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No description available.

The early region 1a oncogene of adenovirus 5 codes for proteins that can activate transcription of viral and cellular genes. This study describes the construction of three deletions and one point mutation that together span the entire coding region of the second exon of E1A. The exon-2 mutants were tested for their ability to activate transcription from the adenovirus early region 3 promoter (E3) in transient expression assays. Dl1116 (dl aa 205-221) did not affect transactivation of E3 in pKCAT-23. Sub1117 (dl exon-2 aa) and dl1115 (dl aa 188-204) were unable to activate transcription. Pm1131 (SER-219 to stop) had a reduced transactivating efficiency but was still able to stimulate transcription. These results define the 3' boundary of a transactivation domain on the E1A proteins as being between positions 188 and 204. Results obtained in our lab define the 5' boundary as being between 138-147 (Jelsma et al., 1988). The mutants that could not transactivate were tested for their ability to block wildtype E1A transactivation of the E3 promoter in assays similar to those described by Glenn and Ricciardi (1987). Dl1115 and sub1117 appeared to block transactivation by WT E1A. In transient expression assays, the fatty acid sodium butyrate was found to stimulate transcription of the CAT gene, when added to the medium of HeLa cells transfected with pKCAT-23. This suggests that sodium butyrate is transactivating the Ad 5 E3 promoter. / Thesis / Master of Science (MS)

The E1A gene of adenovirus 5 carries out a number of functions in infection and oncogenic transformation, including the transcriptional activation of viral and cellular genes, the repression of transcriptional enhancers, and cooperation with the adenovirus E1B gene or with the ras oncegene to transform primary cells. The purpose of this work was to investigate the mechanism of action of E1A, by determining the regions of the proteins that are required for these functions. Deletion and point mutations were made in the region unique to the larger E1A mRNA, by exonuclease digestion and deletion loop mutagenesis respectively. These mutants and a series of mutants which delete sequences spanning the entire coding region, were examined for their effect on transcriptional activation, enhancer repression, and transformation. The region which, when deleted, rendered E1A defective for transcriptional activation was found to be confined to the region unique to the 13s mRNA and the beginning of exon 2. Mutations in three regions, all within the 12s exon 1, affected repression activity. The first two, the N terminal region of the protein, and a region, CR1, conserved between adenovirus serotypes, were essential for repression activity. The third region, at the end of exon 1 of the 12s mRNA, was probably only indirectly involved in repression. Deletions in three regions of exon 1 resulted in a loss of the transforming function of E1A. The first two corresponded to the regions required for repression, suggesting that enhancer repression is a component of transformation. The third region, containing CR2, also conserved between adenovirus serotypes, is functionally distinct from the other two and appeared to affect the morphology of the transformants. These two functions did not operate efficiently when present ion separate plasmids. The 13s unique region and exon 2 were not required for transformation but the loss of the transactivation function of E1A did result in an increased adhesiveness of the transformants. / Thesis / Doctor of Philosophy (PhD)

Many viral transcriptional activators have been shown to activate genes of heterologous systems. To assess the ability of the herpes simplex virus ICP4 trans-activating protein to complement an adenovirus mutant lacking its own trans-activator, the E1a protein, I constructed an adenovirus type 5 vector containing a temperature sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in human cells which are permissive (293) or nonpermissive (KB and R970-5) for E1a⁻ viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus, however it is incapable of complementing adenovirus E1a⁻ mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. My results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators. / Thesis / Master of Science (MS)

The immense global tuberculosis (TB) burden highlights the shortcomings of current vaccination and antibiotic regimens. Novel prophylactic TB vaccines that can either boost or replace BCG entirely remains an active area of research. Additionally, the success of current antibiotic therapies against TB is hindered by their complexity and duration, with large percentages of patients failing to complete treatment. Multi-armed approaches are required to properly and efficiently combat diseases. Besides prophylactic vaccines, development of therapeutic vaccine strategies as an adjunct to antibiotic treatment would represent another major step in TB control. To achieve such a goal, vaccines must consider the pathogen’s life cycle, the immunological responses which they drive, and the populations in which they will ultimately be administered. As such, the purpose of this dissertation is to utilize state-of-the-art molecular cloning techniques to construct novel chimpanzee adenovirus-vectored vaccines that provide prophylactic and therapeutic immunity against pulmonary TB. By considering different phases of the pathogen’s life cycle, we aim to select a collection of antigens that are protective, regardless of disease state. Development of such platforms would lay and bolster the groundwork for improved vaccine strategies against TB. / Dissertation / Doctor of Philosophy (PhD)

