Identification of Lawsonia intracellularis proteins recognized by neutralizing antibodies and use of these proteins to design a subunit vaccine

Lawsonia intracellularis is an obligate intracellular microorganism which causes diseases known as porcine ileitis or porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of L. intracellularis antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to accomplish using conventional microbiological techniques. The overall goal of this thesis is to identify, characterize and evaluate L. intracellularis bacterial proteins as neutralizing antibody targets which may then be suitable candidates for subunit vaccine development. In this thesis, 2-dimensional (2-D) gel electrophoresis coupled with Western-immunoblotting, mass spectrometry, and bioinformatics were used to identify proteins on the surface of the bacteria that interact in vitro with pig intestinal cells (IPEC-1) and have immunogenic properties. Eleven immunogenic bacterial proteins were detected of which LI0710 (annotated as fliC), LI1153 (annotated as Putative protein N), LI0649 (annotated as autotransporter), and LI0169 (OppA; annotated as ABC dipeptide transport system) were predicted to be expressed on the outer membrane. The 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 L. intracellularis lysate established that all four recombinant proteins were immunogenic. To be able to quantify invasion of the vaccine strain of L. intracellularis in McCoy and IPEC-1 cells and to determine whether antibodies specific for the recombinant L. intracellularis proteins inhibit the bacterial attachment and penetration into eukaryotic cells, the bacteria were labeled with cell-permeable fluorescent dye 5’-carboxyfluoroscein succidyl ester (CFSE) prior to cell infection. Flow cytometry was applied to determine the percentage of eukaryotic cells which were infected with fluorescent bacteria. As obligate intracellular bacteria, their replication is dependant on eukaryotic cells and thus qPCR analysis was applied to quantify bacterial growth. qPCR analysis showed increase of bacterial DNA over the course of five days, indirectly showing that bacterial invasion and growth took place. The CFSE+ (i.e. infected) McCoy cells were sorted from the CFSE- (i.e. non-infected) McCoy cells using fluorescence-activated cell sorting (FACS) while confocal microscopy was performed to visually confirm bacterial invasion and cytosolic localization of CFSE-L. intracellularis. Rabbit hyperimmune serum was generated against each recombinant protein and we investigated the effect that hyperimmune sera had on invasion of IPEC-1 cells. Serum antibodies significantly inhibited invasion and replication of CFSE-bacteria, thus indicating that each of the recombinant proteins is a potential neutralizing antibody target and a candidate for subunit vaccine formulation. In conclusion, we used 2-D gel electrophoresis coupled with mass spectrometry to identify four antigens that, when formulated in a vaccine, may lead to production of neutralizing antibodies and disease protection