Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

Does hCG or hCGß play a role in cancer cell biology?

The role that hCG might play in the oncogenic process in cancer is certainly complex. We know that the expression of hCG and its beta subunit is a widespread phenomenon which has been described in many cancer subtypes. However, hCG's involvement in breast cancer has been antithetical: the detection of ectopically expressed hCG(ß) by breast tumors has been employed as a biomarker of malignancy, and hCG has been proposed as a ligand vehicle for toxic drugs, with the aim of targeting the LH/hCG receptor which is reported to be expressed by malignant breast tissue. However, it has also been proposed that hCG is a protective agent against the development of breast cancer, leading some to advocate hCG administration to non-pregnant women as a prophylactic measure against cancer. Nevertheless, suggestions that hCG is involved in the angiogenesis, metastasis and immune escape that are central to cancer progression - are phenomena which clearly apply to breast cancer. Indeed, a tumor vaccine based upon hCG has very recently been shown to protect against mammary tumors in mice. We propose that this apparent paradox is resolved if the free beta subunit of hCG produced by tumors acts as an autocrine anti-apoptotic and angiogenic growth factor, whilst intact heterodimeric hCG, as in pregnancy, is part of developmental signaling that initiates tissue differentiation (including breast ductal tissue development), and hence reduces the population of stem-like cells which are susceptible to oncogenic factors.

Designing a new generation of anti-hCG vaccines for cancer therapy.

The heterodimeric 'pregnancy-specific' hormone human chorionic gonadotropin (hCG) has been used as the basis for a contraceptive vaccine. More recently, the observation that hCG, particularly in the form of the beta-chain expressed in the absence of alpha-chain, is aberrantly expressed in a number of different tumors has opened up a second potential application for such vaccines. Drawbacks of the currently available vaccines are that they are either relatively weakly immunogenic or that they induce antibodies that cross-react with human leuteinizing hormone (hLH). We have explored the possibility of creating mutated versions of the hCG beta-chain with improved immunologic properties.

Vaccines for the control of reproduction--status in mammals, and aspects of comparative interest.

The objective of producing vaccines which target elements of the reproductive system to control fertility has been pursued for many years. Of the many targets for such vaccines, several sperm-associated antigens have been proposed for antibody-mediated intervention before fertilization but the very abundance of antigen to be neutralized has been a barrier. Zona pellucida antigens associated with the surface of the oocyte have also been targeted and used successfully for control of 'wild' elephant populations but worries concerning immunopathologically-mediated tissue damage have been mooted. Vaccines using human chorionic gonadotropin (hCG) which is required for the implantation and maintenance of the fertilized egg, although of interest for the development of fertility control in human populations, has no relevance in the context of the present conference because external fertilization of fish eggs is independent. The pathways by which gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus promote release of luteinizing (LH) and follicle-stimulating hormone (FSH) which govern the physiological maturation and maintenance of the reproductive organs, provide many targets for immunological intervention. Most consistent success has been reported using GnRH-based vaccines which are immunosterilizing in a variety of mammalian species such as pigs, rodents and white-tailed deer. The fact that the structure of the decapeptide, GnRH, has been maintained over so many years of evolution and been conserved across so many animal species, encourages the view that a strategy for control of sexual maturation in fish based upon stimulation of GnRH antibodies may well prove to be a practical proposition, provided the formulation of an appropriate highly immunogenic vaccine can be achieved.

Changes in the reproductive system of male mice immunized with a GnRH-analogue conjugated to mycobacterial hsp70.

Immunosterilization is an attractive alternative to surgical castration. Gonadotropin-releasing hormone (GnRH) controls the production of the gonadotropins thereby having an orchestrating effect on the reproductive hormone cascade and spermatogenesis. Induction of neutralizing antibody can abrogate the effect of the hormone. Current GnRH-based vaccines often require strong adjuvants and/or multiple injections of the vaccines to overcome variability in the response. Heat shock proteins (hsp) have been used as carrier molecules because of their powerful intrinsic ability to enhance an immune response to associated antigens. A GnRH-analogue, GnRH-d6-Lys, was conjugated to recombinant Mycobacterium tuberculosis hsp70. Male BALB/c mice were immunized i.p. with GnRH-hsp70 in the mild adjuvant Ribi or in incomplete Freund's adjuvant (IFA). The initial immunizations were done on pre-pubertal 3-week-old mice, with boosts at 5 and 8 weeks of age. The mice were killed at 10 weeks of age and GnRH-specific antibodies and serum testosterone levels measured. All the immunized mice mounted GnRH-specific antibody responses, with no difference in the mice immunized with GnRH-hsp70/Ribi or with GnRH-hsp70/IFA. There was substantial atrophy of the urogenital complex and significantly (P < 0.0005) reduced levels of testosterone-dependent testicular relaxin-like factor mRNA expression. Mice immunized with GnRH-hsp70/Ribi showed substantially reduced (P < 0.001) serum testosterone levels. These results indicate that hsp70 may serve as a particularly advantageous carrier for GnRH-based vaccines.

