DNA beta-amyloid(1-42) trimer immunization for Alzheimer disease in a wild-type mouse model

Abstract

Context: DNA beta-amyloid(1-42) (Abeta42) trimer immunization was developed to produce specific T helper 2 cell (T(H)2)-type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Abeta42 peptide that occur in the brain of patients with AD.

Objective: To compare the immune response in wild-type mice after immunization with DNA Abeta42 trimer and Abeta42 peptide.

Design and intervention: Wild-type mice received either 4 microg of DNA Abeta42 trimer immunization administered with gene gun (n = 8) or intraperitoneal injection of 100 microg of human Abeta42 peptide with the adjuvant Quil A (n = 8). Titers, epitope mapping, and isotypes of the Abeta42-specific antibodies were analyzed.

Main outcome measures: Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Abeta42-specific antibodies, and T-cell activation.

Results: DNA Abeta42 trimer immunization resulted in antibody titers with a mean of 15 microg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a T(H)2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed T(H)1/T(H)2 immune response (IgG1/IgG2a ratio of 1) (P < .001). No increased T-cell proliferation was observed in the DNA-immunized mice (P = .03).

Conclusion: In this preliminary study in a wild-type mouse model, DNA Abeta42 trimer immunization protocol produced a T(H)2 immune response and appeared to have low potential to cause an inflammatory T-cell response.

Figure 1. Schematic Representation of the Double Plasmid System (GAL4 Activator, UAS/Aβ42 Trimer Responder) Used for Genetic Immunization

Constitutive expression of the GAL4 transcription factor is driven by a cytomegalovirus (CMV) promoter on the activator plasmid. The GAL4 protein binds as a homodimer to the responder plasmid at sites in the upstream activator sequence (UAS), part of a minimal promoter. GAL4 binding enhances transcription of the β-amyloid_1–42 (Aβ42) trimer sequence that has been cloned into a DNA fragment between an adenovirus E3 (early region 3) leader sequence and an endosomal targeting sequence derived from the mouse major histocompatibility complex class II gene H2-DM. These flanking regions are involved in targeting the messenger RNA (mRNA) transcript to the endoplasmic reticulum for protein synthesis and secretion. The simian virus 40 (SV40) polyadenylation (PolyA) sequence on both the activation and responder plasmids stabilize mRNA transcripts.

Figure 2. Different IgG Isotypes in B6SJLF1/J Mice After Peptide and DNA Immunization With Human β-Amyloid_1–42

From a blood sample taken after 4 vaccinations, the ratio of the optical density values of IgG1 to IgG2a was calculated. Mice immunized with DNA (D1–D8) showed a predominant IgG1 response indicating a T helper 2 cell (T_H2) response (mean [SD] IgG1/IgG2a ratio,9.63[1.18]). Mice immunized with peptide (P1–P8) showed both isotypes, IgG1 and IgG2a, indicating a mixed T_H2/T_H1 response (mean [SD] IgG1/IgG2a ratio, 1.23 [0.14]). P<.001 for comparison between the groups. Square data markers indicate group means; error bars indicate SD.

Figure 3. Epitope Mapping of the β-Amyloid_1–42 Antibodies in Mice Immunized With DNA Aβ1–42 Trimer and Aβ1–42 Peptide

A similar pattern of antibody binding sites (epitopes) was found for DNA-immunized mice (D1 to D8) and peptide-immunized mice (P1 to P8). All plasma samples were used in a 1:500 dilution and were analyzed in triplicates. Positive binding was found for full-length Aβ1–42 and Aβ1–15 in DNA- and peptide-immunized plasma samples (A and B). Some plasma samples from the peptide-immunized mice had high titers of antibodies binding to different epitopes, Aβ10–25 and Aβ16–30 (C and D). No binding was found for Aβ4–10 and Aβ21–35 (E and F), as well as for Aβ1–9, Aβ5–14, and Aβ6–20 (data not shown). Each data marker represents a mean of 3 values; error bars indicate standard deviation.

Figure 4. Stimulation Indexes for β-Amyloid₄₂ Restimulated Splenocyte Cultures

Cells were cultured and restimulated with Aβ1–42 peptide (10 ug/mL) 10 days after the final immunization. Incorporation of tritium-labeled thymidine was used to measure cell proliferation and a stimulation index (SI) was calculated using the following formula: counts per minute (CPM) of wells with Aβ42 antigen divided by CPM of wells with no antigen. DNA-immunized mice had an SI of less than 1 (mean [SD], 0.93 [0.12]; n=3) and peptide-immunized mice had an SI of 2 to 7 (mean [SD], 4.77 [1.14]; n=4). P=.03 for comparision between the groups.