Vaccine 28 (2010) 4268–4274
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Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein Xianlan Cui a,∗ , Janine A. Duckworth a , Petra Lubitz b,c , Frank C. Molinia a , Christoph Haller b,c , Werner Lubitz b,c , Phil E. Cowan d a
National Research Centre for Possum Biocontrol at Landcare Research, PO Box 40, Lincoln 7640, New Zealand Department of Medicinal Chemistry, University of Vienna, Vienna, Austria Bird-C GmbH & CoKEG, Hauptstrasse 88, 3420 Klosterneuburg-Kritzendorf, Austria d National Research Centre for Possum Biocontrol at Landcare Research, Private Bag 11052, Palmerston North, New Zealand b c
a r t i c l e
i n f o
Article history: Received 26 October 2006 Received in revised form 12 April 2010 Accepted 15 April 2010 Available online 29 April 2010 Keywords: Zona pellucida Bacterial ghosts (BGs) Immunogenicity Brushtail possum Mucosal immunisation
a b s t r a c t The introduced common brushtail possum (Trichosurus vulpecula) is a major pest in New Zealand and immunocontraceptive vaccines are being developed for biocontrol of possum populations, with bacterial ghosts (BGs) being evaluated as a means of oral delivery. Recombinant BGs expressing possum zona pellucida 3 protein (ZP3) as an L membrane-anchored protein (ZP3-L ) or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) were produced by the expression of the cloned bacteriophage X174 lysis gene E in E. coli NM522. The humoral immune responses of possums immunised with BGs expressing possum ZP3 were investigated following oral, intranasal/conjunctival, parenteral, and intraduodenal administration to evaluate the BG-ZP3 system for possum fertility control. Antibodies to possum ZP3 were detected in the serum, oviduct secretions, and follicular fluid of immunised animals. Intranasal/conjunctival immunisation elicited reliable antibody immune response in serum and at a key effector site, the ovarian follicular fluid. Intraduodenal administration of possum ZP3 BG vaccine as a priming immunisation elicited significant systemic immune responses, but oral immunisation did not, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. The detection of antibodies at elevated levels at target sites in the reproductive tract following mucosal delivery demonstrates, for the first time, the potential of BGs as an effective system for vaccine delivery to wild animals, and intranasal/conjunctival immunisation as a promising means for delivery of immunocontraceptive vaccines to wild animals. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Immunologically based fertility control (immunocontraception) is being developed in New Zealand as a management tool to reduce pest populations of common brushtail possums (Trichosurus vulpecula), an introduced Australian marsupial [1]. Key steps for the successful development of possum immunocontraception include the identification of target antigens critical for possum reproduction, and the development of cost-effective and efficient delivery systems [2]. A range of potential contraceptive targets derived from the possum zona pellucida (egg coat) have been identified and shown to reduce the reproductive performance of possums by 60–80% when zona pellucida (ZP) antigens were administered
∗ Corresponding author. Current address: Animal Health Laboratory, Department of Primary Industries, Parks, Water and Environment, 165 Westbury Road, Prospect, TAS 7250, Australia. Tel.: +61 3 6336 5364; fax: +61 3 6336 5374. E-mail address:
[email protected] (X. Cui). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.04.032
parenterally with Freund’s adjuvants [3–5]. Although these investigations showed that ZP proteins 2 and 3 (ZP2 and ZP3) were promising target antigens for an immunocontraceptive vaccine, administration of the recombinant proteins and adjuvants by injection is impractical for field delivery. Bacterial ghosts (BGs) are being evaluated for their suitability for use in an oral or aerosol delivery system for the dissemination of immunocontraceptive vaccines to possum populations in the wild. BGs are cell envelopes, produced by the expression of the cloned bacteriophage X174 lysis gene E in Gram-negative bacteria [6]. Expression of plasmid-encoded gene E leads to the fusion of the inner and outer membrane, which results in the formation of a transmembrane tunnel in the bacterial cell envelope, through which the cytoplasmic contents are expelled [7] while preserving the integrity of the periplasmic space. The resulting empty BGs share functional and antigenic determinants with their living counterparts [8], which means that all structures presented within the envelope complex of the bacteria are maintained on the resulting BG. E-mediated lysis has been achieved in a vari-
