Adin Sejdic ^1,2 Hans J. Hartling ³ Jon G. Holler ¹ Lars Klingen Gjaerde ⁴ Birgitte Lindegaard ^1,2 Arnold M. Dungu ¹ Filip Gnesin ⁵ Maria Moeller ³ Rebecca S. Teglgaard ³ Carsten Niemann ^2,4 Patrick T. Brooks ³ Charlotte S. Jørgensen ⁶ Kristina T. Franck ⁶ Thea K. Fischer ^2,7 Hanne Vibeke H. Marquart ^2,3 Zitta Barrella Harboe ^1,2* Sisse R. Ostrowski ^2,3

• ¹ Department of Pulmonary and Infectious Diseases, Copenhagen University Hospital, Hilleroed, Denmark
• ² University of Copenhagen, Copenhagen, Capital Region of Denmark, Denmark
• ³ Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Capital Region of Denmark, Denmark
• ⁴ Department of Hematology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Zealand, Denmark
• ⁵ Department of Cardiology, Rigshospitalet, University of Copenhagen, Copenhagen, Capital Region of Denmark, Denmark
• ⁶ Department of Virus and Microbiological Diagnostics, Statens Serum Institut, Copenhagen, Denmark
• ⁷ Department of Clinical Research, Copenhagen University Hospital, Hillerød, Denmark

Vaccine breakthrough SARS-CoV-2 infections are common and of clinical and public health concern. However, little is known about the immunological characteristics of patients hospitalized due to these infections. We aimed to investigate and compare immune cell subpopulations and induced immune responses in vaccinated and non-vaccinated patients hospitalized with severe COVID-19.A nested case-control study on adults (> 18 years) who received at least two doses of a mRNA-COVID-19 vaccine and were hospitalized with SARS-CoV-2 breakthrough infections and severe COVID-19 between January 7, 2021, and February 1, 2022, were eligible for inclusion. Age-and sex-matched non-vaccinated controls were identified. Immunophenotyping was performed using a custom-designed 10-color flow cytometry prefabricated freeze-dried antibody panel (DuraClone, Beckman Coulter (BC), Brea, Calif). TruCulture (Myriad RBM, Austin, USA) was used to assess induced immune response in whole blood, revealing different critical signaling pathways as a proxy for immune function. All samples were obtained within 48 hours of admission.In total, 20 hospitalized patients with severe COVID-19 and a breakthrough SARS-CoV-2 infection were included, ten vaccinated and ten non-vaccinated patients. Vaccinated patients had lower concentrations of CD19 B cells (p = 0.035), naïve CD4 T cells (p = 0.015), a higher proportion of γδ1 T cells (p = 0.019), and higher unstimulated immune cell release of IL-10 (p = 0.015).We observed immunological differences between vaccinated and non-vaccinated patients hospitalized due to severe COVID-19 that indicate that vaccinated patients had lower B cell concentrations, lower concentrations of CD4 naïve T cells, a skewed gamma-delta V1/V2 ratio, and an exaggerated IL-10 response at admission. These results could indicate a suboptimal immune response involved in SARS-CoV-2 breakthrough infections that cause severe COVID-19 in vaccinated adults. However, the sample size was small, and further research is needed to confirm these results.