Identification of New Antigen Targets & the Molecular Dissection of Antigenicity for Possum Immunocontraception

M.S.Harris2, X.Cui3, S.Jones3, C. McCartney2, M.G.O'Rand1

1Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC, USA 27599

Cooperative Research Centre for the Conservation and Management of Marsupials, 2Department of Biological Sciences, Macquarie University, NSW 2109, Australia.

3Landcare Research, PO. Box 69, Lincoln 8152.

Introduction

Immunological interference in reproduction, or immunocontraception, may provide a humane, cost-effective and long-term solution for possum management when used in combination with conventional lethal control methods (Bayliss 1999, Ramsey et al., this proceedings). Ideally antibodies elicited by an immunocontraceptive vaccine would not only block gamete production, fertilisation or embryonic development, but would also be species specific and affect both male and female possums. This paper summarises the progress of the Marsupial CRC and Landcare Research toward identifying sperm antigen targets and dissecting the immunogenic regions of the possum zona pellucida proteins that mediate infertility.

Zona Pellucida Antigens

Mammalian eggs are surrounded by an extracellular coat, termed the zona pellucida (ZP), that has vital roles in the fertilisation process and early embryonic development. Immunisation with whole zona pellucida or its component proteins reduce the fertility of a variety of species, thus the ZP proteins have become the primary focus of immunocontraceptive vaccines for wildlife control (Prasad et al., 1996). The possum zona pellucida is comprised of three proteins: ZP1, ZP2 and ZP3. Whole porcine zona pellucida and possum recombinant ZP2 and ZP3 reduce the fertility of female possums (Duckworth et al., this proceedings). As some regions of the zona pellucida proteins are highly conserved between species ongoing experiments are focussing on dissecting the possum immune response to the ZP proteins to identify possum specific sequences capable of reducing fertility.

When the body mounts an immune response to an antigen, antibody production is mediated by B cells as part of the humoral arm of the immune response. A technique called B-cell epitope mapping can be used to identify individual peptide sequences within a protein that stimulate B-cells and thus a strong antibody response. The individual immunogenic ZP peptides recognized by the sera of female possums immunised with recombinant possum ZP3 and ZP2 are currently being assessed using this technique. Pooled serum from possums immunised with ZP3 recognised several immunogenic peptides. Individual sera are currently being assessed so that immunogenic epitopes can be correlated with the fertility status of the animals. Investigations of pooled and individual sera from animals immunised with possum ZP2 have also identified several immunogenic sequences. These immunogenic regions have been correlated with the fertility status of the immunised animals, revealing one epitope in the N-terminus and two epitopes in the C-terminus of the protein that were associated with infertility. Two of these epitopes appear to be species specific at the sequence level. Future experiments will assess their species specificity and ability to reduce possum fertility in animal trials.

Sperm Antigens

Immunisation with sperm proteins has the potential to reduce the fertility of both male and female possums and thus increase the effectiveness of an immunocontraceptive vaccine (Kerr, 1995).

Possum Sp17

The sperm protein Sp17 is known to mediate eutherian sperm binding to the zona pellucida (Wen et al., 1999). Antibodies against Sp17, or the RSA family of proteins of which it is a part, have been shown to reduce the fertility of a number of eutherian mammals (O'Rand and Irons, 1984, Lea et al., 1998). The possum homologue of Sp17 has been cloned, sequenced and shown to be localised to the sperm tail and acrosome, the sperm organelle involved in interaction with the egg. It is therefore likely that Sp17 plays a similar role in marsupial fertilisation. However any immunocontraceptive vaccine must first be able to elicit a strong immune response. An immunogenicity trial was performed with the possum Sp17 protein. Adult female possums received a subcutaneous injection of 300_g of recombinant Sp17 (rSp17) in Complete Freund's Adjuvant at week 0 and were boosted at weeks 4 and 8 with 300_g of rSp17 emulsified in Incomplete Freund's adjuvant. Control females were treated with saline emulsified in adjuvant only. The results of the immunogenicity trial demonstrated that possum Sp17 elicited a strong serum immune response. Anti-rSp17 antibodies were also present in uterine and vaginal flushings as well as follicular fluid. Binding of these anti-rSp17 to the native Sp17 on possum sperm was also confirmed using a number of immunological techniques. At the end of the immunogenicity trial a small number of treated and control females were superovulated and artificially inseminated (AI) intravaginally (Molinia et al., this proceedings). Eggs were collected 48 h after AI and fertilisation rates assessed. The percentage of fertilised eggs in the Sp17 treated animals was reduced by 64 percent compared to the control animals. These preliminary results suggest that anti-Sp17 antibodies may interfere with the fertilisation process in marsupials and further assessment of its ability to reduce possum fertility is warranted.

Novel Marsupial Sperm Proteins

Immunising female possums with whole possum sperm reduced fertility by 80 percent (Duckworth et al., 1998), thus providing evidence that sperm antigens are able to reduce possum fertility. Analysis of the proteins recognised by the sera of the sperm immunised possums identified a single immunodominant sperm surface protein. N-terminal amino acid sequence indicated that this Infertile Serum Antigen (ISA) was a novel sperm protein. We are concentrating on obtaining more amino acid sequence to facilitate the design of PCR primers for identifying the gene encoding this protein. Another sperm surface protein of interest has been identified in the wallaby. The WSA-1 epididymal protein is synthesized by the epididymis and added to the whole sperm surface during sperm maturation in the epididymis. Antibodies generated against this protein agglutinate wallaby sperm, inhibiting their forward motility. Some epididymal maturation proteins identified in eutherian mammals have a role in the fertilisation process and therefore potential as immunocontraceptive target antigens (Cuasnicu et al., 1990). N-terminal amino acid sequencing of the WSA-1 protein indicated that it was a novel sperm protein and work is continuing to identify its gene and a possum sperm homologue. Such sperm surface antigens are of particular interest for the development of possum immunocontraceptive vaccine because they are accessible to antibody attack in the male and female reproductive tracts as well as during fertilisation, thus maximising the chance of immunological interference in the reproductive process.

