N-glucosylated adhesin peptide-dendrimers: toward a selective apheresis for personalized treatment of multiple sclerosis.

Multiple sclerosis (MS), is a neurodegenerative disease possibly involving also an antibody-mediated mechanism in the damage of myelin sheath surrounding the axons in the central nervous system. We previously demonstrated that the N-linked β-D-glucopyranosyl moieties (N-Glc) on epitopes in non-typeable Haemophilus influenzae adhesin C-terminal fragment HMW1(1205-1526), named HMW1ct and expressed as a mixture of three N-glucosylated variants containing 7, 8, and 9 glucose moieties on Asn in NX(S/T) sequons in a 1:1:1 ratio, were essential for high affinity antibody binding in a subpopulation of MS patients. Although the disease-specific autoantigen remains elusive, molecular mimicry or epitope spreading mechanisms are highly speculated. With the idea in mind that IgMs can be reminiscent of an early triggering infection event and that IgGs could have a prognostic value, the isolation of specific autoantibodies, and particularly the elusive IgMs, is a major goal to assess their role. The main purpose of this thesis work was to develop peptide probes to detect and isolate specific and high affinity autoantibodies from sera of patients suffering from MS. We selected and synthesised three peptides of HMW1(1347-1354) bearing one or two N-Glc respectively on Asn-1349 and/or Asn-1352. The short unstructured nonapeptides (as confirmed by CD and NMR-based conformational analysis) with one or two N-Glc efficiently bind IgG Abs, displaying IC50 (10-8-10-10 M by competitive indirect ELISA), in three representative MS patient sera. We selected the di-glucosylated adhesin peptide Ac-KAN(Glc)VTLN(Glc)TT-NH2 as the shortest sequence up to now able to compete with the highest affinity with anti-N-Glc HMW1ct IgM binding, even though with a high IC50 (≈10 µM). This result was introductory for the development of an octopus-like polymeric structure with the final aim to capture and isolate IgMs and proposing this strategy for a future aphaeresis technique. 40 kDa dextran was activated with glycidyl-propargyl groups and the alkyne-modified dextran was used as a scaffold to conjugate by CuAAC the di-glucosylated adhesin peptide azide analogue. Quantitative NMR characterization allowed to measure the loading in peptides (19.5%), meaning that each peptide-grafted dextran molecule contains ≈100 N(Glc) moieties. This novel polymeric structure was proven to dramatically increase binding potency of IgGs and IgMs in MS sera. Abs from a representative MS serum, were successfully purified on a sepharose resin specifically modified with the adhesin peptide-dextran conjugate, as confirmed by ELISA. This result appears promising as a proof-of-concept of the selective snaring of circulating autoantibodies (possibly perpetuating nonself recognition) that could likely lead to develop a specific apheresis-based device.