Made available in DSpace on 2014-06-12T18:04:40Z (GMT). No. of bitstreams: 2 arquivo6203_1.pdf: 1326366 bytes, checksum: 5cd77ce2de56aff15acaf6fb51f8cd1f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Os papilomavírus constituem um grupo de pequenos vírus de DNA de dupla fita caracterizados por induzirem a formação de lesões que são em geral benignas, podendo regredir naturalmente ou se transformar em tumores malignos. Dentre eles se encontra o papilomavírus bovino (BPV). Até o momento são bem caracterizados seis diferentes tipos de BPV (BPV de 1-6) e recentemente outros dois novos tipos (BPVs 7 e 8) foram descritos. Na região Nordeste do Brasil, em especial no Estado de Pernambuco (região da Zona da Mata), a incidência de papilomatose bovina é muito alta provocando grandes perdas econômicas para os criadores. No momento não há tratamento com eficácia comprovada. A proteção contra infecção é conferida por anticorpos neutralizantes direcionados proteína estrutural L1. Estes anticorpos podem ser eficientemente induzidos por imunização com virus-like particles (VLP), que se formam espontaneamente após a expressão de L1 em vetores recombinantes. A levedura metilotrófica Pichia pastoris emergiu como um sistema heterólogo, eficiente e de baixo custo para a produção de proteínas. Neste trabalho foi avaliada a produção da proteína L1 de BPV-4 por células de P. pastoris. O gene L1 foi amplificado e clonado no vetor de expressão pPICZA (Invitrogen). As células de P. pastoris foram transformadas com pPICZAL1B4. Os transformantes de P. pastoris obtidos foram analisados para expressão do gene L1 por RT-PCR e Dot blot. Os transformantes analisados mostraram transcrição do gene L1 e conseqüente produção da proteína

Après les nombreux succès de la vaccination au XXème siècle, elle se heurte aujourd’hui aux pathogènes émergents qui nécessitent le développement de nouvelles formulations vaccinales, afin d’induire des réponses immunitaires complètes, et particulièrement des réponses CD8+ cytotoxique efficaces. Nous avons développé une stratégie innovante utilisant des pseudo-particules virales (VLP) dérivées d’un rétrovirus murin, le Virus de la Leucémie Murine (MLV), comme plateforme antigénique. Ces rétroVLP sont des candidats vaccins de choix, sécuritaires en raison de l’absence de matériel génomique et facilement manipulables par génie génétique, ce qui permet de les proposer dans différentes stratégies vaccinales, et notamment contre le VIH. Notre approche consiste à utiliser comme vaccin des rétroVLP recombinantes produites ex vivo, ou l’ADN codant les protéines capables de former in situ ces rétroVLP (stratégie dite « plasmoVLP »). Les travaux antérieurs de l’équipe ont montré que la formation des rétroVLP améliorait les réponses immunitaires dirigées contre les antigènes qu’elles véhiculent, et nous avons cherché au cours de cette étude à mieux caractériser les mécanismes expliquant leur immunogénicité. Nous avons montré dans un premier temps que les rétroVLP étaient capables d’activer efficacement les cellules dendritiques in vitro et in vivo, et de déclencher des réponses aussi bien humorales que cellulaires contre les protéines d’enveloppe du VIH et contre des épitopes T insérés dans la capside. Les mécanismes d’activation des cellules dendritiques ont également été précisés par une analyse transcriptomique. De plus, la vaccination plasmoVLP, qui favorise les réponses CD8+, a aussi montré de fortes réponses cellulaires contre des antigènes tumoraux associées à des résultats de protection très encourageants, y compris dans des schémas de vaccination thérapeutiques. Par ailleurs, nous avons développé au cours de ces travaux une nouvelle stratégie consistant à moduler l’immunogénicité des rétroVLP en ajoutant dans leur structure des molécules adjuvantes, notamment des ligands des récepteurs de type Toll (TLR). Ainsi, nous avons conçu et breveté au laboratoire une molécule d’ARN simple brin que nous avons appelée ncRNA. Nous avons mis en évidence l’effet immunostimulant du ncRNA, qui est capable d’activer plus efficacement les cellules dendritiques in vitro, d’orienter les réponses CD4+ vers un profil Th1 et de promouvoir la prolifération des lymphocytes T CD8+ spécifiques des antigènes véhiculés par les rétroVLP in vitro, démontrant qu’il améliore l’efficacité de la présentation croisée. Les réponses vaccinales contre les épitopes T internes et l’enveloppe du VIH sont également augmentées en présence de ncRNA, par un mécanisme impliquant la voie des TLR. Ces travaux ont donc permis de mieux caractériser l’immunogénicité des rétroVLP, mettant ainsi en avant leur potentiel vaccinal, et démontrent l’efficacité d’une stratégie d’adjuvantation innovante et prometteuse utilisant un ligand des TLR. / After a century of success, vaccination now faces new pathogens that require the development of new formulations to induce complete immune responses, especially potent CD8+ cytotoxic responses. We developed a novel strategy based on virus-like particles (VLPs) deriving from the Murine Leukemia Virus (MLV), so-called retroVLPs, as antigen platforms. RetroVLPs are promising vaccine candidates, as they are totally safe due to the lack of any genomic material in their composition, and they can be easily manipulated by genetic engineering, which allows using them in different vaccine strategies, including against HIV. We can use retroVLPs as a vaccine by producing them ex vivo and injecting them as proteins, or by injecting the DNA plasmids that encode the structural proteins that can self-assemble in vivo (“plasmoVLP” strategy). Previous work had shown that the retroVLP formation improves immune responses against antigens they carry, and we aimed in this study to better characterize the mechanisms explaining their immunogenicity. We showed that retroVLPs efficiently activate dendritic cells in vitro and in vivo, and trigger humoral as well as cellular vaccine responses, against HIV envelope and T cell epitopes inserted in the capsid. The mechanisms of dendritic cells activation have also been specified by transcriptome analysis. Moreover, plasmoVLP vaccination, which promotes CD8+ responses, showed strong cellular responses against tumor antigens associated with good protection, including in therapeutic vaccination schemes. Otherwise, we developed during this work a new strategy in order to modulate the immune properties of retroVLPs by adding in their structure adjuvant molecules, and particularly Toll-like receptor (TLR)-ligands. Thus, we designed and patented a single-stranded RNA acting as a TLR-ligand, that we called ncRNA. We demonstrated the immunostimulating properties of ncRNA, which is able to induce a higher activation of dendritic cells in vitro when carried by the retroVLPs, and to promote Th1-biased CD4+ T cell responses as well as proliferation of CD8+ T cell specific for an internal epitope carried into the retroVLP, showing ncRNA improves the efficacy of cross-presentation. Vaccine responses against both internal epitopes and HIV envelope are also improved in the presence of ncRNA, by a TLR-dependent mechanism. This work helped to better characterize the immunogenicity of retroVLP, highlighted their vaccine potential, and demonstrate the effectiveness of an innovative and promising strategy adjuvantisation using a TLR-ligand.

