Identification of autoantigens in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system in which cells from primarily the adaptive immune system infiltrate the brain and spinal cord, leading to inflammation and demyelination. Debuting primarily between 20-40 years of age and with a prevalence in Sweden of ~0.2%, it is one of the leading causes of disability in working-age adults. While the cause is still unknown, the risk of developing MS is influenced by an interplay of both genetic and environmental risk factors. Genetic and immunological data point towards CD4+ T cells being a primary driver of the disease. While some de facto targets, i.e., autoantigens, have been identified, the known autoantigen repertoire still contains considerable gaps. This remains a critical problem for developing autoantigen-targeted diagnostic tools and autoantigen-specific treatment strategies. This thesis aimed to identify novel autoantigens in MS. Paper I addressed a common problem when studying autoantigen-specific T-cell responses: autoreactive T cells are rare, and antigens can be either weak stimulators or contain contaminants that are challenging to remove, resulting in high assay noise and low sensitivity. By covalently coupling recombinant protein antigens to 1 μm paramagnetic polystyrene beads, we show that contaminants can be removed while the ability to stimulate T-cell responses remains. This resulted in a sensitive assay with high signal-to-noise ratios, with a threshold for detection at 1 in 18 000 cells. In Paper II, we used this novel method to examine T cell responses to myelin oligodendrocyte glycoprotein (MOG), an autoantigen for which previous results have conflicted. By examining peripheral blood mononuclear cells (PBMCs) from a cohort of persons with MS (pwMS) and matched healthy controls (HC), MOG-specific CD4+ T cells were detected in approximately half of all pwMS. Additionally, MOG-epitopes were presented by monocytes and restricted to HLA-DR. Lastly, using three different antibody-assays, we could not detect any significant portion of MOG-specific autoantibodies despite the presence of MOG-specific T cells. Paper III addressed the main aim of this thesis, i.e., identifying novel autoantigens. This study combined the antigen-bead method with the Human Protein Atlas recombinant protein epitope signature tag library to screen for T-cell reactivity against a panel of 63 central nervous system-expressed proteins. In a smaller screening cohort, there were increased proinflammatory responses against four novel autoantigens targets: fatty acid binding protein 7 (FABP7), prokineticin-2 (PROK2), reticulon-3 (RTN3), and synaptosome associated protein 91 (SNAP91), as well as the previously described autoantigen MOG in pwMS. The screening results were validated using full-length versions of the targets in two larger cohorts, including pharmacologically untreated pwMS. The autoreactive profiles of individuals were heterogenous, but a panel of several autoantigens could distinguish between MS and non-MS with high accuracy. Immunophenotyping revealed MS-specific autoreactive cells to be mainly HLA-DR-restricted CD4+ T cells and responded with interferon-gamma and granulocyte-macrophage colony-stimulating factor production upon stimulation. The presence of autoantibodies was examined in a large cohort of patients and controls. Still, it was not increased in MS. Immunization of mice with the novel autoantigens induced T cell responses, leading to CNS-leukocyte migration and crossing of the blood-brain barrier, demonstrating encephalitogenic potential. Paper IV explored a possible immunological link between Epstein-Barr virus infection and MS. We examined serological responses to alpha-crystallin B (CRYAB) and Epstein-Barr virus nuclear antigen 1 (EBNA1) in a cohort of 713 pwMS and 722 HC. Anti-CRYAB-antibodies were associated with MS with an odds ratio (OR) of 2.0, which had a synergistic effect with high EBNA1 responses (OR of 9.0). By depleting plasma of anti-EBNA1 antibodies, CRYAB responses were similarly removed, demonstrating cross-reactivity between the two antigens due to an amino acid sequence homology (RRPFF, CRYAB aa11-15 and EBNA1 aa402-406 respectively). In a mouse model, EBNA1-primed T cells were also CRYAB-reactive, and EBNA1 and CRYAB-responsive T cells were highly correlated and increased in natalizumab-treated pwMS, pointing towards a similar cross-reactivity in the T-cell compartment as well. In conclusion, this thesis presents methods for sensitively assessing autoreactive T-cell responses, reexamining and confirming MOG and CRYAB as targets. It considerably expands the knowledge regarding the targets of the autoimmune attack in MS by adding four novel autoantigens to the known repertoire. Further, it demonstrates an underlying heterogeneity of the immunological landscape of MS and provides a mechanistic link between Epstein-Barr virus and MS. It demonstrates a first step in the development of autoantigen-specific methods for diagnostics and introduces novel targets for potentially effective antigen-specific immunotherapy

Identification of four novel T cell autoantigens and personal autoreactive profiles in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid–binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+and human leukocyte antigen–DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS

Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, for which and Epstein-Barr virus (EBV) infection is a likely prerequisite. Due to the homology between Epstein-Barr nuclear antigen 1 (EBNA1) and alpha-crystallin B (CRYAB), we examined antibody reactivity to EBNA1 and CRYAB peptide libraries in 713 persons with MS (pwMS) and 722 matched controls (Con). Antibody response to CRYAB amino acids 7 to 16 was associated with MS (OR = 2.0), and combination of high EBNA1 responses with CRYAB positivity markedly increased disease risk (OR = 9.0). Blocking experiments revealed antibody cross-reactivity between the homologous EBNA1 and CRYAB epitopes. Evidence for T cell cross-reactivity was obtained in mice between EBNA1 and CRYAB, and increased CRYAB and EBNA1 CD4$^{+}$ T cell responses were detected in natalizumab-treated pwMS. This study provides evidence for antibody cross-reactivity between EBNA1 and CRYAB and points to a similar cross-reactivity in T cells, further demonstrating the role of EBV adaptive immune responses in MS development

