The Betanodaviruses caused nerve necrosis in several fishes. There are two RNAs in the Betanodaviruses. RNA1 encodes RNA dependent RNA polymerase and RNA2 encodes capsid protein. I analyzed the RNA2 sequences of the viruses isolated from two species of grouper, Epinephelus malabaricus (MGNNV) and E. lanceolatus (DGNNV). The similarity of two grouper viruses (GNNV) was 99%. Their similarities to DlEV and SJNNV were 87.8% and 78.6%, respectively. The capsid protein was successful expressed and assembled to virus-like particles. Deleting N- and C-termini revealed different impacts on VLP formation. Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted. The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively. Expression the ORF of RNA2 in E. coli formed virus-like particles, indistinguishable from native virus particles in appearance, whereas a mutant of Asp-75 expressed no VLPs. The emergence of a trimer band in mutant D75N as wild type suggested that the Asp-75 mutation could halt the packaging process in the trimer stage, not proceeding to assemble intact VLPs. The D54N mutant remained the ability of VLP formation but lost packaging high molecular weight of RNAs. Another Asp mutant at C-terminus, D335A, lost the ability of VLP assembly. The D335 may play an important role on the instability of VLP structure.

No description available.

Interleukin 15 has great application potential such as in the biological treatment of cancer. It is involved in a variety of immunological processes, the most important of these involve influencing and induction of NK cells and T-lymphocytes proliferation. However, its therapeutic usages are limited by a low stability and short half-life. For this reason, there are various approaches of stabilization and expansion of its biological activity being explored. In this work, we analysed and developed a new approach, which uses viral nanostructures derived from major capsid VP1 protein of mouse polyomavirus as a carrier of IL-15. Moreover, VP1 proteins can be relatively easily modified and they are also capable to penetrate into the tumour cells. There were prepared two variants of IL-15 together with control nanostructures in the baculovirus expression system, one was composed of IL-15 and the other of the IL-15 fusion protein and truncated variant of VP1. Protein constructs were characterized by electron microscopy and biochemical methods. The total protein yield of VP1ΔC-IL15-HIS fusion variant was higher (up to 53 mg/L of complete medium) than IL-15 alone (8,5 mg/L). However, testing of the biological activity of the prepared proteins in vitro did not show any induction of proliferation on Jurkat...

O câncer cervical é a consequência mais séria da infecção por Papilomavírus humano - HPV, constituindo uma das principais causas de morte entre mulheres no mundo, remetendo a um importante desafio para a saúde pública mundial. Desenvolvemos a produção de VLPs contendo as proteínas L1 e L2 de HPV16, utilizando células epiteliais humanas 293-F em suspensão e em meio isento de soro fetal bovino. As células possuem metabolismo intenso, adequado para a expressão de proteínas heterólogas. Apresentam vantagens como a facilidade de cultivo, transfecção e ocorrência de processos como a glicosilação de proteínas, fosforilação, formação de pontes dissulfeto e outras modificações pós-traducionais essenciais para a função das proteínas, facilitando a produção similar às condições in vivo. O sistema de produção de VLPs L1L2 de HPV16 foi estabelecido de maneira eficiente, em células epiteliais humanas em suspensão e com resultados promissores, podendo contribuir para o desenvolvimento de uma vacina profilática de amplo espectro de proteção, com custo reduzido de produção. / Cervical cancer is the most serious consequence of infection by human papillomavirus - HPV, constituting one of the leading causes of death among women worldwide that points to a major challenge to global public health. We have developed the production of VLPs containing L1 and L2 proteins of HPV16, using human epithelial cells 293-F suspended in medium without fetal bovine serum. The cells have intense metabolism, suitable for expressing heterologous proteins. Present advantages such as ease of culture, transfection and occurrence of processes such as protein glycosylation, phosphorylation, formation of disulfide bonds and other essential post-translational modifications to protein function, facilitating the production similar to the conditions in vivo. The VLPs L1L2 of HPV16 production system was established efficiently in human epithelial cells in suspension and with promising results, which may contribute to the development of a prophylactic vaccine broad protection spectrum with reduced production cost.

