The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.

During an adenovirus infection the accumulation of alternatively spliced mRNAs is subjected to a tight temporal regulation. The IIIa protein is a structural protein expressed exclusively late after infection. To study the significance of the restricted IIIa protein expression we used a Tet-ON regulated adenoviral vector to overexpress the IIIa protein during the early phase of infection. The results show that unregulated IIIa protein expression caused a reduction in late viral protein accumulation and a slight block of viral DNA replication. Further, the results indicate that IIIa splicing might be subjected to a regulation via a feed back loop stimulating its own expression. To improve the efficacy of vectors for regulated transgene expression, we constructed binary adenoviral vectors based on the Tet-ON and Tet-OFF systems. These vectors encode both the transcriptional activator and the tetracycline-regulated expression cassette from the same viral unit, ensuring that each infected cell will express both the activator and the reporter gene. In model experiments this system was shown to result in a tight control of gene expression with no detectable background expression of the transgene and induction levels reaching 500-600 fold. Introduction of dsRNA into a cell will induce a sequence specific degradation of the homologous mRNA via a mechanism named RNA interference (RNAi). The adenovirus VA RNAs are short highly structured RNAs that are expressed in large amounts late during an adenovirus infection. Here we showed that both VA RNAI and VA RNAII functions as virus-encoded suppressors of RNAi, by interfering with the activity of Dicer, the enzyme that cleaves the initial dsRNA to short-interfering RNAs (siRNAs) that mediate RNAi. Further, the VA RNAs themselves are substrates for Dicer and are cleaved into siRNAs in vivo that are incorporated into active RNA-induced silencing complexes. There is a great interest in developing novel therapeutic strategies based on RNAi. We constructed adenoviral vectors that express short hairpin RNAs, which in vivo will be cleaved to siRNAs that induce sequence-specific RNAi. We compared the efficiency of RNAi induced by vectors based on the viral VA RNAI and the human U6 promoters. Our results suggest that under conditions where the recombinant virus does not replicate, the VA RNA promoter is more effective in down regulating target gene expression, whereas the U6 promoter was more effective under replicative conditions.

Adenoviruses belong to the most common human pathogens. The severity of infection varies greatly, from subclinical to lethal, depending on the virus type and immune status of the infected host. The 51 known human adenovirus serotypes are divided into six species (A-F) based on characteristics such as tropism. Species B adenoviruses, which are the subjects of this thesis, are further divided into subspecies B:1 that contains Ad3, Ad7, Ad16, Ad21 and Ad50 and subspecies B:2 that contains Ad11, Ad14, Ad34 and Ad35. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes (Ad11, Ad34 and Ad35) are also associated with renal disease. The main aim of this thesis was to identify and characterize cellular receptors for species B adenoviruses. This will ultimately help to understand the diverse tropism shown by different adenoviruses and perhaps contribute to development of antivirals. Also, since adenoviruses are among the most commonly used vector for gene therapy it is of importance to characterize the initial steps of adenovirus life cycle. Members of species B adenoviruses have been shown to utilize both the complement regulating membrane cofactor protein (MCP), i.e. CD46, and a still unknown receptor. CD80 and CD86, usually found on antigen-presenting cells, have also been suggested as receptors We found first that Ad11 used CD46 as a cellular receptor on respiratory A549 cells, and subsequently that CD46 is a cellular receptor for all species B adenovirus serotypes, except for adenovirus types 3 and 7, using cells that represent the tropism of species B adenoviruses, i.e. respiratory, conjunctival and renal epithelial cells. We further compared the relative roles of CD46 with CD80 and CD86 using cells that represent species B adenovirus tropism. Using soluble candidate receptors and antibodies against corresponding receptors to challenge virus binding to and infection of cells, we found that on these cells, CD46 is a cellular receptor for all species B adenoviruses except Ad3 and Ad7, and that CD80 and CD86 do not play an important role. We have further pinpointed the interaction site for Ad11 on CD46 by X-ray crystallography. The extracellular region of CD46 contains four short consensus repeats (SCR1-4) of which the outermost N-terminal SCR1 and SCR2 mediate binding to Ad11. This interaction was confirmed by inhibiting infection and binding of Ad11 to A549 cells using soluble SCR1-2 fragments. Surprisingly the conformation of bound CD46 differs profoundly from its unbound state, with the bent surface structure straightened into an elongated rod. Viral proteins can sometimes undergo large conformational changes upon receptor binding, but this is, to the best of our knowledge, the first example of a virus protein dramatically changing the overall structure of its receptor. CD46 serves as a receptor for a large number of viral and bacterial pathogens and it is structurally and functionally related to other viral receptors such as CD21 and CD55. The mode of interaction presented here may serve as a conceptual framework for studies of many other receptors that are constructed from SCR domains.

