Sustained antigen delivery improves germinal center reaction and increases antibody responses in neonatal mice

Abstract

Neonates and young infants are known to have limited responses to pediatric vaccines due to reduced germinal center formation. Extended vaccine antigen dosing was previously shown to expand germinal center formation and improve humoral responses in adult mice. We report that sustained antigen delivery through sequential dosing overcomes neonatal limitations to form germinal center reactions and improves humoral immunity. Thus, vaccine strategies that extend the release of vaccine antigens may reduce the number of doses, and time needed, to achieve protective immunity in neonates and young infants.

Fig. 1. Extending antigen availability through sequential dosing increases antibody responses after vaccination in neonatal mice.

a Groups of neonatal C57BL/6 mice were immunized with a total of 0.2 μg of PPS14-TT + 25% by volume Imject^TM following the dosing schedules shown and the immune responses were evaluated on the indicated time points. b Serum total IgG as well as IgG1 and IgG2c antibodies against PPS14 were measured by ELISA following s.c. immunization of mice (n = 7 mice per group). c Mice were immunized s.c. with PPS14-TT and boosted 4-weeks later. Seven days after boosting, splenocytes were collected to measure PPS14-specific IgG+ ASCs by ELISPOT. (bolus n = 9; constant n = 12; escalating n = 10). d Bone marrow was harvested 3-months after i.p. immunization with PPS14-TT to measure PPS14-specific IgG+ ASCs by ELISPOT (bolus n = 11; constant n = 9; escalating n = 12; unimmunized n = 7). Data are representative of experiments done twice (b); or in c and d, data were pooled from two independent experiments. Mean $\pm$SEM is shown, and each data point represents an individual mouse. Asterisk indicates statistically different from bolus as determined by Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test. *p $\le$ 0.05, **p$\le$ 0.01, ****p$\le$ 0.0001.

Fig. 2. Sequential dosing increases GC responses after vaccination in neonatal mice.

Mice were i.p. immunized with PPS14-TT. Splenic GC responses were measured by microscopy at 10-days post-vaccination and by flow cytometry 7, 10, and 14-days post-vaccination. a Representative immunofluorescence staining images of spleens from neonatal mice immunized according to bolus, constant, and escalating schedule. Sections were stained with CD4, GL-7, and B220 antibodies in addition to Hoechst counterstaining. Violin plots show mean $\pm$SEM GC center counts. *p $\le$ 0.05. b Representative gating strategy for splenic T_FH, Foxp3^- T_FH, and GC B cells. c Representative contour plots of T_FH cells with mean percent of CD4⁺ T cells (right), and frequency of T_FH cells as a percent of CD4⁺ T cells (left). Cells were pregated on live dump^-CD4⁺ B220^- T cells. d Representative histograms of Foxp3^- T_FH with mean percent of T_FH cells (right), and frequency of Foxp3^- cells (left). Cells were pregated on live dump^-CD4⁺ B220^-PD-1⁺ CXCR5⁺ T cells. In c and d, on Day 7: bolus dose n = 12 mice; constant dose n = 13 mice; escalating dose n = 14 mice. On Day 10: bolus dose n = 13 mice; constant dose n = 13 mice; escalating dose n = 13 mice. On Day 14: bolus dose n = 11 mice; constant dose n = 12 mice; escalating dose n = 11 mice. Data were combined from two independent experiments (c–e). Mean $\pm$SEM is shown, and each data point represents an individual mouse. Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons test was used for statistical evaluations. *p $\le$ 0.05, **p$\le$ 0.01.