Partial nucleotide sequences have been identified and cloned that encode possum acrosomal enzymes likely to be involved in sperm binding to, and penetration of, the zona pellucida. Current experiments are concentrating on identifying the entire nucleotide sequence encoding these proteins before the proteins are expressed and tested for their ability to inhibit possum fertilisation.

Possum Sp17/ZP2 Dual Antigen Constructs

Ultimately it is likely that an immunocontraceptive vaccine would target multiple antigens in an effort to maximise immunocontraceptive efficiency. To this end, "dual antigen" constructs containing different parts of the possum ZP2 and Sp17 nucleotide sequences have been constructed and the proteins expressed in bacteria. The anti-fertility effects of these "dual antigen" proteins will also be assessed in future trials. Experiments performed with similar constructs in the mouse have demonstrated that whilst immunisation with part of mouse ZP2 produced only a modest reduction in fertility, constructs combining parts of the ZP2 sequence with that of Sp17 significantly increased the immunocontraceptive effect (M. O'Rand et al., unpublished results).

Summary

Significant progress has been made toward identifying possum sperm antigen targets and dissecting the immune response of infertile female possums immunised with possum ZP2 and ZP3.

A number of novel possum sperm antigens have been characterised with the potential to reduce the fertility of male and/or female possums. Research is now concentrating on identifying the genes encoding these proteins to facilitate fertility testing. Possum Sp17, the homologue of a known sperm protein with demonstrated immunocontraceptive effects in a number of eutherian species, has been identified. Immunolocalisation, immunogenicity and preliminary fertility trials suggest that Sp17 has potential as an immunocontraceptive target in the possum. This antigen will therefore be further assessed in possum breeding trials.

Recombinant possum ZP3 and ZP2 reduce the fertility of female possums. Current experiments are focussed on identifying which peptide sequences within the whole ZP protein are responsible for stimulating strong antibody responses resulting in infertility. To date, investigations into the ZP2 protein have identified two immunogenic peptide sequences that appear to be both correlated with infertility and species specific. The effectiveness of these antigens will be tested in future possum fertility trials. We have successfully constructed `dual antigen' proteins which contain some sequence from both possum ZP2 and Sp17. These, and other similar multiple antigen constructs, will also be tested for their ability to maximise the anti-fertility affects of immunocontraceptive vaccines.

In conclusion, current and future work is focused on producing species-specific possum immunocontraceptive vaccines that target multiple antigens and affect both male and female possums, thereby maximising the effectiveness of immunocontraception for possum biocontrol.

Acknowledgments

This work is supported by the Australian Government's Cooperative Research Centres Program and the NZ Foundation for Research, Science and Technology (Contract C09X0009). Dr O'Rand was supported by the United States Public Health Service, NIH grant U54HD29099.The authors appreciated the advice of Drs Ying Wen and Richard Richardson in cloning the possum Sp17.

References

Bayliss, P. 1999: Ecological modelling and possum biocontrol in New Zealand. In: Advances in the biological control of possums. The Royal Society of New Zealand Miscellaneous Series 56:19-21

Cuasnicu, P.S.; Conesa, D.; Rochwerger, L. (1990): Potential contraceptive use of an epididymal protein that participates in fertilization. In: N.J. Alexander; J.M. Spieler , G.M.H. Waites (eds): Gamete interaction: prospects for immunocontraception. Wiley-Liss, Inc, New York.pp143-153

Duckworth, J.A.; Buddle, B.M.; Scobie, S. 1998: Fertility of brushtail possums (Trichosurus vulpecula) immunised against sperm. Journal of Reproductive Immunology 37:125-138

Kerr, L.E. 1995: Sperm antigens and immunocontraception. Reproduction, Fertility and Development 7: 825-830

Lea, I.A.; Vanlierop, M.J.C.; Widgren, E.E.; Grootenhuis, A.; Wen, Y.; Vandiun, M.; O'Rand, M.G.(1998): A chimeric sperm peptide induces antibodies and strain-specific reversible infertility in mice. Biology of Reproduction 59: 527-536

O'Rand, M.G; Irons, G.P. (1984): Monoclonal antibodies to rabbit sperm autoantigens. II Inhibition of human sperm penetration of zona-free hamster eggs. Biology of Reproduction 30: 721-9

Prasad, S.V.;Wilkins, B; Dunbar, B.S. 1996: Molecular biology approaches to evaluate species variation in immunogenicity and antigenicity of zona pellucida proteins. In: S.K. Gupta; C. Doberska (eds): Zona pellucida glycoproteins and immunocontraception. Journal of Reproduction and Fertility Supplement 50: 143-149

Wen, Y.; Richardson, R.T.; O'Rand, M.G. (1999): Processing of the sperm protein Sp17 during the acrosome reaction and characterisation as a calmodulin binding protein. Developmental Biology 206: 113-122

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