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Rabies is an important causative agent of disease resulting in an acute infection of the nervous system and death of the individual. Vaccination of wildlife species, which are vectors of the disease, can reduce the number of non-protected animals so that the virus is not sustained in nature. Vaccines currently distributed to North American wildlife do not effectively immunize all at-risk species. This project pursued the development of recombinant human adenovirus type 5 (hAd5) expressing rabies glycoprotein as an effective vaccine vector against rabies. Prevec et al. (1990) had previously described AdRG1 expressing the rabies (ERA) glycoprotein gene from the early 3 region. Low levels of rabies glycoprotein were detected in cultured cells although good titres of rabies neutralizing antibodies were measured in mice and skunks (Prevec et al., 1990; Charlton et al., 1992). Using AdRG1 as a guide, I examined conditions required for optimal rabies glycoprotein expression. Vectors AdSVRG1.3, AdRG1.2, AdRG4, AdRG1.3, and AdHCMV13RG, containing variable exogenous promoters and genomic deletions, were constructed and evaluated. Although no formula was derived to predict optimal vector structure, AdRG1.3, containing rabies glycoprotein and SV40 polyadenylation sequences in parallel orientation to the E3 promoter, demonstrated high levels of glycoprotein expression and elicited high levels of neutralizing antibodies. Rabies glycoprotein shares structural and functional properties with the glycoprotein of vesicular stomatitis virus, also a rhabdovirus. We predicted that chimaeric glycoprotein molecules would structurally resemble the parental glycoproteins and, thus, be useful as vaccines. Five chimaeric DNA sequences of the two glycoproteins were designed and expressed from hAd5 vectors. The chimaeric proteins were expressed at low levels in infected cultured cells yet no anti-rabies nor anti-VSV neutralizing antibodies were detected in serum from immunized BALB/c mice. A block in the post-translational processing pathway of the chimaeric proteins at the level of oligosaccharide modifications may also be a result of blocked movement of proteins. Doctor of Philosophy (PhD)

Thesis Master of Science (MS)

Sequences at the 5' end of the nascent RNA are known to be important as signals for encapsidation of the genome of vesicular stomatitis virus. In order to define the specific sequences involved in this process and to develop an in vitro encapsidation system, in vitro transcription from SP64-based plasmids was used to synthesize RNA molecules corresponding to various portions of the viral 5' plus strand sequence. Some of these RNAs were tested for their ability to bind the capsid N protein in vitro. N protein in this assay was provided either from VSV mRNA programmed reticulocyte lysates or from infected cell extracts or, in collaboration with Dr. Sue Moyer (Gainesville, Florida), purified from viral nucleocapsids. This thesis describes the construction of the SP64-based plasmids and the use of their RNA transcription products in the experiments described above. I also constructed a series of plasmids that could direct the synthesis of RNA molecules which have many of the features of VSV defective particle genomes. Two of the constructs generate a defective-like RNA carrying a reporter gene capable of expressing the bacterial lac Z protein. These RNAs have the potential, after in vitro encapsidation and transfection into mammalian cells, of producing readily detectable helper-dependent virions. Thesis Master of Science (MS)

The potential of human adenovirus (Ad) to serve as a vector for expression of heterologous genes was evaluated. An experimental gene, consisting of sequences coding for the glycoprotein of vesicular stomatitis virus (VSV) attached to the promoter and polyadenylation signal of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, was inserted into early region 3 of adenovirus, in both orientations. The TK promoter was functional in both orientations. The TK promoter was functional in both orientations and responded to trans-activation by HSV infection. Abundant expression of VSV G however depended on the presence of a second transcript. This transcript was present only in the recombinant carrying the insertion in the orientation parallel to the E3 promoter (AdG12) and was initiated upstream of the insertion, within Ad sequences. The potential of Ad recombinants to serve as vaccine vectors was investigated using the recombinant AdG12. Antibody against VSV G was induced in cows, pigs, and dogs in response to infection with AdG12. Protection of mice, immunized with AdG12, against a lethal challenge with VSV demonstrated the biological effectiveness of this immune response. Thesis Master of Science (MS)