Janine A. Duckworth

Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation

In female (XX) mammals one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY)1. X-inactivation in eutherian mammals is mediated by the non-coding RNA Xist2. Xist is not found in metatherians3 and how X-inactivation is initiated in these mammals has been the subject of speculation for decades4. Using the marsupial Monodelphis domestica we identify Rsx (RNA-on-the-silent X), an RNA that exhibits properties consistent with a role in X-inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, where both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-inactivation in mammals and pose questions about the mechanisms by which X-inactivation is achieved in eutherians.In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.

Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula).

In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freunds adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freunds adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n = 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%, P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula)

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.

Could recombinant technology facilitate the realisation of a fertility-control vaccine for possums?

Abstract While lethal control remains the primary tool for possum control in New Zealand, there has been substantial research effort over the last decade into controlling possum populations by reducing the animals’ breeding potential. In particular, the potential of immunological control to reduce fertility has been investigated, whereby the possums’ immune system is induced to react against its reproductive system, in order to block or destroy key components of the reproductive process. Two of the most targeted key components have been the blocking of circulating hormones (e.g. gonadotrophin-releasing hormone) and the inhibition of functional egg surface proteins (e.g. zona pellucida). While some success has been achieved for each approach, three main obstacles remain to the development of a working fertility-control vaccine for possums: first, ensuring that the vaccine remains efficacious in oral-delivery formulation; second, ensuring sufficient levels of fertility reduction; and third, ensuring that the induced immune response is sustained for a duration sufficient for long-term suppression of reproduction. The use of a genetically modified recombinant organism (parasite, bacterium or virus) to deliver a fertility-control vaccine could satisfy these requirements. A strong precedent for this approach has been set already in wildlife biology, namely the oral rabies vaccine, which is based on a recombinant vaccinia virus and which has been used successfully for the last 25 years as a disease-eliminating vaccine in mesocarnivores in Europe and North America. This review outlines the different forms and examples of recombinant organisms with potential for engineering into recombinant fertility-control vaccines to reduce possum reproduction; non-transmissible agents or fully-transmissible vectors are discussed, with shortcomings and benefits outlined for each.

Identification and evaluation of an infertility-associated ZP3 epitope from the marsupial brushtail possum (Trichosurus vulpecula)

Immunologically based fertility control vaccines against zona pellucida (ZP) proteins are being developed in New Zealand for biocontrol of the brushtail possum (Trichosurus vulpecula), an introduced Australian marsupial pest. We have shown that immunization of female possums with recombinant possum ZP3 protein (rZP3) reduced fertility by 79%. To enhance the specificity of possum immunocontraceptive vaccines, B-cell epitopes on possum ZP3 protein were mapped using sera of female possums immunized with possum rZP3 and subjected to a fertility trial. The amino acid sequence of the full-length possum ZP3 protein was used to synthesize a complete set of 83 (12-mer) biotinylated peptides each with an overlap of five amino acids with the neighboring peptides. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with possum rZP3. Sixteen epitopes were identified on the possum ZP3 protein. Comparison of the ELISA binding patterns of these peptides to antibodies in the individual sera with the fertility status of rZP3-immunized possums identified only one epitope (amino acids 156-172) to be associated with infertility. However, female possums immunized with this epitope showed no significant reduction in fertility. The possible reasons for the failure of this potential infertility epitope are discussed.

Vaccinia virus as a vaccine delivery system for marsupial wildlife

Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife.

Development of a contraceptive vaccine for the marsupial brushtail possum (Trichosurus vulpecula) : lack of effects in mice and chickens immunised with recombinant possum ZP3 protein and a possum ZP3 antifertility epitope

Zona pellucida fertility-control vaccines are being developed in New Zealand to control an introduced marsupial pest, the brushtail possum (Trichosurus vulpecula). In this study recombinant possum ZP3 protein (rZP3) and a possum ZP3 peptide (amino acids 334–361) (both known to block fertility in possums) were examined for their potential to induce species-specific, or at least marsupial-specific, infertility. Laboratory mice (a ‘model’ eutherian mammal species) and domestic chickens (a ‘model’ bird species) immunised with possum rZP3 or possum-infertility ZP3 peptide in Freund’s adjuvants showed no reduction in a range of parameters indicative of reproductive performance. The lack of contraceptive effects on mouse and chicken fertility is an encouraging result in terms of rZP3 and ZP3 peptide specificity, and these promising antigens are to be expressed in a bacterial ghost vaccine system for mucosal delivery to possums and the effects on possum fertility evaluated. Ultimately, a much wider range of non-target species will need to be screened and tested once the antigens have been successfully formulated in their final delivery vehicle.

MHC haplotypes and response to immunocontraceptive vaccines in the brushtail possum.

The possum is a major invasive pest in New Zealand. One option for its control is the use of immunocontraceptive vaccines. Initial trials of vaccines have shown individual variation in response. The use of vaccines on wild populations could result in the evolution of a resistant population through selection for possums that remain fertile because of low or no response. Understanding the basis of this variation is therefore important. The major histocompatibility complex (MHC) is an important influence on the nature of immune responses. This study has investigated the relationship between MHC alleles and individual immune responses to immunocontraceptive vaccines comprising zona pellucida peptides. We identified MHC alleles and putative haplotypes, and compared these between individuals with measured responses to immunocontraceptive vaccines. Two haplotypes were found to associate significantly with differences in vaccine response. Possums that carried haplotype 6 showed reduced responsiveness to one vaccine, while possums that carried haplotype 9 showed increased responsiveness to a separate vaccine. The identification of MHC haplotypes associated with different responses to immunocontraceptive vaccines offers the opportunity to understand what factors trigger non-response and the persistence of fertility in some individuals, and may allow vaccines to be optimised to minimise non-responsiveness.