Plasmid and adenoviral vectors have been used to generate
antibodies in mice that resemble human
autoantibodies to the
thyrotrophin receptor. No such studies, however, have been performed for
thyroid peroxidase (TPO), the major
autoantigen in human
thyroiditis. We constructed plasmid and adenovirus vectors for in vivo expression of TPO. BALB/c mice were immunized directly by
intramuscular injection of TPO-plasmid or TPO-adenovirus, as well as by
subcutaneous injection of dendritic cells (DC) infected previously with TPO-adenovirus. Intramuscular TPO-adenovirus induced the highest, and TPO-plasmid the lowest, TPO antibody titres. Mice injected with TPO-transfected DC developed intermediate levels.
Antibodies generated by all three approaches had similar affinities (Kd approximately 10(-9)M) and recognized TPO expressed on the cell-surface. Their
epitopes were analysed in competition assays using monoclonal human
autoantibodies that define the TPO
immunodominant region (IDR) recognized by patients with thyroid
autoimmune disease. Surprisingly, high titre
antibodies generated using adenovirus interacted with diverse TPO
epitopes largely outside the IDR, whereas low titre
antibodies induced by
DNA-plasmid recognized restricted
epitopes in the IDR. This inverse relationship between antibody titre and restriction to the IDR is likely to be due to
epitope spreading following strong antigenic stimulation provided by the adenovirus vector. However, TPO antibody
epitope spreading does not occur in Hashimoto's
thyroiditis, despite high
autoantibody levels. Consequently, these data support the concept that in human thyroid autoimmunity, factors besides titre must play a role in shaping an
autoantibody epitopic profile.