Abstract

The Space and Naval Warfare Systems Center San Diego (SPAWARSYSCEN) trains and cares for approximately one hundred marine mammals, including California sea lions (Zalophus californianus) and Atlantic bottlenose dolphins (Tursiops truncatus). The Navy is dedicated to providing the highest level of care for these animals, including a comprehensive preventive medicine program to protect the animals from disease. Vaccination of individuals within a population is perhaps the single most effective preventive medicine tool. Unfortunately, there are currently no vaccines available for marine mammals. This presents a challenge to our veterinary staff, particularly since infectious diseases are among the most prevalent causes of morbidity and mortality in marine mammals (1,2). Therefore, a primary goal of this project is to investigate the development of safe and effective vaccines for protecting marine mammals from infectious diseases, specifically by utilizing plasmid-based DNA vaccine technology.

Plasmid-based DNA immunization is an effective, low-risk, and relatively inexpensive method of vaccination against common infectious diseases. The ideal plasmid vaccine would encode for a protein product that induced long-lasting, protective immunity against the parent pathogen. To date, several plasmid vaccines have been engineered to contain specific gene sequences that encode for immunogenic, nonpathogenic proteins of infectious organisms, including canine distemper, influenza, and measles (3-5). The uptake and expression of protective proteins from plasmid-based DNA vaccination have been observed in several domestic and exotic animal species, including fish, penguins, pigs, cows, and horses (6-9). We plan to apply plasmid transfection technology to marine mammals, specifically California sea lions and bottlenose dolphins, and assess its value as a tool for vaccine development.

Through collaborative relationships with investigators at Vical, Inc., University of Georgia, University of California at Davis, University of Southern Mississippi, and the Aquarium of the Pacific, six project objectives have been defined and are currently under investigation. First, reagents and assays are being developed to measure both humoral and cellular immune responses to plasmid vaccine administration in bottlenose dolphins and California sea lions (10-11).

Second, potential sites of immunization (i.e. skin, mucous membranes) are being studied for the presence of necessary immune system components for plasmid-based vaccination (12). Third, a reporter gene plasmid is being identified for use in sea lions, and a reporter gene plasmid vaccine is presently being administered to Navy dolphins to demonstrate plasmid transfection. Fourth, major infectious threats to wild and semi-domesticated bottlenose dolphins and California sea lions are being identified, as well as their immunogenic protein gene sequences. Fifth, a plasmid vaccine will be constructed that contains immuno-stimulatory gene sequences of a specified target organism. Finally, the newly constructed plasmid vaccine will be administered and immune response measured with the appropriate assays. The multi-institutional collaboration of scientists and veterinarians greatly strengthens this research effort, and each laboratory is essential in reaching these project goals.

Collaborators

1.  Peter M. Hobart, Gary Hermanson, Mary Wloch
Department of Molecular Biology, Vical, Inc., San Diego, CA 92121

2.  Tracey B. Schock, Tanja S. Zabka, Branson Ritchie
Department of Medical Microbiology and Parasitology, University of Georgia, Athens, GA 30605

3.  Jeff L. Stott, Brian M. Aldridge
Laboratory of Marine Mammal Immunology, University of California, Davis, CA, 95616

4.  Rhonda A. Patterson, Bobby L. Middlebrooks
Department of Biological Sciences, University of Southern Mississippi, USM Box 5018, Hattiesburg, MS 39406-5018

5.  Johanna Sherrill
Aquarium of the Pacific, 100 Aquarium Way, Long Beach, CA 90802

Acknowledgement

The authors thank SPAWARSYSCEN San Diego, the Office of Naval Research (ONR Award #N0001400WX21016), and the National Research Council for their support of this project.

References

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2.  Osterhaus, A.D.M.E., and Visser, I.K.G. 1992. Morbilliviruses and herpes viruses affecting free-living marine mammals. W.A.V.M. 12 th Intemational Symposium: 235-242.

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4.  Fu, T.M., Friedman, A., Ulmer, J.B., Liu, M.A., and Donnelly, J.J. 1997. Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization. J Virol. 71" 2715-2721.

5.  Polack, F.P., Lee, S.H., Permar, S., Manyara, E., Nousari, H.G., Jeng, Y., Mustafa, F., Valsamakis, A., Adams, R.J., Robinson, H.L., and Griffin, D.E. 2000. Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat Med 6:776-81

6.  Kim, C.H., Johnson, M.C., Drennan, J.D., Simon, B.E., Thomann, E., and Leong, J.A. 2000. DNA vaccines encoding viral glycoproteins induce nonspecific immunity and MX protein synthesis in fish. J Virol 74" 7048-7054.

7.  Sherrill, J. 2000. Humoral immune response to DNA-mediated vaccination in African black-footed penguins (Spheniscus demersus). In press.

8.  Van Rooij, E.M., Haagmans, B.L., Glansbeek, H.L., de Visser, Y.E., and de Bruin, M.G. 2000. A DNA vaccine encoding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection. Vet Immunol Immunopathol 74:121-136.

9.  Cantlon, J.D., Gordy, P.W., and Bowen, R.A. 2000. Immune responses in mice, cattle, and horses~to a DNA vaccine for vesicular stomatitis. Vaccine 18" 2368-2374.

10. Zabka, T. S., Anders, B.B., Poet, S.E., Romano, T., and Van Bonn, W. 1999. The basis for assessing the humoral immune response of the Atlantic bottlenose dolphin (Tursiops truncatus) to DNA mediated vaccines. Proceedings 30 th Annual IAAAM Conference: 92-93.

11. Hure, M.C., King, D.P., Aldridge, B.M., Gulland, F., Van Bonn, W., Blanchard, M., and Stott, J.L. 2000. Characterization of monoclonal antibodies to Califomia sea lion immunoglobulin molecules. Proceedings AAZV and IAAAM Joint Conference: 142-143.

12. Zabka, T.S., Poet, S.E., Steffens, W.L., Miller, G., and Romano, T.A. 2000. Immunologic characterization of the skin from the Atlantic bottlenose dolphin (Tursiops truncatus). Proceedings AAZV and IAAAM Joint Conference: 145-146.