Human chrionic gonadotropin (hCG) is a placental glycoprotein hormone, a heterodimeric molecule, consisting of alpha and beta chains. It induces the synthesis of progesterone, which is essential for the maintenance of the fertilized egg. Antibodies directed against hCG can, therefore, prevent pregnancy and serve as a vaccine. hCG belongs to the glycoprotein hormone family and shares the alpha chain with the other members. The beta chain is a hormone-specific subunit that is unique to hCG, but still possesses 85% amino acid homology with the beta chain of luteinizing hormone (LH), which means that prolonged immunization with hCG produces antibodies that cross-react with LH.
We have taken an approach involving the mutation of beta hCG to eliminate cross-reactive epitopes without affecting the natural folding of the polypeptide chain and thus the unique beta hCG-specific epitopes.
Several mutants have been constructed that have maintained the binding to hCG-specific monoclonal antibodies (mAbs) but have lost the ability to bind to a panel of LH cross-reactive mAbs. To investigate the immunogenicity of selected mutants, mice were immunized with expression plasmid DNA, containing the gene for wild-type beta hCG and two mutants: mutant 3, with four amino acid substitutions (68 Arg-->Glu; 74 Arg-->Ser; 75 Gly-->His; 79 Val-->His), and mutant 7, with a single amino acid substitution (68 Arg-->Glu).
Although both mutants were able to elicit antibody responses in at least some animals, the levels were less than those seen with the wild-type beta hCG DNA, and there seems still to be a residual cross-reactivity with LH. Attempts to improve the immunogenicity of the mutants and to further modify the sequence to remove the cross-reactivity are currently underway.
[Show abstract][Hide abstract] ABSTRACT: Following priming and boosting of mice with a DNA vector pEE6DeltaS-hCGss expressing sequences encoding a transmembrane version of the beta-chain of human chorionic gonadotropin (hCGbeta), we failed to detect appreciable levels of specific antibody. However, subsequent challenge with hCG protein in Ribi adjuvant elicited a strong and rapid secondary immune response. This response was of comparable magnitude to that produced following priming, boosting and challenge with protein in adjuvant. Thus, DNA vaccination with this vector is as efficient in generating B cell memory as is conventional immunization, but the memory generation occurs in the absence of an overt effector response. Despite an overall similar level of specific antibody, the DNA-vaccinated mice produced hCG-specific antibodies biased towards IgG2a and IgG2b isotypes, whereas the protein-vaccinated mice produced higher levels of IgG1 antibodies. Both Th1 and Th2 cytokines (interferon-gamma (IFN-gamma) and IL-4) were lower in the spleens of the DNA-immunized animals compared with the protein-Ribi-immunized animals, possibly suggesting a different level of helper T cell response to the two different modes of immunization.
[Show abstract][Hide abstract] ABSTRACT: In this review article, the reader is introduced to recent advances in our knowledge on a subset of the cystine knot superfamily of homo- and hetero-dimeric proteins, from the perspective of the endocrine glycoprotein hormone family of proteins: follitropin (FSH), Iutropin (LH), thyrotropin. (TSH) and chorionic gonadotropin (CG). Subsequent papers will address the structure-function behaviour of other members of this increasingly significant family of proteins, including various members of the transforming growth factor-beta (TGF-beta) family of proteins, the activins, inhibins, bone morphogenic growth factor, platelet derived growth factor-beta, nerve growth factor and more than 35 other proteins with similar topological features. In the present review article, specific emphasis has been placed on advances with the glycoprotein hormones (GPHs) that have facilitated greater insight into their physiological functions, molecular structures and most importantly the basis of the molecular recognition events that lead to the formation of hetero-dimeric structures as well as their specific and selective recognition by their corresponding receptors and antibodies. Thus, this review article focuses on the structural motifs involved in receptor recognition and the current techniques available to identify these regions, including the role of immunological methodology, peptide fragment design and synthesis and mutagenesis to delineate their structure-function relationships and molecular recognition behaviour.
[Show abstract][Hide abstract] ABSTRACT: The beta-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 alpha-chain being shared with the other members of the glycoprotein hormone family but the beta-chain being unique to hCG. Nevertheless, there is sequence homology between the hCG beta-chain and the beta-chain of human luteinizing hormone (hLH) which results in cross-reactive antibodies being produced following immunization with wild-type hCGbeta. To reduce or eliminate such cross-reactions we generated a number of mutants of the hCGbeta-chain. One mutant (hCGbeta(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 BAChCGbeta(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 BAChCGbeta and with the native hCGalphabeta 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 beta-chain.
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