Development of Salmonella-based immunocontraceptive vaccines for the European Red Fox (Vulpes vulpes)

Strategies for the delivery of an anti-fertility or immunocontraceptive vaccine to the feral fox population of Australia are being investigated. Salmonella typhimurium is a priority candidate vaccine delivery system because of its documented ability to elicit a variety of immune responses to carried antigens after immunisation by the oral route. This study investigated immune responses in the female European red fox (Vulpes vulpes), generated by oral immunisation with attenuated, recombinant S. typhimurium strains expressing a model fox sperm antigen, fSPlO. Strategies for enhancing immune responses to fSPIO by co-immunisation with both Escherichia coli heat-labile enterotoxin B subunit (LTB) and canine interleukin-6 (IL-6) were assessed. The feasibility of using an alternative expression system for vaccine presentation and delivery of immune-enhancing molecules, was studied. Finally, the influence of reproductive endocrine status on immune responses in the female reproductive tract was addressed. The aro-attenuated S. typhimurium strain SL3261 expressed fSPIO in vitro under the control of the constitutive trc promoter in plasmid pKK233-2. Oral immunisation with S. typhimurium strains carrying either the recombinant fSP 10-expressing plasmid or the empty vector, induced anti-S. typhimurium lipopolysaccharide (LPS) antibodies in the serum (IgG) and vaginal secretions (IgA and IgG) of all immunised foxes. However, immunisation with S. typhimurium expressing fSPIO induced a low antifSPlO IgG antibody immune response in serum and vaginal secretions of only one out of a total of ten immunised foxes. The antiserum from the one seropositive fox recognised recombinant fSPIO and bound specifically to testis and sperm. Oral co-delivery of S. typhimurium expressing fSPIO and S. typhimurium expressing either LTB or canine IL-6 failed to enhance the immune response to fSPIO. Immunisation with S. typhimurium expressing LTB, induced specific anti-LTB IgG antibodies in serum and vaginal secretions of all immunised foxes. Responses were statistically significant from control animals only after secondary and tertiary immunisations and specific antibody levels were not sustained. No anti-LTB IgA antibodies were detected in the vaginal secretions of any immunised fox. An alternative expression system facilitating extracellular transport of antigens was successfully used for secretion of fSPIO in E. coli. The fSPlO-secreting plasmid was stable in S. typhimurium. The oestrous cycle was shown to influence specific vaginal IgA levels in intact vixens. Anti-LPS vaginal IgA levels were monitored during anoestrus and oestrus in foxes previously immunised with S. typhimurium SL3261. Specific vaginal IgA secretion was suppressed during oestrus. Further, specific anti-fox sperm antigen vaginal IgA but not IgG antibody levels were dramatically reduced by oestradiol-17ß treatment of ovariectomised foxes previously immunised by the Peyer’s patch and intradermal routes. In conclusion, oral immunisation of foxes with attenuated S. typhimurium was effective for the induction of both systemic and mucosal IgG antibodies specific for a carried model bacterial protein. However, further optimisation of S. typhimurium delivery vectors and expression systems is required for the induction of specific vaginal IgA immune responses to heterologous antigens, including fox sperm proteins. Strategies to augment the mechanisms involved in mucosal immune system priming and mucosal effector maintenance must be developed. The endocrine status of foxes must be considered, and can potentially be exploited to improve mucosal immune responses in the female reproductive tract.