Hepatitis B virus genotype G (HBV/G) is a unique genotype of HBV which contains a 36-nucleotide insertion in the Core gene as well as 2 mutations that lead to stop codons in the Pre-Core coding region. Chronic infection with HBV/G is not known to occur without a co-infecting HBV genotype, suggesting that it is defective on its own. This study aims to look at the replication capacity of HBV/G, HBV/A, and HBV/A/G recombinant strains circulating in Canada and to determine the relationship between co-infecting strains. Four full-length HBV genomes were isolated from 2 different patients and transiently transfected into the HepG2 human hepatoma cell line for phenotypic analysis of each strain. HBV/G, HBV/A and HBV/A/G recombinant strains were isolated from Patient 1, while a different HBV/A/G recombinant strain was isolated from Patient 2. HBV replication capacity was measured using a quantitative real time PCR assay. Markers of replication, such as secreted HBsAg and HBeAg, intracellular core particles and replicative DNA intermediates were measured by ELISA, Western blot and Southern blot, respectively. HBV/G demonstrated a higher replicative capability, relative to its co-infecting strains, while both HBV/A/G strains had levels of secreted HBV DNA greater than HBV/A alone, suggesting a modulating effect due to recombination. Replication marker levels revealed possible reasons for a co-infection requirement during HBV/G infection such as HBeAg for chronicity. These observations demonstrate the potential interactions of HBV/G with its co-infecting HBV genotype and provide the first reported phenotypic analysis of a HBV recombinant.

Hepatitis B virus genotype G (HBV/G) is a unique genotype of HBV which contains a 36-nucleotide insertion in the Core gene as well as 2 mutations that lead to stop codons in the Pre-Core coding region. Chronic infection with HBV/G is not known to occur without a co-infecting HBV genotype, suggesting that it is defective on its own. This study aims to look at the replication capacity of HBV/G, HBV/A, and HBV/A/G recombinant strains circulating in Canada and to determine the relationship between co-infecting strains. Four full-length HBV genomes were isolated from 2 different patients and transiently transfected into the HepG2 human hepatoma cell line for phenotypic analysis of each strain. HBV/G, HBV/A and HBV/A/G recombinant strains were isolated from Patient 1, while a different HBV/A/G recombinant strain was isolated from Patient 2. HBV replication capacity was measured using a quantitative real time PCR assay. Markers of replication, such as secreted HBsAg and HBeAg, intracellular core particles and replicative DNA intermediates were measured by ELISA, Western blot and Southern blot, respectively. HBV/G demonstrated a higher replicative capability, relative to its co-infecting strains, while both HBV/A/G strains had levels of secreted HBV DNA greater than HBV/A alone, suggesting a modulating effect due to recombination. Replication marker levels revealed possible reasons for a co-infection requirement during HBV/G infection such as HBeAg for chronicity. These observations demonstrate the potential interactions of HBV/G with its co-infecting HBV genotype and provide the first reported phenotypic analysis of a HBV recombinant.

