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Title: Serum profiling and autoantibodies identification in Multiple Sclerosis using epitope and CSF IgG phage display libraries
Authors: Cortini, Andrea
Supervisore/Tutore: Edomi, Paolo
Issue Date: 27-Mar-2009
Publisher: Università degli studi di Trieste
Multiple sclerosis (MS) is considered the prototype of inflammatory autoimmune diseases of the central nervous system (CNS).
The typical feature of the disease is the plaques of demyelination. The evolution of the plaque lesion in MS implicates an inflammatory phase followed by a recovery of functional myelin; a second step is the chronic progressive disease with axonal loss. The earlier phase of MS may be mediated by an autoimmune reaction.
Whereas the role of T cells in MS pathogenesis is well established, the role of B cells and autoantibodies in demyelination and plaque formation is still unresolved. However several evidences suggest a contribute of autoantibodies in MS pathogenesis. B cells and myelin specific autoantibodies are present in the sclerosis plaques, and there is an increased production of immunoglobulin (Ig) in the cerebrospinal fluid (CSF) of more than 90% of MS patients . Typically these Ig present an oligoclonal pattern and sequencing of oligoclonal IgG showed extensive somatic mutations suggesting B cell clonal expansion and a specific antigen-driven immune response.
The most extensively studied putative autoantigens are components of CNS myelin (myelin basic protein MBP, proteolipid protein PLP, myelin oligodendrocyte glycoprotein MOG). The autoantibodies in MS recognize both linear and conformational epitopes, but at present the conformational epitopes of myelin proteins have not been identified. For example, in MS, the T-cell receptors of autoreactive T lymphocytes recognize various peptides of the MBP, and, in EAE, the anti-MOG antibodies recognize only conformational epitopes. Furthermore, the progression of MS is accompanied by the decline of primary T-cell autoreactivity and by the concurrent emergence of neo-autoreactivity (epitope spreading).
However recent investigation have showed that no myelin antigens, like neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, may also have a role in MS pathogenesis. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis.

Several strategies, involving the phage display technology, have been employed in the attempt to discover the antigen that drives the immune response in MS.
A first strategy depends on the cloning of IgG repertoire of MS patients in a phage display library screened with brain sections or known antigens. Another strategy involves large phage display libraries of random peptides screened with IgG of CSF in order to identify peptides recognized by antibodies present in CSF of MS patients.
Phage display is a technique which involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein-protein interactions and to select antibodies against a wide range of different antigens.

In this project we have proposed:

1. to study the autoimmune response in MS by using the phage display for the expression of antibodies involved in the disease. We wanted to make a ScFv library from B cells of CSF of different multiple sclerosis patients, to employ as tool to select a phage display Human Brain cDNA library for the identification of new antigens recognized by the immune sistem in MS patients.
2. To produce single gene mini library of putative antigens (MBP,PLP, MOG) for the generation of epitope chips to use for serotyping the immune response in different patients
3. To investigate the feasibility to use a single gene phage display mini-library as tool for epitope mapping (both linear and conformational) of novels autoantigens
4. To investigate the role of NSE(neuron specific enolase), a new possible no myelin autoantigen in multiple sclerosis, in the pathogenesis of the disease and the usefulness as possible diagnostic marker.

Scope 1
B-cells from liquor of two MS patients were centrifuged and the total RNA was extracted from the pellets. Total RNA was retrotranscripted and variable region of heavy and light chain of the antibodies were amplified by PCR. Heavy chain and light chain were assorted and assembled before to be cloned in the phagemid vector pDAN 5.
A 2x104 independent clones library was obtained and analyzed by PCR and fingerprinting. A diversity of 30,8% for heavy chain and 72,7% for light chain was established.
ScFv library was used to select a phage display Human Brain cDNA library.
17 clones with an high reactivity were obtained and after sequencing 6 clones on 17 have shown to be the same antigen(antigen A ); the reactivity on other two antigens obtained with the selection (antigen B and C) of CSF from 18 MS patients and 16 patients with other neurological disease (OND) was tested by ELISA to evaluate diagnostic value of this protein. The results shown that SM response was statistically different from OND response; the ELISA test gave a specificity of 94,12% and a significance of 53,85 %.
The reactivity for the antigen B was also evaluated on sera of MS patients and controls. The MS response was statistically different from OND response and shown a specificity of 97,44% and a significance of 58,62 %.

Scope 2&3
We have generated three single gene mini libraries of the major antigens in MS (MBP, MOG and PLP); cDNA of each gene was obtained by RT-PCR and after fragmentation cloned in a phagemid vector (pEP1) to obtain a mini-library for each gene.
We have obtained a 2x105 for MBP, 2.4x104 for MOG and 1.6x106 for PLP independent clones library. MBP and MOG libraries were characterized by PCR and fingerprinting. Sequencing analysis shown that the entire MBP transcript variant 7 mRNA (664-1177 nt) and MOG isoform alpha 1 mRNA (262-918 nt) were represented in the respective library.
To testing the capacity of selecting a single epitope from our libraries, we have performed a selection test with a commercial monoclonal antibody that recognize MBP 82-98 epitope; after three selection panning all selected clones contain the nucleotidic sequence 906- 956 nt (MBP transcript variant 7 mRNA) which encodes the immunogenic epitope recognized by the monoclonal antibody.

Scope 4
The reactivity of sera from 31 MS patients and 14 healthy controls was tested by ELISA on NSE ; statistical analysis of the results shown that the two populations were significantly different.
Ciclo di dottorato: XXI Ciclo
metadata.dc.subject.classification: NEUROSCIENZE
Keywords: Serum profilingEpitope libraryPhage displayMultiple sclerosisCSF single chain library
Type: Doctoral
Language: en
Settore scientifico-disciplinare: BIO/18 GENETICA
NBN: urn:nbn:it:units-7410
Appears in Collections:Scienze biologiche

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