Die Vorliegende Arbeit beschäftige sich mit der Etablierung eines lentiviralen Vektorsystems, mit dem es möglich ist die RNA-Interferenz experimentell zu nutzen. Dafür wurden SEC Sequenzen in den Vektor pGJ3-eGFP kloniert. Nach Optimierung der Transfektions und Transduktionsschritte wurden im Anschluss rekombinante virale Partikel hergestellt. Zur Überprüfung der Effektivität der Induzierten RNA-Interferenz erfolgte die Transduktion einer persistierend mit Masern infizierten Zelllinie (C6SSPE). Ziel der siRNA Sequenzen war dabei die mRNA des N-Proteins, welches eine zentrale Rolle im viralen Replikationszyklus spielt. Die Reduktion der mRNA wurde über quantitative real time RT-PCR nachgewiesen.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17976号 / 医博第3840号 / 新制||医||1001(附属図書館) / 80820 / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 宮地 良樹, 教授 長澤 丘司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

This thesis investigates enhancing the thermal stabilization of a human type 5 adenoviral vector (AdHu5) through spray drying. The spray drying process was used to dry and effectively immobilize the AdHu5 within a mixture of carbohydrate or amino acid excipients into a powder form, resulting in significantly increased thermal stabilization of the viral vector. Spray dried powders were characterized by scanning electron microscopy, differential scanning calorimetry, Karl Fischer titrations, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) to identify the effects of temperature and atmospheric moisture on the immobilizing matrix. The best performing spray dried powder in terms of thermal stability consisted of an excipient blend of mannitol and dextran. Response surface methodology was employed to optimize production of these mannitol/dextran powders; measured responses were those relevant to industrial processing of a therapeutic material, namely powder yield for maximizing quantity, particle size for maximizing production of inhalation-deliverable powders, and adenoviral vector response for minimizing the loss of therapeutic activity. The spray drying process parameters of inlet temperature, spray gas flow rate, liquid feed rate and solute concentration in the feed were optimized resulting in a powder yield of 90%, percentage of ideally-sized particles of 50% and a near-zero viral vector titre loss of 0.25 log loss median tissue culture infectious dose (TCID50). The spray dried mannitol/dextran powders proved to have exceptional thermal stability during long term storage as minimal viral vector activity loss was observed when stored at 20°C for 90 days at low relative humidity (0.7 ± 0.3 log TCID50) in comparison to the liquid control which exhibited complete activity loss under the same storage conditions. Furthermore, viral activity of mannitol/dextran powders was retained over short term exposure (72 hours) to temperatures as high as 55°C whereas the liquid control expectedly lost all AdHu5 activity after 30 minutes. Overall, this work provides a guideline for the production of thermally stable powders and active biopharmaceuticals, such as AdHu5 vectors for vaccine applications, using the spray drying process. / Thesis / Master of Applied Science (MASc) / Many vaccines and their base components inherently deteriorate in function at moderate temperatures. Storage by refrigeration at temperatures ranging between 4°C and -80°C is the norm. Such refrigeration is costly for long term storage and significantly limits where vaccines can be sent. This reduces the availability of vaccines in locations around the world where these storage conditions are infeasible but vaccines are needed most. Spray drying, a process which forms dry powders from solution, was used; the solution contained sugars or amino acids to surround and protect the sensitive vaccine component. The produced powders from this work exhibited enhanced thermal stability compared to the control, reducing the need for refrigeration during storage and transport. The spray drying process was further optimized for industrial use by maximizing the amount of powder recovered and ensuring the particle size was appropriate for inhalable use, but most importantly, minimizing losses in therapeutic effectiveness during processing. This production of a thermally stable vaccine is advantageous because is allows for better world-wide accessibility and reduces overall production and delivery costs.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24839号 / 医博第5007号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上, 治久, 教授 寺田, 智祐, 教授 林, 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Natural exposure to Plasmodium falciparum’s asexual blood-stage results in protection against severe disease, but no vaccine using the widely-studied blood-stage antigens apical membrane antigen 1 (AMA1) or merozoite surface protein 1 (MSP1) has proven convincingly protective in clinical trials. Challenges include antigenic polymorphism, the apparent requirement for exceptionally high antibody concentrations for protection, and clinical-grade production of conformationally-accurate recombinant protein antigens followed by formulation with a human-compatible adjuvant. This thesis describes the generation of viral-vectored vaccines targeting ten less-studied blood-stage antigens, focusing upon antigens implicated in erythrocyte invasion. These vaccines were immunogenic in mice and rabbits. The rabbit antibodies raised were functionally active in the in vitro assay of parasite growth inhibitory activity (GIA). GIA with antibodies against one antigen, RH5, exceeded that achieved with antibodies against the ‘gold standard’ AMA1 or MSP1 antigens. This antigen’s amino acid sequence is relatively conserved between parasite strains. Importantly, and unlike anti-AMA1 and MSP1 antibodies, the GIA effects transcend genetically diverse strains. It was hypothesised that blockade of the interaction of RH5 with its receptor basigin was likely to be a mechanism of action of anti-RH5 antibodies. Vaccine-induced polyclonal anti-RH5 serum was found to be capable of blocking this interaction, as well as merozoite attachment to erythrocytes. A panel of RH5-specific monoclonal antibodies were raised: those which block the RH5-receptor interaction were capable of neutralising parasites. Minimal linear epitopes recognised by these antibodies were mapped, and are likely to be within or close to RH5’s receptor binding site. These data support prompt clinical testing of RH5-based vaccines, and shed light upon the mechanism of action of anti-RH5 antibodies. However substantial challenges remain in establishing whether this antigen, selected on the basis of the in vitro assay of GIA, will be capable of achieving in vivo protection against P. falciparum. Further work presented in this thesis addresses the use of quantitative PCR data to assess blood-stage vaccine efficacy in experimental human challenge with P. falciparum, and the use of surface plasmon resonance to establish more detailed characterisation of vaccine-induced antibody responses. Finally, the results of P. falciparum challenge of RH5-vaccinated Aotus nancymaae non-human primates are presented.

Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.

To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

No description available.

Alzheimer's disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution. These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD.