Gene therapy is a promising treatment option for cancer. Ideally, a therapeutic gene is delivered specifically into tumor cells sparing the neighboring normal cells. For this purpose gene delivery vectors are designed that can recognize structures, which are exclusively expressed on tumor cells (i.e. the tumor-associated antigens -TAA-). Retroviral vectors are commonly used for gene therapy by modifying the envelope protein responsible for the recognition of the target cell. The Vesicular Stomatitis Virus G protein (VSV-G) is a well-liked choice for pseudotyping the retroviral vectors since it confers on the viral particle stability to allow concentration to high titers necessary for the clinical applications. However, the main drawback of VSV-G, the ubiquitously expressed receptor and thus the broad target range, hinders the use of this protein for targeted gene therapy. In this thesis, we aimed to modify the VSV-G for targeted gene therapy against Prostate Stem Cell Antigen (PSCA) -expressing tumors. Therefore we followed two approaches. The first approach comprised of the fusion of a single-chain antibody fragment against PSCA to the N-terminus of VSV-G. In the second approach the VSV-G was modified by insertion of a small epitope. We could demonstrate that two positions in the N-terminal region of VSV-G protein permit insertion of a ten amino acid long epitope. These mutant VSV-G proteins were successfully assembled into retroviral particles. We demonstrated that the mutant retroviral particles can be used for targeting to PSCA-positive cells using nanobeads. The nanobeads were chemically coupled to antibodies against the epitope in the VSV-G protein and PSCA on the tumor cell. These bispecific nanobeads allowed the recruitment of mutant retroviral particles to the PSCApositive cells. Our results point out the potential of these mutant retroviral particles in targeted gene delivery. Further studies will be necessary to assess the efficiency of in vivo targeted gene therapy using these mutant retroviral particles.

Diante da necessidade de responder algumas indagações relacionadas a epidemiologia da Estomatite Vesicular, principalmente aquelas que dizem respeito a ocorrência de surtos em locais onde existem coleções d\'água, foi desenvolvido um modelo de transmissão do VSA utilizando a água como via de transmissão, a tilápia nilótica, inoculada intraperitonealmente, como fonte de infecção e o cobaio como hospedeiro susceptível. O objetivo da utilização deste modelo biológico de transmissão do Vírus da Estomatite Vesicular foi de avaliar o papel desempenhado pelos peixes no ciclo epidemiológico, propor um modelo de ciclo epidemiológico do VSA, destacando o papel da água como via de transmissão e padronizar uma técnica de RT-PCR para a detecção do VSA, em amostra de tecidos. Através do modelo desenvolvido, fica demonstrado que estes peixes eliminaram partículas virais na água, decorridos 13 dias pós-inoculação e que esta última se caracteriza como via de transmissão, possibilitando a infecção dos hospedeiros susceptíveis (cobaios) através de inoculações experimentais em coxim plantar. A tilápia nilótica pode ser considerada como uma fonte de infecção, por ser capaz de eliminar um agente infeccioso no meio ambiente e através de uma via de transmissão este agente alcançou o hospedeiro susceptível; os peixes podem ser inseridos no ciclo epidemiológico da Estomatite Vesicular como fonte de infecção, sendo capazes de eliminar na água partículas virais infectantes, destacando o papel da água como via de transmissão; fica padronizada uma técnica de RT-PCR dirigida ao gene codificador da proteína RNA-polimerase, útil para a detecção direta do Vírus da Estomatite Vesicular Alagoas e Indiana em amostras de tecidos. / A model of transmission of Vesicular Stomatitis was developed to Vesicular Stomatitis Alagoas (VSA) serotype employing water as a way of transmission, the Nile tilapia intraperitoneal inoculated as a source of infection and guinea pigs as susceptible hosts aiming to answer many questions concerning Vesicular Estomatitis epidemiology, as the risk of disease on farms with dose relationship with riverine areas and the role of fishes in the epidemiological cycle of the disease. Furthermore, a RT-PCR assay was developed to detect VSA in tissue samples. According to the experimental transmission, fishes eliminated virus into the water after 13 days pos-infection and a model to VSA epidemiological cycle is proposed in which water was characterized as a way of transmission, carrying the virus to the susceptible host through experimental inoculation and the Nile tilapia should be thought as a source of infection, once it was able to eliminate the infective agent into the environment. A useful tool to the diagnosis of both Indiana and Alagoas serotypes was developed.

Thesis (M.S.)--Brigham Young University. Dept. of Microbiology and Molecular Biology, 2005. / Includes bibliographical references.

Thesis (M.S.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 57 pages. Includes Vita. Includes bibliographical references.

