Um dos principais limitantes ao desenvolvimento e aprovação para utilização em humanos das vacinas de DNA é a falta de um vetor ideal de entrega gênica, que seja ao mesmo tempo eficiente e seguro. Embora mais seguros, os vetores não virais enfrentam uma série de barreiras físicas, enzimáticas e difusionais que limitam a chegada do transgene ao núcleo das células alvo. Dando continuidade ao trabalho desenvolvido em nosso grupo de pesquisa, o principal objetivo desta dissertação de mestrado foi avaliar o desempenho do vetor não viral comercial Lipofectamina e da proteína multifuncional T-Rp3 na entrega do gene da glicoproteína do vírus da raiva (RVGP) a células BHK-21. Primeiramente, o gene RVGP foi inserido no plasmídeo modelo pVAX1. Foram então realizados estudos de transfecção em células BHK-21 (Baby Hamster Kidney), utilizando-se Lipofectamina como agente de transfecção, no sentido de constatar a correta expressão do gene RVGP contido no novo plasmídeo. Como controle positivo, foi utilizado o plasmídeo pCMV-RVGP. Os estudos de PCR quantitativo da transcrição reversa (qRT-PCR) e imunofluorescência indicaram a expressão da glicoproteína pelo pVAX1RVGP, ainda que em valores de expressão menores se comparados com o plasmídeo controle pCMV-RVGP. Foi também desenvolvido com sucesso um método quantitativo de determinação da expressão da RVGP em células utilizando-se citometria de fluxo, que confirmou os resultados anteriores. Devido ao plasmídeo pVAX1RVGP ter apresentado baixa eficiência de expressão da RVGP, buscou-se a elevação da eficiência a partir da adição da sequência de KOZAK no plasmídeo pVAX1RVGP. Nesse caso, ainda que os resultados indiquem um aumento na expressão, não houve confirmação estatística (p<0,05). Os estudos de entrega com a proteína T-Rp3 foram realizados com um lote da T-Rp3 armazenada em ultrafreezer. A proteína demonstrou-se não ser estável após o congelamento em nitrogênio líquido e armazenamento em ultrafreezer pelo tempo de 10 meses. Apesar de ser capaz de complexar o pDNA após esse tempo, não foi eficiente em ensaios de transfecção, tendendo a agregar em relações molares altas e ausência de soro fetal bovino. / One of the major bottlenecks on the development and approval of DNA vaccines in humans is the lack of an ideal gene delivery vector, which must be safe and efficient at the same time. Although safer, the non-viral vectors face a series of physical, enzymatic and diffusion barriers that limits the arrival of the endogenous gene in the nuclei of the target cells. The main goal of this work was the evaluation of the performances of the commercial non-viral vector Lipofectamine, and the recombinant protein T-Rp3, a multifunctional protein, on the delivery of the rabies virus glycoprotein (RVGP) gene to BHK-21 cells. First, the RVGP gene was inserted into the pVAX1 plasmid, and transfections using BHK-21 (Baby Hamster Kidney) cells were performed using the Lipofectamine reagent to verify the correct expression of the RVGP gene present in the new plasmid. As a positive control, the plasmid pCMV-RVGP was used. The quantitative reverse transcription (qRT-PCR) and immunofluorescence studies indicated the expression of RVGP from pVAX1RVGP, although in lower expression values in comparison to the control plasmid. In addition, a flow cytometry quantitative method to quantify and compare the expression of the RVGP in the membrane of the transfected cells was developed, confirming the previous results. With the purpose of increase, the expression of RVGP, the KOZAK consensus sequence was added to the new pVAX1RVGP plasmid, and despite of the apparent increase of RVGP expression, this could not be confirmed statistically. The experiments of gene delivery using the T-Rp3 protein were performed using a protein batch storaged in ultrafreezer for 10 months. However, the protein has shown not being stable after storage for this long period. Moreover, despite of being capable to complex pDNA after this time, T-Rp3 was not efficient in the transfection assays and tended to aggregate in high molar ratios.