At present, the human adenoviruses (Ads) comprise 51 members, which have been classified into six species (A to F). In general, adenovirus (Ad) tissue tropism or disease patterns vary according to species, although adenoviruses from different species can sometimes cause the same symptoms. The current interest in adenoviruses is partly due to the aim of using them as vectors for gene therapy. Hematopoietic cells are attractive targets for gene therapy and the transductions can be performed ex vivo. However, the most commonly used adenovirus vectors, based on Ad2 or Ad5, are inefficient in their transduction of hematopoietic cells since they attach poorly to these cells. Most Ads, including Ad2 and Ad5, appear to use the coxsackie-adenovirus receptor (CAR) (a component of tight junctions), for attachment to host cells. However, species B Ads do not bind to CAR and several studies have indicated that species B-based vectors would be more suitable for hematopoietic cells. Species B Ads can be further divided into species B1 and B2, which display different tissue tropisms. Species B1 Ads mostly cause acute respiratory infections whereas species B2 Ads have been associated with persistent infections of the kidney and urinary tract. One of the key determinants of tropism is believed to be the initial high-affinity attachment of the virion to host cell fiber receptors. By reciprocal blocking experiments and different ways of characterizing the species B attachment receptors, we have shown that the species B2 serotypes Ad11p and Ad35 and the species B1 serotypes Ad3p and Ad7p also differ in receptor usage. There are at least two different Ad species B receptors. Since one of these receptors appeared to be used by all four serotypes, we designated this receptor sBAR (species B adenovirus receptor). The other receptor appeared to be used exclusively by the two species B2 serotypes and was therefore designated sB2AR (species B2 adenovirus receptor). Binding to sBAR can be abolished by EDTA and restored with Mn2+ or Ca2+, whereas binding (of Ad11p and Ad35) to sB2AR is independent of divalent cations. Furthermore, sBAR appears to be trypsin sensitive whereas sB2AR is not. We also identified CD46 as a receptor for Ad11p. Even so, CD46 does not appear to be a functional receptor for Ad7p. Both Ad7p and Ad11p attached to CD46-transfected Chinese hamster ovary (CHO) cells more efficiently than to control CHO cells. However, only Ad11p (selectively) infected CD46-transfected CHO cells. Anti-CD46 antibodies inhibited Ad7p and Ad11p from binding to, and Ad11p from infecting, CD46-transfected CHO cells. However, in human cells, anti-CD46 antibodies had an inhibitory effect only on Ad11p binding (~30%) but did not affect Ad7p binding. In binding experiments with EDTA, divalent cations and pretrypsinized cells, Ad11p and Ad7p showed the same pattern in their binding to CHO-CD46 cells as in the previous study. Since Ad7p interacted almost as efficiently with control CHO cells as with CHO-CD46 cells after addition of Mn2+, it seems that Ad7p mainly addressed an endogenously expressed hamster receptor on CHO-CD46, the properties of which resemble sBAR. In addition, Ad3p and Ad7p attach poorly to PBMCs and CD46 is expressed on all nucleated cells. Thus, CD46 appears to correspond to sB2AR rather than to sBAR. With these differences in receptor usage in mind, we studied the binding and infectious capacity of these species B Ads in various hematopoietic cells. We found that all species B serotypes bound efficiently to CD34+ hematopoietic stem cells (HSCs) and also productively infected HSCs. However, only