Studies on the tropism of canine adenovirus type I

Copeland, Kathryn (2003) Studies on the tropism of canine adenovirus type I. PhD thesis, University of Glasgow.

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Abstract

Adenoviruses have been extensively researched for use as vectors for both gene therapy and vaccination. Gene therapy is the delivery of transgenes in order to treat genetic disease, whereas vaccination with viral vectors is primarily to induce immune responses to transgenes and in some cases the vector itself. While immune reactions against the adenovirus backbone can be desirable in vaccination strategies, this has proved to be a particular problem in gene therapy settings. The most fully investigated human adenoviruses (HAds) are serotypes 2 and 5, which are endemic in the population with most people harbouring neutralising antibodies to them. Quantifying the levels of neutralising antibodies to adenoviral vectors in patients is important as these may impede vector efficiency upon administration. Recent research has shown that this can be overcome by the use of alternative vectors based on less prevalent HAds and adenoviruses from heterologous species. However, before such alternatives can be used it is first necessary to investigate whether these viruses can express genes in cells of the target species. For development as a live vaccine vector their ability to infect cells of the target species is of interest. Canine adenovirus type 1 (CAV-1) is a candidate vector for both gene therapy and vaccination, hi its canine host CAV-1 causes a number of diseases including hepatitis. It has tlie ability to infect a wide range of tissues in the dog and cells from several animal species (Koptopoulos & Cornwell, 1981). In addition, limited investigations have shown CAV-1 capable of infecting human cells (Gehle & Smith, 1969). One of the aims of the presented work was to investigate CAV-1 infection in cells of two candidate species: cats and humans, using sensitive modern techniques including real-time PCR and RT-PCR. This would determine the suitability of CAV-1 as a vector in these species, primarily for vaccination in cats and gene therapy in humans. In feline cells CAV-1 infection was productive with growth kinetics similar to CAV-1 infection of canine cells. In addition, transgene expression was demonstrated from a replication competent CAV-1 vector. DNA replication and transcription were demonstrated in human cells, as was transgene expression. Classical adenoviral cytopathic effect was not observed in human cells, however, in A549 cells CAV-1 infection resulted in the limited production of de novo infectious virus. These findings indicate that CAV-1 has potential for development as a vector for use in both species. One of the features that initially attracted researchers to the development of adenoviral vectors was their ability to infect a wide variety of tissues. However, the promiscuous tropism of these vectors can lead to limited transgene expression in target tissues. Untargeted vectors can cause a number of problems such as toxicity in non-target tissues and high dosage requirements, which can lead to harmful immune responses. The fibre mediates initial adenovirus attachment to host cells and research has shown that adenoviruses can be targeted through modification of this protein. A second aim of this work was proof-of-principle experiments to investigate whether CAV-1 could tolerate the fibre of another adenovirus serotype. CAV-1 vectors that were pseudotyped with CAV-2 and HAd5 were created that grew to high titres and displayed altered tropism in preliminary investigations. As such, a vector system was developed that will facilitate targeting through exploitation of existed targeted HAd5 fibres in their entirety or through knob exchange. In addition, fibre deleted vectors were developed. Infection of MDCK cells with these viruses did not result in cytotoxicity. However, fibre expressing cell lines allowed productive infection. Finally, it is important to determine the levels of pre-existing antibodies in target species and therefore the neutralisation of CAV-1 by human and feline serum samples was determined. None of the feline samples had neutralising activity against CAV-I. Only 22% of human serum samples were able to neutralise CAV-1 compared with the neutralisation of HAd5 by 46% of samples. The same human samples were investigated for the ability to neutralise CAV-1 vector pseudotyped with the HAd5 fibre. The results showed that neutialisation of tliis vector by human sera was related to that of CAV-1 and HAd5. In conclusion these results demonstrate that CAV-1 can express transgenes in feline and human cells, that there is potential to modify its existing tropism and that there are low (or no) neutralising antibodies to it in both species investigated.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Mark Morrision
Keywords: Virology
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71388
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71388

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