Conformational epitopes of myelin oligodendrocyte glycoprotein are targets of potentially pathogenic antibody responses in multiple sclerosis.

BACKGROUND: Myelin/oligodendrocyte glycoprotein (MOG) is a putative autoantigen in multiple sclerosis (MS). Establishing the pathological relevance and validity of anti-MOG antibodies as biomarkers has yielded conflicting reports mainly due to different MOG isoforms used in different studies. Because epitope specificity may be a key factor determining anti-MOG reactivity we aimed at identifying a priori immunodominant MOG epitopes by monoclonal antibodies (mAbs) and at assessing clinical relevance of these epitopes in MS. METHODS: Sera of 325 MS patients, 69 patients with clinically isolated syndrome and 164 healthy controls were assayed by quantitative, high-throughput ELISA for reactivity to 3 different MOG isoforms, and quantitative titers correlated with clinical characteristics. mAbs defined unique immunodominant epitopes distinct to each of the isoforms. RESULTS: In the majority of human samples anti-MOG levels were skewed towards low titers. However, in 8.2% of samples high-titer anti-MOG antibodies were identified. In contrast to anti-MOG reactivity observed in a mouse model of MS, in patients with MS these never reacted with ubiquitously exposed epitopes. Moreover, in patients with relapsing-remitting MS high-titer anti-MOG IgG correlated with disability (EDSS; Spearman r = 0.574; p = 0.025). CONCLUSIONS: Thus high-titer reactivity likely represents high-affinity antibodies against pathologically relevant MOG epitopes, that are only present in a small proportion of patients with MS. Our study provides valuable information about requirements of anti-MOG reactivity for being regarded as a prognostic biomarker in a subtype of MS.

Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis.

The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.

Apoptotic effects of Human Herpesvirus-6A on glia and neurons as potential triggers for central nervous system autoimmunity.

BACKGROUND: Human Herpesvirus type 6 (HHV-6A and/or HHV-6B) has been tentatively associated with multiple sclerosis (MS). However, there is currently no direct proof of pathogenicity. OBJECTIVES: To determine whether exposure to HHV-6 variants is capable of inducing programmed cell death (apoptosis) in representative cell types of the central nervous system (CNS). STUDY DESIGN: HHV-6A and HHV-6B variants were grown on human T cell lines HSB2 and MOLT-3, respectively. Human neuronal (SK-N-SH), astrocytes (CRT), and oligodendrocytes (TC620) cell lines were exposed in vitro to infected T cells in a trans-well system for up to 4 days (5x10(4) cells target cells and 2x10(6) T cells). Apoptosis was measured by a FACS-based method. RESULTS: Exposure to HHV-6A induced apoptosis in a time-dependent manner, while exposure to HHV-6B did not. Three days after exposure, apoptosis was increased compared to normalized controls, by 239% in neurons, 321% in astrocytes, and 326% in oligodendrocytes, respectively. CONCLUSIONS: This study provides the demonstration that exposure to immune cells carrying replicating HHV-6A may injure glial cells and neurons by inducing apoptosis, and direct evidence for a causal association between HHV-6A with MS and related disorders.

Role of nerve growth factor and other trophic factors in brain inflammation.

Inflammation in the brain is a double-edged process that may be beneficial in promoting homeostasis and repair, but can also result in tissue injury through the damaging potential of inflammatory mediators. Thus, control mechanisms that minimize the extent of the inflammatory reaction are necessary in order to help preserve brain architecture and restore function. The expression of neurotrophic factors such as nerve growth factor (NGF) is increased after brain injury, in part mediated by effects on astrocytes of pro-inflammatory mediators and cytokines produced by immune cells. Conversely, cells of the immune system express NGF receptors, and NGF signaling modulates immune function. Multiple sclerosis (MS) and the disease model experimental autoimmune encephalomyelitis are neurodegenerative disorders whereby chronic destruction of the brain parenchyma results from an autoaggressive, immune-mediated inflammatory process and insufficient tissue regeneration. Here, we review evidence indicating that the increased production of NGF and other trophic factors in central nervous system (CNS) during these diseases can suppress inflammation by switching the immune response to an anti-inflammatory, suppressive mode in a brain-specific environment. Thus, trophic factors networks in the adult CNS not only protects axons and myelin but appear to also actively contribute to the maintenance of the brain immune privilege. These agents may represent good targets for therapeutic intervention in MS and other chronic CNS inflammatory diseases.

Approved and future pharmacotherapy for multiple sclerosis.

