The Immunological Properties of Factor VIII

1966 ◽  
Vol 16 (03/04) ◽  
pp. 559-573 ◽  
Author(s):  
L Uszyński
Keyword(s):  
Factor Viii ◽  
Inhibitory Activity ◽  
Hemophilia A ◽  
Severe Form ◽  
Molecular Defect ◽  
Bovine Plasma ◽  
Human Factor Viii ◽  
Antigenic Properties ◽  
Coagulation Tests ◽  
Normal Human

SummaryRabbits immunized against human AHG fibrinogen-free preparations, were shown to produce anti-AHG antibodies. The inhibitory activity of these antibodies was tested by thromboplastin generation test, thrombelastography, and the specific anti-AHG antibodies neutralization test. The latter test permitted quantitative determination of antigenic form of factor VIII. The inhibitory activity of anti-FI-O-Ta serum resulted exclusively from the anti-AHG antibodies which in coagulation tests behaved like circulating anticoagulants directed against factor VIII.The anti-AHG antibodies were neutralizable by normal human serum or plasma even contained only trace of AHG activity after storage. There was no antigenic form of factor VIII in the severely affected patients with hemophilia A, von Willebrand’s disease nor in the normal plasma adsorbed on bentonite. The presented results suggest a molecular defect of factor VIII in patients with hemophilia A. The severe form of this disease depends, probably, on a major impairment of AHG biosynthesis, leading to changes in the antigenic properties of the molecule. The AHG from rabbit, porcine and bovine plasma respectively did not neutralize the anti-AHG antibodies formed in rabbits immunized against human factor VIII preparations.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

Investigations on the Nature of Hemophilia A

1963 ◽  
Vol 09 (01) ◽  
pp. 030-052 ◽  
Author(s):  
Eberhard Mammen
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Barium Sulfate ◽  
Freeze Drying ◽  
Room Temperature ◽  
Hemophilia A ◽  
Normal Human Plasma ◽  
Normal Human ◽  
And Storage ◽  
Prothrombin Activation

SummaryIn this paper an inhibitor is described that is found in hemophilic plasma and serum different from any till now described inhibitor. The inhibitor only inhibits prothrombin activation in the “intrinsic clotting systems”. This inhibitor is probably not present in normal human plasma or serum. It is destroyed by ether and freeze drying, is labile to acid and storage at room temperature. It is stable upon dialysis and has not been adsorbed on barium sulfate, aluminum hydroxide or kaolin. It precipitates at 50% v/v saturation with alcohol. The nature of this inhibitor seems to be a protein or lipoprotein.Factor VIII was isolated from hemophilic plasma. The amount isolated was the same as from normal plasma and the activity properties were not different. Hemophiliacs have normal amounts of factor VIII.

Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A

2001 ◽  
Vol 85 (01) ◽  
pp. 125-133 ◽  
Author(s):  
Huiyun Wu ◽  
Mark Reding ◽  
Jiahua Qian ◽  
David Okita ◽  
Ernie Parker ◽  
...  
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Th2 Cells ◽  
Tween 20 ◽  
Sorbitan Monolaurate ◽  
Human Factor Viii ◽  
Ifn Γ ◽  
Th1 And Th2

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

Identification of a Novel Missense Mutation in Exon 4 of the Human Factor VIII Gene Associated with Sever Hemophilia a Patient

2007 ◽  
Vol 10 (23) ◽  
pp. 4299-4302 ◽  
Author(s):  
Habib Onsori ◽  
Mohammad Ali Hossein . ◽  
Sheideh Montaser-Kou . ◽  
Mohammad Asgharzadeh . ◽  
Abbas Ali Hosseinpou .
Keyword(s):  
Factor Viii ◽  
Missense Mutation ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Exon 4

scholarly journals Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4671-4677 ◽  
Author(s):  
S Connelly ◽  
JM Gardner ◽  
RM Lyons ◽  
A McClelland ◽  
M Kaleko
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
High Dose ◽  
Specific Enzyme ◽  
Adult Mice ◽  
Human Factor Viii

