by developing countries. HPV VLPs are formed from the L1 capsid protein, against which it is given strong humoral immunity. A targeted vaccination strategy for the prevention of cervical cancer in humans is very important to public health;however there are many people already infected with HPV, and therefore a therapeutic approach is needed. In this study was proposed the expression of the L1 gene of HPV-16 in Pichia pastorisyeast cells as the basis for a preventive strategy and the construction of a DNA vaccine based on the HPV-16 E5 protein, identified as a potential tumor antigen for cancer therapy. The optimized L1 gene of HPV-16 was cloned into the expression vector pPGK for integration into the genome of Pichia. The transcript of this gene was confirmed by RT-PCR and L1 protein expression detected by dot blot, colony blot and western blot. The DNA vaccine was constructed by cloning the optimized E5 gene of HPV-16, with the inserted AU1 epitope, in the pCIneo vector. In vitro analysis were performed with HEK-293 cells transfected with the PCI-E5sintH16 vector, including RT-PCR and western blot for the initial evaluation of DNA vaccine functionality. The study of the cancer therapy with the E5 gene of HPV-16 although still unexplored is quite relevant.

Les papillomavirus humains (HPV) oncogènes sont les agents étiologiques des cancers du col utérin. S’il est établi que 30 à 60% des femmes sont infectées par un HPV oncogène, seul 1% des cas d’infection évoluera vers un cancer invasif, les autres se résolvant spontanément, ce qui autorise à penser qu’une vaccination adéquate pourrait permettre de maîtriser, voire de guérir, les lésions (pré)cancéreuses induites par ces virus. A ce titre, les cibles préférentielles sont les protéines virales E6 et E7. En effet, non seulement ces protéines sont à la base du processus de transformation cellulaire et leur présence est requise pour le maintien du phénotype transformé mais, de plus, elles contiennent des épitopes reconnus par les lymphocytes T auxiliaires et cytotoxiques. Nous avons choisi d’orienter nos recherches sur le virus HPV de type 16 car son incidence est majeure. De même, le taux élevé d’expression de la protéine E7 dans les tumeurs nous a conduit à la choisir comme cible. Notre travail a consisté en la construction de vecteurs plasmidiques codant pour des protéines HPV-16 E7 native ou de fusion. Les fusions ont été réalisées afin de modifier la nature de la protéine E7 afin de la cibler vers les voies de présentation en association avec les molécules du complexe majeur d’histocompatibilité de classe I ou de classe II (MHC-I ou MHC-II). Nous avons étudié et comparé les réponses induites par nos vecteurs afin de déterminer le vecteur le plus efficace. Nous avons ensuite étudié le mécanisme d’action de ce vecteur afin de déterminer les points clés de l’élaboration d’une réponse immune anti-HPV-16 E7. Nous avons ainsi déterminé que cibler la protéine HPV-16 E7 vers la voie du MHC-I (protéines de fusion avec l’ubiquitine; Ub) n’augmentait pas son immunogénicité. A l’inverse, le ciblage de cet antigène vers la voie du MHC-II (protéine de fusion avec la chaîne invariante; Ii) permet à une majorité de souris C57BL/6 de résister à l’injection d’une dose léthale de cellules tumorales syngéniques E7-positives. Nous avons également montré que l’injection du vecteur ciblant E7 vers la voie du MHC-II permet l’élimination de cellules présentant un épitope classe I H-2Db. Cette réponse est médiée par des lymphocytes T CD8+. Nous avons également montré que, si la présence des cellules T CD4+ n’est pas nécessaire pour l’élimination proprement dite de cellules E7-positives, la génération des effecteurs de cette élimination requiert la présence de lymphocytes T CD4+. Nos investigations nous ont permis d’émettre l’hypothèse de l’apparition d’une réponse par transfection directe d’APC et présentation d’épitopes par les voies classiques sur MHC-I et –II, le phénomène de cross-présentation semblant ne pas être impliqué dans la genèse de la réponse. Nos résultats suggèrent donc que c’est l’instabilité de E7 qui fait de cet antigène abondamment synthétisé un piètre inducteur de réponses cytotoxiques. En effet, l’ubiquitination N-terminale de E7 provoque sa dégradation rapide et pourrait empêcher sa sécrétion. Lorsque l’on tient compte de la nécessité de l’activation de cellules T auxiliaires dans l’élaboration des réponses anti-E7 enregistrées, cette dégradation pourrait permettre à E7 de ne pas être présenté sur MHC-II et donc de ne pas générer les cellules CD4+ requises pour la génération des cellules CD8+. Finalement, nous avons montré que cibler une protéine E7 délétée de son motif oncogénique majeur vers la voie du MHC-II menait à l’apparition de réponses cytotoxiques anti-E7 efficaces. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished