Solidified saturated fats coating subunit vaccines greatly extended vaccine booster release and contributed to a Th1/Th2 mixed immune response in mice.

Delayed release of vaccine coupled with a soluble vaccine acts as a primer and a booster with only a single administration, which would be very beneficial to livestock producers. We developed a subdermal pellet consisting of solid-phase pure stearic acid (SA) or palmitic acid (PA) that was used to encapsulate a small volume liquid vaccine consisting of fluorescently labeled *Ovalbumin (Cy5-*OVA) formulated with Emulsigen-D +/- Poly I:C (EMP) adjuvants. Mice were also immunized via the subcutaneous route with Cy5-*OVA-EMP (soluble liquid). The vaccine leached out of the pellet with very little dissolution of the fat itself resulting in the sustained subdermal delivery of antigens and adjuvants. Cy5-*OVA was still visible 60 days post administration in mice immunized with stearic acid-coated or palmitic acid-coated pellets. In these mice, persistently high IgG1 and IgG2a antibody titres were detected as well as significant IFNγ production at least 60 days post-injection. These responses were significantly higher than those observed after a single subcutaneous injection of the vaccine. A repeat trial with the pellets alone +/- the soluble vaccine showed comparable immune responses after surgical implantation of the pellet, suggesting that pellet alone may be sufficient. The PA-coated vaccines led to dermal inflammation in the mice that would limit usefulness of this vehicle, but this was largely absent when SA was used to coat the pellets. These data suggest that the SA-coated adjuvanted vaccine prolonged the release of the vaccine and triggered a comparable immune response to the mice that received the two liquid injections, and a single pellet vaccine should be tested as a novel immunization method for livestock.

Immunoproteomic analysis of Lawsonia intracellularis identifies candidate neutralizing antibody targets for use in subunit vaccine development.

Lawsonia intracellularis is an obligate intracellular microorganism and the causative agent of porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to achieve using conventional microbiological techniques. In this work, we used 2-dimensional gel electrophoresis coupled with Western-immunoblotting, mass spectrometry and bioinformatics to identify bacterial proteins that interact in vitro with pig intestinal cells (IPEC-1), have immunogenic properties and the potential to be used as subunit vaccine antigens. We detected eleven immunogenic bacterial proteins from which fliC (LI0710), LI1153 (annotated by NCBI as Putative protein N), and LI0649 (annotated as autotransporter) were predicted to be expressed on the outer membrane while LI0169 (oppA; annotated as ABC dipeptide transport system) was predicted to be periplasmic with a transmembrane domain forming a central pore through the plasma membrane. Genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole Lawsonia lysate established that all four recombinant proteins were immunogenic. Further, rabbit hyperimmune sera generated against the vaccine strain of L. intracellularis and rabbit serum specific for each recombinant protein showed an inhibitory effect on the attachment and penetration of live, avirulent L. intracellularis, thus indicating that each protein is a potential neutralizing antibody target and a candidate for subunit vaccine formulation.