Mucosal immunization of mice with a recombinant Salmonella choleraesuis that expresses a multimeric gonadotropin releasing hormone fusion protein

Various modified Salmonella vaccines have been developed to protect against a variety of disease pathogens. Domestic swine can be protected against salmonellosis by administering an oral vaccine using avirulent Salmonella choleraesuis strain 54 (SC54).;Experiment 1 describes the molecular engineering of the pNS2TrcD-rmGnRH expression plasmid. This plasmid is able to propagate in a wide variety of gram-negative bacteria, thus making it an attractive candidate for use in Salmonella. The recombinant multimeric GnRH (rmGnRH) fusion protein encodes for five GnRH amino acid sequences interspersed with four T-cell sequences from various zoonotic disease organisms, making the protein highly foreign and immunogenic to the host immune system. Results from this experiment showed successfully cloned expression plasmid constructs at ∼500bp (rmGnRH) and ∼3500bp (pNS2TrcD-rmGnRH) observed via agarose gel electrophoresis.;Experiment 2 describes the transformation of pNS2TrcD-rmGnRH into Salmonella choleraesuis strain 54 (SC54) and in vitro induced protein expression that shows stably-expressed rmGnRH fusion protein. After the induction, the cells were lysed and the expressed rmGnRH protein was observed via SDS-PAGE and measured for immunoreactivity using immuno-blot analysis. Results showed positive protein expression after 2 hours of induction and a greater concentration of protein was observed after 4 hours. Immunoblot analysis showed immunoreactivity against purified rabbit anti-rmGnRH antibody. As a control, a second immuno-blot showed immunoreactivity against mouse anti-his antibody to detect the 6X histidine tag connected to the protein and to determine if the protein was completely expressed.;Experiment 3 describes the oral delivery of recombinant rSC54 to CD-1 mice with comparative analyses of serology, sperm motility and concentration, and testicular morphology. Mice were assigned to one of 7 treatment groups including rSC54 oral gavage (n=9), rSC54 nasal lavage (n=9), subcutaneous injection of heat-killed rSC54 (n=9), subcutaneous injection of live rSC54 (n=9), intramuscular injection (IM) of purified rmGnRH protein emulsion positive control (n=9), and oral gavage of non-modified SC54 as a control (n=9) and a no treatment negative control group (n=7). Serum samples were collected monthly for 3 months and analyzed for anti-rmGnRH antibody concentrations via enzyme-linked immunosorbent assay (ELISA). Testicular and epididymal tissue were obtained at necropsy and evaluated for sperm concentration and motility, tissue weight, and histological characteristics. Results from the experiment showed a moderate decrease in sperm concentration and testis size, and an antibody response in mice that were orally gavaged with rSC54 and the positive control group when compared to untreated and control mice. Mice that were treated nasally with rSC54 showed a delayed immune response with antibody titers increasing after a boost was administered. Mice that were injected subcutaneously did not generate a significant antibody response compared to negative control mice.;Experiment 4 describes the development of a drying process capable of stabilizing the live rSC54 bacterium in a solid carbohydrate matrix. The process known as "foamation" uses both vacuum and temperature manipulations to arrest cellular machinery, thereby inhibiting degradation of live biologicals within the solid matrix. Two different bacterial carbohydrate matrices using sucrose and trehalose were exposed to ambient (25°C) and above-ambient (37°C) temperatures for three weeks with testing of bacterial viability at 1 week intervals. Differential scanning calorimetry (DSC) was used to measure the melting points of the matrices to determine the limits of thermal stability. The results showed that the majority of bacteria that were stabilized using the foamation process were successfully protected against a prolonged exposure to elevated temperatures of 37°C, while DSC results showed that the carbohydrate matrices remained intact when exposed to higher than ambient temperatures. (Abstract shortened by UMI.).