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ety of Gram-negative bacteria, including Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Helicobacter pylori, Pasteurella multocida, Mannheimia haemolytica and others [7,9–12]. The BG system can be extended to be carriers of foreign proteins; or in an immunological sense to be carriers of target antigens. Such target antigens have been expressed in the bacterial carriers using anchor sequences, such as E for N-terminal and L for C-terminal anchoring of the target antigen in the inner membrane, or fused to the periplasmic proteins (e.g. MalE) for export to the periplasmic space [13]. An alternative presentation is to design a construct that imbeds the target antigen into the surface-layer (S-layer) proteins SbsA and SbsB of Bacillus stearothermophilus [14–16]. Flexible surface loops in both SbsA or SbsB proteins accept large foreign sequences of up to 600 amino acids [17,18] without losing the self-assembly properties of the S-layer monomers. The S-layer self-assembly structure is not released to the external medium by the E-mediated lysis but is retained within the inner lumen of the cytoplasmic space or, if fused to a signal sequence, is exported to the periplasmic space [9]. Target antigens presented by recombinant BGs can induce both humoral and cellular immune responses [7,19]. Most importantly, BG particles are easily recognised by macrophages and dentritic cells, and they also contain immunostimulating compounds such as lipopolysaccharides, lipid A and peptidoglycan [20]. In addition to their superior immunostimulatory properties, which make the addition of adjuvants obsolete, BGs can be produced inexpensively in large quantities by fermentation, are stable as freeze-dried material for long periods, and do not require the cold-chain storage system. Another advantage is that there is virtually no limitation on the size of foreign protein moieties that can be expressed, which means that multiple epitopes can be presented simultaneously [7]. These potential advantages make BGs an attractive delivery system for biological control of wild pest animals. In this study the immune responses of female possums to two different formulations of BGs expressing possum ZP3, either as membrane-anchored protein or as S-layer fusion protein exported to the periplasmic space, were investigated for their potential as oral vaccines for possum fertility control. 2. Materials and methods 2.1. Bacterial strain and plasmids E. coli strain NM522 [supE thi-1D(mcrB-hsdSM) (rK− mK+) [FˇıproAB lacIqZDM15]] was obtained from Stratagene (La Jolla, USA). The commercially available vector pMAL-p2X (New England BioLabs, Hitchin, Hertfordshire, UK) is designed to produce maltose-binding-protein (MBP) fusions. The malE gene on this vector includes the sequence coding for the amino-terminal signal peptide of MBP, which directs the fusion protein to the periplasm of E. coli. The vector pBSZP3 was kindly provided by K. Mate, University of Newcastle in Australia, and was used as a template for PCR amplification of possum ZP3 gene. Plasmids pKSEL5-2, pMalA, and pML1 [7] were obtained from our plasmid collection.
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used according to the manufacturer’s instructions (MBI Fermentas, Vienna, Austria). 2.3. Cloning and transformation procedures The ZP3 PCR product was purified using a PCR purification kit from Qiagen (QIAquick PCR Purification Kit, Hilden, Germany), digested with the appropriate restriction enzymes for 2 h at 37 ◦ C, and gel purified using a gel extraction kit from Qiagen (QIAquick Gel Extraction Kit, Hilden, Germany). All restriction enzymes were obtained from Roche (Roche Diagnostics GmbH, Vienna, Austria) or NEB (New England BioLabs, Frankfurt on Main, Germany). After digestion with the appropriate restriction enzymes (XhoI/HindIII or SnaBI) inserts were ligated with T4 DNA ligase (Roche Diagnostics Australia Pty Ltd, Castle Hill, NSW, Australia) overnight at 16 ◦ C into the appropriate expression vector, either pKSEL5-2 or pMalA. The resulting vectors are called pKSEL5-2/ZP3 and pMalA/ZP3, respectively. Competent cells of the E. coli strain NM522 were prepared [21]. Competent E. coli NM522 cells were transformed with the ligation products. Aliquots of the transformed