- Email: [email protected]
409. Identifying Functional Adenovirus-Host Interactions Using Tandem Mass Spectrometry
ADENOVIRUS VECTORS: INNATE IMMUNITY directly bind to human C3 protein. After initial pathogen engagement by proteins of the classical and/or alternative complement pathways, C3 acts as a pivotal protein, initiating the subsequent activation of the common complement pathway. Activated complement proteins interact with Kupffer cells, platelets, and endothelial cells, and modulate several innate and adaptive immune response systems. Since several of these same systems have been implicated with Ad associated toxicities, we hypothesized that these toxicities may be significantly diminished by pre-emptive complement system blockade. Ad injection into Ad naïve wild-type (wt) mice confirmed a rapid activation of the murine complement system. High doses Ad vector injections into wt and complement deficient (C3-KO) mice resulted in similar Ad transduction levels of liver hepatocytes but altered the innate toxicity of Ads. For example, the profound thrombocytopenia typically induced after Ad injections into wt mice was completely abolished after identical injections into C3-KO mice. Indeed, complement blockade also resulted in significantly reduced plasma levels of numerous cytokines and chemokines (IL-4, IL-5, IL-6, IL10, mG-CSF, mGM-CSF, KC, and MIP-1α) that are typically induced within hours of Ad injected wt mice. The global importance of Ad interactions with the complement system were further validated by comparisons of liver transcriptome responses 6 hrs after Ad injection into C3-KO and wt mice. Array analysis of up to 30,000 RNA transcripts demonstrated the transcriptome of Ad injected C3-KO livers was strikingly different from the grossly abnormal transcriptome dysregulation induced in Ad injected wt mouse livers being more similar to that noted in mock-injected wt mice (also see Z. Hartman poster this meeting). Specifically, C3-KO transcriptome analysis confirmed a significantly diminished transcriptional dysregulation of numerous murine immune response genes, as well as those genes expressing coagulation, metabolism, apoptosis, and extracellular proteins, as compared to the greater changes induced in these same genes after Ad injections into wt mice. Our results confirm that complement inhibition strategies initiated prior to iv Ad vector administration should be investigated as a potential means to preemptively abrogate numerous Ad capsid induced toxicities and facilitate the known benefits of Ad mediated gene therapy and/or immunization applications.
408. The Kupffer Cell Scavenger Receptor Is Accountable for Hepatic Sequestration of Adenovirus Hidde J. Haisma,1 Anna Rita Bellu,1 Marianne G. Rots,1 Gera Kamps,1 Josee Plantinga,1 Jan Kamps.2 1 Therapeutic Gene Modulation, Groningen University Institute for Drug Exploration, Groningen, Netherlands; 2Pathology and Laboratory Medicine, Groningen University Institute for Drug Exploration, Groningen, Netherlands. Introduction: The development of systemically deliverable vectors that are targeted to specific receptors is a major goal in gene therapy. Adenovirus is the preferred vector for gene therapy because of its superior in vivo gene transfer efficiency. However, systemic administration of adenovirus results in preferential accumulation in the liver. This elimination from the circulation is primarily caused by hepatic Kupffer cells that are also responsible for the inflammatory response. Aim: We hypothesized that the scavenger receptor present on Kupffer cells may serve as a receptor for adenovirus. Blockage of the receptor should increase the circulation time and availability for targetd cells of the adenovirus in vivo and reduce the inflammatory response. Results: In vitro studies showed specific inhibition of Adenovirusinduced gene expression when scavenger receptor antagonists were S158
applied to Kupffer cells. In contrast, prior administration of antagonists were ineffective in Coxsackievirus Adenovirus Receptorpositive HepG2 cells. In vivo, pre-administration of a scavenger receptor antagonist resulted in a transient increase in the blood circulation levels with plasma concentrations increasing 10-fold. This was accompanied by a 5-15-fold higher level of transgene expression in tissues measured at 48 h after virus administration. The administration of a scavenger receptor antagonist did not result in increased toxicity as measured by liver enzymes (ALT, AST). Conclusion: From our results we conclude that the scavenger receptor is responsible for liver sequestration of adenovirus vectors and scavenger receptor antagonists may have applications to reduce the vector dose or improve the level of gene expression from adenoviral vectors. As other viral vectors such as Adeno-Associated Virus (AAV) and Lentivirus are similarly targets for the scavenger receptor, we anticipate that this will have great impact on the development of systemic gene therapy.
