Comparison of Immunospecificity and Immunogenicity of three prototypes of anti-hCG vaccines based upon the recombinant hCG β-chain

ავტორი: ნუნუ მიცკევიჩი
თანაავტორები: N. Gachechilaze, N.Chikadze
საკვანძო სიტყვები: Vaccines, Immunospecificity, Immunogenicity, Human chorionic gonadotropin (hCG).
ანოტაცია:

Introduction: Human chorionic gonadotropin (hCG) is a placental member of the glycoprotein hormone (GPH) family which also includes the pituitary hormones: follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH) (1). They are heterodimers consisting of a common alpha and a hormone-specific beta subunit. The β-subunits are partly homologous to each other. The closest homology is between hCGβ and hLHβ (2). hCG is a pregnancy hormone secreted by the pre-implantation blastocyst and, subsequently, by the trophoblast (3). Since it is responsible for the maintenance of the corpus luteum in early pregnancy, blocking the action of this hormone with antibodies prevents pregnancy. Thus, vaccines based on hCG can be used for immunocontraception (4-6). Later it has been shown that the levels of serum hCG strongly correlate with trophoblastic germ cell tumors, and it was used for diagnosis and monitoring tumor burden and for evaluating the effectiveness of therapeutic intervention (7,8). Elevated serum hCGβ levels and/or tissue expression were since used as an independent predictor of an unfavorable disease outcome and were associated with a more aggressive disease course in renal, colorectal, bladder, and pancreatic cancers (9, 10) It was proposed that hCG may act at different levels to facilitate cancer progression: as a transforming growth factor, an immunosuppressive agent, an inducer of metastasis, and/or an angiogenic factor (11). Therefore, the hCG-based vaccines are been tested in anti-tumor therapy (11,12). Vaccines, which incorporate the full-length hCGβ-chain, stimulate the production of antibodies that cross-react with hLH due to the 85% amino acid sequence homology of the first 110 amino acids of the β-chains of the two hormones. As a strategy aimed at reducing hLH cross-reactivity with hCG several hCGβ-chain mutants were engineered by using oligonucleotide-directed site-specific mutagenesis (12,13). One of the mutant molecules hCGβ(R68E) containing an arginine to glutamic acid replacement at a position 68 in the protein sequence has grossly diminished ability to provoke hLH cross-reactive antibodies whether administered intranasally to mice or intramusculary to rabbits (12,13). Much of anti-hCGβ(R68E) antibodies were re-focused towards normally weakly immunogenic C-terminal peptide (CTP) unique for hCGβ and not shared by hLH. In the mutant hCG molecule CTP acquires the properties of an immunodominant epitope (13, 14). However a vaccine preparation based on the mutant hCGβ(R68E) has not been finalised regarding an optimal carrier molecule and an adjuvant. The aim of this study was to assess immunogenicity of the sera elicited in response to hCGβ(R68E) conjugated to heat shock protein 70 ( HSP 70) or to keyhole limpet hemocyanin (KLH) compared to the hCGβ(R68E) alone. Results and Discussion: In order to study specificity and immunogenicity of the immune sera the titers of anti-hCG, anti-hCGβ and anti-CTP were determined in mice immunized with recombinant protein hCGβ(R68E) conjugated with HSP70 and KLH as carrier molecules, compared to the immunogen hCGβ(R68E) alone. All sera showed appreciable levels of antibodies to the protein studied compared to the control ovalbumine protein. Immunization with hCGβ(R68E)-HSP70 raised equally high titers of antibodies to the native hCGαβ heterodimer, native hCGβ-chain and CTP. The 50% titer of these antibodies was defined as 1:800, whilst the end point titer was detected as 1:12800, indicating a high concentration of the respective antibodies (Figure a). However antibodies raised in response to hCGβ(R68E)-KLH and to hCGβ(R68E) alone (Figure b and c) reacted strongly with CTP (50% titer 1:400 and end-point titer 1:6400), but not with the native hCGαβ or hCGβ (50% of titer 1:200, end point titer-1:3200).Sera from mice immunized with a) hCGβ(R68E)-HSP70 (n=7), b) hCGβ(R68E)-KLH(n=7) and c) hCGβ(R68E) (n=7) were analyzed using direct ELISA as described in Materials and Methods, plates coated with proteins as indicated. The sera was diluted in PBS-T-BSA at 1:50 - 1:25,600 and the results were expressed as optical density, M±SD. Detection of the high titers of the anti-CTP antibodies in experimental sera was expected (Figure 2a,b,c) We have previously shown that a single arginin68 to glutamic acid substitution dramatically alters the antigenicity and immunogenicity of the mutant molecule by re-focusing it to the CTP region of the β-chain (13,14). This makes HSP70 the most efficient carrier for mutant hCGβ(R68E) molecule for a proposed vaccine formulation. The fact that the immunization with this conjugate induced equally strong anti-CTP and anti-native hCGαβ/hCGβ immune responses means that such sera in vivo would bind with high affinity to the endogenous hormone and its beta subunit. This makes hCGβ(R68E)-HSP70 conjugate a good potential candidate for the development of hCG-based vaccine, particularly that both hCG holohormone and its beta subunit have been identified as tumor growth factors (8, 11). Recently we have demonstrated that the immune complexes formed by anti-hCGβ(R68E) rabbit sera and native hCG are successfully phagocytosed in vitro by monocytes and neutrophils, indicating a good clearance capacity in vivo. In conclusion, this study demonstrated that the HSP70 increases immunogenisity of the mutant molecule hCGβ(R68E) since antibodies generated against hCGβ(R68E)-HSP70 conjugate bind with high affinity to the CTP region of hCG, as well as to the native holohormone hCGαβ and its hCGβ subunit. Thus, hCGβ(R68E)-HSP70 is an effective prototype of hCG-based vaccine characterized by a high degree of immunogenicity. References: 1. Stenman UH., Tiitinen A., Alfthan H. , Valmu L. The classification, functions and clinical use of different isoforms of hCG. Human Reproduction Update 2006; 12(6), p.769-784. 2. Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ & Isaacs NW Crystal structure of human chorionic gonadotropin. 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