David W. Silversides

A synthetic luteinizing hormone releasing hormone vaccine. I. Conjugation and specificity trials in BALB/c mice.

The immunobiology of luteinizing hormone releasing hormone (LHRH) was explored, to provide a conceptual and practical basis for the use of LHRH in immunocastration. Cysteine substituted analogues of LHRH were synthesized including Cys1-LHRH (C1-LHRH), Cys6-LHRH (C6-LHRH) and Cys10-LHRH (C10-LHRH). These were reacted to carrier molecules using the heterobifunctional cross-linking reagent m-maleimidobenzoylsulfosuccinimide ester (SMBS), producing peptide-carrier conjugates of known peptide content and conjugation orientation. This reaction regime was found to be rapid, efficient and allowed for easy control of peptide to carrier ratios. Conjugates were used in active immunization trials in BALB/c mice to characterize the murine immune response against LHRH. BALB/c mice were shown to have the capacity to recognise all three cysteine substituted LHRH analogues and to produce antibodies cross-reactive with native LHRH. The specificity of LHRH antisera generated was found to be dependent on the site of conjugation of the peptide to carrier molecule. C1-LHRH generated carboxy terminal directed antibodies, C10-LHRH generated amino terminal directed antibodies, while C6-LHRH could generate amino terminal directed or carboxy terminal directed antibodies, or both within a given animal. No intrinsically immunodominant epitopes were seen within the LHRH molecule.

Monoclonal antibodies against LHRH: development and immunoactivity in vivo and in vitro

Mouse myeloma NS1-Ag4 cells were fused with spleen cells from a BALB/c mouse previously immunized with luteinizing hormone releasing hormone (LHRH) conjugated to serum bovine albumin (BSA). Fused cells were grown in HAT restrictive medium which was screened for LHRH binding ability via a primary binding assay employing [125I]LHRH and cold ethanol precipitation. One clone (hy-USASK/DSIL-LHRH-A1) was selected for further study. Cell culture fluid and ascites fluid bound 30% of [125I]LHRH at 1:4000 and 1:400,000 dilution respectively. A competitive inhibition assay using ascites fluid at 1:2,000,000 dilution and LHRH standards at 0.125-32.0 ng/ml was established. Initial studies using rabbit anti-mouse allotype sera in a horseradish peroxidase (HRP)-ELISA system indicate the antibody is IgG1. A dose of 0.5 ml ascites fluid containing LHRH antibody given intravenously (i.v.) on day 9 of gestation was effective in terminating pregnancy in rats. A 1 cm progesterone implant made of elastomer polymer and placed interperitoneally blocked this effect. Ascites fluid (4.5 ml) containing LHRH antibody, when infused i.v. into mature spayed female dogs induced a precipitous decline in mean luteinizing hormone (LH) levels and reduced LH pulsatility over 4 days. It was concluded that the mouse monoclonal antibody is specific for LHRH, and can interrupt reproductive events in vivo.

A synthetic luteinizing hormone releasing hormone vaccine II. Temporal aspects of titer development and formulation trials in BALB/c mice

Cysteine substituted analogues of luteinizing hormone releasing hormone (LHRH) were coupled to carrier molecules, and the resulting conjugates used to characterize the immune response to native LHRH generated in BALB/c mice and to formulate vaccines in an effort to maximize titer development. In an active immunization trial designed to characterize temporal aspects of anti-LHRH titer development, titers could be detected 1 week after initial immunization. No differences were observed in response between male and female mice. Booster immunizations could enhance the titers against LHRH. The titers developed against the carrier molecule were uniformly higher than the corresponding anti-LHRH titers throughout the course of the trial. In separate trials, vaccines were formulated and tested in BALB/c mice for titer development against LHRH. Adjuvants, carrier molecules, dosage and peptide to carrier ratio were considered. Dosages of 50 micrograms conjugate per immunization per mouse, at conjugation ratios of 3-12 peptides per 10(5) Da carrier molecule, were found to produce immune responses. Adjuvants including Havlogen and dimethyldioctadecylammonium bromide (DDA), and carrier molecules including keyhole limpet hemocyanin (KLH), porcine thyroglobulin (TGB) and equine gamma globulin (EGG) were all found to be effective.