Antigen depot is not required for alum adjuvanticity

Abstract

Alum adjuvants have been in continuous clinical use for more than 80 yr. While the prevailing theory has been that depot formation and the associated slow release of antigen and/or inflammation are responsible for alum enhancement of antigen presentation and subsequent T- and B-cell responses, this has never been formally proven. To examine antigen persistence, we used the chimeric fluorescent protein EαGFP, which allows assessment of antigen presentation in situ, using the Y-Ae antibody. We demonstrate that alum and/or CpG adjuvants induced similar uptake of antigen, and in all cases, GFP signal did not persist beyond 24 h in draining lymph node antigen-presenting cells. Antigen presentation was first detectable on B cells within 6-12 h of antigen administration, followed by conventional dendritic cells (DCs) at 12-24 h, then finally plasmacytoid DCs at 48 h or later. Again, alum and/or CpG adjuvants did not have an effect on the magnitude or sequence of this response; furthermore, they induced similar antigen-specific T-cell activation in vivo. Notably, removal of the injection site and associated alum depot, as early as 2 h after administration, had no appreciable effect on antigen-specific T- and B-cell responses. This study clearly rules out a role for depot formation in alum adjuvant activity.

Figure 1.

Magnitude and kinetics of antigen-specific immune responses are comparable following immunization with alum or CpG adjuvants. A) All adjuvants significantly increased the mean percentage of tg T cells (CD4⁺KJ1.26⁺) expressing CD69, at d 1, 5, and 11. B) Mean percentage of tg T cells at the peak of expansion at d 5 postimmunization. C) Total number of tg T cells expressing the IL-4 reporter was higher early (d 1; P<0.05) with CpG/OVA compared with alum/OVA, although later (d 11) only alum/OVA or alum/OVA/CpG produced significant numbers of GFP-positive antigen-specific cells compared with nonimmunized controls. D, E) Serum was taken from animals 14 d after immunization and assessed for the presence of anti-OVA IgG1 (D) and anti-OVA IgG2a (E) by ELISA. Results are means ± sd of 3 animals/group and are representative of ≥2 independent experiments. *P < 0.05 vs. unimmunized control; 1-way ANOVA.

Figure 2.

Immunization with alum or CpG adjuvants gives rise to a comparable distribution, magnitude, and duration of antigen uptake and presentation in vivo. A–C) Duration and magnitude of antigen persistence were measured as the mean fluorescence intensity (MFI) of EαGFP protein detected at 6, 24, and 72 h postimmunization in B cells (A), CD11c DCs (B), and plasmacytoid DCs (C). D–F) Duration and magnitude of antigen presentation were measured as the MFI of Y-Ae detection of the Eα-MHCII complex at 1, 6, 12, 24, 36, 48, and 72 h postimmunization in B cells (D), CD11c DCs (E), and plasmacytoid DCs (F). Results are means ± sd of 3 animals/group and are representative of ≥3 independent experiments. *P < 0.05 for EαGFP/CpG vs. EαGFP/alum groups; 1-way ANOVA.

Figure 3.

Immunization with alum in comparison with CpG adjuvant results in a short-term persistence of antigen. A) Timeline outlining experiment protocol. B) Mean percentage of tg T cells at 5 d following immunization of mice that received an adoptive transfer of tg T cells 1, 5, 10, 14, and 28 d previously. C) Tg T cells (CD4⁺KJ1.26⁺) were counted and then assessed for CFSE division by flow cytometry. Results are means ± sd of 3 animals/group and are representative of ≥2 independent experiments. *P < 0.05 for OVA/alum vs. OVA/CpG; 1-way ANOVA.

Figure 4.

Ear ablation does not alter the magnitude and kinetics of antigen-specific immune responses following alum immunization. A) Histological analysis demonstrates a discrete subcutaneous depot of alum up to 24 h after injection. Injection sites were subsequently removed immediately, or at 2, 6, or 24 h postimmunization. B–E) Effect of injection site removal on mean percentage of tg T cells expressing CD69 (B), mean percentage of tg T cells in draining lymph nodes (C), mean percentage of tg T cells (CD4⁺KJ1.26⁺) that divided (D), and mean percentage of tg T cells (CD4⁺KJ1.26⁺) expressing GFP, a marker of IL4 production, at 5 d postimmunization (E). F–I) Effect of injection site removal 2 h postimmunization on titers of anti-OVA IgG1 after immunization with OVA/alum (F), alum/CpG and titers of anti-OVA IgG2a (G), OVA/alum (H), and alum/CpG (I). Results are means ± sd of 3 animals/group and are representative of ≥2 independent experiments. *P<0.05 for intact vs. removed; 1-way ANOVA.

Figure 5.

Ear ablation following OVA/alum immunization does not alter the magnitude or phenotype of recall memory responses. A) Mice were immunized with OVA adsorbed to alum, and the injection site was ablated 2 h later. After 2 wk, OVA-specific responses were recalled in control (intact) and ablated mice by immunization with heat-aggregated OVA (HAO). B–E) IgG1 responses were detectable prior to (B) and at 1 wk (C), 2 wk (D) or 3 wk (E) following challenge. Injection site ablation induced a small increase in IgG1 titer at 1 wk postchallenge; no effect of ablation was observed at other time points. F–I) Injection site ablation did not affect the inability of alum/OVA immunized mice to produce IgG2a responses at 1 wk (G), 2 wk (H) or 3 wk (I) postchallenge, compared to prechallenge (F). Results are means ± sd of 5 animals/group. ***P < 0.01.

Figure 6.

Ear ablation does not alter the magnitude and duration of antigen uptake and presentation in vivo following immunization with alum/EαGFP. Effect of injection site ablation at 2 h postimmunization on duration and magnitude of antigen presentation. Lymph nodes were collected at 6, 24, and 48 h postimmunization and antigen presentation by B cells (A), CD11c DCs (B), and plasmacytoid DCs (C) was expressed as Y-Ae mean fluorescence intensity. Results are means ± sd of 3 animals/group and are representative of ≥2 independent experiments. *P<0.05 for intact vs. removed; Student's 2-tailed unpaired t test.