Low concentrations of anti-Aβ antibodies generated in Tg2576 mice by DNA epitope vaccine fused with 3C3d molecular adjuvant do not affect AD pathology

Abstract

It has been demonstrated that an active vaccination strategy with protein- or DNA-based epitope vaccines composed of the immunodominant self B cell epitope of amyloid-β₄₂ (Aβ₄₂) and a non-self T helper (Th) cell epitope is an immunotherapeutic approach to preventing or treating Alzheimer's disease (AD). As a DNA-based epitope vaccine, we used a plasmid encoding three copies of Aβ(1-11) and Th cell epitope, PADRE (p3Aβ(1-11)-PADRE). We have previously reported that three copies of component of complement C3d (3C3d) acts as a molecular adjuvant significantly enhancing immune responses in wild-type mice of the H2(b) haplotype immunized with p3Aβ(1-11)-PADRE. Here, we tested the efficacy of p3Aβ(1-11)-PADRE and the same vaccine fused with 3C3d (p3Aβ(1-11)-PADRE-3C3d) in a transgenic (Tg) mouse model of AD (Tg2576) of the H2(bxs) immune haplotype. The overall responses to both vaccines were very weak in Tg2576 mice despite the fact that the 3C3d molecular adjuvant significantly enhanced the anti-Aβ response to 3Aβ(1-11)-PADRE. Importantly, generation of low antibody responses was associated with the strain of amyloid precursor protein Tg mice rather than with a molecular adjuvant, as a p3Aβ(1-11)-PADRE-3C3d vaccine induced significantly higher antibody production in another AD mouse model, 3xTg-AD of the H2(b) haplotype. Finally, this study demonstrated that low concentrations of antibodies generated by both DNA vaccines were not sufficient for the reduction of Aβ pathology in the brains of vaccinated Tg2576 animals, confirming previous reports from preclinical studies and the AN-1792 clinical trials, which concluded that the concentration of anti-Aβ antibodies may be essential for the reduction of AD pathology.

FIG. 1.

3Aβ_1–11-PADRE epitope vaccine with and without 3C3d molecular adjuvant induced low titers of anti-Aβ antibodies in Tg2576 mice. Preventive immunizations of Tg2576 mice (initiated at 3–4 months of age and carried up to 17–18 months) with p3Aβ_1–11-PADRE induced poor production of anti-Aβ antibodies. Addition of 3C3d molecular adjuvant to p3Aβ_1–11-PADRE plasmid (p3Aβ_1–11-PADRE-3C3d) significantly enhanced humoral immune response (***p < 0.001); however, the strength of the humoral immune response in both immunized groups was low.

FIG. 2.

Isotype profile of Aβ-specific antibodies generated in transgenic Tg2576 mice. (A) Immunizations of Tg2576 mice with DNA epitope vaccines (p3Aβ_1–11-PADRE-3C3d or p3Aβ_1–11-PADRE) induce production of IgG1, IgG2a^b, and IgG2b isotypes. Interestingly, a high production of IgM isotypes was detected in both groups of experimental mice. (B) Calculation of the IgG1/IgG2a^b ratio determined a slight Th2-biased humoral immune response in the group immunized with p3Aβ_1–11-PADRE, whereas addition of 3C3d molecular adjuvant induced a stronger Th2-type of immune response (group p3Aβ_1–11-PADRE-3C3d).

FIG. 3.

Immunizations with either vaccine candidate did not change Aβ plaque load in the brains of Tg2576 mice. Analysis of Aβ plaque load was performed on brain sections of transgenic mice terminated at 17–18 months of age. Preventive immunizations did not affect plaque deposition in the brains of mice from both vaccinated groups. Representative staining images of the distribution of Aβ plaques stained with 6E10 monoclonal antibody in the cortex of nonimmunized and p3Aβ_1–11-PADRE-3C3d- and p3Aβ_1–11-PADRE-immunized mice are presented. Scale bar: 200 μm at 4× magnification.

FIG. 4.

Biochemical analysis of brain tissues revealed no changes in soluble and insoluble Aβ levels in both immunized and nonimmunized groups of Tg2576 mice. Low titers of anti-Aβ antibodies generated after immunization with p3Aβ_1–11-PADRE-3C3d or p3Aβ_1–11-PADRE were not sufficient for inhibition of both soluble and insoluble Aβ₄₀ and Aβ₄₂ peptide levels in the brain. Analysis of neuropathological changes was performed in the brains of vaccinated and control (nonimmunized) mice at the age of 17–18 months.