Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

Adeno-associated viral (AAV) vectors have been shown to be potential vectors for the treatment of diseases, including protocols using RNA interference (RNAi). AAV vector production in insect cells using the baculovirus vector expression system has been a major advance in furthering their use. A major limitation of AAV vector production at high cell densities is a reduction in cell specific yield, which is thought to be caused by nutrient limitations. Nutrient consumption profiles after infection, however, have still not been fully characterized, probably due to the difficulty of characterizing consumption patterns based on increases in cell density, which are minimal after infection. It is known, however, that cells increase in size after infection; therefore, the driving hypothesis of this thesis was that biovolume, or the total volume enclosed by the membrane of viable cells, which accounts for both cell density and cell size, could be used to characterize nutrient consumption patterns both before and after infection. The relationships between nutrient consumption and change in cell density and biovolume were examined by statistical correlation analysis. It was found that in uninfected cultures, no significant correlation differences, using either cell density or biovolume, were observed since cell size remained relatively constant; however, in infected cultures, more than half of the nutrients were found to be better correlated with biovolume than with cell density. When examining the nutrient and metabolite concentration data on a biovolume basis, nutrient consumption remained relatively constant. It is hypothesized that since it has been reported that the rate of cell respiration increases after infection, a more complete oxidation of nutrients occurs to satisfy increased energy needs during infection. By having a basis to base nutrient consumption, we can better assess the needs of the culture. This will allow the development of feeding strategies based on cellular requirements instead of supplying the cultures with generic nutrient cocktails. It is expected that different nutrient mixtures can be used to target different goals such as 1) enhancing cell growth (before infection) and 2) improving the production of recombinant products (after infection). This will not only increase the efficiency of AAV vector production, but will also reduce the cost of production and make the process more economical by eliminating the addition of unnecessary nutrients. Although promising, some limitations of using biovolume still exist. A first limitation is the biovolume measure itself. This measure requires a device that measures cell size, such as a Coulter Counter Multisizer (Beckman-Coulter, Miami, FL, USA), which can be expensive. Capacitance probes can be a more cost effective tool to estimate biovolume; however, the availability of capacitance probes is still not common. A second limitation is the interpretation of the biovolume profiles, which can depend strongly on the fraction of cells in culture that are infected. If the culture is infected asynchronously, then there will be many different cell populations in the culture. Future work may require separating the cell size distribution into populations of viable and non-viable cells to get a better biovolume measure as opposed to assuming that viability is well distributed over the entire range of cell sizes. In infected cultures where the viability may be low, it is likely that the cell size distribution of non-viable cells will be concentrated at the lower end of the distribution (smaller diameter) rather than being well distributed over the whole range. If this is the case, for the infected cultures with low viability, the mean cell diameter calculated will be underestimated, which will lead to an overestimation of nutrient consumption for cultures with low viability. This will certainly affect the accuracy of the nutrient consumption profiles. By separating cell size distribution data into different cell populations of viable and nonviable, the accuracy can be improved.

The amino-terminal 33 amino acids of the baculovirus integral membrane protein, ODV-E66, are sufficient for localization of fusion proteins to viralinduced intranuclear microvesicles (MV) and occlusion derived virus envelopes during infection, and has been termed the sorting motif (SM). When abundantly expressed, SM-fusions are also detected in the inner nuclear membrane (INM), outer nuclear membrane and endoplasmic reticulum of infected cells, suggesting proteins with the SM use the same trafficking pathway as cellular INM proteins to traffic to nuclear membranes. This study identifies the essential characteristics required for sorting of the SM to the INM of uninfected cells, and the MV and ODV envelopes of infected cells. These features are an 18 amino acid transmembrane sequence that lacks polar and charged amino acids (a.a.) with a cluster of charged a.a. spaced 5-11 residues from the end of the transmembrane sequence. A comparison of the a.a. sequence of these SM features with cellular INM proteins shows the features are conserved. The model of INM protein sorting and localization predicts the only known sorting event during INM protein trafficking is immobilization/retention in the INM. This study uses confocal microscopy and fluorescence recovery after photobleaching to compare the localization and mobility of lamin B receptor (LBR) fusions (which contain SM-like sequences) to a viral SM fusion when expressed in either mammalian or insect cells. The results show that immobilization is not necessarily required for accumulation of proteins in the INM. Furthermore, the results from infected cells show that an active sorting event, likely independent of immobilization, can distinguish the viral SM from cellular sequences similar to the SM. The results of this study show that sorting of proteins to the INM can be mediated by the viral SM or INM protein SM-like sequences that can function either independent of, or in addition to, immobilization. These data combined with recent reports suggest that in addition to diffusion:retention a signal mediated mechanism for sorting and localization to the INM can occur.

