Gene vaccination to bias the immune response to amyloid-beta peptide as therapy for Alzheimer disease

Abstract

Background: The amyloid-beta (Abeta) peptide has a central role in the neurodegeneration of Alzheimer disease (AD). Immunization of AD transgenic mice with Abeta(1-42) (Abeta(42)) peptide reduces both the spatial memory impairments and AD-like neuropathologic changes in these mice. Therapeutic immunization with Abeta in patients with AD was shown to be effective in reducing Abeta deposition, but studies were discontinued owing to the development of an autoimmune, cell-mediated meningoencephalitis. We hypothesized that gene vaccination could be used to generate an immune response to Abeta(42) that produced antibody response but avoided an adverse cell-mediated immune effect.

Objective: To develop an effective genetic immunization approach for treatment and prevention of AD without causing an autoimmune, cell-mediated meningoencephalitis.

Methods: Mice were vaccinated with a plasmid that encodes Abeta(42), administered by gene gun. The immune response of the mice to Abeta(42) was monitored by measurement of (1) antibody levels by enzyme-linked immunosorbent assay (ELISA) and Western blot and (2) Abeta(42)-specific T-cell response as measured by interferon-gamma enzyme-linked immunospot (ELISPOT) assay.

Results: Gene-gun delivery of the mouse Abeta(42) dimer gene induced significant humoral immune responses in BALB/c wild-type mice after 3 vaccinations in 10-day intervals. All 3 mice in the treated group showed significant humoral immune responses. The ELISPOT assay for interferon-gamma release with mouse Abeta(42) peptide and Abeta(9-18) showed no evident cytotoxic T-lymphocyte response. We further tested the responses of wild-type BALB/c mice to the monomer Abeta(42) gene vaccine. Western blot evaluation showed both human and mouse Abeta monomer gene vaccine elicited detectable humoral immune responses. We also introduced the human Abeta(42) monomer gene vaccine into AD double transgenic mice APPswe/PSEN1(A246E). Mice were vaccinated with plasmids that encode Abeta(1-42) and Abeta(1-16), or with plasmid without the Abeta gene. Treated mice showed significant humoral immune responses as demonstrated by ELISA and by Western blot. These mice also showed no significant cellular immune response as tested by ELISPOT. One of the treated mice was killed at 7 months of age for histological observations, and scattered amyloid plaques were noted in all layers of the cerebral cortex and in the hippocampus in both Abeta(42)- and control-vaccinated mice. No definite difference was discerned between the experimental and control animals.

Conclusions: Gene-gun-administered genetic immunization with the Abeta(42) gene in wild-type BALB/c and AD transgenic mice can effectively elicit humoral immune responses without a significant T-cell-mediated immune response to the Abeta peptide. This immunotherapeutic approach could provide an alternative active immunization method for therapy and prevention of AD.

Figure 1

Gene immunization vector contains SP72, a synthetic mammalian expression promoter; an Aβ₄₂ gene sequence fused between a human α-antitrypsin secretory signal and a major histocompatibility complex class (MHC) II–targeting peptide sequence, and the ampicillin-resistance gene, Aβ_1–42 monomer or dimer gene, followed by an MHC II–targeting sequence.

