Alzheimer disease is a neurodegenerative disorder for which immunotherapy has treatment potential using either active or passive Aβ42 immunizations to reduce Aβ42 peptide levels in the brains of patients with AD.27
To verify that DNA immunization is a safe way to proceed in the treatment of AD patients, results from a direct comparison of the gene gun–mediated DNA Aβ42 immunization in mice were compared with the results from mice that had been immunized with Aβ42 peptide and Quil A adjuvant. The immune response was assessed 2 ways. First, we analyzed the humoral response in the 2 groups of mice. DNA immunization with the double plasmid system produced a mean antibody titer of 15 µg/mL, which was much less when compared with peptide immunization (1.2 mg/mL) but significantly increased compared with previous studies using an Aβ42 monomer (1-copy) plasmid.18–20
Although antibody levels derived from DNA immunization were lower than levels produced by peptide immunization, the achieved levels are most likely effective, and the antibody isotype is biased to TH
2, indicating a low probability to cause inflammation. A predominant TH
2 response is a more important objective in developing an effective and safe therapy for AD than increased antibody levels alone.
The antibody isotypes obtained are a direct reflection of the cytokine production during an immune response. TH
1 cytokines like IFN-γ promote immunoglobulin class switching from IgM to IgG2a, whereas TH
2 cytokines like IL-4 and IL-5 lead to immunoglobulin isotype switching to IgG1.25,28
A predominant IgG1 antibody response (TH
2 type) in the DNA-immunized mice and a mixed IgG1/IgG2a (TH
1) response after peptide vaccination is consistent with results published from other groups that show that Aβ42 DNA vaccination leads indeed to a polarized TH
Also in the peptide immunization protocols, it is well established that the type of adjuvant used has a major influence on the elicited immune response.32,33
Qs21, a more highly refined derivative of Quil A and a known TH
1-type adjuvant, was used in the clinical trial.12–16,33
Immunization with Aβ1–42 peptide and Quil A yielded a broad cross-reactive IgG1/IgG2a immune response as compared with the solely IgG1 isotype (TH
2 type) in the DNA Aβ42 trimer–immunized mice.
The binding sites (epitopes) of Aβ42 antibodies analyzed in an AD mouse model showed that antibodies directed against an N-terminal region (epitope), amino acids 1–15 of the 42 amino acids of the peptide (Aβ1–15), were more effective in plaque clearance than antibodies detecting C-terminal epitopes,34
which is consistent with the antibody binding site (epitope) mappings for DNA and peptide immunization at Aβ1–15 in this study. In the same study,34
the authors also showed an important impact of the antibody isotypes: IgG2a antibodies were the most effective antibodies to reduce Aβ42 levels, likely through their high affinity for Fc receptor binding (FcγRI) on phagocytic microglia cells. However, it is also believed that TH
1-type antibodies contributed to the inflammation in the clinical trial.12,13
In a different study, it was shown that the highest activity levels for reduction of Aβ42 levels by microglia cultures were found for IgG1 antibodies (TH
Several antibody actions that result in reducing brain levels of Aβ42 peptide are implicated, including the opsonization of Aβ42 to increase phagocytosis, the interference of antibodies with Aβ aggregation, and the function of antibodies as a peripheral sink without entering the brain.36
The observed broader reactivity of the antibodies from peptide-immunized mice is a characteristic feature of a TH
1 immune response. In some respects, the breadth of the immune responses, an increased number of loci or epitopes of antibody binding to the Aβ42 peptide seen following peptide immunization, is analogous to the process of epitope spreading (antibody binding to increased number of regions on a particular peptide), seen in the SJL mouse model of experimental autoimmune encephalomyelitis, a mouse model for multiple sclerosis. In that model, epitope spreading is associated with disease worsening and progression.37
Thus, the highly focused and TH
2 predominant response seen following DNA immunization indicates a reduced risk for inflammation in AD patients.
Analyzing the T-cell response in both the DNA Aβ42– and peptide-immunized mice was essential in pursuing an immunization approach for AD treatment. Circulating Aβ-reactive T cells are present in patients with AD without any previous immunizations.14
In the experiment, the T-cell reactivity, response of T cells to Aβ42 peptide in cell culture, was very low in the DNA-immunized mice. However, it is clear that T cells had been involved earlier in the immunization procedure and is demonstrated by the switching of the immunoglobulin isotypes from IgM to IgG1 (TH
2 help). The lack of Aβ42-reactive T cells after their initial help in B-cell activation and respective antibody production might be favorable in regard to the immunization of patients with AD by this method, because without Aβ42-specific T cells (and lack of IFN-γ), the risk of inflammation is low. Different from other DNA Aβ42 immunization approaches in which only parts of the Aβ peptide were included,31,38–40
we used full-length DNA Aβ42 trimer containing both B- and T-cell epitopes and still were able to show nonreactive T cells to restimulation with Aβ42 peptide, again indicating a low probability of an inflammatory T-cell response when administered to patients with AD. Further detailed analysis of the suggested exhaustion of a specific T-cell response after DNA Aβ42 trimer immunization is of high interest.
Finding the cytokine IL-10 in DNA-immunized mice is of particular interest, since IL-10 is described as an anti-inflammatory cytokine that down-regulates TH
IL-10 up-regulation (4000 pg per milliliter of plasma) has been shown in mice immunized with an 11x-Aβ1–6 DNA adenovirus vector, and Kim et al43
suggested that IL-10 production drives the TH
2 response in their immunization protocol. Since this particular cytokine was found in the DNA-immunized mice, further analysis of IL-10 is indicated.
The parallel comparison of gene gun–delivered DNA Aβ42 immunization and intraperitoneal injections of Aβ42 peptide showed the characteristic features of genetic immunizations. DNA Aβ42 trimer immunization of wild-type mice resulted in a strongly polarized TH2 immune response that has high probability to be noninflammatory and therefore safe as therapy to delay AD.
If DNA Aβ42 trimer immunization is able to reduce Aβ 42 peptide levels in the brains of patients with AD by one-half as it does in the transgenic mouse brain,16–21
it may be possible to slow progression of AD for several years. The overall potential of DNA Aβ42 immunization may be considerable, and effort should be undertaken to test it thoroughly.