Baculoviruses are versatile viruses that can be used as biopestisides, or for the production of recombinant protein and vaccines. Baculoviruses have also been found to be able to transfer genes to mammalian cells. This finding opened the door for the application of baculovirus vectors in human gene therapy. However, the mass production of clinical grade baculovirus vectors is challenging. Downstream processing has now become the bottle-neck of the manufacturing process. In this work, an anion exchange chromatography-based process was investigated for the purification of recombinant baculovirus vectors using a novel hydrogel based membrane (Natrix Separations Ltd.). Crude recombinant baculovirus supernatant from infected insect cell cultures was first subjected to a clarification process consisting of centrifugation and filtration. The pH of the viral solution was adjusted and then passed through a fast protein liquid chromatography system consisting of the ion exchange membrane. After washing weakly bound impurities, the captured baculoviruses are recovered by an elution step. Overall, baculoviruses strongly associated with the membrane; however, this interaction which was much physical as it was chemical, could not be entirely reversed and baculovirus was lost in the process. Product purity has also been evaluated and up to 85% of total protein reduction was determined. The significant losses of baculovirus observed have indicated major limitations in using this membrane for the purification of baculovirus.

The human kallikrein family is a family of proteolytic enzymes, classified as serine proteases, that derive from chromosome 19, locus 13.3-13.4. These enzymes are widespread in pathophysiological processes such as cancer and neurodegenerative diseases; hence studies of catalytic sites and inhibitors are important in relation to the longer term of design of therapeutic drugs. One member of the family, human kallikrein 4 (hK4) which is thought to carry out crucial functions in the prostate, was expressed in this study as a secreted protein in a baculovirus expression system, bearing a His-tag and V5-epitope that were used for purification and detection respectively. Its mass was estimated to be 35kDa, ~2kDa less than the equivalent product expressed in monkey kidney cells. The protein was purified to 50-90% purity with a yield of 0.93mg/L-4.8mg/L based on methods derived from computational prediction of its properties, such as pI. Computational analysis was extended by applying high-performing computing techniques, such as molecular dynamics, and flexible ligand docking, to predict antigenic regions, the likely substrate specificity and putative inhibitors. These results show that hK4 has a loop, between Leu83-Ser94 that shows promise as a specific segment that can be exploited for generation of antibodies. Preferred substrates were also predicted to bear hydrophobic residues at the P'-region of the scissile bond and amphiphilic residues at the P-region. At the S-region, hK4 potentially involves its unique PLYH-motif in recognizing the P4/P5 position from the substrate. Flexible ligand-docking studies indicate that hK4 can be inhibited by inhibitors that carry a modified bulky hydrophobic sidechain with a guanidinium group at the P1-position and its own putative autoactivation region residues at the P2, P1' and P2' position. The computational study was extended to other members of the kallikrein family, predicting distinctions between these that could be used for future studies. These results show that 8 of the fifteen kallikrein members are very homologous in terms of specificity bearing typical trypsinlike activity and specificity, except for hK2, hK3, hK4, hK5, hK7, hK9, hK15 that retain certain distinct signatures in the binding pocket in terms of secondary specificity. The principles of substrate-specificity analysis that were developed were further applied on three metzincins, MMP-3, ADAM-9 and ADAM-10. These three enzymes are metalloproteases, which are involved in tissue remodeling, intracellular