Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

Die Gentherapie hämatopoetischer Stammzellen (HSCs) besitzt das Potenzial, verschiedene erbliche, nur symptomatisch behandelbare, Erkrankungen dauerhaft zu heilen. Die Mehrheit der aktuell angewandten Verfahren dazu, basiert auf der Isolation von hämatopoetischen Stammzellen, der ex vivo Modifikation dieser Zellen durch retrovirale Vektoren und der Reinfusion der modifizierten Zellen in den immunsupprimierten Patienten. Dieser Ansatz ist mit einer Reihe von Nachteilen verbunden, unter anderem einem teilweisen Verlust des Rekonstitutionsvermögens der Stammzellen nach ex vivo Kultur oder der Gefahr der Transformation durch Integration des retroviralen Vektorgenoms. Darüber hinaus sind aktuelle Gentherapieansätze mit hohen Kosten und großem logistischem Aufwand verbunden, was den Zugang zu diesen Behandlungen für potentielle Patienten stark einschränkt. Die vorliegende Arbeit verfolgt einen neuen Ansatz zur Gentherapie von HSCs, der auf der Mobilisierung von Stammzellen aus dem Knochenmark in den peripheren Blutstrom und der Transduktion dieser Stammzellen mit adenoviralen Vektoren basiert. Hierbei codieren die Vektoren sowohl ein Transgen als auch eine Integrationsmaschinerie. Der erste Teil der Arbeit belegt in einem humanen CD46-transgenen Mausmodell, dass adenovirale Vektoren der ersten Generation in der Lage sind, mobilisierte HSCs im Blut zu transduzieren und dass es den so transduzierten Stammzellen möglich ist, zurück ins Knochenmark zu migrieren und dort das Transgen zu exprimieren. Allerdings wurde im Verlauf von zwei Wochen ein Rückgang der Transgenexpression beobachtet. Um dies zu umgehen, wurde ein adenovirales Vektorsystem der dritten Generation genutzt, das eine hochaktive Sleeping Beauty Transposase, zum Zweck der Transgenintegration, codiert. Dieses System ermöglichte die stabile Genmodifikation mobilisierter hämatopoetischer Stammzellen nach intravenöser Injektion. Die Expression des Transgens konnte über längere Zeitspannen (bis 12 Wochen) beobachtet werden. Die modifizeirten Stammzellen waren darüber hinaus in der Lage, genmodifizierte Kolonien in vitro zu bilden und das hämatopoetische System letal bestrahlter Mäuse nach Knochenmarkstransplantation zu rekonstituieren. Es wurde somit gezeigt, dass HSCs nach in vivo Modifikation weiterhin funktional waren. / The gene therapy of hematopoietic stem cells holds the potential for curative treatment of several otherwise incurable inherited diseases. The majority of current gene therapy treatments relies on the collection of hematopoietic stem cells, their ex vivo modification with retroviral vectors and their transplantation into a myeloconditioned patient. This approach entails several disadvantages, including a reduction of stem cell engraftment potential after ex vivo culture and the potential danger of integrational mutagenesis. In addition, the high costs and complex logistics of this approach limit the access of patients to gene therapeutic regimens. This work explores an alternative approach to hematopoietic stem cell (HSC) gene therapy, termed stem cell in vivo transduction. This approach is based on the mobilization of HSCs from the bone marrow into the peripheral blood and the transduction of the stem cells with adenoviral vectors delivering a transgene as well as a transgene integration machinery. In the first part of this work, it was shown that first-generation adenoviral vectors could be used for the transduction of mobilized HSCs in the periphery of human CD46-transgenic mice. Further, the transduced HSCs were able to home back to the bone marrow and express the transgene. However, over the course of 14 days, a loss of transgene expression in HSCs was observed. To ameliorate these shortcomings, helper-dependent adenoviral vectors encoding a hyperactive Sleeping Beauty transposase for transgene integration were used for stable gene modification of hematopoietic stem cells following intravenous vector administration in mobilized human CD46-transgenic mice. Using this improved vector platform, gene marking of bone marrow HSCs could be observed for extended periods of time (up to 12 weeks). Further, the functionality of the modified HSCs was demonstrated both in colony-forming progenitor assays as well as through the transplantation of gene-modified HSCs into lethally irradiated recipients. Transplantation of modified HSCsled to long-term multi-lineage reconstitution showing that gene-modified stem cells were fully functional. Subsequently the safety of systemic vector administration in mobilized hosts as well as of the Sleeping Beauty-mediated transgene integration was assessed in human CD46- transgenic mice. Lastly, the stem cell in vivo transduction approach was employed in NOG mice transplanted with human CD34+ cells, as well as in Macaca nemestrina non-human primates.