Thèse ou mémoire avec insertion d’articles / La complexité et le coût de production à grande échelle des rAAV présentent un sérieux obstacle à la commercialisation des thérapies géniques. Des améliorations dans la fabrication des vecteurs rAAV sont réalisables à l'aide de lignées cellulaires d'empaquetage ou productrices, qui, dans le cas des rAAV, sont difficiles à générer en raison de la toxicité des protéines AAV Rep et des gènes auxiliaires d'adénovirus nécessaires à la production. Le gène AAV REP code pour quatre protéines Rep (Rep -78, -68, -52, -40) qui ont de multiples fonctions et rôles qui se chevauchent dans la production d'AAV. Les gènes auxiliaires adénoviraux utilisés dans la production de rAAV sont E2A, E4 et VA et jouent des rôles uniques dans le cycle de vie de l'AAV. Pour générer une lignée cellulaire d'empaquetage de AAV, tous ces composants doivent être intégrés de manière stable dans le génome des cellules. L'expression doit être étroitement régulée en raison de la cytotoxicité et lorsque l'expression est induite, les niveaux doivent être adéquats pour soutenir la production de rAAV. Une telle lignée cellulaire d'empaquetage serait un point de départ pour créer une lignée cellulaire productrice pour la production d'un sérotype spécifique d'AAV et d'un gène thérapeutique spécifique en intégrant un gène CAP d'intérêt et une séquence thérapeutique flanquée d'ITR. Le travail présenté dans cette thèse décrit le processus de génération et de caractérisation des lignées cellulaires d'empaquetage 293SF-CymR/λR-GyrB AAV exprimant les protéines Rep et les conceptions et tentatives de création d'une lignée cellulaire d'empaquetage Rep/Helper. Nous avons identifié et intégré avec succès une combinaison de deux protéines Rep (Rep68 et Rep40) exprimées à partir de deux promoteurs inductibles par le cumate et la coumermycine. Les lignées cellulaires créées ont démontré leur capacité à produire des titres à des niveaux comparables aux productions par transfection transitoire avec les lignées cellulaires parentales et se sont avérées stables en culture sans sélection. Nous décrivons également notre conception et nos travaux de recherche sur la faisabilité pour générer des lignées cellulaires d'emballage Rep/Helper où les séquences codant pour les protéines E4orf6 et E2A DBP sont également exprimées à partir de promoteurs inductibles par le cumate et la coumermycine. / The complexity and cost of large-scale rAAV production present a serious obstacle in commercialization of rAAV gene therapies. Improvements in rAAV vector manufacturing are achievable using packaging or producer cell lines, which in case of rAAVs are difficult to generate due to the toxicity of AAV Rep proteins and adenovirus helper genes required for production. The AAV REP gene encodes four Rep proteins (Rep -78, -68, -52, -40) which have multiple overlapping functions and roles in AAV production. The adenoviral helper genes used in rAAV production are E2A, E4 and VA and serve unique roles in the AAV lifecycle. To generate an AAV packaging cell line all these components must be stably integrated into the genome of the cells. Expression must be tightly regulated due to cytotoxicity and when expression is induced, the levels must be adequate to support rAAV production. Such a packaging cell line would be a starting point to create a producer cell line for production of a specific serotype of AAV and specific therapeutic gene by integrating a CAP gene of interest and ITR flanked therapeutic sequence. The work presented in this thesis describes the process of generating and characterizing 293SF-CymR/λR-GyrB AAV packaging cell lines expressing the Rep proteins and the designs and attempts of creating a Rep/Helper packaging cell line. We successfully identified and integrated a combination of two Rep proteins (Rep68 and Rep40) expressed from two cumate and coumermycin inducible promoters. The created cell lines demonstrated ability to produce titers at levels comparable to transient transfection productions with the parental cell lines and were shown to be stable in culture without selection. We also describe our design and investigation into the feasibility of Rep/Helper packaging cell line generation where the E4orf6 and E2A DBP protein coding sequences are also expressed from cumate and coumermycin inducible promoters.

Cette thèse actualise les connaissances de l'évaluation de l'allergénicité des aliments et son application au diagnostic de l'allergie alimentaire. Après la définition, les caractéristiques et la classification des allergènes alimentaires, les phénomènes physico-chimiques modifiant l'allergénicité des aliments ainsi que les réactivités croisées sont décrites. Ainsi sont introduits les outils cliniques et biologiques utiles au diagnostic de l'allergie alimentaire et à la détection des traces d'allergènes alimentaires. Une collaboration étroite entre cliniciens et chercheurs biologistes, permet d’optimiser la prise en charge diagnostique et thérapeutique de l’allergie alimentaire. Cette démarche se concrétise par la mise à disposition et l'utilisation de divers outils (développement d’allergènes recombinants, dosage de contaminants alimentaires dans des médicaments ou aliments, ...) et est illustrée par diverses mises en situation clinique réelles. / The aim of this thesis consists in reviewing the advanced knowledge about the evaluation of food allergenicity and the different applications to the diagnosis of food allergy. Definition, characteristics and classification of food allergens precede the description of physical and chemical factors involved in the modification and cross-reactivity of food allergens. This review leads to the current tools used for the in vivo and in vitro diagnosis of food allergy as well as the recent procedures which have been developed for the detection of allergen traces in industrial food. A close collaboration between clinicians and biologists improves the diagnosis procedures. This cooperation is illustrated by various clinical situations in which new tools (i.e. specific IgE to recombinant allergens, food allergen traces identification in meals or drugs) have improved the diagnostic procedures and consequent therapeutic measures.

