Mimotopes for pathogenic AQP4-specific Antibodies in NMO

Neuromyelitis optica (NMO) is an acute inflammatory demyelinating disease of the central nervous system (CNS). Hallmark of this disease are astrocytopathic lesions in spinal cord and optic nerves, and the presence of so-called NMO-IgG in the vast majority of patients. NMO-IgG are antibodies directed against the water channel aquaporin 4 (AQP4). These antibodies are not only a diagnostic marker of NMO, but they are pathogenic. Unfortunately, the triggers for the production of pathogenic NMO- IgG or for the increase in AQP4-specific antibodies in established NMO remain essentially unknown. Similarly, we do not yet have any biomarkers available, which could reveal the size and quality of the AQP4-specific antibody repertoire in patients, or help to predict disease course, severity or treatment responses. The pathogenic AQP4-specific antibodies of NMO patients recognize conformational epitopes formed by extracellular loops of AQP4. However, antibodies specific for conformational epitopes can also recognize linear peptides mimicking these epitopes (= mimotopes). Therefore, the identification of linear peptides binding to AQP4-specific antibodies of NMO patients could help us to specifically address the open issues: If such linear peptides are found in naturally occurring antigens to which NMO patients could have been exposed, these antigens might play a role in the formation and/or expansion of AQP4-specific antibodies in NMO; if they are not found in these sources, but cross-react with AQP4-specific antibodies simply due to structural similarity of a random amino acid sequence to the conformational epitope, such mimotopes could represent ideal tools to identify subsets of AQP4-specific antibodies, and to probe the AQP4-specific antibody repertoire of individual patients. In the current proposal, we plan to continue our mimotope search for NMO-IgGs, to apply them to study the AQP4-specific antibody repertoire of patients, and to analyze whether there are dominating anti-AQP4-reactivities in NMO patients, and whether patients with specific mimotope- binding patterns of their AQP4-antibody repertoire differ from each other, e.g. in terms of disease severity, titers, or treatment responses.

Pathogenic autoantibodies against the water channel aquaporin 4 (AQP4) are found in most patients suffering from neuromyelitis optica spectrum disorders (NMOSD). When these antibodies bind to AQP4 on astrocytes in the central nervous system (CNS), they initiate damage to these cells, which eventually causes the formation of large tissue destructive lesions in the CNS. As a result of the antibody action, untreated patients may become permanently paralyzed or blind. Such patients would very much profit from studies on the induction and breakage of tolerance to AQP4. At the time when we started our project, all attempts to create suitable animal models by active sensitization with AQP4 have failed. However, it had been observed that substantial numbers of NMOSD patients developed clinical symptoms of their disease in sequel to infections. This raised the possibility that mimicry processes between bacterial/viral proteins and AQP4 could be involved. We addressed this challenge and identified peptides, which mimic the conformational AQP4 epitopes recognized by pathogenic antibodies of NMOSD patients. Immunization of Lewis rats with these mimicking peptides (=mimotopes) induced the production of AQP4-reactive antibodies in these animals. At the time of the project start, it was also completely unclear whether the systemic presence of AQP4-reactive antibodies alone could induce any damage to the central nervous system (CNS). To address this point, we systemically applied such antibodies over prolonged period of time to rats. We showed that AQP4-specific antibodies can enter the CNS on their own, via circumventricular organs and meningeal or parenchymal blood vessels. Moreover, depending on mode and site of antibody entry, different types of lesions with AQP4 loss developed. We could also show that established lesions have short and far reaching effects on blood vessels and their branches, and that AQP4-antibodies have also profound effects on the AQP4 expression in peripheral tissues. Cumulatively, the results of this project provide a conceptual framework for the formation of AQP4-specific antibodies in NMOSD patients, and established an immunization strategy for the creation of animal models suitable for tolerance studies in this devastating disease. Our results also showed that the pathological changes caused by AQP4-specific antibodies inside and outside the CNS drive the evolution of this disease.