In previous experiments, the sperm-specific isozyme of lactate dehydrogenase (LDH-C) had been purified from mouse testes and shown to suppress the fertility of female baboons by 70% compared to controls. Although these results demonstrated the feasibility of this approach for contraceptive vaccine development, it is not practical to purify enough of the protein from natural sources for human use. Therefore, a need exists to develop a contraceptive vaccine based on synthetic peptides. In the current study, baboon LDH-C cDNA was amplified by the reverse transcriptase-polymerase chain reaction technique. The amino acid sequences of human and baboon LDH-C were 99.3% identical, indicating that the human LDH-C would be an effective antigen in nonhuman primates. The immunodominant epitope of human LDH-C was identified, synthesized, and conjugated to diphtheria toxoid (DT). This construct was used to immunize 15 female baboons; 15 control animals were immunized with DT alone. The fertility of the experimental group was reduced by 75% as compared to the controls (p < 0.02). One year after the last immunization, the contraceptive effect was completely eliminated (no statistical difference between the groups). These results show that a synthetic peptide based on the sequence of human LDH-C is effective in preventing pregnancy in nonhuman primates. The effect is completely reversed 1 yr after the last immunization. The contraceptive effect is not related to serum antibody titers, and human LDH-C is only slightly more effective than mouse LDH-C in female baboons.

Sera from patients with known or suspected immunological infertility were used to screen a human testis cDNA library. A total of 59 sera detected 38 unique cDNA inserts of which four were testis specific by Northern blot analyses. One of these is a testis-specific isoform of calpastatin. Five additional clones, although not testis specific, were found to be testis abundant. The number and type of clones identified by these human sera suggests a possible aetiology for immunologic infertility. The testis-specific clones will be further characterized to establish their usefulness as contraceptive vaccine candidates.

A panel of three synthetic peptides based on the 310-327 region of mouse LDH-C4 was used to examine the effect of peptide conformation on immunogenicity. The peptides, without prior conjugation to carrier molecules, were injected into outbred mice and the antisera were assayed for peptide- and LDH-C4-reactive antibodies by ELISA. An 18-residue random coil peptide (alpha-N) and an 18-residue amphipathic alpha-helix peptide (alpha-1) were weakly immunogenic. A conformationally stable 40-residue alpha-alpha fold peptide (alpha-3) was highly immunogenic. The antibodies elicited by alpha-3 reacted strongly with the native molecule by ELISA. Solution-phase binding assays were used to further characterize the specificity of the sera from two mice immunized with alpha-3. Antibodies from one of the mice appeared to recognize the helical portion of the peptides, while antibodies from the other mouse reacted only with the immunogen and may be specific for the non-natural beta-bend residues or possibly a topographic determinant peculiar to the anti-parallel helices. Serum from neither mouse was able to recognize the native molecule in solution. Peptides intended to mimic topographic determinants for the purpose of synthetic vaccine development may have to be more complex than those used in this study in order to induce high-affinity antibodies capable of exerting a significant biological effect.

A protein which binds the insect juvenile hormone has been isolated from the hemolymph of the fourth instar tobacco hornworm, Manduca sexta (Lepidoptera). Bioassay and chemical characterization of the bound ligand from the purified binding protein indicates that this molecule is the primary macromolecule responsible for juvenile hormone transport in the hemolymph of this insect. The juvenile hormone binding protein has been purified using gel filtration, ion exchange chromatography and preparative polyacrylamide gel electrophoresis. The protein is a single polypeptide chain of about 28,000 daltons with a sedimentation coefficient of 2.2S and an isoelectric point of 5.0. Binding analysis using ahydroxyapatite batch assay indicates that the juvenile hormone binding protein has one binding site with a Ka of 1.2 × 107 M−1 at 4°C.