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Orientadores: Hyun Mo Yang, Silvia Martorano Raimundo / Dissertação (mestrado) - Universidade Estadual de Campinas, Insituto de Matemática, Estatística e Computação Científica / Made available in DSpace on 2018-08-18T12:55:04Z (GMT). No. of bitstreams: 1 Sabino_MarcioRodrigues_M.pdf: 1310178 bytes, checksum: a2f78a77882031aeada8b32f6276f07e (MD5) Previous issue date: 2011 / Resumo: O interesse em modelar a dinâmica de transmissão da Tuberculose (TB) é principalmente pelo fato de que a TB representa um grande problema de Saúde Pública em todo o mundo. Segundo a Organização Mundial de Saúde, a TB é a principal causa de mortalidade por doenças infecciosas, sendo responsável por mais de 2 milhões de óbitos a cada ano (WHO, 2007). Outro motivo, muito importante desse estudo é o fato da descoberta da "vacina gênica (ou vacina de DNA)" pelo grupo de pesquisas do pesquisador Célio Lopes Silva, coordenador do Laboratório de Vacinas Gênicas da Faculdade de Medicina de Ribeirão Preto da USP. A vacina, ainda em fase de experimento e padronização pode se tornar a maior promessa de combate a doenças infecciosas para as quais até hoje não existe prevenção segura, como é o caso da TB. Neste trabalho apresentam-se estudos epidemiológicos da TB, as principais características da ação da vacina na epidemiologia da doença e, posteriormente, a formulação de um modelo matemático para descrever a dinâmica da transmissão da TB usando a vacina gênica como estratégia de controle da doença. A suposição básica do modelo é que a vacinação é adotada não somente como proteção para os indivíduos suscetíveis, mas também como tratamento para os indivíduos infectados e infecciosos. Apresenta-se uma análise do modelo geral determinando-se os pontos de equilíbrio do sistema e as condições de estabilidades destes pontos: analiticamente para o equilíbrio trivial, e numericamente, para o equilíbrio não trivial. Através de uma simplificação do modelo geral, foi também possível mostrar analiticamente a estabilidade global dos pontos de equilíbrio trivial e não trivial através da função de Lyapunov. Foram estimados alguns parâmetros do modelo, utilizando-se os dados que descrevem o cenário da TB no Brasil entre 1980 e 2007. Por fim, através deste ajuste, determinou-se os valores críticos de vacinação para o controle da doença / Abstract: The interest in modeling the transmission dynamics of the Tuberculosis(TB) is mainly due to the fact that TB is a major public health problem worldwide. According to World Health Organisation, TB is the leading cause of mortality from infectious diseases, accounting for more than 2 million deaths each year (WHO, 2007). Another reason, very important to this study is the discovery of "genetic vaccines" (or "DNA vaccine") by the research group of the researcher Celio Lopes Silva, coordinator of the Laboratory of genetic vaccines of the Faculty of Medicine of Ribeirao Preto, USP. The vaccine, still under experimentation and standardization, is a great promise for combating infectious diseases for which today there is no safe prevention, such as TB. This work presents epidemiological studies of TB, the main characteristics of the action of the vaccine on the epidemiology of the disease and then, a mathematical model formulation to describe the dynamics of TB transmission by using the genetic vaccine as a strategy for disease control. The basic assumption of the model is that vaccination is adopted not only as protection for susceptible individuals, but also as a treatment for infected and infectious individuals. An analysis of the general model is presented, by determining the equilibrium points of the system and the conditions of stability of these points: analytically for the trivial equilibrium point, and numerically for the endemic equilibrium point. By simplifying the general model, it was also possible to show analytically the global stability of the trivial and nontrivial equilibrium points with Lyapunov functions. Were estimated parameters of the model, using the data describing the scenario of TB in Brazil between 1980 and 2007. Finally, with these settings, critical values of vaccination for disease control were determined / Mestrado / Matematica Aplicada / Mestre em Matemática Aplicada

No description available.

