To facilitate the characterization of vaccines against tuberculosis, we have developed a murine in vitro functional assay for evaluating vaccine-induced antituberculosis activity. We reasoned that inhibition of intramacrophage growth of M. tuberculosis
was a direct and relevant end point for assessing the potency of TB vaccines. With this functional assay, multiple protective mechanisms likely contribute to the in vitro reduction in mycobacterial proliferation, including both innate and adaptive immune responses. Importantly, we demonstrated that the vaccine-induced in vitro growth inhibition activity detected in our coculture assay significantly correlates with the protective immunity seen postvaccination in our mouse model of pulmonary tuberculosis and is relevant for estimating the in vivo potency of TB vaccines. In addition, we have shown that the protective responses evoked by different types of vaccines can be assessed using this assay. In the human in vitro functional studies reported thus far, only BCG-induced activity has been evaluated (4
). Our results suggest that the antituberculosis protective immunity evoked by subunit, viral vector, or DNA vaccines should be detectable in similar assays designed to measure vaccine-induced immune responses in human clinical trials. An unexpected finding from our in vivo and in vitro protection studies was the moderate antituberculosis activity detected with the heat-killed BCG vaccine preparation. Although this result was surprising, it is consistent with earlier data reported by Opie and Freund, who showed that injection of heat-inactivated tubercle bacilli was nearly as effective as live BCG immunization in protecting rabbits against infection with M. tuberculosis
A major goal of tuberculosis vaccine research during the past 2 decades has been to identify the correlates of protective immunity against M. tuberculosis
. The identification of protective correlates of immunity would clearly facilitate the evaluation and characterization of new TB vaccines both in preclinical studies and clinical trials. In vaccine studies, molecules that are upregulated (or downregulated) after the immunization are candidates as protective correlates. In our studies, using the in vitro coculture assay, cytokine profiles associated with antituberculosis protective activity were identified. Importantly, the patterns of cytokines that are up or downregulated after immunization with active TB vaccines were similar. For most of the vaccines, GDF15, IFN-γ, IL-21, IL-27, and TNF-α expression levels were upregulated while BMP1, IL-1 and Tnfsf14 expression levels were downregulated (relative to naives) in cocultures of infected BMM
and immune splenocytes. In contrast, the expression of these same cytokines was usually not differentially regulated for in vitro assays using control splenocytes. This result suggests that different types of TB vaccines evoke similar patterns of protective immune mediators in a mouse model of tuberculosis. Interestingly, the BCG-induced cytokine patterns detected with the in vitro assay resembled the cytokine responses seen in the lungs of BCG-vaccinated mice at 10 days after an aerogenic challenge with M. tuberculosis
). In both the in vitro and in vivo experiments, IFN-γ, IL-21, IL-27, and TNF-α were upregulated (after exposure to a M. tuberculosis
infection) in the cells of animals immunized with BCG. The IFN-γ and TNF-α results are not surprising since these cytokines have been shown to be critical components of protective immunity against M. tuberculosis
). However, the roles of IL-21 and IL-27 during a mycobacterial infection are less certain. IL-21 is a type I cytokine which is produced largely by antigen-activated T cells. Although its major functions are to activate CD8 T cells and NK cells and to stimulate B-cell immunoglobulin production, IL-21 can also suppress dendritic cell activity (2
). Based on this immunosuppressive activity of IL-21, it has been suggested that this cytokine plays a pivotal role in the regulation of pathogen-induced immune responses. It has recently been shown that the combination of IL-21 and TGF-β induces proinflammatory Th17 cells (36
). It would be of interest to determine whether TH17 cell-promoting activity is also detected when IL-21 is combined with GDF15, another TGF-β family member that was shown to be upregulated in this study. Similar to IL-21, IL-27 may have an important immune regulatory capacity since it has both proinflammatory and antiinflammatory properties. While IL-27 has been shown to promote inflammation, Th1 responses, and IFN-γ production, it can also inhibit inflammatory responses (19
). Surprisingly, animals deficient in the IL-27 receptor were able to limit M. tuberculosis
infections more effectively than controls and neutralization of IL-27 in vitro led to enhanced antituberculosis activity in human monocytes (26
). Clearly, further studies will be needed to define the precise roles of IL-21 and IL-27 in mediating protective immunity against M. tuberculosis
. Besides allowing the identification of contrasting immune responses to different vaccines, these data permit comparisons of the cytokine responses between highly active and less active vaccine formulations in the mouse model. It is surprising that the only consistent difference we observed in the cytokine profiles for the highly active and moderately effective vaccines was the increased IFN-γ levels seen for the most potent vaccines. We are currently investigating whether the elevated IFN-γ levels are associated with greater protection because of the increased expression of IFN-γ-inducible genes such as the CXCL9 and CXCL10 chemokines.
In addition to identifying cytokine patterns that correlate with vaccine-induced protection, this murine in vitro system should be useful for defining cell subsets and cellular immune mechanisms which contribute to antituberculosis protective immunity. Using a similar murine coculture assay, Cowley and Elkins have demonstrated that double-negative T cells, membrane-bound TNF, and IFN-γ-independent processes partially mediate the anti-Francisella
protective immunity induced by the live vaccine strain of Francisella tularensis
). In preliminary experiments, we found that purified splenic T cells from BCG-vaccinated mice inhibited mycobacterial growth in vitro and that the depletion of CD4 cells but not CD8 cells largely abrogated the protective effect of BCG vaccine (K. Kolibab and S. Derrick, unpublished results). These data are consistent with earlier in vivo and in vitro results which indicated that the BCG vaccine induces a strong CD4 antituberculosis protective response (24
). It should be emphasized that cells from other relevant organs can be utilized in this assay. For example, we have shown that cells from the lymph nodes of BCG-vaccinated mice also inhibit the intramacrophage growth of M. tuberculosis
in the coculture assay (M. Parra, unpublished results). Future experiments with the coculture assay will further examine the relative importance of T-cell subsets from the different relevant organs in mediating the protective immunity induced by the various types of TB vaccine preparations.
In summary, we have described the development of a murine in vitro coculture assay to characterize the protective activity induced by TB vaccines. We established the relevance of the assay by showing that in vivo and in vitro vaccine-induced protection results and cytokine patterns were similar. Given this correlation between in vivo and in vitro activity, we anticipate that the coculture assay will be useful for screening and comparing new TB vaccine preparations and for elucidating antituberculosis protective immune mechanisms. Moreover, as TB vaccines progress through clinical trials, this assay could be adapted to evaluate manufacturing consistency and vaccine stability and to potentially bridge preclinical data to human clinical trial results.