the sB2AR binding Ad serotypes Ad11p and Ad35 could attach primary PBMCs efficiently. Our results regarding the subsequent steps in infection of PBMCs suggest that both Ad11p and Ad35 enter PBMCs and deliver viral DNA to the nuclei of most PBMC cell types. However, productive infections were only clearly detected in stimulated T-cells (most frequently) and monocytes, whereas Ad infection seemed eclipsed in unstimulated lymphocytes. Replication of Ad DNA seemed seriously impaired in at least T-cells, indicating limited production of infectious particles in PBMCs. The capacity of species C Ads to establish persistent infections in lymphatic tissues has been described previously. These Ads also persistently infect various transformed hematopoietic cell lines in vitro. Our studies indicate that replication of the species B2 Ads is also restricted in cells of hematopoietic origin (both in primary and transformed cells). Taken together, the results indicate that species B2 Ads (as compared to other Ads) seem to enter and infect most hematopoietic cells efficiently, which is in line with the persistent nature of these Ads. They would presumably act as suitable vectors for efficient transduction of most cells of hematopoietic origin, as has already been shown for e.g. HSCs and dendritic cells. The finding that replication of Ads in T-cells appears to depend on the level of T-cell activation, strengthens the hypothesis that T-cells may serve as a reservoir for human Ads and raises possible safety issues for usage of species B-based vectors in hematopoietic cells.

Fibrosis is a sequel of chronic inflammation and is defined as an excessive deposition of collagen that ultimately leads to organ dysfunction. To date there are no effective treatments for fibrosis. The main cell type involved in collagen deposition and organization is the myofibroblast. In the first study we examined how myofibroblasts differentiate in human fibrotic conditions and in experimental animal models. Human tissues were stained with antibodies that recognize integrin receptors and in addition we also stained for α-SMA, a myofibroblast marker. We found a co-localization between these two markers in stromal cells and hypothesized that integrin α1 is important for the acquisition of the myofibroblast phenotype. To tests this hypothesis we used knockout animals for this integrin subunit. These animals showed a reduction of α-SMA positive fibroblasts, indicating that the α1 integrin subunit is required for proper myofibroblast differentiation. In the second study we used a neuroblastoma tumor model to study tumour growth when a drug targeting the synthesis of cellular NAD was administered. In treated animals an expansion of the nonvascular stroma was observed compared to controls. Normalization of the vasculature was observed in treated tumors together with a decrease in hypoxia. Moreover, this was followed by a decrease in stromal PDGF-B and VEGF expression, suggesting a deactivation of the stroma. In the third study the effects of over-expression of the two pro-fibrotic growth factors TGF-β and PDGF-B in skin was evaluated. We observed that both growth factors induced fibrosis. Over time, a decrease in blood vessel density was observed in both treatment groups. Both factors also stimulated an expansion of the connective tissue cell population originating from the microvascular pericyte, but the phenotype of these cells differed in the different treatments with regards to expression of markers. Furthermore, in tissue over-expressing PDGF-B but not TGF-β, the fibrotic process was partially reversible.

