Alzheimer's Disease: IgGs against β-amyloid conformational epitopes exposed by engineered phages as novel biomarkers for state/stage diagnosis

Alzheimer's Disease (AD) is a chronic and progressive neurodegenerative disease and is characterized by the presence of neurotoxic Aβ plaques in the brain. These plaques are formed by monomeric Aβ spontaneously assembling into soluble oligomers, which cluster together to form insoluble fibrils. The solubility of Aβ, and the quantity of Aβ in different pools, may be more closely related to disease state. The composition of these pools of Aβ reflects different populations of amyloid deposits, and has definite correlates with the clinical status of the patient. However, a confirmed diagnosis of AD can only be obtained through post-mortem identification of neurofibrillary tangles and/or abnormal plaque deposits in the brain. The confirmatory diagnosis of AD in early stages could facilitate early therapeutic intervention and treatment of the disease. Conformation-dependent antibodies have been reported to recognize a generic epitope that is specific to many types of amyloid fibrils and not soluble monomer regardless of their sequences. It is possible to hypothesize that different aggregational states are also present in patients with AD and that these evoke a conformation- dependent antibody response. However, it is not possible to precisely define the aggregation state of the protein and its misfolding. About that, various possible conformational misfondings of proteins similar to Aβ already present have been searched, eg. in bacterial proteins. Thus, in our work a set of proteins having conformational motifs homologous to Aβ-42 through bioinformatics tools have been screened. Among microbial proteins, the epitopic region of Yersinia pestis F1 capsular antigen (Caf 1) showed the most significant structural homology with the fibrillar form of Aβ. In order to search for IgG autoantibodies in AD sera against a potential “generic conformational antigen” of Aβ-42 in vivo and to evaluate the possibility to use them as state/stage biomarkers of AD, monoclonal antibodies directed against Caf 1 were used in alternate biopanning cycles, the so-called “double binding” phage display selection. Thanks to the phage display technology it is possible to select “conformational mimotopes” that can be recognized by conformation-dependent antibodies. The reactivity of the phage clones isolated were evaluated in ELISA assay against monoclonal Ab that recognized Caf1 of Y. pestis and IgGs of AD-patients. From the selected clones, one clone, named 12III1, detected a significant level of IgG for discrimination between AD and non-AD subjects. Furthermore, this phage clone was able to interfere with Aβ-42 fibrillation in vitro and to promote its disaggregation in SH-SY5Y cells. The data obtained suggested that the peptides displayed, although selected also by the monoclonal antibody specific for Caf1, probably mimicked several conformational epitopes recognized by circulating IgG from patients with AD. Thus, in order to investigate the immune response for a state/stage diagnosis of AD, different phage clones resulting from the same double binding selection were investigated. The ability to interact with amyloid plaques in AD mouse brain sections with high significance, confirmed that the peptides were amyloid mimotopes recognized by conformation-dependent antibodies. In addition, to detect conformational antibodies present in the different stages and states of the disease, the peptides were used in a new combinatorial diagnostic micro-array besed on Phage mediated Immuno PCR CDC Biochip, able to discriminate AD from healthy sera. The data would pave the way for the use of these Aβ-42 conformational mimotopes to determine the status and stage of AD. This could be very useful for a personalized pharmacological treatment, avoiding the progression of the disease. Since these peptides were able to interact with amyloid plaques in brain sections, they could also be used as detection probes for imaging. Furthemore, their ability to interact and disaggregate Aβ-42 fibrils could lead to their use in therapeutic practices.