RSS-Feed abonnieren
PDF herunterladen
DOI: 10.1055/a-2736-5418
Free Nucleic Acids in the ChAdOx1 nCov-19-S Adenovirus Vector Vaccine Contribute to an Anti-platelet Factor 4 Antibody Response
Autoren
Funding Information This work was supported by a starting grant of the “Forschungsverbund Molekulare Medizin” by a scholarship of the “Gerhard-Domagk Masterclass” of the University Medicine Greifswald and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Project Number 493623784 and Gerhard Domagk Nachwuchsförderprogramm (Clinician Scientist Program Rural_Age Scholarship) to F.S. This research was funded by Deutsche Forschungsgemeinschaft (DFG), grant numbers 514598754, 374031971-TRR240, GR 2232/9-1, SCHO 2052/1-1, TH 2320/3-1. L.M. received initial funding (Anschubfinanzierung) by Universitätsmedizin Greifswald. L.S. was supported by the Else Kröner-Fresenius Stiftung, by the American Society of Hematology with the ASH Global Research Award and within the Gerhard Domagk Research Program by the Universitätsmedizin Greifswald. Some parts of the study were funded by EMA service contract No. EMA/2021/17/TDA. The views expressed in this report are the personal views of the authors and may not be understood as representing the position of the European Medicines Agency or one of its Committees or Working Parties. AstraZeneca provided the vaccination stock and the dilution buffers.
Abstract
Background
Vaccination against SARS-CoV-2 was instrumental in controlling the COVID-19 pandemic. Rare cases of vaccine-induced immune thrombocytopenia and thrombosis (VITT) emerged following vaccination with the adenovirus vector-based vaccines ChAdOx1 nCov-19-S and Ad26.COV2.S. VITT is mediated by high-titer IgG anti-platelet factor 4 (PF4) antibodies that activate platelets, leading to thrombosis and thrombocytopenia. Similar antibodies have been detected following natural adenovirus infections, suggesting a common immunological trigger. This indicates that a constituent of adenovirus is relevant. Adenovirus is a DNA virus. Virion-unbound viral DNA is present in natural adenovirus infections.
Objectives
To identify whether free virion-unbound DNA is present in ChAdOx1-nCoV19 vaccine and whether adenoviral DNA enhances the immune response to PF4 in mice.
Methods
We assessed ChAdOx1 nCov-19-S for virion-unbound DNA and differentiated free human and free adenovirus DNA by sequencing. We immunized mice with ChAdOx1 nCov-19-S and its fractions, in which we removed proteins by proteinase K and/or DNA by DENERASE.
Results
Using ultracentrifugation and proteinase K digestion, we isolated and characterized free nucleic acids, confirming the presence of both adenoviral and host cell-derived DNA in ChAdOx1 nCov-19-S. Mice immunized with PF4 in combination with ChAdOx1 nCov-19-S or its virion-free supernatant—but not with PF4 alone—developed a strong anti-PF4 IgG response, an effect completely abolished by nuclease (DENARASE) treatment.
Conclusion
Virion-unbound DNA in ChAdOx1 nCov-19-S contributes to anti-PF4 antibody formation. This highlights the potential of reducing virion-unbound DNA in vaccine formulations to mitigate unintended immune responses to PF4.
Data Availability Statement
Animal study data and DNA analysis data may be found in the data supplement available with the online version of this article. Additional data can be obtained from the corresponding author upon reasonable request.
Contributors' Statement
F.S., J.V.D.B., and L.M.: established the animal model and performed all mouse experiments; F.S.: established the anti-Spike IgG ELISA and performed the histology; A.R. and S.M.: prepared the vaccine fractions and performed proteomic and nucleic acid analysis; A.R. and J.H.: recombinantly produced mPF4; R.P.: performed the DLS experiments; J.W.: performed mPF4 ELISAs; L.S. and T.T.: contributed to study design and VITT pathogenesis concept; N.E., U.V., and A.G.: supervised the study and analyzed data.
‡ These authors share first authorship.
