In this work, we showed that it is possible to use the IFN-γ-inducing ability of CFP/CpG immunization to design a new vaccine against TB. Our data confirm that a change from a homologous immunization schedule with three doses of CFP/CpG to a prime-boost strategy using CFP/CpG to boost BCG vaccination (BCG-CFP/CpG) conferred significant protection against experimental TB. Aside from establishing this protective effect, which is not observed with homologous immunization, we also verified that heterologous immunization improved the protection conferred by a single BCG immunization. Indeed, BCG-CFP/CpG immunization sustained the restriction of bacillus growth 70 days after challenge, while vaccination with BCG only did not.
BCG immunization is the gold standard to evaluate the protective efficacy of a vaccine in experimental models. Moreover, considering that a BCG homologous booster in humans does not affect protection induced by this vaccine, a new vaccine that can boost immunity in BCG-vaccinated individuals should be very beneficial. It could certainly have an impact on the number of new TB cases in the adult population, especially given that the exclusion of BCG vaccination could have a negative effect on the control of severe forms of childhood TB.
A major impact on vaccine-induced protection in our study was associated with the order of stimulation. When we changed the order of immunization and used a CFP/CpG prime followed by a BCG booster (CFP/CpG-BCG), we noted no additional protection compared to that provided by a single BCG immunization. Considering the larger antigen diversity in live microorganisms than in a protein suspension, we hypothesize that a BCG prime expands a high number of specific T-cell clones and, following a CFP/CpG booster, only CFP-specific clones will be selected. In contrast, when we used a CFP/CpG prime and a BCG booster, the diversity of expanded T-cell clones was more restricted than that of clones which would be expanded following the booster.
An important issue worth mentioning is our concern about Koch's phenomenon (40
). Hernandez-Pando et al. reported the occurrence of Koch's phenomenon when BALB/c mice were vaccinated with large doses of killed Mycobacterium vaccae
but not with low doses (24
). Based on this experimental evidence, we assumed that previous contact with CFP antigens in the presence of a powerful inflammatory property adjuvant (CpG oligodeoxynucleotides) would result in extensive inflammation and necrosis, as observed with a homologous immunization schedule (16
). Indeed, homologous immunization increased the production of IFN-γ locally but did not modify TNF-α secretion. Aside from a Th1 and inflammatory response, we also observed increased levels of IL-4 and IL-5 in the lungs. When we reduced CFP antigen exposure by using a single dose to boost BCG, the magnitude of the inflammatory response decreased, and the pattern shifted from mixed Th1/Th2 to Th1. We suggest that IFN-γ plays a key role in exacerbating inflammation in the presence of IL-4 and IL-5. Indeed, IFN-γ has also been associated with the development of mycobacterium-induced caseous necrosis, with excessive amounts of this cytokine contributing to the pathology observed in tuberculous patients (2
According to Rook et al., progressive TB occurs because the potentially protective Th1 response is converted to an immunopathological response, normally associated with IL-4, which fails to eliminate the bacteria (39
). In experimental murine TB, the presence of at least 10% of IL-4-producing cells exacerbates the toxic effects of TNF-α, including fibrosis and necrosis (25
). Furthermore, in vitro studies have demonstrated that Th2 cytokines mediate an alternative pathway of macrophage activation not aimed at the elimination of intracellular pathogens, because these macrophages increase the expression of transferrin and DC-SIGN receptors, increase IL-10 and TGF-β secretion, and decrease TNF-α and IL-12 production (26
). It has also been reported that Th2 cytokines inhibit apoptotic and autophagic control of intracellular M. tuberculosis
induced by IFN-γ secretion (20
). The interaction of IL-4 and IL-13 with IL-4 receptor alpha induces a signaling via IRS-1 (insulin receptor substrate-1) and STAT6. The IRS-1 signaling activates the Akt pathway, which inhibits the autophagy in murine and human macrophages. The STAT-6 signaling upregulates Bcl-2, which inhibits apoptosis and sequestrates beclin-1, impairing the initiation of autophagy (20
). IL-4 also impairs the effector mechanisms of cytotoxic T cells (35
). In addition, IL-4 also influences the apoptosis of mycobacterium-reactive human lymphocytes in the presence of TNF-α (42
). The increased expression of IL-4 in M. tuberculosis
-activated lymphocytes promotes CD30 expression, which sensitizes the lymphocytes to TNF-α-mediated apoptosis via TRAF2 depletion, and this may be associated with the immunopathology in human TB.