Dichapetalum cymosum (poison leaf) is a very common problem plant in southern Africa. Fluoroacetic acid, believed to be the poisonous entity in the plant, is produced by the plant, but the micro-organisms associated with this plant may also play a role in the production thereof. A previous study on Burolderia cepacia, an endophyte of D. cymosum showed active metabolism of fluoroacetate by this endophyte. The isolated endophytes from D. cymosum were studied to determine whether they synthesise any fluorinated compounds. It seemed from preliminary results that symbionts might play a role in the synthesis of the poisonous entities in D. cymosum, but further investigation is required. The detection of glandular lesions on the abaxial side of the leaf led to closer examination and the cross sections revealed unusually deformed epidermis cells with adjacent cells containing vacuoles filled with phenolic-like crystals. Transmission electron microscopy (TEM) of the spongy parenchyma cells directly above the glandular lesions indicated the presence of clusters of small, virus-like particles (VLPs) in the chloroplasts. Observations by TEM showed that these VLPs have analogous structures to phytoferritin. Tapura fischeri (leafberry tree) is a tree member of the same family, and it was found to also contain a fluorinated compound. Endophytes were also found in the plant and similar glandular lesions with analogous VLPs were observed at these sites. This might indicate that endophytes have a share in the biosynthesis of the fluorinated compounds found in Dichapetalaceae. Numerous factors ought to be considered in order to fully understand the chemical ecology of the intricate system regarding the endophytes and the possible toxicity of the family Dichapetalaceae. / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