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

Crossed Concanavalin A Affinoimmunoelectrophoresis Of Factor VIII - Related Antigen In Whole Plasma

1981 ◽  
Author(s):  
J S Krauss ◽  
M Sheard
Keyword(s):  
Factor Viii ◽  
Concanavalin A ◽  
Human Factor ◽  
Von Willebrand Disease ◽  
Con A ◽  
Factor Viii Related Antigen ◽  
Crossed Immunoelectrophoresis ◽  
Human Factor Viii ◽  
Normal Human ◽  
Von Willebrand

Normal human factor VIII is precipitated by the lectin concanavalin A (con A). In order to study this interaction pooled normal plasma has been subjected to crossed affinoimmunoelectrophoresis with con A in the first dimension and commercial antiserum to factor VIII in the second dimension. Both decreased anodal migration and decreased precipitin height of the factor VIII - related antigen (FVIIIR:ag) confirmed the precipation of FVIIIR:ag by con A.This technique, performed in conjunction with crossed immunoelectrophoresis (CIEP) of FVIIIR:ag, should prove useful in the analysis of von Willebrand disease (vWd) variants whose FVIIIR:ag has decreased carbonhydrate and, therefore, is poorly precipitated by con A.

Correction of the coagulation defect in hemophilia A mice through factor VIII expression in skin

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2799-2805 ◽  
Author(s):  
Steven S. Fakharzadeh ◽  
Yue Zhang ◽  
Rita Sarkar ◽  
Haig H. Kazazian
Keyword(s):  
Transgenic Mice ◽  
Factor Viii ◽  
Hemophilia A ◽  
Systemic Disease ◽  
Factor Viii Activity ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Transgenic Lines ◽  
Rag 1 ◽  
Involucrin Promoter

To test the hypothesis that factor VIII expressed in the epidermis can correct hemophilia A, we generated transgenic mice in a factor VIII–deficient background that express human factor VIII under control of the involucrin promoter. Mice from 5 transgenic lines had both phenotypic correction and plasma factor VIII activity. In addition to the skin, however, some factor VIII expression was detected in other tissues that have stratified squamous epithelia. To determine whether an exclusively cutaneous source of factor VIII could correct factor VIII deficiency, we grafted skin explants from transgenic mice onto mice that are double knockouts for the factor VIII and RAG-1 genes. Two graft recipients had plasma factor VIII activity of 4% to 20% of normal and improved whole blood clotting compared with factor VIII–deficient mice. Thus, expression of factor VIII from the epidermis can correct hemophilia A mice, thereby supporting the feasibility of cutaneous gene therapy for systemic disease.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Differential Immunodominance of Human and Porcine Factor VIII in Hemophilia A Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 40-40 ◽  
Author(s):  
John F. Healey ◽  
Ernest T. Parker ◽  
Rachel T. Barrow ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
High Titer ◽  
Domain Specificity ◽  
Chi Square ◽  
C2 Domains ◽  
Intravenous Injections ◽  
Domain Specific ◽  
Human Factor Viii ◽  
Chi Square Test