Figure 2

Amyloid-β₄₂ (Aβ₄₂)–specific immune responses in BALB/c wild-type mice immunized with mouse Aβ₄₂ dimer gene vaccine. A, Western blot shows that all 3 mice in the vaccinated group produced specific anti-Aβ₄₂ antibodies that detected mouse recombinant glutathione S-transferase–Aβ₄₂ (GST-Aβ₄₂) dimers (lower arrow in lane P) and tetramers (upper arrow in lane P) (lanes m1, m2, m3). Mice in the control group (vector lacking Aβ gene) were negative (lane C). Recombinant GST-Aβ₄₂ protein in Escherichia coli extracts was shown in lane P stained with Coomassie blue. Serum samples were obtained 14 days after a third vaccination at 10-day intervals with dilution of 1:2000. kDa indicates kilodalton. B, Same serum samples as in A were tested by enzyme-linked immunosorbent assay for Aβ₄₂ peptide and show that all 3 mice (m1, m2, m3) vaccinated with Aβ₄₂ dimer gene showed specific antibody against human Aβ₄₂ peptide with 1:2000 dilution. C, Enzyme-linked immunospot assay demonstrated that no detectable cellular immune response was observed in Aβ₄₂ dimer gene–vaccinated mice. Concanavalin A (Con A) was added as a positive control. P–indicates no peptide added in peripheral blood T-cell culture; P+, a mixture of Aβ_9–18 and Aβ_1–42 peptide was added to the culture for 36 hours for specific antigen stimulation for T cells to release interferon-γ (INF-γ).

Figure 3

Western blot to show humoral immune response of BALB/c wild-type mice against monomer Aβ₄₂. Mice were immunized with monomer human (amyloid-β [hAβ] protein) and mouse (mAβ) Aβ_1–42 gene vaccine by gene gun. Human Aβ induced a higher immune response compared with mAβ in these mice in short schedule (3 immunizations in a 10-day interval and blood was drawn 2 weeks after the third shot) immunizations (panel A), but responses were similar with a long-schedule vaccination (additional 3 shots in a 1-month interval and blood was drawn 2 weeks after the last shot) (panel B). Administration of granulocyte-monocyte colony-stimulating factor (GMSF) (performed with mAβ₄₂ immunizations) seemed to enhance antibody production. This result demonstrated that genetic vaccination can break the self-tolerance in mice for mAβ₄₂ protein and also elicit strong immune responses to hAβ₄₂.

Figure 4

Human amyloid-β 42 (Aβ ₄₂)–specific immune responses in transgenic (Tg) mice immunized with human Aβ ₄₂ gene vaccine. A, Anti-Aβ peptide antibody titer assayed by enzyme-linked immunosorbent assay in Tg mice immunized with both Aβ _1–42 and Aβ _1–16 gene construct for 4 times in 2-week intervals. The serum sample was obtained 2 weeks after the last immunization and titers were tested against Aβ peptide 1–16, 17–28, 29–42 fused to glutathione S-transferase (GST) protein produced in Escherichia coli. A higher response against Aβ _1–16 was seen. B, The same serum sample tested with Western blot shows a similar result; both mouse 1 (m1) and 2 (m2) in the treated group show a higher response against Aβ peptide. A higher titer is achieved for the m1 than for the m2. Control mice are negative for antibodies. Lane 1; Aβ _1–16, lane 2; Aβ _17–28, lane 3; Aβ _29–42 fused to GST was loaded and probed with serum in a 1:2000 dilution. The third treated mouse showed no detectable humoral response (data not shown). C, Enzyme-linked immunospot assay shows that no significant cellular immune response was observed in human Aβ ₄₂ gene–vaccinated Tg mice. Peripheral blood T cells were pooled from the vaccinated and control groups of mice and the cells were cultured in quadruplicate (2 × 10⁵ cells per well in a 96-well microplate format) in the presence of peptide or absence of peptide using a mixture of Aβ _1–42 and Aβ _9–18 peptide at 10 μg/mL for 36 hours and further processed for detection of released interferon-γ.

Figure 5

Preliminary examination of brains of transgenic (Tg) mice 5 months after immunization. Mice were killed 5 months after immunization. Brains were fixed in paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylineosin (A) and evaluated by immunohistochemistry for amyloid- β (Aβ ) (B) and glial fibrillary acid protein (C). Scattered amyloid plaques were noted diffusely in the brains in both the control- and Aβ gene vaccine–immunized mice without evidence of lymphocytic inflammation. A brisk astrocytic response to plaque deposition was identified. Analysis of older animals is necessary to assess for differences between the DNA-vaccinated and control vector–vaccinated mice for amyloid deposition and other parameters.