A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under nonhemophilic conditions was maintained by programmed death (PD) ligand 1–expressing (PD-L1–expressing) regulatory T cells (Tregs) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Tregs and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1⁺ Tregs and reengaged removal of inhibitor-forming B cells. We also demonstrated the existence of FVIII-specific Tregs in humans and showed that such Tregs upregulated PD-L1 in patients with hemophilia after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1–mediated B cell tolerance against FVIII operated in healthy individuals and in patients with hemophilia A without inhibitors, and that ITI reengaged this mechanism. These findings may impact monitoring of ITI success and treatment of patients with hemophilia A.

In Belgium, a total of three Vaccinium species with a shrub or dwarf shrub growth form can be found: Vaccinium myrtillus L., Vaccinium vitis-idaea L., and Vaccinium uliginosum L. Vaccinium uliginosum is the only one of these for which the extent of clonality is unknown. Therefore, the clonal structure of two V. uliginosum populations was inferred from random amplified polymorphic DNA (RAPD) markers. Among the 47 sampled patches, 61 clones were identified. The mean values of the number of clones divided by the number of samples (G/N), the Simpson's index (D), and the genotypic evenness (E) were 0.28, 0.95, and 0.96, respectively. Vaccinium uliginosum exhibited a typical phalanx growth strategy that resulted in structured populations at the ramet level, that is, ramets belonging to the same clone were closely associated and formed distinct clumps. However, at the clone level, populations were not structured, that is, genetic distances between pairs of clones were not correlated with the spatial distances between the clones within a population. Genetic diversity was as high as that in nonclonal species (mean value of Shannon's diversity index (H_o) = 0.647). In accord with the life history traits of V. uliginosum (long-lived species with a mixed breeding system and potentially high seed dispersal), most of the genetic variation was found within populations.Key words: genetic diversity, genetic structure, seedling recruitment, vegetative propagation, Ericaceae.

Replacement therapy in severe hemophilia A leads to factor VIII (FVIII) inhibitors in 30% of patients. Factor VIII gene (F8) mutation type, a family history of inhibitors, ethnicity and intensity of treatment are established risk factors, and were included in two published prediction tools based on regression models. Recently investigated immune regulatory genes could also play a part in immunogenicity. Our objective is to identify bio-clinical and genetic markers for FVIII inhibitor development, taking into account potential genetic high order interactions. The study population consisted of 593 and 79 patients with hemophilia A from centers in Bonn and Frankfurt respectively. Data was collected in the European ABIRISK tranSMART database. A subset of 125 severely affected patients from Bonn with reliable information on first treatment was selected as eligible for risk stratification using a hybrid tree-based regression model (GPLTR). In the eligible subset, 58 (46%) patients developed FVIII inhibitors. Among them, 49 (84%) were “high risk” F8 mutation type. 19 (33%) had a family history of inhibitors. The GPLTR model, taking into account F8 mutation risk, family history of inhibitors and product type, distinguishes two groups of patients: a high-risk group for immunogenicity, including patients with positive HLA-DRB1*15 and genotype G/A and A/A for IL-10 rs1800896, and a low-risk group of patients with negative HLA-DRB1*15 / HLA-DQB1*02 and T/T or G/T for CD86 rs2681401. We show associations between genetic factors and the occurrence of FVIII inhibitor development in severe hemophilia A patients taking into account for high-order interactions using a generalized partially linear tree-based approach.

Fidanacogene elaparvovec is an adeno-associated virus (AAV)-based gene therapy that enables endogenous factor IX (FIX) expression in hemophilia B, potentially eliminating the need for prophylactic FIX replacement. Post-treatment monitoring focuses on FIX durability, hepatic safety, and immune-mediated loss of expression. A 40-year-old man with severe hemophilia B received a single intravenous dose of fidanacogene elaparvovec in the phase 3 BENEGENE-2 trial (NCT03861273). FIX activity was assessed by one-stage and chromogenic assays. Liver enzymes, clinical parameters, and adverse events were monitored. Prednisolone was initiated for liver enzyme elevation and later switched to budesonide in response to declining FIX levels. Follow-up continued for 4 years. FIX activity peaked at week 6 (~30 IU/dL) and remained stable until week 16. Moderate ALT elevation (peak ~70 U/L) responded to prednisolone. Between weeks 22–28, FIX levels declined without ALT elevation. Budesonide (9 mg/day) was started at week 25 for 3 months and gradually tapered, stabilizing FIX activity at 10-20 IU/dL. This level has been maintained for 4 years without bleeding episodes or FIX replacement. Fidanacogene elaparvovec provided sustained, clinically meaningful FIX expression. Budesonide may offer an effective hepatocyte-targeted alternative to systemic corticosteroids while supporting long-term transgene expression.

Introduction: The standard therapy for hemophilia A patients is the replacement of deficient factor (F) VIII with plasma-derived or recombinant FVIII concentrates. This replacement therapy can be complicated due to the potential occurrence of (neutralizing) antibodies and subsequent short half-life or even completely diminished activity of FVIII. Several strategies have been followed to optimize the pharmacokinetics of therapeutic FVIII, including coupling to polyethylene glycol (PEG). Presumably, the use of PEG should increase the half-life of FVIII and may reduce the frequency of administration to the patients. Method: For detection and characterization of antidrug antibodies (ADAs), an in-house multiplex microsphere-based immunoassay based on Luminex (LumiTope), an in-house ELISA, and a flow cytometry assay were established. The specificity of the identified ADAs was confirmed by competitive immunoassays. Based on a modified Nijmegen-Bethesda assay (NBA), the neutralizing nature of the ADAs was determined. Results: A mild hemophilia A patient developed ADAs after treatment with an extended half-life FVIII product PEGylated with a 40-kDa PEG. Our in-house immunoassays detected ADAs raised against both, the FVIII and PEG fraction of the therapeutic FVIII. The anti-FVIII antibodies were of immunoglobulin G (IgG) isotype only, whereas IgG, IgM, and IgA anti-PEG antibodies were detected. Competitive assays confirmed the respective specificity of the ADAs against FVIII or PEG. NBA revealed that anti-PEG antibodies were neutralizing while anti-FVIII antibodies were nonneutralizing. The PEG antibody cross-reacted with other PEGylated FVIII modified with different PEG reagents. The ADAs against PEG were persistent and could be detected several months after discontinuation of treatment with the PEGylated FVIII. Conclusion: Inhibitors against the PEG moiety of FVIII therapeutics may abolish the efficacy of the product. Accordingly, screening for antibodies against PEGylated FVIII proteins may be useful before and during the treatment. Article published online: 26 October 2022 © 2022. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany