Objective-The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis.Methods-Fluorescence activated cell sorting and single-cell reverse transcriptase PCR were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE).Results-Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis and complement and Ig deposition.Interpretation-Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly

Objective-Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory Bcell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic.Methods-We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy-and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections.Results-Whereas humanized control rAbs derived from anti-myelin hybridomas and antimyelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immuno-reactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells.Interpretation-The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.Address correspondence to Dr Gilden, Department of Neurology, University of Colorado Denver School of Medicine, 12700 East 19th Avenue, Mail Stop B182, Aurora, CO 80045. don.gilden@ucdenver.edu. Gregory Owens and Jeffrey Bennett contributed equally to this work.Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article. NIH Public Access Subjects and Methods Multiple Sclerosis and Control PatientsCSF (approximately 20ml) was collected from MS patients (see supplemental Table 1) after informed consent was given. MS diagnosis was made using established international criteria. 14, 15 CD138 + plasma cells and, in some patients, CD19 + B lymphocytes were sorted, and H-and L-chain V regions were amplified, sequenced, and analyzed as described elsewhere. 8,10 Construction of Human IgG1 Recombinant AntibodiesFull-length bivalent IgG1 rAbs expressing an H-chain C-terminal Flag epitope ( Supplementary Fig 1) were produced from H-and L-chain V-region sequences of selected CD138 + and CD19 + clones as described previously. 16 Cloned V-region inserts were sequenced to ensure fidelity of the rAbs. Construction of Chimeric Monoclonal AntibodiesAnti-human MBP (5E3 monoclonal antibody [mAb]) and anti-human MOG monoclonal antibodies (6D7 and 2B7 mAbs) were derived from BALB/c mice immuni...

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), the etiology of which is poorly understood. The most common laboratory abnormality associated with MS is increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands (OCBs) in the brain and cerebrospinal fluid (CSF). However, the major antigenic targets of these antibody responses are unknown. The risk of MS is increased after infectious mononucleosis (IM) due to EBV infection, and MS patients have higher serum titers of anti-EBV antibodies than control populations. Our goal was to identify disease-relevant epitopes of IgG antibodies in MS; to do so, we screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. We identified two phage peptides that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays. Using a highly sensitive phage-mediated immuno-PCR assay, we determined specific bindings of the two EBV epitopes to IgG from CSF from 46 MS and 5 inflammatory control (IC) patients. MS CSF IgG have significantly higher bindings to EBNA1 epitope than to EBNA2 epitope, whereas EBNA1 and EBNA2 did not significantly differ in binding to IC CSF IgG. Further, the EBNA1 epitope was recognized by OCBs from multiple MS CSF as shown in blotting assays with samples separated by isoelectric focusing. The EBNA1 epitope is reactive to MS intrathecal antibodies corresponding to oligoclonal bands. This reinforces the potential role of EBV in the etiology of MS.

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Background Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), the etiology of which is poorly understood. The most common laboratory abnormality associated with MS is increased intrathecal IgG synthesis and the presence of oligoclonal bands (OCBs) in the brain and cerebrospinal fluid (CSF). However, the major antigenic targets of these antibody responses are unknown. The risk of MS is increased after infectious mononucleosis (IM) due to EBV infection, and MS patients have higher serum titers of anti-EBV antibodies than control populations. Objectives To identify disease-relevant epitopes of IgG antibodies in MS. Methods We screened phage-displayed random peptide libraries (12-mer) with total IgG antibodies purified from the brain of a patient with acute MS. We identified and characterized the phage peptides for binding specificity to intrathecal IgG from patients with MS and from controls by ELISA, phage-mediated Immuno-PCR, and isoelectric focusing. Results Two phage peptides were identified that share sequence homologies with EBV nuclear antigens 1 and 2 (EBNA1 and EBNA2), respectively. The specificity of the EBV epitopes found by panning with MS brain IgG was confirmed by ELISA and competitive inhibition assays. Using a highly sensitive phage-mediated immuno-PCR assay, we determined specific bindings of the two EBV epitopes to IgG from CSF from 46 MS and 5 inflammatory control (IC) patients. MS CSF IgG have significantly higher bindings to EBNA1 epitope than to EBNA2 epitope, whereas EBNA1 and EBNA2 did not significantly differ in binding to IC CSF IgG. Further, the EBNA1 epitope was recognized by OCBs from multiple MS CSF as shown in blotting assays with samples separated by isoelectric focusing. Conclusions The EBNA1 epitope is reactive to MS intrathecal antibodies corresponding to oligoclonal bands. This reinforces the potential role of EBV in the etiology of MS.