The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein.

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freund's adjuvant. A single booster immunisation in incomplete Freund's adjuvant was administered 3-5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1-3-month intervals for the next 33 months. Forty-three koalas were recaptured at least three times allowing reliable assessments of their fertility. Fourteen animals were observed never to have a pouch young. Of the remaining 29 animals the reproductive productivity of PZP treated females was reduced compared with control and recBP-ZP3 treated females, in terms of both total number of young produced, and failure to produce further young in females of proven fertility. One month after the initial immunisation, serum antigen-specific antibody titres were higher in animals immunised with PZP or recBP-ZP3 compared to controls, and reached a plateau by 4 months. Antibody against the relevant immunising antigen was also detected in ovarian follicular fluid, uterine fluid and vaginal secretions. Epitope analysis suggested that immune responses other than antibodies directed against the ZP3 amino acid sequence were responsible for mediating infertility. The results demonstrate that the fertility of female koalas can be compromised by immunisation against zona pellucida antigens. However, unlike in the eastern grey kangaroo and the brushtail possum, immunisation with bacterial recombinant brushtail possum ZP3 did not compromise fertility in the koala.

Immunocontraception of Eastern Grey kangaroos (Macropus giganteus) with recombinant brushtail possum (Trichosurus vulpecula) ZP3 protein.

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 microg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos (n=7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

Immunisation of the male tammar wallaby (Macropus eugenii) with spermatozoa elicits epididymal antigen-specific antibody secretion and compromised fertilisation rate.

Immunocontraception has been proposed as an effective and humane means of controlling overabundant kangaroo populations in Australia. We have examined the feasibility of using a sperm-based vaccine for this purpose using a model macropod species, the tammar wallaby (Macropus eugenii). This study has demonstrated immunocontraception in a marsupial species following immunisation of males with homologous spermatozoa. Serum anti-sperm IgG titres were associated with a significant reduction in fertilisation rates following mating with superovulated female wallabies. Antigen-specific IgG penetrated the reproductive tract at the rete testis and bound spermatozoa in vivo. IgG was detected bound to the acrosome and midpiece regions of both epididymal and ejaculated spermatozoa. The absence of adverse testicular pathology and sperm movement effects suggests that contraception may have been achieved by antibody-mediated blocking of sperm surface antigens essential for fertilisation. This study demonstrates that a contraceptive vaccine targeting sperm antigens has potential for fertility control in male macropods.