Abstract Hemophilia A inhibitor patients and patients with acquired hemophilia A recognize immunodominant epitopes in the A2 and C2 domains of human factor VIII (fVIII). Hemophilia A mice also recognize A2 and C2 domain epitopes when immunized with human fVIII using a dosing schedule that mimics clinical use. We compared the immune responses of hemophilia A mice to human and porcine fVIII using a domain specific ELISA. In this assay, monoclonal antibodies are tested against a panel of six single human fVIII domain hybrid human/porcine fVIII molecules that contain the human A1, A2, ap, A3, C1 or C2 domains. With anti-human antibodies, a positive signal with one of the single human domain proteins identifies domain specificity, whereas loss of signal indicates domain specificity of anti-porcine fVIII antibodies. Exon16 (E16) - disrupted hemophilia A mice (n = 3) received six weekly μ10 g/kg intravenous injections of recombinant B-domain deleted human fVIII and a final 25 μg/kg boost. To obtain comparable inhibitor titers, E16 mice (n = 3) received six weekly injections of μ40 g/kg of recombinant B-domain deleted porcine fVIII. Spleens from high titer mice were fused with NS1 mouse myeloma cells and 485 of the resulting hybridomas were analyzed for fVIII domain specificity (Table). Only two hybridomas secreted antibodies specific for the ap domain. Human fVIII elicited a significantly greater number of antibodies to the A2 domain, whereas porcine fVIII elicited a significantly greater number of antibodies to the A1 and A3 domains (p < 0.01, chi square test). The greater number of anti-C2 antibodies to human fVIII was not significant at the 95% confidence level (p = 0.08). The differential immunodominance of human and porcine fVIII epitopes suggests that it may be possible to design a recombinant hybrid human/porcine fVIII molecule that is less immunogenic than human fVIII in the treatment of patients with hemophilia A. Domain Specificity of Anti-FVIII MAbs Mouse ID: Immunogen No. of MAbs A1 A2 A3 C1 C2 CR & MD CR: Cross Reactive MD: Multidomain 1- Human fVIII 95 2 16 2 7 21 23 & 24 2- Human fVIII 126 13 23 1 2 27 39 & 21 3- Human fVIII 54 1 15 2 1 10 9 & 15 4- Porcine fVIII 123 39 7 19 8 16 33 & 0 5- Porcine fVIII 27 13 5 0 0 4 2 & 3 6- Porcine fVIII 60 9 6 12 1 9 13 & 10

Immunomodulation of Transgene Responses Following Naked DNA Transfer of Factor VIII into Hemophilia A Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1279-1279
Author(s):  
Peiqing Ye ◽  
David J. Rawlings ◽  
Arthur R. Thompson ◽  
Hans D. Ochs ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Immune Responses ◽  
Factor Viii ◽  
Hemophilia A ◽  
Dna Transfer ◽  
Secondary Response ◽  
Inhibitory Antibody ◽  
Naked Dna ◽  
Human Factor Viii

Abstract Naked DNA transfer of liver-specific, high-expressing plasmid pBS-HCRHPI-FVIIIA in Rag2(−/ −) SCID mice produced persistent high-level gene expression of human factor VIII (hFVIII) (Miao, Hum. Gene Ther. 2003). However, in immunocompetent hemophilia A mice, a robust humoral immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity (Ye, Mol. Ther. 2004). Transient immunomodulation strategies were explored to prevent the formation of inhibitory antibody formation. Eight groups of mice (n=8) were treated by naked DNA transfer of plasmid pBS-HCRHPI-FVIIIA. Each group were subjected to treatment with single or combined immunosuppressive regimen: CyclosporineA (CSA) daily for 14 days; Rapamycin daily for 14 days; Mycophenylate mofetil (MMF) daily for 14 days; combination of CSA and MMF; combination of Rapamycin and MMF; a monoclonal antibody (MR1) against murine CD40 ligand on days -1, 1, 2, 7, & 14; recombinant murine Ctla4Ig on days 1 & 2; and combination of MR1 and Ctla4Ig. Combination regimens were given using the same combined schedule and dosages. All animals treated with immunosuppression had delayed or no immune responses against hFVIII except the group treated with CSA only. The most effective treatment was observed in animals treated with the combination of Ctla4Ig and MR1. Seven of 8 animals failed to develop detectable inhibitors. One animal developed transient low-titer antibodies. This group of animals produced persistent, therapeutic levels of hFVIII gene expression for over 6 months. Tolerized animals were subsequently challenged by the T dependent antigen, bacteriophage Φx174, and exhibited a normal primary and secondary response including amplification and isotype switch. These results strongly suggest that transient immunomodulation strategies to disrupt B- and T- cell interactions at the time of plasmid injection is effective to promote long-term immune tolerance that is specific for FVIII without altering subsequent immune responses to other T cell dependent antigens.

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