In this study, to verify the immunomodulatory effect of rapamycin, metformin and aspirin on TB subunit vaccine, we treated the mice with rapamycin, metformin and aspirin separately during the course of subunit vaccine LT69/LT70 immunization and then monitored the vaccine-specific memory T cell responses and long-term protective ability against mycobacterial organism. It was found that rapamycin and metformin treatments enhanced the vaccine-specific T-cellular immune memory responses, and the potentiating effect of rapamycin is better than metformin, while aspirin had no effect. Especially, we found that rapamycin treatment promoted the development of more TCM like cells and enhanced the vaccine’s long-term protective efficacy.
At the 32th week, 28 weeks following immunization, the mice that received LT70 and rapamycin generated significantly higher levels of IFN-γ and IL-2 than the mice received LT70 alone (Fig. 1), whereas the difference was not observed 8 weeks post immunization. The reason might be that rapamycin treatment promoted the formation of vaccine-specific TCM cells which could survive for a long period of time, and their anamnestic response is quick and strong. As a result, the late-secretions of IFN-γ and IL-2 in the mice that received rapamycin maintain a high level. On the contrary, the mice that only received LT70 did not produce enough TCM cells, so that the late-secretions of IFN-γ and IL-2 was low along with the contraction of effector T cells or TEM cells.
In the immunoassay for the detection of the long-term TCM-like response, we observed that the frequencies of IFN-γ+ CD4+ T cells (p < 0.01) and IFN-γ+ CD8+ T cells (p < 0.05) following twice stimulation in the rapamycin treated mice were significantly higher than that in the un-treated mice (Fig. 2). These findings lend the notion that rapamycin treatment during the course of subunit vaccine vaccination indeed favored the generation of vaccine-specific TCM-like cells. In addition, 30 weeks post vaccination, rapamycin treatment in combination with the vaccine resulted in a 0.8 ~ 0.9-log10 CFU better decline in the number of mycobacterial organisms in the lungs than those that received vaccine alone. These results suggest that the mice that received rapamycin plus vaccine conferred superior long-term protective efficacy against infection, which is consistent with the results of the TCM-like cell response. The correlation between TCM and long-term protective immunity against infection also has been shown in other studies [6, 8, 24]. This study also has its limitation that, due to biosafety facility limitations, as we performed M. bovis BCG challenge instead of M. tuberculosis to evaluate protective efficacy. Future studies are needed to further confirm the protective efficacy by M. tuberculosis challenge.
Rapamycin has been reported to increase the quantity and quality of memory CD8+ T cells by promoting the generation of memory precursors at expansion phase and accelerating the effector to memory cell transition at contraction phase. In this study, long-duration of rapamycin treatment (total 53 days) generated stronger vaccine-specific memory response than short-duration treatment (total 25 days) (Fig. 3). The reason might be that the long-duration of rapamycin treatment covered both the expansion and contraction phases of T cell response during vaccination. However, we did not find the difference of protective efficacy between the long-duration and the short-duration treatment 30 weeks post vaccination (Fig. 4). The reason might be that at 30 weeks post vaccination to evaluate the protective ability of TCM cells was not long enough.
Metformin treatments during vaccination enhanced the TB subunit vaccine-specific long-term immunological IFN-γ rather than IL-2 recall response against antigen stimulation, but its enhancement was less than rapamycin (Fig. 1). Moreover, metformin did not promote the long-term TCM-like cell response (Fig. 2). The results indicate that metformin plus TB subunit vaccine only established limited T-cell immune memory response. The results between the mice that received LT70-aspirin and LT70 alone had none difference (Fig. 1, 2), which suggests that aspirin has no improvement on the T-cell immune memory response for the subunit vaccine.
Rapamycin administration has been reported to enhance the long-term protective efficacy of BCG vaccine and tumor vaccines[19–21]. In this study, we observed that rapamycin treatment promoted the development of TCM like cells and hence enhanced the long-term protective efficacy of TB subunit vaccine. Therefore, rapamycin might be used in designing vaccine regimens or as potential adjuvant to enhance vaccine-induced memory response and to prolong the longevity of vaccine’s protective efficacy. Also, repurposing rapamycin as an adjuvant in vaccination can avoid the vast majority of drug development costs. Nonetheless, rapamycin administration is fraught with side effect, such as immunosuppressive effect. Therefore, further studies are required to optimize the delivery system, dosage and timing of rapamycin treatment.
In summary, our findings demonstrate that rapamycin has better effect than metformin on the regulation of T-cellular long-term immune memory responses for TB subunit vaccine. A long duration of low-dose rapamycin treatment promoted the generation and persistence of subunit vaccine-induced TCM-like cells that led to long-term T-cell memory and high protection. These findings might have important implications for developing new vaccination strategies for TB subunit vaccines.