Children with hemophilia A lack clotting factor VIII (FVIII) due to a genetic mutation. It is well known that administration of FVIII concentrate leads to immunological tolerance for the FVIII protein in the majority of children. In 30% of these children tolerance is not achieved leading to the development of anti-FVIII antibodies (i.e. inhibitors). Our knowledge on the underlying immunological mechanisms leading to tolerance is limited. Recently, Non-Factor Therapy (NFT) has become available for prevention of bleeding in patients with hemophilia, i.e. prophylaxis. Currently, many children with severe hemophilia A use NFT as the subcutaneous administration of NFT is very convenient. In children on NFT prophylaxis, intravenous FVIII concentrate is exclusively used on-demand for treatment of bleeding. As NFT is very effective in the prevention of bleeds, patients may not be exposed to the deficient FVIII protein for periods up to a year or longer. It is currently not known how robust immunological tolerance is in the absence of exposure to a deficient antigen. The infrequent exposure to FVIII, enabled by NFT, provides an opportunity to study the immunological tolerance mechanisms for FVIII in children with hemophilia A. The aim of SPIRIT is to investigate the mechanisms of the immunological tolerance to FVIII in patients with hemophilia A aged younger than 18 years using NFT for prophylaxis. In this observational cohort study, children (aged \<18 years) with congenital hemophilia A, who are treated with non-factor therapy as prophylaxis, will be longitudinally followed. Participants will have blood drawn anually, during the regular clinic visits, and additionally following FVIII exposure. Feces samples will be collected and analyzed in children aged \<12 years, following the same scheme as blood sampling. The main study endpoint are the immunological mechanisms underlying tolerance to FVIII, including presence, titers, subtypes and affinities of FVIII-specific (non-)neutralizing antibodies, FVIII-specific T and B cell responses and the role of gut microbiota.
Study Type
OBSERVATIONAL
Enrollment
500
Amsterdam UMC, locatie AMC
Amsterdam, North Holland, Netherlands
FVIII-specific non-neutralizing antibody development
Patients will be longitudinally monitored for the development of FVIII-specific non-neutralizing antibodies (NNAs). The development of FVIII-specific NNAs will be assessed with a direct enzyme-linked immunosorbent assay (ELISA)(Optical Density (OD)), by reporting the presence of FVIII-specific NNAs (Yes/No). Incidence will be calculated as the proportion of patients who develop newly detectable FVIII-specific NNAs during the study period.
Time frame: From enrollment up to 5 years
Characterization of FVIII-specific antibodies (Immunoglobulin isotypes)
FVIII-specific antibodies will be characterized by measuring immunoglobulin isotypes (IgA, IgM, and IgG) over time using ELISA.
Time frame: From enrollment up to 5 years
Characterization of FVIII-specific antibodies (IgG subclasses)
FVIII-specific antibodies will be characterized by measuring IgG subclasses (IgG1, IgG2, IgG3, and IgG4) over time using ELISA.
Time frame: From enrollment up to 5 years
Characterization of FVIII-specific antibodies (affinity)
FVIII-specific antibodies will be characterized by measuring the affinity (KA \[M-1\]) over time using ELISA.
Time frame: From enrollment up to 5 years
Characterization of FVIII-specific antibodies (titer)
FVIII-specific antibodies will be characterized by measuring inhibitor titers (Bethesda Units (BU)/mL) over time using the Nijmegen-modified Bethesda assay.
Time frame: From enrollment up to 5 years
FVIII inhibitor development (neutralizing antibodies)
Participants will be longitudinally monitored for the development of FVIII-specific neutralizing antibodies using the Nijmegen-modified Bethesda assay (BU/mL). Inhibitor development will be defined as a titer ≥ 0.6 BU/mL confirmed on at least two consecutive measurements. Incidence will be calculated as the proportion of patients who develop confirmed FVIII inhibitors during the study period.
Time frame: From enrollment up to 5 years
FVIII-specific T and B cell responses
FVIII-specific T- and B-cell responses will be characterized by assessing differential gene expression profiles and immune activation signatures over time using RNA sequencing. FVIII-specific T- and B-cell responses will be compared between patients with FVIII-specific NNAs and/or inhibitors and patients without FVIII-specific NNAs and/or inhibitors.
Time frame: From enrollment up to 5 years
Immunomodulatory microbial metabolites (in children <12 years)
In participants younger than 12 years of age, concentrations of immunomodulatory microbial metabolites (i.e. short chain fatty acids, including butyrate and/or tryptophan catabolites (umol/g)) in fecal samples will be assessed over time using high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Concentrations of immunomodulatory microbial metabolites in fecal samples will be compared between patients with FVIII-specific NNAs and/or inhibitors and patients without FVIII-specific NNAs and/or inhibitors.
Time frame: From enrollment up to 5 years
Gut microbiota composition (in children <12 years)
In participants younger than 12 years of age, microbial composition, including diversity indices and diversity indices, in fecal samples will be assessed over time using 16S rRNA gene sequencing. Gut microbiota composition in fecal samples will be compared between patients with FVIII-specific NNAs and/or inhibitors and patients without FVIII-specific NNAs and/or inhibitors.
Time frame: From enrollment up to 5 years
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