409. Identifying Functional Adenovirus-Host Interactions Using Tandem Mass Spectrometry Anuj Gaggar,1,3 Martin Sadilek,2 Andre Lieber,1,3 Dmitry M. Shayakhmetov.3 1 Department of Pathology, University of Washington, Seattle, WA; 2 Department of Chemistry, University of Washington, Seattle, WA; 3 Department of Medicine, University of Washington, Seattle, WA. The initial steps of viral infection are critical determinants of viral tropism and pathogenesis. Intravenously applied Ad-based vectors were found to be cleared rapidly from the blood and transduce liver tissue, subsequently causing severe inflammatory responses. While virus interactions with blood factors, rather than with known Ad attachment receptors, have been suggested to mediate both virus clearance and liver cell transduction, the identity of factors in the blood interacting with Ad remains unclear. To identify proteins involved in functional Ad-host interactions following intravenous virus application, we describe a systematic, high-throughput approach based on the binding of putative cellular receptors/host factors to a structural Ad protein immobilized on Ni2+-agarose beads. Following expression in E. coli, purified Ad5 or Ad35 fiber knob domains were conjugated to agarose beads and mixed with freshly purified mouse plasma. After several washes, proteins bound to the Ad fiber knob domains were eluted with urea for subsequent analysis by tandem mass spectrometry. Using this approach, we identified a number of plasma proteins including C4-binding protein (C4BP), fibrinogen, and complement proteins C3 and C4, which efficiently bind to Ad fiber knob domains (MS/MS Mowse scores >35). To further confirm that these MS/MS-identified proteins are capable of direct binding to Ad fiber knob domains, we immobilized available plasma proteins on a nitrocellulose membrane and used the Ad fiber knob domains in a virus-overlay protein blot assay. This analysis revealed that both the Ad35 and Ad5 fiber knob domains efficiently bind to the proteins identified using our technique. Furthermore, we demonstrated that the infectivity of Ads can be modulated by interactions with C4BP but not other plasma proteins. Importantly, we observed a dramatic increase in Ad infectivity on a human hepatocarcinoma cell line that was dose dependent and required physiological concentrations of C4BP. To study whether blood factor binding to the Ad fiber knob domain can interfere with virus binding to known cellular attachment receptors we infected CHO-CAR (expressing CAR) or CHO-C2 (expressing CD46) cells with Ad5 or Ad5/35 vectors respectively. While infection of CHO-CAR with Ad5 in the presence of physiological concentrations of C4BP or other plasma proteins did not change virus infectivity, C4BP could efficiently compete with Ad5/35 infection of CHO-C2 cells. Here we demonstrate that by using a straightforward high-throughput approach, novel functional interactions of a virus with host factors Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
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
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408. The Kupffer Cell Scavenger Receptor Is Accountable for Hepatic Sequestration of Adenovirus Hidde J. Haisma,1 Anna Rita Bellu,1 Marianne G. Rots,1 Gera Kamps,1 Josee Plantinga,1 Jan Kamps.2 1 Therapeutic Gene Modulation, Groningen University Institute for Drug Exploration, Groningen, Netherlands; 2Pathology and Laboratory Medicine, Groningen University Institute for Drug Exploration, Groningen, Netherlands. Introduction: The development of systemically deliverable vectors that are targeted to specific receptors is a major goal in gene therapy. Adenovirus is the preferred vector for gene therapy because of its superior in vivo gene transfer efficiency. However, systemic administration of adenovirus results in preferential accumulation in the liver. This elimination from the circulation is primarily caused by hepatic Kupffer cells that are also responsible for the inflammatory response. Aim: We hypothesized that the scavenger receptor present on Kupffer cells may serve as a receptor for adenovirus. Blockage of the receptor should increase the circulation time and availability for targetd cells of the adenovirus in vivo and reduce the inflammatory response. Results: In vitro studies showed specific inhibition of Adenovirusinduced gene expression when scavenger receptor antagonists were S158
