Solid phase immunoadsorption for therapeutic and analytical studies on neuropathy-associated anti-GM1 antibodies.

Autoimmune neuropathies including Guillain-Barré syndrome are frequently associated with anti-GM1 ganglioside antibodies. These are believed to play a pathogenic role and their clearance from the circulation would be predicted to produce therapeutic benefit. This study examines the conditions required for effective immunoadsorption of anti-GM1 antibodies using glycan-conjugated Sepharose as a matrix. In solution inhibition studies using a range of GM1-like saccharides in conjunction with mouse and human anti-GM1 antibodies, the whole GM1 pentasaccharide beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal-(1-4)-beta-Glc was the favored ligand for maximal inhibiton of antibody-GM1 interactions in comparison with monosaccharides, Gal-(1-3)-beta-GalNAc-betaOMe, and synthetic GM1 mimetics. Immunoadsorption studies comparing binding of mouse monoclonal anti-GM1 antibodies to GM1-Sepharose and beta-Gal-(1-3)-beta-GalNAc-Sepharose confirmed the preference seen in solution inhibition studies. GM1-Sepharose columns were then used to adsorb anti-GM1 immunoglobulin G and immunoglobulin M antibodies from human neuropathy sera. Anti-GM1 antibodies subsequently eluted from the columns often showed a striking monoclonal or oligoclonal pattern, indicating that the immune response to GM1 is restricted to a limited number of B-cell clones, even in the absence of a detectable serum paraprotein. These data support the view that immunoadsorption plasmapheresis could potentially be developed for the acute depletion of serum anti-GM1 antibodies in patients with neuropathic disease, and also provide purified human anti-GM1 antibodies for analytical studies.

Characterisation of the immunoglobulin variable region gene usage encoding the murine anti-ganglioside antibody repertoire.

Neuropathogenic murine antibodies reactive with terminal disialylgalactose epitopes are innate and preferentially encoded by the VH7183.3b gene. Here we have studied antibodies reactive with internal galactose-linked disialosyl epitopes and the terminal trisaccharide of GT1b. Antibodies were of moderate affinity and unmutated. Anti-GD1b antibodies were often encoded by the VH10.2b heavy and gj38c light chain genes. Anti-GT1b antibodies with broader glycan binding patterns were encoded by VHQ52 and VHJ558 family genes. These data indicate that the discrete specificities of ganglioside-binding antibodies are dictated by particular patterns of V gene usage residing within the innate B cell repertoire.

Innate murine B cells produce anti-disialosyl antibodies reactive with Campylobacter jejuni LPS and gangliosides that are polyreactive and encoded by a restricted set of unmutated V genes.

In Guillain-Barré syndrome following Campylobacter enteritis, anti-lipopolysaccharide antibodies cross-react with neural gangliosides, thereby precipitating autoimmune neuropathy. We examined the properties of 15 murine anti-LPS/ganglioside mAbs specific for NeuAc(alpha2-8)NeuAc-Gal disialosyl epitopes. Many mAbs displayed features of an innate B cell origin including polyreactivity (13/15), hybridoma CD5 mRNA expression (5/15), predominance of IgM (9/15) or IgG3 (3/6) isotype, low affinity, and utilisation of unmutated VH and VL VDJ rearrangements. Antibody specificity resided in highly selective V gene usage, with 6/15 mAbs being encoded by the VH7183.3b gene. These data indicate that neuropathogenic antiganglioside autoantibodies can arise from the natural autoantibody repertoire.

Synthetic disialylgalactose immunoadsorbents deplete anti-GQ1b antibodies from autoimmune neuropathy sera.

Acute and chronic autoimmune neuropathies, including Guillain-Barré syndromes (GBS) are often characterized by the presence of autoantibodies that react with neural gangliosides. Evidence from human and animal studies indicates that anti-ganglioside antibodies play a primary neuropathogenic role, and their rapid elimination from the circulation through specific immunoadsorption therapy thus has the potential to ameliorate the course of the disease. Here we have tested this therapeutic principle in the Miller Fisher variant of GBS that is associated serologically with acute phase anti-GQ1b ganglioside immunoglobulin G (IgG) antibodies, and in chronic ataxic neuropathies associated with persistently elevated immunoglobulin M (IgM) antibodies that react with GQ1b, GD3 and other disialylated gangliosides. Human and mouse anti-GQ1b IgG and IgM antibodies may also react with GD3, suggesting the shared terminal disialoside epitope could be involved in antibody binding. We thus synthesized the terminal trisaccharide, NeuAc(alpha2-8)NeuAc(alpha2-3)Gal common to GQ1b and GD3, and conjugated it to bovine serum albumin (BSA). This disialylgalactose glycoconjugate (DSG-BSA) binds anti-GQ1b antibodies in 32/58 (55%) human sera containing IgG or IgM anti-GQ1b antibodies at titres up to 1/130 000; it also binds a wide range of mouse monoclonal anti-GQ1b and -GD3 antibodies. When conjugated to Sepharose as mock therapeutic immmunoaffinity columns, the immobilized trisaccharide (DSG-Sepharose) eliminates anti-GQ1b antibodies from positive sera in proportion to their level of binding to DSG-BSA. Oligosaccharide-specific immunoadsorption therapy thus provides a new therapeutic approach to anti-GQ1b antibody-associated syndromes that could be applied to clinical practice. Furthermore, modification of the immobilized oligosaccharide epitopes to incorporate other glycan structures may allow this approach to be adapted to other forms of autoimmune neuropathy associated with uniform anti-glycolipid antibody profiles.

Tolerance to self gangliosides is the major factor restricting the antibody response to lipopolysaccharide core oligosaccharides in Campylobacter jejuni strains associated with Guillain-Barré syndrome.

Guillain-Barré syndrome following Campylobacter jejuni infection is frequently associated with anti-ganglioside autoantibodies mediated by molecular mimicry with ganglioside-like oligosaccharides on bacterial lipopolysaccharide (LPS). The regulation of antibody responses to these T-cell-independent antigens is poorly understood, and only a minority of Campylobacter-infected individuals develop anti-ganglioside antibodies. This study investigates the response to gangliosides and LPS in strains of mice by using a range of immunization strategies. In normal mice following intraperitoneal immunization, antibody responses to gangliosides and LPS are low level but can be enhanced by the antigen format or coadministration of protein to recruit T-cell help. Class switching from the predominant immunoglobulin M (IgM) response to IgG3 occurs at low levels, suggesting B1-cell involvement. Systemic immunization results in poor responses. In GalNAc transferase knockout mice that lack all complex gangliosides and instead express high levels of GM3 and GD3, generation of anti-ganglioside antibodies upon immunization with either complex gangliosides or ganglioside-mimicking LPS is greatly enhanced and exhibits class switching to T-cell-dependent IgG isotypes and immunological memory, indicating that tolerance to self gangliosides is a major regulatory factor. Responses to GD3 are suppressed in knockout mice compared with wild-type mice, in which responses to GD3 are induced specifically by GD3 and as a result of polyclonal B-cell activation by LPS. The anti-ganglioside response generated in response to LPS is also dependent on the epitope density of the ganglioside mimicked and can be further manipulated by providing secondary signals via lipid A and CD40 ligation.