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410. Adenovirus Activates Mouse Platelets and Induces Platelet Leucocyte Association
ADENOVIRUS VECTORS: INNATE IMMUNITY can be identified. Despite the complexity of the mouse plasma protein composition, we were able to rapidly identify several new Adinteracting partners, including one with immediate applications to Ad vector development. The approach of using a structural protein to identify host factors critical in viral infection, immune evasion, and tissue specificity can be universally applied to a variety of viral and non-viral pathogens and can reveal new targets for prospective therapies.
410. Adenovirus Activates Mouse Platelets and Induces Platelet Leucocyte Association Maha Othman,1 Andrea Labelle,1 David Lillicrap.1 Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada.
1
Adenovirus vectors have been used extensively in pre-clinical and clinical studies as agents for gene delivery. Acute, transient thrombocytopenia is a known adverse effect of adenovirus vector administration with a mechanism that currently remains unresolved. The fall in platelets appears 24 hours following IV administration of adenovector and is vector dose dependent. We have initiated experiments to investigate the mechanism of Ad induced thrombocytopenia. We examined the expression of the platelet activation marker P-selectin (CD-62p) on mouse platelets after incubation with adenovirus in vitro by means of flow cytometry. An indirect immunoflourescence approach was used with anti- mouse CD 62p monoclonal antibody and an FITC labeled rat antimouseIgG. An appropriate negative isotype control and positive platelet activation control using thrombin were incorporated into these studies. The index of platelet activation (IPA), was used to quantify the activation effect of Ad on the platelets. This is the product of the mean channel fluorescence and the percentage of positive cells. We also examined Platelet leucocyte association (PLA) using the direct immunoflourescence and double colour staining of platelets and leucocytes using FITC labeled anti-mouse CD41 (platelets) and PE labeled anti-mouse CD 45 (common leucocyte antigen) respectively. Our study has shown that the mean IPA for platelets stimulated by adenovirus was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. PLA visualisation was also confirmed by means of direct fluorescence microscopy. These preliminary in vitro results indicate that the Adenovirus activates mouse platelets as it induces degranulation and expression of P-selectin and supports the recruitment of leucocytes presumably through an interaction with the leucocyte P-selectin glycoprotein ligand-1. This may have a role in mediating the accelerated platelet clearance by the reticuloendothelial system following adenovirus administration. In addition, the interaction with leucocytes may also stimulate procoagulant microparticle generation and thus lead to systemic hemostatic activation. To our knowledge, this is the first report that identifies Adenovirus as an inducer for mouse platelet activation and as a promoter for platelet leucocyte association. However, further in vitro and in vivo studies are still needed to better understand the mechanism of Ad induced thrombocytopenia; a complication that significantly limits the application of Ad mediated gene therapy.
411.
Trapping of Adenovirus Vectors in Blood
Daniel Stone,1 Shaoheng Ni,1 Zong-Yi Li,1 Dmitry M. Shayakhmetov,1 Andre Lieber.1,2 1 Division of Medical Genetics, University of Washington, Seattle, WA; 2Department of Pathology, University of Washington, Seattle, WA. In an attempt to circumvent the limitations of Adenovirus (Ad) vectors derived from group C serotype Ad5 we recently developed vectors based on group B serotype Ad11. These vectors are benefited by the low prevalence of Ad11-neutralizing antibodies in humans, and the CD46-dependent tropism of Ad11. CD46 is expressed at high levels in cell types including hematopoietic stem cells, dendritic cells and metastatic tumor cells. Following systemic intravenous delivery of an Ad5 vector, a chimeric Ad5 vector possessing the CD46 interacting Ad11 fiber (Ad5/11), and an Ad11 vector to CD46 transgenic mice we discovered that the kinetics of vector clearance from blood are different. Using real time quantitative PCR to detect viral genomes we found that the number of Ad11 vector genomes in mouse serum at 3 minutes post tail vein injection was 2 orders of magnitude lower than for Ad5 and Ad5/11 vectors, but at 15 and 120 minutes post injection the number of vector genomes was comparable between Ad5, Ad5/11 and Ad11. To investigate the differences between the numbers of vector genomes in serum at 3 minutes post injection the association of vector genomes with blood cells was also monitored by real time quantitative PCR at all time points. The number of vector genomes associated with blood cells was more than one order of magnitude