BACKGROUND: Pharmacotherapy for relapsing-remitting multiple sclerosis (MS) advanced with the demonstration that interferon beta and glatiramer acetate improve the clinical course of this disease. Mitoxantrone is the first drug approved by the Food and Drug Administration for treatment of secondary progressive MS. Despite this progress, the agents presently available are only partially effective, are difficult to administer, and may have significant side effects. Several orally administered immunomodulatory agents are presently being evaluated for treatment of MS. One class of drugs, HMG CoA inhibitors (statins), is safe and well-tolerated and could become another mainstay of MS therapy. REVIEW SUMMARY: This article reviews the clinical evidence for approved MS therapies and discusses their mechanisms of action. Furthermore, the clinical and laboratory data suggesting a potential role for statins in MS therapy are discussed. CONCLUSIONS: Although treatment with interferon beta, glatiramer acetate, and mitoxantrone, the approved therapies, provide important treatment options for patients with relapsing-remitting MS and secondary progressive MS, the potential benefits of other medications, including statins, should be explored in controlled clinical trials.

Transient increases in anti-aquaporin-4 antibody titers following rituximab treatment in neuromyelitis optica, in association with elevated serum BAFF levels.

Rituximab is increasingly used for prevention of relapses of neuromyelitis optica (NMO), a condition that is highly associated with serum anti-aquaporin-4 (AQP4) antibodies. However, B-cell depletion also induces systemic B-cell activating factor (BAFF), which may promote antibody production. We collected serial serum samples from a total of seven patients with NMO prior to, and following, treatment with rituximab. The samples were analyzed for anti-AQP4 antibody titer using a cell-based assay and serum BAFF levels were measured on available samples by standard enzyme-linked immunosorbent assay. Anti-AQP4 antibody levels decreased after 4 weeks to 12 weeks from the first injection of rituximab, but they increased transiently in several patients at 2 weeks after the first injection, in association with a parallel increase in serum BAFF levels. Although anti-AQP4 antibodies appear to decrease overall following rituximab treatment, our findings raise concern over the potential for an early BAFF-mediated worsening of patients with NMO receiving rituximab.

Relevant antibody subsets against MOG recognize conformational epitopes exclusively exposed in solid-phase ELISA.

A pathogenic role for circulating anti-myelin antibodies is difficult to establish in multiple sclerosis (MS). Here, we unravel a broad heterogeneity within the anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in humans and non-human primates, and demonstrate that detection of important epitopes of MOG within the pathogenic repertoire is exclusively dependent on presentation on a solid-phase MOG conformer. Results of ELISA and those of a liquid-phase assay were compared using a MOG protein with identical sequence but different conformations. We tested sera from 50 human subjects, plasma of Callithrix jacchus marmosets known to contain antibodies reactive to either conformational or linearized MOG, and monoclonal, conformation-dependent anti-MOG antibodies. We have found no antibody reactivity against the soluble MOG conformer in human serum, and show that this lack of detection is not due to technical artifacts. Rather, dominant epitopes of MOG are not displayed in soluble phase, as shown by a complete lack of binding of conformation-dependent mAb. In MP4-immune marmosets that exhibit demyelinating pathology due to spreading of antibody determinants to myelin-embedded MOG, only ELISA can detect pathogenic circulating anti-MOG antibodies. Thus, the accurate detection of important subsets of pathogenic anti-MOG antibodies requires methods in which MOG is displayed similarly to its natural conformation in myelin.

Restrictive and diversifying elements of the anti-myelin/oligodendrocyte glycoprotein antibody response in primate experimental allergic encephalomyelitis.

Autoantibody responses against conformational epitopes of myelin/oligodendrocyte glycoprotein (MOG) possess myelin destructive potential, as demonstrated in the marmoset model of human multiple sclerosis (MS) and in some rodent models of experimental allergic encephalomyelitis. We have previously characterized monoclonal Fab fragments specific for conformational epitopes of MOG that were derived from a combinatorial antibody library generated from a MOG-immune marmoset. In this paper, we address the molecular heterogeneity of humoral responses against MOG in this outbred model of MS by studying additional antibody clones derived from a genetically unrelated animal. We find that all MOG-specific IgGkappa Fab fragments, unrelated to genetic make-up, utilize a restricted set of variable region genes, IGHV1 and IGHV3 for the H chain and IGKV1, IGKV3, and IGKV5 for the L chain. Despite these restricting factors, diversity within these antibody repertoires can be observed, predominantly within the H-chain CDR3 regions. Our findings suggest that only a limited set of Ig genes is necessary to launch a diverse, destructive humoral immune response against a single CNS antigen in primates. These results are the first to contribute to a better understanding of how myelin-directed and potentially destructive autoantibody responses may develop in human MS.

