M. Salahuddin

Immunogenicity of three C-terminal synthetic peptides of the beta subunit of human chorionic gonadotropin and properties of the antibodies raised against 45-amino acid C-terminal peptide

Immunological studies were carried out in rhesus monkeys and rabbits on three C-terminal synthetic peptides of beta-hCG (115-145; 111-145 and 101-145) after conjugating these to tetanus toxoid (TT). The immunogenicity of the peptide conjugates was comparatively poorer with reference to Pr-beta-hCG-TT conjugates at similar doses and immunization schedule. Amongst the three peptides, the best response was obtained with the 45-amino acid c-terminal peptide (45-CTP; 101-145). The anti-45-CTP recognized native hCG and was devoid of cross-reaction with hLH. hCG-induced testosterone production by mouse Leydig cells was inhibited by anti-45-CTP antiserum, although its neutralization capacity decreased more rapidly upon dilution than anti-beta-hCG sera of comparable titres. Immune complexes formed by the anti-45-CTP with hCG had a lower sedimentation value than those formed by anti-beta-hCG antisera with hCG, suggesting the presence of a limited number of immuno-determinant regions in the 45-amino acid C-terminal synthetic peptide.

Kinetics of antibody response in animals injected with processed beta-HCG conjugated to tetanus toxoid (Pr-β-HCG-TT)

Abstract Processed beta-subunit of HCG conjugated covalently to tetanus toxoid (Pr-β-HCG-TT) was found to be immunogenic in a number of species of animals tested. Single or multiple injections of the conjugate were required depending on the adjuvant used. The conjugate gives rise to the concomitant formation of antibodies against both HCG and tetanus toxoid. In rhesus monkeys, the antibodies appeared after a lag period of a few weeks, and increased progressively to attain plateau levels anywhere between 9 and 20 weeks. The duration of antibody response after primary immunization in most monkeys was beyond one year. The response was reversible and antibody titers declined to insignificantly low levels in course of time. A single repeat injection was adequate to evoke a powerful secondary immune response. The conjugate elicits the formation of antibodies with fairly high affinity constants for binding with HCG. Ka values ranged from 10 9 to 10 10 L/mole.

Immunological reactivity of antibodies produced by Pr-β-HCG-TT with different hormones

Abstract Antisera produced by injection of Pr-β-HCG-TT in rhesus monkeys ( Macaca mulatta ) and goat have been analysed for reactivity with various hormones. All antisera bound 125I-HCG. β-HCG competed with labelled HCG for binding to the sera in all cases. No cross-reactivity of human placental lactogen (HPL) and human growth hormone (HGH) was seen up to 1μg quantities per assay tube as gauged by competitive inhibition assays. Human follicle stimulating hormone (HFSH), human luteinizing hormone (HLH) and human thyroid stimulating hormone (HTSH) did not compete with 125I-HCG at the normal maximum physiological concentration of these hormones. The concentrations in radioimmunoassay system at which the competition appears with other hormones have also been determined.

Investigations on the ability of antisera produced by Pr-β-HCG-TT to neutralize the biological activity of HCG

Abstract The ability of antibodies generated by the vaccine Pr-β-HCG-TT in two species of animals to neutralize the biological activity of HCG has been investigated. The antisera from an immunized goat and serveral rhesus monkeys (Macaca mulatta) abolished the HCG induced rise in ventral prostate weight of immature male rats and of uterine weight in prepubertal mice. These were also found to be effective in preventing the binding of 125 I-HCG to receptors in rat testes and goat corpora lutea preparations. The antisera also blocked the HCG induced synthesis and secretion of progesterone in corpus luteum slices in vitro. These investigations show that the antibodies elicited by the conjugate (Pr-β-HCG-TT) form a complex with HCG, which is biologically inactive.

Clinical and immunological responses with Pr-β-hCG-TT vaccine

Abstract Six different batches of Pr-β-hCG-TT vaccine have been evaluated in 23 women for the antibody response. The anti-sera formed against these conjugates were capable of reacting immunologically with the whole hCG in radioimmunoassays and also neutralized the biological activity of hCG in radioreceptor assays. The antibody titres attained peak levels 4–6 months after the first injection. The peak titres could not be sustained and most of the subjects showed a spiky pattern. One subject was considered as non-responsive, two others had fairly low titres. Amongst the conjugates tested, batch 108 with SPLPS (sodium pthalate denatured with lipo-polysaccharides from S. entritides ) seemed more promising, but the addition of the adjuvant was found to be pyrogenic and unlikely to be accepted clinically.

