The factors which lead to selection of dominant antigenic sites concentrated in discreet regions of proteins and polypeptides are important to the development of antigen-specific immunotherapies for autoimmune diseases and for vaccine design. In this study, the main immunogenic regions of the immunopathogenic autoantigen, retinal S-antigen, have been identified by examination of the specificity of antibody responses of different species. Using cyanogen bromide and synthetic peptides in western blots and the ELISA, the specificities of antisera from rabbits, guinea pigs, rats and 19 inbred strains of mice were tested. All animals produced high titers of antibody to S-antigen with the exception of PL/J mice. Antibodies which bound epitopes contained in peptide CB46, a 46 amino acid-containing peptide located at the C-terminus of S-antigen, were dominant in all species and strains tested. The epitopes in CB46 were multiple, overlapping, and concentrated in a stretch of approximately 30 residues. Two overlapping synthetic peptides from that region substantially competed the anti-CB46 response of all animals. Antibodies which recognized peptide CB47, a 47 residue peptide from the N-terminus, comprised the next most common group. This epitope was similar in all mice and overlapped the epitope defined by rat antibodies. All anti-CB47 antibodies mapped to an 11 residue region of CB47. Eleven strains of mice did not respond to CB47 after one immunization with S-antigen; however, multiple immunizations readily converted all animals so tested to CB47 responders. Rabbits and guinea pigs exhibited very weak responses to CB47 following one immunization; multiple immunizations increased the response minimally. Rats produced a strong antibody response to peptide CB123, which contains the known uveitogenic sites, while very little activity to CB123 was raised in rabbits and guinea pigs. Only 3 murine strains, LP, LP.R3, and B10.R3-71, responded with antibodies to CB123 and the epitope was mapped to a 30 residue region which in rats also contains two distinct pathogenic sites and an antibody epitope. Only rats and rabbits made antibody to the CB35 peptide; the epitopes were contained within an 18 residue sequence. The results show that a main immunogenic region is located in S-antigen near the C-terminus and is independent of species or MHC. Less dominant, species and strain-dependent immunogenic regions were found in three other areas, i.e. peptides CB47, CB123 and CB35.
Bibliographical noteFunding Information:
We thank Colin Campbell, David Asomaning, Wesley Obritsch, Henry Lee and Shiv Prasad for excellent technical assistance, Gary Fletcher for histology preparations and Dr's Laura Smith and Stella Robertson, Alcon Labs, for mAb 505.47. We thank Steve Fling for helpful suggestions on the manuscript. This work was supported in part by NIH grants EY-03660 (DSG), EY-05417 (DSG), EY-05095 (LAD) and by funds from Research to Prevent Blindness. Dr. Gregerson is a Senior Scientific Investigator of Research to Prevent Blindness.