Antibody formation and specificity in Bethesda‐negative brother pairs with haemophilia A

Antibodies directed towards non‐neutralizing epitopes on the factor VIII protein (FVIII) may be detected in patients with haemophilia A. We evaluated the prevalence of non‐neutralizing antibodies, in 201 inhibitor‐negative brother pairs with severe haemophilia A, enrolled in the Malmö International Brother Study and the Haemophilia Inhibitor Genetics Study. To evaluate binding specificity of the antibodies, ELISA plates were coated with two recombinant full‐length (FL) FVIII‐products and one recombinant B‐domain‐deleted (BDD) product. Seventy‐nine patients (39.3%) had a history of positive inhibitor titre measured by Bethesda assay, and FVIII antibodies were detected in 20 of them (25.3%). Additional 23 samples from subjects without a history of FVIII inhibitors were ELISA‐positive corresponding to a frequency of non‐neutralizing antibodies of 18.9%. The antibody response towards the different FVIII products was heterogenous, and was raised not only towards the non‐functional B‐domain but also towards both FL‐rFVIII and BDD‐rFVIII. In patients considered successfully treated with immune tolerance induction, 25.4% had remaining FVIII antibodies. The number of families with an antibody response in all siblings was increased when the total antibody response was taken into account, further supporting the concept of a genetic predisposition of the immune response. Further studies and careful monitoring over time are required to appreciate the immune response on the risk of inhibitor development or recurrence in the future.     

Product‐dependent anti‐factor VIII antibodies

The development of anti‐factor (F)VIII antibodies in haemophilia A (HA) subjects undergoing replacement therapy has been well documented. The correlation between antibody development and the FVIII product used for replacement therapy remains a subject of discussion. The aim of this study was to evaluate the presence of anti‐FVIII antibodies towards three commercial rFVIII products in 34 HA subjects’ plasmas. Antibodies were quantitated by a Multiplex Fluorescence Immunoassay. All plasmas contained anti‐FVIII antibodies at variable concentrations ranging from 50 nm to 570 μm . Eleven of the 20 HA subjects treated with one (r)FVIII product contained inhibitory anti‐FVIII antibodies (0.8‐3584 BU). The inhibitory antibody titre and the molar concentrations of total antibody were mildly correlated (r² = 0.6). Pronounced differences in antibody recognition with the three rFVIII products were observed. For the group treated with Product ‘A’, the titre towards this product was 2.4‐fold higher than that observed with another full‐length rFVIII‐containing product (Product ‘B’) and almost four‐fold higher than that measured with a B domain‐less rFVIII product (Product ‘C’). For the group of 14 HA subjects treated with FVIII other than Product ‘A’, only one showed higher antibody titre when measured with this product. Our data suggest that the development of anti‐FVIII antibodies is biased towards the product used for treatment and that a significant fraction of antibodies bind to the B domain of FVIII.     

Inhibitor development after switching of FVIII concentrate in multitransfused patients with severe haemophilia A

Switching between different therapeutic FVIII concentrate types has been postulated as a possible cause of inhibitor development in patient with haemophilia A. In this single‐centre, retrospective study, the incidence, titre and duration of inhibitor development in multitransfused patients, defined as patients with more than 150 exposure days (ED), were analysed from January 1970 to December 2007 in relation to ED and the number of switches between different products. Inhibitor titre was assessed by Bethesda assay (before 1998) or Nijmegen assay (after 1998). Medical records of 167 patients were screened, of which 97 patients met the inclusion criteria. Fourteen products of plasmatic origin (different purities) and five recombinant (three generations) were used. Nine patients (9%) developed inhibitors, all transient, low‐titre (1.41 ± 0.54 BU) after 323 ± 287 ED in average. Seventeen patients had no product switches of which four patients (23%) developed inhibitors (97 ED in average), whereas 13 patients (77%) did not (ED: 230). Fifty patients switched between plasmatic products only (median: 10 changes) of which five patients (10%) developed inhibitors (ED: 503), whereas 45 patients did not (ED: 932). Five patients switched between recombinant products only (seven changes) of which no patient developed inhibitors (748 ED). Twenty‐five patients switched between plasmatic and recombinant products (13 changes) of which no patient developed inhibitors (ED: 1654). No statistically significant differences between patient groups were observed. Neither the number of different FVIII products administered nor the switching of products influenced the incidence of inhibitor in multitransfused patients.     

