Made available in DSpace on 2014-08-20T14:37:54Z (GMT). No. of bitstreams: 1 tese_sergio_jorge.pdf: 2951290 bytes, checksum: 56f3ca5434b1ecaa09d8776156f09097 (MD5) Previous issue date: 2014-03-06 / Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a contagious respiratory disease that affects swine production worldwide. M. hyopneumoniae colonizes the ciliated epithelial cells of the respiratory tract, damaging the cells and predisposing the infected animals to secondary infections, causing significant economic losses. The commonly used vaccines to control this disease consist of inactivated whole cells (bacterins). These bacterins provide only partial protection and do not prevent the colonization of M. hyopneumoniae on the epithelial cells. Efforts to develop a more effective vaccine against mycoplasmas have been proposed and vaccines developed using recombinant DNA technology represents a promissing alternative. Although the genomes of four strains of. M hyopneumoniae have been sequenced, few recombinant antigens have been evaluated as candidate vaccines. Our research group produced and evaluated the immunogenicity and antigenicity of 35 secreted recombinant proteins and 6 transmembrane recombinant proteins. Some of these proteins were identified as having the potential to be used as vaccine antigens, including the molecular chaperone DnaK (P42 heat shock protein). The aim of this study was to assess the potential of recombinant P42 in vaccine preparations against EP, using swine animal model housed under field condictions in a M. hyopneumoniae-positive farm. Both, humoral and cellular immune responses were elicited when rP42 was delivered in Phosphate buffer saline, when combined to an oil based adjuvant, and when added to a whole cell vaccine preparation. The results indicate that immunization with rP42 emulsified in oil based adjuvant is able to induce antibodies against M. hyopneumoniae as well as the expression and the anti-inflammatory cytokine IL-10 in pigs. These immunogenic properties make recombinant antigen P42 a promising candidate for a recombinant subunit vaccine against EP. / Mycoplasma hyopneumoniae é o agente etiológico da Pneumonia Enzoótica Suína (PES). A bactéria coloniza e paralisa as células epiteliais do trato respiratório, predispondo o hospedeiro a infecções secundárias, causando significativas perdas econômicas. As vacinas atualmente disponíveis são compostas por células bacterianas inteiras inativadas (bacterinas), as quais proporcionam apenas uma proteção parcial aos suínos. Elas são capazes de reduzir as lesões pulmonares e os sinais clínicos, mas não impedem a colonização pelo agente. Com a finalidade de desenvolver uma vacina mais eficiente contra a PES, o uso da tecnologia do DNA recombinante representa uma estratégia promissora. Após o sequenciamento e análise proteômica de quatro cepas de M. hyopneumoniae, nosso grupo de pesquisa produziu e avaliou a imunogenicidade e antigenicidade de 35 proteínas recombinantes secretadas e 6 proteínas transmembrana. Algumas destas proteínas apresentaram potencial para serem usadas como antígenos vacinais, onde se destacou a chaperona molecular DnaK P42 (proteína de choque térmico). O objetivo deste estudo foi avaliar a proteina recombinante P42 em formulações vacinais contra a PES. Para tanto, foram realizados ensaios utilizando suínos mantidos em condições de campo numa granja com status de positiva para M. hyopneumonia. Foram avaliadas as respostas humoral e celular dos grupos de animais imunizados com a rP42 em tampão fosfato salina, com a rP42 emulsificada em adjuvante oleoso e com a rP42 associada à bacterina comercial. Os resultados mostraram que a imunização com rP42 emulsificada em adjuvante oleoso é capaz de induzir anticorpos contra M. hyopneumoniae além da expressão da citocina anti-inflamatória IL-10. Estas propriedades imunogênicas tornam o antigeno rP42 um candidato para o desenvolvimento de uma vacina de subunidade recombinante contra a PES.

