Adenoviruses (Ad) are double-stranded (ds) DNA, non-enveloped viruses. There are seven species (A-G) of human Ads with 52 knownserotypes to date. Human Ads cause a broad range of pathologies, ranging from upper respiratory tract infections to persistent urinary tract infections. The main determinant for Ads tropism in vitro is the protruding, antenna-like, fiber protein. The fiberknob is responsible for the main interaction with the attachment receptor of the host cell. Most Ad species use the coxsackie- adenovirus receptor (CAR) as their main attachment receptor. Most species B Ads, however use CD46. CD46 is a cell surface complement regulatory protein, which is expressed on all nucleated cells in humans. Species B Ads exhibit a low seroprevalenc in the human population, making these Ads promising vector candidates for gene therapy. We have studied human Ad species B members, serotypes 7 and 11 (Ad7 and Ad11), as well as their interaction with CD46. Our first experiments showed that all species B Ads use CD46 as their main attachment receptor, with the exception of Ad3 and Ad7. Second, we performed mutational studies of recombinant Ad11p fiberknobs. These studies showed that arginine 279 in the Ad 11 fiberknob is necessary for CD46 binding. Finally we studied the effect of Ad11 binding to CD46. The results indicate that CD46 is rapidly downregulated on the cell surface after Ad11 binding. These results may provide a further understanding of the basic biology and pathology of species B Ads and may also be useful in construction of gene therapy vectors based on species B Ads.

Recombinant adenoviruses, commonly used in gene therapy and vaccine applications, compromise the expression and function of hepatic CYP3A for 14 days. When given with docetaxel (DTX), plasma clearance of DTX (3.38 ± 0.22 l/kg.h) was significantly lower than those given DTX alone (6.41 ± 1.10 l/kg.h). The area under the plasma concentration-time curve of DTX in rats given virus (2,987.37 ± 197.97 ng/ml.h) was significantly greater than those given drug alone (1,666.59 ± 317.04 ng/ml.h). The virus extended the half-life of DTX three-fold. This may explain why adenoviral vectors improve chemotherapy. PEGylation of the virus reduced interleukin-6 (IL-6), IL-12, tumor necrosis factor alpha (TNF-α), aspartate transaminase (AST) and lactate dehydrogenase (LDH) levels in mice and non-human primates. PEGylation dramatically reduced transduction efficiency of virus in the baboon liver and did not alter hepatic transgene expression in the mouse. Unmodified and PEGylated virus (3 x 1012 vp/kg) reduced hepatic CYP3A4 protein by 60% and 40%, respectively 96 hours after virus administration. Catalytic activity was decreased by 55% and 45% with respect to an untreated control by the native and PEGylated viruses respectively. This suggests that changes in hepatic CYP3A during infection is not entirely due to the immune response and these observed effects most likely occur in humans. The effects of adenovirus on hepatic CYP3A expression and function in mice, however, resolved at a faster rate than that in baboons. HC-04 cells are a suitable in vitro model to study virus infection and hepatic CYP3A function. A panel of adenoviruses inhibited CYP3A catalytic activity and induced changes in expression and distribution of retinoid X receptor alpha (RXRα), pregnane X receptor (PXR) and constitutive androstane (CAR) receptors. Virus (1.5 x 1011 vp) inhibited CYP3A in the mouse. When the ability of the virus to bind to integrins was removed, changes in CYP were not detected. Treatment with a RGD peptide, that binds to integrins, reduced CYP3A activity in a manner similar to the virus. Silencing of β3 and β5 integrins also resolved changes in CYP3A activity during infection, suggesting that simple engagement of integrin receptors can initiate changes in CYP3A. / text

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Wyoming, 2007. / Title from PDF title page (viewed on Nov. 4, 2008). Includes bibliographical references (p. 47-49).