Long‐term tolerability,immunogenicity and efficacy of Nuwiq® (human‐cl rhFVIII) in children with severe haemophilia A

Introduction

Nuwiq^® (human‐cl rhFVIII, simoctocog alfa) is a 4th generation recombinant human FVIII, without chemical modification or fusion with any other protein, produced in a human cell line.

Aim/Methods

This study (GENA‐13) was an extension of the GENA‐03 study in which previously treated children aged 2‐12 years with severe haemophilia A received Nuwiq^® prophylaxis for ≥6 months. GENA‐13 examined long‐term tolerability, immunogenicity and efficacy of Nuwiq^® prophylaxis in children.

Results

Of 59 patients enrolled in GENA‐03, 49 continued Nuwiq^® prophylaxis in GENA‐13 for a median (range) of 30.0 (9.5‐52.0) months. No patient withdrew due to drug‐related adverse events or developed inhibitors. Only 2 of 20 518 infusions were associated with possibly related adverse events (dyspnoea, fever). The estimated annualized bleeding rate (ABR) was 0.67 (95% CI: 0.44, 1.02) for spontaneous and 2.88 (95% CI: 1.86, 4.46) for all bleeds. Younger children (2‐5 years) had lower ABRs than children aged 6‐12 years. Annualized bleeding rates were reduced in GENA‐13 vs GENA‐03, especially for spontaneous bleeds in younger children (71% reduction; ABR ratio 0.29 [95% CI: 0.11, 0.74]). Nuwiq^® efficacy was rated as excellent/good in the treatment of 83.0% of 305 evaluated breakthrough bleeds. Surgical prophylaxis with Nuwiq^® was rated as excellent for all 17 assessed procedures.

Conclusion

Long‐term treatment with Nuwiq^® for the prevention of bleeds in children with severe haemophilia A was well tolerated, effective and reduced spontaneous bleeding by up to 70% compared with GENA‐03.     

A flow cytometry evaluation of anti‐FVIII antibodies: correlation with ELISA and Bethesda assay

Summary. In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme‐linked immunosorbent assay (ELISA) that detects both inhibitory (I‐Ab) and non‐inhibitory (NI‐Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I‐Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII‐m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control‐m). Captured anti‐FVIII antibodies were detected using anti‐human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I‐Ab, <0.5 BU mL^?1); 13 PI (patients with I‐Ab, 1.1–8200 BU mL^?1). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII‐m and control‐m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI‐Ab by FC, and two of them developed high levels of I‐Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 μL of plasma or serum is required especially making it useful for paediatric patients.     

Immunoglobulin isotypes and functional anti‐FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice

Summary. Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti‐factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16‐disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T‐ and B‐cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti‐FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII‐treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII‐treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro‐environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain‐dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.     

Genotype and phenotype of haemophilia A in Thai patients

To study genotype and phenotype correlation of haemophilia A in Thai patients, molecular defects of the factor VIII (FVIII) gene were examined and their correlation with clinical phenotypes were evaluated. The molecular pathologies of FVIII in Thai patients were found to be heterogeneous. The most common mutation was FVIII intron 22 inversion accounting for about 30% of the severe cases while gene deletion was rare. Sixteen point mutations were identified, comprising two nonsense mutations (R-5X and R1966X), five missense mutations (T233I, D542Y, G1850V, W2229S and G2325C), five nucleotide deletions (1145delT, 1187-8delACAC, 1191-4delA, 1458delGA and 1534delA), three nucleotide insertions (1439-41insA, 1934insTA and 2245insACTA) and one splicing defect (IVS15+1G>T). Nine mutations (T233I, D542Y, 1145delT, 1458delGA, 1534delA, 1934insTA, W2229S, 2245insACTA and G2325C) were novel, firstly identified in Thai patients. The genotypes were found to correlate with clinical phenotypes in a majority of cases. However, in five patients the molecular defects did not correlate with clinical severity and FVIII:C level. Cellular and molecular mechanisms were proposed to be responsible in amelioration of clinical severity caused by deleterious mutations. Carrier detection by direct mutation analysis was also demonstrated.     

