Design, structural, and immuno‐analytical properties of antigenic bioconjugates comprising a β‐amyloid‐plaque specific epitope

Abstract

Immunotherapeutic approaches are investigated for treatment of neurodegenerative diseases of the Alzheimer's dementia (AD) type. The identification of a beta-amyloid-plaque specific epitope, Abeta(4-10) (4FRHDSGY10), recognized by therapeutically active antibodies from transgenic AD mice could provide the basis for the development of AD vaccines. Here we report on the synthesis, structural and immuno-analytical characterization of bioconjugates comprising the beta-amyloid(4-10) epitope as new vaccine lead structures against Alzheimer's disease. To produce antigenic bioconjugates, potential immunogens, the epitope peptide elongated by a cysteine residue or a cysteinyl-pentaglycine hexapeptide unit either at the N- or C-terminus was attached via a thioether bond to synthetic oligopeptide carriers, such as oligotuftsin derivatives, sequential oligopeptide carrier, or lysine dendrimer. The antigenic properties of these constructs were determined by enzyme-linked immunosorbent assay (ELISA) using an anti-Abeta(1-17) monoclonal antibody. Our results indicate that the major factors which influence the antibody binding of the Abeta(4-10) epitope are (i) the epitope topology and (ii) the presence of a spacer moiety between the carrier and the epitope peptide. Interestingly, the carrier type had no marked effect on the binding of the antibody to the epitope-conjugates. The conformational preferences of the conjugates were examined by circular dichroism spectroscopy in water and in trifluoroethanol. In water, the conjugates adopt random coil conformation independently on their primary structure. However, differences related to the attachment site of the epitope to the carriers were determined in TFE, conjugates in which the epitope was attached to the carrier through the N-terminus exhibiting more ordered secondary structure.

Epitope Mapping Immunoassay Analysis of the Interaction between β-Amyloid and Fibrinogen

“…Additionally, our evidence suggests that the Aβ–Fbg interaction might proceed by a new mechanism independent of the known Fbg knob-hole interaction. These previous findings supported a basis for the present study, which was focused on the identification of the essential amino acids within the Aβ4–10 [17] and Aβ17–40 [14] and Aβ17–42 [14] epitope sequences, and on the evaluation of their relative contribution to the interaction with Aβ-specific mono- and polyclonal antibodies [18].…”

Seropositivity and Antibody Profiling of Patients Are Dramatically Impacted by the Features of Peptides Used as Immunosorbents: A Lesson from Anti–Citrullinated Protein/Peptide Antibody

“…Our data not only sustain conclusions obtained by others with citrullinated or noncitrullinated Ags (12,34,39,40) but also raised the question of which peptide characteristic had a major impact in the ELISA results. Indeed, only a few works have investigated the influence of the biotin position or the epitope-biotin distance, on single epitope recognition (11,34,40). With various peptides encompassing a single epitope in different conformations we showed that biotin-epitope distance was the major significant parameter that influenced epitope recognition.…”

“…A fter the development of ELISA in the 70s (1,2), the discovery of the high avidin-biotin affinity (3,4) allowed researchers to precisely manipulate proteins/peptides in numerous ELISA constructs in the 90s (5). Today, although ELISA is used worldwide, for instance to detect and quantify Abs, some studies highlighted the influence of experimental parameters on the outputs of such assay (6)(7)(8)(9)(10), reporting that a small change in the structure of an epitope-presenting peptide could impact its recognition by Abs (11)(12)(13)(14) and thus our perception of the analyzed biological processes. This is particularly important for autoimmune diseases such as rheumatoid arthritis (RA) in which autoantibody profiling, specificity, and titers can be associated with pathophysiology mechanisms and impact diseases diagnosis or therapy management (15)(16)(17)(18)(19)(20).…”

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

“…Bard et al also reported that immunization with a full length Aβ peptide is not required to trigger an efficacious antibody response and that antibodies produced by immunization with small epitope peptides also significantly reduced neuritic pathology. Furthermore, Manea et al showed that bioconjugates generated by attachment of multiple copies of the Aβ(4‐10) epitope to carriers consisting of branched chain polypeptides displayed increased antigenicity and thus might be potential immunogens …”

“…In contrast, the mass spectrometric analysis of the mutant Aβ(4-10) peptides upon affinity binding to a monoclonal anti-Aβ(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aβ(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) to the monoclonal Aβ antibody provided a binding constant K D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ-specific antibodies with improved therapeutic efficacy.…”