Carrapatos são parasitas hematófagos transmissores de doenças para humanos e animais, e responsáveis por prejuízos econômicos de bilhões de dólares para os sistemas pecuários mundiais. A emergência de carrapatos multirresistentes a acaricidas torna urgente o desenvolvimento de vacinas efetivas contra este parasita. Neste trabalho, nós utilizamos dados de sialotranscriptomas de carrapatos R. microplus para selecionar antígenos secretados pelo parasita para serem testados como uma vacina multicomponente. Os antígenos produzidos de forma recombinante em E. coli foram utilizados como imunógenos em animais suscetíveis à carrapatos para testes de proteção vacinal contra infestações por R. microplus em bovinos e por R. sanguineus em cães. No primeiro ensaio, bovinos imunizados com oito antígenos recombinantes adjuvantados com um polímero sintético apresentaram indução de IgG sérica específica para cinco antígenos, e redução de 22% na infestação por carrapatos R. microplus. No segundo ensaio, a imunização de bovinos com nove antígenos adjuvantados com sais de alumínio gerou IgG específica para todos os antígenos inoculados, e proteção vacinal crescente de 70% e 75% em duas infestações sucessivas com carrapatos R. microplus, sem revacinação, indicando boost natural pela infestação. No terceiro ensaio, a imunização de cães com nove antígenos recombinantes associados à hidróxido de alumínio induziu a soroconversão de IgG dos animais para todos os antígenos, e proteção vacinal de 36% contra a infestação por carrapatos R. sanguineus. Todas as formulações afetaram principalmente o número de fêmeas ingurgitadas após a infestação. O efeito protetor de antígenos derivados de carrapatos R. microplus sobre carrapatos R. sanguineus (proteção cruzada) indica ser possível o desenvolvimento de uma vacina multicomponente efetiva contra os dois parasitas. / Ticks are hematophagous parasites which transmit diseases to humans and animals, and are responsible for billions of dollars of damage to the world\'s livestock systems. The emergence of ticks resistant to multiple acaricides makes urgent the development of effective vaccines against this parasite. In this work, we used data from the R. microplus tick sialotranscriptome to select antigens secreted by the parasite to be tested as a multicomponent vaccine. Recombinant antigens expressed in E. coli were used as antigens in tick\'s susceptible hosts to evaluate it efficacy in protect animals against R. microplus and R. sanguineus infestations. In the first assay, bovines immunized with eight recombinant antigens adjuvanted with a synthetic polymer presented the induction of serum IgG reactive for five antigens, and reduction of R. microplus infestation in 22%. In the second trial, immunization of cattle with nine antigens adjuvanted with aluminum salts generated serum IgG against all antigens, and vaccine protection against R. microplus parasitism was calculated in 70% and 75% for each infestation. In the third trial, the immunization of dogs with recombinant antigens adjuvanted with aluminum hydroxide induced IgG seroconversion against all antigens, and a 36% of protection against infestation by R. sanguineus ticks in immunized animals. All formulations reduced mainly the number of engorged females recovered from infestation. The cross reactive protection induced by R. microplus derived antigens R. sanguineus tick infestation indicate that it is possible to develop a unique multicomponent vaccine against the two parasites.

Carrapatos são parasitas hematófagos transmissores de doenças para humanos e animais, e responsáveis por prejuízos econômicos de bilhões de dólares para os sistemas pecuários mundiais. A emergência de carrapatos multirresistentes a acaricidas torna urgente o desenvolvimento de vacinas efetivas contra este parasita. Neste trabalho, nós utilizamos dados de sialotranscriptomas de carrapatos R. microplus para selecionar antígenos secretados pelo parasita para serem testados como uma vacina multicomponente. Os antígenos produzidos de forma recombinante em E. coli foram utilizados como imunógenos em animais suscetíveis à carrapatos para testes de proteção vacinal contra infestações por R. microplus em bovinos e por R. sanguineus em cães. No primeiro ensaio, bovinos imunizados com oito antígenos recombinantes adjuvantados com um polímero sintético apresentaram indução de IgG sérica específica para cinco antígenos, e redução de 22% na infestação por carrapatos R. microplus. No segundo ensaio, a imunização de bovinos com nove antígenos adjuvantados com sais de alumínio gerou IgG específica para todos os antígenos inoculados, e proteção vacinal crescente de 70% e 75% em duas infestações sucessivas com carrapatos R. microplus, sem revacinação, indicando boost natural pela