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77 hKGF Gene Transfer for the Prevention of Radiation Induced Salivary Hypofunction Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S30 ADEN[.]
ADENOVIRUS AND OTHER DNA VIRUS VECTORS I Thereafter, the heads of mice were irradiated and after weeks (single dose and fractionated schemes) or months (single dose only) salivary ow rates were measured Mice that received a single dose of IR and no treatment exhibited an ∼ 60% decrease of salivary output after weeks; whereas, mice administered the AdhMnSOD vector exhibited an ∼ 20% decrease of salivary output (p≤0.01) Mice treated with a control Ad5 vector (AdLacZ) secreted low levels of saliva similar to the untreated and irradiated mice Impressively, this AdhMnSODmediated protection of salivary output was generally maintained for months Mice that received daily fractions of Gy alone, or treated with AdLacZ, had a reduction in salivary ow of ∼65-70% after weeks Conversely, the salivary output of mice receiving the same IR regimen but treated with AdMnSOD was decreased by ∼40%, approaching statistical signicance (p acinar) Next, we evaluated if hKGF gene transfer led to submandibular epithelial cell proliferation For this experiment, mice were administered either the AdLTR2EF1a-hKGF or AdControl vector (Zheng et al, Clin Cancer Res, 2009) at -48 h At time zero these mice, plus a group of untreated Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ADENOVIRUS AND OTHER DNA VIRUS VECTORS I mice, were irradiated (15 Gy) At +24 h all mice were administered bromodeoxyuridine (BrdU), then at +48 h submandibular glands were harvested and BrdU incorporated in salivary epithelial cell nuclei was quantied BrdU-positive cells/eld were comparable for the non-irradiated and irradiated, AdLTR2EF1a-hKGF-treated, glands and both were signicantly greater than the values seen in mice that were irradiated and either untreated or treated with AdControl vector We are currently assessing the intracellular KGF signaling pathway(s) involved Finally, we tested whether hKGF gene transfer affected the growth of SCC tumors in these mice (SCC VII/SF; Cotrim et al, Clin Cancer Res, 2007) For these studies, 2x10e5 viable tumor cells were injected subcutaneously into the right hind leg of each mouse When tumors were ∼0.7mm diameter, mice were randomly divided into four groups (no treatment; IR to the tumor bearing leg alone, salivary hKGF gene transfer alone, hKGF gene transfer plus IR) Submandibular gland hKGF gene transfer had no effect on tumor growth ± IR, i.e., both endogenous tumor growth rates and IR regrowth delay Collectively, these results support the potential clinical application of salivary hKGF gene transfer to preserve salivary gland function in head and neck cancer patients 78 Generation of Replication-Defective Adenovirus Vector Expressing HCV Structural Gene CE1E2 and Comparative Immunogenicity in Mice with Different Delivery Routes Jie Guan, Yao Deng, Xiao Yin, Wenjie Tan, Xiaobing Wu National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China Purpose: To construct recombinant viral vectors expressing HCV structural gene CE1E2, and evaluated the immunogenicity via different delivery routes This study was an attempt to lay a foundation for HCV vaccine development in the future Method: Constructed the recombinant replication-defective adenovirus serotype vectors named rAd5-CE1E2 expressing the HCV CE1E2 gene (aa1-746 HCV 1b serotype) and recombinant TianTan Vaccinia vectors named rTTV-CE1E2 expressing the HCV CE1E2 gene (aa1-746 HCV 2a serotype) Groups of female BalB/c mice were immunized at 6-8 week of age Mice in three groups were immunized once with rAd5-CE1E2 given by Intramuscular injection (i.m.) or Intranasal injection(i.n.) or Intradermal injection (i.d.) Mice of three homologous immune groups were immunized with rAd5-CE1E2 prime immunized by i.m or i.m or i.n and boost by i.m or i.n or i.m respectively at 6-week intervals Mice in one group of heterologous immunity were prime-immunized with rAd5-CE1E2 and boost with rTTV-CE1E2 at 6-week intervals Mice immunized with PBS were as control Viral doses were 5×109vp for rAd5-CE1E2 or 5×107pfu for rTTVCE1E2 per mouse The cellular immunity responses were tested by ELISPOT or ICS weeks after immunization or for long-term about 20 weeks after boost Mice in the rAd5-CE1E2-immunized groups and control group were challenged by 107 pfu rTTV-CE1E2 per mouse given by i.p weeks after the last injection Then the mice were killed days after challenge (peak of vaccinia titer in ovaries), and their ovaries were harvested After freeze-thawing and homogenization procedure, vaccinia titer was determined by plaque assay Results: Splencoytes were harvested