Short or Long Interval between Priming and Boosting: Does It Impact on the Vaccine Immunogenicity?

Abstract

Characterizing the impact of the vaccination schedule on the induction of B and T cell immune responses is critical for improving vaccine immunogenicity. Here we compare the effect of a short (4 weeks) or a long (18 weeks) interval between priming and boosting in mice, using a model vaccine formulation based on the chimeric tuberculosis vaccine antigen H56 combined with alum. While no significant difference was observed in serum antigen-specific IgG response and the induction of antigen-specific T follicular helper cells into draining lymph nodes after the two immunization schedules, a longer interval between priming and boosting elicited a higher number of germinal center-B cells and H56-specific antibody-secreting cells and modulated the effector function of reactivated CD4+ T cells. These data show that the scheduling of the booster immunization could affect the immune response elicited by vaccination modulating and improving the immunogenicity of the vaccine.

Keywords: B cell response; T cell response; alum; antibodies; immunization; prime–boost schedules.

Figure 1

Experimental design. Two different immunization schedules (A,B) were tested and compared for the induction of serum antibodies (in blood), germinal center B cells, and antigen-specific T helper cells (in draining iliac lymph nodes, ILN), and cytokine production (in the spleen, SPL). C57BL/6 mice were subcutaneously immunized with the H56 antigen and alum and boosted with the antigen 4 (A) or 18 (B) weeks apart. Mice immunized with H56 antigen alone, or Phosphate Buffered Saline (PBS) were used as controls. Blood samples, ILN, and SPL were collected at the time points indicated by symbols. W, weeks; D, days.

Figure 2

Antigen-specific IgG response. C57BL/6 mice were subcutaneously immunized, as summarized in Figure 1, and humoral response was analysed after the short (a) and long (b) immunization protocol. H56-specific IgG serum titers were analyzed 0, 2, 4, 7, 11, 15, 18 weeks following priming, and 10, 28, and 49 days after boosting by ELISA. Values are reported as GMT ± 95% CI of 12 mice from 2 different experiments. Antibody titers were expressed as the reciprocal of the dilution of the sample, reporting an optical density value double with respect to the background. The Mann–Whitney test for multiple pairwise comparisons was used for assessing statistical differences for each time point between groups. ** p ≤ 0.001; *** p ≤ 0.0001.

Figure 3

B-cell response. C57BL/6 mice were subcutaneously immunized, as summarized in Figure 1. (A) Germinal center B cells were identified as GL-7 + CD95+ among B220 + B cells in iliac lymph nodes 10 days after boosting. Dot plots are shown from a single animal representative of the group, and numbers indicate frequencies of CD95 + GL-7 + respect to B220 + cells B–C. Time-course analysis of the frequencies of GC- cells, with respect to B220+ B cells (B) and absolute numbers of GC-B cells per ILN (C) reported as mean ± SEM of six mice per group. (D) Number of H56-specific IgG-secreting cells per million splenocytes, reported as mean ± SEM of six mice per group. The Mann–Whitney test for multiple pairwise comparisons was used to assess the statistical difference between the short (4W) and long (18W) schedules for each vaccine formulation. * p ≤ 0.05.

Figure 4

Induction of Ag85B-specific CD4+ T cells. C57BL/6 mice were subcutaneously immunized, as summarized in Figure 1, and lymph nodes draining the sites of immunization (ILN) were collected 10 days after boosting. Ag-specific T cells were identified by staining with Ag85B-specific MHC class II tetramers (Tet-Ag85B). Tetramer+ T cells, detected as CD44high Tet-Ag85B+ cells, gated on live CD4+ lymphocytes (A), and the follicular helper T cells (Tfh), identified as CXCR5+ PD-1+ among tetramer-binding CD4+ T cells (B), are shown from a single animal representative of the group. Absolute numbers of Tet-Ag85B+ CD44+ T cells (C) and follicular helper T cells (D) per ILN elicited by the two schedules assessed, reported as mean ± SEM of six mice per group. The Mann–Whitney test for multiple pairwise comparisons was used to assess the statistical difference between the short (4W) and long (18W) schedules for each vaccine formulation. * p ≤ 0.05.

Figure 5

Helper T cell multifunctional response. Multifunctional profiles of CD4+ T cells detected by the different immunization protocols as reported in Figure 1. (A) Histograms represent the frequency of CD4+ CD44+ T cells producing different combinations of cytokines after the short and long schedule. Responses are grouped and color-coded according to the functionality (orange for single cytokine, light blue for two or three cytokine production). Values are reported as mean ± SEM of six mice per group. The Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparison was used to assess statistical differences among groups (*p ≤ 0.05). (B) Pie charts represent the portion of CD4+ T cells producing three cytokines (orange), two cytokines (light blue), or a single one (grey). Frequencies are reported within each slice.