signalling and cell-to-cell mediation. The substrate-specificity analysis was carried out on all three metzincins using the structure of a crystallized complex of the MMP-3 enzyme with the TIMP-1 natural inhibitor as template. In this specific enzyme-substrate complex, the challenge was to model and suggest a possible orientation of the P-region, which is not known. The interactions on the P/S-region are therefore unclear and need to be clarified. In order to suggest the arrangement of the enzyme-substrate complex and the undefined S-subsites, four new residues were added in an extended beta-sheet conformation to the P1' residue (derived originally from the TIMP-1 inhibitor) to create a full-length modeled substrate spanning P4'-P4. This new modeled region, in particular, was bound through backbone H-bonds with the enzyme at position 169 (MMP nomenclature) suggesting a new crucial residue for substrate binding, and satisfied steric and chemical restraints in the S'-region of the enzyme. This modeling approach also indicated a putative presence of an S2/S3-pocket on these metzincins which is composed of different residues for MMP-3, ADAM-9 and ADAM-10, and which could prove useful for future drug design projects. Furthermore, the data argue against the involvement of a polarizable water molecule in catalysis, a mechanism that has been postulated by various groups. A new catalytic mechanism is suggested to involve an oxyanion anhydride transition state. This study is a demonstration of the power of combining bioinformatics with wet-lab biochemistry.

O circovírus suíno tipo 2 (PCV2) é o principal agente da síndrome multissistêmica do definhamento do suíno (SMDS), uma doença mundialmente disseminada e que provoca perdas econômicas significativas para a suinocultura. Visando contribuir no diagnóstico da síndrome, o presente trabalho padronizou e comparou testes para a detecção do PCV2. Para isso, foram utilizadas as técnicas de amplificação por círculo rolante (ACR) e variações da PCR (convencional, tempo-real e competitiva). Utilizando a ACR foi possível obter a amplificação total de genomas do PCV2, os quais foram clonados, sequenciados e agrupados no genótipo PCV2b. Os genomas clonados foram isolados, recircularizados e transfectados em células PK-15. Este procedimento possibilitou a recuperação do vírus infeccioso em títulos de até 105,55 DICC50/mL. Portanto, a ACR foi uma ferramenta útil em estratégias de isolamento e sequenciamento do vírus. No entanto, a ACR foi menos sensível que a PCR para fins de detecção do PCV2. No segundo estudo, buscando métodos auxiliares no diagnóstico da SMDS, dois ensaios para a quantificação do PCV2 foram desenvolvidos. Estes ensaios foram baseados nas técnicas de PCR competitivo (cPCR) e de PCR em tempo real. Visando determinar qual seria o mais adequado para estimar a carga viral do PCV2, os dois métodos foram comparados. Ambos os ensaios foram capazes de detectar diferenças significativas entre o número de cópias de DNA de PCV2 encontradas em tecidos de animais saudáveis e acometidos pela SMDS (≥ 2,5 log10). No entanto, uma diferença média de 1,8 log10 na carga viral foi encontrada entre ensaios, onde as maiores cargas virais foram detectadas pela PCR em tempo real. Outro objetivo deste trabalho foi gerar vacinas baseadas na proteína do capsídeo (Cap) do PCV2. Assim, no terceiro estudo, três baculovírus recombinantes foram construídos de modo a expressar a proteína Cap. Em dois recombinantes, a seqüência de nucleotídeos do peptídeo sinal (PS) da glicoproteína I do herpesvírus bovino (BoHV-gI) foi inserida na extremidade 5’ do gene cap (ORF2). Além disso, um recombinante contendo a seqüência de nucleotídeos do PS foi construído sem o sinal de localização nuclear (NLS) de proteína Cap. Através do ensaio de imunoperoxidase em monocamada (IPMA), antígenos de PCV2 foram detectados em células Sf21 infectadas pelos três vírus recombinantes. Este resultado sugere que os recombinantes construídos são potenciais candidatos vacinais, uma vez que eles foram