infestação. No terceiro ensaio, a imunização de cães com nove antígenos recombinantes associados à hidróxido de alumínio induziu a soroconversão de IgG dos animais para todos os antígenos, e proteção vacinal de 36% contra a infestação por carrapatos R. sanguineus. Todas as formulações afetaram principalmente o número de fêmeas ingurgitadas após a infestação. O efeito protetor de antígenos derivados de carrapatos R. microplus sobre carrapatos R. sanguineus (proteção cruzada) indica ser possível o desenvolvimento de uma vacina multicomponente efetiva contra os dois parasitas. / Ticks are hematophagous parasites which transmit diseases to humans and animals, and are responsible for billions of dollars of damage to the world\'s livestock systems. The emergence of ticks resistant to multiple acaricides makes urgent the development of effective vaccines against this parasite. In this work, we used data from the R. microplus tick sialotranscriptome to select antigens secreted by the parasite to be tested as a multicomponent vaccine. Recombinant antigens expressed in E. coli were used as antigens in tick\'s susceptible hosts to evaluate it efficacy in protect animals against R. microplus and R. sanguineus infestations. In the first assay, bovines immunized with eight recombinant antigens adjuvanted with a synthetic polymer presented the induction of serum IgG reactive for five antigens, and reduction of R. microplus infestation in 22%. In the second trial, immunization of cattle with nine antigens adjuvanted with aluminum salts generated serum IgG against all antigens, and vaccine protection against R. microplus parasitism was calculated in 70% and 75% for each infestation. In the third trial, the immunization of dogs with recombinant antigens adjuvanted with aluminum hydroxide induced IgG seroconversion against all antigens, and a 36% of protection against infestation by R. sanguineus ticks in immunized animals. All formulations reduced mainly the number of engorged females recovered from infestation. The cross reactive protection induced by R. microplus derived antigens R. sanguineus tick infestation indicate that it is possible to develop a unique multicomponent vaccine against the two parasites.

O desenvolvimento de vacinas multivalente constitui urna das prioridades na pesquisa de vacinas modernas. A utilização de vetores vivos para apresentação de antígenos heterólogos mostra-se bastante atraente, e eliminaria a necessidade de várias doses para se alcançar uma proteção máxima. Este tipo de vacina poderia ser administrado em dose única, o que poderia aumentar a cobertura vacinal. Neste trabalho foi explorado o potencial do Mycobacterium bovis BCG recombinante (rBCG) vivo expressando antígenos de Bordetella pertussis, como futuro componente de urna vacina tetravalente rBCG-DTP contra a tuberculose, tétano, difteria e pertussis. Os antígenos de pertussis utilizados foram a subunidade S1 mutada e atóxica da Toxina Pertussis (SI-P1) e o fragmento CRD, um domínio imunogênico da proteína FHA (Adesina Hemaglutinina Filamentosa). PT é o principal fator de virulência de B. pertussis e tanto sozinho como combinado com outros antígenos é o principal componente de todas as vacinas acelulares desenvolvidas até o momento. FHA é um importante fator de aderência da bactéria às células ciliada alvo do hospedeiro, sendo o CRD (domínio de reconhecimento a carboidrato) o responsável pelo reconhecimento de carboidratos em receptores de células do hospedeiro. Os genes detses antígenos foram clonados em vetores de expressão micobacterianos e expressos em BCG sob o controle do promotor mutado da β-lactamase de Mycobacterium fortuitum, pBlaF*, em fusão com o peptídeo sinal da β-lactamase. Estes vetores possuem como marcador de seleção um gene de resistência a kanamicina. Camundongos foram imunizados com rBCG expressando S1-PT e os respectivos esplenócitos mostraram elevada produção de IFN-γ e baixa produção de IL-4, caracterizando uma forte resposta celular Th1 antígeno-específica dominante. rBCG-S1PT induziu uma baixa resposta humoral contra PT. Camundongos imunizados com rBCG-S1PT mostraram elevado nível de proteção contra um desafio intracerebral com uma cepa virulenta de B. pertussis. Animais imunizados com rBCG expressando CRD revelaram a presença de anticorpos anti-FHA no soro. Ensaios com camundongos imunizados com a combinação destas duas vacinas estão sendo realizados. Uma nova abordagem para obtenção de rBCG sem o uso de genes de resistência a antibióticos como marcador de seleção, foi investigada, utilizando a complementação em BCG auxotrófico. Uma cepa de rBCG auxotrófico para lisina foi transformada com vetores de expressão contendo o gene de complementação para lisina e os antígenos de pertussis sob controle do mesmo promotor: a