The existing seasonal influenza vaccine does not provide broad long-term protection against seasonal influenza and must be remanufactured yearly due to frequens mutations and reassortment of theinfluenza genes. A universal influenza vaccine with the ability to raise long lasting immunity is the focus of several studies, including the Edufluvac project. Edufluvac is based on virus-likeparticles, a modern recombinant platform wellsuited for vaccinatin applications. Redbiotec's rePAX® technology allows the generation of multivalent recombinant baculovirus which generatesvirus-like particles presenting multiple proteins on the surface in insect cell culture. Any effects oninsect cell culture protein expression brought on by the regulatory elements controlling each gene in the baculovirus, or by the genome position of the baculovirus genes, could affect the composition of the virus-like  particles. The ai of this thesis was to elicit a better understanding of the protein expression by analysing multi-protein influenza virus-like particles and virus-like particles encoding a reporter gene regulated by different promoter and terminator combinations. Different bivalent and tetravalent influenza gene bacmids were cloned as well as seven bacmids encoding a YFP gene regulated by different promoter and terminator combinations. Spodopera frugiperda cells weretransfected with the bacmids and harvested recombinant baculovirus was used to perform testexpressions in High-Five™ cells. The resulting protein expression levels from the bivalent andtetravalent recombinant baculovirus were analyzed and compared by Western blots and ELISA assays. The expression of YFP in infected Spodoptera and High-Five™ cells was monitored byfluorescence microscopy and measured with FACS to quantify protein expression differencesbetween the seven promoter and terminator combinations. Analysis of the bivalent constructs indicated that the order of the genes in a recombinant baculovirus does not affect the protein expression in High-Five™ cells. The analysis of the tetravalent constructs revelaed positionalvariations in expressin of the H1 and M1 genes, but the number of test expressions and recombinant baculovirus construct clones included in the analysis were not hogh enough to allow a definitive conclusion. Of the different promoter and terminator constructs highest mean fluorescence intensity was reached with the reference combination. The early promoter yielded mean fluorescent intensitites that were close to the values of the negative control in both cell lines.

Nanotechnology and its potential for biomedical applications has become an area of increasing interest over the last few decades. Specifically, ultrasmall nanoparticles, ranging in size from 5 to 50 nm, are highly sought after for their physical and chemical properties and their ability to be easily transmitted though the bloodstream. By adjusting the material properties, size, surface potential, morphology, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug and gene delivery, biodetection of pathogens or proteins, and tissue engineering. The aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs) using a quick and easy nanoprecipitation method1, with some modifications, for general use in various biomedical areas. Nanoprecipitation of two solutions – PLGA dissolved in acetonitrile and aqueous poly(vinyl alcohol) (PVA) – at varying concentrations produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. By the data collected from this study, a selection method can be used to choose a desired PLGA nanoparticle size given a potential biomedical application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. Size of the ultrasmall PLGA NPs was characterized by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Spherical morphology of the PLGA NPs was also proved by TEM. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs will be able to be used in various biomedical applications depending on the goal of the furthered study. As an example of potential application, ~15 to 20 nm PLGA NPs were consistently fabricated for use as virus-like particle (VLP) scaffolds. Following formation, PLGA NPs were introduced to modified human papillomavirus (HPV) protein during protein refolding and assembly into virus-like particles (VLPs) via buffer exchange. The size of the VLPs was monitored with and without PLGA nanoparticles present in solution during the refolding process and TEM images were collected to confirm encapsulation. / Master of Science / Nanotechnology, the manipulation of materials on an atomic or molecular scale, and its potential for biomedical applications has become an area of increasing interest over the last few decades. Nanoparticles, spherical or non-spherical entities of sizes approximately one-billionth of a meter, have been used to solve a wide variety of biomedical problems. For reference, a human hair is about 80,000 to 100,000 nm in size and the nanoscale typically ranges in size from 1 to 1000 nm. This size range is not visible to the naked eye, so methods of analysis via scientific equipment becomes paramount. Specifically, this study aims to fabricate ultrasmall nanoparticles, ranging in size from 5 to 50 nm, which are highly sought after for their physical and chemical properties and their ability to easily travel though the bloodstream. By adjusting the material properties, size, shape, surface charge, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug delivery, detection of viruses, and tissue engineering. The specific aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs), a type of polymer, using a quick and easy nanoprecipitation method1, with some modifications. Nanoprecipitation occurs by combining two liquid solutions – PLGA and aqueous poly(vinyl alcohol) (PVA) – which interact chemically to form a solid component – a polymer nanoparticle. These two solutions, at varying concentrations, produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. Data collected from this study can be used to select a desired nanoparticle size given a potential application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs can be used for a variety of biomedical applications depending on the goal of the furthered study. Two PLGA NP example applications are tested for in this work – in DNA loading and in encapsulation of virus-like particles (VLPs), which are synthetically produced proteins that can be neatly folded to resemble a virus. These VLPs can be used to as an alternative to live vaccines and they can be designed to stimulate the immune system. Positive initial results from this study confirm the potential of these nanoparticles to have a wide impact on the biomedical field depending on specific tailoring to a given application.

