'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single ("classical") or multiple ("complex") anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for effective immune-specific therapy of organ-specific autoimmune diseases associated with complex and dynamic pathogenic autoimmunity, such as MS; our data further demonstrate that the "multi-epitope-targeting" approach to therapy is optimized through specifically designed multi-epitope-proteins, rather than myelin peptide cocktails, as "multi-epitope-targeting" agents. Such artificial multi-epitope proteins can be tailored to other organ-specific autoimmune diseases.

Pathogenic T cells in MOBP-induced murine EAE are predominantly focused to recognition of MOBP21F and MOBP27P epitopic residues.

Myelin-associated oligodendrocytic basic protein (MOBP) is a central nervous system (CNS)-specific myelin constituent important in stabilizing the unique multi-layered structure of the CNS-myelin sheath. Although autoimmunity against MOBP has been associated with multiple sclerosis pathogenesis, anti-MOBP pathogenic T cells have been poorly investigated as compared to T cells against other encephalitogenic myelin proteins. We have recently determined MOBP15-36 as the major encephalitogenic epitope of MOBP for SJL/J mice. In this study, epitope-specificity was dissected to the level of residues crucial for activation/control of MOBP-specific encephalitogenic T cells. Structural bioinformatic analysis of MOBP15-36/I-A(s) interaction and experimental data have determined MOBP21F and MOBP27P of I-A(s)-binding nonameric core epitope (MOBP20-28) as TCR-contact residues critical for functional activation of encephalitogenic MOBP-specific T cells. Accordingly, the non-encephalitogenic/nonstimulatory pMOBP16-21A27A-33 inhibited encephalitogenic MOBP-reactive T cells, shifted their cytokine secretion profile, and effectively suppressed pMOBP15-36-induced EAE. Moreover, pMOBP16-21A27A-33 fully reversed ongoing clinical EAE induced by whole MOBP as well as by pMOBP15-36. Data show that encephalitogenic MOBP-reactive T cells are predominantly focused to recognition of MOBP21F and MOBP27P, and suggest that both their selection and control are governed by MOBP21F and MOBP27P epitopic residues. Such focused epitopic recognition may affect profoundly on peripheral self-tolerance to MOBP and on potential altered peptide ligand-mediated therapy of MOBP-related autoimmune pathogenesis.

OSP/claudin-11-induced EAE in mice is mediated by pathogenic T cells primarily governed by OSP192Y residue of major encephalitogenic region OSP179-207.

Pathogenic autoimmunity against oligodendrocyte-specific protein (OSP/claudin-11), recently implicated in multiple sclerosis (MS) pathophysiology, has been poorly investigated as compared to that against other myelin encephalitogens. Using recombinant soluble mouse OSP (smOSP) and overlapping peptides thereof, we show that smOSP-induced chronic EAE in C57BL/6J mice is primarily associated with CD4(+) T cells reactive against OSP179-207 and OSP22-46, the major and minor encephalitogenic regions, respectively, and with a predominant B cell response against OSP22-46. The encephalitogenic OSP179-207-specific T cells recognized OSP190-202 as minimal stimulatory epitope, while minimal encephalitogenic sequence was OSP191-199. Further delineation and structural bioinformatic analysis of the major encephalitogenic region suggested four overlapping potential I-A(b) core epitopes, predicting OSP192Y as major TCR-contact residue shared by OSP 188-196, OSP190-198, and OSP191-199 cores, albeit at different MHC-II pockets. Accordingly, substitution at OSP192Y yielded OSP188-192A-202, a non-stimulatory/non-encephalitogenic altered peptide ligand (APL) that was antagonistic for OSP188-202-specific encephalitogenic T cells. Systemic administration of OSP188-192A-202 suppressed OSP188-202-induced EAE and fully reversed smOSP-induced EAE. These data suggest that a single epitopic residue (OSP192Y) governs the selection and control of most pathogenic T cells associated with smOSP-induced EAE in H-2(b) mice. This may impact profoundly on peripheral self-tolerance to OSP and on potential APL-mediated therapy of OSP-related autoimmune pathogenesis.