capazes de produzir antígenos de PCV2. / Porcine circovirus type 2 (PCV2) is the major agent of postweaning multisystemic wasting syndrome (PMWS), a worldwide spread disease that causes significant economic losses to the swine productive chain. Aiming to contribute in the diagnosis of the syndrome, this thesis compared and developed tests for PCV2 detection. For this, multiply-primed rolling-circle amplification (MPRCA) and PCR-based assays (conventional, real-time and competitive) were tested. The MPRCA allowed amplifying the full-length PCV2 genomes, which were cloned, sequenced and grouped on PCV2b genotype. The cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 105.55 TCID50/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes in sight of sequencing and virus isolation strategies. However, it was less sensitive than PCR for diagnostic purposes. In the second study, searching for methods in support to PMWS diagnosis, two PCR assays were developed: a competitive PCR (cPCR) and a SYBR green real-time PCR. The quantitative PCR methods were compared to determine which would be more suitable to estimate the PCV2 DNA load. Both assays were able to detect significant differences between the numbers of PCV2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥2.5 log10). However, a mean difference of 1.8 log10 on the viral load was found between assays, where the highest viral loads were detected by SYBR green real-time PCR. In the work outlined herein, another purpose was to generate vaccine candidates based on PCV2 capsid protein (Cap). Therefore, in the third study, three types of recombinant baculoviruses were constructed to express the Cap protein. In two recombinants, the nucleotide sequence from the signal peptide (SP) of bovine herpesvirus glycoprotein I (BoHV-gI) was inserted at the 5’ end of the cap gene (ORF2). Additionally, one recombinant containing the SP nucleotide sequence was constructed lacking the nuclear localization signal (NLS) of Cap protein. Through immunoperoxidase monolayer assay (IPMA), the PCV2 antigen was detected in Sf21 cells infected by the three recombinant viruses. This result suggests that the recombinants here constructed are potential vaccine candidates, once they were able to produce PCV2 antigens.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-09-04T16:57:00Z No. of bitstreams: 1 Tiago Landim d'Avila Administração de plasmídeos....pdf: 1447180 bytes, checksum: b65ad3c9ece2b6f29cc032b2fa22c49a (MD5) / Made available in DSpace on 2012-09-04T16:57:00Z (GMT). No. of bitstreams: 1 Tiago Landim d'Avila Administração de plasmídeos....pdf: 1447180 bytes, checksum: b65ad3c9ece2b6f29cc032b2fa22c49a (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A interleucina-12 (IL-12) é uma glicoproteína heterodímerica, codificada por dois genes distintos co-expressados na mesma célula. IL-12 precisa ser glicosilada para exibir atividade funcional. Essa glicoproteína promove a diferenciação de células T e é capaz de estimular a proliferação de células T ativadas e células NK. Além disso, IL-12 promove a produção IFN-γ por células NK e o aumento da atividade lítica de células NK e linfócitos T CD+ citotóxicos. Várias das atividades de IL-12 são desempenhadas ou são amplificadas pela associação com IL-2. Em nosso laboratório, foi gerada uma construção plasmideal capaz de expressar IL-12 canina, na forma de proteína de fusão de cadeia única (pcDNA3.1-scca-IL-12), e outra construção plasmideal capaz de expressar IL-2 canina (pcDNA3.1-ca-IL-2) em células de mamífero. Experimentos preliminares, a combinação de IL-12 e IL-2 expressas em sobrenadantes de células COS-7 mostrou-se capaz de promover produção de IFN-γ em células mononucleares de sangue periférico (CMNSP) de cães sadios. Visando a avaliação do potencial do uso de IL-12 e/ou IL-2 para o desenvolvimento de vacina ou método imunoterapico, no presente trabalho, grupos de cães sadios foram injetados três vezes, com intervalo de 2 dias entre cada duas doses consecutivas, com plasmídeo pcDNA3.1-scca-IL-12 e/ou pcDNA3.1-ca-IL-2 