Successful human adenovirus (HAdV) replication relies on multiple protein-protein interactions between viral and host proteins. HAdV type 5 (HAdV-5) pVI is a multifaceted protein necessary for viral endosomal escape, activation of viral protease, as well as nuclear shuttling of certain viral proteins. Preliminary mass spectrometry experiments indicated that pVI can bind cellular importins and histone chaperones, of which many are considered novel pVI targets. Here, the binding of the pVI protein to cellular importins was validated, and preliminary studies were done to characterize whether HAdV-5 infection changes importin levels in the infected cells. The validation studies were inconclusive, but it was observed that the accumulation of the importin proteins was not altered in during HAdV-5 infection. In addition, the role of NAP1L1 and NAP1L4, two ubiquitously expressed histone chaperones, was examined during HAdV-5 infection and their effect on HAdV-5 genome structure. Here, it is shown that NAP1L1 knockdown affected viral mRNA and protein as well as hindered viral histone-like protein pVII deposition onto viral DNA during the late stage of infection. In contrast, the NAP1L4 protein was shown to co-localize to viral replication centers (VRCs), and its elimination promoted the pVII protein deposition on virus DNA. These results suggest that NAP1L1 is involved in viral transcription and chromatin assembly, whereas NAP1L4 has anti-viral properties during the assembly process.

Auf Adenoviren (Ads) basierende Vektoren wurden als ein gezielter Anti-Krebs-Wirkstoff entwickelt, der erfolgversprechende Resultate in prä-klinischen Studien erzielen konnte. Solche onkolytischen Ads sind zwar in klinischen Studien generell als sicher eingestuft worden, konnten jedoch die therapeutischen Erwartungen nicht erfüllen. In dieser Doktorarbeit konnte, unter Verwendung der Genexpressionsprofile von Ovarialkarzinom-Zellen, der epitheliale Phänotyp als Hindernis für allgemein verwendete onkolytische Ads, die auf den Coxsackie- und Adenovirusrezeptor (CAR) oder CD46 ausgerichtet sind, identifiziert werden. Der Zugang zu den Virus-Rezeptoren war zwingend an Zelldepolarisation und den Verlust der epithelialen Zonulae occludens und adherens gekoppelt, was Merkmale der Epithelial-zu-Mesenchymal-Transition (EMT) darstellt. Bedeutsam ist in diesem Zusammenhang, dass Tumore in situ als auch Xenograft-Tumore zum größten Teil aus Epithelzellen oder epithelial/mesenchymalen (E/M) Hybrid-Zellen bestehen. Diese E/M Hybrid-Zellen sind die einzigen Zellen, welche an Zellkulturbedingungen adaptieren, wo sie durch EMT während weiterem Passagieren in Mesenchymzellen differenzieren. Bemerkenswert ist hierbei die Tatsache, dass nur Mesenchymzellen und E/M Hybrid-Zellen, die sich im EMT-Prozess befanden, sensitiv zu viraler Onkolyse waren. In Versuchen, die festgestellte Resistenz zu überwinden, wurde herausgefunden, dass bisher nur wenig erforschte Adenovirus-Serotypen (Ad3, Ad7, Ad11 und Ad14), welche einen anderen Rezeptor als CAR oder CD46 auf Zellen benutzen, besser geeignet sind, um polarisierte Epithelzellgewebe zu infizieren. Diese Ads induzierten EMT-ähnliche Prozesse in Ovarialkarzinom-Kulturen mit epithelialem Phänotyp, was zu deren effizienter Onkolyse führte. Die vorliegende Arbeit trägt somit zur Aufklärung der Diskrepanz zwischen der Virustherapie-Effizienz in vivo und in vitro bei und bietet Anhaltspunkte für die Konstruktion von zukünftigen onkolytischen Ads. / Vectors based on adenoviruses have been designed as targeted anti-cancer therapeutics that showed promising results in pre-clinical applications. In clinical trials, these oncolytic adenoviruses have generally been proved safe in patients, but have fallen short of their expected therapeutic value. In this thesis the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses was studied in order to identify cellular mechanisms that confer resistance to virotherapy. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, it was discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie- and adenovirus receptor (CAR) or CD46. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-mesenchymal transition (EMT). Importantly, tumors in situ as well as xenograft tumors derived from primary ovarian cancer cells mostly contained epithelial cells and cells that are in an epithelial/mesenchymal (E/M) hybrid stage. These E/M cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT to differentiate into mesenchymal cells. Notably, only mesenchymal cells and E/M cells in the process of EMT were susceptible to viral oncolysis. In attempts to overcome the observed resistance, it was found that thus far little explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14), which use cellular receptor(s) other than CAR and CD46, have superior oncolytic abilities on polarized epithelial tissue. This study therefore contributes to the clarification of observed discrepancies between virotherapy performances in vitro and in vivo and gives a rationale for the construction of future oncolytic adenoviruses.