higher for Ad11 than Ad5 or Ad5/11 at all time points tested with the highest difference seen at 3 minutes post injection (∼2 orders of magnitude higher). To study which type of blood cells associate with Ad11 vectors, CD46 transgenic mice were injected in the tail vein with 3H-[thymidine] labeled Ad vector. Individual cell types were separated from blood collected at 3 minutes post injection and the highest levels of tritium labeled Ad11 were associated with the platelet fraction. Subsequent analyses revealed that Ad5, Ad5/11 and Ad11 vectors associate with platelets within minutes after intravenous injection but that Ad11 vectors associate at higher levels. Electron microscopic analysis of platelets isolated 3 minutes after injection of Ad5, Ad5/11 or Ad11 vectors revealed that Ad particles were associated with the platelet surface and structures of the surface-connected canalicular system. We also noticed that Ad11 vectors associate with mononuclear cells more efficiently than Ad5/11 vectors. When taken together, the observations that Ad11 associates with platelets and mononuclear cells more efficiently than Ad5/11 suggests that capsid proteins other than fiber may mediate blood cell interactions. We are currently investigating the mechanism(s) by which Ad capsids and blood cells interact. The roles of the serotype specific (Ad5 or Ad11) capsid proteins hexon, penton and fiber in Ad/blood cell interactions will be presented. Our aim is to develop new Ad vectors that are not trapped in blood upon systemic intravascular administration.
412. Renal Pathophysiology after Systemic Administration of a Recombinant Adenovirus: Changes in Drug Metabolism Based upon Vector Dose Michael P. Boquet,1 Hong T. Le,1 Erin A. Clark,1 Shellie M. Callahan,1 Maria A. Croyle.1 1 Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, TX. In addition to its ability to maintain water and electrolyte balance and efficiently remove waste products from the circulation, the kidney actively metabolizes many drugs, hormones and xenobiotics through the cytochrome P450 (CYP) enzyme system. Considering that adenovirus (Ad) significantly alters the expression and function
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
S159
410. Adenovirus Activates Mouse Platelets and Induces Platelet Leucocyte Association Maha Othman,1 Andrea Labelle,1 David Lillicrap.1 Pathology and Molecular Medicine, Queen’s University, Kingston, ON, Canada.
1
Adenovirus vectors have been used extensively in pre-clinical and clinical studies as agents for gene delivery. Acute, transient thrombocytopenia is a known adverse effect of adenovirus vector administration with a mechanism that currently remains unresolved. The fall in platelets appears 24 hours following IV administration of adenovector and is vector dose dependent. We have initiated experiments to investigate the mechanism of Ad induced thrombocytopenia. We examined the expression of the platelet activation marker P-selectin (CD-62p) on mouse platelets after incubation with adenovirus in vitro by means of flow cytometry. An indirect immunoflourescence approach was used with anti- mouse CD 62p monoclonal antibody and an FITC labeled rat antimouseIgG. An appropriate negative isotype control and positive platelet activation control using thrombin were incorporated into these studies. The index of platelet activation (IPA), was used to quantify the activation effect of Ad on the platelets. This is the product of the mean channel fluorescence and the percentage of positive cells. We also examined Platelet leucocyte association (PLA) using the direct immunoflourescence and double colour staining of platelets and leucocytes using FITC labeled anti-mouse CD41 (platelets) and PE labeled anti-mouse CD 45 (common leucocyte antigen) respectively. Our study has shown that the mean IPA for platelets stimulated by adenovirus was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. PLA visualisation was also confirmed by means of direct fluorescence microscopy. These preliminary in vitro results indicate that the Adenovirus activates mouse platelets as it induces degranulation and expression of P-selectin and supports the recruitment of leucocytes presumably through an interaction with the leucocyte P-selectin glycoprotein ligand-1. This may have a role in mediating the accelerated platelet clearance by the reticuloendothelial system following adenovirus administration. In addition, the interaction with leucocytes may also stimulate procoagulant microparticle generation and thus lead to systemic hemostatic activation. To our knowledge, this is the first report that identifies Adenovirus as an inducer for mouse platelet activation and as a promoter for platelet leucocyte association. However, further in vitro and in vivo studies are still needed to better understand the mechanism of Ad induced thrombocytopenia; a complication that significantly limits the application of Ad mediated gene therapy.
