Comparison of Corynebacterium parvum and Bordetella pertussis with Freund's complete adjuvant as immunopotentiators for beta-human chorionic gonadotropin linked to an atoxic fragment of tetanus toxin.

Corynebacterium parvum and Bordetella pertussis were compared with Freund's complete adjuvant (FCA) for their abilities to potentiate the immune response to haptenic beta-human chorionic gonadotropin covalently coupled to an atoxic 54,000-molecular-weight fragment of tetanus toxin (beta-hCG-TTII). The ability of each adjuvant to enhance production of antibodies to hCG in rabbits was measured by 125I-hCG radioimmunoassay. At sera dilutions of 1:10,000, analysis of variance for the 8-week postimmunization course showed that the mean 125I-hCG binding capacities of the C. parvum group was significantly greater overall than the B. pertussis group (P = .0002) and that the FCA-treated group had the greatest binding capacity overall (P less than .018). The mean binding capacities at 1:40,000 dilution again showed the FCA-treated group to have significantly higher anti-hCG titers overall (P less than .0015), with C. parvum potentiating a greater overall antibody response than B. pertussis (P = .001). These results indicate that FCA is the most efficacious of the three tested adjuvants in potentiating antibody production to the hapten component of beta-hCG-TTII. C. parvum was also effective at promoting an anti-beta-hCG response, although not to the same degree as FCA. B. pertussis had only minimal potentiating effect compared to FCA or C. parvum.

A candidate carrier protein for beta-human chorionic gonadotropin: 54,000-molecular-weight fragment of tetanus toxin.

As an alternative to intact tetanus toxoid as a carrier for beta-human chorionic gonadotropin (beta-hCG), a fragment of tetanus toxin was sought that had a relatively low molecular weight, yet was highly immunogenic. Purified culture filtrate tetanus toxin was subjected to limited enzymatic digestion with papain, and the resulting fragments separated by column chromatography on Sephadex G-150. Four fractions were thus identified. Fraction II was found to have a molecular weight of 54,000 by SDS-polyacrylamide gel electrophoresis. This fragment was covalently linked to the beta-subunit of hCG (beta-hCG-TTII) using carbodiimide hydrochloride. The ability of beta-hCG-TTII to stimulate production of anti-hCG sera in rabbits was measured by 125I-hCG radioimmunoassay. At sera dilutions of 1:40,000, an average 125I-hCG binding capacity of 34.7 +/- 5.86% (mean +/- SD) was observed 8 weeks after the final immunization. Tetanus toxin Fragment II has the potential for future application in active immunization studies involving hormone-carrier conjugates.

Comparison of Corynebacterium parvum with Freund's complete adjuvant as a potentiator of the immune response to beta-human chorionic gonadotropin linked to tetanus toxoid.

Corynebacterium parvum was compared with Freund's complete adjuvant (FCA) for potentiation of the rabbit immune response to beta-human chorionic gonadotropin linked to tetanus toxoid (beta-hCG-TT). With each adjuvant, antibodies to hCG were detected using passive hemagglutination. Higher antibody titers were produced by FCA-treated animals. The ability of antisera to beta-hCG-TT to neutralize the biological action of native hCG was determined by the rat uterine weight assay. Anti-beta-hCG-TT sera from C. parvum-treated rabbits were not significantly different (p greater than 0.10) from FCA potentiated anti-beta-hCG-TT sera in neutralizing hCG-induced uterine weight gain. C. parvum has potential for future application in active immunization studies of fertility regulation.

Effect of carrier preimmunization on neutralizing antibody response of rabbits immunized with beta-human chorionic gonadotropin coupled to tetanus toxoid.

Antisera to the conjugate of beta-human chorionic gonadotropin and tetanus toxoid (beta hCG-TT) generated in rabbits receiving TT carrier preimmunization were characterized and compared with antisera from rabbits receiving the conjugate without preimmunization. Double immunodiffusion and immunoelectrophoresis were used to identify individual antisera components to the conjugate and its substructures. 125IhCG radioimmunoassay indicated similar antibody titers (P = 0.447) regardless of whether the rabbits were preimmunized with TT. However, bioneutralization of native hCG as measured by inhibition of prepubertal rat uterine weight increase was significantly more effective (P = 0.025) with antisera from rabbits which received carrier presensitization. These results demonstrate that carrier preimmunization provides a method for increasing the efficacy of neutralizing antibody produced against haptenic beta hCG.

The effectiveness in rhesus monkeys of an antifertility vaccine based on neutralization of chorionic gonadotropin.

PIP: Rhesus monkeys were immunized against the beta subunit of sheep luteinizing hormone (LH; oLHbeta) as an animal model for immunity against chorionic gonadotropin for fertility control. oLHbeta has been demonstrated to cross-react with rhesus chorionic gonadotropin (rhCG) but not rhesus LH. 13 multiparous monkeys received 50 mcg oLHbeta in complete Freund's reagent at 4 sc sites, repeated in 3 weeks and occasionally afterward. All developed circulating antibodies that bound hCG and oLH. RhCG was assayed in a competitive ligand binding assay using receptors from rat testis. Monkey antiserum bound hCG, 12.5% of which was displaced by 100 mcg rhLH. Antisera from 2 monkeys neutralized rhCG bioassayed in the immature rat uterine weight assay in a dose-related manner. After 2 immunizations 11 of the 13 macaques has regulat ovulatory menstrual cycles. 10 of them were mated 28 times, 1-5 each, at ovulation. There was only 1 pregnancy after 1 year in a monkey whose antibody titer had declined. In comparison, 5 of 10 controls, mated once each at ovulation, became pregnant. The mechanism by which immunization prevented pregnancy is unknown. Furthermore, use of a heterologous antigen in lieu of rhCG whose structure is unknown, may have minimized possible adverse effects.^ieng

Biologic effects following active immunization with delta 5-3 beta-hydroxysteroid dehydrogenase.

Immunization of female rats with crude preparations of delta 5-3 beta-hydroxysteroid dehydrogenase combined with complete Freund's adjuvant resulted in suppression of the endogenous enzyme activity in the ovarian interstitial tissue and the corpora lutea. As a result, estrous cyclicity, nidation, or maintenance of pregnancy was adversely affected in proportion to the level of the antibody to the enzyme following the series of immunizations. On the basis of the biologic response, it can be assumed that progesterone synthesis was reduced as a result of neutralization of enzyme action in the ovary by the formation of antibodies to the delta 5-3 beta-hydroxysteroid dehydrogenase. Further studies are required to verify this assumption.

Impairment of spermatogenesis and libido through antibodies to lutenizing hormone.

PIP: To histologically and biologically assess the impairment of testicular function after active immunization with LH, adult male rats (49) and rabbits (9) were actively immunized with bovine LH and then compared with saline-injected controls. When the measured antibody titer was high, the mating behavior of the males was altered. The males mated only 41.8% of the time when exposed to cycling females. The control group mated 90% of the time. Testicular weights were unchanged but the seminiferous tubules of the experimental group showed vacuolation in the Sertoli cells with some of the spermatids being multinucleated. Passive immunization of rats with anti-LH sera diminished mating capacity to a greater extent than active immunization. Immunization of rabbits led to a rapid decrease in libido. Uniform interruption of spermatogenesis was noted along with a loss of testicular weight of immunized rabbits. The probable mechanism of the LH inhibition of spermatogenesis is the interference with androgen production in the interstitial tissue.^ieng