1088 toll like receptors activation in the innate and adaptive immune response to helper dependent adenoviral vectors

1088 Toll Like Receptors Activation in the Innate and Adaptive Immune Response to Helper Dependent Adenoviral Vectors flow cytomctry analysis to study capsid and EGFP specific T cell responses Histolo[.]

flow cytomctry analysis to study capsid and EGFP specific T cell responses Histologic analyses of EGFP expression, T cell infiltration, and pathology as well as molecular characterizations of vector genomes in the livers were carried out Our data revealed that, at day 8, efficient EGFP transduction was established in all livers When compared to the results in mouse liver, ss vector transduced macaque liver more efficiently but sc vector performed equivalently in both species Virtually no clinical pathology, liver infiltration and EGFP specific T cell responses were noticeable at day However, animals followed for 38 days demonstrated transient transaminase elevations EGFP specific cytotoxic T cell response became evident as early as week in PBMCs, and secondary lymphoid and non-lymphoid tissues at the necropsy A complete loss of EGFP expression and manifestation of CD8 infiltration were noticed in all livers harvested at day 38 Capsid T cell responses were undetectable in all pre- and post- treatment PBMCs Analysis of vector gcnomes in the livers suggested molecular pathways similar to what we described in previous studies In summary, both ss and scAAV217 vector accomplished efficient but transient EGFP gene transfer in macaque liver The EGFP expression in hepatocytes appeared to be quickly terminated by the destructive T cell response directed to the transgene, This contrasts with the results obtained in mice where GFP expression is stable and transgene specific T cells arc low to absent Our findings emphasize the possible negative impact of transgene immunity on AAV vector mediated gene transfer to primate liver 1087 Transient Blockade of Icas CoStimulatory Pathways Induced Long-Term Tolerance to Factor VIII Following Nonviral Gene Transfer of Factor VIII into Hemophilia A Mice Baowei Peng,' Peiqing Ye,' Bruce R Blazer.' Hans D Ochs,' Carol H Miao.' 'Department ofPediatrics, Children Hospital and Regional Medical Center and University ofWashington, Seattle, WA; }Department ofPediatrics, University ofMinnesota, Minneapolis, MN s Approximately 2S-30% of hemophilia A patients produce inhibitory antibodies in response to factor VIII (FVIII) protein replacement therapy Potential gene therapy techniques used to treat hemophilia A have also resulted in a significant humoral immune response to FVIII in murine models Nonviral gene transfer of a FVIII plasmid into hemophilia mice induces strong humoral responses through predominantly TH2 signals, representing a unique and useful model for testing various immunomodulation strategies In this study, we tested if transient blockade of the inducible costimulator (ICOS)ICOS ligand (ICOSL) pathway can modulate the immune response following gene therapy Two separate groups ofmice (n=S and n=11 per group , respectively) were subjected to administration of FVIII plasmid via hydrodynamics-based tail-vein injection , and transient immunosuppressive regimen using anti-ICOS monoclonal antibody (mAb) (16 treatments in a week period) None of the anti-ICOS mAb treated mice developed inhibitory antibodies against FVIII and all produced persistent, high-level FVIII (100-300nglml) for at least ISO days (experimental period), whereas control FVIII plasmid-treated mice all developed high-titer inhibitory antibodies T cells isolated from spleen, blood, and lymph nodes of anti-I COS mAb treated and untreated mice were further analyzed at different time points The CD4+ T cells isolated from the spleen of tolerized mice did not proliferate in response to FVIII stimulation in vitro, whereas cells from plasmid only-treated mice proliferated significantly In the first couple of weeks post plasmid treatment, induction of tolerance was associated with significantly increased percentage (but not absolute numbers due to CD4+ T cell depletion) ofCD4+CD2S highFOXP3 +regulatory T cells in the peripheral blood , Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety o r Gene Therapy spleen , and lymph nodes of tolerizcd mice compared to untreated and plasmid only-treated mice A decrease ofCD62L and CD4SRB expression and an increase of GITR expression distinguished the activated Treg cell subset observed in anti-ICOS mAb treated mice from Treg cells in mice receiving only FVIII plasmid Furthermore, at week time point and afterwards, the expansion of Treg cells was only observed in the lymph nodes of tolcrizcd mice but not in spleen and peripheral blood , suggesting that Treg cells prolifcrated selectively in the lymph nodes where FVIII antigen is being presented by APCs during the maintenance of tolerance Adoptive transfer ofTreg cells from tolerized mice can delay and/or reduce the production of inhibitors against FVIII in recipient hemophilia A mice treated with FVIII plasmid Anti-ICOS mAb treatment has the potential for a novel immunomodulatory strategy to prevent the formation of inhibitory antibodies against FVIII following gene therapy Furthermore, we show that the CD4+CD2S highFOXP3+ regulatory T cells play an important role in the induction and maintenance of long-term tolerance post anti-ICOS immunotherapy 1088 Toll-Like Receptors Activation in the Innate and Adaptive Immune Response to Helper Dependent Adenoviral Vectors Vincenzo Cerullo, I Michael Seiler,' Daniel Wang; Viraj Mane,' Christian Clarke, I Brendan Lee I ,2 'Molecular and Human Genetics, Baylor College ofMedicine, Houston, TX; }Holl'ard Hughes Medical Institute, Baylor College ofMedicine, Houston, TX Toll-like Receptors (TLRs) are innate receptors that sense microbial products and trigger dendritic cell (DCs) maturation and cytokine production, thus bridging innate and adaptive immunity Cellular activation by most members of the TLR family involves a signaling cascade that proceeds through myeloid differentiation primary response gene 88 (MyD88), interleukin-l (IL-I) receptor-activated kinase (IRAK) and tumour-necrosis factor receptor (TNFR)-associated factor (TRAF6), and culminates in the activation of several transcription factors, including nuclear factor-kB (NF-kB) These transcription factors directly upregulate cytokine/ chemokine production Since the major obstacle to adenoviral vector gene delivery is the dose-dependent innate toxicity following the intravenous administration, we sought to characterize it from a cellular base standpoint We first asked whetherTLRs are involved in the immune response following the intravenous injection of adenovirus vector To test that, we injected high dose (SxI0 12VP/Kg) of helper dependent adenoviral vector (HD-Ad) expressing a reporter gene into MyD88-/- mice and measured the eytokines and chemokines production hours post-injection MyD88-/- mice showed a drastic reduction of proinflammatory molecules (IL6, RANTES , MCP-I , p