cells were plated onto LB agar plates supplemented with ampicillin at a final concentration of 100 (g/ml. Following overnight culture at 28 ◦ C, transformant colonies were analysed for recombinant constructs, using restriction analysis. Plasmid DNA was isolated from positive clones using a Peqlab miniprep kit (EZNA Plasmid Miniprep Kit I, Peqlab-Biotechnologie GmbH Erlangen, Germany) according to the manufacturer’s instructions. Possum ZP3 amino acids 23–342 were expressed as fusion protein with the C-terminal membrane-anchor sequence L attached to the inner membrane (ZP3-L ), or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) [8]. Expression analyses of both fusion proteins were performed using SDS-PAGE and Western blot. The ZP3-L fusion protein derived from the vector pKSEL5-2/ZP3 was confirmed to be ∼49.5 kDa in size. The vector pMalA/ZP3 produced an MBP-SbsA-ZP3 fusion protein of ∼217 kDa. 2.4. Preparations of E. coli cell extracts, SDS-PAGE and Western blot Expression from the ZP3 constructs pKSEL5-2/ZP3 and pMalA/ZP3 was tested in E. coli NM522. Flasks containing 30 ml LB medium supplemented with ampicillin at a final concentration of 100 (g/ml were inoculated with 500 l overnight culture of E. coli containing the different expression plasmids. The bacteria were grown at 37 ◦ C and the OD600 of the growing culture was measured every 30 min. At an OD600 of ∼0.2, expression of the fusion proteins was induced by addition of 3 mM isopropyl -dthiogalactopyranoside (IPTG) (MBI Fermentas, Vienna, Austria). Samples (1.5 ml) for SDS-PAGE and Western blot were removed before and 30, 60 and 90 min after induction of ZP3-L or MBPSbsA-ZP3 expression. SDS-PAGE and Western blot were performed as described previously [12] and rabbit anti-recombinant possum ZP3 (1:1000) [22] was used in Western blot. 2.5. Production of E. coli NM522 ghosts carrying ZP3-L or MBP-SbsA-ZP3 fusion proteins
2.2. PCR amplification of ZP3 Primers for PCR were constructed to amplify the possum ZP3 from nucleotides 67 to 1164 (corresponding amino acids 23–342). The restriction sites XhoI (5 ) and HindIII (3 ) were included in the PCR primers to allow directional cloning of the ZP3 fragment into vector pKSEL5-2, whereas SnaBI sites were selected for cloning the fragment into corresponding SnaBI sites of the vector pMalA. In the PCR reactions the proof-reading DNA polymerase Pfu was
E. coli NM522 (pKSEL5-2/ZP3) and E. coli NM522 (pMalA/ZP3) were made competent as described above. Competent cells from both samples were transformed with E-lysis plasmid pML1 as described previously [7]. E. coli NM522 cells containing a combination of lysis and expression plasmids were employed for small-scale BG production. Transformants were grown at 28 ◦ C in 25 ml LB medium supplemented with ampicillin and kanamycin at concentrations
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of 100 g/ml and 50 g/ml, respectively. When an OD600 of 0.2 was reached, the expression of ZP3 fusion protein was induced with IPTG at a final concentration of 3 mM. Following 90 min of expression, gene-E-mediated lysis was induced by increasing the temperature from 28 ◦ C to 42 ◦ C. The course of lysis was monitored by measuring the OD600 every 15 min (Shimadzu UV-160, Kyoto, Japan) as well as by observation of the cells using light microscopy. To follow the efficiency of lysis, viable cell counts were determined throughout the growth and lysis cycle using a spiral plater (WASPsystem, Don Whitely Scientific, USA) with the appropriate software. Samples for Western blot experiments (1.5 ml) were collected prior to induction and during the time course of lysis. Unlysed reference cultures were used to prepare glycerol stocks serving as workingstock for large-scale fermentation. The working stocks were stored at −70 ◦ C. E. coli NM522 cells containing a combination of lysis and expression plasmids, pKSEL5-2/ZP3/pML1, MalA/ZP3/pML1, and pML1, respectively, were employed in a volume of 10 l for large-scale BG production. Gene-E expression was allowed to proceed for 120 min, reaching a lysis efficiency of 99.9%, at which time an antibiotic mix of gentamycin and streptomycin (at final concentrations of 40 g/ml and 200 g/ml, respectively) was added with a further incubation for 60 min to reduce colony-forming units to zero. Thereafter the cells were harvested by centrifugation at 5000 × g. The BG pellets were washed once with 9 l of distilled water and three additional washes with 4.5 l, 1.2 l and 0.4 l of distilled water. The final pooled pellet was lyophilised for long-term storage. BGs expressing possum ZP3 were imported to New Zealand for testing under permit (MAF #2000009353). 