To date, classical vaccination approaches have not been sufficient to develop highly effective vaccines against tuberculosis. Consequently, many recent experimental immunization strategies against this disease have focused on combining partially effective vaccines to achieve superior levels of efficacy relative to individual vaccination regimens. In particular, heterologous prime/boost regimens have been shown to induce strong and broad cellular immunity and an enhanced quality of T-cell responses (2
). In this study, we showed that a heterologous prime/boost regimen using a TB fusion protein (E6-85) formulated in DDA/MPL adjuvant as a prime and an MVA recombinant virus expressing ESAT6, antigen 85B, antigen 85A, Hsp60, and Mtb39 with IL-15 as an adjuvant (MVA/IL-15/5Mtb) as the boosting agent elicited antituberculosis immunity in the mouse model which was equivalent to the protective responses induced by BCG vaccine. Moreover, using multiparameter flow cytometry, we showed that the E6-85/MVA/IL-15/5Mtb prime/boost regimen evoked diverse CD4 and CD8 T-cell responses. The most protective vaccines in this study, the E6-85/MVA/IL-15/5Mtb combination and live BCG vaccine, induced significantly elevated levels of multifunctional CD4 and CD8 T cells expressing IFN-γ and TNF-α or IFN-γ, TNF-α, and IL-2. These potent vaccines also induced a high frequency of monofunctional cells expressing only IFN-γ. In contrast, the less-protective E6-85/adjuvant formulation generally elicited a more modest MFT-cell and monofunctional response than the more protective vaccination regimens. Interestingly, the median IFN-γ response for the MFT cells detected in these experiments was at least 3-fold higher than the median levels of IFN-γ in cells making only this cytokine. Earlier reports also indicated that IFN-γ concentrations were severalfold higher in MFT cells than in monofunctional T cells (6
). The induction of MFT cells by the E6-85/MVA/IL-15/5Mtb heterologous prime/boost schedule is of interest because recent studies have suggested that T cells which exert multiple effector functions, including concurrent expression of IFN-γ, TNF-α, and IL-2, are functionally superior to monofunctional cells (14
). In mice, the induction of MFT cells by immunization correlated with the extent of protection against Leishmania major
). In humans, the presence of MFT cells has been associated with the control of viremia in nonprogressive HIV infections (1
). Several immune mechanisms have been suggested to explain the presumed superiority of MFT cells (25
). First, MFT cells seem to secrete more IFN-γ than monofunctional cells. Since IFN-γ is a critical cytokine in the defense against mycobacterial infections, elevated IFN-γ concentrations should enhance infection control. Second, the secretion of IFN-γ and TNF-α, another important antimycobacterial immune modulator, from the same cell may increase local intracellular killing. Finally, IL-2 secretion from MFT cells should promote T-cell expansion and enhance long-term survival of memory CD4 cells in vivo
A clear difference in the T-cell response profile evoked by prime/boost immunization with the E6-85/MVA/IL-15/5Mtb vaccine regimen compared to BCG vaccination was the significantly elevated percentages of splenic CD4 T cells making both TNF-α and IL-2 (Fig. ). Lindenstrøm et al. have also reported that a TB subunit vaccine preparation induces long-lived TNF-α/IL-2 double-positive cells, while fewer cells with the same phenotype were identified in the spleens of BCG-vaccinated animals (17
). These data strongly suggest that the type of vaccine administered can impact the quality and magnitude of the vaccine-induced T-cell memory response. The induction of TNF-α/IL-2-positive MFT cells and cells that make only IL-2 in response to MVA/IL-15/5Mtb vaccinations may explain the persistence of the MVA response seen in the 16-month study with the MVA/IL-15/5Mtb vaccine. The failure of BCG to induce this double-positive phenotype may contribute to the waning BCG protective response detected at 16 months postvaccination in the same study. This intriguing observation suggests that further studies should explore the role of the vaccine-induced TNF-α/IL-2-positive T-cell subset in the persistence of antituberculosis protective immunity.
Recent vaccination studies have indicated that increasing the intervals between immunizations may permit enhanced maturation of effector memory cells to more-potent central memory cells prior to boosting, which ultimately should lead to improved vaccine efficacy (4
). In our studies using the MVA/IL-15/5Mtb vaccine candidate as a boosting agent, no impact on the antituberculosis protective responses was observed when the time interval between priming and boosting was increased. With both homologous (1-month versus 7-month boosts) and heterologous (2-month versus 6-month boosts) regimens, the MVA/IL-15/5Mtb vaccine-induced antituberculosis protection was not dependent on the immunization schedule. Even extending the postboosting vaccination-challenge interval to 11 months (Table ) did not reduce the protective effectiveness of the MVA/IL-15/5Mtb vaccine. Interestingly, in humans, the interval between an initial BCG immunization and an MVA85 booster vaccination did not influence the magnitude of the antituberculosis immune response (20
). Since MVA does not replicate in mammalian cells, viral persistence cannot explain the longevity of MVA/IL-15/5Mtb-induced immunity. It is more likely that the capacity of the MVA/IL-15/5Mtb vaccine to induce strong and broad cellular immunity (including IFN-γ, IL-17, and IL-27 expression) (Table ) and to effectively boost memory cell populations diminished any impact increasing (or decreasing) immunization time intervals may have on the induction of protective immunity.
In sum, we have shown that vaccination with our MVA/IL-15/5Mtb vaccine induces highly persistent antituberculosis protective responses. Furthermore, immunization with a heterologous prime/boost regimen using a TB fusion protein formulated with DDA/MPL adjuvant followed by boosting with MVA/IL-15/5Mtb induces substantial protective immunity in a mouse model of pulmonary tuberculosis. Most clinical efforts related to TB vaccines are currently focusing on the development of vaccines to boost BCG responses. While this approach sidesteps concerns about discontinuing BCG use, it also does not resolve safety issues related to BCG immunization in areas to which HIV is endemic and the continued uncertainty about the overall effectiveness of BCG-induced immunity. This heterologous E6-85 protein/MVA/IL-15/5Mtb prime/boost regimen provides an alternative strategy for combating the devastating TB epidemic.