Abstract

Traditional vaccination protocols have had limited success, at best, in preventing infectious diseases in marine mammals. Thus, there is a critical need for the development of effective, safe vaccines for use in marine mammals. Nucleic acid vaccines are an effective new way to safely and economically protect humans and animals from diseases caused by viruses, bacteria and parasites. They consist of plasmid DNA containing a sequence encoding for an immunogenic protein. The plasmid is taken up by host cells, where the encoded protein is made and presented to the immune system. Unfortunately, very little is known about how marine mammals will respond to DNA vaccines.

In order to evaluate the humoral immune response in T. truncatus to this type of inoculation, a DNA vaccine was produced using a bacterial β-galactosidase reporter gene expression plasmid. The reporter gene system allows expression of an immunogenic protein that is foreign to the host but is non-pathogenic. In a pilot study, two Navy dolphins were used. One animal was vaccinated with 50µg of pCMVβ, containing theβ-galactosidase gene, and one received 50 µg of an empty, control plasmid, pcDNA3.1+. Each treatment was administered in phosphate buffered saline via intramuscular injection. Blood was collected and serum was harvested prior to inoculation and then every 14 days for one year. Three repeat vaccinations were given every 4 weeks. The samples were screened for β-galactosidase antibodies using indirect enzyme-linked immunosorbent assay (ELISA) with a rabbit anti-T. truncatus Ig secondary antibody and an anti-rabbit antibody conjugated with horseradish peroxidase (HRP). Each sample was analyzed in triplicate. T. truncatus anti-β-galactosidase antibody titers were calculated as the reciprocal of the highest dilution that was two standard deviation above the average optical density of the negative control, a non-vaccinated dolphin.

Results of the pilot study suggest that β-galactosidase antibodies were not produced by the test cetacean, as there was not a significant difference in the 13-galactosidase antibodies produced by the control cetacean. Therefore, another study was conducted changing the amount of the vaccine to 500 µg of plasmid and the route of inoculation to an ultrasound guided intramuscular injection in the longissimus muscle near the cervical lymph node. The vaccination schedule was identical to the initial pilot study. Again, the samples were analyzed by ELISA for 13-galactosidase antibody production. The animal receiving the test plasmid demonstrated high 13-galactosidase antibody titers; however testing archived serum samples from this animal prior to vaccination also showed high titers to β-galactosidase. This high background titer has been seen in other aquatic species, and β-galactosidase may not be the best reporter gene for these studies. Studies are currently underway investigating alternative strategies and reporter genes for nucleic acid transfection in marine mammals.