2.6. Quantification of ZP3-L and MBP-SbsA-ZP3 in E. coli NM522 ghosts From lyophilised ghost preparations of E. coli NM522, containing either ZP3-L anchored in the inner membrane or MBP-SbsA-ZP3 imbedded within the periplasmic space, the amount of recombinant ZP3 (rZP3) was determined per milligram of freeze-dried ghosts. About 50 g freeze-dried ZP3-L and MBP-SbsA-ZP3 BGs were resuspended in distilled water and proteins separated, as described previously [12], on 12% and 7.5% polyacrylamide gels, respectively. To obtain a standard curve, 0.25, 0.5 and 1 g of purified rZP3 protein was applied to the same gel. Western immuno-blotting was performed and the membranes were probed with anti-ZP3 sera. The intensities of the specific ZP3-L and MBPSbsA-ZP3 bands were determined by densitometry (Chemidoc XRS, Bio-Rad, Hercules, USA; Software: Quantity One). The standard curve allowed the calculation of the concentration of rZP3 contained in ZP3-L and MBP-SbsA-ZP3 freeze-dried ghosts. ZP3-L BGs were calculated to have ∼1.67 g rZP3 per mg BG. The immunisation dose for ZP3-L BGs was 20 mg, thus equivalent to ∼30 g rZP3 per dose. MBP-SbsA-ZP3 BGs were calculated to carry ∼20 g of rZP3 per mg BG, providing an immunisation dose (16 mg) equivalent to ∼320 g rZP3. 2.7. Animals and immunisation Mature female possums that weighed >2.5 kg were trapped from the Canterbury Region of the South Island, New Zealand. They were housed indoors in individual cages and offered fresh apples, vegetables and cereal-based possum pellets and fresh water ad libitum. Animals were acclimatised to captivity for at least 6 weeks [23] before they were randomly assigned to treatment groups. Possums were anaesthetised by inhalation of 5% halothane (Fluothane, ICI, UK) administered in pure oxygen (2 l/min) for immunisation, surgery and collection of blood samples. All the procedures in this study were approved by the Animal Ethics Committee, Landcare
Research, Lincoln, and were performed in accordance with the Animal Welfare Act 1999 of New Zealand. Possum ZP3 was expressed in E. coli as a single protein consisting of amino acid residues 23–342 [22]. Female possums (n = 20 females per group) were immunised with possum rZP3 vaccine or PBS in Freund’s adjuvants (Difco Laboratories, Detroit, MI, USA), three times at weeks 0, 3 and 6 and fertility was assessed following superovulation and artificial insemination [24,25]. Detailed results of this trial will be published elsewhere but, in summary, there were no significant differences between control and rZP3-immunised groups in the number of follicles ≥2 mm remaining (p = 0.95). However, there were significant reductions in the numbers of ovulation sites (p = 0.03), eggs (p = 0.01), and embryos (p = 0.001) of rZP3 immunised animals when compared to those of the control animals. The fertility of female possums immunised with rZP3 prepared in Freund’s adjuvant was reduced by 79% compared to that of control animals. Serum antibody titres at week 9 differed significantly between the control and immunised animals (Mann–Whitney U-test, p < 0.001). Antibodies against rZP3 were detected in the sera of 18 possums that had antibody titre ranging from 102 to 106 at week 9 and also had a significant reduction in fertility when compared to control animals, whereas the other two animals with low levels of antibody titres to rZP3 responded poorly to artificial insemination treatment, indicating that there was a significant reduction in fertility of possums with high rZP3 antibody titres. In Trial 1, eight female possums were immunised orally and four by the intranasal/conjunctival route (eye/nose) with 20 mg of ZP3L BGs (equivalent to a dose of ∼30 g rZP3), respectively. Eight female possums were immunised orally and another eight female possums by the intranasal/conjunctival route with 20 mg of negative control BGs, respectively. An identical dose was given 2 and 4 weeks later. For oral immunisations, BGs were resuspended in 1 ml of PBS and administered into the back of the oral cavity with a needleless syringe. For the intranasal/conjunctival immunisation BGs were suspended in 400 l PBS, and ∼30 l applied drop-wise into each eye and the remainder into the external nares of each nostril. As a positive control, four possums were injected subcutaneously with 20 mg of ZP3-L BGs (30 g rZP3/dose) in complete Freund’s adjuvant (Difco Laboratories, Detroit, MI, USA) at