Submitted by Luciane Willcox (luwillcox@gmail.com) on 2016-09-12T17:13:48Z No. of bitstreams: 1 Nayara Final.pdf: 4140088 bytes, checksum: ab360755584995cab3cab99f56557007 (MD5) / Approved for entry into archive by Luciane Willcox (luwillcox@gmail.com) on 2016-09-12T18:50:42Z (GMT) No. of bitstreams: 1 Nayara Final.pdf: 4140088 bytes, checksum: ab360755584995cab3cab99f56557007 (MD5) / Made available in DSpace on 2016-09-12T18:50:42Z (GMT). No. of bitstreams: 1 Nayara Final.pdf: 4140088 bytes, checksum: ab360755584995cab3cab99f56557007 (MD5) / Fiocruz / Instituto Carlos Chagas, Fiocruz-PR, Curitiba, PR, Brasil / Sistemas convencionais para expressão de anticorpos diméricos consistem em geral na co-transfecção de dois plasmídeos, cada um codificando uma das duas cadeias do anticorpo. Este trabalho teve por objetivo construir novos vetores para expressão simultânea das duas cadeias do anticorpo e outras proteínas oligoméricas a partir de um único plasmídeo, buscando melhorar o nível de expressão de proteínas heterodiméricas. A eficiência desses vetores foi testada para expressão do anticorpo dimérico constituído pelo Fab do anticorpo MEDI-493 fusionado à fração cristalizável de uma IgG1 humana selecionada devido à sua maior estabilidade. A escolha deste anticorpo foi baseada no seu potencial para o tratamento e diagnóstico rápido de infecções pelo Vírus Sincicial Respiratório Humano (hRSV). Os sistemas de expressão desenvolvidos neste projeto foram usados também para expressar partículas tipo-vírus (VLPs) de hRSV. As VLPs foram reconstituídas utilizando a combinação das proteínas G e M, ou F0 e M de hRSV. Sequências de aminoácidos do anticorpo e das VLPs foram usadas para gerar genes sintéticos que foram subclonados nos vetores de expressão. A expressão do anticorpo foi conduzida em células HEK293/HEK293T. Os melhores resultados foram obtidos com o plasmídeo baseado em dois cassetes de expressão. O anticorpo recombinante foi purificado a partir dos sobrenadantes da cultura celular por cromatografia de afinidade utilizando proteína A/G imobilizada em uma fase sólida e a expressão foi confirmada por Western Blot. O reconhecimento da F1, que é a proteína do hRSV que contém o epítopo alvo deste anticorpo, foi demonstrado inicialmente por Western Blot contra a proteína F1 recombinante. A atividade do anticorpo recombinante foi subsequentemente testada utilizando amostras de paciente infectados com o vírus hRSV por Western Blot em também por ensaios de ELISA. O anticorpo recombinante foi capaz de reconhecer as amostras clínicas com especificidade equivalente a do anticorpo comercial MEDI-493, confirmando seu potencial para aplicação em diagnóstico de infecções causadas pelo hRSV. / Conventional systems for expression of dimeric antibodies rely usually on cotransfection of two plasmids, each one coding one of the two antibody chains. The aim of this work was to construct new vectors for simultaneous expression of both antibodies chains and of other oligomeric proteins from a single plasmid, aiming to improve the level of expression of heterodimeric proteins. The efficiency of these vectors was tested for expression of a dimeric antibody constructed by using the Fab fragment of the antibody MEDI-493 with the crystallizable fraction of a human IgG1 selected based on its higher stability. This antibody was chosen based on its potential for treatment and rapid diagnostic of Respiratory Syncytial Virus (hRSV) infections. The expression system developed in this project was also used to express virus-like particles (VLPs) of hRSV. The hRSV VLPs were reconstituted using G and M, or F0 and M protein combinations. The amino acid sequences of this antibody and VLPs were used to generate nucleotide sequences. The respective genes were acquired from a gene synthesis company and subcloned into the new expression vectors. Expression of the recombinant antibody was conducted in HEK293/HEK293T cells in parallel experiments to compare the performance of the three different genetic constructs. Best results were obtained with a plasmid containing two expression cassettes, one for each antibody chain. The recombinant antibody was purified from cell culture supernatants by affinity chromatography using protein A/G immobilized on a solid phase and its presence was confirmed by Western Blot. Interaction of the recombinant antibody with its target on the hRSV F1 protein was determined by Western Blot. The activity of the recombinant antibody was tested using clinical specimens from patients infected with hRSV by Western Blotting and ELISA assays. The recombinant antibody was able to detect the F1 protein the clinical specimens with efficiency similar to the commercial antibody MEDI-493. This result indicates the recombinant antibody can be used for diagnosis of infections caused by hRSV.