La prévalence de l’allergie alimentaire est en constante augmentation. Parallèlement les consommations alimentaires et les aliments eux même évoluent. L’aliment, de plus en plus soumis aux traitements industriels, peut présenter une allergénicité modifiée. L’objet de cette thèse sera d’évaluer l’influence des différents procédés industriels sur l’immunoréactivité des protéines alimentaires modifiées. Les différentes études présentées dans l’ouvrage aborderont l’effet de la texturation sur les protéines de soja ou de l’extrusion sur les protéines de lin (traitement thermique), l’effet de l’incorporation de nouveaux ingrédients dans l’alimentation moderne (inuline, lin). L’allergénicité des protéines recombinantes (process biotechnologique) sera abordée au travers de deux études : l’une proposera les protéines recombinantes de l’arachide comme outil diagnostique, en particulier pour le test d’activation des basophiles ; L’autre cherchera à démontrer l’innocuité allergénique d’un lysozymes recombinant humain, qui sera utilisé en alimentation humaine. L’étude de ces propriétés allergéniques sera réalisée par les tests in vitro (prick tests et test de provocation orale) et par les tests in vitro (IgEs). Des études par immuno-empreinte (immunoblot et dot blot) et par spectrométrie infrarouge permettront d’identifier les protéines modifiées. Enfin des tests cellulaires (test d’activation des basophiles) seront également réalisés. / The prevalence of the food allergy is in constant increase. At the same time food consumptions and food they even evolve. The food, more and more subjected to the industrial treatments, can present a modified allergenicity. The aim of this thesis will be to estimate the influence of the various industrial processes on the immunoreactivity of modified food proteins. The various studies presented in the work will approach the effect of the texturisation on proteins of soy or the extrusion on flaxseed proteins (heat treatment), the effect of the incorporation of new ingredients in the modern food (inuline, flaxseed). The allergenicity of recombinant proteins (biotechnological process) will be approached through two studies: the one will propose peanut recombinant proteins as a diagnostic tool, in particular for the basophil activation test (BAT); other one will try to demonstrate the allergenic harmlessness of a recombinant human lysozyme, which will be used in human food. The study of these allergenic properties will be realized by the in vivo tests (prick tests) and by the in vitro tests (IgEs). Studies by immunoblot and by infrared spectrometry will allow identifying modified proteins. Finally cellular tests (BAT) will be also performed.

PhD / HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

PhD / HIV-1 is the cause of the majority of global HIV infections. Not only being more virulent, and relatively easily transmitted than HIV-2, HIV-1 is also more extensively studied. HIV-1 is known for its highly recombinogenic nature, together with an extreme genetic variety, both attributable to an error-prone reverse transcriptase which gives rise to heterozygous virion. Sequence diversity of HIV-1 has resulted in identification of 9 subtypes of HIV-1 M group, as well as 43 circulating and a number of other unique recombinant forms of HIV-1. The extensive heterogeneity of HIV-1 has become the main consideration in vaccine development, mainly due to the inherent variability of HIV-1 and the frequent generation of new recombinant forms, which subsequently makes the effort to control the HIV-1 pandemic more challenging. The inter-subtype recombination event is a common phenomenon observed in Malaysia whereby there is a co-circulation of multiple HIV-1 subtypes; CRF01_AE and subtype B. Therefore, it becomes crucial to widen the knowledge of currently emerging CRF01_AE/B inter-subtype recombinants, in order to assist the future regional vaccine design and also to prevent wider spread of these strains. Concurrently, with a better understanding on the characteristics of HIV-1 CRF01_AE/B recombinant forms, further diversification of these strains can possibly be thwarted. The objectives of this study included, firstly to study the molecular epidemiology pattern of different HIV-1 strains, as well as to observe their frequency and distribution. Our second aim was to identify possible derivative from CRF33_01B, and also other new CRF01_AE/B inter-subtype recombinant forms in Malaysia. Thirdly, we aimed to identify possible biological advantages of the CRF33_01B isolates over its parental