multiple sites across the possum’s back, followed by booster immunisations with an identical dose in incomplete Freund’s adjuvant (Difco Laboratories, Detroit, MI, USA) at weeks 2 and 4. In Trial 2, eight female possums were immunised orally and eight by the intranasal/conjunctival route with 16 mg of MBP-SbsAZP3 BGs (equivalent to a dose of ∼320 g rZP3), respectively. An identical dose was given 2 and 4 weeks later. Another eight animals received an intraduodenal primary immunisation to assess the effects of passage through the stomach on the immune responses. About 16 mg (320 g rZP3/dose) of MBP-SbsA-ZP3 BGs were suspended in 1 ml PBS and injected directly into the duodenum. These animals then received two oral booster immunisations with 16 mg MBP-SbsA-ZP3 BGs at weeks 2 and 4. Eight female possums were immunised orally and another eight female possums by the intranasal/conjunctival route with 20 mg of negative control BGs, respectively. Intraduodenal vaccination was performed using a modified laparoscopic procedure developed previously for intrauterine insemination of possums [24]. Briefly, anaesthesia was induced by 5% halothane in 1.5 l of oxygen after a pre-injection of 0.3 ml of Atropine (Phoenix Pharm, Auckland, New Zealand) to reduce salivation. Once anaesthetised each possum was restrained on its back with the head above the abdomen at an angle of approximately 30◦ . A 7-mm laparoscope was inserted into the abdominal cavity via a 2-mm incision midway between sternum and pouch. A 5-mm trocar was also inserted 2 cm to the left of the midline for inflation of the abdominal cavity with air and for introduction of
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Fig. 1. Antibody titres (mean ± SE) in possum sera (a) and binding of antibodies in the ovarian follicular fluid and secretions of the reproductive tracts (b) in Trial 1, following immunisation with ZP3-L bacterial ghosts (BGs). Control possums received negative control BGs. Antibody titres are expressed as log2 of the reciprocal of the highest serum dilution and arrows indicate the times of immunisation at weeks 0, 2 and 4 (a).
grasping forceps. The BG vaccine was loaded into a 1-ml syringe and injected through the body wall into the duodenum using a 25-mm-long 23-gauge needle. In Trial 3, 20 female possums were immunised by intranasal/conjunctival route with 16 mg (320 g rZP3/dose) of MBP-SbsA-ZP3 BGs, and another 20 female possums by the same route with PBS only, followed by booster immunisations at weeks 2 and 4. Animals were killed 7–8 weeks for reproductive-tract assessment [24] after primary immunisation. 2.8. Sampling In all trials, 3–6 ml of blood was collected from the ventral tail vein of each possum at weeks 0, 2, 4 and at post-mortem, and the sera harvested. At the end of the trial, animals were euthanased by overdose of pentobarbitone (Chemstock, Animal Health, Christchurch, New Zealand) under anaesthetic. Reproductive secretions were collected by flushing the uterus, oviduct, and lateral vagina with 1 ml PBS containing protease inhibitors. Where possible, follicular fluid was collected by aspiration from two or three ovarian follicles from each animal. 2.9. ELISA The rZP3 ELISAs were carried out as described previously [25]. Immuno-Plate MaxiSorb F96 flat-bottomed plates (Nunc, Roskilde, Denmark, cat. No. 4-42404) were coated with 100 l of 5 g/ml recombinant possum ZP3 purified from E. coli [21] diluted in coating buffer (50 mM Tris–HCl, 150 mM NaCl, pH 9.0). Subsequent steps were identical to those of the rZP2 ELISA protocol [25]. The absorbance at 450-nm wavelength measured using a microplate autoreader (Bio-Tek Instrument Inc., Winooski, VT, USA). The ELISA titre of each serum was expressed as the log2 of the reciprocal of the highest positive serum dilution that gave an OD at least 1.5 times
greater than the mean of the OD values of sera from control animals. Secretions with at least 1.5 times the mean of the OD value of the control animal secretions were defined as positive [26–28]. 2.10. Data analysis Data from the antibody titres in sera were expressed as log2 of the reciprocal of the serum dilution. Absorbance values of antibody binding to rZP3 in mucosal secretions and ovarian follicular fluid are presented as mean values ± SE. The differences of antibody titres at week 6 and antibody binding of mucosal secretions and ovarian follicular fluid were analysed using the non-parametric Mann–Whitney U-test between the immunised possums and the corresponding control possums [29]. p value 0.05).