In this way, confirming this detrimental role of IL-4, our data show that in IL-4 KO mice the CFP/CpG homologous immunization conferred significant protection compared to that in nonimmunized mice and also to that in CFP/CpG-immunized WT mice. However, this effect seems not to be mediated by an increase in IFN-γ secretion, once the production levels of this cytokine were similar among all IL-4 KO mice: nonimmunized, CFP/CpG immunized, and BCG-CFP/CpG immunized. It is possible that other factors, such as cytokine receptor modulation or lipid mediator production, might be involved in this IL-4-mediated detrimental role.
Our experiments suggest that the presence of IL-4 in the microenvironment corrupted the Th1 immune response. We hypothesized that, for the reasons described above, the IL-4 secretion impaired the clearance of bacilli that is dependent on the cooperation of Th1 and Th17 immune responses. The persistence of mycobacteria exacerbated the inflammatory response and in addition to the increased influx of IL-4-producing Th2 cells generated a process like a feedback of mixed immune responses. The excess inflammatory response caused tissue damage without bacterial clearance. On the other hand, BCG-CFP/CpG-immunized mice did not exhibit significant IL-4 secretion. We believe that, in this group, low concentrations of IL-4 allowed the effective elimination of the bacteria. This hypothesis can be explained by the fact that we found low ex vivo concentrations of Th1, Th17, and inflammatory cytokines in this group.
Therefore, the protection induced by a BCG-CFP/CpG vaccine was not associated with increased IFN-γ and IL-17 concentrations ex vivo, but it was associated with decreased IL-4, IL-5, IL-10, TNF-α, and TGF-β concentrations. To evaluate whether the reduction in IFN-γ detected ex vivo in lung tissue was due to a reduced migration or a deficiency of the functional ability of Th1 antigen-specific cells, we performed a lung cell culture. Seventy days after challenge, in vitro production of IFN-γ and IL-17 was sustained only in the BCG-CFP/CpG-vaccinated group. In contrast, lung cells of the CFP/CpG group secreted more IL-4 and IL-5 and less IFN-γ than BCG-CFP/CpG-immunized mice did. These data show that the specific Th1 and Th17 cells migrated into the lungs of BCG-CFP/CpG-immunized mice and that the CFP/CpG booster may have acted to sustain the recruitment and activation of these cell populations in a later phase of infection.
Although IFN-γ represents the best protective biomarker, the role of Th17 cells in TB has been described only recently. A protective role against this infection has been attributed to IL-17, one that is strongly associated with the recruitment of Th1-specific cells to the lung in the initial and late phases of infection (27
It is important to emphasize that the low ex vivo detection of IFN-γ and IL-17 in the BCG-CFP/CpG group might be due to the very low production of Th2 and regulatory cytokines locally. In this microenvironment, even low concentrations of IFN-γ could sustain the functional activity of this cytokine and the elimination of bacilli, effects that could impair further and unnecessary IFN-γ and IL-17 production. On the other hand, in the CFP/CpG group, the effects of IFN-γ may have been corrupted by Th2 and regulatory cytokines, and the persistence of bacilli likely induced more IFN-γ secretion.
Finally, experiments employing immunization of IL-4 KO mice confirmed the detrimental role of this cytokine, as the lack of IL-4 appeared to be protective in CFP/CpG-immunized mice after challenge. Thus, beyond IFN-γ, the characterization of additional immunological parameters associated with protection, or which may predict progression of infection, is essential to define levels of protection conferred by distinct vaccine formulations. In this study, we confirmed the essential role of IFN-γ, and we suggest that IL-17 may contribute indirectly to restrict the growth of bacilli, while IL-4 may not. We believe that a better understanding of mechanisms underlying protection against TB, as well as the mechanisms involved in the failure of unsuccessful vaccines, will facilitate the rational design of new TB vaccines.