por via muscular por eletroporação. Em seguida, os cães foram submetidos à avaliação clinica, clinico-laboratorial (hemograma, determinação da concentração de transaminases, proteínas, ureia e creatinina no soro) e ensaios foram realizados para a detecção da expressão das proteínas recombinantes (sensibilização de CMNSP para produção de IFN-γ e determinação da concentração de IL-2 no soro). Não foram observadas diferenças nos parâmetros avaliados entre os grupos de animais. Visando a produção de IL-12, células BTI-Tn-5B1-4 foram infectadas com baculovírus recombinante contendo cDNA scca-IL-12 e capazes de adicionar uma cauda de 6 histidinas na extremidade carboxila. Para isso, duas construções diferentes em baculovírus foram elaboradas nosso laboratório, sendo uma delas no presente trabalho. Células BTI-Tn-5B1-4 infectadas com as construções em baculovírus apresentaram a proteína recombinante: a) parcialmente degradada e em grande quantidade no sedimento celular e b) integra e em baixa quantidade no sobrenadante da cultura; de acordo com os resultados de obtidos por SDS-PAGE e Western blot. / Interleukin-12 (IL-12) is a heterodimeric glycoprotein, encoded by two distinct genes co-expressed in the same cell. IL-12 needs to be glycosylated to display functional activity. This glycoprotein promotes differentiation of T cells and is capable of stimulating proliferation of activated T cells and NK cells. In addition, IL-12 promotes IFN-γ production by NK cells and increased lytic activity of NK cells and cytotoxic Tlymphocyte CD +. Several of the activities of IL-12 are held or amplified by the association with IL-2. In our laboratory, a plasmideal construction was generated to express canine IL-12 like single-chain fusion protein (pcDNA3.1-scca-IL-12), and other construction to express canine IL-2 (pcDNA3.1-CA-IL-2) in mammalian cells. Preliminary experiments, the combination of IL-12 and IL-2 supernatants expressed in COS-7 cells was able to promote IFN-γ in peripheral blood mononuclear cells (PBMC) of healthy dogs. To assess the potential of using IL-12 and/or IL-2 for the development of vaccine or immunotherapy method, in this study, groups of healthy dogs were injected three times with an interval of 2 days between each two consecutive doses with plasmid pcDNA3.1-scca-IL-12 and/or pcDNA3.1-ca-IL-2 intramuscularly by electroporation. Then the dogs were submitted to clinical evaluation, clinical and laboratory (blood count, determination of the concentration of transaminases, proteins, urea and serum creatinine) and tests were performed to detect the expression of recombinant proteins (PBMC sensitization to produce IFN-γ concentration and determination of serum IL-2). No differences were observed in all evaluated parameters between the groups of animals. Aiming the production of IL-12 cells, BTI-Tn-5B1-4 were infected with recombinant baculovirus containing cDNA scca-IL-12 and capable of adding a tail of 6 histidines at the carboxyl end. For this, two different constructs were prepared in our laboratory baculovirus, one of them in this work. Cells BTI-Tn-5B1-4 infected with the constructs presented in the recombinant baculovirus: a) partially degraded in large quantities in the sediment cell and b) incorporates and in low quantities in the culture supernatant, according to the results obtained by SDS-PAGE and Western blo

O circovírus suíno tipo 2 (PCV2) é o principal agente da síndrome multissistêmica do definhamento do suíno (SMDS), uma doença mundialmente disseminada e que provoca perdas econômicas significativas para a suinocultura. Visando contribuir no diagnóstico da síndrome, o presente trabalho padronizou e comparou testes para a detecção do PCV2. Para isso, foram utilizadas as técnicas de amplificação por círculo rolante (ACR) e variações da PCR (convencional, tempo-real e competitiva). Utilizando a ACR foi possível obter a amplificação total de genomas do PCV2, os quais foram clonados, sequenciados e agrupados no genótipo PCV2b. Os genomas clonados foram isolados, recircularizados e