The work presented herein focuses on the activation of the adaptive immune system in order to develop T cell-based immunotherapy for viral infections and cancer. The main goal was to identify and activate viral or tumoral antigen-specific T cells by using different identification, isolation and stimulation techniques. One such approach was that we modified dendritic cells (DCs) with an adenoviral vector encoding the full length pp65 antigen from cytomegalovirus (CMV). Through strategic modification techniques we demonstrate that it is possible to obtain DCs presenting antigen-specific peptides both on major histocompatibility complex (MHC) class I and MHC class II molecules for simultaneous CD8+ and CD4+ T cell activation. We also demonstrate that it is possible to generate prostate antigen-specific CD8+ T cells from a naïve repertoire of T cells by using DCs and HLA-A2-restricted peptides derived from prostate tumor-associated antigens or by using an adenoviral vector encoding the full length prostate tumor-associated antigen STEAP. We further demonstrate that CD8+ T cells directed against several prostate-specific peptide epitopes can be found in peripheral blood and in the prostate tumor area of prostate cancer patients. Furthermore, we have characterized a number of prostate-derived cell lines in terms of HLA haplotype and tumor-association antigen expression. We concluded that our methods for generating T cells restricted to CMV antigen have the ability to be applied for adoptive T cell transfer to patients with CMV disease and that prostate antigen-specific T cells can be found within prostate cancer patients, which enables future development of immunotherapeutic strategies for prostate cancer.

Improvements in the use of traditional breast cancer therapies have improved the overall survival of women with early stage disease. Remarkable advances in research have created a unique opportunity for developing active vaccination strategies that engage the bodys own immune system in the fight against breast cancer. Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. HER-2/neu-targeted DNA-based and fiber-modified dendritic cell (DC)-based vaccines are both analyzed as potent elements in eliciting HER-2/neu specific antitumor immune responses. A HER-2/neu-expressing DNA plasmid (pcDNA/neu) coadministered with the appropriate adjuvant vector was the first study looking at improving vaccine efficacy and enhancing immune responses. Various protection and prevention studies, using FVB/N (wild-type) and FVB/neuN [transgenic (Tg)] mice and Tg1-1 tumor cells, derived from a spontaneous tumor from Tg mice, are used to help narrow down the large panel of adjuvant vectors. Results showed the adjuvant vector pcDNA/TNF-α, when coadministered with pcDNA/neu, induced more efficient protective tumor-specific immunity and significantly delayed breast cancer development in Tg mice.<p>Another study utilized an<i>in vivo</i> murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient fiber-modified adenovirus (AdV) containing neu (AdV(RGD)neu), to form DC(RGD)neu, and non-modified DCneu. DC(RGD)neu displayed an upregulation of immunologically important molecules and inflammatory cytokine expression through FACS Analysis, and more importantly increased expression of neu, when compared to DCneu. DC(RGD)neu stimulated a higher percentage of HER-2/neu-specific CD8+ T cells, a stronger neu-specific CTL response, and induced a much stronger Th1- and Th2-type immune response than DCneu. Furthermore, vaccination with DC(RGD)neu induced enhanced protective tumor-specific immunity compared to DCneu in wild-type and Tg mice.<p>Overall the construction of recombinant vectors containing two transgenes (HER-2/neu and TNF-α), can not overcome the induction of HER-2/neu-directed immune tolerance. The fiber-modified (RGD) DCneu vaccine induced enhanced anti-HER-2/neu immunity compared to non-modified DCneu in the prevention of breast cancers.

The helper-dependent adenoviral (HD-Ad) vector is the latest generation of Ad vectors. It ameliorates the vector-associated immunogenic problems with increased capacity for carrying DNA because all viral coding genes are removed. I hypothesize that HD-Ad vectors can be effective vehicles for retinal gene delivery. The objectives of this study are to determine if HD-Ad vectors can deliver reporter genes, GFP or lacZ, driven by a CMV or a MOPS promoter, into specific retinal layers. Subretinal injections were performed and eyes removed at time points from 1 week to 3 months, processed for fluorescent microscopy, X-gal staining, and H&E staining. Transgene expression was detected for at least 3 months. A dose dependent relationship was revealed between the level of transgene expression and viral vector dose. Distinctively, the MOPS promoter drove photoreceptor cell specific expression. Notably, no sign of inflammation or tissue toxicity was detected, demonstrating the benefits of the HD-Ad vector.

