Abstract

Immunization of aquatic mammals presents several considerable challenges. Nevertheless, for those caring for marine mammals in public, private and governmental facilities, vaccine research and development is becoming a necessary part of preventive medicine efforts. In addition to the many species related immunological hurdles, there are significant safety issues to consider. Adverse affects associated with off-label use of conventional veterinary vaccines are considered by many clinicians and caretakers an unacceptable risk. Conventional vaccine strategies have been investigated, though with varying success immunologically, and with respect to tolerability. Live-attenuated vaccines pose the risk of untoward illness, while killed and subunit vaccines rely heavily on chemical adjuvants for which no safety data exists in marine mammals. Molecular-based vaccines may provide an advantage in this respect. Immunostimulatory (ISS) DNA, also referred to as CpG DNA, has become an attractive new molecular-based immunotherapeutic strategy for use in disparate species. The rationale for this is simple. CpG-oligodeoxynucleotides (ODNs) interact with the mammalian immune system by interacting with the cell surface receptor known as toll-like receptor 9 (TLR 9). TLR 9 is an evolutionarily conserved mechanism among all mammals for pathogen recognition. CpG-ODN stimulate innate immunity through the production of proinflammatory cytokines and the induction of B lymphocyte and NK cell activity. In this way they function as a molecular adjuvant, and when used in combination with antigenic protein, can significantly enhance adaptive immune responses. CpG-ODN may therefore be uniquely suited as a strategy that combines safety, immunogenicity and flexibility across a broad species range. In order to determine the utility of ISS DNA in aquatic mammals, NMMP performed in vitro studies using CpG motifs already recognized as stimulatory in human and murine models. Our goal was to demonstrate three broad phenomenon already established in these terrestrial mammals: 1) that CpG-ODN are biologically active in the cetacean immune system as demonstrated by dose responsiveness, 2) that CpG-ODN are recognized by dolphin innate immune components parallel to those observed for human and rodents and 3) that these responses, so elicited can be correlated with TLR 9 expression. Our results show that CpG-ODNs are indeed immunogenic in dolphins, inducing the production of key cytokines and B cell proliferation in a dose-dependent manner, and that this activity, so elicited, correlated with TLR 9 expression. If proven efficacious, this molecular adjuvant may be put to use in a new vaccine strategy that can be applied broadly among the marine mammal species for which immunization is indicated.