Work Packages:
Work Package 1 (WP1):
In WP1, severely immunodeficient B2m-NOG mice will be used as recipients for transplantation by human peripheral blood monocytes (PBMC) to generate “humanized” immune mice. First, we will analyze the genes of patients with relapsing-remitting MS (RRMS) that attend the MS clinic of the Aiginitio Hospital University Hospital, and recruit patients as well as healthy individuals that carry the DRBI*1501 MS susceptibility gene of the human major histocompatibility complex into the study after obtaining their written consent. We will isolate PBMC from fresh blood samples taken from both patients and healthy individuals, profile the immune cell populations by flow cytometry and use the cells to transplant groups of B2m-NOG mice. The engraftment of transplanted mice by human immune cells will be monitored over time using samples of mouse blood by flow cytometry. The success of reconstitution of the mice by the human immune system will be measured.
Work Package 2 (WP2):
In WP2, we will use PBMC isolated from DRB1*1501- positive RRMS patients and DRB1*1501-positive healthy participants to transplant B2-NOG mice. Before the transplantation, we will carefully select the RRMS patient donors from our cohort based on the following three main criteria; 1) those having a highly active disease -relapse, 2) those having the DRB1*1501 genotype, 3) those having a disease relapse upon treatment with Tysabri, which traps immune cells in the blood. Groups of transplanted mice will be further subdivided into mice allowed to develop any spontaneous disease symptoms (spontaneous sDR2.MS model) and mice immunized for the induction of experimental autoimmune encephalomyelitis (EAE), so that altered susceptibility to experimental demyelination can be detected (inducible iDR2.MS model). All mice will be monitored daily for neurological and non-neurological symptoms with care to identify signs of graft-versus-host disease, for level of engraftment by a human immune system using blood samples and flow cytometry, and by neuropathological analysis when animals are sacrificed at the end of the experiment.
Work Package 3 (WP3):
In WP3 we will synthesize and characterize sterile peptide analogues based on epitopes of myelin proteins that are recognized by T cells in MS patients and are useful for the induction of ΕΑΕ in mice. Peptide analogues will also be coupled to an oxidized form of the mannan polysaccharide (OM) that circumvents activation of the complement system. Vianex will synthesize the new immunotherapy candidate developed by our team, named ELMog, which comprises human MOG35-55 peptide coupled to OM. Mouse ELMog (OM-MOG) induces strong antigen-specific immune tolerance and protection against EAE in mice after prophylactic or therapeutic administration, and human ELMog represents a promising experimental drug for selectively inactivating myelin-specific immune responses in MS patients.
Work Package 4 (WP4):
In WP4, we aim to define the precise immune mechanisms by which OM-conjugated myelin peptides such as ELMog protect mice against experimental demyelination. ELMog induces strong antigen-specific immune tolerance, but the mechanism of action is not yet known. Specifically, we will investigate whether OM-peptides induce T cell anergy, one mechanism by which antigen-specific T cells are known to be inactivated. We will also investigate whether OM-peptides induce immune tolerance via the induction of bone marrow-derived myeloid suppressor cells (MDSC). MDSC massively expand in the blood and lymphoid organs under conditions of inflammation, autoimmunity and cancer and have immunosuppressive properties in models of cancer and EAE. Identified immune mechanisms will be further probed using cutting edge methodologies to identify the molecular (single cell RNA sequencing) and cellular (conditional gene targeting) basis of protection in mice. Finally, we seek to obtain proof-of-concept that the humanized mice generated in this project represent improved mouse models for MS, by testing one drug that is already approved for use in human MS where it targets and inactivates B cells (Ocrelizumab), and another experimental drug that selectively targets myelin-specific T cells (ELMog) and has shown promising pre-clinical results in wild-type and humanized HLA-DR2 transgenic mice in the EAE model.
Work Package 5 (WP5):
In WP5, a Technical Feasibility Study will be prepared, which will evaluate and analyze the potential outcomes of this project. Recording of the advantages, weaknesses, opportunities, and threats of the project will identify new strategies to ensure the sustainability of the results of the project upon completion. The results of this analysis will support the decision making of the partnership. Finally, based on the data produced, a Business Plan will be prepared which will lead to the further development and utilization of emerging research products.