Prevalence and epitope specificity of non-neutralising antibodies in a large cohort of haemophilia A patients without inhibitors

2011 ◽  
Vol 105 (06) ◽  
pp. 954-961 ◽  
Author(s):  
Hervé Chambost ◽  
Christine Biron-Andreani ◽  
Pierre-Emmanuel Morange ◽  
Christophe Combescure ◽  
Alain Marques-Verdier ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Haemophilia A ◽  
Neutralising Antibodies ◽  
Epitope Specificity ◽  
Iatrogenic Complication ◽  
Severity Of The Disease ◽  
Recombinant Fviii

SummaryAntibodies (inhibitors and non-neutralising antibodies [NNA]) directed against factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Inhibitors reduce FVIII procoagulant properties, whereas NNA are directed against non-functional epitopes. NNA are poorly studied and their prevalence, epitope specificity and physiopathology inadequately defined. The aim of this study was first to evaluate NNA prevalence in a French retrospective multicentric series of 210 patients without inhibitors, then to determine their epitope specificity (against the heavy chain [HC] or the light chain [LC] of FVIII) and particularly to assess the prevalence of anti-B domain NNA using specifically designed x-MAP assays. NNA occurred in 18.1% of patients (38/210) and their prevalence was not influenced by the severity of the disease. Among the 38 patients with NNA, 73.7% had anti-FVIII Abs against the HC, 13.2% against the LC and 13.2% had anti-FVIII Abs against both chains. There is thus a clear immuno-dominance of the HC of FVIII in the epitope profile of NNA, whatever the severity of HA. The proportion of NNA that recognised the B domain was 18.4% (n=7/38). A multivariate analysis did not highlight differences in NNA occurrence between patients treated with recombinant FVIII or with plasma-derived FVIII (19.6% vs. 14.9%, p=0.53).

Longitudinal Analysis of Factor VIII Inhibitors in a Previously Untreated Mild Haemophilia A Patient with an Arg593→Cys Substitution

1999 ◽  
Vol 81 (05) ◽  
pp. 723-726 ◽  
Author(s):  
Simone Timmermans ◽  
Ellen Turenhout ◽  
Christine Bank ◽  
Karin Fijnvandraat ◽  
Jan Voorberg ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Longitudinal Analysis ◽  
Factor Viii Inhibitor ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Initial Development ◽  
Inhibitor Development ◽  
Epitope Specificity ◽  
A2 Domain

SummaryRecent studies suggest that certain missense mutations associated with mild to moderate haemophilia A predispose to inhibitor development. In this study, we present a longitudinal analysis of the epitope specificity of an inhibitor that developed in a mild haemophiliac with an Arg593→Cys mutation. Immunoprecipitation studies revealed the presence of antibodies directed towards the light chain and A2 domain of factor VIII. Limited reactivity was observed with metabolically labelled C2 domain. Almost complete inhibitor neutralization was achieved upon addition of A2 domain. Binding of the inhibitor was not affected by the presence of the Arg593→Cys substitution in the recombinant A2 fragment. Evaluation of the epitope specificity of anti-factor VIII antibodies in plasma samples obtained at different time-points of inhibitor development revealed initial development of a low titre inhibitor directed towards the A2 domain and factor VIII light chain. A second period of factor VIII replacement therapy resulted in a dramatic rise in factor VIII inhibitor titre, which maintained their original epitope specificity. Based on the results of this and previous studies (Fijnvandraat et al., 1997; Thompson et al., 1997) it is argued that inhibitor development in patients with the Arg593→Cys mutation may proceed via a similar mechanism.

scholarly journals The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3663-3671 ◽  
Author(s):  
Richard Prescott ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Inge Scharrer ◽  
Inga Marie Nilsson ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Epitope Specificity ◽  
Extensive Analysis ◽  
Number Of Patients ◽  
Recombinant Fviii ◽  
Inhibitor Titer

Abstract Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

Von Willebrand Factor Modulates Factor VIII Immunogenicity: Comparative Study of Different Factor VIII Concentrates in a Haemophilia A Mouse Model

2002 ◽  
Vol 88 (08) ◽  
pp. 221-229 ◽  
Author(s):  
Mathias Behrmann ◽  
John Pasi ◽  
Jean-Marie Saint-Remy ◽  
Ronald Kotitschke ◽  
Michael Kloft
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Major Complication ◽  
Epidemiological Studies ◽  
Haemophilia A ◽  
Epitope Specificity ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe development of an immune response towards factor VIII (FVIII) remains the major complication of haemophilia A replacement therapy. Product-related risk factors have recently been identified on the basis of epidemiological studies, but the mechanism is not understood. To this end, various commercially available FVIII concentrates were administered by the IV route to FVIII-knockout mice and the resulting immune response was characterised. Significantly higher inhibitor titres (Bethesda assay) were observed for one recombinant FVIII and one plasma-derived FVIII product depleted in von Willebrand factor (VWF). Inhibitor titres were reduced upon pre-incubation of FVIII with purified VWF. Epitope specificity of anti-FVIII IgG was characterised using FVIII-fragments produced in E. coli. Concentrates with no or reduced VWF-level elicited antibodies recognising predominantly the acidic a1 and a3 regions. Addition of VWF prior to injection also modified the epitope specificity. FVIII concentrates, therefore, show qualitative and quantitative variations in immunogenicity, which are at least partly modulated by VWF.

