Mechanisms of maternal antibody interference with rotavirus vaccination

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  Figure 1. Maternal antibodies interfere with pup immune responses. (A) Schematic diagram of experimental breeding and infection protocol using C57BL/6 female and BALBc male mice. (B) Serum rotavirus-specific IgG in pups as measured by ELISA from 4 to 10 weeks of age; p = 0.01 at 4 wk and p < 0.0001 at 6, 8, and 10 wk for presence vs absence of MatAbs at vaccination. (C, D) Differentiation of maternal and pup antibodies by (C) IgG1a and (D) IgA at 10 weeks of age; p = 0.006, and p = 0.003 for MatAbs only vs vaccine only, and vaccine only vs vaccine + MatAbs, respectively (C) and p < 0.0001 for MatAbs only vs vaccine only, and vaccine only vs vaccine + MatAbs in (D); n = 8, n = 12, and n = 15 for MatAbs only, vaccine only, and vaccine + MatAbs, respectively. (E) Stool IgA titers in the presence or absence of MatAbs; p < 0.0001, p = 0.004, p = 0.001, and p = 0.002 at 4, 6, 8, and 10 wk, respectively, for presence vs absence of MatAbs at vaccination. (F) Longitudinal analysis of serum IgG by ELISA from pups vaccinated with a heterologous rotavirus vaccine strain; p < 0.0001 and p = 0.002 at 4 and 6 wk, respectively. (G) Comparison of IgG quantified by ELISA and serum neutralization titers in…

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  Figure 2. Maternal antibodies limit the formation of germinal centers (GCs) in draining lymph nodes. (A) Flow cytometry of GCs in mesenteric lymph nodes (MLNs) from pups with (n = 6) or without (n = 6) MatAbs at 20 days post vaccination, with representative flow cytometry plots and scatterplot presenting all mice, p = 0.0001. (B, C) H&E staining (B) and immunofluorescence (C) of MLN sections from mice vaccinated in the presence/absence of MatAbs. Scale bars: 1000 µm. (D) Flow cytometry of T follicular helper (TfH) cells in MLNs from pups with (n = 6) or without (n = 6) MatAbs at 20 days post vaccination. The gating strategy of a representative mouse used to identify TfH cells is presented in Fig. EV1. Data information: Statistical significance was determined by unpaired two-tailed t-tests (***p = 0.0001). Source data are available online for this figure.

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  Figure EV1. Gating strategy for identification of Tfh cells and GC B cells by flow cytometry. (A) Plot for forward versus side scatter, and mesenteric lymph node (MLN) cells gate. (B) Plot of forward scatter area as a function of forward scatter height within the MLN cells gate, and resulting single cells gate. (C) Viability dye (L/D) exclusion plot within the singlet gate, and resulting live cell gate. (D) Gating of CD4 cells, then gating of T follicular helper (Tfh) cells. (E) identified by PD-1 and CXCR5 within the CD4 gate. This panel is presented as a representative mouse in Fig. 2D. (F, G) Gating of B220+ B cells (F), then gating of germinal center (GC) B cells (G), identified by Fas and GL7 cells within the B220+ gate.

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  Figure 3. Maternal antibody-mediated interference is robust with low antibody doses. (A) Longitudinal measurement of dam rotavirus-specific antibody by ELISA for a low MatAbs (n = 1) and a high MatAbs (n = 1) mouse. (B, C) Longitudinal measurement of serum rotavirus-specific IgG (B) and IgA (C) in pups receiving high, low, or no MatAbs; p = 0.0025, p = 0.05, p = 0.005 at 4, 8, and 10 wk, respectively, for no MatAb vs high MatAbs and p = 0.0001, p = 0.03 at 4 and 10 wk, respectively, for no MatAb vs low MatAb in (B), and p = 0.03, p = 0.01 for low or high MatAb, respectively, vs no MatAb. (D) Quantification of rotavirus-specific IgG and IgA at the time of pup vaccination in the serum and milk of dams following natural infection (n = 1) or receiving 50 μL (n = 1) or 250 μL (n = 1) of pooled serum from infected mice intraperitoneally (IP). (E, F) Longitudinal measurement of rotavirus-specific serum IgG (E) and stool IgA (F) in pups receiving high, low or no MatAbs; p = 0.005, p = 0.0003, p = 0.005 at 6, 8, and 10 wk, respectively, for 250 vs 0 µL serum and p = 0.003, p = 0.0002, p = 0.0004 at 6, 8, and 10 wk, respectively, for 50 vs 0 µL serum in (E); p = 0.05, p = 0.003, p = 0.02, p = 0.02…

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  Figure EV2. Quantification of IgA in mouse stool and serum samples in the presence or absence of MatAbs. (A) Longitudinal serum IgA titers following vaccination at 7 days old with different volumes of rotavirus-seropositive serum passively transferred to dams; n = 3, n = 4, and n = 9 for 250, 50, and 0 µL serum, respectively. (B) Stool IgA titers in adult mice from experiment (A) 14 days after challenge with rotavirus; n = 5, n = 7, n = 4, and n = 3 for naïve control, and 0, 50, and 250 µL, respectively. (C) Rotavirus-specific IgG and IgA ELISA of pooled serum used for passive transfer experiments, mean of n = 2 technical replicates shown. Data information: error bars indicate SEM. Source data are available online for this figure.

