Previous reports suggest that a plague vaccine capable of inducing cell-mediated and humoral immunity would be effective against pneumonic plague [13
]. Therefore, in this current study, DNA vaccines were constructed encoding plague V-Ag or a F1-V fusion protein in combination with IL-12 as a molecular adjuvant. IL-12 is produced by APCs, indicating its crucial role for protection against intracellular pathogens through induction of natural killer (NK) cells and Th1 cell responses [17
]. IL-12 has also been adapted as a molecular adjuvant for development of vaccines against intracellular pathogens, such as human immunodeficiency virus (HIV) [25
] and Mycobacterium
], and these reports suggest that protective efficacies are associated with activation of proliferating T cells and IFN-γ production upon Ag-specific stimulation. In this study, although IL-12 DNA vaccinations showed only partial protection against pneumonic plague, IL-12 DNA vaccinations induced significantly greater IgG1 and IgG2a subclass responses to F1- and V-Ag, as well as production of IFN-γ. These results show that IL-12 can be used as a molecular adjuvant for vaccines to enhance protective immunity against pneumonic plague.
In this study, i.m. IL-12 DNA vaccinations induced significant Ab responses to F1- and V-Ags by week 6, but while the IgG anti-V-Ag titers continued to increase, serum IgG anti-F1-Ag Ab titers did not. Boosting nasally with F1-Ag protein was required to induce robust IgG responses. These results were consistent with previous observations that DNA immunization effectively primes the host [26
], and the combination of DNA and Ag immunizations represents one means to effect optimal immunity to plague. Indeed, our results also showed that IL-12 DNA vaccination with nasal boosting with F1-Ag protein afforded greater protection than immunization with F1-Ag protein only. Thus, IL-12 DNA vaccines provided effective priming that ultimately led to protective immunity against plague.
To obtain more effective protection against pneumonic plague, induction of immune responses in the respiratory tract may be required. Indeed, our previous study showed that oral administration of Salmonella
vaccine resulted in stimulation of distal mucosal sites, afforded effective protection, and > 80 % survival against pneumonic plague [21
]. In this study, i.m. IL-12 DNA vaccinations also induced significant Ab responses at mucosal sites, including NP. However, the protective efficacies conferred by i.m. IL-12 DNA vaccinations were less than those conferred by our oral Salmonella
] but better than the same IL-12 DNA vaccine given nasally [20
]. To obtain better protective immunity, enhancing the IL-12-based DNA vaccines may be required. Others have shown that the immunogenicity of DNA vaccines for plague varies depending on the type of eukaryotic promoter/enhancer used or mode of Ag expression, e.g., polymeric form, [29
]. Previous reports have also shown that DNA immunization with a gene gun induces greater Ab responses and more effective protection against plague than by conventional injection methods [29
]. Additional studies using IL-12 DNA vaccine platform to enhance protective immunity against pneumonic plague are warranted.
Our results also showed that IL-12 DNA vaccination induced significantly elevated IgG1 responses, as well as IgG2a Ab responses. A previous report suggests that elevated IgG1 Abs to F1- and V-Ags responses are deemed important since enhanced IgG1 subclass titers to F1- and V-Ag correlated with protection against plague [32
]. In this study, mice were primed with IL-12 DNA vaccine and subsequently boosted with F1-Ag protein plus CT. CT is a mucosal adjuvant capable of inducing Th2-type responses [33
]. Using this vaccination regimen, a mixed Th cell response was induced, as evidenced in our IgG subclass responses and cytokine analyses. Depending upon immunogen used, cytokine responses can vary [15
]. Immunization with live yersiniae has been shown to stimulate elevated IFN-γ responses by immune T cells, and adoptive transfer of these T cells could confer protective immunity against pneumonic plague [15
]. Although other Th cell cytokines may have also been produced, these were not reported [15
], and this further suggests that IFN-γ-producing T cells can contribute to protection to plague. On the other hand, using an attenuated Salmonella
vaccine vector, it has been shown that a mixed Th cell response is obtained against V- and F1-Ags despite the presumed bias imparted by Salmonella
]. It has been suggested that the expression of capsular F1-Ag on the cell surface of Salmonella
may redirect host responses to passenger Ags, thus, eliciting the mixed Th cell phenotype [21
]. Nonetheless, the described IL-12 DNA vaccines also impart a mixed phenotype, which may be, in part, contributed by the nasal co-delivery of F1-Ag with CT adjuvant, a known potent Th2 cell-promoting adjuvant [33
]. Thus, the combination of IL-12 with CT may temper the IL-12-driven Th1 cell responses. An important note is that the amount of IL-12 produced by the DNA vaccines, at best, is in the nanomolar range, whereas, typically soluble IL-12 is given in the micromolar range [35
], resulting in enhanced Th1 cell responses, but also greater toxicity to the host [36
]. Each of these variables could account for the observed mixed Th cell phenotype obtained with the described IL-12 DNA vaccines.
On the other hand, Brandler et al. reports variable Ab responses among different inbred mouse strains, as well as among outbred mice, immunized with plague DNA vaccines, and suggests caution be used in interpreting DNA immunization studies that rely on data obtained from a single mouse strain [37
]. However, BALB/c mice, as in our study, are responsive to DNA vaccines when boosted with proteins, suggesting that the combination of DNA and a protein vaccine approach is required to induce optimal promotion in both humoral and cellular immunity in all mice strains [37
]. Outbred Swiss-Webster mice are unresponsive to any DNA vaccinations [37
]. Our study also showed that the combination of DNA vaccination priming followed by protein boosts induces optimal immune responses against plague.
In this study, priming with IL-12/V-Ag or IL-12/F1-V conferred similar protection against pneumonic plague challenge as long as F1-Ag protein boosts were included in the vaccination regimen. Although these results are discrepant with previous observations that a combination or fusion of F1- and V-Ag has an additive protective effect when used to immunized mice against plague [9
], vaccination with each protein alone also indicates sufficient protection against both bubonic and pneumonic plague [7
]. Our results also showed that Ab responses to F1- and V-Ag in sera and nasal washes from mice immunized with IL-12/V DNA vaccine were elevated or better than mice immunized with IL-12/F1-V DNA vaccine.
In summary, by using a bicistronic plasmid encoding the molecular adjuvant, IL-12, plus the vaccine encoding V- or F1-V-Ag, we showed effective priming using the IL-12 DNA vaccine followed by booster immunizations with recombinant F1-Ag protein. Although these vaccines provided partial protection against pneumonic plague, Th1, Th2, and Th17 cell responses were induced locally, as well as systemically. These results suggest that IL-12 can be used as a molecular adjuvant to allow inclusion of a cell-mediated component to enhance protective immunity against pneumonic plague.