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. 2024 Feb 22:15:1355813.
doi: 10.3389/fimmu.2024.1355813. eCollection 2024.

The spectrum of neutralizing and non-neutralizing anti-FVIII antibodies in a nationwide cohort of 788 persons with hemophilia A

Ilja Oomen #  1   2 , Marieke Verhagen #  3   4   5 , Mariarosaria Miranda  2 , Peter Allacher  6 , Erik A M Beckers  7 , Nicole M A Blijlevens  3   4 , Johanna G van der Bom  8 , Michiel Coppens  9 , Mariëtte Driessens  10 , Jeroen C J Eikenboom  11 , Karin Fijnvandraat  1   2 , Shermarke Hassan  8   12 , Waander L van Heerde  3   4   13 , H Louise Hooimeijer  14 , Joop H Jansen  5 , Paul Kaijen  2 , Frank W G Leebeek  15 , Daniëlle Meijer  5 , Helmut Paul  6 , Sanna R Rijpma  5 , Frits R Rosendaal  11 , Cees Smit  9   11 , Lize F D van Vulpen  16 , Jan Voorberg  2   8 , Saskia E M Schols #  3   4 , Samantha C Gouw #  1   8
Affiliations

The spectrum of neutralizing and non-neutralizing anti-FVIII antibodies in a nationwide cohort of 788 persons with hemophilia A

Ilja Oomen et al. Front Immunol. .

Abstract

Objectives: Anti-factor VIII (FVIII) antibodies have been reported to exhibit both neutralizing and non-neutralizing characteristics. This is the first study investigating the full spectrum of FVIII-specific antibodies, including non-neutralizing antibodies, very-low titer inhibitors, and inhibitors, in a large nationwide population of persons with hemophilia A of all severities.

Methods: All persons with hemophilia A (mild (FVIII > 5-40 IU/dL)/moderate [FVIII 1-5 IU/dL)/severe (FVIII < 1 IU/dL)] with an available plasma sample who participated in the sixth Hemophilia in the Netherlands study between 2018 and 2019 were included. The presence of anti-FVIII antibodies of the immunoglobulin A, M, and G isotypes and IgG subclasses, along with antibody titer levels, were assessed using direct-binding ELISAs. FVIII specificity was assessed using a competition-based ELISA approach. The inhibitor status was determined using the Nijmegen ultra-sensitive Bethesda assay (NusBA) and the Nijmegen Bethesda assay (NBA).

Results: In total, 788 persons with hemophilia A (336 (42.6%) mild, 123 (15.6%) moderate, 329 (41.8%) severe hemophilia) were included. The median age was 45 years (IQR 24-60), and the majority (50.9%) had over 150 exposure days to FVIII concentrates. Within our population, 144 (18.3%) individuals had non-neutralizing FVIII-specific antibodies, 10 (1.3%) had very low-titer inhibitors (NusBA positive; NBA negative), and 13 (1.6%) had inhibitors (both NusBA and NBA positive). IgG1 was the most abundant FVIII-specific antibody subclass, and the highest titer levels were found for IgG4. In individuals without a reported history of inhibitor development, no clear differences were observed in antibody patterns between those who were minimally or highly exposed to FVIII concentrates. IgG4 subclass antibodies were only observed in persons with a reported history of FVIII inhibitor or in those with a currently detected (very low-titer) inhibitor.

Conclusion: In this cross-sectional study, we identified non-neutralizing antibodies in a relatively large proportion of persons with hemophilia A. In contrast, in our population, consisting of persons highly exposed to FVIII concentrates, (very low-titer) inhibitors were detected only in a small proportion of persons, reflecting a well-tolerized population. Hence, our findings suggest that only a small subpopulation of non-neutralizing FVIII-specific antibodies is associated with clinically relevant inhibitors.

Keywords: ELISA; antibody; factor VIII; hemophilia A; immunoglobulin A; immunoglobulin G; immunoglobulin M.

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Conflict of interest statement