Antibodies to native myelin oligodendrocyte glycoprotein are serologic markers of early inflammation in multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOG-specific IgGs were most frequently found in serum of clinically isolated syndromes (P < 0.001) and relapsing-remitting MS (P < 0.01), only marginally in secondary progressive MS (P < 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P < 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressed in vivo are early targets for pathogenic Abs; (ii) these Abs, which are not detected in solid-phase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model.

Poly(ADP-ribose) polymerase-1 activation in a primate model of multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated disabling neurological disorder involving inflammation, demyelination, axonal damage, and neurodegeneration. Poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme linked to DNA repair, has been shown to regulate the cellular inflammatory response through interactions with nuclear factor-kappaB. Extensive PARP-1 activation can, by separate mechanisms, also cause cell death. PARP-1 activation in brain occurs in several settings associated with oxidative stress and DNA damage, and PARP-1 inhibition has been shown to attenuate inflammation and improve neuronal survival in these settings. Here we studied the pattern of PARP-1 activation in a nonhuman primate model of MS, marmoset (Callithrix jacchus) experimental allergic encephalomyelitis (EAE). Characteristic of this model is relapsing and remitting focal demyelination typical of human MS. Immunostaining for poly(ADP-ribose), the enzymatic product of PARP-1, showed PARP-1 activation specifically in plaque areas of EAE brains. Robust immunostaining was found in astrocytes surrounding demyelinated EAE plaques and in scattered nearby microglia, oligodendrocytes, and neurons. The immunostaining also suggested PARP-1 activation in occasional endothelial cells surrounded by microglia or infiltrating peripheral blood cells. Given the importance of PARP-1 in both inflammation and cell death processes, these findings suggest that PARP-1 activation may be a significant factor in the pathogenesis of MS.

Antibody responses against galactocerebroside are potential stage-specific biomarkers in multiple sclerosis.

BACKGROUND: Galactocerebroside, the major glycolipid of central nervous system myelin, is a known target for pathogenic demyelinating antibody responses in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). OBJECTIVE: To address the importance of anti-galactocerebroside (alpha-GalC) antibodies in MS and to evaluate them as biomarkers of disease. METHODS: alpha-GalC IgGs were quantified from sera of patients with MS and in marmoset EAE by a new immunosorbent assay. RESULTS: We report a significant difference in serum alpha-GalC IgG titers between patients with relapsing-remitting (RR)-MS and healthy controls (HCs; P < .001). The frequencies of alpha-GalC antibody-positive subjects (alpha-GalC titers > or = mean HC titers+3 SD) are also significantly elevated in RR-MS compared with HC (40% vs 0%; P = .0033). Immunoaffinity purified alpha-GalC IgGs from human serum bind to cultured human oligodendrocytes, indicating that the ELISA detects a biologically relevant epitope. Corroborating these findings, alpha-GalC antibody responses in marmoset EAE were similarly found to be specifically associated with the RR forms and not the peracute or progressive forms, in contrast with other anti-myelin antibodies (P = .0256). CONCLUSION: (1) alpha-GalC antibodies appear MS-specific and are not found in healthy subjects, unlike antibodies against myelin proteins; (2) when present, alpha-GalC antibodies identify mostly RR-MS and may be an indicator of ongoing disease activity. This novel assay is a suitable and valuable method to increase accuracy of diagnosis and disease staging in MS.

The human T cell response to myelin oligodendrocyte glycoprotein: a multiple sclerosis family-based study.

Myelin oligodendrocyte glycoprotein (MOG) is an encephalitogenic myelin protein and a likely autoantigen in human multiple sclerosis (MS). In this work, we describe the fine specificity and cytokine profile of T cell clones (TCC) directed against MOG in three nuclear families, comprised of four individuals affected with MS and their HLA-identical siblings. TCC were generated from PBMC by limiting dilution against a mixture of eleven 20-mer overlapping peptides corresponding to the encephalitogenic extracellular domain of human MOG (aa 1-120). The frequency of MOG peptide-reactive T cells was surprisingly high (range, 1:400 to 1:3,000) and, unexpectedly, cloning efficiencies were highest at low seeding densities of 10(2) or 10(3) PBMC per well. A total of 235 MOG peptide-reactive TCC were produced, all of which were CD4(+)CD8(-)TCRalphabeta(+)TCRgammadelta(-). All 11 MOG peptides were recognized by the TCC, and different epitopes of MOG appeared to be immunodominant in the HLA-identical siblings. The patterns of cytokine secretion by TCC from single individuals were generally similar. The healthy individuals exhibited Th2-, Th0-, and T regulatory cell 1-like cytokine profiles, whereas TCC from one sibling with MS had a striking Th1-like phenotype, producing high levels of IFN-gamma and TNF-alpha, and low IL-4 levels. Thus, MOG-reactive T cells appear to constitute an important part of the natural T cell repertoire, a finding that could contribute to the development of autoimmunity to this protein.

