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The Cysteine-Rich Amino Terminus of the Thyrotropin Receptor Is the Immunodominant Linear Antibody Epitope in Mice Immunized Using Naked Deoxyribonucleic Acid or Adenovirus Vectors

Autoimmune Disease Unit (L.S.-L., P.N.P., C.-R.C., C.P., B.R., S.M.M.), Cedars-Sinai Research Institute and UCLA School of Medicine, Los Angeles, California 90048; Department of Pharmacology 1 (Y.N.), Nagasaki University School of Medicine, Nagasaki 852-8523, Japan; and Division of Endocrinology and Metabolism (J.C.M.), Department of Medicine, Mayo Clinic, Rochester, Minnesota 55902

Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org.

Experimental Graves’ disease is more effectively produced by immunization approaches involving in vivo TSH receptor (TSHR) expression than by conventional immunization with TSHR protein and adjuvant. Unlike conformational epitopes that are extremely difficult to define, linear epitopes can be readily assessed using synthetic peptides. TSHR linear epitopes are well characterized in conventionally immunized animals, but there is no information for animals vaccinated with TSHR DNA in plasmid or adenovirus vectors. We used synthetic peptides to characterize linear epitopes in mice immunized by in vivo expression of TSHR DNA. TSHR adenovirus-injected mice had higher antibody levels than TSHR DNA-vaccinated mice. However, the dominant peptide recognized in both groups was the TSHR cysteine-rich N terminus (residues 22–41). Sera from TSHR adenovirus-immunized (but not TSHR DNA-vaccinated) mice interacted to a lesser extent with peptides encompassing residues 352–401, which include the region deleted following TSHR cleavage as well as the ectodomain juxta-membrane region. Although antibodies characterized using synthetic peptides are probably TSH blockers or nonfunctional, stimulating antibodies may recognize linear components in a conformational epitope. The cysteine-rich TSHR N terminus is functionally important in the action of stimulating TSHR autoantibodies in humans. The immunodominance of the same region in immunized mice suggests that this region may also be immunodominant in humans.

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