411.
Trapping of Adenovirus Vectors in Blood
Daniel Stone,1 Shaoheng Ni,1 Zong-Yi Li,1 Dmitry M. Shayakhmetov,1 Andre Lieber.1,2 1 Division of Medical Genetics, University of Washington, Seattle, WA; 2Department of Pathology, University of Washington, Seattle, WA. In an attempt to circumvent the limitations of Adenovirus (Ad) vectors derived from group C serotype Ad5 we recently developed vectors based on group B serotype Ad11. These vectors are benefited by the low prevalence of Ad11-neutralizing antibodies in humans, and the CD46-dependent tropism of Ad11. CD46 is expressed at high levels in cell types including hematopoietic stem cells, dendritic cells and metastatic tumor cells. Following systemic intravenous delivery of an Ad5 vector, a chimeric Ad5 vector possessing the CD46 interacting Ad11 fiber (Ad5/11), and an Ad11 vector to CD46 transgenic mice we discovered that the kinetics of vector clearance from blood are different. Using real time quantitative PCR to detect viral genomes we found that the number of Ad11 vector genomes in mouse serum at 3 minutes post tail vein injection was 2 orders of magnitude lower than for Ad5 and Ad5/11 vectors, but at 15 and 120 minutes post injection the number of vector genomes was comparable between Ad5, Ad5/11 and Ad11. To investigate the differences between the numbers of vector genomes in serum at 3 minutes post injection the association of vector genomes with blood cells was also monitored by real time quantitative PCR at all time points. The number of vector genomes associated with blood cells was more than one order of magnitude higher for Ad11 than Ad5 or Ad5/11 at all time points tested with the highest difference seen at 3 minutes post injection (∼2 orders of magnitude higher). To study which type of blood cells associate with Ad11 vectors, CD46 transgenic mice were injected in the tail vein with 3H-[thymidine] labeled Ad vector. Individual cell types were separated from blood collected at 3 minutes post injection and the highest levels of tritium labeled Ad11 were associated with the platelet fraction. Subsequent analyses revealed that Ad5, Ad5/11 and Ad11 vectors associate with platelets within minutes after intravenous injection but that Ad11 vectors associate at higher levels. Electron microscopic analysis of platelets isolated 3 minutes after injection of Ad5, Ad5/11 or Ad11 vectors revealed that Ad particles were associated with the platelet surface and structures of the surface-connected canalicular system. We also noticed that Ad11 vectors associate with mononuclear cells more efficiently than Ad5/11 vectors. When taken together, the observations that Ad11 associates with platelets and mononuclear cells more efficiently than Ad5/11 suggests that capsid proteins other than fiber may mediate blood cell interactions. We are currently investigating the mechanism(s) by which Ad capsids and blood cells interact. The roles of the serotype specific (Ad5 or Ad11) capsid proteins hexon, penton and fiber in Ad/blood cell interactions will be presented. Our aim is to develop new Ad vectors that are not trapped in blood upon systemic intravascular administration.
412. Renal Pathophysiology after Systemic Administration of a Recombinant Adenovirus: Changes in Drug Metabolism Based upon Vector Dose Michael P. Boquet,1 Hong T. Le,1 Erin A. Clark,1 Shellie M. Callahan,1 Maria A. Croyle.1 1 Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, TX. In addition to its ability to maintain water and electrolyte balance and efficiently remove waste products from the circulation, the kidney actively metabolizes many drugs, hormones and xenobiotics through the cytochrome P450 (CYP) enzyme system. Considering that adenovirus (Ad) significantly alters the expression and function
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
S159