Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations.

Animal models of Graves' disease have been generated in recent years with various vaccination protocols using wild-type TSH receptor. In this study, we report the findings of genetic immunization of Swiss outbred mice with three different mutated human TSH receptor plasmids, each containing one constitutive activating mutation located at the ectodomain (S281N), exoloop (I486F), and transmembrane segment (D633H) respectively. Although the overall rate of thyrotoxicosis in the mice was < 10%, anti-TSH receptor antibodies could be detected in many animals by flow cytometry, radioreceptor assay, and functional bioassays using recombinant human TSH receptor. Mice injected with plasmids harboring activated mutants (S281N and D633H) showed production of predominantly stimulating antibodies, whilst those treated with wild-type receptor plasmids generated mainly blocking sera. Most of these antibodies displaced radiolabeled bovine TSH, and their epitopes, independent of functional characteristics, were mapped to the first 271 amino acids of the TSH receptor. This supports recent findings that binding of stimulatory or blocking antibodies lie in close proximity within the leucine-rich repeat region.