Abstract

Immunization using DNA, or nucleic acid vaccination, represents a potentially powerful, low-risk method for protection of valuable aquatic species from common diseases associated with high mortality or morbidity. Studies investigating immune responses to DNA-based vaccines in a variety of animals, including mice, fish, pigs, chickens, cattle, and non-human primates, show great promise for this technology as a new weapon for the prevention of infectious disease. Animals kept in aquaria and zoos may be exposed to a variety of pathogens that can ultimately result in fatalities and endemic infections, sometimes with severe economic implications. Factors contributing to successful plasmid vaccination of certain aquatic species, specifically elasmobranchs, penguins, pinnipeds, and cetaceans, are not well-defined to date. This study investigates humoral responses of African black-footed penguins (Spheniscus demersus) and chain dogfish (Scyliorhinus retifer) inoculated with a plasmid encoding β-galactosidase.

Humoral response to plasmid vaccination was investigated in twelve adult penguins (six males and six females). On day 0, the diluent control group (n=6) received sterile phosphate-buffered saline (PBS) by IM injection, the test plasmid group (n=3) received 50 µg of the test plasmid pCMV-β (containing the gene for β-galactosidase) in PBS, and the control plasmid group (n=3) received 50 µg of pCI (lacking the gene for β-galactosidase) in PBS. Serum was harvested weekly for antibody screening. After 3 weeks, the six birds that received saline were divided equally, assigned to the other two groups, and given the appropriate injection of test or control plasmid. This approach was used to achieve ultimate group sizes of six birds for each of the three conditions, while minimizing the total number of animals (12) devoted to the study. Blood samples were obtained weekly for 6 weeks post-inoculation with plasmid from all birds. On day 21, a booster vaccine (identical to the first vaccine) was given to the 12 plasmid-vaccinated birds.

Penguin sera were screened in duplicate for antibodies against β-galactosidase using ELISA. Anti-chicken IgG, shown to have an acceptable binding efficacy to penguin serum immunoglobulins, was employed as secondary antibody. The geometric mean of ELISA titers for all six penguins of each group (saline, pCI, or pCMV-β) was calculated for each day serum was collected. Results show that penguins inoculated with the pCMV-β construct produced measurable anti-β-galactosidase antibodies when compared to control animals. On days 21, 28, and 42, there was a statistically significant difference (using single factor ANOVA) between the mean of log-transformed titers of control birds (saline- or pCI-inoculated) versus birds vaccinated with the pCMV-β reporter plasmid. These results demonstrate that African black-footed penguins, directly transfected with the gene encoding β-galactosidase, are capable of mounting a humoral response against the in vivo expressed protein.

The portion of the study examining antibody responses of adult chain dogfish to IM plasmid vaccination is in progress. On day 0 and day 21, two groups of dogfish received either 50 µg of the control plasmid pCI (n=3), or 50 µg of the test plasmid pCMV-β (n=4), respectively. Serum was harvested every 7–21 days for 18 weeks post-inoculation. ELISA is being used to screen the dogfish sera for anti-β-galactosidase antibodies, using anti-sandbar shark IgM as secondary antibody. Initial results indicate that β-galactosidase as an antigen may not elicit strong immune responses in chain dogfish. Although DNA vaccination has been investigated in several teleost species, plasmid vaccination in an elasmobranch species has not been reported; thus, data from this trial represent an exciting preliminary look at the humoral immune response of elasmobranchs to nucleic acid vaccines.

This study has demonstrated that African black-footed penguins are capable of immunologically responding to foreign proteins that are transferred in the form of plasmid DNA. Analysis of immune response to DNA-mediated vaccination in chain dogfish is still underway. As an extension of the study reported here, humoral responses to DNA vaccination in pinnipeds (Mirounga angustirostris and Phoca vitulina) and cetaceans (Tursiops truncatus) are currently being investigated at the Marine Mammal Center (Sausalito, CA, USA) and the U.S. Navy Marine Mammal facility (San Diego, CA, USA), respectively. Research at these separate facilities using protocols similar to those reported here should yield intriguing discoveries in the fields of aquatic animal medicine and vaccinology.

Once proven to induce an immune response to well-characterized protein antigens, plasmid vaccination can be used as a tool for screening genes encoding potentially protective antigens from various bacterial, viral, or parasitic pathogens. Knowledge gained from this work will be integral in the future development of nucleic acid vaccines against infectious diseases of aquatic species in both captive and wild environments.

Acknowledgments

This project was supported in part by the Sea Research Foundation in Mystic, CT, USA. The authors wish to thank the members of the Mystic Aquarium husbandry staff whose time and efforts made this study possible, as well as Vickie Burnley and Chanagun Chitmanat of the University of Georgia for excellent technical assistance. We are also grateful to Dr. John Marchalonis at the University of Arizona for his time and the donation of anti-sandbar shark IgM.