In this study, to obtain an effective DNA vaccine against pneumonic plague, several constructs expressing the individual plague Ags or coexpressing them as a F1-V fusion protein in combination with IL-12 DNA as a molecular adjuvant were generated. Since Y. pestis
is an intracellular pathogen, M. A. Parent et al. and Philipovskiy and Smiley suggested that plague vaccines should be designed to maximally prime both cellular and humoral immunity for effective protection (23
). IL-12 was selected as a molecular adjuvant because past studies have shown that IL-12 could exhibit both Th1- and Th2-type properties and enhance IgA production when applied mucosally (4
). IL-12 is produced by antigen-presenting cells, indicating its crucial role for protection against intracellular pathogens through the induction of NK cell activity and Th1 cell responses (1
). IL-12 has also been adapted as a mucosal adjuvant for development of mucosal vaccines against intracellular pathogens, such as human immunodeficiency virus (5
) and Mycobacterium tuberculosis
). For the development of an effective plague vaccine, we tested IL-12 as a mucosal adjuvant against Y. pestis
. Interestingly, our results showed that mice nasally immunized with the IL-12 DNA vaccine, without encoding F1- and V-Ags, but with a F1-Ag protein boost, showed better survival against pneumonic plague than those mice immunized with F1-Ag protein (plus CT) only. These results show that IL-12 can be used as a mucosal adjuvant for vaccines to enhance protective immunity.
Ab responses in mice immunized with IL-12(Low)/F1-Ag, IL-12(Low)/V-Ag, or IL-12/F1-V began to increase by week 6. Although three DNA immunizations were insufficient to elevate the anti-F1-Ag and anti-V-Ag Ab responses, robust Ag-specific responses were induced in mice nasally boosted with F1-Ag protein. These results were consistent with previous observations that DNA immunization effectively primes the host (7
), and the combination of DNA and Ag immunizations represents one means to effect optimal immunity to plague. Other studies have shown the effectiveness of DNA vaccines to plague, but these were all applied parenterally, either via intramuscular injection (9
) or with a gene gun (9
). As with our nasal immunization, multiple deliveries were required. These studies also showed that the immunogenicity of DNA vaccines for plague varied depending on the mode of Ag expression, e.g., polymeric form (30
). Such an approach may be required to enhance F1-Ag's immunogenicity. However, none of these studies evaluated different molecular adjuvants as described in this current study. Thus, our results showed that mucosal IL-12 DNA vaccines provide sufficient priming that leads to protection, and nasal application of recombinant F1-Ag alone is insufficient to confer protection. This priming effect was partly enhanced by IL-12 alone, since priming mice with IL-12(Low)/β-Gal was as effective in conferring protection as immunizing mice with IL-12(Low)/F1-Ag. As anticipated, immunity to pneumonic plague also requires anti-V-Ag immunity. Immunizing mice with IL-12(Low)/V-Ag and boosting with F1-Ag protein were not as effective as immunizing mice with IL-12(Low)/F1-V DNA vaccine and boosting with F1-Ag protein, again suggesting that the DNA-encoded F1-Ag must be priming the host to improve protective immunity.
Our results showed that IL-12 DNA vaccination induces higher IgG1 Ab titers to F1-Ag than the other IgG subclasses. In this study, mice were primed with IL-12 DNA vaccines and subsequently boosted with F1-Ag protein plus CT; such adjuvant combinations have been previously tested (18
). CT, a well-known mucosal adjuvant that induces Th2-type responses (19
), has been used mucosally in combination with recombinant IL-12 (18
). Such combinations induce significant amounts of IgG2a, as well as IgG1 and IgG2b Ab titers, suggesting that IL-12 is an effective mucosal adjuvant to induce Th1 responses (18
); however, such Th1 cell bias was not evident in our study, and it was not evident when F1- and V-Ags were delivered by a Salmonella
vaccine vector (36
). This finding suggests that the potency obtained with the IL-12(Low)\F1-V DNA vaccine is less than when using the recombinant IL-12 (4
). Nonetheless, elevated IgG1 Abs to F1- and V-Ags were induced, which has been previously deemed important, since enhanced IgG1 subclass titers to F1- and V-Ag correlates with protection against plague (34
). Given our findings, the IL-12(Low)/F1-V DNA vaccine mediates a mixed Th cell phenotype, as evidenced in our CFC analyses, and may further push this bias using Th2 cell-promoting adjuvants, as with booster immunizations using F1-Ag protein plus CT, to enable protection against pneumonic plague. On the other hand, Brandler et al. reported variable Ab responses between different inbred mouse strains, as well as outbred mice, immunized with plague DNA vaccines, and suggested caution be used in interpreting DNA immunization studies that rely on data obtained from a single mouse strain (6
). However, BALB/c mice, as in our study, were responsive to DNA vaccines when boosted with protein, suggesting that the combination of DNA and protein vaccine approach was required to induce optimal promotion in both humoral and cellular immunity in all mouse strains (6
). Outbred Swiss-Webster mice were unresponsive to any DNA vaccination (6
). Our study also showed that the combination of DNA vaccination priming followed by protein boosts induced optimal immune responses against plague.
Our results showed that IL-12 DNA(Low) vector encoding F1-Ag plus V-Ag induces greater protection than those encoding only F1-Ag or V-Ag. These results are consistent with previous observations that a combination or fusion of these Ags has an additive protective effect when used to immunized mice against plague (12
). The F1-Ag and V-Ag are considered the most effective candidates for vaccines against plague, and vaccination with each protein alone is sufficient for protecting mice against both bubonic and pneumonic plague (16
In this study, two IL-12 DNA vaccines encoding F1-V fusion protein differed in the amounts of IL-12 produced by 10-fold. Since IL-12p40 has both antagonistic and agonistic effects via binding IL-12Rβ1 (14
), IL-12p40/p70 expression ratios were determined, and no significant differences were noted, suggesting all of the polypeptide was intact IL-12p70. When the efficacies of these vaccines were compared, it was anticipated that the IL-12(High)/F1-V DNA vaccine would show improved protection, but instead the efficacy was lost compared to protection conferred by the IL-12(Low)/F1-V DNA vaccine. Thus, the best protection was obtained using the DNA vaccine for F1-V fusion protein in combination with the low-dose IL-12. It was unclear why the IL-12(High)/F1-V DNA vaccine was less protective, since similar Ab and Th cell responses were induced by both vaccines. Subtle differences were evident in the distribution of IgA AFCs, particularly, in the NALT, SMGs, and PPs, which may contribute to enhanced protection. In addition, perhaps differences in innate immune responses may have contributed to the observed differences in protection.
In summary, this is the first description of a nasal DNA immunization regimen that applies DNA vaccines for pneumonic plague. Using a bicistronic plasmid encoding the molecular adjuvant, IL-12, plus the vaccine encoding F1-V-Ag, we show effective priming using the IL-12(Low)/F1-V DNA vaccine followed by booster immunizations with recombinant F1-Ag protein resulting in protection against pneumonic plague. Both Th1 and Th2 cell responses were induced locally as well as systemically. Although a definitive correlate of protective efficacy to discriminate between the IL-12(Low)/F1-V DNA and IL-12(High)/F1-V DNA vaccines could not be defined, these results suggest that IL-12 can be used as a mucosal adjuvant to allow inclusion of a cell-mediated component to enhance protective immunity against pneumonic plague, albeit the amount of IL-12 is dose dependent.