Le Grapevine fanleaf virus (GFLV) est le principal agent de la maladie du court-noué de la vigne. Sa protéine de capside (CP) permet la formation des virions indispensables à la protection du génome viral, au mouvement de cellule à cellule au sein de tubules formés par la protéine de mouvement (MP) du virus, et à la transmission du GFLV par son nématode vecteur Xiphinema index. Principaux résultats : 1. un motif exposé à la surface de la CP dont la nature est critique pour transmission du GFLV par X. index a été identifié et pourrait constituer un déterminant de la spécificité de transmission. 2. Des tubules fluorescents ont été produits de façon constitutive in planta. Ils permettent de complémenter en trans un GFLV dépourvu de MP. 3. L’expression transitoire de la CP conduit à la production de pseudo-particules. Celles-ci sont modifiables à façon et font de la capside du GFLV une plateforme biotechnologique unique. De plus, c’est un puissant outil pour étudier la biologie du virus. / Grapevine fanleaf virus (GFLV) is the main agent of grapevine fanleaf degeneration disease. Its coat protein (CP) self-assembles in virions necessary for viral genome protection, for cell-to-cell movement using tubules formed by the movement protein (MP) of the virus, and for the transmission of GFLV by its nematode vector Xiphinema index.Main results: 1. An outer surface-exposed CP motif has been identified as critical for GFLV transmission by X. index and could be a determinant of transmission specificity. 2. Fluorescent tubules have been produced by constitutive expression in planta. They allow the complementation in trans of a GFLV deleted of its MP coding sequence. 3. Transient expression of the GFLV CP leads to the production of virus-like particles. They can be easily modified and show that GFLV capsid is a unique biotechnology platform. In addition, they are a powerful tool to study the biology of the virus.

Die Proteinhülle von Pflanzenviren besteht aus Protein-Untereinheiten, die das Genom des Virus verkapseln und transportieren. Der zugrunde liegende self-assembly-Mechanismus kann für nanotechnologische Anwendungen genutzt werden, indem Fremdmoleküle in den Viren verkapselt werden. Diese sogenannten Virus-ähnlichen Partikel (VLP) können zum Wirkstofftransport in der Krebstherapie, für Gentherapie oder als Kontrastmittel bei bildgebenden Verfahren verwendet werden. Auch die Oberfläche der Proteinhülle kann chemisch oder molekularbiologisch funktionalisiert werden. Ein großer Vorteil von Pflanzenviren ist, dass sie keine Gefahr für den Menschen darstellen und einfach in natürlichen Wirtspflanzen hergestellt werden können. In dieser Arbeit wird am Beispiel des Cowpea Chlorotic Mottle Virus (CCMV) gezeigt, wie diese Nanomaterialien effizient hergestellt und nutzbar gemacht werden können. Im ersten Kapitel wurde eine Affinitätsextraktion basierend auf einem neuen Peptid-Aptamer entwickelt, um das CCMV selektiv aus dem Extrakt der infizierten Pflanze zu isolieren. Das Reinigungsprotokoll wurde validiert und eine finale Reinheit von 98,4% mittels Umkehrphasen-HPLC bestimmt. Im zweiten Kapitel wurden CCMV-VLPs durch rekombinante Expression und anschließende in vitro Assemblierung der Kapsidproteine hergestellt. Die Monodispersität der VLPs wurde durch Transmissionselektronenmikroskopie (TEM) bestätigt. Zusätzlich wurde eine Mutante der CCMV-VLPs hergestellt, die physiologischen Bedingungen standhält und für den Einsatz in vivo geeignet ist. Im dritten Kapitel wurden CCMV und dessen VLPs für nanotechnologische Anwendungen genutzt. Gold-Nanopartikel wurden in CCMV-VLPs verkapselt und im TEM sichtbar gemacht. Das CCMV-Kapsid wurde mit einem Modellpeptid funktionalisiert, um das Potenzial dieses Nanomaterials als chemische Plattform zu demonstrieren. / The protein shell of plant viruses consists of protein subunits that encapsulate and transport the viral genome. The underlying self-assembly mechanism can be utilized for nanotechnological applications by encapsulating foreign molecules within the viruses. These so-called virus-like particles (VLPs) can be used for drug delivery in cancer therapy, gene therapy, or as contrast agents in imaging techniques. Additionally, the surface of the protein shell can be functionalized chemically or via molecular biology techniques. A significant advantage of plant viruses is that they pose no threat to humans and can be easily produced in natural host plants. This work demonstrates how these nanomaterials can be efficiently produced and utilized using the example of Cowpea Chlorotic Mottle Virus (CCMV). In the first chapter, an affinity extraction based on a novel peptide aptamer was developed to selectively isolate CCMV from the extract of the infected plant. The purification protocol was validated, achieving a final purity of 98.4% using reverse-phase HPLC. In the second chapter, CCMV-VLPs were produced through recombinant expression and subsequent in vitro assembly of the capsid proteins. The monodispersity of the VLPs was confirmed by transmission electron microscopy (TEM). Additionally, a mutant of the CCMV-VLPs was produced that withstands physiological conditions and is suitable for in vivo applications. In the third chapter, CCMV and its VLPs were used for nanotechnological applications. Gold nanoparticles were encapsulated within CCMV-VLPs and visualized using TEM. The CCMV capsid was also functionalized with a model peptide to demonstrate the potential of this nanomaterial as a chemical platform.