transfectados em células PK-15. Este procedimento possibilitou a recuperação do vírus infeccioso em títulos de até 105,55 DICC50/mL. Portanto, a ACR foi uma ferramenta útil em estratégias de isolamento e sequenciamento do vírus. No entanto, a ACR foi menos sensível que a PCR para fins de detecção do PCV2. No segundo estudo, buscando métodos auxiliares no diagnóstico da SMDS, dois ensaios para a quantificação do PCV2 foram desenvolvidos. Estes ensaios foram baseados nas técnicas de PCR competitivo (cPCR) e de PCR em tempo real. Visando determinar qual seria o mais adequado para estimar a carga viral do PCV2, os dois métodos foram comparados. Ambos os ensaios foram capazes de detectar diferenças significativas entre o número de cópias de DNA de PCV2 encontradas em tecidos de animais saudáveis e acometidos pela SMDS (≥ 2,5 log10). No entanto, uma diferença média de 1,8 log10 na carga viral foi encontrada entre ensaios, onde as maiores cargas virais foram detectadas pela PCR em tempo real. Outro objetivo deste trabalho foi gerar vacinas baseadas na proteína do capsídeo (Cap) do PCV2. Assim, no terceiro estudo, três baculovírus recombinantes foram construídos de modo a expressar a proteína Cap. Em dois recombinantes, a seqüência de nucleotídeos do peptídeo sinal (PS) da glicoproteína I do herpesvírus bovino (BoHV-gI) foi inserida na extremidade 5’ do gene cap (ORF2). Além disso, um recombinante contendo a seqüência de nucleotídeos do PS foi construído sem o sinal de localização nuclear (NLS) de proteína Cap. Através do ensaio de imunoperoxidase em monocamada (IPMA), antígenos de PCV2 foram detectados em células Sf21 infectadas pelos três vírus recombinantes. Este resultado sugere que os recombinantes construídos são potenciais candidatos vacinais, uma vez que eles foram capazes de produzir antígenos de PCV2. / Porcine circovirus type 2 (PCV2) is the major agent of postweaning multisystemic wasting syndrome (PMWS), a worldwide spread disease that causes significant economic losses to the swine productive chain. Aiming to contribute in the diagnosis of the syndrome, this thesis compared and developed tests for PCV2 detection. For this, multiply-primed rolling-circle amplification (MPRCA) and PCR-based assays (conventional, real-time and competitive) were tested. The MPRCA allowed amplifying the full-length PCV2 genomes, which were cloned, sequenced and grouped on PCV2b genotype. The cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 105.55 TCID50/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes in sight of sequencing and virus isolation strategies. However, it was less sensitive than PCR for diagnostic purposes. In the second study, searching for methods in support to PMWS diagnosis, two PCR assays were developed: a competitive PCR (cPCR) and a SYBR green real-time PCR. The quantitative PCR methods were compared to determine which would be more suitable to estimate the PCV2 DNA load. Both assays were able to detect significant differences between the numbers of PCV2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥2.5 log10). However, a mean difference of 1.8 log10 on the viral load was found between assays, where the highest viral loads were detected by SYBR green real-time PCR. In the work outlined herein, another purpose was to generate vaccine candidates based on PCV2 capsid protein (Cap). Therefore, in the third study, three types of recombinant baculoviruses were constructed to express the Cap protein. In two recombinants, the nucleotide sequence from the signal peptide (SP) of bovine herpesvirus glycoprotein I (BoHV-gI) was inserted at the 5’ end of the cap gene (ORF2). Additionally, one recombinant containing the SP nucleotide sequence was constructed lacking the nuclear localization signal (NLS) of Cap protein. Through immunoperoxidase monolayer assay (IPMA), the PCV2 antigen was detected in Sf21 cells infected by the three recombinant viruses. This result suggests that the recombinants here constructed are potential vaccine candidates, once they were able to produce PCV2 antigens.

