Conformation-sensitive Antibodies against Alzheimer Amyloid-β by Immunization with a Thioredoxin-constrained B-cell Epitope Peptide

Nadia Moretto,Angelo Bolchi,Claudio Rivetti,Bruno P Imbimbo,Gino Villetti,Vladimiro Pietrini,Luciano Polonelli,Steven Del Signore,Karen M Smith,Robert J Ferrante,Simone Ottonello

doi:10.1074/jbc.m609690200

Abstract

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.

Highlights

Immunotherapy against the amyloid-␤ (A␤) peptide is a valuable potential treatment for Alzheimer disease (AD)
The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained A␤ fragment repeat and identify Trx(A␤15)4 as a promising new tool for AD immunotherapy
As revealed by this study, Trx(A␤15)4 is a soluble derivative of A␤ with good immunogenicity even when formulated with a moderate strength adjuvant, such as alum, that promotes an anti-inflammatory T helper 2-polarized immunoresponse

Summary

The abbreviations used are

A␤, amyloid-␤; AD, Alzheimer disease; APP, amyloid-␤ precursor protein; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; Trx, bacterial thioredoxin; TTR, human transthyretin. We reasoned that a similar conformational effect, besides an enhanced peptide stabilization and immunogenicity [37, 38], might be obtained with the use of a scaffold protein, such as thioredoxin, with the ability to constrain the structure of short peptides inserted within its surface-exposed active site loop (38 – 40) and to stimulate T-cell proliferation [41, 42]. This kind of recombinant antigen would be highly desirable because of its expected safety, ease of construction, and large scale production in a chemically homogeneous form. We have further documented the ability of the anti-Trx(A␤15) antibody to bind A␤ aggregates in human brain and to ameliorate AD pathology in APP transgenic mice

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION