Gonadotrophin Releasing Hormone (GnRH), a small decapeptide synthesised in the hypothalamus,stimulates the release of the gonadotrophin hormones from the pituitary. GnRH is not naturally immunogenic but when it is conjugated or co-expressed with other immunogenic carrier proteins (hereafter referred to as GnRH constructs), antibodies against GnRH are stimulated. Additional adjuvants may be required to enhance the immune response. Currently, most GnRH immunocontraceptive vaccines available for mammals are injectable formulations. For broad scale application to free-ranging wildlife populations, an orally deliverable form would be more practical. However, at least two major challenges must be overcome to achieve successful oral delivery of immunocontraceptive vaccines: they must be protected from degradation by gastric acids and enzymes in the gastrointestinal tract (GIT) and there must be sufficient uptake by the mucosal immune sites (e.g. Peyer's patches) of the GIT to stimulate an immune response. This thesis aimed to: 1) Assess various GnRH constructs for their immunogenicity and potential for oral administration and 2) Assess a particulate delivery system for its ability to deliver antigenic proteins to the mucosal immune sites of the GIT.
Initially, several GnRH constructs without added adjuvants were assessed via oral,intranasal and intramuscular routes over a twelve week period in male Sprague Dawley rats. These constructs induced a low and variable immune response by the intramuscular route only. GnRH constructs with added adjuvants (CpG oligonucleotides with and without alum, squalene emulsions and immunostimulating complexes, ISCOMs) were then assessed in male Balb/C mice after subcutaneous injection. All mice given GnRH conjugated to the carrier protein Keyhole Limpet Haemocyanin (KLH), and with ISCOMs as the added adjuvant, generated antibodies against GnRH. At twelve weeks, changes in reproductive tissues, including lower testis weights and absence of active spermatogenesis within the seminiferous tubules were observed in all mice receiving this treatment. This combination of GnRH construct with adjuvant may be suitable for oral delivery.
Amorphous silica nanoparticles (SiNPs) have previously been shown to provide partial protection of proteins from the gastric acids and enzymes in the GIT, however their ability to target and be taken up by the mucosal immune sites was unknown. SiNPs were assessed for their ability to deliver protein and peptide antigens via the oral route in female Balb/C mice.
Initially, a biodistribution study was undertaken using SiNPs (~350 nm and ~850 nm in diameter) which were either unmodified or PEGylated. PEGylation alters the surface characteristics of the particles and may enhance attachment at mucosal sites. A fluorescent and a gold marker were incorporated for detection of SiNPs in vivo. SiNPs were delivered into the stomach by oral gavage and mice were killed at 3 h, 6 h, 9 h, 12 h and 24 h. Markers were detected in the small intestine, Peyer's patches, spleen, liver and lung tissues up to 24 hours after delivery. Differences in uptake due to particle size and/or surface modification were minimal. For all particle types, high proportions (up to 40 %) of the gold marker were detected in the faeces and urine indicating not all SiNPs were absorbed across the GIT. Overall, the biodistribution study showed that the SiNPs were taken up by various lymphoid tissues after oral delivery.
Tetanus toxoid (TT) was then incorporated into SiNPs (~350 nm) to assess whether an immune response against TT could be induced after oral delivery. Mice were given three doses of a particular treatment at two week intervals and blood was collected for analysis of antibodies. No TT specific antibodies were generated in the plasma of any mice receiving oral doses of either blank SiNPs or 280 μg TT. However, two of six mice generated TT specific antibodies when they received 280 μg of TT in SiNPs. By comparison, all mice that received any TT treatment subcutaneously generated strong antibody responses against TT.
In a second study, half or double the original TT dose was incorporated into SiNPs. Three of six mice receiving 140 μg of TT in SiNPs generated TT specific antibodies. Only one of six mice receiving 560 μg TT generated antibodies. As in the initial trial, two of six mice responded to 280 μg TT in SiNPs. These results indicated that a lower dose was more effectively taken up than a higher dose. Factors influencing the variability of responses may include particle and antigen degradation within the GIT, aggregation of the particles and/or poor uptake the particles at the mucosal immune sites.
Results from this thesis have shown that of the GnRH constructs and adjuvants assessed, GnRH:KLH with ISCOMs was the most promising preparation in mice. While further studies are warranted, it has also shown that amorphous silica nanoparticles have potential as an oral vaccine delivery system.