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.

Age-related macular degeneration (AMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). Current treatment strategies for AMD include laser photocoagulation, photodynamic therapy (PDT) and excision of neovascular membranes, but have met with limited success. In our previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin (VS) attenuated the corneal neovascularization in animals. The primary objective of this study was to investigate gene delivery of vasostatin (VS) attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. However, systematic expression of angiogenesis inhibitor may bring adverse effects to physiological processes. The feasibility, efficiency and safety of gene delivery with systemic and local routes were evaluated. Intramuscular polymer-based gene delivery had no side effect such as virus vector and revealed the safety. Recombinant adenovirus (Ad) was used gene delivery system because of its high titer, wide host range, and transduction efficiency. Adeno-associated virus (AAV) represents highly efficient that can facilirate long-term transduction. We propose to improve the efficacy and safety of VS gene delivery, and to search for the effective delivery route and other adjuvant therapy in conjunction with VS for treatment of CNV. Recently, PDT with veteporfin is an established treatment for subfoveal CNV secondary to AMD. We tried to compare the effect and safety of standard and reduced-dose light application PDT in an animal mocel of CNV. The 180-residue VS and its 48-residue (VS48) inhibited the migration and tube formation in cultured endothelial cells. Topical VS application suppresses the progression of laser-induced CNV via angiogenesis ihhibition, as well as in VS48. VS-48 inhibited the growthof vessels in arota rings. Electroretinograms (ERG) analysis revealed that topical VS-48 application for 21 days had no effect on rat retinal functions. Topical VS-48 treatment significantly reversed the CNV-induced alterations in ERG. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Rats treated with intrmuscular injection with PVP-VS also showed a significant reduction in the CNV lesions for at least 42 days. Subconjunctival injection with Ad vector revealed no retinal toxicity in ERG. Ad-luciferase via subconjunctival injections showed ocular expression for as long as 112 days by using bioluminescence image analysis in rodent. AAV-luciferase via subconjunctival injections showed ocular expression for as long as 365 days by using bioluminescence image analysis in mice, and AAV serotype 5-luciferase even showed expression lasting for 2 years. Suppression of laser photocoagulation¡Vinduced CNV by Ad-VS was documented in rat model. Combination therapies are important as treatment options. We demonstrated that PDT could effectively attenuate CNV in a rat model, and reduced doses, worked just as well as the standard dose. In the preliminary study of PDT combined topical VS application, treatment led to CNV attenuation more than alone with PDT. The above experiments would enable us to demonstrate that the vasostatin delivery might be a promising strategy for the treatment of AMD and other retinal or ocular disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

Nuclear factor-κB (NF-κB) is one of the main regulators of inflammatory and immune responses. It is a family of transcription factors composed of five members: RelA, RelB, cRel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Homo- and hetero-dimers of family members are inhibited by inhibitor of &klappaB (IκB) family members and activated by IκB kinase (IKK) family members. The IKK family is comprised of IKKα, IKKΒ, and IKKγ. The focus of my dissertation delves into the role of NF-κB activation by IKKΒ in both an immunotherapy setting and its role in T cell mediated anti-tumor immune responses. A central focus of immunotherapy is to develop vaccine adjuvants that are capable of enhancing a protective adaptive immune response. Microbial adjuvants in vaccines activate key transcription factors, including NF-κB and interferon response factors (IRF). The individual role of these transcription factors in successful vaccines is not clear. We used constitutively active IKKΒ (CA-IKKΒ) expressed in an adenoviral vector (AdIKK) to determine the role of classical NF-κB activation in a vaccine-induced immune response. In an in vivo model, AdIKK induced rapid and sustained NF-κB-driven inflammation in the lungs compared to the control virus. AdIKK infection had no impact on the magnitude of T cell activation as measured by IFN-γ production; however pulmonary inflammation resulted in increased cellularity of draining lymph nodes (LN) at early timepoints resulting in increased early antibody and T cell responses. Taken together, these findings show that IKKΒ-induced NF-κB activation of an inflammatory response affects the kinetics, but not the magnitude of the adaptive immune response. NF-κB is activated in many tumor types and contributes to the progression of cancer by suppressing apoptosis, and enhancing proliferation, angiogenesis and metastasis. NF-κB is also activated in other cells within the tumor microenvironment and promotes inflammation initiated by neutrophils and macrophages. In addition to inflammatory cells, T cells can be found within the tumor microenvironment and are associated with improved patient survival. Using CA-IKKΒ, we sought to determine if NF-κB activation in tumor cells could promote T cell mediated tumor immunity. In both primary tumors and a metastatic tumor model, we found that NF-κB expression in tumors rendered immunogenic through expression of Kb-OVA led to tumor rejection or growth suppression. Tumor regression was mediated by increased CD8 T cell recruitment by chemokines. Microarray results showed increases in T cell chemokines, including CCL2 and CCL5. Knock-down of CCL2 by Lentiviral shRNA in LLC-OVA-IKK cells resulted in abrogation of tumor regression. These results suggest that NF-κB is capable of promoting immune surveillance in tumors through increased recruitment of T cells. Overall, my dissertation highlights beneficial roles of IKKΒ-induced activation of NF-κB in two separate systems: vaccine induced immune responses and tumor immune surveillance.

