Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice

Abstract

Background: In prior work we detected reduced anti-Abeta antibody titers in Abeta-vaccinated transgenic mice expressing the human amyloid precursor protein (APP) compared to nontransgenic littermates. We investigated this observation further by vaccinating APP and nontransgenic mice with either the wild-type human Abeta peptide, an Abeta peptide containing the "Dutch Mutation", E22Q, or a wild-type Abeta peptide conjugated to papillomavirus virus-like particles (VLPs).

Results: Anti-Abeta antibody titers were lower in vaccinated APP than nontransgenic mice even when vaccinated with the highly immunogenic Abeta E22Q. One concern was that human Abeta derived from the APP transgene might mask anti-Abeta antibodies in APP mice. To test this possibility, we dissociated antigen-antibody complexes by incubation at low pH. The low pH incubation increased the anti-Abeta antibody titers 20-40 fold in APP mice but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-Abeta titers were still lower in transgenic mice vaccinated with wild-type Abeta or E22Q Abeta relative to non-transgenic mice. Importantly, the dissociated anti-Abeta titers were equivalent in nontransgenic and APP mice after VLP-based vaccination. Control experiments demonstrated that after acid-dissociation, the increased antibody titer did not cross react with bovine serum albumin nor alpha-synuclein, and addition of Abeta back to the dissociated serum blocked the increase in antibody titers.

Conclusions: Circulating human Abeta can interfere with ELISA assay measurements of anti-Abeta titers. The E22Q Abeta peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against Abeta abrogate the effects of Abeta self-tolerance.

Figure 1

Transgenic APP mice immunized with Aβ vaccines have increased titers when incubated at pH 2.5. Results are from sera obtained 14 days after the fourth vaccination. Aliquots from each sample were incubated at either pH 2.5 or pH 7.0 for 20 minutes, separated by centrifugation through 10,000 MW filters and brought to neutral pH for standard ELISA assay. Data presented are mean ± sem. * Indicates P < 0.001 compared to pH 7.0 (values at pH 7 are 200 and 400 for WT and VLP respectively). Sample size is 5–6 per group.

Figure 2

Nontransgenic mice immunized with Aβ vaccines do not have increased titers when incubated at pH 2.5. Results are from sera obtained 14 days after the fourth vaccination. Aliquots from each sample were incubated at either pH 2.5 or pH 7.0 for 20 minutes, separated by centrifugation through 10,000 MW filters and brought to neutral pH for standard ELISA assay. Data presented are mean ± sem. Sample size is 5–6 per group.

Figure 3

Comparison of anti-Aβ titers of Aβ vaccinated APP and nontransgenic mice after acid dissociation of Aβ and anti-Aβ antibody. Results are from sera obtained 14 days after the fourth vaccination. All samples were incubated at pH 2.5, centrifuged through 10,000 MW filters and neutralized before ELISA assay. So the effects of genotype could be directly compared in the three vaccine conditions, the nontransgenic titer value for each vaccine group was set to 100%. Data presented are mean ± sem. * P < 0.05 compared to APP mice given the same vaccine. Sample size is 5–6 per group.

Figure 4

Specificity of increased antibody binding after acid-dissociation of sera from Aβ immunized APP transgenic mice. Sera were collected from transgenic mice, diluted 1:400 and preincubated at either pH 7 (open bars) or pH 2.5 (solid bars) as described in methods. The sera were then incubated on ELISA plates coated with Aβ1-42, phosphate buffered saline (PBS; a "no protein" control), bovine serum albumin (BSA), recombinant alpha-synuclein (α-SYN) or Aβ peptide amino acids 11–20. ELISA assays were completed as described in Methods and the optical density at 450 nm used to estimate the amount of antibody binding to the ELISA plate. Results are presented as mean ± sem.

Figure 5

Competition with Aβ1-42 against the increased antibody binding found after acid dissociation of sera from APP mice vaccinated with Aβ. Sera were collected from transgenic mice, diluted 1:1000 and preincubated at either pH 7 (open bars) or pH 2.5 (solid bars) as described in methods. After centrifugation and reconstitution of serum volume, the sera were incubated with Aβ1-42 at the indicated concentrations. Because the Aβ was initially dissolved in DMSO, some samples were also incubated with DMSO at the concentrations found in the 100 μg/ml Aβ incubation (2% DMSO). ELISA assays were completed as described in Methods and the optical density at 450 nm used to estimate the amount of antibody binding to the ELISA plate. Results are presented as mean ± sem.