3.2. Immunogenicity of MBP-SbsA-ZP3 BGs In MBP-SbsA-ZP3 BG trial (Trial 2), the titres of ZP3 antibodies in sera of all eight possums immunised by intranasal/conjunctival route with MBP-SbsA-ZP3 BGs reached 1:2048 to 1:32,768 at week 6 after two booster immunisations, with four possums having titres of 1:256 to 1:2048 at week 4 after the first booster immunisation (Fig. 2a). Only three of eight orally immunised possum had antibody responses, with titres of 1:256 to 1:8192 at week 6 after two booster immunisations, and two animals responded with titres of 64–1024 at weeks 2 and 4. Six out of eight immunised possums in the surgical group had antibodies against ZP3 at serum dilutions of 1:256 to 1:16,384 at week 6, and three animals had titres of 1:256 to 1:32,768 at weeks 2 and 4. One negative control animal had the titre of 1:256 at week 6. There was a significant difference in serum antibody titres at week 6 between the immunised animals and
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the corresponding control animals by the intranasal/conjunctival route (Mann–Whitney U-test, p < 0.0002), but not by oral route (Mann–Whitney U-test, p = 0.38). Possums immunised by intraduodenal route had significantly higher antibody titres than control possums by oral route (Mann–Whitney U-test, p = 0.021) (Fig. 2a). All possums (8/8) in the intranasal/conjunctival group and about half the possums in the oral (5/8) and surgical groups (4/8) had ZP3 antibodies in the ovarian follicular fluid. There were no significant differences in antibody binding in vagina and uterus secretions between the corresponding control animals and the immunised animals by the oral and intranasal/conjunctival routes (Mann–Whitney U-test, p > 0.05), but there was a significant difference in antibody binding in oviduct secretions by the oral route (Mann–Whitney U-test, p = 0.02), and in oviduct secretions (Mann–Whitney U-test, p = 0.02) and follicular fluid (Mann–Whitney U-test, p = 0.0002) by the intranasal/conjunctival route (Fig. 2b). In Trial 3, the titres of ZP3 antibodies in 18 sera of 20 possums immunised by intranasal/conjunctival route with MBP-SbsA-ZP3 BGs reached 1:256 to 1:4096 at week 6 after two booster immunisations, with 13 possums having titres of 1:256 to 1:4096 at week 4 and six possums with titres of 1:256 to 1:4096 at week 2. There was a very significant difference in serum antibody titres at week 6 between the control animals and the animals immunised by intranasal/conjunctival route (Mann–Whitney U-test, p < 0.0001) (Fig. 2c). Oviduct secretions in 9 of 20 and ovarian follicular fluid in 9 of 11 BG-immunised possums had ZP3 antibodies. There were significant differences in antibody binding of secretions in the vagina, oviduct, and follicular fluid between the control animals and immunised animals (Mann–Whitney U-test, all p < 0.05), but not in the uterus (Mann–Whitney U-test, p = 0.18) (Fig. 2d).