Molecular characterization of antibody specificities against myelin/oligodendrocyte glycoprotein in autoimmune demyelination.

Myelin/oligodendrocyte glycoprotein (MOG) is a target antigen for myelin-destructive Abs in autoimmune central nervous system demyelinating disorders. Little is known about the molecular and structural basis of these pathogenic Ab responses. Here, we have characterized anti-MOG Ab specificities in the marmoset model of experimental allergic encephalomyelitis, by means of a combinatorial IgG-Fab library. We found that a diverse population of Ig genes encodes for auto-Abs that exclusively recognize conformation-dependent antigenic targets on MOG. These antigenic domains correspond to exposed epitopes in vivo, as the Fab fragments recognize native MOG in situ in marmoset brain tissue. The Ab fragments described here represent Ab specificities that are common constituents of the humoral immune repertoire against MOG in outbred populations, as demonstrated by their ability to displace native anti-MOG Abs present in sera from MOG-immune marmosets and patients with multiple sclerosis. Furthermore, neuropathological analysis and characterization of Ab epitope specificities in animals immunized with MOG or MOG-derived peptides revealed that only conformation-dependent Abs are associated with demyelinating activity, suggesting that epitope recognition is an important factor for Ab pathogenicity. Our findings provide novel and unexpected knowledge on the diversity of anti-MOG Ab responses in nonhuman primates and humans, and will permit the dissection of pathogenic auto-Ab properties in multiple sclerosis.

Frontline: Epitope recognition on the myelin/oligodendrocyte glycoprotein differentially influences disease phenotype and antibody effector functions in autoimmune demyelination.

Preliminary observations of humoral immunity against the myelin oligodendrocyte glycoprotein (MOG) in experimental allergic encephalomyelitis (EAE) and human multiple sclerosis (MS) suggest that a subset of anti-MOG autoantibodies directed against conformational epitopes is of pathogenic predominance. Here, we provide proof that in marmoset EAE, autoantibodies reactive against conformational epitopes of MOG are not only responsible for aggravating demyelination, but also an essential factor for disease dissemination in space within the central nervous system, a hallmark for typical forms of human MS. In terms of effector mechanisms, IgG deposition and complement activation occur exclusively in association with presence of these conformational antibodies, while microglial/macrophage activation appears to be a common immunopathological finding regardless of the fine determinant specificity of anti-MOG antibodies. These findings highlight for the first time the complex heterogeneity of function and pathogenicity in the polyclonal anti-MOG antibody repertoire of outbred species. Because the linear and conformational antibody determinants of MOG are shared between marmosets and humans, these results are directly relevant to understanding effector mechanisms of organ damage in MS.

Marmoset fine B cell and T cell epitope specificities mapped onto a homology model of the extracellular domain of human myelin oligodendrocyte glycoprotein.

Aberrant association of autoantibodies with myelin oligodendrocyte glycoprotein (MOG), an integral membrane protein of the central nervous system (CNS) myelin, has been implicated in the pathogenesis of multiple sclerosis (MS). Sensitization of nonhuman primates (Callithrix jacchus marmosets) against the nonglycosylated, recombinant N-terminal domain of rat MOG (residues 1-125) reproduces an MS-like disease in which MOG-specific autoantibodies directly mediate demyelination. To assess the interrelationship between MOG structure and the induction of autoimmune CNS diseases and to enable structure-based rational design of therapeutics, a homology model of human MOG(2-120) was constructed based on consensus residues found in immunoglobulin superfamily variable-type proteins having known structures. Possible sites for posttranslational modifications and dimerization have also been identified and analyzed. The B cell and T cell epitopes have been identified in rat MOG-immunized marmosets, and these sequences are observed to map primarily onto accessible regions in the model, which may explain their ability to generate potent antibody responses.