strains; CRF01_AE and subtype B. Currently, the HIV-1 epidemic in Malaysia is in a concentrated phase with evidence of predominance of both CRF01_AE and subtype B found among heterosexuals and injecting drug users, respectively. There is urgent necessity to apply a more detailed and continuous molecular characterization and epidemiological monitoring of these recombinant forms in Malaysia. We obtained plasma samples from 115 HIV-1-infected patients who attended HIV clinic at the University Malaya Medical Centre in Kuala Lumpur, Malaysia. The HIV-1 PR-RT, gp120-env and gp41-env genes were amplified and sequenced from 50 samples, while the remaining 65 samples were successfully studied at either one or two HIV-1 specific genomic regions. Cloning, phylogenetic analyses, together with bootscanning methods were employed to assign subtypes and to identify inter-subtype recombination based on all three genomic regions. From the plasma-derived sequences of 50 patients, 46% were found to harbour CRF01_AE, 10% and 6% had subtype B and B’, and a total of 18% of the patients were infected with CRF33_01B, while the remaining 18% of patients was found to have unique recombinant forms. As for the other 65 patients, majority of them harboured CRF01_AE and subtype B. This study shows that co-circulation of multiple HIV-1 subtypes and their recombinant strains are frequent in the Malaysian population, while capable of spreading to different HIV-1 risk groups. Possible recombination hotspots in CRF01_AE/B recombinants are suggested to be within the HIV-1 PR-RT gene region. Further, this study highlights the need to characterize and monitor the molecular epidemiology of these recombinant forms. The ideal environment for the inter-subtype recombination event to take place is created by the co-circulation and dual infections of both CRF01_AE and subtype B. With more HIV-1 CRF01_AE/B recombinant forms emerging and shaping the nature of HIV epidemic in Malaysia, certainly it will complicate the timely diagnosis of these molecularly altered HIV-1 forms. The recent identification of the novel CRF33_01B suggests the emergence of other new CRF01_AE/B inter-subtype recombinant forms in Malaysia, as preliminarily demonstrated in some HIV-1 patients identified in the first part of this study. The peripheral blood mononuclear cells (PBMCs) of these HIV-1 patients were co-cultured with those of healthy donors, which we then isolated the proviral genomic DNA. The nested long-range PCR was performed to obtain seven overlapping viral genome fragments that made up the whole viral genome. The detailed phylogenetic, as well as bootscan analyses confirmed the mosaic compositions and recombinant structures of the newly emerging CRF01_AE/B recombinant forms derived from CRF01_AE and subtype B. One of them in particular; HIV-1 isolate 06MYKLD46 is structurally similar to CRF33_01B, except for an extra subtype B fragment within the env region. It also has close phylogenetic relationship and similar breakpoints with CRF33_01B, mainly at the PR-RT region. Furthermore, the other three distinct HIV-1 recombinants; isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 also display near full-length genomes composed of the backbone of CRF01_AE, with insertions of subtype B fragments at different gene regions. These results indicate the high possibility of second generation of minor recombinant forms derived from CRF33_01B, as well as the continuous evolution and rapid dispersal of CRF01_AE/B recombinants in Malaysia. The high prevalence of newly emerging CRF33_01B (CRF01_AE/B inter-subtype recombinant) may cause a possible epidemiologic shift, attributable to its altered virologic characteristics and possible transmission advantages compared to its parental strains. Two major determinants; the viral factor and host factor have influenced the progress of a productive HIV-1 infection upon virus entry into the host cells. We have assessed the two main viral factors; the in vitro viral replication capacity and the viral fitness of the circulating HIV-1 strains in Malaysia. We have determined that CRF33_01B primary isolate (07MYKLVik) replicates better in activated whole PBMCs and CD4+ T-lymphocytes and is ‘fitter’ than one of its parental strain; CRF01_AE (07MYKLNBL) but not subtype B (07MYKLAfik). Subtype B has more advanced ability to produce a progressive infection in all cell types, including MDMs, and has a comparable viral fitness to that of CRF33_01B. We also investigated the role of host factors in a productive HIV-1 infection, by determining the viral