scholarly journals The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3663-3671 ◽  
Author(s):  
Richard Prescott ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Inge Scharrer ◽  
Inga Marie Nilsson ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Epitope Specificity ◽  
Extensive Analysis ◽  
Number Of Patients ◽  
Recombinant Fviii ◽  
Inhibitor Titer

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

scholarly journals Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain

1999 ◽  
Vol 81 (01) ◽  
pp. 39-44 ◽  
Author(s):  
R. Laub ◽  
Di Giambattista ◽  
P. Fondu ◽  
H.-H. Brackmann ◽  
H. Lenk ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hepatitis A ◽  
Hemophilia A ◽  
Hepatitis A Virus ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Inhibitor Titer

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Prevalence and Epitope Specificity of Non-Neutralizing Antibodies in Haemophilia A Patients without Inhibitors Using a Multiplex x-MAP Technology.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3493-3493
Author(s):  
Aurélien Lebreton ◽  
Priscilla Lapalud ◽  
Hervé Chambost ◽  
Christine Biron-Andréani ◽  
Elodie Boissier ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Factor Viii ◽  
Prospective Studies ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Simultaneous Detection ◽  
Haemophilia A ◽  
Epitope Specificity ◽  
Immuno Assay ◽  
Recombinant Fviii

Abstract Abstract 3493 Poster Board III-430 Background and Objectives Antibodies (Abs) directed against Factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Anti-FVIII Abs neutralize procoagulant activity of FVIII. They are called inhibitors. Non-neutralizing antibodies (NNA) have also been described in hemophiliacs treated by FVIII concentrates. Their role and prevalence are still under debate. NNA could form immune complexes with FVIII and be responsible of an increased FVIII clearance inducing a shortened half-life. The aim of this retrospective study was to evaluate the prevalence of NNA in a large cohort of HA patients without inhibitors and to determine their epitope specificity against the two chains of FVIII or the B domain, using a x-MAP technology. Indeed, this approach has many advantages: it is fast, requires small volumes of plasma, and is suitable for multiplexing immuno-assay. Methods Samples of 187 patients (mean age 29 years, range [1-86]) with severe (n= 133), moderate (n= 31) or mild (n= 23) HA from three haemophilia centers were studied. All patients have been previously treated and were inhibitor-free at the time of the test (<0.6 Bethesda Units/mL). Among them 33% (n=61) were treated by plasma-derived FVIII while 67% (n=126) had received recombinant FVIII (rFVIII). Two assays based on x-MAP technology were used: (i) the first determined the NNA reactivity with the heavy chain (HC) or the light chain (LC) of FVIII (Lavigne-Lissalde et al. 2008); (ii) the second identified the NNA directed towards the B-domain, by differential binding to full-length rFVIII or B-domain deleted rFVIII. Results The observed NNA prevalence in the 187 patients under study was 24% (n=45): 13.3% (n=6) of NNA were directed against the LC, 62.2% (n= 28) were directed against the HC and 24.5% (n=11) against both chains. In the NNA positive population, the proportion of NNA directed toward the B-domain was of 9% (n=4). No difference was observed in the prevalence between the 3 centers. The prevalence of NNA in patients grouped into four categories of age [1-15 yrs] (n=51, NNA prevalence: 27%), [16-30 yrs] (n=65; 20%), [31-45 yrs] (n=35; 20%) and [>45 yrs] (n=33; 27%) was not statistically different. Conclusion This retrospective study, using the x-MAP technology, found a prevalence of 24% NNA in HA patients, comparable to other reports in the literature. Most of the NNA were directed against the HC of FVIII. In 9% of the cases, NNA recognized the B domain. Further prospective studies will allow to better explain the role of NNA, overall on the FVIII pharmacokinetics and particularly those binding to the B domain. Other prospective studies could highlight a difference according to the type of treatment. Lavigne-Lissalde G. et al. (2008). “Simultaneous detection and epitope mapping of anti-factor VIII antibodies.” Thromb Haemost99(6): 1090-6 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Heterogeneity of factor VIII antibodies: further immunochemical and biologic studies

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 807-817 ◽  
Author(s):  
MB Hultin ◽  
FS London ◽  
SS Shapiro ◽  
WJ Yount
Keyword(s):  
Factor Viii ◽  
Isoelectric Focusing ◽  
Heavy Chain ◽  
Light Chain ◽  
Major Peak ◽  
Igg Antibodies ◽  
Multiple Peaks ◽  
Light Chain Type ◽  
Apparent Homogeneity ◽  
Chain Type

Abstract Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.

scholarly journals The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene

2007 ◽  
Vol 15 (10) ◽  
pp. 1856-1862 ◽  
Author(s):  
Lingxia Chen ◽  
Fuxiang Zhu ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Viii Gene ◽  
Split Delivery

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1036-1036
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Kenichi Ogiwara ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Heavy Chain ◽  
Light Chain ◽  
Early Phase ◽  
Factor Viia ◽  
Clotting Factors ◽  
Initiation Phase ◽  
A1 Domain

Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.

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