Made available in DSpace on 2014-06-12T23:13:26Z (GMT). No. of bitstreams: 2 arquivo2933_1.pdf: 6870658 bytes, checksum: acbcf9d7334f444ca5bd229930ce06f2 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer cervical é a segunda maior causa de mortes entre mulheres no mundo. Esta neoplasia maligna está relacionada com a presença do Papilomavírus Humano (HPV), sendo o tipo 16 responsável por 60% dos casos. O HPV infecta o tecido epitelial mucoso ou cutâneo e é responsável pelo aparecimento de verrugas ou papilomas benignos que tendem a regredir naturalmente na maioria dos casos, mas que ainda assim causam prejuízos aos indivíduos infectados e aos sistemas públicos de saúde, sendo a papilomatose considerada a doença sexualmente transmissível mais prevalente no mundo, tornando essencial a aplicação de estratégias de combate à esta infecção. Vacinas baseadas em Virus-like particles (VLPs), formadas a partir das proteínas capsidiais L1 e L2 - que induzem a formação de anticorpos neutralizantes - já estão comercialmente disponíveis. Contudo, possuem um preço elevado considerando, principalmente, os países em desenvolvimento. A imunidade humoral é direcionada contra os epítopos conformacionais da proteína L1 que compõe 90% da estrutura capsidial. Para produção das VLPs os genes das proteínas capsidiais são expressos em sistemas heterólogos e o produto resultante é purificado. A escolha de um sistema mais simples e barato para essa expressão é fundamental para a redução do custo das vacinas. Uma alternativa promissora é a levedura Pichia pastoris. Este trabalho propôs a produção extracelular da proteína L1 de HPV16, que teve seu gene inserido no vetor pPCIZAα. A construção pPICZAαL1H16 foi integrada no genoma da levedura Pichia pastoris e a transcrição do gene L1 e a expressão da proteína foram confirmadas por RT-PCR e imunodetecção em Dot Blot. A expressão extracelular da proteína L1 de HPV 16, em células de P. pastoris, é uma etapa essencial na busca do desenvolvimento de uma estratégia vacinal mais economicamente viável baseada em VLPs

Porcine reproductive and respiratory syndrome (PRRS) is the most significant infectious disease currently affecting the swine industry worldwide. Several inactivated and modified live vaccines (MLV) have been developed to curb PRRSV infections. The unsatisfactory efficacy and safety of these vaccines, drives for the development of new generation PRRS universal vaccines. Virus like particles (VLPs) based vaccines are gaining increasing acceptance compared to subunit vaccines, as they present the antigens in more veritable conformation and are even readily recognized by the immune system. Hepatitis B virus (HBV) core antigen (HBcAg) is very well studied and has been successfully used as a carrier for more than 100 other viral sequences. In this study, hybrid HBcAg VLPs are generated by fusion of the conserved protective epitopes of PRRSV and expressed in E. coli. An optimized purification protocol that overcomes issues from ultracentrifugation is developed to obtain hybrid HBcAg VLP protein from the inclusion bodies. This hybrid HBcAg VLP protein self assembled to 23nm VLPs that were shown to block virus infection of susceptible cells when tested on MARC 145 cells. Therefore, the safety of non-infectious and non-replicable VLPs and production through low-cost E. coli fermentation may make this vaccine competitive against current vaccines on both efficacy and cost. / Master of Science

