Our previous studies demonstrated that LcrV DNA vaccines could elicit protective immunity against lethal intranasal Y. pestis
challenge in a Balb/C model [18
]. Significant levels of LcrV-specific antibodies in serum were detected in immunized animals. Preliminary analysis of antibody isotypes has suggested that a Th1-type antibody response may be important in providing better protection [18
]. In the current study, we further examined whether T cell immune responses could be elicited against any of the predicted T cell epitopes. Two positive CD8+ T cell epitopes were identified from animals immunized with the LcrV DNA vaccine. T cell immune responses against these two epitopes were very high, as confirmed by both ELISPOT and ICS assays. More importantly, depletion of CD8+ T cells in LcrV DNA vaccine-immunized mice led to reduced levels of protection when a Y. pestis
intranasal challenge was conducted. This would be the first time that CD8+ T cell epitopes have been identified for the LcrV antigen when CD8+ T cell immune responses were elicited by the Y. pestis
DNA vaccine and also that these epitopes play an important role in protection against lethal Y. pestis
It was somewhat unexpected to see the limited effect of CD4+ T cell depletion on the outcome of the challenge. CD4+ T cells must play a key role in the development of LcrV-specific antibody responses. It is possible that once such antibody responses are established, CD4+ T cells may not directly contribute to the protection of immunized mice at challenge. In the current study, mainly CD8+ T cell epitopes were studied. A more specific analysis on CD4+ T cell epitopes may yield information related to the epitope specificity of CD4+ T cell immune responses.
The role of T cell immune responses in protective immunity against bacterial infections is less understood particularly compared to their role against viral infections. Antibody responses against bacterial toxins are classical examples of the critical protective immunity against infectious diseases caused by exotoxin-producing microorganisms, such as tetanus and diphtheria. For intracellular bacterial infections, such as Listeria
, protective immunity often relies upon the development of cell-mediated immune responses to control the intracellular bacteria [48
]. Although animal tissues obtained at the late stages of the infection revealed aggregates of extracellular bacteria, Y. pestis
has long been considered a facultative intracellular pathogen that replicates within the macrophage phagolysosome in the mammalian host [49
]. Therefore, it is not unexpected that cell-mediated immune responses may be generated during Y. pestis
infection. However, as an acute virulent disease, antibody protection has received much attention as the main mechanism of protective immunity while Y. pestis
-specific T cell responses have been less studied in the past.
In 1970s, Wong et al. demonstrated, for the first time, that T cells isolated from immune mice could produce soluble factors protecting phagocytes from cytolysis upon subsequent encounters with Y. pestis in vitro
]. Later studies reported that these soluble factors may include the Th1 cytokines, IFN-γ and tumor necrosis factor alpha (TNF-α) [25
]. The pre-treatment of phagocytes with these cytokines could reduce bacterial intracellular replication and pre-injecting mice with IFN-γ and TNF-α protects against septicemic plague [25
]. Recent studies demonstrated that cell-mediated immune responses, induced in B cell deficient mice using live Gram-negative Y. pestis
, contributed to the protection against Y. pestis
intranasal infection and transfer of T cells from immune mice to naïve mice could also confer protection [26
]. Reports also demonstrated that T cell responses might participate in vaccine-mediated protection against subcutaneous Y. pestis
]. Collectively, these results strongly support the hypothesis that cell-mediated immunity can contribute to the protection against plague.
Results from the current report provide further evidence to demonstrate the direct link between CD8+ T cells and protection in a well-established mucosal challenge model. Identification of two CD8+ T cell epitopes proved the effectiveness of DNA vaccines to elicit high-quality T cell immune responses and provided useful biomarkers to monitor CD8+ T cell responses in future plague vaccine development. Our data justify more studies using gene-based vaccination approaches, such as DNA vaccines, to elicit protective CD8+ T cell responses, which are difficult to elicit by either killed virus vaccines or recombinant protein-based plague vaccines. A more complete profile of cytokines should be mapped to understand whether Y. pestis-specific CD8+ T cell responses are polyfunctional, i.e., whether multiple cytokines are elicited with the LcrV antigen. Protection studies should also be conducted in non-human primates to establish if gene-based plagues vaccines can elicit better protection against pneumonic plague challenges, an important benchmark for plague vaccine development. T cell immunity, especially epitope-specific CD8+ T cell immune responses should be measured in such non-human primate studies to provide guidance to later human studies.