Figure 4 |. Subversion of primary addiction by heterologous SARS-CoV-2 boosting.

(a) Schematic representation of immunization strategies. (b) Anti-WH1 (left) and BA.1 RBD IgG titers (middle) and NT50 of BA.1 pseudovirus (right) 2 weeks after indicated dose in homologously (WWW) or heterologously (WBB) immunized S1pr2-Igk^Tag/Tag mice. P-value: 2-tailed T test. FC, fold-change. (c) Evolution of anti-WH1 and BA.1 RBD tag-specific titers. Thin lines represent individual mice, thick lines link medians of log transformed titer values. (d) Comparison of FLAG and Strep anti-RBD titers shown in (c) at 2 weeks after 3^rd immunization (left) along with primary addiction index (right). P-values: 2-tailed T test. (e) Anti-full S tag-specific titers and primary addiction index for the same samples as in (d). Results in (b-d) are from 2 independent experiments, with the number of mice in each group indicated. (f) Comparison of de novo FLAG⁺ antibody responses in WBB mice versus mice in which the 1^st dose and fate-mapping were omitted (ØBB). ØBB data are for 10 mice from 2 independent experiments. WBB data are from (c), remeasured in the same assay as ØBB. (g) Schematic representation of antibody fractionation. (h) BA.1 NT50 titers for WBB mice at the time point as in (d). Post-depletion NT50s normalized as described in Methods. P-values: 1-tailed paired T test. (i) Potency of anti-RBD BA.1 Strep⁺ (FLAG-depleted) and FLAG⁺ (Strep-depleted) fractions. Raw NT50 values for each fraction were divided by their respective RBD ELISA titers. P-values: 2-tailed paired T test. (j) WH1 RBD structure (PDB: 6MOJ) with amino acid changes in BA.1 highlighted in red. (k) DMS of serum samples obtained from 3 WBB mice 2 weeks after 3^rd immunization (d). Antibody binding sites on RBD are shaded according to escape fraction. The positions most highly targeted by each serum fraction are indicated (BA.1-specific residues in parentheses).