seleção dos recombinantes é realizada em meio sem lisina. Estas construções permitiram a expressão estável dos antígenos e serão avaliadas quanto a indução de uma resposta imunológica efetiva contra pertussis. / The development of combined vaccines constitutes one of the priorities in modem vaccine research. The use of live vectors for heterologous antigen presentation is desirable, as it could eliminate the necessity of several doses to reach a maximum protection and increase vaccine coverage. In this work, the potential of recombinant Mycobacterium bovis BCG (Bacillus Calmette and Guerin) (rBCG), expressing Bordetella pertussis antigens was investigated. The antigens used were the genetically detoxified S1 subunít of pertussis toxin (S1-PT) and the CRD fragment of FHA (Filamentous hemagglutinin. The antigen genes were cloned into mycobacterial expression vectors under control of the upregulated M. fortuitum β-lactamase promoter, pBlaF*, in fusion with the β-lactamase signal sequence. Mice were immunized with rBCG expressing S1-PT and the respective splenocytes induced specific production of INF-γ and low IL-4, characterizing a strong antigen-specific Th1-dominant cellular response. The rSCG-S1 PT induced a low humoral response against PT. Mice immunized with rSCG-S1 PT strains displayed high-level of protection against an intracerebral challenge with live B. pertussis. Animals immunized with rBCG expressing CRD induced anti-FHA antibodies production. Protection induced by the combination of these two strains is being evaluated. A new approach for production of rBCG without the use of antibiotic resistance markers was also investigated, using complementation in auxotrophic BCG. A lysine auxotrophic rSCG strain was transformed with expression vectors containing the complementation gene for lysine and the pertussis antigens: selection of recombinant clones was carried in media without lysine. These constructs allowed steady expression of the antigens and will be evaluated for the induction of an immunological response against pertussis. These strains would be appropriate for clinical evaluation in humans.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

O desenvolvimento de vacinas multivalente constitui urna das prioridades na pesquisa de vacinas modernas. A utilização de vetores vivos para apresentação de antígenos heterólogos mostra-se bastante atraente, e eliminaria a necessidade de várias doses para se alcançar uma proteção máxima. Este tipo de vacina poderia ser administrado em dose única, o que poderia aumentar a cobertura vacinal. Neste trabalho foi explorado o potencial do Mycobacterium bovis BCG recombinante (rBCG) vivo expressando antígenos de Bordetella pertussis, como futuro componente de urna vacina tetravalente rBCG-DTP contra a tuberculose, tétano, difteria e pertussis. Os antígenos de pertussis utilizados foram a subunidade S1 mutada e atóxica da Toxina Pertussis (SI-P1) e o fragmento CRD, um domínio imunogênico da proteína FHA (Adesina Hemaglutinina Filamentosa). PT é o principal fator de virulência de B. pertussis e tanto sozinho como combinado com outros antígenos é o principal componente de todas as vacinas acelulares desenvolvidas até o momento. FHA é um importante fator de aderência da bactéria às células ciliada alvo do hospedeiro, sendo o CRD (domínio de reconhecimento a carboidrato) o responsável pelo reconhecimento de carboidratos em receptores de células do hospedeiro. Os genes detses antígenos foram clonados em vetores de expressão micobacterianos e expressos em BCG sob o controle do promotor mutado da β-lactamase de Mycobacterium fortuitum, pBlaF*, em fusão com o peptídeo sinal da β-lactamase. Estes vetores possuem como marcador de seleção um gene de resistência a kanamicina. Camundongos foram imunizados com rBCG expressando S1-PT e os respectivos esplenócitos mostraram elevada produção de IFN-γ e baixa produção de IL-4, caracterizando uma forte resposta celular Th1 antígeno-específica dominante. rBCG-S1PT induziu uma baixa resposta humoral contra PT. Camundongos imunizados com rBCG-S1PT mostraram elevado nível de proteção contra um desafio intracerebral com uma cepa virulenta de B. pertussis. Animais imunizados com rBCG expressando CRD revelaram a presença de anticorpos anti-FHA no soro. Ensaios com camundongos imunizados com a combinação destas duas vacinas estão sendo realizados. Uma nova abordagem para obtenção de rBCG sem o uso de genes de resistência a antibióticos como marcador de seleção, foi investigada, utilizando a complementação em BCG auxotrófico. Uma cepa de rBCG auxotrófico para lisina foi transformada com vetores de expressão contendo o gene de complementação para lisina e os antígenos de pertussis sob controle do mesmo promotor: a seleção dos recombinantes é realizada