Figure 5.

Scaffold-specific antibody responses can competitively inhibit antigen-specific responses when scaffold is provided in excess

(A) (Left) Schematic representation of the scaffold competition experimental design where mice were immunized with either varying doses of RBD-I53-50 nanoparticle immunogens (blue triangle) or varying doses of RBD-I53-50 nanoparticle immunogens co-delivered with excess I53-50 scaffold (blue triangle + orange rectangle). (Right) Schematic depicting the study timeline and blood collection time points that each data panel represents.

(B–G) Post-prime (week 2) (B–D) and post-boost (week 6) (E–G) anti-SARS-CoV-2 spike (B and E) and anti-I53-50 scaffold (C and F) serum IgG binding titers in BALB/c mice immunized with the protein doses indicated at the bottom of (E)–(G), measured by ELISA and plotted as the area under the curve (AUC) for each serum dilution series. Each symbol represents an individual animal, and the geometric mean AUC from each group is indicated by the bar (N = 5 mice/group).

(D and G) Ratio of the post-prime (week 2) (D) and post-boost (week 6) (G) spike-specific to I53-50 scaffold-specific binding antibody titers (AUC). The black dashed line indicates a ratio of 1.

(H and I) Post-boost (week 6) spike-specific (H) and I53-50 scaffold-specific (I) serum IgG ELISA curves in BALB/c mice. Each line represents the geometric mean absorbance at 450 nm (N = 5 mice/group).

(J) Post-boost serum pseudovirus neutralization titers using a vesicular stomatitis virus (VSV) pseudotyped with the Wuhan-Hu-1 S glycoprotein harboring the D614G substitution (G614) and VeroE6 cells stably expressing TMPRSS2.⁸⁴ Each symbol represents the reciprocal half-maximal inhibitory dilution (IC₅₀) of an individual animal. GMT for each group is indicated by a bar (N = 5 mice/group). Representative data from duplicate measurements made with distinct batches of pseudoviruses are shown. The dotted horizontal line represents the lower limit of detection of the assay. Raw data curves are shown in Figure S7E.

p values between groups were determined by Brown-Forsythe and Welch one-way ANOVA test, with Dunnett’s T3 multiple comparisons test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.