Assessment of the Humoral Immune Response to Nucleic Acid Vaccination in the Bottlenose Dolphin (Tursiops truncatus)
Tracey B. Schock1; William Van Bonn2; Tracy A.
Romano3; Steven E. Poet4, DVM; Branson W. Ritchie5
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.