Radiolabeled somatostatin analogues based on octreotide have proven useful in the management of somatostatin receptor subtype 2 (sstr2)-positive tumours in clinical trials. The aim was to compare the potency and evaluate the combination of 111In- and 177Lu-DOTATOC with a double-deleted version of vaccinia virus (ddVV), an oncolytic virus for inhibiting the growth of sstr2-expressing human embryonic kidney (HEK-293) cells or MC-38 murine colon cancer cells grown as monolayers or as spheroids. Cytotoxicity studies were carried out using ddVV, 111In-DOTATOC and 177Lu-DOTATOC, individually or in combination on MC-38 spheroids, HEK-293 cells and spheroids. HEK-293 cell growth in spheroids was reduced to 17.2 ± 4.9% and 26.5 ± 6.3 % with 111In-DOTATOC and 177Lu-DOTATOC alone and 13.1 ± 7.1% and 0% in combination, respectively. MC-38 spheroids showed greater toxicity in combination treatment. Combination of ddVV with 111In- or 177Lu-DOTATOC is only advantageous in monolayer culture. No advantage was observed in spheroid models.

Univ., Diss., 2010--Würzburg.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 59 pages. Includes Vita. Includes bibliographical references.

abstract: Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology. / Dissertation/Thesis / M.S. Biochemistry 2012