effect on the host cell morphological features. We found that CRF33_01B (07MYKLVik) culture displayed more large syncytia (multinucleated giant cells) with multiple nuclei compared to subtype B (07MYKLAfik) culture, while no snycytia was observed in CRF01_AE (07MYKLNBL) culture. Generally, the cells within CRF33_01B and subtype B cultures appeared to be morphologically distinct from CRF01_AE cultures. This may indicate a more productive HIV-1 infection of CRF33_01B and subtype B, similar to our finding from the in vitro viral replicative capacity and viral fitness assays of these HIV-1 strains. We also studied the effect of different HIV-1 strain infections on host differential gene expression profiles, by using the PCR Array, which detects a total of 84 genes known to be involved in the host response to HIV-1 infection. It was observed that the in vitro infection with CRF33_01B isolates resulted in a more damaging effect on host cells and caused more apoptotic death within the infected cultures, compared to the isolates of its parental subtypes. Moreover, subtype B isolates resulted in a poorer cell response upon viral infection, compared to CRF01_AE/B isolate. Concurrently, it also gave less productive spread of viral infection within the infected cultures, in comparison to CRF01_AE/B isolate. We speculate that if the same scenario is reflected in vivo, CRF01_AE/B inter-subtype recombinant including CRF33_01B would have a better survival rate within the host upon their infection, in comparison to their parental strains. This again strengthens our presumption that CRF33_01B has potential ability to disseminate widely in the Malaysian population and gives a progressive change of the current molecular epidemiological trend by gradually replacing the current predominance of CRF01_AE in the country.

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-13T12:05:33Z (GMT). No. of bitstreams: 1 Goes_LidianaCristinade_M.pdf: 4033239 bytes, checksum: 07d68044592d217e51b3096b35496c87 (MD5) Previous issue date: 2009 / Resumo: Este trabalho visou investigar o efeito dos quelatos IDA-Ni(II), CM-Asp-Ni(II), TED-Ni(II) e TREN-Ni(II) na purificação de pró-insulina humana recombinante com cauda de poli(histidina) (PIS) a partir de solução clarificada, obtida após solubilização dos corpos de inclusão e sulfitólise, proveniente do processo de produção de insulina (BIOMM, MG). Dentre os adsorventes estudados, Sepharose-TREN-Ni (II) apresentou a maior capacidade de adsorção em termos de PIS, cerca de 50% da proteína alimentada. Os demais adsorventes apresentaram a seguinte ordem de capacidade de adsorção IDA > HisTrap > CM-Asp > TED. Em termos de seletividade, os adsorventes com CM-Asp-Ni (II) e TEDNi (II) imobilizados apresentaram maior seletividade. Os dados de adsorção de PIS no equilíbrio a 25°C foram bem representados pelo modelo de Langmuir-Freundlich., fornecendo valores de capacidade máxima de adsorção entre 43,14 mg e 154,56 mg de PIS/g de adsorvente e valores de constante aparente de dissociação entre 10-5 a 10-7 M. O estudo termodinâmico para adsorção de PIS nos adsorventes estudados na faixa de 4 a 25°C (exceto para TED-Ni(II), faixa de temperatura de 25 a 45°C) mostrou diminuição dos valores de Kd com o aumento da temperatura, fornecendo valores de ?H° positivos para todos os casos, indicando que a adsorção de PIS nos quelatos estudados é um processo endotérmico. Os valores de ?Gº foram negativos para todos os sistemas, indicando que a adsorção é espontânea. Os valores de ?Sº encontrados foram positivos e pouco alterados com o aumento de temperatura em todos os casos, favorecendo a complexação proteína-íon metálico. / Abstract: This work sought to investigate the effect of the chelating groups IDA-Ni(II), CMAsp- Ni(II), TED-Ni(II) and TREN-Ni(II) in the purification of a recombinant human proinsulin poly-histidine tagged (PIS) starting from clarified solution, obtained from the dissolution of inclusion bodies and oxidative sulfitolysis, originating from the process of insulin production (BIOMM, MG). Among of the studied adsorbents, Sepharose-TREN-Ni (II) it presented the largest adsorption capacity in terms of PIS, about 50 % of the fed protein. The other adsorbents presented the following order in adsorption capacity IDA > HisTrap > CM-Asp > TED. In selectivity terms, the adsorbents with the chelating groups CM-Asp-Ni (II) and TED-Ni (II) immobilized presented a larger selectivity. The data of adsorption of PIS in the equilibrium to 25°C were well represented by the model of Langmuir-Freundlich, supplying