In this dissertation the investigation of potential applications of hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) harboring CTL epitopes for anticancer vaccine development is presented. The objective of this study was to investigate the potential of recombinant HaPyV VP1 based VLPs for anticancer vaccine generation in model systems, including investigation of VP1 applicability for heterologous CTL epitopes insertions, VLPs assembly and ability to induce insert specific immune response in vivo. HaPyV VP1 VLPs carrying CLT epitopes derived from different proteins were generated, most suitable positions for insertion into VP1 protein were selected, the ways to improve assembly and yield of the chimeric VLPs were determined and new VLPs purification procedure was created allowing to purify VLPs cheaper, faster and more efficiently. HaPyV VP1 based VLPs ability to induce CTL immune response in vivo was evaluated for the first time. It was demonstrated that model chimeric VLPs were able to stimulate antigen specific CTL cells in vitro and in vivo, induced insert specific humoral and CTL immune response in vivo and protected mice from insert specific virus infection and antigen-specific tumor growth. Presented data confirmed that HaPyV protein VP1 is universal carrier for CTL epitopes, capable to tolerate insertions, to form VLPs and to induce effective, long lasting immune response against inserted antigens in vivo. / Disertacijoje yra aprašomas perspektyvų panaudoti žiurkėno poliomos viruso (HaPyV) pagrindinio struktūrinio baltymo VP1 formuojamas į virusus panašias daleles priešvėžinių vakcinų kūrimui tyrimas. Pagrindinis disertacijos darbo tikslas buvo modelinėse sistemose parodyti rekombinantinių HaPyV VP1 baltymų formuojamų į virusus panašių dalelių panaudojimo priešvėžinių vakcinų kūrimui galimybes, įvertinant svetimų CTL epitopų įterpimo į VP1 baltymą toleravimą, VPD formavimosi efektyvumą bei sukeltą įterptam antigenui specifinį imuninį atsaką. Disertacijoje atlikta tyrimo srities literatūros apžvalga, smulkiai aprašomi darbe naudoti metodai, atlikti eksperimentai, pateikiami bei analizuojami gauti rezultatai. Darbe pirmą kartą buvo nuodugniai ištirtos HaPyV viruso VP1 baltymo formuojamų VPD savybės, parodytas jų tinkamumas būti CTL epitopų nešikliais, ištirtos įterpimui palankiausios VP1 baltymo vietos, išbandyti nauji VPD gavimo ir gryninimo būdai, pagerinantys chimerinių VPD formavimąsi bei išeigas. Panaudojant modelines chimerines VPD in vivo buvo ištirtas chimerinių HaPyV VP1 pagrindu sukonstruotų VPD sukeliamas humoralinis ir ląstelinis imuninis atsakas. Gauti rezultatai parodė, kad HaPyV VP1 baltymas yra vienas iš nedaugelio virusų struktūrinių baltymų, kurie ne tik formuoja VPD, bet pasižymi ir universaliomis baltymo – nešiklio savybėmis, o in vivo sukelia efektyvų, ilgalaikį, įterptam epitopui specifinį imuninį atsaką.

The aim of this thesis is to investigate the targeting potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as vectors for directed cell delivery of therapeutic or diagnostic compounds. Major capsid protein VP1 of MPyV is able to selfassemble into the noninfectious VLPs. Our main goal is to retarget these VLPs from its native receptor to the prostatic cancer cells by changing the receptor binding site in the surface-exposed loop of VP1. We introduced a peptide ligand CTITSKRTC, which binds prostate-specific membrane antigen (PSMA), by insertion or substitution into BC loop of VP1. These modifications did not change the stability of the particles and genetic substitution prevented the native receptor binding. PSMA-specific binding of modified VLPs was tested by pull-down assay and surface plasmon resonance. In order to further utilize these VLPs, we tested several approaches for preparation of VLPs as vehicles for compounds delivery into eukaryotic cells. Although the method for encapsidation of the DNA into the VLPs in cellular nuclear extracts, which mimic the in vivo conditions, did not enabled us to produce pseudocapsids, we successfully optimized procedure for dissassembly and reassembly of purified particles. This method will be use for encapsidation of molecules into the...

