Molecular Immunology 57 (2014) 138–140 Contents lists available at ScienceDirect Molecular Immunology jo u r n al homep age: www.elsevier.com/locate/molimm Antigenic epitopes of MAP2694 homologous to T-cell receptor gamma-chain are highly recognized in multiple sclerosis Sardinian patients Davide Cossu a , Speranza Masala a , Jessica Frau b , Giuseppe Mameli a , Maria Giovanna Marrosu b , Eleonora Cocco b , Leonardo Antonio Sechi a,∗ a Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Università di Sassari, Italy b Centro Sclerosi Multipla, Dipartimento di Scienze Cardiovascolari e Neurologiche, Università di Cagliari, Italy a r t i c l e i n f o Article history: Received 19 July 2013 Received in revised form 28 August 2013 Accepted 3 September 2013 Keywords: Mycobacterium avium ss. paratuberculosis Multiple sclerosis Molecular mimicry ELISA Peptide library a b s t r a c t Mycobacterium avium ss. paratuberculosis (MAP) is an intracellular pathogen recently associated with multiple sclerosis (MS). Aiming to identify immunodominant epitopes belonging to MS related protein MAP2694 (UniProt accession no. Q73WG6), we investigated the binding activity of selected peptides against MS Sardinian sera. An overlapping 9-mers peptide library was synthesized spanning the entire aminoacidic sequence of the protein. Peripheral blood was collected from 47 MS patients and 42 sex and age matched healthy volunteers and subjected to enzyme-linked immunosorbent assay (ELISA) in order to investigate the reaction against the linear peptides generated. Two out of 58 synthetic 9-mers were strongly recognized by MS patients’ antibodies compared to controls. A competitive inhibition assay demonstrated that these two epitopes are immunodominant antibody targets within MAP2694 protein, as sera pre-adsorbed with these peptides were able to significantly block the antibody reaction to the MAP2694 protein, even if at a lesser extent than MAP2694 protein itself. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of Johne’s disease, an enteric granulomatous disease in ruminants and it has been associated with different autoimmune diseases such as Crohn’s disease, type 1 diabetes and more recently multi- ple sclerosis (MS) (Cossu et al., 2013). MS is a chronic inflammatory demyelinating disease of the central nervous system (CNS) with unknown etiology. However, MS is probably the consequence of an intricate interaction between genetic and environmental fac- tors, which induce an abnormal immune response (Ascherio et al., 2012). Recently it was reported an association between MAP and MS in Sardinia, proven both with the detection of bacterial DNA in the peripheral blood of MS patients, and with the finding of a strong humoral response against some MAP proteins (Cossu et al., 2011, 2012). One of these antigens, MAP2694, is a transmembrane protein sharing sequence homologies with the T-cell receptor gamma- chain and the complement component 1q (C1q) of the host. In a pilot study conducted in a Sardinian cohort of 50 MS patients and ∗ Corresponding author at: Dipartimento di Scienze Biomediche, Università di Sassari, Viale San Pietro 43 b, 97100 Sassari, Italy. Tel.: +39 079228462. E-mail address: sechila@uniss.it (L.A. Sechi). 56 healthy controls (HCs), this antigen gave strong ELISA values in 32% of the MS patients and only in 2% of the HCs (Cossu et al., 2011). Besides, in a different study carried out on a larger sample size, MAP2694 positivity was detected in 33.7% of 436 MS patients and 3.8% of 264 HCs respectively (Frau et al., 2013). In both studies it was tested the whole protein, which was fused with the maltose binding protein tag. Hence, this study was set up in order to identify the most immunogenic region of MAP2694 and to better understand the antigenic structure of the protein. Firstly, it was performed a screening by ELISA to identify the most antigenic peptides and to determine the location of the relevant epitopes. Once identified, the inhibition activity of the positive peptides was assessed by a competitive inhibition assay. In view of these results, two immunodominant peptides were identified within the MAP2694 sequence: MAP2694 97–105 (PGQPFGPGG) and MAP2694 295–303 (ADVTIADPT). Interestingly one of the epitopes is located within the high homology region with the T-cell receptor gamma-chain. 2. Materials and methods 2.1. Subjects In this study, 47 MS patients [26 females and 21 males (F/M: 1.2/1)] free-therapy for at least 6 months and 42 age and sex 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.09.001