values of maximum adsorption capacity between 43, 14 mg and 154, 56 mg of PIS / g adsorbent and values of apparent constant of dissociation among 10-5 to 10-7 M. The thermodynamic study for adsorption of PIS in the adsorbents considering the range from 4 to 25°C (except for TED-Ni (II), temperature range from 25 to 45°C) showed decrease of the values of Kd with the increase of temperature, providing positive values of ?H° for all of the cases, indicating that the adsorption of PIS in the studied chelating groups is a endothermic process. The values of ?Gº were negative for all of the systems, indicating that the adsorption is spontaneous. The values of ?Sº founded were positive and had little alteration due to temperature increase in all of the cases, favoring the formation of complex protein-metallic ion. / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Titre de l'écran-titre (visionné le 5 juin 2023) / Les AAV constituent des vecteurs viraux très utilisés en thérapie génique. Cependant une limite à leur utilisation est leur large tropisme, soit leur capacité à infecter plusieurs types de cellules. Pour y remédier, il a été question dans ce projet de produire des AAV2 recombinants dont la capside a été modifiée par l'insertion d'un anticorps à domaine unique (single domain antibody, sdAb) pour permettre une transduction spécifique de cellules du cancer de l'ovaire : les SKOV3. Ces cellules expriment à leur surface le récepteur tyrosine kinase Axl et l'anticorps inséré dans la protéine VP1 de la capside virale, lui est spécifique. Les AAV contenant l'anticorps (AAV-sdAb) ont été produits par transfection transitoire de cellules HEK293SF-3F6, puis purifiés par ultracentrifugation avec différents gradients d'Iodixanol et concentrés par filtration tangentielle avec des membranes à fibres creuses (Hollow fibers). L'insertion de l'anticorps dans la capside de l'AAV2 a été faite en utilisant 2 types de séquences de liaison : une courte et une longue. La production des AAV-sdAb avec courte séquence de liaison et exprimant la protéine fluorescente verte (GFP) comme gène rapporteur s'est avéré difficile alors que celle avec la plus longue séquence a permis d'obtenir des titres vingt fois plus élevés. Pour démontrer une transduction spécifique des cellules cibles SKOV3, les plasmides permettant de produire les AAV-sdAb ont été mutés pour inhiber le site d'attachement des AAV2 au sulfate d'héparine, principal récepteur auquel il se lie à la surface des cellules hôtes. Les résultats obtenus ont permis de démontrer que le tropisme des AAV2 a été altéré par les mutations et que les AAV-sdAb infectent de façon spécifique les cellules SKOV3 qui expriment le récepteur Axl auquel l'anticorps inséré est spécifique. Ce projet a ainsi démontré qu'il est possible de modifier le tropisme des AAV à l'aide d'anticorps à domaine unique. / The use of adeno-associated viruses (AAV) as vectors for gene therapy has increased in recent years. However, a major drawback to their use is the large tropism, allowing the infection of many types of cells. To overcome that issue, we incorporated in this project a single-domain antibody (sdAb) into the capsid of AAV serotype 2 (AAV2) to enable it to specifically bind to a receptor tyrosine kinase Axl expressed on the surface of ovarian cancer cells SKOV3. The sdAb against Axl was inserted into the VP1 capsid subunit of AAV2. In order to investigate their targeting efficacy, AAV with the modified capsid (AAV-sdAb) were produced by transient transfection of HEK293SF-3F6 cells, purified by ultracentrifugation using Iodixanol step-gradients and concentrated by tangential-flow filtration using Hollow fiber membranes. In this study, two types of linker sequences, short and long were used for the insertion of the sdAb into VP1. Production of infectious AAV particles expressing GFP as a reporter proved to be difficult when using the short linker sequence, while production using the longer linker led to titers twenty times higher. To prove a specific transduction of Axl-expressing cells (SKOV3), plasmids required for AAV-sdAb production were mutated to inhibit the binding of AAV2 to its main receptor on cell surface: heparan sulfate. Characterization of those AAV-sdAb showed that the mutations led to an altered tropism for AAV2's natural receptor and more importantly, the viral vectors infect specifically the Axl-expressing cells SKOV3 as a result of the inserted anti-Axl antibody. Therefore, this study proved that it is possible to modify the tropism of AAV by using a single-domain antibody.