Made available in DSpace on 2014-12-17T15:01:15Z (GMT). No. of bitstreams: 1 AndreaFA.pdf: 1749500 bytes, checksum: 3468acf35904221d81b3482eef39fc68 (MD5) Previous issue date: 2005-04-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / In vivo production of viral biopesticides is the major source of viral insecticides currently in the marketplace. However, this system presents limitations during production scale-up. For the Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV), the insect used for replication has cannibalistic characteristics, thus production is even more difficult. Insect cells are commonly used for in vitro baculovirus production. Most of these cell lines are derived from Lepidoptera species. The Sf21 cell line is derived from Spodoptera frugiperda caterpillar ovarian tissue, and its clonal isolate Sf9 has been used for biopesticide production due to its ease of growth in suspension cultures. In this work, the in vitro production capabilities of a Brazilian SfMNPV isolate obtained from cornfields was evaluated. Comparison of polyhedra production was carried out using both Sf21 and Sf9 cells, based on volumetric and specific yields. Both cell lines were cultivated in Hyclone medium supplemented with different fetal bovine serum concentrations (2,5 and 5%). The best results were obtained using Sf9 cells supplemented with 5% serum. These results were further confirmed quantitively through kinetic parameter estimation for both cells lines and different serum concentrations. After seven successive passages, this system still presented high specific polyhedra production / A produ??o in vivo de biopesticidas virais ? a maior fonte destes inseticidas presentes no mercado atualmente. Entretanto, o sistema in vivo apresenta limita??o em rela??o ao escalonamento de produ??o. No caso do v?rus Spodoptera frugiperda nucleopoliedrovirus (SfMNPV), o inseto usado para sua multiplica??o tem caracter?sticas canibais, o que dificulta ainda mais a sua produ??o in vivo. As c?lulas de inseto s?o comumente utilizadas para produ??o in vitro de baculov?rus. V?rias linhagens destas c?lulas s?o derivadas principalmente da esp?cie Lepidoptera. A linhagem Sf21 ? derivada do tecido ovariano da lagarta Spodoptera frugiperda e um clone isolado da linhagem original, denominado Sf9, tem sido utilizado para produ??o de biopesticidas, por apresentar f?cil crescimento em cultivo em suspens?o. Neste trabalho, foi testada a viabilidade de produ??o in vitro de um isolado viral brasileiro de SfMNPV obtido em lavouras de milho. A produ??o de poliedros, em c?lulas Sf21 e Sf9, foi determinada com base na avalia??o comparativa da produtividade volum?trica e espec?fica destes poliedros. Ambas as linhagens celulares foram cultivadas, em suspens?o, em meio HyClone suplementado com diferentes concentra??es de soro fetal bovino (2,5 e 5%). A c?lula Sf9 suplementada com 5% de soro apresentou os melhores resultados de produ??o. Os resultados foram confirmados quantitativamente atrav?s dos par?metros cin?ticos estimados para as duas linhagens e diferentes concentra??es de soro. O sistema demonstrou, ap?s sete passagens sucessivas, uma alta produ??o espec?fica de poliedros

Comparadas com células de mamífero, as células de inseto são mais fáceis de cultivar, não acumulam quantidade significativa de sub-produtos tóxicos e apresentam maiores rendimentos na expressão de proteínas heterólogas, porém apresentam uma menor capacidade de realizar modificações pós-traducionais. Células de inseto podem ser empregadas na produção in vitro de baculovírus, usados como pesticidas biológicos. As células Sf-9 estão entre as células de inseto com uso mais difundido. Entender o metabolismo destas células permitirá melhorias nos processos que as empregam, entretanto, ainda há relativamente pouca informação sobre o assunto. Considerando mais especificamente o uso dessas células para produção de baculovírus, também é necessário mais entendimento sobre o processo infectivo e parâmetros que o afetam. Este trabalho teve como objetivo estudar a influência da suplementação do meio de cultivo com diferentes aminoácidos no desenvolvimento das células Sf-9 e determinar a concentração de oxigênio dissolvido ideal para o cultivo destas células, visando elaborar uma metodologia de cultivo em biorreator otimizada e, paralelamente, estudar o processo de infecção de cultivos dessas células com o baculovírus Spodoptera frugiperda (SfMNPV) em diferentes escalas. Para estudar a influência que a adição de aminoácidos ao meio tem sobre o crescimento celular, células Sf-9 foram cultivadas em frascos schotts de 100 e 500 mL, com 20 mL do meio SF900III SFM (serum free medium) suplementados com