Lukas Page ^1* Kevin Dennehy ¹ Katharina Mueller ² Philipp Girl ^2,3,4 Eva Loell ¹ Hellen Buijze ¹ Johanna-Maria Classen ⁵ Helmut Messmann ⁵ Christoph Roemmele ⁵ Reinhard Hoffmann ¹ Sebastian Wurster ⁶ Andre Fuchs ⁵

• ¹ Institute for Laboratory Medicine and Microbiology, Augsburg University Hospital, Augsburg, Baden-Württemberg, Germany
• ² Bundeswehr Institute of Microbiology, Munich, Bavaria, Germany
• ³ Institute of Infection Medicine and Zoonoses, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, München, Bavaria, Germany
• ⁴ Central Institute of the Bundeswehr Medical Service Munich, Munich, Germany
• ⁵ Internal Medicine III – Gastroenterology and Infectious Diseases, Augsburg University Hospital, Augsburg, Baden-Württemberg, Germany
• ⁶ Department of Infectious Diseases, Infection Control and Employee Health, University of Texas MD Anderson Cancer Center, Houston, Texas, United States

Pre-existent pools of coronavirus-specific or cross-reactive T cells were shown to shape the development of cellular and humoral immune responses after primary mRNA vaccination against SARS-CoV-2. However, the cellular determinants of responses to booster vaccination remain incompletely understood. Therefore, we phenotypically and functionally characterized spike antigen-specific T helper (Th) cells in healthy, immunocompetent individuals and correlated the results with cellular and humoral immune responses to BNT162b2 booster vaccination over a six-month period. To that end, blood of 30 healthy healthcare workers was collected before, 1, 3, and 6 months after their 3rd BNT162b2 vaccination. Whole blood was stimulated with spike peptides and analyzed using flow cytometry, a 13-plex cytokine assay, and nCounter-based transcriptomics. Spike-specific IgG levels at 1 month after booster vaccination correlated with pre-existing CD154+CD69+IFN-γ+CD4+ effector memory cells as well as spike-induced IL-2 and IL-17A secretion. Early post-booster (1-month) spike IgG levels (r=0.49), spike-induced IL‑2 (r=0.58), and spike-induced IFN‑γ release (r=0.43) correlated moderately with their respective long-term (6-month) responses. Sustained robust IgG responses were significantly associated with S-specific (CD69+±CD154+±IFN-γ+) Th-cell frequencies before booster vaccination (p=0.038), especially double/triple-positive type-1 Th cells. Furthermore, spike IgG levels, spike-induced IL‑2 release, and spike-induced IFN‑γ release after 6 months were significantly associated with increased IL‑2 & IL‑4, IP‑10 & MCP1, and IFN‑γ & IP‑10 levels at 1 month post-booster, respectively. On the transcriptional level, induction of pathways associated with both T-cell proliferation and antigen presentation was indicative of sustained spike-induced cytokine release and spike-specific IgG production 6 months post-booster. Using support vector machine models, pre-booster spike-specific T-cell frequencies and early post-booster cytokine responses predicted sustained (6-month) responses with F1 scores of 0.80-1.00. In summary, spike-specific Th cells and T-cellular cytokine signatures present before BNT162b2 booster vaccination shape sustained adaptive cellular and humoral responses post-booster. Functional T-cell assays might facilitate early identification of potential non-responders.