Conditionally-replicating adenoviruses (CRAds) have generally demonstrated only modest therapeutic efficacy in human clinical trials, in part due to their poor ability to spread throughout a tumor mass. In these studies, I first examined whether inclusion of an intact early region 3 (E3) and the p14 fusion-associated small transmembrane (FAST) protein in a CRAd vector can enhance oncolytic efficacy by improving viral spread. E3 encodes the adenovirus death protein (ADP), which enhances virus progeny release from infected cells, while p14 FAST can allow spread of the virus through cell-cell fusion. I generated viruses with (CRAdRC109) or without (CRAdRC111) an intact E3 region, which encoded the p14 FAST gene between the fiber coding sequence and E4 region of their viral genomes. In the A549 human lung cancer cell line, both CRAdRC109 and CRAdRC111 expressed p14 FAST at very low levels when compared to CRAdFAST, a similar virus that expressed the protein from within the E3 deletion, and thus had a relatively poor ability to mediate cell-cell fusion. Although inclusion of E3/ADP in CRAdRC109 did result in larger plaques and increased virus spread relative to CRAdRC111, neither virus showed improved oncolytic activity relative to CRAdFAST. I subsequently developed CRAdRC116, in which the E3 region of the viral genome was replaced with a bicistronic expression cassette containing the p14 FAST and ADP coding sequences separated by a self-cleaving 2A peptide sequence. This virus co-expressed p14 FAST and ADP and caused extensive cell-cell fusion in A549 cells. However, expression of ADP from CRAdRC116 did not increase cancer cell killing nor virus spread, and thus did not enhance oncolytic efficacy relative to CRAdFAST. These studies suggest that p14 FAST and ADP do not exhibit synergy when co-expressed from a CRAd vector. Future studies should instead focus on combining other methods of improving viral spread in conjunction with expression of ADP or FAST proteins from CRAd.

Adenovirus (Ad) has been one of the most commonly used vectors in gene therapy for many years. One of the limitations of using Ad for cancer therapy is that Ad does not spread within a tumor well. As such, fusogenic proteins have been incorporated into these vectors in an attempt to improve spread through cell-cell fusion. Fusogenic protein mediated cell fusion is also associated with syncytiosome release and cell death, which promote an immune response and suggests that fusogenic proteins may be effective as a sole therapeutic for cancer treatment. In this study, I examined whether the reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein can improve Ad efficacy in cancer models in vitro and in vivo. Ad-mediated FAST protein expression induced cell-cell fusion in human embryonic kidney 293 cells and in human lung adenocarcinoma A549 cells. Infected cells had decreased membrane integrity, cellular metabolic activity and increased cleaved caspase-3 expression. In an A549 xenograft nude mouse model, AdFAST did not induce tumor cell fusion or promote mouse survival compared to control mice. In murine 4T1 mammary carcinoma cells, FAST protein expression had modest effects on cell fusion and metabolic activity in vitro. When AdFAST was administered intratumorally in a 4T1 syngeneic mouse model, there were no obvious signs of cell fusion or improved mouse survival. As FAST protein expression promotes membrane permeability, whether AdFAST could enhance the bystander effect of cancer therapies or promote uptake of anti-cancer drugs was investigated in vitro. FAST protein expression in combination with thymidine kinase/ganciclovir or etoposide administration did not have an enhanced effect. Addition of Smac-mimetic compound (SMC) was not effective in the SMC resistant A549 cell line, but the combinational therapy was able to decrease metabolic activity in SMC susceptible SNB75 cells. AdFAST and administration of the membrane impermeable chemotherapeutic drug bleomycin decreased metabolic activity and promoted cleaved caspase-3 expression in A549 cells. Overall, my results suggest that FAST protein expression could improve adenovirus efficacy and could be used with other anti-cancer treatments.