ERA Biotech S.A. és una empresa que desenvolupa la seva pròpia tecnologia per a la producció de proteïnes i pèptids d’alt valor afegit. El mètode de producció i acumulació de proteïna recombinant es basa en el mecanisme natural d’acumulació de proteïnes de reserva de blat de moro (zeïnes) en orgànuls densos derivats de reticle endoplasmàtic anomenats cossos proteics. La tecnologia Zera® empra el domini ric en prolina de l’extrem N-terminal de la γ-zeïna per induir la formació de novo de cossos proteics heteròlegs en teixits i cèl•lules eucariotes. L’elevada densitat que presenten aquests orgànuls permet una recuperació i enriquiment de la proteïna de fusió d’interès, mitjançant tècniques d’homogeneïtzació i fraccionament cel•lular. Originàriament dissenyats per permetre la purificació per afinitat del proteïna d’interès, els elements de fusió també poden ajudar a mantenir l’estabilitat, el plegament i la solubilitat del producte. Malgrat tot, per a algunes aplicacions posteriors es requereix la producció de la proteïna nativa, amb el qual són necessàries etapes de proteòlisi mitjançant endoproteases específiques. En un context industrial l’addició d’una proteasa exògena suposa l’etapa més costosa en el procés de producció. A més, les condicions de processament poden arribar a interferir amb l’activitat biològica del component purificat. Un dels objectius principals de moltes empreses dedicades a la producció de proteïnes recombinants, tracta de cercar alternatives al processament convencional per addició de proteases exògenes. L’objectiu general d’aquest treball s’ha centrat en el desenvolupament i aplicació de dues estratègies de processament aplicades a proteïnes recombinants de fusió produïdes mitjançant la tecnologia Zera®, en diferents sistemes d’expressió. La primera aproximació ha consistit en la producció d’enteroquinasa bovina (EK), endoproteasa específica, mitjançant la tecnologia Zera®. La producció d’enteroquinasa pròpia evitaria l’adquisició comercial de la mateixa, amb el qual es reduirien els costos globals de producció. S’han dissenyat diferents construccions amb el domini catalític de l’enteroquinasa fusionat al domini Zera® per tal de permetre la seva acumulació en cossos proteics heteròlegs. Degut a la conformació catalítica que adopta, observàrem que el seu extrem N-terminal havia de romandre lliure per tal de mantenir la seva activitat proteolítica. La fusió del domini Zera® al extrem C-terminal d’EK va resultar incompatible amb la viabilitat de les cèl•lules de mamífer o del teixit foliar de tabac, indicant cert grau de citotoxicitat promogut per la proteasa activa. D’altra banda, el bloqueig de l’extrem N-terminal d’EK mitjançant la fusió del domini Zera®, requeria d’una etapa prèvia de processament per tal d’activar la proteasa in vitro. Els baixos nivells d’expressió assolits en cèl•lules de mamífer, juntament amb la baixa idoneïtat de l’estratègia, varen motivar l’exploració d’altres alternatives de processament. La segona aproximació descrita en aquest treball es basa en l’estudi de l’autoprocessament de proteïnes de fusió mitjançant inteïnes. Les inteïnes són elements proteics naturals capaços de promoure l’splicing de proteïnes a través d’una sèrie de reaccions que permeten la seva auto-excissió i la unió dels fragments que les flanquegen o exteïnes. Certes modificacions genètiques en residus clau de la seqüència de les inteïnes, han permès modular la seva activitat per permetre l’autoprocessament in vitro de forma controlable. S’ha estudiat l’aplicació de dos tipus diferents d’inteïnes induïbles per al processament de proteïnes de fusió Zera®, en cèl•lules de mamífer, en cèl•lules d’insecte, i en planta de tabac. El rendiment global del procés de producció de rhGH (com a proteïna model), fou analitzat i comparat emprant dos sistemes de processament diferents sobre la proteïna de fusió