L’apoptose, mort cellulaire observée suite à l’activation des caspases effectrices, est un moyen de défense contre les pathogènes, en particulier les virus. Le virus de la vaccine (VACV) est un virus contenant un grand génome à ADN codant pour environ 200 protéines, dont plusieurs inhibent l’apoptose. Cette apparente redondance fonctionnelle complique l’étude des protéines antiapoptotiques du virus dans un contexte d’infection virale. Dans ce travail, nous comparerons les propriétés antiapoptotiques des protéines B13, F1, GAAP et N1 de VACV. Cette comparaison sera établie dans un premier temps en dehors de toute infection virale. En utilisant des vecteurs lentiviraux, nous avons obtenu des lignées cellulaires stables (U2-OS) exprimant ces protéines en isolation. Nous avons alors pu tester les capacités antiapoptotiques de ces protéines en réponse à des stimuli provoquant l’apoptose extrinsèque et intrinsèque. Les résultats ont montré que B13 était la plus puissante molécule inhibitrice de l’apoptose intrinsèque et qu’elle était la seule à inhiber l’apoptose extrinsèque. Ensuite nous avons tiré avantage d’un virus de la vaccine déficient (vv811) qui ne possède aucune de ces protéines antiapoptotiques, capable à lui seul d’induire l’apoptose, en l’absence de toute autre stimulus. En infectant nos lignées cellulaires exprimant les molécules in trans avec vv811, nous avons pu montrer que B13 inhibait cette apoptose induite par le virus beaucoup plus efficacement que F1. GAAP et N1 dans ce contexte n’ont pas démontré de propriétés antiapoptotiques. Enfin, nous avons construit par mutagénèse des virus vv811 recombinants exprimant les molécules étudiées in cis. Suite à l’infection par ces virus de cellules U2-OS et Hela, B13, de nouveau, et F1 ont montré des capacités d’inhibition importantes de l’apoptose. L’action de GAAP s’est révélée dépendante du type cellulaire et N1 n’a pas pu inhiber l’apoptose induite par ce virus déficient dans aucune des cellules testées. En utilisant ces différentes approches, nous avons pu nous affranchir des problèmes de redondance et comparer 4 molécules antiapoptotiques du virus de la vaccine, y compris dans un contexte d’infection virale. Les résultats ont confirmé que toutes les protéines étudiées possédaient des propriétés antiapoptiques et ont clairement montré que B13 était la plus puissante / Apoptosis, which occurs following activation of effector caspases, can restrict the replication of intracellular pathogens, especially viruses. Vaccinia virus (VACV) is a large dsDNA virus encoding approximately 200 proteins, several of which inhibit apoptosis. This redundancy of viral anti-apoptotic proteins complicates the study of these proteins in the context of viral infection. Here a comparative study of the anti-apoptotic proteins B13, F1, GAAP and N1 with and without virus infection is presented. Firstly, using lentiviral constructs, we generated transduced cell lines expressing the anti-apoptotic proteins in isolation and we analysed their ability to protect against extrinsic and intrinsic apoptosis induced by different drugs. In that context B13 was the most potent inhibitor of intrinsic apoptosis and the only protein to inhibit both extrinsic and intrinsic apoptosis. We then used a deficient VACV strain, vv811, that lacks the genes coding for the four anti-apoptotic proteins. Infection with vv811 can induce apoptosis without the need for any other stimulus. After vv811 infection of cell lines expressing the anti-apoptotic proteins in trans, B13 and to a lesser extent F1, inhibited apoptosis. Finally, we introduced each gene separately into vv811 by genetic recombination. Using these recombinant viruses to induce apoptosis, B13 and F1 were very potent inhibitors. The protection conferred by GAAP was cell type dependant and N1 failed to protect any of the tested cells from the virus induced apoptosis. Using these different approaches, we have been able to overcome the redundancy issue to compare 4 anti-apoptotic proteins from VACV, including in the context of viral infection. The results illustrate that vv811 is a useful tool to determine the role of VACV anti-apoptotic proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 is most potent.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) has been used as a vaccine, yet safety concerns remain due to its viral immunoevasive properties. Among these, VV infection of antigen presentation cells (APC) perturbs MHC class II-mediated antigen (Ag) presentation. The goals of this project include: 1) to define mechanisms by which VV disrupts class II presentation; and 2) to examine whether disruption of the class II pathway by VV alters T cell responses in vitro and in vivo. A significant reduction in the expression of the class II chaperone, invariant chain (Ii), was observed during the late stage of VV infection. Yet surface expression of MHC class II molecules was maintained along with cell viability. To examine whether VV acts solely to disrupt host protein synthesis, B cells were treated with an inhibitor of translation-cycloheximide (CHX). Like VV, CHX negatively regulated Ii protein expression and class II presentation. Ii proteolysis also contributed in part to reduce Ii expression in VV infected and CHX treated APC. Yet only VV infection altered lysosomal protease expression, potentially influencing Ii degradation. Over-expression or ectopic-expression of Ii partially protected cells from VV-induced class II dysfunction. These studies suggest VV destabilizes class II molecules by disrupting Ii expression. To examine the presentation of viral Ags by class II, CD4 T cells from VV-primed mice were used. Viral proteins were presented by class II shortly after APC exposure to low concentrations of VV. The presentation of VV Ags correlated temporally with reductions in exogenous peptide presentation. At higher MOI (≥ 1), class II presentation of VV Ags was reduced. To examine the in vivo effects of VV on Ag presentation, a mouse model of ovalbumin-induced airway hypersensitivity was used. Th2 cytokine production was reduced, while a novel inflammatory cytokine Interleukin-17 (IL-17) production was enhanced in asthmatic VV-infected mice. In health mice, repeated VV infections lead to enhanced CD8 T cell production of Interferon-γ (IFN-γ) and IL-17. Finally, antibodies to a viral protein H3 were generated and shown to preserve class II presentation. Together these studies suggest VV disruption of the class II pathway may blunt T cell responses to VV.

No description available.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...

