The immunological potency and therapeutic potential of a prototype dual vaccine against influenza and Alzheimer's disease

Abstract

Background: Numerous pre-clinical studies and clinical trials demonstrated that induction of antibodies to the β-amyloid peptide of 42 residues (Aβ42) elicits therapeutic effects in Alzheimer's disease (AD). However, an active vaccination strategy based on full length Aβ42 is currently hampered by elicitation of T cell pathological autoreactivity. We attempt to improve vaccine efficacy by creating a novel chimeric flu vaccine expressing the small immunodominant B cell epitope of Aβ42. We hypothesized that in elderly people with pre-existing memory Th cells specific to influenza this dual vaccine will simultaneously boost anti-influenza immunity and induce production of therapeutically active anti-Aβ antibodies.

Methods: Plasmid-based reverse genetics system was used for the rescue of recombinant influenza virus containing immunodominant B cell epitopes of Aβ42 (Aβ1-7/10).

Results: Two chimeric flu viruses expressing either 7 or 10 aa of Aβ42 (flu-Aβ1-7 or flu-Aβ1-10) were generated and tested in mice as conventional inactivated vaccines. We demonstrated that this dual vaccine induced therapeutically potent anti-Aβ antibodies and anti-influenza antibodies in mice.

Conclusion: We suggest that this strategy might be beneficial for treatment of AD patients as well as for prevention of development of AD pathology in pre-symptomatic individuals while concurrently boosting immunity against influenza.

Figure 1

Preparation of chimeric virus: (A) Schematic presentation of the rescue strategy of WSN-Aβ_1-10 chimeric virus. (B) SDS-PAGE and coomassie staining of purified chimeric (WSN-Aβ_1-10) and wild-type (WT) viruses. (C) WB analysis of purified virus using anti-Aβ antibody revealed the chimeric HA-Aβ_1-10 protein of the correct size. (D) Proteins corresponding to NP, HA and M1 were detected in WB analysis of purified virus using anti-WSN polyclonal serum.

Figure 2

Expression of β-amyloid B cell epitopes by chimeric influenza virus WSN (WSN-Aβ_1-10 and WSN-Aβ_1-7). MDCK cells infected with WSN-Aβ_1-10 and WSN-Aβ_1-7 were positive for immunostaining with anti-Aβ and anti-HA antibodies, whereas cells infected with WSN-WT were positive only with anti-HA antibody.

Figure 3

Anti-HA antibodies inhibited agglutination of RBC by both wild-type and chimeric influenza viruses, while anti-Aβ antibodies only inhibited agglutination of RBC by the chimeric virus.

Figure 4

Mice immunized with killed WSN-Aβ_1-10 virus generated significantly higher anti-Aβ₄₂ specific antibodies compared with that in mice immunized with WSN-Aβ_1-7. Anti-Aβ antibody responses were measured in sera of individual mice immunized 3 times with indicated viruses at dilution 1:200. Lines represent the average (n = 5, *P < 0.05; **P < 0.01).

Figure 5

Anti-Aβ and anti-WSN immune responses in mice immunized with different doses of WSN-Aβ_1-10 and WSN-WT: Anti-Aβ (A) and anti-WSN (B, C) antibodies were analyzed in sera of individual mice immunized 3 times with indicated doses of killed WSN-Aβ_1-10 and WSN-WT viruses formulated in Quil A. Lines and error bars indicate the average ± s.d. (n = 6 for groups immunized with 5 and 25 μg and n = 16 for groups immunized with 50 μg killed viruses (*P < 0.05; ***P < 0.001).

Figure 6

Kinetics of anti-Aβ (A) and anti-WSN-WT antibody responses (B) in mice immunized with 50 μg/mouse of WSN-Aβ_1-10 and WSN-WT viruses. Concentration of anti-Aβ antibodies and half-maximal titers (HMAT) of anti-WSN-WT antibodies were analyzed in individual mice. HMAT was determined in the sera of individual mice by dividing the highest OD₄₅₀ value in the dilution range of each sample by two. Initial dilution of sera in these experiments was 1:500 and they were serially diluted up to 1:500000. Error bars indicate the average ± s.d. n = 16 and n = 8 in groups immunized with WSN-Aβ_1-10 and WSN-WT viruses respectively (**P < 0.01, ***P < 0.001).

Figure 7

Therapeutic potency of anti-Aβ antibody generated in mice immunized with WSN-Aβ_1-10: (A) Immune sera generated after immunization with killed WSN-Aβ_1-10 (at dilution 1:600) bound to the brain sections of cortical tissues from an AD case and (B) this binding was blocked by pre-absorption of sera with Aβ₄₂ peptide. (C) Immune sera generated after immunization with killed WSN-WT (at dilution 1:600) did not bind to the brain sections of cortical tissues from an AD case. Original magnification was ×4 and scale bar was 200 μm.

Figure 8

Antibodies generated in mice immunized with dual vaccine, WSN-Aβ_1-10 bind to Aβ₄₂ and inhibit its neurotoxicity: (A) Sera isolated from WSN-Aβ_1-10, but not WSN-WT vaccinated mice at dilution 1:200 bound to all species of Aβ₄₂ peptide, including oligomers recognized by A11 oligomer-specific antibodies. Control monoclonal 6E10 antibody bound to all forms of Aβ₄₂ peptide. (B) Anti-Aβ_1-10 inhibits Aβ₄₂ fibrils- and oligomer-mediated toxicity. Human neuroblastoma SH-SY5Y cells were incubated with Aβ₄₂ oligomers and Aβ₄₂ fibrils, in the presence or absence of anti-Aβ_1-10 antibody or irrelevant mouse IgG. Control cells were treated with the vehicle, and cell viability was assayed in all cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data were collected in four replicate and was expressed as a percentage of control ± s.d.

Figure 9

Antibodies generated in mice immunized with dual vaccine, WSN-Aβ_1-10 neutralize both WSN-WT (A) and WSN-Aβ_1-10 (B) viruses. Titers of HI antibody against WSN-WT (A) or WSN-Aβ_1-10 (B) viruses were measured in individual mice (n = 6/per group) after 3 immunizations. The statistical difference between each group was determined (*P < 0.05; **P < 0.01).

Figure 10

Virus neutralization titers of sera generated after 2, 3 and 4^th immunizations with dual vaccine and WSN-WT are the same. HI titers against WSN-WT (A) and WSN-Aβ_1-10 (B) were evaluated in sera of individual mice immunized after 2, 3, and 4 immunizations with WSN-WT (close sq) or WSN-Aβ_1-10 (open sq). Error bars indicate the average ± s.d. for mice immunized with WSN-Aβ_1-10 (n = 16) or WSN-WT (n = 8) (*P <0.01; **P < 0.01).