em meio sem lisina. Estas construções permitiram a expressão estável dos antígenos e serão avaliadas quanto a indução de uma resposta imunológica efetiva contra pertussis. / The development of combined vaccines constitutes one of the priorities in modem vaccine research. The use of live vectors for heterologous antigen presentation is desirable, as it could eliminate the necessity of several doses to reach a maximum protection and increase vaccine coverage. In this work, the potential of recombinant Mycobacterium bovis BCG (Bacillus Calmette and Guerin) (rBCG), expressing Bordetella pertussis antigens was investigated. The antigens used were the genetically detoxified S1 subunít of pertussis toxin (S1-PT) and the CRD fragment of FHA (Filamentous hemagglutinin. The antigen genes were cloned into mycobacterial expression vectors under control of the upregulated M. fortuitum β-lactamase promoter, pBlaF*, in fusion with the β-lactamase signal sequence. Mice were immunized with rBCG expressing S1-PT and the respective splenocytes induced specific production of INF-γ and low IL-4, characterizing a strong antigen-specific Th1-dominant cellular response. The rSCG-S1 PT induced a low humoral response against PT. Mice immunized with rSCG-S1 PT strains displayed high-level of protection against an intracerebral challenge with live B. pertussis. Animals immunized with rBCG expressing CRD induced anti-FHA antibodies production. Protection induced by the combination of these two strains is being evaluated. A new approach for production of rBCG without the use of antibiotic resistance markers was also investigated, using complementation in auxotrophic BCG. A lysine auxotrophic rSCG strain was transformed with expression vectors containing the complementation gene for lysine and the pertussis antigens: selection of recombinant clones was carried in media without lysine. These constructs allowed steady expression of the antigens and will be evaluated for the induction of an immunological response against pertussis. These strains would be appropriate for clinical evaluation in humans.

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 dissertacao_gabriel_klafke.pdf: 1619582 bytes, checksum: dcdfb7127ef88b680dc43b41887f2c86 (MD5) Previous issue date: 2010-03-18 / During the past decades, the development of genetically modified plants became a consolidated reality. Taking advantage of the genetic engineering process, it is possible to obtain modified plants to use as bioreactors in the production of tissue or organs expressing antigens which can be easily used as vaccines. The plant-based expression systems as tomato and lettuce, which attend as models for that process, present innumerous advantages such as conservation of eukaryotic machinery, which promote pos-translational modifications, possibility of large-scale production and development of safer and economically more attractive vaccines. Taking use of that strategy, the Swine mycoplasm hyopneumoniae (SMP) disease can be one important and possible target to either be eradicated or controlled. The SMP, caused by fastidious bacterium Mycoplasma hyopneumoniae, is one of the most important respiratory disease in swine breeding, due to its very high prevalence coupled with associated losses all over the whorld, and has in the recombinant DNA technology a viable alternative in the development of more effective and safe vaccines The objective of my work was to genetically manipulate tobacco plants in order to use them as bioreactor in the production of an antigen against PMS. Tobacco leaves and internodes were cultured in different concentrations of BAP and AIA hormones. The best regeneration results for both explants were seen with 1,5mg.L-1 BAP and 0,1mg.L-1 AIA. The selection test with the kanamycin antibiotic appeared to be highly effective, showing a total inhibition of regeneration with 30 mg.L-1 and 100 mg.L-1 for leaves and internodes respectively. The recombinant colonies of A. tumefaciens, containing ltb-r, were co-cultivated with the internodes and leaves from plants germinated in vitro. The next step, the explants were transferred to the selection medium in order to induce the selection of the putatively transformed cells. The genomic DNA from regenerated and putatively transformed plants were extracted and amplified by PCR, where it was detected the presence of a band referent to ltb r1. The analyses of the integration and the transcription of ltb-r1 were carried out by Southern blot and RT-PCR, respectively. In both techniques, it was possible to confirm the presence of one band which corresponds to the expected size of ltb-r1, supporting the integration and expression of the gene. However, with the tests used here, it was not possible to detect with accuracy the