In this study, to obtain an effective DNA vaccine against pneumonic plague, two DNA vaccines were constructed co-expressing the V-Ag or F1-V fusion protein in combination with LTN DNA as a molecular adjuvant. Since Y. pestis
is a facultative intracellular pathogen, Parent M. A. and co-workers suggested that plague vaccines should be designed to maximally prime both cellular and humoral immunity for effective protection [13
]. LTN was selected as a molecular adjuvant because past studies have shown that LTN exhibits both Th1- and Th2-type properties when applied mucosally and parenterally [18
]. LTN is produced by CD8+ T cells, NK cells, and γδ TCR+
IEL, indicating induction of protection immunity against tumors through chemotaxis of T cells and natural killer (NK) cells [32
]. LTN has also been adapted as a molecular adjuvant for development of vaccines against pathogens, including human immunodeficiency virus (HIV) [34
] and avian coccidiosis [35
]. For the development of an effective plague vaccine, we tested LTN as a molecular adjuvant against Y. pestis
. In this study, the mucosal adjuvant effect by LTN to stimulate protective immunity was not as apparent when given nasally. Although nasal immunization with LTN/βgal DNA vaccine plus F1-Ag did appear to confer improved protection against pneumonic plague challenge, this was not significantly different from any of the vaccinated groups. Likewise, for i.m. DNA vaccinated mice, protection conferred by the LTN/βgal DNA vaccine was not significantly different from the LTN/V or LTN/F1-V immunized mice. However, these results show that i.m. immunization with LTN DNA vaccines showed significant improved protection against pneumonic plague challenge than nasally immunized mice. Examination of the supportive Th cells revealed a spectrum of Th1-, Th2-, and Th17-type cytokines. I.m. immunization influenced the production of Th17 cell responses, further supporting the notion that LTN can be used as a molecular adjuvant for vaccine to enhance protective immunity against plague.
In mice immunized with LTN DNA vaccine by either i.n. or i.m. route, Ab responses to F1- and V-Ag began to increase by wk 6. Although three DNA immunizations were insufficient to elevate the anti-F1- and -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 [25
], and the combination of DNA and protein immunizations offers one means to effect optimal immunity to plague. Our results also showed that i.n. and i.m. LTN DNA vaccinations provide sufficient priming effect on induction of immunity to F1- and V-Ag in the peripheral immune compartment, resulting in improved efficacy when compared to nasal application of recombinant F1-Ag alone. Thus, LTN DNA vaccines provide effective priming that ultimately leads to protective immunity against plague.
The stimulation of neutralizing Abs when using LTN adjuvant was less apparent when applied nasally. Nasal LTN DNA vaccinations conferred less protection than the same vaccines given by the i.m. route. These results were unexpected, since we previously showed that Salmonella
] and IL-12-based DNA vaccines [25
] were effective against pneumonic plague challenge. Our results also showed, although serum Ab responses to F1- and V-Ag between i.n. and i.m. LTN DNA-vaccinated mice were similar after boosting with F1-Ag protein, Ab responses induced during the priming phase by the nasal LTN DNA vaccines were slightly lower than those Abs obtained by i.m.-vaccinated mice. Moreover, nasal immunization with LTN/F1-V produced less robust nasal Ab responses when compared to mice similarly immunized via the i.m. route. Although there did appear to be some tissue specificity, the cytokine analysis revealed the Th cell responses to V-Ag in the nasally DNA immunized mice were dampened, particularly the Th1 cell responses, when the same Th cell responses were compared to i.m. immunized mice. Such differences could account for the dampened efficacy by the nasally immunized mice. Thus, the molecular adjuvant, LTN, when given as a DNA vaccine, seems to perform better when given parenterally and provides better protection against pneumonic plague than the same vaccines given nasally. These data differ from that previously shown for the LTN protein when applied nasally with Ag [24
No differences in IgG subclass responses were observed in mice nasally vaccinated with LTN DNA vaccines. However, IgG1 and IgG2a anti-F1-Ag responses were significantly greater than IgG2b responses in i.m.-immunized mice with LTN/V-Ag and LTN/F1-V DNA vaccines. In fact, the LTN/F1-V vaccine stimulated greater IgG2a than IgG1 anti-F1-Ag Abs from those mice primed with the LTN/V vaccine, although both groups were subsequently boosted with F1-Ag protein plus CT. CT is a well-known mucosal adjuvant that stimulates Th2-type responses [38
]. Elevated IgG1 Abs to F1- and V-Ag were induced, which has been previously deemed important since enhanced IgG1 subclass titers to F1- and V-Ag correlated with protection against plague [40
]. Thus, using the described vaccination regimens, mixed Th cell responses were induced supporting the varied IgG subclass responses.
Our results show that immunity to both V- and F1-Ags are required for protection against pneumonic plague evident by the similar levels of protection conferred by mice vaccinated i.m. with LTN/V or LTN/F1-V DNA vaccines plus F1-Ag boosts. These results are consistent with previous observations that a combination or fusion of these Ags has an additive protective effect when used to immunize mice against plague [9
]. In addition, others have also reported that the F1- and V-Ag are considered the most effective candidates for vaccines against plague, although vaccination with each protein alone is sufficient for protecting mice against plague challenges [7
]. Indeed, our Ab results in mice immunized with LTN DNA vaccine expressing V-Ag only or F1-V were consistent with Ab responses obtained in these other studies. Therefore, DNA vaccine expressing a combination of F1- and V-Ag, or as a fusion F1-V-Ag protein, is able to effectively prime for protection against plague.
In summary, this is the first description of LTN as a molecular adjuvant that tests DNA vaccines mucosally and parenterally for plague. Using a bicistronic plasmid encoding LTN plus the vaccine encoding V-Ag or F1-V-Ag, we showed effective priming by i.m. delivery of LTN DNA vaccine followed by booster immunizations with recombinant F1-Ag protein, resulting in protection against pneumonic plague. Th1, Th2, and Th17 cell responses were induced either by mucosal or parenteral vaccination; however, i.m. immunization with the LTN DNA vaccine markedly enhanced Th17 cell immunity when compared to the same vaccines administered nasally. These results suggest LTN can be used as a molecular adjuvant to allow inclusion of a cell-mediated component to enhance protective immunity against plague.