Abstract

DNA plasmid vaccines represent one of the latest advances in medical technology. They, compared to traditional vaccines, provide a safe, efficacious, multifaceted, thermally stable and inexpensive means of inducing a complete immune response in vivo to protect against specific bacterial, viral and parasitic infections. This technology also can be used to screen for genes encoding protective antigens for pathogenic organisms acquired from the environment or carriers and potentially to treat chronic carriers or immunocompromised patients. Finally, it will contribute to our understanding of the host species antibody response to vaccines. To date, there are no investigations of its application to cetaceans, of which many are a valuable component of captive collections and/or have a threatened or endangered status in the wild. According to the successful application of DNA plasmid vaccines in a variety of other animals, including aquatic species, we hypothesize that the Atlantic bottlenose dolphin (Tursiops truncatus) will mount an immune response to a DNA mediated vaccine as well. Furthermore, we hypothesize that the course of this immune response can be traced using an indirect enzyme-linked immunoassay in which the dolphin serum antibodies will be detected by a rabbit anti-dolphin IgG. Thus, the objectives of this study were to define the immunoassay technique for and assess the safety of the materials used in an innocuous DNA plasmid vaccine for T. truncatus.

The test subjects were two adult, male Atlantic bottlenose dolphins housed at SPAWARSYSCEN SD, California. The animals were clinically evaluated as healthy, both prior to and during this study. One dolphin received an intramuscular injection of a 1 ml sterile phosphate-buffered saline (PBS) solution containing 50 μg of a DNA plasmid. This plasmid (pCMV-β) was composed of the reporter gene encoding for the protein, β-galactosidase, and the promoter/enhancer sequence of the human cytomegalovirus. To serve as the control, the second dolphin was similarly inoculated, but the plasmid (pCDNA-3.1) did not contain the reporter gene. Serum samples were collected biweekly, and repeat vaccinations were administered at four week intervals to ensure adequate uptake into the host cell. In the first part of this study, serum samples from a random dolphin and bovine calf served as the antigen in the indirect ELISA. The results prove that the rabbit anti-dolphin antibody binds specifically and sensitively to the dolphin serum antibodies. In the second part of the study, the antigen for the indirect ELISA was β-galactosidase, because it would bind with any dolphin serum antibodies that were generated against the in vivo expressed β-galactosidase. Thirteen replicate wells of the random dolphin serum were run to determine the cut-off value, which indicated the absence of anti-β-galactosidase antibodies. This value was calculated by averaging the thirteen optical density values at the starting dilution and adding two standard deviations. Each of the inoculated dolphin serum samples was run in triplicate. The anti-β-galactosidase antibody titers were reported as the reciprocal of the serum dilution at which the similarly calculated optical density value was just higher than the cut-off value. The preliminary results may suggest that the test dolphin mounted a subtle immune response when challenged by the pCMV-β vaccine, and the control dolphin did not. This study successfully establishes the laboratory protocol for assessing the efficacy of, supports the safety of the materials used in, and promotes further study of DNA mediated vaccines for T. truncatus.