Question: SARS-CoV-2 spike (S) specific T-cell and antibody (AB) responses after primary BNT162b2 vaccination partially depend on the phenotypic composition of pre-existing T-helper (Th) cell populations (Saggau et al., 2022). However, adaptive cellular responses and their impact on sustained immune protection after booster vaccination are incompletely understood. We monitored cellular and humoral immune responses before and up to 6 months after the 3^rd BNT162b2 vaccination to identify early determinants of sustained responses.

Methods: Blood of 30 healthy subjects was collected before (T0), and 1 and 6 months after (T1, T6) their 3^rd BNT162b2 vaccination. Serum samples were tested for nucleocapsid (N), S, and neutralizing AB responses. Subjects developing N IgG or those with incomplete follow-up were excluded. Whole blood was stimulated with S peptides (0.6 nMol/peptide) as previously described (Weis et al., 2020, Lauruschkat et al., 2021) and subsequently analyzed by flow cytometry (CCR7, CD3, CD4, CD45RO, CD69, CD154, IFN-γ) and 13-plex multiplex cytokine assays. Significant associations between readouts were identified by multiple testing-adjusted rank correlation analysis.

Results: We found moderate correlation between S IgG (r=0.47), S-induced IL‑2 (r=0.58) and IFN‑γ (r=0.43) at T1 and T6 (Fig. 1A). When comparing the top and bottom half of S IgG responders at T6, sustained robust IgG responses were significantly associated with S-specific (CD69⁺ ± CD154⁺ ± IFN-γ⁺) Th-cell frequencies at T0 (p=0.038), especially multifunctional type-1 Th cells. Furthermore, S IgG, S-induced IL‑2, and S-induced IFN‑γ at T6 were significantly associated with increased IL‑2 & IL‑4, IP‑10 & MCP1, and IFN‑γ & IP‑10 levels at T1, respectively (Fig. 1B). Using support vector machine models, pre-booster S-specific T-cell frequencies and cytokine responses at T1 predicted T6 responses with F1 scores of 0.82-1.00.

Conclusions: Pre-existing S-specific Th cells and T-cellular cytokine signatures shape sustained adaptive cellular and humoral responses after BNT162b2 booster vaccination. Functional T-cell assays might facilitate early identification of potential non-responders.