The major hormone-specific discontinuous epitopes on human chorionic gonadotrophin.

The hormone human chorionic gonadotrophin (hCG) shows extensive sequence homology with LH. Thus, many of the antigenic epitopes on hCG are shared with LH, leading to immunological cross-reaction between these two hormones. Anti-fertility and anti-cancer vaccines based upon hCG should ideally target only the hCG-specific epitopes. The hCG-unique linear epitopes located in the C-terminal peptide of the hCG beta-chain are well characterised. In contrast, the hCG-specific discontinuous epitopes, termed beta1, beta6 and beta7, have remained poorly defined. By generating hCG beta-chain molecules containing single amino acid substitutions we have identified R10, N13, R60 and Q89 as being important in the formation of the beta1 epitope, with R60 providing a particularly dominant residue. We also show that the amino acid residue Q89 contributes to the beta7 epitope, whilst D61 plays a role in both the beta6 and beta7 epitopes.

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells.

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.

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) beta-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 hCGbeta, a mutated hCGbeta-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 hCGbeta 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.

A low budget luminometer for sensitive chemiluminescent immunoassays.

We have designed a simple luminometer based on a reasonably priced Peltier-cooled charge-coupled device (CCD) camera, housed in a light-tight box, with straightforward lens imaging and a simple platform for a microtitre or other assay format. The quantitative readout of the CCD image is recorded on a PC using customised software. The instrument can be assembled in a standard university workshop for under pound3000, compared with the cheapest commercial instruments retailing at pound10,000 and above. Consistent with the general view on chemiluminescent assays, the sensitivity is 10-100 times greater than that obtained with parallel ELISA's using a chromogenic substrate. A unique feature of the CCD format is that it enables assays to be carried out on arrays of minidots and even nanodots of antigen on the floor of each microtitre well. This permits direct comparison and standardisation of reactivity of a single sample against several antigens and economy in the use of reagents, test sample and technician time; finger-prick samples of blood can be analysed. The instrument should have widespread applicability in developing countries and, indeed, in any laboratories with hard-pressed budgets.

The effect on immunoglobulin glycosylation of altering in vivo production of immunoglobulin G.

The effect on murine immunoglobulin G (IgG) glycosylation of altering IgG production in vivo was assessed in interleukin (IL)-6 transgenic and CD4 knockout mice. C57BL/6 mice carrying the IL-6 transgene showed increased levels of circulating IgG. This was associated with decreased levels of galactose on the IgG oligosaccharides. No decrease in beta4-galactosyltransferase mRNA or in enzyme activity was seen in IL-6 transgenic mice. MRL-lpr/lpr mice normally have elevated levels of circulating IgG, again accompanied by decreased levels of IgG galactose. Disruption of the CD4 gene in MRL-lpr/lpr mice led to a substantial decrease in the concentration of circulating IgG, but IgG galactose levels remained low. Thus, an enforced decrease in IgG levels in the lymphoproliferative MRL-lpr/lpr mice did not alter the percentage of agalactosyl IgG in these mice, suggesting that agalactosyl IgG production is not simply caused by excessive IgG synthesis leading to an insufficient transit time in the trans-Golgi, but rather to a molecular defect in the interaction between galactosyltransferase and the immunoglobulin heavy chain.

DNA vaccination favours memory rather than effector B cell responses.

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.

Dual specificity antibodies using a double-stranded oligonucleotide bridge.

The covalent conjugation of oligonucleotides to antibody Fab' fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics.

The effect on IgG glycosylation of altering beta1, 4-galactosyltransferase-1 activity in B cells.