While adenovirus (Ad) vectors have been shown to elicit potent antigen-specific T cell responses, the kinetics and nature of antigen-specific mucosa! and systemic T-cell responses has rarely been examined, especially following mucosal administration of Ad-based vectors. In the present studies, the phenotypic and functional characterization of antigen-specific CD8+ T cell responses following intrarectal (i.r.) vaccination with an Ad vector expressing Gallus gallus ovalbumin (OVA) was conducted. The frequencies of OVA-specific CD8+ T cells was maximal at 2 weeks post-vaccination in all tissues examined and then declined, demonstrating normal expansion and contraction kinetics. CD8+ T cells induced in the course of immunization exhibited phenotypic characteristics of effector memory T cells including up-regulation of the cell surface molecules CD43, CD44 and a low level of expression of CD127 at both local and systemic sites. While the discordance between the number of tetramer-reactive and cytokine-producing OVA-specific CD8+ T cells was observed, CD8+ T cells appeared to be fully functional in vivo. Upon secondary antigen exposure, the CD8+ T cell population expanded dramatically, particularly at the mucosa! surfaces. In addition, the CD8+ T cell response generated in the course of i.r. priming protected mice from intravaginal (i. vag.) vaccinia virus one month after immunization, thus underscoring the importance of inducing a tissue-resident effector memory T cell subset for protection against pathogens at mucosal surfaces. In developing future vaccines for mucosal diseases, the induction of a tissue-resident effector memory T cell subset should be one of the immunization objectives. Lentiviral vectors represent an attractive mode of genetic vaccination. Most commonly used, vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentiviral vectors do not efficiently infect epithelial cells from the apical side, and, therefore, are not suitable as mucosa! vaccines. In the present studies, Ebola Zaïre strain glycoprotein (EboZ)-pseudotyped lentiviral vectors, which have been previously used to deliver transgene to the lung epithelium, were delivered i.r. and evaluated as a mucosal booster vaccine. Rectal delivery of EboZ-pseudotyped lentiviral vectors expressing β-galactosidase (β-gal) had resulted in low, but detectable levels of β-gal expression 2 weeks after administration. When delivered on its own, EboZ-pseudotyped lentivirus did not prime detectable antigen-specific immune response. However, when delivered i.r. 30 days after i.r. Adβ-gal immunization, a significant enlargement (boost) of β-gal-specific CD8+ T cell responses, especially in the colonic lamina propria (LP), was observed as compared to the delivery of EboZ-pseudotyped vector encoding different transgenes or VSVG-pseudotyped lentivirus expressing β-gal. When these animals were i. vag. challenged with vaccinia virus expressing β-gal, a dramatic expansion of β-gal-specific CD8+ T cells, especially in the vaginal tract, was observed. In addition, this prime and boost strategy protected the mice from i. vag. vaccinia virus challenge. Therefore, i.r. Ad-based priming followed by i.r. EboZ-pseudotyped lentiviral boosting was an effective strategy for eliciting protective mucosal CD8+ T cell responses. / Thesis / Doctor of Philosophy (PhD)