The helper-dependent adenoviral (HD-Ad) vector is the latest generation of Ad vectors. It ameliorates the vector-associated immunogenic problems with increased capacity for carrying DNA because all viral coding genes are removed. I hypothesize that HD-Ad vectors can be effective vehicles for retinal gene delivery. The objectives of this study are to determine if HD-Ad vectors can deliver reporter genes, GFP or lacZ, driven by a CMV or a MOPS promoter, into specific retinal layers. Subretinal injections were performed and eyes removed at time points from 1 week to 3 months, processed for fluorescent microscopy, X-gal staining, and H&E staining. Transgene expression was detected for at least 3 months. A dose dependent relationship was revealed between the level of transgene expression and viral vector dose. Distinctively, the MOPS promoter drove photoreceptor cell specific expression. Notably, no sign of inflammation or tissue toxicity was detected, demonstrating the benefits of the HD-Ad vector.

Els adenovirus oncolítics són una estratègia molt prometedora pel tractament del càncer, però l'augment de la seva potència i dispersió intratumoral és imprescindible per tal que aquests vectors presentin una eficàcia òptima pel seu ús en humans. Una estratègia molt prometedora per tal d'augmentar l'eficàcia dels adenovirus oncolítics és la inserció de transgens terapèutics en el genoma adenoviral. En aquest treball es presenta la generació i evaluació d'adenovirus oncolítcs armats amb dos transgens diferents: les glicoproteïnes fusogèniques de membrana (FMG) per una banda, i la hialuronidasa PH20 per l'altre. Les FMG són altament citotoxiques per les cèl·lules tumorals degut a la seva capacitat de fusionar les cèl·lules en grans sincitis multinucleats. En aquest treball hem generat dos adenovirus fusogenics salvatges: l'AdwtRGD-F, que expressa la proteïna F del paramixovirus SV5 i l'AdwtRGD-GALV que expressa la proteïna de l'envolta del virus de la leucèmia de gibbó (GALV). L'expressió de la proteïna F pel virus AdwtRGD-F va resultar en la formació de sincitis petits, però la fusió induïda per la proteïna F no va ser capaç d'augmentar la potència de l'adenovirus AdwtRGD-F. En canvi, l'expressió de la proteïna GALV pel virus AdwtRGD-GALV va resultar en la formació de grans sincitis i en la millora de la potència citotòxica de l'adenovirus in vitro. A continuació, el gen de la glicoproteïna GALV va ser inclós en el genoma d'un adenovirus de replicació selectiva de tumor: l'ICOVIR5. Per tal d'insertar el gen de la glicoproteïna GALV en el genoma de l'ICOVIR5 sense afectar la capacitat d'encapcidació de l'adenovirus vam eliminar una part del genoma d'aquest virus (els ORFS 1,2 i 3 de la regió E4) per tal de generar l'ICOVIR9dE4. L'expressió de la glicoproteïna GALV per part de l'ICOVIR9dE4 va augmentar significativament la capacitat citotòxica del virus respecte l'ICOVIR5 in vitro. Tot i això, les diferents modificacions realitzades en l'ICOVIR9dE4 van resultar en una disminució de la producció viral i en una potència antitumoral in vivo molt similar a la presentada per l'ICOVIR5. A continuació, vam insertar el gen de la glicoproteïna GALV en el genoma de l'ICOVIR15 (un adenovirus oncolític molt similar a l'ICOVIR5 però amb un genoma més curt) per tal de generar l'ICOVIR16. La inserció del gen GALV en l'ICOVIR16 no va requerir l'eliminació de cap gen viral. In vitro, l'adenovirus fusogènic ICOVIR16 va presentar una elevada capacitat citotòxica i una producció viral similar a la de l'ICOVIR15. In vivo, l'administració d'ICOVIR16 (tan per via intratumoral com sistèmica) va controlar el creixement tumoral més eficientment que l'adenovirus control en tots els models tumorals analitzats. Per una altra banda, per tal que l'adenovirus sigui