13. Selected Modification of the Adenovirus Type 5 Hexon Modulates Interaction with Coagulation Factor X and Hepatocyte Transduction In Vivo

AbstractA major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c+, ER-TR7+, and MAdCAM-1+ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46+ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

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Human Coagulation Factor X-Adenovirus Type 5 Complexes Poorly Stimulate an Innate Immune Response in Human Mononuclear Phagocytes

Journal of Virology ◽

10.1128/jvi.03576-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2884-2891 ◽

Cited By ~ 12

Author(s):

Karsten Eichholz ◽

Franck J. D. Mennechet ◽

Eric J. Kremer

Keyword(s):

Coagulation Factor ◽

Human Adenovirus ◽

Adenovirus Type ◽

Natural Host ◽

Innate Immune ◽

Mononuclear Phagocytes ◽

Factor X ◽

Dynamic Interactions

ABSTRACTOne of the first lines of host defense against many viruses in vertebrates is the innate immune system, which detects pathogen-associated molecular patterns (PAMPs) using pathogen recognition receptors (PRR). The dynamic interactions between pathogens and hosts create, in some cases, species-specific relationships. Recently, it was shown that murine factor X (mFX)-armored human adenovirus (HAd) stimulated a mFX-Toll-like receptor 4 (TLR4)-associated response in mouse macrophagesin vitroandin vivo. Given the importance of studies using animals to better understand host-pathogen interactions, we asked if human FX (hFX)-armored HAd type 5 (HAd5) was capable of activating innate immune sensors in primary human mononuclear phagocytes. To this end, we assayed human mononuclear phagocytes for their ability to be stimulated by hFX-armored HAd5 via a TLR/NF-κB pathway, in particular, a TLR4 pathway. In our hands, we found no significant interaction, activation, or maturation of human mononuclear phagocytes caused by the presence of hFX-armored HAd5.IMPORTANCEAnimals, and mice in particular, are often used as informative and powerful surrogates for how pathogens interact with natural host systems. When possible, extended and targeted studies in the natural host can then be performed. Our data will help us understand the differences in preclinical testing in mice and clinical use in humans in order to improve treatment for HAd diseases and Ad vector effectiveness.

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Coagulation factor X mediates adenovirus type 5 liver gene transfer in non-human primates (Microcebus murinus)

Gene Therapy ◽

10.1038/gt.2011.87 ◽

2011 ◽

Vol 19 (1) ◽

pp. 109-113 ◽

Cited By ~ 26

Author(s):

R Alba ◽

A C Bradshaw ◽

N Mestre-Francés ◽

J-M Verdier ◽

D Henaff ◽

...

Keyword(s):

Gene Transfer ◽

Coagulation Factor ◽

Adenovirus Type ◽

Microcebus Murinus ◽

Factor X ◽

Liver Gene ◽

Adenovirus Type 5

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Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement

Nature Medicine ◽

10.1038/nm.3107 ◽

2013 ◽

Vol 19 (4) ◽

pp. 452-457 ◽

Cited By ~ 99

Author(s):

Zhili Xu ◽

Qi Qiu ◽

Jie Tian ◽

Jeffrey S Smith ◽

Gina M Conenello ◽

...

Keyword(s):

Coagulation Factor ◽

Adenovirus Type ◽

Natural Antibodies ◽

Factor X ◽

Adenovirus Type 5

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Manipulating Adenovirus Hexon Hypervariable Loops Dictates Immune Neutralisation and Coagulation Factor X-dependent Cell Interaction In Vitro and In Vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1004673 ◽

2015 ◽

Vol 11 (2) ◽

pp. e1004673 ◽

Cited By ~ 28

Author(s):

Jiangtao Ma ◽

Margaret R. Duffy ◽

Lin Deng ◽

Rachel S. Dakin ◽

Taco Uil ◽

...

Keyword(s):

Coagulation Factor ◽

Cell Interaction ◽

Factor X ◽

Dependent Cell

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In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications

Viruses ◽

10.3390/v13081483 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1483

Author(s):

Emily A. Bates ◽

John R. Counsell ◽

Sophie Alizert ◽

Alexander T. Baker ◽

Natalie Suff ◽

...