The adenovirus (Ad) family consists of 52 different human types, which are divided into seven species (A-G). Human Ads cause disease in the respiratory tract, lymphoid tissue, intestine, urinary tract, and/or in the eye. Most, but not all Ads have been demonstrated to use the coxsackie-adenovirus receptor (CAR) as an efficient receptor in vitro, but CAR has been questioned as an in vivo-receptor for various reasons. Thus, there are reasons to believe that Ads use other mechanisms for binding to target cells. In an attempt to investigate the impact of tear fluid during in vitro infection of ocular Ads (i.e. Ad37), using corneal cells, we found that human tear fluid promoted infection of an Ad with pronounced respiratory tropism (i.e. Ad5) used here as a control, but surprisingly not of Ad37. Furthermore using a virus overlay protein blotting assay we found that Ad5 bound to several tear fluid proteins. One of these, human lactoferrin (hLf) which is a component that belongs to the innate immune system in various body fluids, was alone able to promote both binding and infection of all species C Ads (Ad1, Ad2, Ad5, Ad6) in epithelial cells. hLf was also found to promote gene delivery (GFP) from an Ad5-based vector. Further we have identified lactoferricin (Lfcin), the N-terminal part of hLf, as to be responsible for this effect. We also show that plasma, saliva, and tear fluid promote infection of Ad5 in respiratory and ocular epithelial cells, and that plasma promotes infection of Ad31. The component in plasma that is responsible for this effect is likely to be coagulation factor IX (FIX) and X (FX), since both these factors were able to promote binding and infection of Ad5 and/or Ad31 in epithelial cells. Finally, we show that the excess of fiber production from initial Ad infection and the release of fibers before the particle itself is released caused masking of the tropism-specific receptors in both infected and non-infected surrounding cells. This means that the overproduction of fibers affects the ability of Ad to spread within tissues. We conclude that soluble components in body fluids, such as hLf, FIX, and FX have the ability to mediate binding and infection of selected human Ads (species C and Ad31) in epithelial cells that represent the tropism of these Ads. We suggest that these components may serve as bridges between the virion and the cell surface. This is contributes to the knowledge about Ad lifecycle, and might help to improve the de-/retargeting of gene therapy based on Ad vectors.

No description available.

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.

No description available.

Adenoviral CD40L gene therapy (AdCD40L) is a strong inducer of anti-tumor immune responses via its activation of dendritic cells (DCs). Activated DCs can in turn activate T cells, which are key players in an efficient anti-tumor response. This thesis includes three papers that focus on different aspects of AdCD40L gene therapy. In the first paper, the infiltration of suppressive CD11b+Gr-1+ cells in orthotopic MB49 bladder tumors was investigated and found to be significantly reduced while activated T cells were increased when the tumors had been treated with local AdCD40L gene therapy. Further, AdCD40L could tilt the cells in the tumor microenvironment in favor of an efficient anti-tumor immunity (M1 macrophages and activated T cells) instead of an immunosuppressive environment (CD11b+Gr-1int/low myeloid cells and M2 macrophages). Immunotherapy combined with chemotherapy has shown promising results, and the second paper investigates the combination of AdCD40L gene therapy together with the chemotherapeutic drug 5-Fluorouracil (5-FU). A synergistic effect of the combination treatment on orthotopic MB49 bladder tumors could be demonstrated. The combination therapy resulted in decreased tumor growth, increased survival and systemic MB49-specific immunity, whereas AdCD40L or 5-FU therapy alone had a poor effect on tumor growth. Efficient AdCD40L therapy is dependent on high transduction efficiency in both cancer cells and cells present in the tumor microenvironment. In an attempt to enhance the transduction efficiency, and thereby the therapeutic efficacy, a modified adenovirus was developed for paper three. This modified Ad5PTDf35(mCD40L) could, in comparison with the unmodified Ad5(mCD40L), demonstrate increased transduction capacity of a variety of murine cells. Further, the ability of antigen presenting cells (APCs) to present antigens to T cells was improved after transduction with Ad5PTDf35(mCD40L).

There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease. Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types. To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity. This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.

Adenovirus (Ad) is a widely studied DNA virus, but the nucleoprotein structure of the viral genome in the cell is poorly characterized. Our objective is to study Ad DNA-protein associations and how these affect the viral life cycle. Most of the viral DNA condensing protein, protein VII, is lost within a few hours of infection and this loss is independent of transcription. Cellular histones associate with the viral DNA after removal of protein VII, with a preferential deposition of H3.3. Micrococcal nuclease accessibility assays at 6 hpi showed laddering of the viral DNA, suggesting the genome is wrapped in physiologically spaced nucleosomes. Although viral DNA continues to associate with H3.3 at late times of infection, the overall level of association with histones is greatly reduced. Knockdown of the H3.3 chaperone HIRA had no effect on the viral life cycle suggesting that other H3.3 chaperones are involved. Our studies have begun to elucidate the nucleoprotein structure of Ad DNA in the infected cell nucleus.