IO was supported by an unrestricted research grant from SOBI, the EAHAD research grant, the Heimburger Award, and the Martin Villar research grant. MM received funding from the European Community H2020-MSCA-ITN-2019 project 859974 EDUC8. SG received unrestricted research funding from SOBI, EAHAD, CSL Behring Heimburger Award, Grifols Martin Villar Award, and the Netherlands Organization for Scientific Research NWO. KF has received unrestricted research grants from CSL Behring, SOBI, and Novo Nordisk and consultancy fees from SOBI, Novo Nordisk, and Roche all fees to the institution. FL has received grants/research funding from CSL Behring, UniQure, SOBI, and Takeda and consultancy fees from Biomarin, CSL Behring, Takeda, and Uniqure all fees to the institution. FL also served as a DSMB member for a study sponsored by Roche. JE received research funding from CSL Behring funds to the institution. MC has received financial support for research from Anthos, Bayer, CSL Behring, Novo Nordisk, and Roche, as well as an honoraria for lecturing or consultancy from Alexion, Bayer, CSL Behring, Daiichi Sankyo, Octapharma, Pfizer, SOBI, and Viatris. Nonfinancial conflict of interest: member of the gene therapy working group of the European Association for Haemophilia and Allied Disorders EAHAD, member of the European Reference Network ERN EuroBloodNet, chair of the working group thrombosis and hemostasis of the Dutch Society of Vascular Medicine NVIVG, part of the Dutch Internist’s Society NIV. JV is listed as an inventor on a patent application on ADAMTS13 variants. SS has received a research grant from Bayer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic overview of the study methods to assess the prevalence and characteristics of non-neutralizing antibodies, very low-titer inhibitors, in our population. ELISA, enzyme-linked immunoassay; NusBA, Nijmegen ultra-sensitive Bethesda Assay. Participants were recruited from the sixth Hemophilia in the Netherlands cohort; all had an available plasma sample. Laboratory analysis consisted of ELISA approaches to detect FVIII-specific antibodies (direct-binding ELISA approaches were used to detect FVIII-binding antibodies and to assess the antibody titer levels, and FVIII specificity was confirmed by using a competition-based ELISA approach), and the Nijmegen ultra-sensitive Bethesda assay and Nijmegen Bethesda assay were used to detect very low-titer inhibitors or inhibitors. Icons were created using BioRender.
Figure 2
Figure 2
Prevalence of non-neutralizing FVIII-specific antibodies, very low-titer inhibitors, and inhibitors in the study population of 788 persons with haemophilia A. N, number; FVIII, factor VIII; ELISA, enzyme-linked immunoassay; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M. It should be mentioned that in the total cohort, 165 FVIII-specific antibodies were detected by ELISA. These 165 antibodies were classified as NNA (n = 144; both NusBA and NBA negative), very low-titer inhibitor (n = 7/10 ELISA positive, all NusBA positive), and inhibitor (n = 11/13 ELISA positive, all NusBA and NBA positive), and three persons with ELISA-positive results were unclassified due to missing NusBA and NBA data (n = 3). For non-neutralizing antibodies, the antibody isotype is composed of IgA (n = 17; 11.8%), IgG (n = 114; 79.2%), IgM (n = 6; 4.2%), IgG and IgA (n = 4; 2.8%), or IgG and IgM antibodies (n = 3; 2.1%). For very low-titer inhibitors, the antibody isotype included IgA (n = 1; 10.0%) and IgG (n = 6; 60.0%), and three (30.0%) persons were ELISA negative. For inhibitors, the antibody isotype included IgG (n = 10; 76.9%) and IgG and IgA antibodies (n = 1; 7.7%), and two (15.4%) persons were ELISA negative.
Figure 3
Figure 3
Characteristics of non-neutralizing FVIII-specific antibodies, very low-titer inhibitors, and inhibitors. y, year; n, number of participants; N, total number of participants; CED, cumulative exposure days; FVIII, factor VIII; ITI, immune tolerance induction. Titer levels are presented with a median and an interquartile range. Each dot represents one positive measurement. Some participants only have one antibody present, whereas others have multiple antibodies in their sample. For non-neutralizing antibodies (A); antibody isotype and subclass and titer levels are presented according to different subgroups of participants. (A) Left panel: data of individuals without a history of inhibitor development, divided into different parts; data of persons with < 50 CED to FVIII (n = 51, left part); persons with 50–150 CED to FVIII (n = 13, middle part); and persons highly exposed to FVIII (CED > 150) (n = 59; right part). No IgG4 subclass antibodies were present in persons with NNA without a history of inhibitor development. For persons with non-neutralizing antibodies, the table presents data for 782 participants, as data on ITI therapy were missing for six participants (one mild (< 50 CED), one moderate (>150 CED), and four severe (> 150 CED) hemophilia A). Middle panel: NNA data of persons with a history of inhibitor development who did not receive ITI treatment. Right panel: NNA data for persons with a history of inhibitor development who did not receive ITI treatment. (B) Antibody titer levels obtained in ELISA approaches of seven (seven of 10) participants with very low-titer inhibitors, as the remaining three participants were ELISA negative (but NusBA positive). (C) Antibody titer levels obtained in ELISA approaches of 11 (11/13) participants with inhibitors, as the remaining two participants were ELISA negative (but NusBA and NBA positive).
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