recombinant protein / Nas últimas décadas, o desenvolvimento de plantas geneticamente modificadas tornou-se uma realidade consolidada. Nesse sentido, utilizando-se da engenharia genética, é possível obter plantas servindo como biorreatores na produção de tecidos ou orgãos expressando antígenos que podem ser facilmente utilizados como vacina. Os sistemas de expressão em plantas como tomate, alface, tabaco que servem como modelos desse processo, apresentam várias vantagens, entre elas, a conservação da maquinaria eucariótica que promove as modificações póstraducionais das proteínas e ainda a possibilidade de produção em larga escala. Dentro desta estratégia de produção de proteínas, pode-se citar a pneumonia micoplásmica suína (PMS), causada pelo agente Mycoplasma hyopneumoniae, uma das principais doenças de suínos que provoca elevadas perdas econômicas em todo mundo, e tem, na tecnologia do DNA recombinante, uma alternativa de desenvolvimento de vacinas mais efetivas. O objetivo do trabalho foi transformar plantas de tabaco para sua utilização como biorreator na produção de um antígeno vacinal contra a PMS. Folhas e entrenós foram cultivados em diferentes concentrações de BAP e AIA. As melhores taxas de regeneração foram encontradas utilizando 1,5 mg.L-1 de BAP e 0,1 mg.L-1 de AIA para segmentos de folhas e entrenós. O teste de seleção utilizando canamicina mostrou-se altamente eficiente, obtendo-se a supressão da regeneração com 30 mg.L-1 e 100 mg.L-1 para segmentos de folhas e entrenós, respectivamente. Colônias recombinantes de A. tumefaciens contendo ltb-r1 foram co-cultivadas com entrenós e segmentos foliares de plantas germinadas in vitro. Após esta etapa, os explantes foram transferidos para meios de seleção, visando selecionar células possivelmente transformadas. O DNA genômico das plantas regeneradas e putativamente transformadas foi extraído e amplificado por PCR, na qual foi possível visualizar uma banda referente ao ltb-r1. A detecção da integração e transcrição do gene foi realizada por Southern blot e RTPCR, respectivamente. Em ambas as técnicas, foi possível verificar a presença de uma banda do tamanho esperado para ltb-r1, demonstrando assim, a integração e expressão do gene. Entretanto, não foi possível detectar com precisão, através dos testes utilizados, a proteína recombinante.

The development of a safe and effective HIV vaccine remains challenging due to its high antigenic variability. Poxviruses are large, stable, and have a track record of use as human vaccine candidates. Recombinant fowlpox virus 9 (rFP9), a highly attenuated host range-restricted poxvirus strain, has been safely administered to humans with no ill effects, and is known to be immunogenic. This thesis describes the construction of complex rFP9 encoding various sequences of SIVmac239. The SIVmac239/macaque model is widely used for HIV vaccine development. The ultimate aim of this work was to combine the advantages of FP9 with those of live attenuated SIV to produce a safe yet hopefully effective model HIV vaccine candidate. Transfer plasmids for five different insertion sites within the FP9 genome were designed and constructed. Homologous recombination (HR) of adjacent FP9 sequences was employed to facilitate the integration of SIVmac239 sequences into the FP9 genome. Positive rFP9 were identified by blue colouration in presence of X-gal using a transient colour selection (TCS) technique, and the final markerless pure recombinants were confirmed by PCR. Expression of the target SIV proteins in the presence of T7 polymerase has been demonstrated by immunocytochemical (ICC) staining and Western blotting (WB) assays. Expression was also quantified by enzyme-linked immunosorbent assay (ELISA) in various cell lines at multiple time points. Five different unique rFP9 have been constructed through this project. All SIVmac239 open reading frames (ORFs) save nef have been integrated into the FP9 genome, and protein expression demonstrated where possible. Moreover, a single rFP9 vector expressing the defective SIVmac239 genome driven by T7 RNA polymerase has been successfully constructed and validated using a green fluorescent protein marker.rFP9 showed appropriate transgene expression in both avian and mammalian cells, although at different levels. The expression efficiency of rFP9 was finally compared to another attenuated poxvirus vector, modified vaccinia Ankara (MVA). Comparing the protein expression levels between rFP9 and rMVA was quite difficult because different poxvirus promoters (early/late in rFP9; intermediate in rMVA) were used to direct the transcription of the T7 RNA gene. Given this limitation, although generally higher levels of expression were seen with rFP9, this cannot be attributed to the FP9 with any certainty.