Severe haemophilia A patients have reduced numbers of peripheral memory B cells

The development of inhibitors is a complication of replacement treatment in Haemophilia. Loss of factor VIII-specific memory B cells in the spleen is associated with down regulation of antibodies in mice treated with high doses of FVIII, but changes in B cell memory have not been described in haemophilic patients. The aim of this study was to evaluate the phenotype of circulating lymphocytes in severe haemophilia A. Twenty patients with inhibitors (PI), 22 without inhibitors (P), nine patients during immune tolerance induction (ITI) treatment and 20 healthy donors (HD) were included. Peripheral blood lymphocytes were examined using flow cytometry. Anti-FVIII antibodies were measured using Bethesda and flow cytometry. Percentages of T subsets and B lymphocytes were similar in all groups. In contrast, memory B cells (CD27+) were decreased in PI and P compared with HD, but the level of significance was higher in PI (P = 0.001) than P (P = 0.01). PI with high level of anti-FVIII antibodies presented the lowest B memory values. CD70 expression was also lowest in PI. Non-switched CD27+ subpopulation (IgD+) was prevalent in PI, but did not show statistical significance. When ITI failed, the percentages of CD27+ B cells after 12 months of ITI were lowest. In a longitudinal study performed in four patients, an increased percentage of CD27+ and CD70+ B cells during ITI was found. This work suggests that different peripheral lymphocyte markers, such as CD27 and CD70 on B cells, may be helpful to evaluate anti-FVIII response and to monitor the success of ITI.     

A qualitative and quantitative analysis of von Willebrand factor contained in a very high-purity plasma-derived FVIII concentrate

Background and Objectives We studied the structural and functional properties of von Willebrand factor (VWF) molecules present in a very high‐purity plasma‐derived factor VIII concentrate (VHP pdFVIII – Factane^®) because several observations suggest that the presence of VWF in factor VIII (FVIII) preparations may decrease their immunogenicity. Materials and Methods Ten marketed batches of VHP pdFVIII (Factane^®) with levels of VWF ranging from 15 to 39 IU/100 IU FVIII were analysed. The VWF multimeric pattern was studied by agarose gel electrophoresis. The binding of VWF to FVIII was studied by gel filtration and ELISA. The binding of VWF to GPIb was analysed by ELISA. Results The results showed that high‐molecular‐weight multimers of VWF were present in VHP pdFVIII (Factane^®). VWF subunits maintain a triplet structure similar to that of normal plasma. Regardless of the VWF content, all FVIII molecules of each batch were co‐eluted with VWF, and no free FVIII was detectable. By immunoassays, VWF was found to be able to bind to FVIII and platelet GPIb in a similar manner to that of VWF in normal plasma. Conclusions In all the VHP pdFVIII (Factane^®) batches studied, regardless of the level of VWF, the structure and capacity of VWF binding to FVIII and to platelet GPIb were fully preserved.     

Practical and cost‐effective measurement of B‐domain deleted and full‐length recombinant FVIII in the routine haemostasis laboratory

The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product‐specific laboratory standard is recommended when measuring Refacto‐AF^R activity in plasma with a one‐stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one‐stage and a chromogenic assay calibrated either with pooled normal plasma or a product‐specific laboratory standard. Results obtained with the one‐stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product‐specific laboratory standard and plasma can be established. In patients' plasma, Refacto‐AF levels measured with a one‐stage FVIII assay calibrated with plasma or a product‐specific laboratory standard diverged from ?58% to ?17% and from ?25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full‐length rFVIII measured with the one‐stage assay calibrated with plasma were 6–49% lower than with the chromogenic assay. In a monocentric setting, the long‐term stability of the calibration curves allows the implementation of a practical and cost‐effective approach to determine rFVIII:C levels.     

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.