Long lived protection against pneumonic tularemia is correlated with cellular immunity in peripheral, not pulmonary, organs

Abstract

Protection against the intracellular bacterium Francisella tularensis within weeks of vaccination is thought to involve both cellular and humoral immune responses. However, the relative roles for cellular and humoral immunity in long lived protection against virulent F. tularensis are not well established. Here, we dissected the correlates of immunity to pulmonary infection with virulent F. tularensis strain SchuS4 in mice challenged 30 and 90 days after subcutaneous vaccination with LVS. Regardless of the time of challenge, LVS vaccination protected approximately 90% of SchuS4 infected animals. Surprisingly, control of bacterial replication in the lung during the first 7 days of infection was not required for survival of SchuS4 infection in vaccinated mice. Control and survival of virulent F. tularensis strain SchuS4 infection within 30 days of vaccination was associated with high titers of SchuS4 agglutinating antibodies, and IFN-γ production by multiple cell types in both the lung and spleen. In contrast, survival of SchuS4 infection 90 days after vaccination was correlated only with IFN-γ producing splenocytes and activated T cells in the spleen. Together these data demonstrate that functional agglutinating antibodies and strong mucosal immunity are correlated with early control of pulmonary infections with virulent F. tularensis. However, early mucosal immunity may not be required to survive F. tularensis infection. Instead, survival of SchuS4 infection at extended time points after immunization was only associated with production of IFN-γ and activation of T cells in peripheral organs.

Figure 1

Protection of vaccinated mice against pulmonary SchuS4 infection. Mice were immunized (n=10/group) subcutaneously once with LVS (200 CFU). Unvaccinated mice (n=5/group) served as negative controls. Animals were intranasally challenged 30 or 90 days after the last vaccination with approximately 25 CFU F. tularensis strain SchuS4 and survival of infection was monitored. * = p<0.05. Data is representative of three experiments of similar design.

Figure 2

Replication and dissemination of SchuS4 in the lungs and spleens of vaccinated animals. Mice (n=5/group) were vaccinated with LVS and challenged as described above. Unvaccinated, SchuS4 challenged mice (n=5/group) served as negative controls. At the indicated time points lungs and spleens were assessed for bacterial loads following serial dilution of organ homogenates on MMH agar plates. * = significantly different from unvaccinated (p<0.05). Error bars represent SEM. Data is representative of two experiments of similar design.

Figure 3

SchuS4 specific antibodies in vaccinated mice. Mice were vaccinated with LVS as described above. Unvaccinated mice served as negative controls. At the indicated time points, serum was collected from mice (n=3–5/group) via the lateral tail vein. (A) Serum was assessed for IgM, IgG, IgG1 and IgG2a directed against SchuS4 whole cell lysate (WCL) by CBA. (B) Serum was assessed for agglutination of viable SchuS4. Each symbol represents individual mice. Bars represent median titer. Data is representative of three experiments of similar design.

Figure 4

SchuS4 specific production of cytokines in vaccinated animals. Mice were vaccinated with LVS (n=5/group) as described above. Unvaccinated mice (n=5/group) served as negative controls. Thirty and 90 days after the last vaccination lung and spleen cells from vaccinated and naïve mice were added to cultures of resting peritoneal macrophages that had been exposed to SchuS4 WCL the night before. Lung cells and splenocytes added to macrophages that did not receive SchuS4 WCL (no Ag) served as negative controls. Culture supernatants were collected 72 hours later and assessed for IFN-γ, IL-12p70, IL-5 and IL-10 by ELISA. * = significantly different from unvaccinated animals (p<0.05). Error bars represent SEM. Data is representative of two experiments of similar design.

Figure 5

IFN-γ producing NK cells in the lungs and spleens of vaccinated mice after SchuS4 challenge. Mice (n=5/group) were immunized with LVS as described above. Unvaccinated, uninfected mice (naïve) and unvaccinated, SchuS4 infected mice (unvaccinated) served as negative controls. Mice were infected with approximately 25 CFU SchuS4 30 or 90 days after vaccination. Three days after infection splenocytes were collected and were added, in the presence of brefeldin A (10μg/ml) for 5 hours, to cultures of resting peritoneal macrophages that had been exposed to SchuS4 WCL the night before. Cells were stained for surface expression of CD49b and intracellular IFN-γ and analyzed by flow cytometry. * = significantly unvaccinated controls (p<0.05). Bars represent SEM. Data is the result of two independent experiments pooled together.

Figure 6

Presence of activated T cells in vaccinated animals after SchuS4 challenge. Mice (n=5/group) were immunized with LVS as described above. Unvaccinated, uninfected mice (n=5) and unvaccinated SchuS4 infected mice (−) (n=5) served as negative controls. Mice were infected with approximately 25 CFU SchuS4 30 and 90 days after the last vaccination. Four days after infection, lungs and spleens were assessed for presence of CD4⁺CD44^hi and CD8⁺CD44^hi cells by flow cytometry. * = significantly different from controls (p<0.05). Symbols represent individual mice. Data is representative of two experiments of similar design.

Figure 7

IFN-γ producing T cells in the lungs and spleens of vaccinated mice after SchuS4 challenge. Mice (n=5/group) were immunized with LVS as described above. Unvaccinated, uninfected mice (n=4) and unvaccinated, SchuS4 infected mice (−) (n=5) served as negative controls. Mice were infected with approximately 25 CFU SchuS4 30 and 90 days after the last vaccination. Four days after infection, lung cells and splenocytes were collected and, in the presence of brefeldin A (10μg/ml), were added to cultures of resting peritoneal macrophages that had been exposed to SchuS4 WCL the night before. Five hours later, cells were stained for surface expression of CD4 and CD8 and intracellular IFN-γ and analyzed by flow cytometry. * = significantly different from controls (p<0.05). Symbols represent individual mice. Bars represent mean number of cells positive for both the indicated cell surface receptor and IFN-γ. Data is representative of two experiments of similar design.