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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
Authors
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.
Publication History
Received: 23 September 2025
Accepted after revision: 01 November 2025
Accepted Manuscript online:
03 November 2025
Article published online:
13 November 2025
© 2025. Thieme. All rights reserved.
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