The aim of this study was to develop a system for easy production of different veterinary chimeric vaccines based on stable mouse polyomavirus (MPyV) structures. The system is designed for antigens that are problematic in production or stability. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers based on the major capsid protein VP1 were designed to be exploited as vaccines against other pathogens. The different strategies used in this study are based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by inserting them into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, thus forming giant pentamers of a chimeric protein. Candidate vaccine antigens against porcine circovirus 2 (PCV2), the causative agent of porcine circovirus 2 systemic diseases (PCV2-SD) which causes significant economic losses in swine breeding, were prepared using the constructed vectors. All candidate vaccines induced the production of antibodies against the capsid protein of PCV2 after immunization of mice. The candidate vaccine Var C based on fusion of MPyV and PCV2 capsid proteins, is able to induce production of antibodies with...

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two of the most prevalent swine pathogens that have impacted the global swine industry for decades. Both are RNA viruses with increasing heterogeneity over the years, making a vaccine solution ever so challenging. Modified live-attenuated vaccines (MLVs) have been the most common approach, but the long-term safety regarding their potential for pathogenic reversion still needs to be addressed. Subunit based vaccines have been the focus of numerous development studies around the world with renewed interest in their promising prospects in both safety and efficacy. Our lab has developed a unique approach to use hepatitis B virus core capsid protein (HBcAg) as a vaccine delivery vehicle for either PRRSV or PEDV viral epitope antigens. Recombinantly produced HBcAg forms an icosahedral capsid virus-like particle (VLP) that has 240 repeats in a single assembled particle. By inserting different epitope antigens from these porcine pathogens into the particle, we can achieve repetitive antigen presentation to the host's immune system by taking advantage of the polymeric nature of VLP. The first animal study evaluated the efficacy of 4 VLP based vaccine candidates against PRRSV in mice. These 4 vaccines incorporated 2 B-cell epitopes (61QAAIEVYEPGRS72 and 89ELGFVVPPGLSS100) and 2 T-cell epitopes (117LAALICFVIRLAKNC131 and 149KGRLYRWRSPVIIEK163) from PRRSV structural proteins GP3 and GP5 respectively. Candidate GP3-4 was able to stimulate a significant viral neutralizing response in mouse sera against two PRRSV strains, one being heterologous, demonstrating its potential of cross-protection against PRRSV. The second animal study took an optimized VLP vaccine candidate against PEDV from previous development studies in mice, and assessed its efficacy through a comprehensive pregnant gilt vaccination and neonatal piglet challenge model. The vaccine candidate incorporated B-cell epitope 748YSNIGVCK755 from the PEDV spike protein. It was able to elicit significant viral neutralization antibody titer in gilt milk at 3 days post-farrowing (DPF), and provided nursing piglets with clinical relief in terms of morbidity, viral shedding, small intestinal lesions, and 10 days post-challenge (DPC) survival rate. / Doctor of Philosophy / Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two pathogens that infect pigs, resulting in immense economic losses to the global pork production industry every year. Both viruses have large diversity with various strains due to mutations that have occurred over the years. This makes vaccine development that aims at combating the pathogens even more challenging. One common vaccine strategy has been immunizing animals with modified live viruses with decreased pathogenicity. Naturally, long term safety of this option has been a concern. A much safer vaccine approach that is purely protein based has attracted renewed interest around the world. Protein based vaccines lack genetic materials from the viruses and are not able to replicate inside the host. Our lab has developed a platform that uses protein-based particles (VLPs) originated from the hepatitis B virus (HBV), and incorporates short pieces of proteins from either PRRSV or PEDV to train host's immune system to recognize these pathogens, and hopefully to prevent future infection. For the first animal study, we tested 4 VLP vaccine candidates against PRRSV in mice and discovered that mouse serum from one candidate GP3-4 was able to prevent infection of 2 distinct PRRSV strains in petri dishes, paving the way for further development. For the second animal study, we took an optimized VLP vaccine candidate against PEDV from previous mouse studies, and evaluated its performance in pigs. We immunized pregnant mother pigs with the vaccine before they gave birth, then experimentally infected newborn piglets with the virus. Piglets from the vaccinated mothers showed improved clinical signs and faster recovery from the infection.