Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid

Abstract

The role of adjuvant on the T(h)1 and T(h)2 immune responses to Abeta-immunotherapy (Abeta(42 )peptide) was examined in wild-type mice. Fine epitope analysis with overlapping oligomers of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The 6-20 peptide was recognized only weakly by antisera from mice administrated with Abeta(42) peptide formulated in complete Freund's adjuvant (CFA), alum or TiterMax Gold (TMG). However, mice immunized with Abeta(42) mixed with QS21 induced a significant antibody response to the 6-20 peptide. The only T cell epitope found was within the 6-28 sequence of Abeta(42). QS21 and CFA induced the strongest humoral response to Abeta, alum was intermediate, and TMG the weakest adjuvant. Analysis of antibody isotypes specific for Abeta indicates that alum induces primarily T(h)2-type immune response, whereas TMG, CFA and QS21 shift the immune responses toward a T(h)1 phenotype. Stimulation of splenocytes from Abeta-immunized mice with Abeta(40) peptide induced strikingly different cytokine expression profiles. QS21 and CFA induced significant IFN-gamma, IL-4 and tumor necrosis factor-alpha expression, whereas alum induced primarily IL-4 production. As T(h)1-type immune responses have been implicated in many autoimmune disorders, whereas T(h)2-type responses have been shown to inhibit autoimmune disease, the choice of adjuvant may be critical for the design of a safe and effective immunotherapy for Alzheimer's disease.

Fig. 1

Aβ₄₂-specific immune responses in mice immunized with fibrillar Aβ₄₂ peptide and different adjuvants. Mice were boosted 1 (A), 2 (B) or 3 (C) times with immunogen formulated in specified adjuvants. Control mice (mock) were immunized with PBS + CFA and boosted with PBS + IFA. Pooled sera (A and B, each with n = 12) or individual sera (C, SD represent n = 8) were used in ELISA.

Fig. 2

IgG2a (T_h1) and IgG1 (T_h2) profiles of humoral immune responses in mice immunized with fibrillar Aβ₄₂ formulated in different adjuvants. Sera for this assay were pooled and diluted 1:500 prior to the detection of IgG1 and IgG2a isotypes. Mean values were detected spectrophotometrically and the IgG1:IgG2a ratio was calculated as described in Methods. Sera were collected from mice boosted 2 (A) or 3 (B) times.

Fig. 3

Mapping of B cell epitopes recognized by anti-Aβ₄₂ antibodies generated in mice immunized and boosted 3 times with immunogen and four different adjuvants. The relative binding of 1:2000 diluted antisera to six overlapping peptides and Aβ₄₂ are presented. Two independent experiments were performed (SD represent n = 8).

Fig. 4

Mapping of B cell epitopes by competition assay. (A) Pooled sera from immune mice (after third boost) were collected and pre-incubated with a mixture of Aβ_{1–15 + 6–20}^, Aβ_{11–25 + 16–30 + 21–35 + 26–42} or Aβ₄₂ peptides (concentration of each peptide 2.5 μM) before binding to Aβ₄₂-coated wells. In order to standardize anti-Aβ₄₂ antibody level we used different dilutions of antisera. (B) Inhibition studies with indicated concentrations of Aβ_1–15, Aβ_11–25 and Aβ₄₂ peptides. The sera from mice immunized with fibrillar Aβ₄₂ and different adjuvants were diluted 1:4000 (final dilution 1:8000) and pre-incubated with peptides Aβ_1–15, Aβ_11–25 or Aβ₄₂ before binding to Aβ₄₂-coated wells. The percent of inhibition by small peptides as well as by control full-length peptide was calculated considering the binding of sera without competing peptides to Aβ₄₂ as a 100%. These experiments were repeated twice with similar results.

Fig. 5

Mapping of T cell epitopes in mice immunized and boosted 3 times with immunogen mixed with alum, TMG, QS21 or CFA/IFA. (A) Proliferation of T cells (stimulation index) was determined after in vitro activation of individual mouse splenocyte cultures (SD represent n = 8) with 10 μg/ml of overlapping peptides or Aβ₄₀. (B) Proliferation of T cells was determined in five mice immunized with Aβ₄₂ formulated in CFA/IFA. The stimulation index was determined after in vitro stimulation of individual mouse splenocyte cultures (n = 5) with 10 μg/ml of Aβ₂₈ or Aβ₄₀.

Fig. 6

Detection of IFN-γ (T_h1) and IL-4 (T_h2) lymphokines, and TNF-α pro-inflammatory cytokine in mice immunized and boosted 3 times with Aβ₄₂ formulated in alum, QS21 or CFA/IFA. Splenocytes from immunized mice (2 × 10⁵/well) were incubated with Aβ₄₀ peptide or medium alone in ELISPOT plates as described in Methods. Colored spots were counted after 24 h incubation. ANOVA has been used to compare production of each cytokine in the culture of splenocytes incubated with (experiment) or without (control) Aβ₄₀ peptide (see Methods). Results are shown as mean number of spots/10⁶ cells ± SD for stimulated (closed bars) versus unstimulated (open bars).