A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but their tendency to display low immunogenicity in humans has hindered their usage, despite excellent tolerability and safety profiles. Various approaches have been used to improve the immunogenicity of DNA vaccines. Recent human study data re-established the value of DNA vaccines, especially in priming high-level antigen-specific antibody responses. Data suggests that innate immune responses to the DNA vaccine plasmid itself contribute to the immunogenicity of DNA vaccines, however the underlying mechanisms responsible remain unclear. In this dissertation, we investigate the role of innate immunity in shaping antigen-specific adaptive immune responses following DNA vaccination. The current belief is that the cytosolic DNA sensing pathways govern DNA vaccine immunogenicity. To date, only the type I interferon inducing STING/TBK1 regulatory pathway has been identified as required for DNA vaccine immunogenicity. Surprisingly, neither the upstream receptor nor the downstream signaling molecules in this pathway have been characterized. I therefore investigated a candidate cytosolic DNA receptor, as well as the downstream transcription factors required for generation of antigen-specific immune responses. Additionally, the effects of pro-inflammatory signaling on DNA vaccine immunogenicity have yet to be comprehensively studied. Previous studies have only provided indirect evidence for the role of inflammatory v signaling in DNA vaccination. As such, I also investigated the role of the DNA sensing AIM2 inflammasome in DNA vaccination. My data indicates that AIM2 is a key modulator in DNA vaccination via a previously unrecognized connection to type I interferon. Importantly, this marks the first time a DNA vaccine sensor has been identified. Of note, this dissertation represents a departure from many published works in the field. Whereas previous studies have mostly utilized model antigens and only focused on the adaptive immune responses generated, I analyzed the effects on innate immunity as well. Using various innate gene knockout murine models, I quantified antigen-specific humoral and T cell responses, as well as serum cytokine and chemokines following immunization with a clinically relevant DNA vaccine. Overall, this data provides a basis for understanding the mechanisms of DNA vaccination, allowing for the design of more effective vaccines.

Paracoccidioides brasiliensis é um fungo dimórfico patogênico agente etiológico da paracoccidioidomicose (PCM), uma micose endêmica no Brasil. A busca por alternativas para reduzir o tempo de tratamento da PCM levou ao desenvolvimento de uma vacina de DNA contendo a sequência do peptídeo P10 de P. brasiliensis. Neste trabalho, avaliamos a geração de células T de memória e células T reguladoras em camundongos imunizados com esta vacina de DNA antes e após o desafio com o fungo, através da análise de seus esplenócitos e linfócitos pulmonares por citometria de fluxo. Os resultados mostram um aumento no percentual de células T reguladoras e de memória no baço e pulmões dos animais imunizados antes e depois de 30, 60 e 120 dias do desafio em comparação com os grupos controle e não imunizado. Outro experimento revelou que o modelo experimental da PCM in vivo é capaz de induzir a expressão de ROR&#947t. Este estudo mostra que nossa vacina de DNA contra a PCM gera células com fenótipo de reguladoras e de memória, caracterizando seu potencial para o tratamento desta micose. / Paracoccidioides brasiliensis is a dimorphic fungal pathogen that is the etiological agent of paracoccidioidomycosis (PCM), a mycosis endemic in Brazil. The search for new alternatives to reduce the duration of PCM treatment led to the development of a DNA vaccine encoding the peptide P10 of P. brasiliensis. Presently, we evaluated the generation of memory and regulatory T cells in mice immunized with this DNA vaccine, before and after the challenge with the fungus by analizing their splenocytes and pulmonary lymphocytes by flow cytometry. The results show an increase in the percentage of regulatory and memory T cells on spleens and lungs of immunized mice before and after 30, 60 and 120 days of challenge compared with the control and untreated groups. Another experiment revealed that the PCM in vivo infection model is capable of inducing ROR&#947t expression. This study demonstrates that our DNA vaccine against PCM generates cells with a regulatory and memory phenotype, which shows its potencial in the treatment of this mycosis.

