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The Human Chorionic Gonadotropin-ß Arginine⁶⁸ to Glutamic Acid Substitution Fixes the Conformation of the C-Terminal Peptide

Department of Immunology and Molecular Pathology (M.C.-D., N.T., J.D.M., P.K., I.M.R., P.J.D., T.L.), University College London, London W1T 4JF, United Kingdom; School of Biosciences (N.T.), University of East London, London E15 4LZ, United Kingdom; Department of Molecular Biology (P.M.M., J.J.), University of Aarhus, DK-8000 Aarhus, Denmark; Institute for Biomedical Aging Research (P.B.), Austrian Academy of Sciences, A-6020 Innsbruck, Austria; Chemistry Department (A.L.), University of Glasgow, Glasgow GI2 8QQ, Scotland, United Kingdom; and Department of Oral Immunology (C.K.), Kings College London, London SE1 9RT, United Kingdom

Address all correspondence and requests for reprints to: Dr. Torben Lund, Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London W1T 4JF, United Kingdom. E-mail: t.lund{at}ucl.ac.uk.

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine⁶⁸ of the ß-subunit (hCGß) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCGß-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCGß-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCGß-specific and anti-hCGß/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCGß-R68E was eliminated, mAbs reacting with hCGß-specific epitopes bound to hCGß and hCGß-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCGß-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCGß-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.