Fig. 1: Antibody-independent protection in K18-hACE2 transgenic mice.

a, Experimental setup. Ab⁺ (n = 5) and Ab⁻ (n = 3–7) K18-hACE2 mice were primed with 2 × 10⁵ TCID₅₀ of SARS-CoV-2 D614G and rechallenged with a higher dose (1 × 10⁶ TCID₅₀) of SARS-CoV-2 B.1.1.529 (Omicron). Ab⁺ (n = 4–9) and Ab⁻ (n = 4–7) naïve mice, unexposed to the primary challenge, were infected with 1 × 10⁶ TCID₅₀ of SARS-CoV-2 B.1.1.529. PBS-exposed mice were used as controls. Blood was collected 7, 14 and 21 days after the first infection. Blood, lung, NT and mediastinal lymph node (mLN) were collected 4 days after rechallenge. b, Anti-S1 RBD IgG levels in the plasma after the first challenge. c,d, SARS-CoV-2 RNA in the NT (c) and lung (d). RNA values as copy number per ng of total RNA and the LOD as a dashed line. e, Viral titers in the lung were determined by TCID₅₀. f, Immunohistochemical micrographs of lung sections from PBS-, naïve- and primed-Ab⁺ and Ab⁻ mice. N-SARS-CoV-2-positive cells in brown. Scale bars, 100 μm. g–l, Flow cytometry plots (g and j), frequency (h and k) and absolute number (i and l) of CD8⁺ T cells (g–i) or CD4⁺ T cells (j–l) expressing IFN-γ and TNF in the lungs upon in vitro stimulation with a pool of SARS-CoV-2 peptides. Plots pregated as live⁺/B220⁻/CD19⁻/CD4⁻/CD8⁺ (g–i) or CD8⁻/CD4⁺ (j–l). m, Anti-S1 RBD IgG levels in the plasma 4 days after rechallenge. n,o, Flow cytometry plots (n) and frequency (o) of RBD-specific B cells detected by RBD-tetramers in the lungs (pregated on live⁺/CD4⁻/CD8⁻/B220⁺/CD19⁺). p,q, Flow cytometry histogram (p) and geometric mean fluorescence intensity (gMFI) (q) of surface markers expressed by RBD-specific B cells in the lung of Ab⁺ primed mice. As control, B cells negative for RBD-tetramer staining (gray). gMFI as log₂(fold change) over control B cells. Data are expressed as mean ± s.e.m. and are representative of at least two independent experiments. Data in b–d are pooled from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; Kruskal–Wallis test followed by uncorrected Dunn’s test; each comparison stands alone (c–e, m and o). Two-way ANOVA, Fisher’s LSD test (each comparison stands alone; b, h, i, k and l). LSD, least significant difference.

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