applied to Kupffer cells. In contrast, prior administration of antagonists were ineffective in Coxsackievirus Adenovirus Receptorpositive HepG2 cells. In vivo, pre-administration of a scavenger receptor antagonist resulted in a transient increase in the blood circulation levels with plasma concentrations increasing 10-fold. This was accompanied by a 5-15-fold higher level of transgene expression in tissues measured at 48 h after virus administration. The administration of a scavenger receptor antagonist did not result in increased toxicity as measured by liver enzymes (ALT, AST). Conclusion: From our results we conclude that the scavenger receptor is responsible for liver sequestration of adenovirus vectors and scavenger receptor antagonists may have applications to reduce the vector dose or improve the level of gene expression from adenoviral vectors. As other viral vectors such as Adeno-Associated Virus (AAV) and Lentivirus are similarly targets for the scavenger receptor, we anticipate that this will have great impact on the development of systemic gene therapy.
409. Identifying Functional Adenovirus-Host Interactions Using Tandem Mass Spectrometry Anuj Gaggar,1,3 Martin Sadilek,2 Andre Lieber,1,3 Dmitry M. Shayakhmetov.3 1 Department of Pathology, University of Washington, Seattle, WA; 2 Department of Chemistry, University of Washington, Seattle, WA; 3 Department of Medicine, University of Washington, Seattle, WA. The initial steps of viral infection are critical determinants of viral tropism and pathogenesis. Intravenously applied Ad-based vectors were found to be cleared rapidly from the blood and transduce liver tissue, subsequently causing severe inflammatory responses. While virus interactions with blood factors, rather than with known Ad attachment receptors, have been suggested to mediate both virus clearance and liver cell transduction, the identity of factors in the blood interacting with Ad remains unclear. To identify proteins involved in functional Ad-host interactions following intravenous virus application, we describe a systematic, high-throughput approach based on the binding of putative cellular receptors/host factors to a structural Ad protein immobilized on Ni2+-agarose beads. Following expression in E. coli, purified Ad5 or Ad35 fiber knob domains were conjugated to agarose beads and mixed with freshly purified mouse plasma. After several washes, proteins bound to the Ad fiber knob domains were eluted with urea for subsequent analysis by tandem mass spectrometry. Using this approach, we identified a number of plasma proteins including C4-binding protein (C4BP), fibrinogen, and complement proteins C3 and C4, which efficiently bind to Ad fiber knob domains (MS/MS Mowse scores >35). To further confirm that these MS/MS-identified proteins are capable of direct binding to Ad fiber knob domains, we immobilized available plasma proteins on a nitrocellulose membrane and used the Ad fiber knob domains in a virus-overlay protein blot assay. This analysis revealed that both the Ad35 and Ad5 fiber knob domains efficiently bind to the proteins identified using our technique. Furthermore, we demonstrated that the infectivity of Ads can be modulated by interactions with C4BP but not other plasma proteins. Importantly, we observed a dramatic increase in Ad infectivity on a human hepatocarcinoma cell line that was dose dependent and required physiological concentrations of C4BP. To study whether blood factor binding to the Ad fiber knob domain can interfere with virus binding to known cellular attachment receptors we infected CHO-CAR (expressing CAR) or CHO-C2 (expressing CD46) cells with Ad5 or Ad5/35 vectors respectively. While infection of CHO-CAR with Ad5 in the presence of physiological concentrations of C4BP or other plasma proteins did not change virus infectivity, C4BP could efficiently compete with Ad5/35 infection of CHO-C2 cells. Here we demonstrate that by using a straightforward high-throughput approach, novel functional interactions of a virus with host factors Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
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