Diante da necessidade de responder algumas indagações relacionadas a epidemiologia da Estomatite Vesicular, principalmente aquelas que dizem respeito a ocorrência de surtos em locais onde existem coleções d\'água, foi desenvolvido um modelo de transmissão do VSA utilizando a água como via de transmissão, a tilápia nilótica, inoculada intraperitonealmente, como fonte de infecção e o cobaio como hospedeiro susceptível. O objetivo da utilização deste modelo biológico de transmissão do Vírus da Estomatite Vesicular foi de avaliar o papel desempenhado pelos peixes no ciclo epidemiológico, propor um modelo de ciclo epidemiológico do VSA, destacando o papel da água como via de transmissão e padronizar uma técnica de RT-PCR para a detecção do VSA, em amostra de tecidos. Através do modelo desenvolvido, fica demonstrado que estes peixes eliminaram partículas virais na água, decorridos 13 dias pós-inoculação e que esta última se caracteriza como via de transmissão, possibilitando a infecção dos hospedeiros susceptíveis (cobaios) através de inoculações experimentais em coxim plantar. A tilápia nilótica pode ser considerada como uma fonte de infecção, por ser capaz de eliminar um agente infeccioso no meio ambiente e através de uma via de transmissão este agente alcançou o hospedeiro susceptível; os peixes podem ser inseridos no ciclo epidemiológico da Estomatite Vesicular como fonte de infecção, sendo capazes de eliminar na água partículas virais infectantes, destacando o papel da água como via de transmissão; fica padronizada uma técnica de RT-PCR dirigida ao gene codificador da proteína RNA-polimerase, útil para a detecção direta do Vírus da Estomatite Vesicular Alagoas e Indiana em amostras de tecidos. / A model of transmission of Vesicular Stomatitis was developed to Vesicular Stomatitis Alagoas (VSA) serotype employing water as a way of transmission, the Nile tilapia intraperitoneal inoculated as a source of infection and guinea pigs as susceptible hosts aiming to answer many questions concerning Vesicular Estomatitis epidemiology, as the risk of disease on farms with dose relationship with riverine areas and the role of fishes in the epidemiological cycle of the disease. Furthermore, a RT-PCR assay was developed to detect VSA in tissue samples. According to the experimental transmission, fishes eliminated virus into the water after 13 days pos-infection and a model to VSA epidemiological cycle is proposed in which water was characterized as a way of transmission, carrying the virus to the susceptible host through experimental inoculation and the Nile tilapia should be thought as a source of infection, once it was able to eliminate the infective agent into the environment. A useful tool to the diagnosis of both Indiana and Alagoas serotypes was developed.

HIV infection represents a major health and socioeconomic challenge worldwide. Despite significant advances in therapy, a cure for HIV continues to be elusive. The design of novel curative strategies will require targeting and elimination of cells that constitute the latent HIV-1 reservoir. However, such an approach is impeded by the inability to distinguish latently HIV-infected cells from uninfected cells. The type-I interferon (IFN-I) response is an integral antiviral defense mechanism, but is impaired at multiple levels during productive HIV infection. Interestingly, similar global impairments in IFN-I signaling have been observed in various human cancers. This led to the development of IFN-sensitive oncolytic viruses, including the recombinant Vesicular Stomatitis Virus (VSV 51) and Maraba virus (MG1), as virotherapy designed to treat various cancers. Based on this, it was hypothesized that IFN-I signaling is impaired in latently HIV-infected cells (as observed in productively infected cells) and that VSV 51 and MG1 may be able to exploit such intracellular defects to target and eliminate latently HIV-infected cells, while sparing healthy cells. First, using cell line models of HIV-1 latency, intracellular defects in IFN-I responses, including impaired IFN / production and expression of IFNAR1, MHC-I, ISG15, and PKR, were demonstrated to represent an important feature of latently HIV-infected cells. Consistent with this, the latently HIV-infected cell lines were observed to have a greater sensitivity to VSV 51 and MG1 infection, and MG1-mediated killing, than the HIV-uninfected parental cells. Next, the ability of oncolytic viruses to kill latently HIV-infected human primary cells was demonstrated using an in vitro resting CD4+ T cell model of latency. Interestingly, while both VSV 51 and MG1 infection resulted in a significant reduction in inducible p24 expression, a dose-dependent decrease in integrated HIV-1 DNA was only observed following MG1 infection. In keeping with this, MG1 infection of memory CD4+ T cells from HIV-1 infected individuals on HAART also resulted in a significant decrease in inducible HIV-1 gag RNA expression. By targeting an intracellular pathway that is impaired in latently HIV-infected cells, the findings presented in this dissertation highlight a novel, proof-of-concept approach to eliminate the latent HIV-1 reservoir. Given that VSV 51 and MG1 are currently being studied in cancer clinical trials, there is significant potential to translate this work to in vivo studies.

Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%. In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer. Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.

No description available.

Scope and Contents: Virus specific RNA components of the cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis virus were examined. Studies were carried out both in the presence and absence of defective particle interference. / Thesis / Master of Science (MSc)

No description available.