Keyword(s):

Viral Vector ◽

Ex Vivo ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cell Types ◽

In Vivo Evaluation ◽

In Vivo Biodistribution ◽

Adenovirus Type 5

The human adenovirus phylogenetic tree is split across seven species (A–G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D—such as their cellular tropism, receptor usage, and in vivo biodistribution profile—remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)—a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

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Genomic footprinting detects factors bound to major late and IVa2 promoters in adenovirus-infected HeLa cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1534-1539.1988 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1534-1539

Author(s):

G Albrecht ◽

B Devaux ◽

C Kedinger

Keyword(s):

Protein Interactions ◽

Adenovirus Type ◽

Nuclease Activity ◽

Dnase I ◽

Promoter Activation ◽

Promoter Elements ◽

Dnase I Footprinting ◽

Infected Cells ◽

Adenovirus Type 5

We used DNase I footprinting assays on nuclei isolated from adenovirus-infected cells to examine the nucleoprotein configuration of a 250-base-pair segment which encompasses the adenovirus type 5 major late (ML) and IVa2 promoters. At 12 and 20 h postinfection (p.i.), fine DNase I digestion mapping of wild-type adenovirus-infected cells revealed specific sequences protected from digestion which corresponded to promoter elements required for expression of the ML gene in vivo. At 12 h p.i., a G+C-rich region which lies upstream of the IVa2 cap site and is important for maximal IVa2 activity was also found masked to nuclease activity. At 20 h p.i., however, this element became more sensitive to nuclease attack, while the ML promoter elements stayed protected. No major changes in DNA-protein interactions were detected in the region spanning the ML and IVa2 cap sites upon promoter activation, suggesting that the binding properties of the cognate factors for this region are not modified during the process.

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Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽

10.1182/blood.v66.6.1302.bloodjournal6661302 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1302-1308 ◽

Cited By ~ 1

Author(s):

W Kisiel ◽

KJ Smith ◽

BA McMullen

Keyword(s):

Human Factor ◽

Coagulation Factor ◽

Factor Ix ◽

Light Chains ◽

Factor X ◽

Amino Terminal ◽

Coagulation Factor Ix ◽

Human Factor Ix ◽

Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

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The Low-Density Lipoprotein Receptor-Related Protein (LRP) Mediates Clearance of Coagulation Factor Xa In Vivo

Blood ◽

10.1182/blood.v91.2.555 ◽

1998 ◽

Vol 91 (2) ◽

pp. 555-560 ◽

Cited By ~ 20

Author(s):

Masaaki Narita ◽

Amy E. Rudolph ◽

Joseph P. Miletich ◽

Alan L. Schwartz

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Factor Xa ◽

Coagulation Factor ◽

Lipoprotein Receptor ◽

Factor X ◽

Receptor Associated Protein ◽

Density Lipoprotein Receptor ◽

Lipoprotein Receptor Related Protein

Abstract Blood coagulation factor X plays a pivotal role in the clotting cascade. When administered intravenously to mice, the majority of activated factor X (factor Xa) binds to α2-macroglobulin (α2M) and is rapidly cleared from the circulation into liver. We show here that the low-density lipoprotein receptor-related protein (LRP) is responsible for factor Xa catabolism in vivo. Mice overexpressing a 39-kD receptor-associated protein that binds to LRP and inhibits its ligand binding activity displayed dramatically prolonged plasma clearance of 125I-factor Xa. Preadministration of α2M-proteinase complexes (α2M*) also diminished the plasma clearance of125I-factor Xa in a dose-dependent fashion. The clearance of preformed complexes of 125I-factor Xa and α2M was similar to that of 125I-factor Xa alone and was also inhibited by mice overexpressing a 39-kD receptor-associated protein. These results thus suggest that, in vivo, factor Xa is metabolized via LRP after complex formation with α2M.

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Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis

Blood ◽

10.1182/blood.2020007155 ◽

2020 ◽

Author(s):

Georg Obermayer ◽

Taras Afonyushkin ◽

Laura Goederle ◽

Florian Puhm ◽

Waltraud C. Schrottmaier ◽

...

Keyword(s):

Therapeutic Potential ◽

Coagulation Factor ◽

Mechanistic Explanation ◽

Factor X ◽

Thrombotic Risk ◽

Igm Antibodies ◽

Plasmatic Coagulation ◽

Natural Igm

Thrombosis and the complications associated with it are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural IgM antibodies targeting these structures. Here, we investigated whether natural IgM antibodies, which are known to have important anti-inflammatory house-keeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis.

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