4. Discussion In a previous study (to be published elsewhere), the injection of female possums with a possum rZP3 vaccine prepared in Freund’s adjuvant reduced the number of embryos produced following superovulation and artificial insemination by 79%. This proof-ofprinciple study encouraged us to carry on in the development of an immunocontraceptive vaccine for the brushtail possum. Crucial to the success of immunocontraception is the development of an effective and safe delivery system for dissemination of vaccines to wild animals in the field. Conventional possum control uses poisons that are delivered in baits and reliably reach a very high proportion (80–95%) of wild possums [30]. Orally delivered vaccines in baits could also reach a high proportion of animals and could target the mucosal surfaces of the mouth and gastrointestinal tract. Alternatively, feeding devices that trigger the administration of an aerosol spray into the face of possums may also be suitable to immunise possums via mucosal surfaces of the eyes and nose. From the results reported in this study, intranasal/conjunctival and oral application of BGs carrying ZP3 antigen can induce humoral and mucosal immune responses in the target animal, the brushtail possum, and could be utilised for bait or aerosol delivery. BGs have been successfully used to express a wide range of different recombinant target proteins capable of eliciting protective immunity against different kinds of pathogenic organisms (see reviews in [7,19]). In the present study, the first to apply the technology for use in wild animals, we showed that BGs are effective carriers for ZP3 target antigens inducing specific immune responses in possums, and therefore have potential as a safe non-living bait delivery system for possum fertility control vaccines. The type of BG influenced the immune response. This could be due to the presentation of the antigen and/or the amount of antigen expressed. The estimated amount of rZP3 protein in Trials 1
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and 3 with ZP3-L BGs was 30 g per dose (20 mg BG) and in Trials 2 and 3 with MBP-SbsA-ZP3 BGs was 320 g per dose (16 mg BG). The increased anti-ZP3 titres in sera and a higher incidence of positive antibody binding in the secretions of the reproductive tract in MBP-SbsA-ZP3-BG-immunised animals could be interpreted as a result of the 10-fold higher load of the target antigen ZP3 per dose given to the ZP3-L -BG-immunised animals. It also may reflect the differences in antigen recognition when recombinant ZP3 is coexpressed within the MBP-SbsA protein complex. Further increases in the total amount of antigen administered, by either increasing the amount of the antigen per ghost or increasing the number of BGs per dose, may lead to higher specific anti-ZP3 titres. As possum zona was fixed with Bouin’s reagent in this study and fixation with Bouin’s reagent has been shown to almost completely destroy the ZP structure of mammalian oocytes [31], it is therefore not possible to comment on the in vivo binding of antibodies to ZP of treated possums. However, since the immunised possums with reasonably high titres had significantly lower fertility in the possum rZP3 fertility trial, the rZP3 antibody titres of immunised possums in this study may indicate the potential to reduce possum fertility. Oral immunisation has many advantages over other immunisation routes, including easier administration to wild-living animals and the potential for frequent boosting of the immune response. In this study, oral immunisation with ZP3 BGs gave lower immune responses than the nasal/conjunctival route. It is well known that hostile environmental conditions in the stomach may limit the efficacy of an orally delivered vaccine [32]. In possums, when cholera toxin was administered directly into the ileum, the proportion of animals that responded and the magnitude of the antibody responses in serum and reproductive tract secretions were greater than those following intragastric administration [33]. Similarly in this study the systemic immune response was improved by primary immunisation with MBP-SbsA-ZP3 BGs by the intraduodenal route. This implies that protection of orally delivered BG vaccines from degradation by gastric acidity and gastrointestinal secretions may enhance the immune response and this is currently under investigation. Both oral and nasal/conjunctival immunisations with MBP-SbsA-ZP3 BGs favoured immune responses in the key effector sites of the oviduct and follicular fluid. The mucosal-associated lymphoid tissue (MALT) generally includes the bronchus-associated lymphoid tissue (BALT), nasalassociated lymphoid tissue (NALT), the gut-associated lymphoid tissue (GALT), and the male and female genital tracts [34]. Mucosal delivery of vaccines stimulates both mucosal and systemic immune responses [35]. Intranasal immunisation is the most promising among the mucosal immunisation routes, including oral, intravaginal, and intrarectal [36,37] particularly when targeting the female reproductive tract [38]. Intranasal and eye-drop immunisation of chickens with viral vaccines is widely used to protect against pathogens [39–42]. Intranasal immunisation of rodents and human and non-human primates has been associated with induction of strong secretory IgA and T-cell responses in the upper respiratory tract, mammary glands, and also female genital tract [43]. The ocular immune system consists of the conjunctival-associated and lacrimal-drainage lymphoid tissue [44] and immune stimulation appears to rely on antigenic clearance through the nasolacrimal duct and stimulation of the interconnecting NALTs and GALTs [45]. The intranasal/conjunctival route in the trials in this study gave the best humoral immune responses among the mucosal immunisation routes, particularly in response to the MBP-SbsA-ZP3 BGs. Antibodies against possum ZP3 were detected in sera and the target sites (oviduct and follicular fluid) of immunised possums. The effects of immunisations of ZP3 BGs on fertility could not be assessed as unexpectedly few animals in the treatment and control groups responded to superovulation treatment. However, results from a recent fertility trial demonstrate that immunisation by the
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