Six transgenic and six nontransgenic mice for each group were vaccinated beginning at 9 months of age and sacrificed at 12 months of age, 14 days after the fourth inoculation. but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-A titers were still lower in transgenic mice vaccinated with wild-type A or E22Q A relative to non-transgenic mice. Importantly, the dissociated anti-A 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 A back to the dissociated serum blocked the increase in antibody titers. Conclusions Circulating human A can interfere with ELISA assay measurements of anti-A titers. The E22Q A peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against A abrogate the effects of A self-tolerance. Background Vaccines directed against the A peptide reduce amyloid loads in amyloid precursor protein (APP) transgenic mice [1] and protect mice from amyloid-associated memory impairments [2,3]. Although a fraction of patients in a clinical A vaccination trial developed adverse reactions [4,5], there are indications that some patients benefited from the immunization [6]. Thus, although reformulation may be necessary, some form of anti-A immunotherapy may still be a useful treatment for Alzheimer’s Disease (AD). Several groups, including our own, have noted reduced antibody titers in mice transgenic for human APP compared to nontransgenic mice [7-9]. Typically, this was attributed to some form of self-tolerance that could be partially overcome with additional immunizations. One approach to overcoming B cell tolerance to self proteins when producing vaccines has been to conjugate the self-antigen at high density to SBI-425 papillomavirus virus-like particles (VLPs; [10]). To examine whether self-tolerance plays a role in diminishing immune responses against A in APP Tg mice, and whether VLP conjugation could overcome tolerance, we compared the ability to induce anti-A Ig responses using wild type human A peptide and VLP conjugated A. In order to detect Ab in the Tg mice, we developed a technique to detect anti-A antibodies that are masked by circulating A. In addition, we examined the immunogenicity of a human A variant believed responsible for hereditary SBI-425 cerebral hemorrhage with amyloidosis Dutch-type (DM A peptide) [11], in both WT A and non-transgenic backgrounds. Results and Discussion Antibody responses in APP Tg mice vaccinated with Rabbit polyclonal to AFP (Biotin) WT or VLP-conjugated A were examined by ELISA using our standard procedures [12] and anti-A antibody titers were almost undetectable using both protocols (Fig. ?(Fig.1,1, pH 7.0 results). However, nontransgenic mice did exhibit readily measurable titers (Fig. ?(Fig.2,2, pH SBI-425 7.0 results). For the DM A vaccine high anti-A titers were detected in both genotypes, albeit lower in the transgenic animals. This prompted us to test whether circulating human A might be masking anti-A antibodies in the transgenic mice. When we compared several methods of dissociating antibodies from their antigens, including dithiothreitol (100 mM), -mercaptoethanol (0.5%) and reduced pH (pH 2.5 as described in methods), we found the acid dissociation procedure resulted in the greatest increase in anti-A antibody titers (4 SBI-425 fold greater than any of the other treatments). We then tested if antigen-antibody dissociation would increase the apparent anti-A titers by comparing incubation of the sera at pH 2.5 versus pH 7.0. Open in a separate window Figure 1 Transgenic APP mice immunized with A 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 5C6 per group. Open in a separate window Figure 2 Nontransgenic mice immunized with A 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 5C6 per group. The low pH dissociation procedure caused a dramatic elevation of the apparent anti-A antibody titers in sera collected from transgenic mice (Figure ?(Figure1).1). The titers increased from values near zero in the pH 7.0 incubation to roughly 8000 in the pH 2.5 incubation (t-test; P < 0.001; WT and VLP vaccines). Samples from mice inoculated with the DM A peptide showed only.

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