capaç d'eliminar la totalitat de la massa tumoral es necessari que el virus sigui capaç de distribuir-se eficientment pel tumor. En aquest treball hem analitzat si l'eliminació de la matriu extracel·lular mitjançant l'enzim hialuronidasa facilita la dispersió d'adenovirus oncolítics. Primer vam demostrar que la coadministració intratumoral de hialuronidasa soluble i un adenovirus oncolític en tumors xenografts de ratolí millora l'activitat antitumoral de l'adenovirus oncolític en monoteràpia. A continuació, vam construir l'adenovirus AdwtRGD-PH20, un adenovirus salvatge que expressa una forma soluble de la hialuronidasa testicular PH20. L'administració de l'AdwtRGD-PH20 a ratolins amb tumors de melanoma humà va resultar en la degradació de l'àcid hialurònic present en el tumor, una millor distribució del virus per la massa tumoral i la regressió de tots els tumors tractats. Finalment, vam insertar el cDNA de la hialuronidasa PH20 en el genoma de l'adenovirus oncolític ICOVIR15 per generar l'adenovirus ICOVIR17. L'ICOVIR17 va demostrar una millor eficàcia i distribució antitumoral que l'ICOVIR15 després de ser administrat via intratumoral o sistèmica en ratolins amb tumors humans preestablerts. Els resultats obtinguts en aquesta tesi indiquen que els adenovirus ICOVIR16 i ICOVIR17 són bons candidats per ser analitzats en assajos clínics de fase I. / Oncolytic adenoviruses hold considerable promise for treating solid tumors, although the potency and spread of the virus needs improvement if its full clinical potential is to be realized. On the one hand, we tested here whether the insertion of the gene encoding the gibbon ape leukemia virus envelope (GALV) glycoprotein into the genome of a replication competent adenovirus is able to increase adenovirus potency. The GALV glycoprotein efficiently kills tumor cells by inducing fusion of neighboring cells to form multinucleated syncytia. The in vitro characterization of the fusogenic adenovirus (AdwtRGD-GALV) showed that this virus can induce massive syncytia formation, leading to a significantly increased tumor cytotoxicity compared with a nonfusogenic virus. Next, the GALV gene was inserted into an oncolytic adenovirus which selectively kills pRb pathway-defective tumor cells. The antitumoral activity of the oncolytic adenovirus expressing GALV (ICOVIR-16) was compared to that of the parental virus ICOVIR-15 in nude mice with pre-established human tumors. GALV expression greatly improved the antitumor efficacy of ICOVIR16. When injected directly to the tumor, ICOVIR16 induced tumor regression in 70% of treated tumors, whereas only 30% of tumors treated with ICOVIR15 regressed. When injected systemically, ICOVIR16 showed a greater reduction in tumor progression that was significant compared with the parental virus, ICOVIR15. On the other hand, we tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix, could enhance the intratumoral distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in nude mice bearing tumor xenografts improves the antitumor activity of an oncolytic adenovirus. Next, we constructed a replication competent adenovirus (AdwtRGD-PH20) and an oncolytic adenovirus (ICOVIR17) expressing a soluble form of the human sperm hyaluronidase (PH20). The antitumoral activity of both adenoviruses expressing PH20 was compared to that of the parental viruses AdwtRGD and ICOVIR-15. Both AdwtRGD-PH20 and ICOVIR-17 showed more antitumor efficacy following intratumoral administration in mice with pre-established tumors, along with an improved spread of the virus within the tumor mass. Importantly, a single intravenous dose of ICOVIR-17 induced tumor regression in 60% of treated tumors. Our results indicate that ICOVIR-16 and ICOVIR-17 are promising candidates for clinical testing.