cisteína, prolina, serina ou asparagina. Os resultados foram comparados com cultivos feitos sem suplementação (controles). A condição que apresentou o melhor resultado em frasco schott foi replicada em biorreator de 1 L de volume útil. Para estudar a influência do oxigênio dissolvido (O.D.) foram testados diferentes setpoints de O.D. em cultivos em biorreator. Em tais ensaios foram testadas as concentrações de O.D de 10%, 30% e 50% da saturação com o ar. Para o estudo do processo de infecção, foram realizadas infecções em frascos schotts de 500 mL, com 20 mL de cultivo, e em biorreator de 1 L. Também foram realizadas infecções em garrafas T-25 como forma de controle de virulência do inóculo viral e do vírus produzido. As principais variáveis analisadas foram µmáx, Xvmáx YX/Glc. Nos ensaios de influencia de O.D., analisou-se também qO2 e qCO2 e, nos ensaios de infecção, a porcentagem de células contendo poliedros. A suplementação com prolina foi prejudicial ao cultivo. A adição de asparagina não teve qualquer influência no desenvolvimento celular. Os resultados das adições de cisteína e serina não foram muito conclusivos, em alguns ensaios houve aumento de Xvmáx, já em outros não foi notado efeito significativo. Nos ensaios em biorreator, todos os valores de O.D. testados apresentaram resultados semelhantes, já a adição de cisteína ao meio em biorreator foi bastante maléfica ao crescimento celular. Os ensaios de infecção mostraram que células Sf-9 são bastante susceptíveis à infecção pelo baculovírus Spodoptera frugiperda e boas produtoras de poliedros virais, e que a vazão gasosa tem efeito negativo na concentração viral na fase líquida dos cultivos em biorreator (título viral). / Compared to mammalian cells, insect cells are easier to culture, do not accumulate significant amounts of toxic byproducts and are capable of higher heterologous protein yields, but have a lower ability to perform post-translational modifications. Insect cells may be employed in the in vitro production of baculoviruses, used as biological pesticides. Sf-9 cells are among the most used insect cell lines. Understanding the metabolism of these cells would allow improvements in the processes that employ them, however, Howeverreports on the metabolism and physiology of Sf-9 cells and insect cells in general are scarce. When considering the use of these cells for baculovirus production, it is also necessary more understanding about the infective process and parameters that can affect it. This work aimed to study the influence that the supplementation of the culture medium with different amino acids have on the development of Sf-9 cells and to determine the ideal dissolved oxygen concentration for the culture of these cells, aiming to elaborate an optimized bioreactor culture methodology and, in parallel, to study the infection process of these cells with the Spodoptera frugiperda baculovirus (SfMNPV) at different scales. To study the influence that the addition of amino acids to the medium has on cell growth, Sf-9 cells were cultured in 100 and 500mL schott flasks with 20mL SF900III SFM (serum free medium) supplemented with cysteine, proline, serine or asparagine. The results were compared with cultures without supplementation (controls). The condition that presented the best result in schott flasks was replicated in a 1L bioreactor. To study the influence of dissolved oxygen (O.D.), experiments with different values of O.D. were conducted at a 1L bioreactor. The O.D. tested were 10%, 30% and 50% of air saturation. To study the infection process, infections were carried out in 500 mL schotted flasks, with 20 mL of culture, and in a 1L bioreactor. Infections were also carried out in 25 cm² T-flasks as a form of virulence control of the viral inoculum and virus produced. The main variables analyzed were µmax, Xvmax YX/Glc. In the O.D. tests, qO2 and qCO2 were also analyzed and, in the infection assays, the percentage of cells containing polyhedra. Proline supplementation was detrimental to the culture. The addition of asparagine had no influence on cellular growth. The results of cysteine and serine additions were not very conclusive, in some studies there was an increase of Xvmax, while in others no significant effect was observed. In the bioreactor trials, all O.D. tested showed similar results and the addition of cysteine to the medium was quite harmful to cell growth. Infection assays showed that Sf-9 cells are quite susceptible to infection by the Spodoptera frugiperda baculovirus and good producers of viral polyhedra, and that the gas flow has a negative effect on the viral concentration in the liquid phase of the bioreactor cultures (viral titer).