Aim of this thesis was to study the contribution of the hosts immune system during tumor regression. A wild-type rejection model was studied in which tumor regression is mediated through an adaptive, T cell host response (Research article 1). Additionally, the relationship between VACV infection and cancer rejection was assessed by applying organism-specific microarray platforms to infected and non-infected xenografts. It could be shown that tumor rejection in this nude mouse model was orchestrated solely by the hosts innate immune system without help of the adaptive immunity. In a third study the inflammatory baseline status of 75 human cancer cell lines was tested in vitro which was correlated with the susceptibility to VACV and Adenovirus 5 (Ad5) replication of the respective cell line (Manuscript for Research article 3). Although xenografts by themselves lack the ability to signal danger and do not provide sufficient proinflammatory signals to induce acute inflammation, the presence of viral replication in the oncolytic xenograft model provides the "tissue-specific trigger" that activates the immune response and in concordance with the hypothesis, the ICR is activated when chronic inflammation is switched into an acute one. Thus, in conditions in which a switch from a chronic to an acute inflammatory process can be induced by other factors like the immune-stimulation induced by the presence of a virus in the target tissue, adaptive immune responses may not be necessary and immune-mediated rejection can occur without the assistance of T or B cells. However, in the regression study using neu expressing MMC in absence of a stimulus such as a virus and infected cancer cells thereafter, adaptive immunity is needed to provoke the switch into an acute inflammation and initiate tissue rejection. Taken together, this work is supportive of the hypothesis that the mechanisms prompting TSD differ among immune pathologies but the effect phase converges and central molecules can be detected over and over every time TSD occurs. It could be shown that in presence of a trigger such as infection with VACV and functional danger signaling pathways of the infected tumor cells, innate immunity is sufficient to orchestrate rejection of manifested tumors. / Ziel dieser Arbeit war, die Beteiligung des Wirts-eigenen Immunsystems bei der Tumoregression zu analysieren. Mittels eines Wildtyp-Regressionsmodells, wurde der Anteil des adaptiven Immunsystems studiert (Research-Artikel 1). Mit Hilfe von Organismus-spezifischen Mikroarrays und Genexpressionsanalysen konnte in einem Nacktmausmodell gezeigt werden, dass erfolgreiche, durch onkolytische VACV-vermittelte Tumortherapie auch ohne Beteiligung des adaptiven Immunsystems möglich ist (Research Artikel 2). In einer dritten Studie wurden 75 humane Tumorzelllinien auf ihren intrinsischen Entzündungsstatus hin getestet und bezüglich eines Zusammenhanges von diesem mit der Replikationsfähigkeit von VACV und Adenovirus 5 (Ad5) analysiert (Manuskript für den Research-Artikel 3). Obwohl Xenografts allein kein ausreichendes „Gefahrsignal“ geben und durch das Fehlen einer pro-inflammatorischen Stimulierung keine akute Entzündung verursachen können, ist die Infektion mit onkolytischem VACV ausreichend, um den Gewebe-spezifischen „Trigger“ darzustellen. In diesem Fall wird die Immunantwort aktiviert und nach der Hypothese des „Immunologic Constant of Rejection“ (ICR) geschieht dies, wenn eine chronische in eine akute Inflammation verändert wird. In dem beschriebenen onkolytischen Regressionsmodell ist die Präsenz des Virus ausreichend, um das Immunsystem zu aktivieren, d.h. die chronische Entzündung im Tumor in eine akute umzuwandeln. Dabei ist die adaptive Immunität mit T- und B-Zell-Aktivierung nicht notwendig für die Rückbildung des Tumors. In Abwesenheit eines solchen Stimulus, wie in der ersten Studie mit neu-exprimierenden MMCs, wird die Spezifität der adaptiven Immunantwort benötigt, um die akute Inflammation anzustoßen und die Tumorregression voranzutreiben. Zusammengefasst unterstützt diese Arbeit die Hypothese, dass die Mechanismen, die zu „tissue specific destruction“ (TSD) führen, in verschiedenen immunologischen Erkrankungen zwar divergieren, der Effektor-Mechanismus aber stets der Gleiche ist. Es zeigte sich, dass in Anwesenheit eines „triggers“, wie z.B. der VACV-Infektion und intakten „danger signaling pathways“ der Tumorzellen, die angeborene Immunität allein ausreicht, um die Tumorrückbildung zu vermitteln.