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. Author manuscript; available in PMC: 2022 Dec 8.
Published in final edited form as: Nature. 2022 Jun 8;606(7915):769–775. doi: 10.1038/s41586-022-04816-9

Extended Data Fig. 3: Lm-specific IgG titer, subclass distribution, neutralization efficiency, and lectin staining profile comparisons in virgin compared with pregnant mice.

Extended Data Fig. 3:

(a) IgG titers against UV-inactivated Lm bacteria or purified LLO protein for WT female pregnant/postpartum mice preconceptually primed with ΔActA Lm, ΔActA Lm-primed virgin mice, or naive control mice. (b) OD450 for each antibody isotype or subclass in sera from ΔActA Lm-primed virgin or pregnant mice. All sera diluted 1:100. Background subtracted from staining using naive sera. (c) LLO-induced hemolysis of sheep erythrocytes in media containing anti-Lm IgG from virgin (vIgG) or late gestation/early postpartum pregnant (pIgG) mice. (d) Relative vIgG and pIgG binding to each lectin with defined carbohydrate-specificity for LLO-specific IgG. (e,f) SNA-agarose fractionation of sialylated (SNA Bound) compared with non-sialylated (SNA Unbound) anti-Lm vIgG. SNA lectin staining confirmed successful separation (e). Lm- and LLO-specific IgG was titered from each fraction (f). (g, h) Anti-Lm IgG titer in neonatal mice transferred each type of enzymatically-treated pIgG (g). Lectin staining to confirm de- and resialylation of pIgG: SNA signal indicates presence of terminal sialic acid and ECA signal indicates presence of galactose uncovered by terminal sialic acid removal (h). (i-k) Anti-Lm IgG titer in neonatal mice transferred glycoengineered vIgG (i). SNA and ECA Lectin staining to confirm de- and resialylatoin of vIgG with sialic acid variants (j). PToV virolectin probe for 9-O-acetylated sialic acid and PToV with mutated binding site (nonbinder) demonstrating specificity for vIgG resialylated with each sialic acid variant or deacetylated with SIAE treatment (k). Representative data from at least 2-3 independent experiments (a-f) or combined data from 3 independent experiments with 3-5 mice per group per experiment (g-k) are shown. Bar, mean ± standard error. P values between key groups are shown as determined by one-way ANOVA adjusting for multiple comparisons. Dotted lines, limit of detection.