Discriminatory effect of anti-Pr-β-hCG-TT antibodies on the neutralization of the biological activity of placental and pituitary gonadotropins

In 2 test systems, serum from monkeys and human subjects immunized with an anti-human chorionic gonadotropin (hCG) vaccine, Pr-beta-hCG-TT, were analyzed for their capacities to neutralize hCG/luteinizing hormone (LH)-induced biological effects. hCG-induced ovulation was completely blocked by the monkey antiserum in mice, but the same amount of antiserum, and even 2-fold greater concentrations, did not reduce the number of ovulating animals when primed with ovine LH. Competency of both the immunized monkey and human sera to neutralize the hCG-induced testosterone production in Leydig cells was shown. All monkey and 7/12 human sera did not interfere with the human LH action on Leydig cells. 5 human sera, however, showed varying degrees of inhibition of human LH-induced sterodogesnesis by this sensitive Leydig cell bioassay. Nevertheless, these subjects maintained regular menstrual cycles, and serum progesterone levels during the luteal phase were consistent with ovulation. Normal hormone profiles were constructed from a subject whose serum had shown a fairly high degree of cross-reaction with human LH by the Leydig cell bioassay when estradiol, human LH, and progesterone levels were determined on different days throughout the menstrual cycle.

Antibody response to Pr-β-HCG-TT vaccine in human subjects

Abstract The ability of Pr-β-HCG-TT vaccine to elicit anti-HCG antibodies have been investigated in four human subjects who were in reproductive age and of proven fertility. All subjects responsed positively by formation of anti-HCG and anti-tetanus toxoid antibodies. The anti-HCG antibodies were detectable after a lag period of 6 to 8 weeks and plateau levels were attained at about five months after primary immunization with the vaccine. The anti-HCG antibody titers though declining were still high in three subjects after nearly 11 months of immunization. In one of the subjects the titers declined to near zero levels after 16 months indicating the reversibility of antibody response with this vaccine. The antibodies reacted immunologically with the whole HCG molecule and were competent to neutralize the biological activity of HCG in radioligand receptor assay.

Differential affinity of anti-Pr-β-hCG-TT antibodies for hCG and hLH

Sera from four women immunized with the vaccine Pr-β-hCG-TT have been analysed for binding with hCG and hLH. Resolution of Scatchard plots showed the presence of more than one population of antibodies in these sera. In each case the Association Constants (Ka) of a population of antibodies for binding with hCG were distinctly higher than those for hLH. Results indicate the likely presence in β-hCG of determinants and/or conformations immunologically unique to hCG besides common regions.

Influence of HCG and tetanus toxoid injections on the antibody titers in a subject immunized with Pr-β-HCG-TT

N.D., a subject immunized 11 months earlier with Pr-β-HCG-TT in whom the anti-HCG titers were on the decline, was injected with 2,000 IU and 4,000 IU of HCG intramuscularly at 24-hour intervals. The anti-HCG titers in blood fell in response to HCG administration but remained above zero. No evidence for the presence of free HCG was found in circulation. HCG administration did not alter the anti-tetanus toxoid titers. The anti-HCG titers in the blood sample drawn 21 days after the load test showed a return to nearly about the initial values. No clear cut booster effect of HCG injection on anti-HCG titers was noted. On the other hand injection of tetanus toxoid vaccine increased the antitetanus toxoid antibodies in the subject.

Processing of the preparations of β-subunit of human chorionic gonadotropin for minimization of cross-reactivity with human luteinizing hormone

The immunological reactivity of various components of beta-human cho rionic gonadotropin (beta-HCG) preparations with antisera developed against HCG and human luteinizing hormone (HLH) was assessed. Anti-beta-HCG reactivity was primarily located in 2 or 3 adjacent fractions. Anti-HLH reactivity was partly found in a fraction co-migrating with 1 of the anti-beta-HCG fractions, and also in a fraction migrating more slowly than the anti-beta-HCG fractions. Preparations with no, or minimal, cross-reactivity with anti-HLH were obtained upon absorption of beta-HCG with anti-ovine LH.