Publikationsverlauf
Eingereicht: 23. September 2025
Angenommen nach Revision: 01. November 2025
Accepted Manuscript online:
03. November 2025
Artikel online veröffentlicht:
13. November 2025
© 2025. Thieme. All rights reserved.
Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany
-
References
- 1 Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA, Eichinger S. Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination. N Engl J Med 2021; 384 (22) 2092-2101
- 2 Schultz NH, Sørvoll IH, Michelsen AE. et al. Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination. N Engl J Med 2021; 384 (22) 2124-2130
- 3 Scully M, Singh D, Lown R. et al. Pathologic antibodies to platelet factor 4 after ChAdOx1 nCoV-19 vaccination. N Engl J Med 2021; 384 (23) 2202-2211
- 4 Greinacher A, Warkentin TE. Thrombotic anti-PF4 immune disorders: HIT, VITT, and beyond. Hematology (Am Soc Hematol Educ Program) 2023; 2023 (01) 1-10
- 5 Pavord S, Scully M, Hunt BJ. et al. Clinical features of vaccine-induced immune thrombocytopenia and thrombosis. N Engl J Med 2021; 385 (18) 1680-1689
- 6 Jaax ME, Krauel K, Marschall T. et al. Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4. Blood 2013; 122 (02) 272-281
- 7 Phung Q, Lin MJ, Xie H, Greninger AL. Fragment size-based enrichment of viral sequences in plasma cell-free DNA. J Mol Diagn 2022; 24 (05) 476-484
- 8 Bazemore K, Permpalung N, Mathew J. et al. Elevated cell-free DNA in respiratory viral infection and associated lung allograft dysfunction. Am J Transplant 2022; 22 (11) 2560-2570
- 9 Greinacher A, Selleng K, Palankar R. et al. Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia. Blood 2021; 138 (22) 2256-2268
- 10 Michalik S, Siegerist F, Palankar R. et al. Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines. Haematologica 2022; 107 (04) 947-957
- 11 Warkentin TE, Greinacher A. Laboratory testing for heparin-induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia antibodies: a narrative review. Semin Thromb Hemost 2023; 49 (06) 621-633
- 12 Greinacher A, Warkentin TE. Platelet factor 4 triggers thrombo-inflammation by bridging innate and adaptive immunity. Int J Lab Hematol 2023; 45 (Suppl. 02) 11-22
- 13 Schönborn L, Esteban O, Wesche J. et al. Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure. Blood 2023; 142 (26) 2305-2314
- 14 See I, Lale A, Marquez P. et al. Case series of thrombosis with thrombocytopenia syndrome after COVID-19 vaccination—United States, December 2020 to August 2021. Ann Intern Med 2022; 175 (04) 513-522
- 15 Wang JJ, Schönborn L, Warkentin TE. et al. Antibody fingerprints linking adenoviral anti-PF4 disorders. N Engl J Med 2024; 390 (19) 1827-1829
- 16 Nicolson PLR, Montague SJ, Buka RJ. et al. Anti-PF4 mediated thrombocytopenia and thrombosis associated with acute cytomegalovirus infection displays both HIT-like and VITT-like characteristics. Br J Haematol 2025; 206 (06) 1737-1742
- 17 Cohen TS, Kelly EJ, Nylander S. et al. Serum levels of anti-PF4 IgG after AZD1222 (ChAdOx1 nCoV-19) vaccination. Sci Rep 2022; 12 (01) 7961
- 18 Kristyanto H, Slaets L, Braams E. et al. Assessment of antibodies against platelet factor 4 following vaccination with adenovirus type 26-vectored vaccines. J Thromb Haemost 2024; 22 (12) 3532-3541
- 19 Wang JJ, van der Neut Kolfschoten M, Rutten L. et al. Characterization of reverse-engineered anti-PF4 stereotypic antibodies derived from serum of patients with VITT. Blood 2024; 143 (04) 370-374
- 20 Treverton J, Arnold DM, Ivanov DG. et al. Monoclonal antibodies in the pathogenesis of heparin-induced thrombocytopenia. N Engl J Med 2025; 393 (09) 879-886