Screening Random Peptide Libraries with Subacute Sclerosing PanencephalitisBrain-Derived Recombinant Antibodies Identifies Multiple Epitopes in the C-Terminal Region of the Measles Virus Nucleocapsid Protein

Abstract

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.Panning of phage-displayed random peptide libraries allows an unbiased selection of antibody epitopes/mimotopes without preconceptions about the nature of the target antigens (reviewed in reference 23). In central nervous system (CNS) inflammatory diseases, where access to active diseased tissue is limited or where the levels of tissue antigen may be extremely low, phage peptide panning provides an alternative and sensitive avenue for antigen identification. Panning of phage-displayed random peptide libraries has successfully identified rheumatoid factor-specific mimotopes (22) and allergen mimotopes (19) and has mapped both linear and discontinuous viral epitopes recognized by antibodies specific for various infectious agents (5,10,11,16,20).Infectious and inflammatory diseases of the CNS are often characterized by increased intrathecal immunoglobulin G (IgG) synthesis that is seen as discrete bands of oligoclonal IgG when cerebrospinal fluid (CSF) or brain IgG is separated by isoelectric focusing. In CNS infectious diseases, such as subacute sclerosing panencephalitis (SSPE), neurosyphilis, mumps meningitis, progressive rubella panencephalitis, cryptococcal meningitis, and varicella zoster virus vasculitis, the oligoclonal IgG is directed largely against the infectious agent that causes the disease (reviewed in reference 12). Increased CNS IgG synthesis is accompanied by an elevated number of CD19 ϩ B cells and the app...

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