Each year rabies kills more than 50,000 people and millions of animals worldwide . Although vaccination of dogs, elimination of strays and control of wild animals have been effective in preventing most cases of human rabies in North America and Western Europe, the same approaches have been difficult to implement in parts of the developing world because of cultural, economic and political reasons. Therefore, a pressing need for safe, effective and affordable human rabies vaccines still exists in some developing countries. Plasmid DNA (pDNA) vaccines could fill this gap due to their demonstrated safety, speed and ease of development, and their inherent stability allowing for long-term stockpiling and simplified distribution.
Rabies vaccines of nerve tissue origin have been available for post-exposure use in humans since the end of the 19th century. These vaccines are inexpensive but of relatively low potency requiring a difficult vaccination schedule of 14 to 21 inoculations with two booster doses for post-exposure treatments. In addition, serious side effects are frequently associated with these vaccines, which limit their suitability for pre-exposure prophylaxis. Many developing countries still depend on nerve tissue-derived vaccines. Safe and highly efficacious rabies vaccines produced in cell culture have been available for almost 30 years in affluent countries. Three rabies vaccines are currently available in the United States for human use: human diploid cell vaccine (HDCV); rabies vaccine adsorbed (RVA); and purified chick embryo cell culture vaccine (PCECV). All three vaccines can be used in both pre- and post-exposure applications. In spite of the efficacy and safety profile of cell culture vaccines, manufacturing, storage and distribution costs make them prohibitive for widespread use in many poorer countries. Other rabies vaccine approaches under evaluation include the use of vaccinia virus [2–4] and pDNA [5, 6] to deliver rabies antigens.
The feasibility of pDNA rabies vaccines has been demonstrated in several animal models including mice , dogs , cats , horses  and non-human primates [6, 10]. Interestingly, a single administration of rabies pDNA vaccine in mice was as effective as five doses of a cell culture derived vaccine . Several routes of administration have been tested including intramuscular [5, 9], intradermoplantar  and intradermal [5, 7]. pDNA has also been delivered using the Biolistic® system  as well as by needle injection. Rabies pDNA vaccines have been delivered in PBS [9, 11], Adju-Phos® (aluminum phosphate) , monophosphoryl lipid A  as well as cationic lipid formulations . The choice of formulation can be critical for the effectiveness of the vaccine, as shown by Fischer and co-workers  who demonstrated that a cationic lipid-formulated rabies pDNA vaccine was more effective in eliciting a humoral response than aluminum phosphate or PBS formulation. The proof of concept of pDNA rabies vaccines in animal models suggests that development of an effective pDNA vaccine for human immunization should be achievable.
Protection against rabies infection depends primarily on humoral immunity . Intramuscular injection of pDNA in PBS has been shown to elicit potent cellular but relatively low antibody responses [15, 16]. Therefore, the use of formulations that drive robust antibody responses would be expected to improve the performance of a pDNA rabies vaccine beyond that achievable with pDNA in PBS. Complexation of immunogen-encoding pDNA with cationic lipid systems, such as DMRIE-DOPE and Vaxfectin™  offers a potential enhancement to the relatively weak humoral response elicited by pDNA in PBS. For instance, Hartikka and co-workers  showed a 20-fold increase in antibody titers against influenza nucleoprotein when formulating pDNA with Vaxfectin™ compared to titers obtained with pDNA in PBS. Recently, Hermanson and co-workers  demonstrated the successful protection of rabbits against a lethal dose of aerosolized anthrax spores after immunization with a pDNA:DMRIE-DOPE vaccine. Fischer and collaborators  were able to elicit protective titers in horses after a single injection of pDNA:DMRIE-DOPE. In addition, Fischer demonstrated that titers obtained after administration of rabies pDNA:cationic lipid were higher than those obtained after administration of pDNA:Adju-Phos® and no detectable antibodies were obtained after one injection of pDNA in PBS.
Previous published work [9, 19] indicates that pDNA:cationic lipid formulations could prove successful in raising protective rabies antibody titers in larger mammals. Particularly, Fischer's data on horse pDNA vaccinations  indicate that DMRIE-DOPE could be used as a component of such a vaccine; moreover, pDNA:DMRIE-DOPE vaccines are currently being evaluated in human trials . However, there are no published data on the effect of different cationic lipid formulations on rabies titers. Our results suggest that the cationic lipid Vaxfectin™ appears to be a better candidate than DMRIE-DOPE for formulating a pDNA rabies vaccine. In addition, higher titers were observed at low vaccine doses in mice when DMRIE-DOPE was used at a 4:1 molar ratio (pDNA:cationic lipid) as opposed to the 2.5:1 ratio reported previously . Vaccination of rabbits with pDNA:DMRIE-DOPE resulted in high levels of neutralizing antibodies that persisted for a minimum of 195 days; moreover, data from rabbit studies indicate that protective titers can be obtained with low doses (10 μg) of pDNA:cationic lipid. The fact that protective antibodies were observed 21 days after pDNA administration for all the cationic lipid formulations tested supports the use of rabies pDNA vaccine for post-exposure therapy. In summary, our data suggest that the choice of cationic lipid system as well as the pDNA:cationic lipid ratios can offer significant enhancement to the humoral response to rabies pDNA vaccines.