Designing a Multimer Allergen for Diagnosis and Immunotherapy of Dog Allergic Patients

<div><p>Background</p><p>Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.</p><p>Objective</p><p>To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.</p><p>Methods</p><p>A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.</p><p>Results</p><p>The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.</p><p>Conclusion</p><p>We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.</p></div

Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, for which and Epstein-Barr virus (EBV) infection is a likely prerequisite. Due to the homology between Epstein-Barr nuclear antigen 1 (EBNA1) and alpha-crystallin B (CRYAB), we examined antibody reactivity to EBNA1 and CRYAB peptide libraries in 713 persons with MS (pwMS) and 722 matched controls (Con). Antibody response to CRYAB amino acids 7 to 16 was associated with MS (OR = 2.0), and combination of high EBNA1 responses with CRYAB positivity markedly in-creased disease risk (OR = 9.0). Blocking experiments revealed antibody cross-reactivity between the homolo-gous EBNA1 and CRYAB epitopes. Evidence for T cell cross-reactivity was obtained in mice between EBNA1 and CRYAB, and increased CRYAB and EBNA1 CD4+ T cell responses were detected in natalizumab-treated pwMS. This study provides evidence for antibody cross-reactivity between EBNA1 and CRYAB and points to a similar cross-reactivity in T cells, further demonstrating the role of EBV adaptive immune responses in MS development

Splenocyte proliferation assay in immunized mice.

<p>Proliferative responses after stimulation of splenocytes from immunized mice (immunization allergen denoted as individual graph heading), stimulated with equimolar amounts of rCan f 1, rCan f 2, rCan f 4, rCan f 6, an equimolar mix or Can f 1-2-4-6 (x-axis) and measured by [3H] thymidine incorporation (y-axis). Boxes with median values and horizontal bars denote 50% of values and 1 standard deviation respectively. * p<0.05, ** p<0.01, *** p<0.001 and ns (non significant) p>0.05, analyzed with repeated measurements ANOVA with Tukey's multiple comparison test</p

Biochemical analysis of the linked molecule.

<p>(A) SDS-PAGE of Can f 1-2-4-6 or the single recombinant lipocalins visualized by coomassie staining under reducing conditions. MW markers (lane 1,7, MW (kDa)), rCan f 1 (lane 2), rCan f 2 (lane 3), rCan f 4 (lane 4), rCan f 6 (lane 5) and Can f 1-2-4-6 (lane 6). (B) Analytical size exclusion chromatography of rCan f 1-2-4-6 and the single lipocalin allergens. Molecular weight markers, bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and Ribonuclease A (13.7 kDa). (C) Far-UV CD analysis of Can f 1-2-4-6 or an equimolar mix of the single recombinant allergens Can f 1, Can f 2, Can f 4 and Can f 6. The spectra are expressed as mean residue ellipticities (θ) at a given wavelength.</p

Can f 1-2-4-6 structure based on solution small angle X-ray scattering data.

<p>(A) Experimental SAXS data from Can f 1-2-4-6 in solution (blue dots with error bars), computed fits from the CORAL (red line) and the GASBOR (grey line) models. (B) Respective pair distance distribution function. Maxima marked by vertical lines correspond to distances between the individual domains. (C) The rigid body model of Can f 1-2-4-6 reconstructed by the program CORAL resembles beads on a thread with each lipocalin forming a bead (cartoon representation) interspaced by extended linkers (light blue spheres). This model is in a good agreement with the <i>ab initio</i> GASBOR model (dummy residues represented by grey spheres).</p

Antibody responses after immunization with allergens.

<p>(A) quantification of IgE-responses in mice (n = 6) immunized with each rCan f 1, rCan f 2, rCan f 4, rCan f 6, an equimolar mix or Can f 1-2-4-6 (x-axis in graphs) to plates coated with anti-mouse IgE, mouse antiserum and biotinylated allergen (graph heading) by sandwich ELISA. (B) Comparison of IgG1 and IgG2a-antibodies to each lipocalin induced by solid phase Can f 1-2-4-6 or a mix. Immunoglobulin responses are presented as OD405 nm, y-axis. Boxes with median values and horizontal bars denote 50% of values and 1 standard deviation respectively. * p<0.05, ** p<0.01, *** p<0.001, analyzed with Kruskal-Wallis with Dunn's multiple comparison test (A, B, C) and Mann Whitney test (D).</p

Biologic responses to Can f 1-2-4-6 among dog allergic patients.

<p>Allergen-specific basophil degranulation monitored by addition of serial dilutions of rCan f 1 or equimolar amounts of either rCan f 2, rCan f 4, rCan f 6, an equimolar mix or Can f 1-2-4-6 (x-axis) to whole blood from three dog allergic patients. Positivity was defined by analysis of CD63 and CD203c double positive cells (y-axis) using flow cytometry.</p