At present, 56 types of human adenovirus (HAdVs) have been identified and found to be associated with a variety of clinical features in the respiratory tract, eye, gastrointestinal tract, and other organs. In additions, HAdVs are able to establish persistent and latent infections in humans. Most of the work which has been carried out recently is related to adenovirus vectors and little has been done in other areas such as the nature and mechanisms of adenovirus persistence and latency in human tissues. Another area needing more investigation is the stability of the adenovirus genome which is useful for the development of adenovirus vectors and vaccines and for better understanding of adenovirus evolution especially with conflicting views about this issue.Recombination between two types of adenovirus can happen when the hexon epitope from one type and the fibre epitope from another type are found (intermediate strains). These recombinants can be detected by the conflicting results for serum neutralization (SN) and haemagglutination inhibition (HI) tests or by sequencing and phylogenetic analysis of the hexon and fibre regions of the adenovirus genome. The first part of this study is related to the stability and evolution of different adenovirus species. A total of 31 clinical isolates from AIDS patients previously typed in the hexon L2 region and the fibre knob region were analysed. These isolates were found to be from species D adenovirus (HAdV-D) and 28 of them had contradictory typing results in these two regions so they are clearly intermediate strains. Two isolates appear to be completely new and one isolate (Aids32) was typed as HAdV-D23 variant in both hexon L2 and fibre knob regions. Sequencing and phylogenetic analysis of the hexon L1, fibre shaft and penton regions of these adenoviruses revealed that no intragene recombination events occurred between the hexon L1 and L2 regions or between the fibre knob and fibre shaft regions. Sequencing and phylogenetic analysis of the penton showed that some of the intermediate strains had sequences from a third type of adenovirus in these regions. The penton analysis showed also that intragene recombination between penton HVR and RGD loop regions was common. New types of adenovirus were detected and sequential infection with different adenovirus variants was observed in some patients which indicates that the genome of HAdV-D from AIDS patients are not stable. Full genome sequencing and analysis was carried out for three isolates, two of them appeared to be new types of HAdV-D and the result of multi-recombination events and the third isolate appeared to be a variant of HAdV-D23.The stability of species B adenovirus (HAdV-B) was also analysed. A total of 96 isolates collected from the Manchester area typed previously by serum neutralization (SN) were analysed in five genome regions. Most of these isolates were HAdV-B3 and HAdV-B7 collected during a 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 collected in different years following the outbreak in addition to other adenovirus types isolated from different years. The phylogenetic analysis results of all the isolates in the structural regions: hexon L2, penton and fibre knob were found to be consistent and no mismatches (hexon from one type and fiber from another type) were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable in general. The last part of this study is investigating adenovirus persistence and latency in human tissues. Tonsils and adenoids (106 right and left tonsils and 10 adenoids) were obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty four (72.41%) tonsils and adenoids samples were positive for HAdV by real-time PCR. The viral load was not the same in the right and left tonsils in most of the cases and ranged from 280 to more than 2.6 x 106 copies/107 cells. Seventy eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C types were detected in 82% of the samples followed by species B (7.7%), HAdV-E4 (7.7%) and HAdV-F41 (2.56%). No DNA methylation was detected in the major late promoter (MLP) and E1A promoter regions of six tonsils and adenoids samples and two clinical isolates.

No description available.

At late times during the infection of human KB cells with Adenovirus type 2 (Ad 2), the ultraviolet (uv) sensitivity of transcription was assayed by DNA-RNA hybridization across the length of the genome. Assuming that the majority of the nuclear transcripts at this time of infection are read from the rightward transcribing DNA strand, the resulting plot of the surviving fraction of viral transcription vs. genome position indicates two transcription units are responsible for the transcription from selected DNA restriction fragments of the genome were also obtained. The uv inactivation cross sections generated from such curves identified a long, uv sensitive transcript originating from the major late promoter at approximately 17 map units, and a shorter, less uv sensitive from approximately 63 map units on the genome. The shorter transcription unit accounts for about one third to a half of the viral nuclear RNA synthesized from the right hand 30 to 40% of the genome. The majority of the late viral nuclear transcripts, however, originated at approximately 17 map units and terminated at around 60-70 map units. Similar experiments examining the uv sensitivity of cytoplasmic poly A RNA production at various sites across the length of the genome are consistent with two rightward transcribing transcription units expressed during late Ad 2 infection. The transcriptional organization of late Ad 2 gene expression was also approached through uv transcription unit mapping experiments by examining the uv sensitivities of the synthesis of late Ad 2 proteins for which the approximate gene locations are known. The effect of uv on Ad 2 nuclear transcription was also reflected at the polypeptide level indicating two transcription units are responsible for the synthesis of mRNA coding for late viral proteins. The differential radiosensitivities of late protein synthesis confirmed the relative gene positions on the Ad 2 genome. / Thesis / Master of Science (MS)

The Ad5 E1B antigens of MW 58000 and 19000 are known to be involved in oncogenic transformation of mammalian cells. To obtain sufficient quantities of these proteins for biochemical studies on their mechanism of action, I have attempted to express the Ad5 E1B genes in Escherichia coli. Using the strategy developed by Guarente et al. (1980), I have constructed plasmids which have the trancriptional and translational controls of the E. coli lac operon linked 5' of the 19k and 58k coding sequences. One plasmid was shown to synthesize high levels of a stable, immunoreactive 19k analogue consisting of 19k with 29 adenovirus-coded ribosome-binding sequence is functional in directing translation of this protein. Synthesis of 58k was not demonstrated, perhaps the result of [protein instability in E. coli. However, immunoreactive proteins which may correspond to the amino terminal region of 58k were demonstrated. / Thesis / Master of Science (MS)