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  Figure 4. Impact of FcγRIIB signaling on maternal antibody interference. (A) Serum IgG responses of wildtype (WT) and FcγRIIB knockout (KO) dams following rotavirus infection as shown by ELISA. n = 5 per group. (B) Evaluation of seroconversion by KO pups to rotavirus vaccination in the presence/absence of maternal antibodies by serum IgG ELISA; p = 0.02 and p < 0.0001 at 4 and 8 wk, respectively. (C) Serum IgG, serum IgA and stool IgA responses to rotavirus vaccination in pups vaccinated 24 h after dams were administered rotavirus-positive serum intraperitoneally (IP); p = 0.03 and p = 0.03 at 6 and 8 wk, respectively for serum IgA and p = 0.003 at 8 wk for stool IgA. Data information: Statistical significance was determined by two-way ANOVA with repeated measures. Significant Tukey’s adjusted pair-wise comparisons between vaccination in the absence or presence of MatAbs or passively transferred serum are shown (*p ≤ 0.05 and **p < 0.0001); error bars indicate SEM. Source data are available online for this figure.

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  Figure 5. Combined clearance of vaccine and maternal antibodies, with no long-term effects on immunity. (A) qPCR of rotavirus in stool samples collected after vaccination in pups with and without MatAbs (n = 2 litters each condition, samples from each litter pooled at each timepoint). (B, C) Rotavirus-specific IgG was quantified by ELISA in mice receiving MatAbs that did (2 litters, n = 15) or did not (1 litter, n = 8) receive a vaccine at 7 days old, in serum samples collected at 4 weeks (p < 0.0001) (B) or 10 weeks (p = 0.001) (C) of age. (D, E) IgG (D) or IgA (E) were quantified in stool samples by ELISA in 9-day old pups +/– rotavirus vaccination; p = 0.02 for vaccine + MatAbs vs vaccine only (D) and p < 0.0001, p = 0.0002, p = 0.01 for vaccine + MatAbs vs vaccine only, vaccine + MatAbs vs MatAbs only, and vaccine only vs MatAbs only, respectively (E); n = 6, n = 4, and n = 3 for vaccine + MatAbs, MatAbs only, and vaccine only, respectively. (F) Stool samples were pooled from each litter, and IgA was quantified by ELISA. (G, H) Adult mice born +/– MatAbs were challenged with rotavirus when 10–12 weeks of age; rotavirus shedding was quantified by viral-antigen ELISA in (G) mice born to…

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  Figure EV3. Total IgG and IgA in serum, stool, and milk samples. (A) Total IgG quantified by ELISA in serum samples from 4-week- and 10-week-old pups with MatAbs +/– rotavirus vaccination; n = 7 and n = 14 in unvaccinated and vaccinated pups at 4 wk and n = 8 and n = 14 in unvaccinated and vaccinated pups at 10 wk. (B) Total IgA quantified by ELISA in the same serum samples as in (A); n = 7 and n = 14 in unvaccinated and vaccinated pups at 4 wk and n = 8 and n = 14 in unvaccinated and vaccinated pups at 10 wk. (C) Total IgA ELISA in stool samples collected from pups 0 to 8 days after rotavirus vaccination. (D) Rotavirus-specific IgA and total IgA were quantified by ELISA in milk samples collected from dams at 0, 3, and 7 days after pup vaccination. Data information: error bars indicate SEM. Source data are available online for this figure.

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  Figure 6. Single-cell transcriptomics during maternal antibody interference. (A) Schematic diagram of experimental protocol. (B) Integrated uniform manifold approximation and projection (UMAP) for all samples. Individual cells plotted. (C) Relative expression of canonical and marker genes (x-axis) for immune cells across clusters (y–axis); dots indicate average expression and percentage of cells detected with expression (color and size, respectively). (D) Average proportion of cell types in vaccine + MatAb and vaccine only samples (left) and their change in proportion between vaccine + MatAb and vaccine only (right). (E) Counts of differentially expressed (DE) genes (y–axis) per cell type, comparing vaccine + MatAb and vaccine only with absolute log2 fold change >1 and adjusted p value ≤0.05. (F) GSEA results showing downregulation of hallmark interferon alpha and gamma response in major clusters. Dots are sized to denote significance; the x-axis indicates NES. (G) Distribution and expression of selected interferon-stimulated genes from mesenteric lymph nodes isolated from pups vaccinated in the presence (vaccine + MatAbs, n = 2) or absence of MatAbs (vaccine only, n = 2). Source data are…

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  Figure EV4. Single-cell transcriptomics of the draining mesenteric lymph node. (A) Violin plot showing quality control metrics (from top to bottom: transcripts per cell, genes per cell, percent mitochondrial reads per cell, S score, G2M score, and R score) for each sample (n = 4, x-axis) processed on the 10x Genomics Chromium instrument. (B) Violin plot showing quality control metrics for each cell type (x-axis) from MLN from pups vaccinated in the absence (n = 2) or presence (n = 2) of MatAbs.