Zera® expressada en planta de tabac: el mediat per la inteïna MxeGyrA, i el de la proteasa comercial enteroquinasa. Tot i que els costos globals de producció de rhGH resultaren similars per ambdós processos, el rendiment de producció fou notòriament major en el procés emprant la inteïna. L’èxit de l’aplicació de les inteïnes a la tecnologia Zera®, ha comportat un avenç en el procés de downstream, facilitant de manera significativa la recuperació de la proteïna nativa d’interès. / ERA Biotech S.A. is a biotechnology company whose technology permits high-level production of recombinant proteins and peptides through application of the Zera® assembler peptide. The Zera® domain originates from a maize storage protein (gamma-zein), which naturally accumulates in maize grains in the form of dense protein bodies to elevated levels. The Zera® assembler peptide when fused to a protein of interest triggers the formation in vivo of protein bodies in eukaryotic cells, effectively converting the cells into dense storage organelles. Due to its physicochemical properties, the downstream steps and recovery of the recombinant proteins are extremely efficient. For some applications in the biopharmaceutical industry, fusion or affinity tags need to be cleaved off by site-specific endoproteases in order to recover the native target protein. At a manufacturing scale, the removal of the fusion tag is the most costly step in protein production (cost and specificity/efficiency issues), and can interfere with the biological activity of the purified component. Therefore novel cleavage options which permit specific, efficient and scalable protein production processes are required. In the present study we describe two different cleavage strategies that have been adopted for Zera® fusion proteins expressed in different host expression systems. The first approach was to produce a conventional site-specific endoprotease in house through Zera® technology. Different constructs were designed for the easy and active production of bovine enterokinase (EK) catalytic subunit in mammalian cells and transgenic tobacco plants. Active conformation of this protease was adopted when the N-terminus of the protein was free of any fusion tag, however, proteolytic activity of this protease resulted in cytotoxicity in both host cell systems tested. The fusion of the Zera® domain in the N-terminus of EK and its expression in mammalian cells resulted in the formation de novo of protein bodies accumulating the target fusion protein. Isolation of protein bodies and subsequent downstream steps for protein recovery were designed and set up for this new host system. For the EK activation, a cleavage step by another endoprotease was included, but the low expression levels achieved for this fusion protein, resulted in non-conlcusive data from the activity test. Considering the biochemical properties of this protease its recombinant production for large scale manufacturing results technically cost-unfriendly, so alternative cleavage methods were explored. The second approach described in the present work, consisted in the use and application of self-cleavable elements for the specific cleavage of Zera® fusion proteins. Inteins are naturally occurring protein elements capable of post-translational self-excission from a precursor protein through a process known as protein splicing. MxeGyrA and SspDnaB mini-inteins have been engineered to yield a controllable N-terminal and C-terminal autocleavage induced under certain controlled conditions. Both inteins have shown activity when fused to Zera® and to a protein of interest in mammalian cells (CHO), insect cells (Sf9) and transgenic tobacco plants. The success of the intein application to the Zera® technology has evolved into a faster and more user friendly downstream step leading to the recovery of a native protein of interest.