Neste trabalho nós estudamos qual seria o impacto da indução de resposta imune a epítopos CD8 subdominantes na imunidade gerada pela vacinação genética. Durante a infecção experimental apenas um epítopo imunodominante presente no antígeno ASP-2 é reconhecido. Já os linfócitos T CD8+ induzidos nos animais vacinados com o gene da ASP-2 são capazes de reconhecer além deste mais dois outros epítopos (subdominantes). A identificação desses epítopos permitiu que estudássemos o papel da resposta imune a epítopos subdominantes na imunidade protetora. Após imunização genética com o gene da ASP-2 mutado, sem resposta para o epítopo dominante, confirmamos que a resposta imune aos epítopos subdomiantes pode contribuir na proteção contra a infecção experimental. Apesar do papel critico dos linfócitos T CD8+ na resposta imune protetora induzida pela vacinação genética do tipo imunização e reforço heterólogo, não se sabe ao certo se após o desafio experimental estes linfócitos T CD8+ necessitam proliferar ou recircular para mediar a imunidade protetora. Nossos resultados desafiam o paradigma de ação das vacinas tradicionais de que a imunidade é dependente da proliferação e não da recircular dos linfócitos T de memória e para mediar a imunidade protetora. / In the present study, we evaluated the impact of the immune response to sub-dominant CD8 epitopes on immunity generated by genetic vaccination. During experimental infection only a single dominant epitope is recognized on the antigen ASP-2. In contrast, the CD8+ T lymphocytes induced in animals genetically vaccinated with ASP-2 recognized, in addition to the dominant epitope, two other epitopes (sub-dominants). The identification of these epitopes allowed us to test the role of immune response to sub-dominant epitopes in protective immunity. After genetic vaccination with ASP-2 mutated gene, without the response to dominant epitope, we concluded that the immune response to the sub-dominant epitopes can be important to protective immunity. In spite of the critical role of CD8+ T lymphocytes in protective immune response induced by genetic vaccination using the heterologous prime-boost regimen, it is unclear whether after the experimental challenge these CD8+ T lymphocytes need to proliferate or recirculate to mediate protective immunity. Our results challenge the paradigm of action of traditional vaccines that immunity is dependent on the proliferation of memory T lymphocytes and that these cells do not need to recirculate.

Processos biotecnológicos dependem significativamente das técnicas de separação e purificação utilizadas, para manter boa relação custo-benefício na produção em escala industrial de produtos biotecnológicos com fins comerciais, industriais e terapêuticos. A aplicação do sistema micelar de duas fases aquosas (SMDFA) é proposta como alternativa para purificação de biomoléculas/biopartículas, pois permite sua separação e análise, muitas vezes sem que essas percam sua atividade ou propriedades desejadas. Com essa técnica é possível realizar uma partição seletiva que possibilita altos rendimentos. Esse trabalho destinou-se a estudar o emprego dessa metodologia na extração e purificação de Adenovírus em sistema micelar de duas fases aquosas formado por Triton X-114/Suspensão viral. Os ensaios foram realizados em sistemas de 5 g seguindo um planejamento fatorial completo (23) com 4 pontos centrais. Os fatores estudados foram temperatura de extração, pH da suspensão viral e concentração do tensoativo. Sistemas contendo massas de 3g, 10g e 40g foram avaliados. Foi avaliado o efeito do processo de extração com SMDFA sobre a integridade e infectividade de Adenovírus. Alguns dos parâmetros avaliados no processo foram a recuperação da potência viral (RPv) e a recuperação da potência viral específica (RPvø). Esses dois parâmetros avaliam a inativação do Adenovirus pelo processo de extração e ambos apresentaram melhoras quando comparados com a própria suspensão viral para alguns dos sistemas estudados (i.e RPv:341 % e RPvø 1466 %). Esses resultados indicam que o SMDFA foi capaz de particionar seletivamente as partículas virais infecciosas. De acordo com os resultados do planejamento é possível aumentar ainda mais esses resultados controlando as variáveis concentração de tensoativo, pH da suspensão viral e temperatura de extração. / Biotechnological processes depend significantly on separation and purification techniques used to maintain cost-effective industrial-scale production of biotechnological products for commercial, industrial and therapeutic uses. The application of the aqueous two-phase micelar system (ATPMS) is proposed as an alternative for purification, since it allows the separation and analysis of biomolecules /bioparticles, often without loses of activity or their properties. This allows to perform a selective partition that enables high yields. This work aims to study the use of this methodology in the extraction and purification of adenovirus in micelle aqueous two-phase formed by TritonX-114/Viral suspension. All assays were performed in 5 g systems following a full factorial design (23) with four central points. The studied factors were extraction temperature, pH of the viral suspension and concentration of the surfactant. Systems containing masses of 3g, 10g and 40g were evaluated. Extraction procedure effects over integrity and infectivity of adenovirus were also evaluated. Some of the parameters evaluated in the viral recovery process were viral potency (RPv) and recovery of viral specific potency (RPvø). These two parameters measure the inactivation of Adenovirus by the extraction process and both showed improvement when compared with the viral suspension for some of the systems studied (i.e RPv: 341% and RPvø 1466%). These results show that ATPMS selectively partition the infectious viral particles. According to the results of the experimental design is possible to increase, even further, these results controlling the surfactant concentration, viral suspension pH and temperature of extraction.