This dissertation describes two different projects. The first is the development of an oral DNA vaccine delivery system for fish. A novel oral DNA vaccine delivery system was developed for Rainbow Trout by combining non-viral vectors (polycationic liposomes or polycationic polymer) to facilitate the DNA vaccine's uptake by cell membranes along with enteric-coated protection of the DNA embedded in microparticles to prevent DNA degradation in the gastrointestinal tract. Spray drying and spray coating bead techniques were employed in the preparation of the DNA vaccine microparticles. The spray drying technique allowed production of spherical shape enteric-coated microparticles with a particle size range of 0.18 to 20 ��m. Larger particle sizes of 40-50 mesh were obtained from the spray-coated bead technique. The resultant DNA vaccine microparticles were granulated with regular fish feed and given to fish to investigate the efficacy of the delivery system in providing protection against IHNV, and to demonstrate the ease of administration in fish. An in vivo fish trial experiment showed improvement in fish survival rate when fish were immunized with larger particle size DNA vaccine microparticles. Further research to find effective vector carriers for the DNA vaccine delivery system and to seek modifications of the delivery system that will still prevent the denaturation of plasmid DNA that will also facilitate membrane uptake of the DNA vaccine is needed in order to develop a safe, effective, and commercially viable vaccine to control the outbreak of IHNV. The second project of the dissertation is prediction of in vitro drug release profiles from a novel matrix tablet spray-coated with a barrier membrane using mathematical and statistical models. Tablets were prepared by direct compression followed by spray coating. The relationship of the amount of hydrophilic materials in the core tablets and barrier thickness on drug release mechanism was investigated using factorial design and regression analysis. Drug release characteristics were influenced and can be controlled by modifying the amount of hydrophilic materials in the core tablet and the barrier thickness. Mathematical equation generated from regression analysis of n-value, lag time, and percent drug release as a function of the amount of hydrophilic material and the amount of coating material applied can now be used as a tool for predicting and optimizing in vitro drug release from matrix tablets spray-coated with a barrier membrane. / Graduation date: 2003

Non-viral delivery of plasmid DNA has traditionally relied upon physical forces applied directly to target tissues. These physical methods typically involve contact between an applicator and the target tissue and often cause transient patient discomfort. To overcome the contact-dependent limitations of such delivery methodologies, an atmospheric direct current plasma source was developed to deposit ionized gas molecules onto localized treatment sites. The deposition of charged species onto a treatment site can lead to the establishment of an electric field with strengths similar to those used for traditional electroporation. In vitro experiments proved that this technology could transiently permeabilize cell membranes and that membrane restabilization followed first order kinetics. Optimum delivery of tracer molecules to cell suspensions occurred after 10 minutes of plasma exposure and was attained without adversely effecting cell viability. In vivo testing of the plasma discharge demonstrated the capability of this system to deliver plasmid DNA to murine skin. Initial experiments involved the injection of plasmid DNA encoding luciferase into the dermis of C57BL/6J mice and then exposing the tissue to plasma discharge for 10 mintues. Delivery by this method resulted in increased luminescence that was as much as 19-fold greater than DNA injection alone. Follow-up optimization experiments demonstrated it was possible to obtain luminescence results that were similar in magnitude to those obtained using electroporation, which under optimum conditions resulted in about a 40-fold increase in peak luminescence. Finally, optimum conditions were used to deliver a plasmid DNA encoding for the 120 kilodalton glycoprotein present on the surface of a macrophage tropic HIV. Results from this vaccination experiment indicated this method was capable of producing antigen specific humoral immune responses at similar levels as when electroporation was utilized as the delivery method.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.