Linear and conformation specific antibodies in aged beagles after prolonged vaccination with aggregated Abeta

Abstract

Previously we showed that anti-Abeta peptide immunotherapy significantly attenuated Alzheimer's-like amyloid deposition in the central nervous system of aged canines. In this report we have characterized the changes that occurred in the humoral immune response over 2.4years in canines immunized repeatedly with aggregated Abeta(1-42) (AN1792) formulated in alum adjuvant. We observed a rapid and robust induction of anti-Abeta antibody titers, which were associated with an anti-inflammatory T helper type 2 (Th2) response. The initial antibody response was against dominant linear epitope at the N-terminus region of the Abeta(1-42) peptide, which is identical to the one in humans and vervet monkeys. After multiple immunizations the antibody response drifted toward the elevation of antibodies that recognized conformational epitopes of assembled forms of Abeta and other types of amyloid. Our findings indicate that prolonged immunization results in distinctive temporal changes in antibody profiles, which may be important for other experimental and clinical settings.

Fig. 1

Immunization of aged canines with aggregated Aβ_1–42 induces a robust and relatively uniform antibody response. (A) Time line of injections (black arrows) and blood collection (black arrowheads) in older canines. (B) Anti-Aβ antibody levels detected with ELISA reached a plateau after 4 injections and were maintained until the end of the study. (C) Anti-Aβ antibodies induced by vaccination were primarily of the IgG2 (anti-inflammatory) isotype. Open circles represent the data for individual animals, lanes are the value of a mean. (T= blood collection time points). Bars represent means, and error bars represent SEM.

Fig. 2

Fine epitope mapping of anti-Aβ antibodies with short linear peptides revealed a major linear epitope induced against the N-terminus (peptide 1–15) of the Aβ_1–42 peptide. Linear epitopes were detected by ELISA after 2 (A), 4 (B) and 25 (C) injections. (D) Immune dog serum does not detect canine APP in a Western blot assay, while it recognizes Aβ_1–42 peptide. Brain extracts from three different dogs were loaded on a gel at 5 or 25μg per well. (E) membrane from (D) was striped and re-probed with monoclonal 6E10 antibody to demonstrate the presence of APP protein. Open circles represent the individual animals data, lines are the value of the mean.

Fig. 3

Conformational sensitive antibodies were induced in dogs after 4 injections with aggregated Aβ_1–42 peptide and persisted until the end of the experiment. (A) Canine immune serum after 2, 4 or 25 injections was pre-incubated with short linear peptides (see experimental design section) or Aβ_1–42 peptide and the % of inhibition of binding to Aβ_1–42 on an ELISA plate was measured. (B) Aβ_1–42 peptide in CCB forms high molecular weight aggregates, which were detected with monoclonal antibodies 6E10 and 4G8 in a Western blot assay. (C) Immune canine serum at T4 was incubated with short linear peptides or left without inhibition, and probed against monomeric and aggregated Aβ_1–42 peptide spotted on a membrane. As controls, N-terminal Aβ specific mouse monoclonal 20.1 antibody or serum collected from a PBS injected dog reveal positive and negative staining, respectively. (D) Resulting dot blots from (C) were analyzed using Image J software and presented as a percent of inhibition of binding to monomeric or aggregated Aβ with short linear peptides. Pictures of representative dot blots are shown. (CCB= carbonate coating buffer; T=blood collection time points; mono= monomeric Aβ_1–42 peptide; aggr= aggregated Aβ_1–42 peptide). Open circles represent the individual animals data, lines and bars are the value of the mean, error bars are the SEM.

Fig. 4

After 25 injections, dogs were categorized into 4 separate groups according to the conformational specificity of the antibodies produced: (A) monomeric Aβ-selective antibodies (n=1); (B) Aβ-specific antibodies independently of conformation (n=2); (C) monomeric and fibrillar Aβ forms selective antibodies (n=3); and (D) aggregated proteins sensitive antibodies (n=2). (E) Control oligomer-specific A11 antibody or N-terminal Aβ specific mouse monoclonal antibody 20.1 show predicted binding to pre-fibrillar proteins (A11) or all Aβ forms (20.1) respectively. Canine antibodies (A–D) as well as control antibodies (E) were analyzed using dot blots spotted with monomeric Aβ_1–42, pre-fibrillar Aβ_1–42, fibrillar Aβ_1–42 forms as well as pre-fibrillar forms of a-synuclein and islet APP. The percent of binding of immune serum to monomeric Aβ_1–42 was considered to be at 100%. The percent of binding to other Aβ-related and non-related aggregates was calculated after Image J analysis of the dot blots. Pictures of representative dot blots are shown. (mAβ_1–42= monomeric Aβ_1–42 peptide; pF Aβ_1–42= pre-fibrillar Aβ_1–42 peptide; F Aβ_1–42= fibrillar Aβ_1–42 peptide; pF α-Syn= pre-fibrillar alpha-synuclein protein; pF iAPP= pre-fibrillar islet amyloid polypeptide). Bars represent means, the error bars represent SEM.

Fig. 5

Progression of conformational sensitive antibodies in defined groups during the course of immunization. (A) Monomeric Aβ-selective antibodies. (B) Aβ-specific antibodies independent of conformation. (C) Monomeric and fibrillar Aβ-selective antibodies. (D) Aggregated proteins sensitive antibodies (independent of sequence). Canine immune serum after 2, 4 or 25 injections was analyzed by dot blots. The percent of binding of immune serum to monomeric Aβ_1–42 was considered as 100%. The percent of dog antibody binding to other Aβ-related and non-related aggregates was calculated after Image J analysis of the dot blots. Pictures of representative dot blots are shown. (mAβ_1–42= monomeric Aβ_1–42 peptide; pF Aβ_1–42= pre-fibrillar Aβ_1–42 peptide; F Aβ_1–42= fibrillar Aβ_1–42 peptide; pF α-Syn= pre-fibrillar α-synuclein protein; pF iAPP= pre-fibrillar islet amyloid polypeptide). Bars represent means, the error bars represent SEM.

Fig. 6

Immune canine serum detects amyloid plaques from a mouse model of AD with differential patterns of immunostaining, depending on the type of antibodies produced. (A) Monomeric Aβ-selective antibodies. (B) Aβ-specific antibodies independent of conformation. (C) monomeric and fibrillar Aβ-selective antibodies. (D) Aggregated proteins sensitive antibodies. Representative serum from the defined groups was pre-incubated with competing short peptides or left without competition and then used for the immunodetection of amyloid plaques in 18 months old APP Tg2576 mouse brains. Thioflavin S was used to identify dense core plaques. 40× original magnification. Red – dogs immunoglobulins, green – Thioflavin S, yellow – merged.

Fig. 7

Proposed scheme of the maturation of conformational selective antibodies in dogs after prolonged immunization with aggregated Aβ_1–42 peptide. Initial injections induced mainly monomeric Aβ-selective antibodies (human clinical trials were postponed at this stage). Subsequent injections induced Aβ-specific antibodies independently of conformation, additional injections stimulated the development of either monomeric and fibrillar Aβ-selective antibodies or antibodies against aggregated proteins independent of protein sequence.