Die in Europa und Asien verbreiteten humanpathogenen Hantaviren sind mit dem "Hämorrhagischen Fieber mit renalem Syndrom" (HFRS) assoziiert, während die humanpathogenen Hantaviren in Nord- und Südamerika das "Hantavirale Pulmonale Syndrom" hervorrufen. In Mitteleuropa kommen die humanpathogenen Hantavirus-Spezies Puumalavirus (PUUV) und Dobravavirus (DOBV) vor. Infektionen mit dem PUUV führen zu milden klinischen Verläufen des HFRS, die auch als "Nephropathia epidemica" bezeichnet werden. Für das DOBV konnte in Mitteleuropa die sympatrische Existenz von 2 genetischen Linien, DOBV-Aa und DOB-Af, nachgewiesen werden, die von unterschiedlichen Nagetierwirten (Apodemus agrarius und A. flavicollis) getragen werden und möglicherweise unterschiedlich virulent für den Menschen sind. Chimäre Hepatitis B Virus-Corepartikel und Hefe-exprimiertes, rekombinantes PUUV-Nukleokapsid (N)-Protein stellen vielversprechende Impfstoffkandidaten dar, die in der Rötelmaus, dem natürlichen Wirt und Überträger des PUUV, eine schützende Immunantwort induzieren können. Eine hauptprotektive Determinante konnte im N-Protein des PUUV zwischen den Aminosäuren (AS) 1-45 lokalisiert werden; eine zweite, schwach protektive Determinante ist zwischen AS 75-119 lokalisiert. Die schwache Protektivität der 45 aminoterminalen AS des PUUV-N-Proteins (Stamm Vranica/Hällnäs) konnte durch Verwendung eines 120 AS-langen Segments deutlich verbessert werden. Ein Hefe-exprimiertes PUUV-N-Protein mit aminoterminalem Hexahistidinschwanz induzierte bei Verwendung von komplettem Freund´schen Adjuvans in allen vakzinierten Rötelmäusen eine schützende Immunität. Bei Verwendung von Aluminiumhydroxid, einem für humane Anwendungen zugelassenen Adjuvans, wurden alle immunisierten Tiere mindestens partiell, darunter 75 % der Tiere sogar komplett, vor einem nachfolgenden Viruschallenge geschützt. An der Induktion einer schützenden Immunität sind wahrscheinlich nicht nur zelluläre, sondern auch humorale N-Protein-spezifische Immunantworten beteiligt. / Human infections with European hantaviruses are associated with the "Haemorrhagic fever with renal syndrome" (HFRS), whereas North and South-American hantaviruses cause in human the "Hantavirus pulmonary syndrome". In Central Europe two hantavirus species, Puumala virus (PUUV) and Dobrava virus (DOBV), are pathogenic to human. PUUV infections result in milder clinical courses of HFRS, designated also as "Nephropathia epidemica". For DOBV the sympatric occurrence of two genetic lineages, DOBV-Aa and DOBV-Af, has been detected. These lineages are carried by two different rodent hosts (Apodemus agrarius and A. flavicollis) and might be of different pathogenicity to human. Chimaeric hepatitis B virus core particles and yeast-expressed recombinant PUUV nucleocapsid (N) protein are promising vaccine candidates. They are able to induce a protective immune response in bank voles, the natural host of PUUV. A major protective determinant has been localized between amino acids (aa) 1-45; a second, minor protective determinant is located between aa 75-119. The low protectivity of the 45 amino-terminal aa of the N protein of PUUV (strain Vranica/Hällnäs) can be overcome by the use of a larger, 120 aa-long segment of this N protein. A yeast-expressed PUUV N protein harboring an amino-terminal hexahistidin tag is able to induce a protective immunity in all immunized bank voles when applied with Freund´s complete adjuvant. Using alum, an adjuvant certified for human use, all immunized animals were protected at least partially, 75% even completely, against a subsequent virus challenge. Likely, the protective immunity is mediated not only by cellular but also by humoral immune responses against the N protein.

Production of vaccines and pharmaceutical proteins in plants is a promising nascent technology with a great potential to provide high-quality, safe and non-expensive production and delivery platform. In this work we studied the experimental vaccine against human papillomavirus based on modified plant pathogen - Potato virus X (PVX). The experimental vaccine is based on PVX virus particles decorated with genetically fused HPV-E7 oncoprotein. These chimeric virus particles should be able to activate strong and specific cellular immune response. However the modification of the PVX coat protein with such relatively large fused protein might influence its ability to form particles. In this work we have characterized some properties of such chimeric virus particles like solubility or ability infect host plant. (In Czech)

BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...