An absence of galactose on the N-linked oligosaccharides of immunoglobulin G (IgG) has been shown to affect the functional activity of the antibody molecule. In patients with rheumatoid arthritis there is an increased proportion of IgG which lacks galactose and correspondingly lower levels of beta1, 4-galactosyltransferase (beta4Gal-T) activity. The recent demonstration of several expressed beta4Gal-T genes in man raises the possibility that the enzyme responsible for the decreased IgG galactose is not the "classical" beta4Gal-T (beta4Gal-T1). To directly address the question of whether reduced beta4Gal-T1 would lead to reduced IgG galactose, the level of beta4Gal-T1 in a human IgG-secreting B cell line was specifically altered using stable transfection with sense (SpcDNA3-Gal-T1) or antisense (ASpcDNA3-Gal-T1) human beta4Gal-T1 cDNA. SpcDNA3-Gal-T1 B cell transfectants expressed up to a 2.5-fold higher level of beta4Gal-T enzyme activity for the exogenous neoglycoconjugate acceptor GlcNAc-pITC-BSA than did ASpcDNA3-Gal-T1 transfectants. Flow cytometric analysis with Ricinus communis agglutinin I (RCAI) revealed an overall greater number of Galbeta1,4GlcNAc structures in the fixed and permeabilized SpcDNA3-Gal-T1 B cell transfectants compared with the ASpcDNA3-Gal-T1 transfectants. Moreover, there was increased galactosylation of IgG secreted from the SpcDNA3-Gal-T1 transfectants relative to the ASpcDNA3-Gal-T1 B cell transfectants. Alteration of the level of the "classical" beta4Gal-T (beta4Gal-T1) in B cells therefore affects IgG glycosylation.

Antibody recognition of epitopes on wild-type and mutant beta-(1-->4)-galactosyltransferase-1.

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.

The effect of low-profile serine substitutions in the V3 loop of HIV-1 gp120 IIIB/LAI on the immunogenicity of the envelope protein.

Many microbial antigens contain powerful hypervariable epitopes that fail to induce broadly protective immunity because they dominate the immune response at the expense of more conserved but weaker epitopes. If the undesired B cell epitopes are eliminated, the immune system could be focused on the conserved epitopes and produce a stronger antibody response to conserved parts of the protein and thus become a more efficacious immunogen for a vaccine. We examined this possibility using the human immunodeficiency virus envelope glycoprotein (gp)120 IIIB/LAI and selectively replaced the amino acids from the V3 region and analyzed the overall immunogenicity of the mutant proteins after nucleic acid immunization in mice. The most variable residues of the human immunodeficiency virus type 1 gp120 V3 loop sequence were replaced with serine, which has a small uncharged hydrophilic side chain and therefore is likely to be less immunogenic than amino acids found in wildtype V3 sequences. The serine substitutions did not affect the ability of soluble CD4 to bind the mutant molecules compared with wildtype gp120 and monoclonal antibodies against both linear and discontinuous epitopes located in the V1/V2, C1, and C4 regions of the molecule. These data suggest that the V3 loop substitutions did not grossly affect the overall conformation of the envelope molecule. Immunization of CBA x BALB/c F1 mice with DNA expression plasmids for the wild-type gp120 sequence induced a predominantly IgGI antibody response with end point titers of 10(4)-5 x 10(4). The antibodies reacted only with conformationally intact gp120. Serine replacements targeted to both sides of the V3 loop had a major impact on gp120 immunogenicity, with a markedly reduced response in the majority of animals tested. Analysis of the epitope specificity of the responses suggests that N-terminal amino acids in the V3 loop contribute to the major immunodominant epitope and provides no evidence that their removal enhances immunogenicity of the conserved regions.

Epitopes of human chorionic gonadotropin and their relationship to immunogenicity and cross-reactivity of beta-chain mutants.

PROBLEM: 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. METHOD OF STUDY: 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. RESULTS: 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). CONCLUSIONS: 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.

Immunological analysis of epitopes on hCG.

The heterodimeric glycoprotein hormone, human chorionic gonadotropin, has been extensively characterized in terms of its recognition by mouse monoclonal antibodies. A number of different approaches have led to the definition of several epitope clusters on the surface of the molecule. These include epitopes located solely on the alpha- or beta-chain, some of which are masked when the two chains associate to form the holo-hormone. Additional epitopes comprise amino acids contributed by both the chains. In contrast to the extensive knowledge regarding B cell epitopes, the characterization of T cell epitopes on hCG has only recently begun to be explored.

The role of glycosylation in autoimmune disease.

Oligosaccharide structures play a key role in the antigenicity of a number of clinically important antigens such as blood group determinants. Interest in glycobiology has increased dramatically amongst immunologists during the last few years due to the fact that oligosaccharides also play a central role in adhesion and homing events during inflammatory processes (1), comprise powerful xenotransplantation antigens (2), and may provide targets for tumor immunotherapy (3). Additionally, alterations in glycosylation are now known to occur in a number of autoimmune diseases. This review will first discuss some general aspects of protein glycosylation and then explore some of the autoimmune diseases in which the role of glycosylation has been examined.