Elimination of luteinizing hormone cross-reactive epitopes from human chorionic gonadotropin.

The β-chain of human chorionic gonadotropin (hCG) has been shown to have efficacy in clinical trials when used as a contraceptive vaccine. This hormone is a heterodimer, the α-chain being shared with the other members of the glycoprotein hormone family but the β-chain being unique to hCG. Nevertheless, there is sequence homology between the hCG β-chain and the β-chain of human luteinizing hormone (hLH) which results in cross-reactive antibodies being produced following immunization with wild-type hCGβ. To reduce or eliminate such cross-reactions we generated a number of mutants of the hCGβ-chain. One mutant (hCGβ(R68E)), containing an arginine to glutamic acid replacement at position 68, has been expressed as a recombinant protein in High Five™ insect cells. The recombinant BAChCGβ(R68E) form of this molecule was used to immunize rabbits and the antibody response compared to the response following immunization with the recombinant wild-type protein BAChCGβ and with the native hCGαβ heterodimer isolated from pregnancy urine. The mutant elicited the production of antibodies which avidly recognize native hCG. Compared to immunization with wild-type hCG, the response showed very little cross reactivity with hLH. This is demonstrated to be due to a radically altered epitope usage in the response to the mutant, which now focuses mainly upon the C-terminal region of the β-chain

Refocusing of B-cell responses following a single amino acid substitution in an antigen.

Intranasal immunization of BALB/c strain mice was carried out using baculovirus-derived human chorionic gonadotrophin (hCG) β-chain, together with Escherichia coli heat-labile enterotoxin. Gonadotrophin-reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross-reactive antibodies when holo-hCG is used as an immunogen. In contrast to wild-type hCGβ, a mutated hCGβ-chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross-react with LH. Furthermore, the epitopes utilized in the B-cell response to the mutated hCGβ shifted away from the immunodominant region of the parent wild-type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross-reactive epitope region and towards a normally weakly immunogenic but antigen-unique area. Similar mutational strategies for epitope-refocusing may be applicable to other vaccine candidate molecules