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In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

Introduction The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII in on-demand treatment, increasing the inhibitor risk. Aim To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. Method HA rats were divided into two groups: one group (n = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg−1 rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding antibodies were analysed using an enzyme-linked immunosorbent assay and neutralizing antibodies using a Bethesda-like assay. Results Rats in the knee-bleed group developed a significantly faster inhibitor response and reached significantly higher inhibitor levels. In the knee-bleed group, 80% developed inhibitors vs. 33% in the control group, demonstrating a 2.4 times higher inhibitor risk when treating concurrent with bleeds. Conclusion FVIII treatment in relation to a bleed potentiates inhibitor development compared to FVIII treatment alone in this HA rat, indicating that bleeding is a potential danger signal. Our results support the theory that FVIII replacement therapy concurrent with a bleeding episode increases the inhibitor risk, which to the best of our knowledge, has not been confirmed in an animal model before.

Background: Neutralizing antibodies towardFVIII replacement therapy (inhibitors) are the most seri-ous treatment-related complication in hemophilia A(HA). A rat model of severe HA (F8/) has recentlybeen developed, but an immunological characterization isneeded to determine the value of using the model forresearch into inhibitor development. Objectives: Charac-terize the antibody response towards recombinant humancoagulation factor VIII (rhFVIII) in the HA rat, follow-ing a human prophylactic dosing regimen. Methods: Twoidentical studies were performed, which included a totalof 17 homozygous HA rats (F8/, 0% FVIII activity),12 heterozygous rats (F8+/), and 12 wild-type (F8+/+)rats. All rats received intravenous injections of rhFVIII at50 IU kg1twice weekly for 4 weeks. Predosing bloodsamples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1–7. Results: In both studies,antibodies developed after 4–6 administrations ofrhFVIII, and neutralizing antibodies reached levels simi-lar to human patients (range 1–111 BU, median 6.0 BU)at the end of the study. There was no significant differ-ence between the two studies or between genotypes intime to response or levels reached for binding and neu-tralizing antibodies. Interestingly, early spontaneousbleeds were associated with a faster antibody response.Conclusions: Following intravenous administration ofhuman FVIII, according to a clinical prophylaxis regi-men, a robust and reproducible antibody response is seenin this HA rat model, suggesting that the model is usefulfor intervention studies with the aim of suppressing,delaying, or preventing the inhibitor response. Also,bleeds seem to have an adjuvant effect on the immuneresponse., Background: Neutralizing antibodies towardFVIII replacement therapy (inhibitors) are the most seri-ous treatment-related complication in hemophilia A(HA). A rat model of severe HA (F8/) has recentlybeen developed, but an immunological characterization isneeded to determine the value of using the model forresearch into inhibitor development. Objectives: Charac-terize the antibody response towards recombinant humancoagulation factor VIII (rhFVIII) in the HA rat, follow-ing a human prophylactic dosing regimen. Methods: Twoidentical studies were performed, which included a totalof 17 homozygous HA rats (F8/, 0% FVIII activity),12 heterozygous rats (F8+/), and 12 wild-type (F8+/+)rats. All rats received intravenous injections of rhFVIII at50 IU kg1twice weekly for 4 weeks. Predosing bloodsamples were analyzed for binding and neutralizing anti-rhFVIII antibodies at weeks 1–7. Results: In both studies,antibodies developed after 4–6 administrations ofrhFVIII, and neutralizing antibodies reached levels simi-lar to human patients (range 1–111 BU, median 6.0 BU)at the end of the study. There was no significant differ-ence between the two studies or between genotypes intime to response or levels reached for binding and neu-tralizing antibodies. Interestingly, early spontaneousbleeds were associated with a faster antibody response. Conclusions: Following intravenous administration ofhuman FVIII, according to a clinical prophylaxis regi-men, a robust and reproducible antibody response is seenin this HA rat model, suggesting that the model is usefulfor intervention studies with the aim of suppressing,delaying, or preventing the inhibitor response. Also,bleeds seem to have an adjuvant effect on the immuneresponse.