Les rotavirus et les norovirus sont les principaux agents étiologiques des gastro-entérites en Tunisie. Pendant l’hiver 2011-2012, nous avons collecté les selles et les salives de 114 enfants âgés de moins de 6 ans, souffrant de gastro-entérites et admis à l’Hôpital Fattouma Bourguiba de Monastir. L’analyse des salives a montré que la cohorte se répartissait entre 79% de sécréteur et 21% de non-sécréteur (absence d’antigène dans les salives). Parmi les sécréteurs, les individus du groupe O étaient les plus représentés (42%) suivis des groupes A (30%), B (21%) et AB (7%) alors que 96% des patients étaient positifs pour l’antigène Lewis. Pour 98 patients, l’analyse génétique du sang a montré que le gène FUT2 se répartissait entre 77.6% de sécréteur (Se+/Se+ et Se+/se-) et 22.4% de non-sécréteurs (se-/se-, N=22).L’analyse des fèces a montré que les rotavirus et les norovirus étaient responsables respectivement de 22% et 31% des cas, les infections mixtes représentant 6% des cas. Parmi les norovirus, le génotype GII.3 était prédominant (69% de tous les NoV) tandis que le génotype G9P[8] était le plus fréquemment détecté de tous les rotavirus (37,5%). Les rotavirus ont été détectés chez les individus sécréteurs (N=28) mais aussi chez 4 patients non-sécréteurs (3 souches G9P[8] et une souche G3P[8]). Nous n’avons pas observé de distribution particulière des rotavirus en fonction des antigènes A, B et H parmi les enfants sécréteurs. En revanche, nous avons constaté que l'infection à rotavirus ne s’était produite que chez les individus positifs pour l’antigène Lewis (P=0.017). La présence de génotype P[8] chez des non-sécréteurs est inédite, elle a été confirmée par le séquençage du segment correspondant à VP8* de ces rotavirus.La majorité des infections à norovirus a été détectée chez les patients sécréteurs et cela sans distribution particulière en fonction des antigènes A, B, H et Lewis. Cinq GII.3, un GII.1 et un norovirus de génotype GII.7 ont été détectés chez les non-sécréteurs, Lewis-positifs. La production de particules de synthèse (VLP) de norovirus GII.3 en baculovirus à partir des selles d’un des patients non-sécréteurs nous a permis de tester les échantillons salivaires de toute la cohorte. L’absence d’attachement de ces VLP sur les salives des non-sécréteurs montre que la présence ou l’absence des antigènes de groupe ne reflète pas nécessairement le pouvoir infectieux des norovirus chez les jeunes enfants. Les résultats obtenus sur les rotavirus et les norovirus suggèrent qu’ils existent des voies alternatives aux antigènes de groupe sanguin pour l’attachement des rotavirus et des norovirus dans l’intestin. / Rotavirus and norovirus are the main aetiological agents of gastroenteritis in Tunisia. Stool specimens and saliva were collected from children younger than 6 years of age, admitted to the Fattouma Bourguiba Hospital (Monastir, Tunisia) for gastroenteritis during the winter 2011-12. Saliva analysis showed that 79% and 21% patients had secretor and non-secretor phenotypes, respectively. Group O blood type was predominant (42%) followed by groups A (30%), B (21%) and AB (7%), whilst 96% of the patients were positive for Lewis antigen. For 98 patients, blood samples were available and were used for FUT2 genotyping. 77.6% of the cohort were secretor (Se+/Se+ and Se+/se-) and 22.4% were non-secretor (se-/se-).Rotavirus and norovirus were found alone in 22% and 31% of the stool specimens, respectively. Mixed rotavirus-norovirus infections accounted for 6% of the cases. GII.3 noroviruses were predominant among the noroviruses whilst the G9P[8] genotype was predominant for the rotaviruses.Rotaviruses were detected in secretor (N=28) as well as in non-secretor individuals (three G9P[8] strains and one G3P[8]). No significant association was found between ABO antigens or the secretor status and RV infection. Inversely, we observed that RV infection always occurred in Lewis-positive patients (P=0.017). The presence of the P[8] genotype was confirmed by sequencing part of the VP8* coding region.There was no significant association between norovirus infection and ABO antigens and the FUT2 genotype. Five GII.3, one GII.1 and one GII.7 noroviruses were found in Lewis-positive non-secretor patients. Virus-like particles from a GII.3 norovirus infecting a non-secretor patient from the cohort were expressed in baculovirus and used for binding assay with the 114 saliva samples of the study group. VLP binding with non-secretor saliva was negative and suggested that saliva binding assay might not reflect norovirus infectivity. Overall, our data suggested that rotavirus and norovirus infection might involve non-HBGA binding pathways as well as the canonical HBGA ligands.