Prime-boost strategies in mucosal immunization affect local IgA production and the type of th response

Abstract

Combinations of different delivery routes for priming and boosting represent vaccination strategies that can modulate magnitude, quality, and localization of the immune response. A murine model was used to study T cell clonal expansion following intranasal (IN) or subcutaneous (SC) priming, and secondary immune responses after boosting by either homologous or heterologous routes. T cell primary activation was studied by using the adoptive transfer model of ovalbumin-specific transgenic CD4(+) T cells. Both IN and SC immunization efficiently elicited, in the respective draining lymph nodes, primary clonal expansion of antigen-specific CD4(+) T cells that disseminated toward distal lymph nodes (mesenteric and iliac) and the spleen. After boosting, a significant serum IgG response was induced in all groups independent of the combination of immunization routes used, while significant levels of local IgA were detected only in mice boosted by the IN route. Mucosal priming drove a stronger Th1 polarization than the systemic route, as shown by serum IgG subclass analysis. IFN-gamma production was observed in splenocytes of all groups, while prime-boost vaccine combinations that included the mucosal route, yielded higher levels of IL-17. Memory lymphocytes were identified in both spleen and draining lymph nodes in all immunized mice, with the highest number of IL-2 producing cells detected in mice primed and boosted by the nasal route. This work shows the critical role of immunization routes in modulating quality and localization of immune responses in prime-boost vaccine strategies.

Keywords: T cell priming; cytokines; mucosal immune response; nasal immunization; prime-boost; subcutaneous immunization.

Figure 1

Clonal expansion of transgenic CD4⁺ T cells after IN or SC priming. C57BL/6J mice, adoptively transferred with CFSE-labeled OT-II CD4⁺ T cells, were primed by the IN or SC routes with OVA mixed with the adjuvant CpG ODN. OVA-specific proliferation was analyzed in T cells collected from cervical (CLN), mediastinal (MedLN), axillary (AxLN), iliac (ILN), mesenteric (MLN) lymph nodes, and spleen (SPL) at different time points. (A) OVA-specific proliferation of OT-II CD4⁺ T cells detected by CFSE dilution in lymphoid organs 3 days following IN (bottom histogram) or SC (middle histogram) priming or in untreated mice (top histogram). (B) Time-course analysis of the clonal expansion of OT-II CD4⁺ T cells following IN (filled circle) or SC (empty square) priming with OVA and CpG ODN. The clonal expansion was analyzed on days 0, 3, 5, and 7 post-immunization. Values reported on the y axis indicate the percentage of proliferating OT-II cells compared to the total transgenic cells detected in each lymph node or spleen. Values are expressed as means ± SEM of 2 independent experiments, each performed with three animals. Two-tailed Student’s t-test was used for comparing values of proliferating T cells between nasal and subcutaneous primed mice at each time point. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Figure 2

OVA-specific serum antibody response. BALB/c mice were immunized with OVA mixed with the adjuvant CpG ODN at days 0 and 10 by the IN or SC route followed by a boost at week 7 by the homologous or heterologous route. Antibody response was assessed by ELISA on individual serum samples. (A) OVA-specific IgG response assessed in samples collected at weeks 0, 3, 5, 7, 9, 11, and 13. Antibody titers were expressed as the reciprocal of the highest dilution with an OD value ≥0.2 after background subtraction. Values are reported as GMT ± SEM. (B) Ratio of OVA-specific IgG1 and IgG2a subclasses assessed in final serum of each animal. Data are reported as mean ± SEM for each group.

Figure 3

OVA-specific local IgA. BALB/c mice were immunized with OVA mixed with the adjuvant CpG ODN at days 0 and 10 by the IN or SC route followed by a boost at week 7 by the homologous or heterologous route. OVA-specific IgA were assessed in nasal washes collected at sacrifice (week 13). Bars represents the mean value of OVA-specific IgA concentration per each group. One-way ANOVA and Tukey’s post test for multiple comparisons were used for comparing antibody response in different groups. ^*P < 0.05, ^**P < 0.01, and^***P < 0.001.

Figure 4

OVA-specific lymphocyte response determined by IL-2 ELISPOT assay. BALB/c mice were immunized with OVA mixed with the adjuvant CpG ODN at days 0 and 10 by the IN or SC route followed by a boost at week 7 by the homologous or heterologous route. Lymphocytes were obtained from spleens (A) and pooled cervical and mediastinal lymph nodes (B) at week 13, and the IL-2 production was evaluated following OVA restimulation by ELISPOT assay. The number of antigen-specific spot forming units (SFU) of OVA-restimulated (black histograms) and unstimulated (white histograms) cells is shown. Bars represent the mean number of SFU/10⁶ cells ± SEM of triplicate samples. One-way ANOVA and Tukey’s post test for multiple comparisons was performed for all groups. The numbers above the error bars indicate which groups (1–5) are statistically different. Statistical significance was defined as P < 0.05.

Figure 5

Cytokine production in culture supernatant of splenocytes. IFN-γ (A), IL-4 (B), and IL-17A (C) were detected by Bio-Plex immunoassay in culture supernatants of pooled splenocytes restimulated with OVA (as described in Materials and Methods). Bars represent the mean concentration ± SD of duplicate samples.