at weeks after immunization and stimulated with HCV peptide pool representing core protein or E1 protein or C-terminal of E2 protein IFN-γ-positive SFU against the core or E1 or E2 peptide pool were detected in all immunized groups, whereas litter spots in control group Cellular immune responses against E1 were much stronger than those against core or E2 Among the once-immunized groups, SFU in the group immunized by i.n were signicantly lower than those by i.m and i.d whereas the i.m and i.d group have no statistical difference Among the prime-boost groups, SFU in heterologous immune group were higher than those in homologous immune groups, and SFU in the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy group prime by i.n also lower than others with signicant statistical difference The cellular immune activity in all the prime-boost groups remained high level until experiment was end in the 26th week after primary immunity Conclusions: The recombinant viral vectors were able to elicit cellular immune activities against HCV core, E1, E2 peptide pool in mice and remained high-level for a long time, which were protective in HCV vaccinia virus challenge model Our results suggest that rAd5-CE1E2 is a promising vaccine candidate against HCV infection 79 Interaction between Adenoviral VectorTransduced Dendritic Cell-Based Cancer Vaccine and T Regulatory or T Helper 17 Cells Adele Y Wang,1 Sarah Q Crome,1 Kristy M Jenkins,2 Jeffrey A Medin,3 Jonathan L Bramson,2 Megan K Levings.1 Surgery, University of British Columbia and Immunity and Infection Research Centre, VCHRI, Vancouver, BC, Canada; Pathology and Molecular Medicine Centre for Gene Therapeutics, McMaster University, Hamilton, ON, Canada; 3Stem Cell and Developmental Biology, University Health Network, Toronto, ON, Canada As host-derived tissues, tumours benet from natural immune tolerance mechanisms and are poorly immunogenic Many tumourspecic T cells are deleted in the thymus, and T cells that escape this process are often subjected to suppression by T regulatory (Treg) cells In fact, the induction and expansion of Treg cells is thought to be a major barrier to the development of successful strategies to enhance anti-tumour immunity Recent evidence has linked the development of Treg cells to that of pro-inammatory Th17 cells, with the ultimate balance depending on the local cytokine milieu Currently, however, it is unclear whether Th17 cells are benecial or detrimental in the context of tumour immunotherapy In order to dene how dendritic cell (DC)-based cancer vaccines inuence the balance of tolerance versus immunity, we investigated how adenoviral transduction alters the functional consequences of interactions between human monocyte-derived DCs, and ex vivo Treg or Th17 cells We found that exposure of untransduced or transduced DCs to Treg cells caused a signicant reduction in the expression of co-stimulatory molecules, such as CD80 and CD86, on DCs In contrast, exposure of DCs to ex vivo Th17 cells caused a signicant increase in the expression of costimulatory molecules The presence of Th17 cells in the co-cultures, however, did not prevent Treg-mediated down-regulation of CD80 and CD86 on transduced DCs, indicating that suppression dominates over immunity Interestingly, Treg cells exposed to transduced, but not untransduced DCs had increased production of IL-17 and expression of IL-23 receptor Indeed, transduced DCs produced enhanced levels of IL-1β and IL-6, two of the crucial Th17-promoting cytokines, compared to untransduced DCs These data suggest that adenoviral transduction may specically promote Treg plasticity by inuencing DC cytokine prole, and experiments are on going to determine the functional consequences of altered cytokine production on Treg suppressive capacity In order to better understand the potential of cell-based vaccines to boost effective anti-tumour immune responses it is critical to understand the mechanistic bases for these effects The ndings from this study will ultimately help to dene clinicallyapplicable populations of antigen presenting cells that can efciently stimulate T effector cell immunity S31 ... pathway(s) involved Finally, we tested whether hKGF gene transfer affected the growth of SCC tumors in these mice (SCC VII/SF; Cotrim et al, Clin Cancer Res, 2007) For these studies, 2x10e5 viable tumor... these results support the potential clinical application of salivary hKGF gene transfer to preserve salivary gland function in head and neck cancer patients 78 Generation of Replication-Defective... alone, hKGF gene transfer plus IR) Submandibular gland hKGF gene transfer had no effect on tumor growth ± IR, i.e., both endogenous tumor growth rates and IR regrowth delay Collectively, these