While most TB vaccines currently in clinical trials target early expressed antigens, targeting latency-associated antigens, like HspX, has recently gained momentum in the field of TB vaccine development.12, 13, 14, 15, 26
In the present study, we tested the ability of HspX protein to induce short- and long-term protective immunity both as a prophylactic vaccine and as a booster to BCG vaccination. We found that native HspX conferred both short- and long-term protection, with long-term protection superior to that conferred by BCG. In fact, native HspX boosted BCG both in terms of protective efficacy and immunogenicity. The protective effect of the native HspX subunit vaccine was concentration-dependent, which also affected the quality of the immune response and extent of immunopathology in the lungs. Interestingly, rHspX was ineffective against infection, possibly due to chaperoning and/or folding differences between the native and recombinant forms of the protein.
Owing to the nature of TB infection, an effective TB vaccine should protect prophylactically, as well as against reactivation of latent TB. HspX is a logical vaccine target antigen because it is produced both during latency and, as our results support, during the early phase of infection in vivo
. Here, the subunit form of the vaccine induced protective immunity within the first 30 days of infection (), suggesting that HspX protein is produced by the bacilli and is detected by the immune response during the early stage of infection, thereby rendering HspX an effective prophylactic vaccine candidate. Futhermore, the concentration of native HspX protein used per dose to vaccinate the animals affected the ability of the vaccine to protect. These data suggest that the concentration of a vaccine antigen may determine its ability to protect and that higher concentrations do not provide better protection. As in the lungs, the 0.1
μg concentration was ineffective in the spleen, indicating that the concentration of the vaccine affected the dissemination of bacilli, and that the optimal concentration for preventing dissemination was 1.0
μg. Compellingly, the native HspX vaccine was able to generate long-term protective immunity that rivaled that of BCG (, P
The vaccinating dose of native HspX also affected the quality of the immune response. Induction of cell-mediated immunity is essential for protection against TB and CD4+
T cells are thought to impart protection by producing T helper cell type 1 cytokines.27
The native HspX vaccine used in the present study, therefore, is a promising vaccine candidate because it induced IFNγ-producing cells both in the short- and long-term models (). The marked increase in the number of HspX-specific spleen cells producing IFNγ 30 days post vaccination compared with almost 10-times as many cells observed after 6 months suggests that the native HspX vaccine induced T helper cell type 1 memory immunity that correlated with a significant reduction in the bacterial load following immunization with the 1.0
μg dose. Expansion of fewer HspX-specific cells in the BCG-vaccinated animals may be explained by the fact that BCG, as a live replicating vaccine, induces a very diverse T-cell pool, and therefore the number of HspX-specific T cells in these mice may be diluted compared with vaccination with a single protein. Moreover, it has recently been shown that multifunctional T cells, that is, T cells capable of simultaneously producing IFNγ, IL-2 and TNFα, are functionally superior to single cytokine-producing T cells,28, 29
and are correlated with protection against TB.30, 31
As shown in , multifunctional CD4+
T cells could be detected in the spleens of BCG- and HspX-vaccinated mice after 30 days and their occurence was concentration-dependent. Only the BCG group maintained these cells over time, which were not necessarily HspX-specific as the cells were stimulated ex vivo
with antibodies against CD3 and CD28. As such, it is not unexpected that BCG would induce a greater pool of multifunctional T cells specific to a wide variety of different antigens.
Besides affecting the quality of the immune response, the concentration of native HspX protein used to vaccinate mice also affected the pulmonary pathology following infection with M. tuberculosis.
Immunization with the 1.0
μg concentration of native HspX resulted in the least amount of pathology (). Although the composition of the infiltrates was similar in all the groups, the inflammatory response in the lungs of the mice vaccinated with the 0.1
μg and 10
μg concentrations (, respectively) was more severe than in the BCG- or 1.0
μg HspX-vaccinated mice, with larger lesions resembling those observed in the saline- and adjuvant-control groups (, respectively). These histological data may lend insight into the observation that, while both the 1.0
μg and 10
μg concentrations of native HspX conferred protection, only the 1.0
μg concentration decreased dissemination of the bacilli to the spleen.
Here, we tested the ability of the native HspX vaccine to boost the protective efficacy of BCG and found that this approach significantly increased the protective efficacy of BCG vaccination alone. Boosting BCG with the native HspX vaccine significantly increased the number of HspX-specific, IFNγ-producing cells in mice, which may have been, in part, the reason the prime-boost approach worked better than BCG alone. Overall, our results confirm data in the literature indicating that HspX, particularly when used in conjunction with BCG, is a promising target for developing vaccine regimens effective against the multiple stages of M. tuberculosis
infection. It has been proposed that the failure of BCG to sterilize the lungs of M. tuberculosis
may be due to its inability to induce long-lived T-cell responses against latency-associated antigens like HspX.32
That said, replacing BCG seems unlikely at this point as it is the most widely distributed vaccine in the world,33
and it confers protection in children. Several investigators have recently tested different vaccine strategies aimed at inducing both prophylactic and therapeutic protection against M. tuberculosis
. Aagaard et al.26
recently tested a ‘multistage' vaccine regimen directed against both early-secreted and dormancy-associated antigens and found it to be protective prophylactically, as well as against reactivation TB disease. Others have employed HspX-based vaccines as a means to improve upon the protective effect of BCG in mice.
Based on these data combined with those from other laboratories, incorporating HspX into vaccine regimens may prove a powerful strategy. Our finding, however, that the rHspX was ineffective against pulmonary TB ( and ), indicated that the engineering of this protein (and possibly others) must be considered in vaccine strategy design. The HspX protein is a member of the α-crystallin-like heat shock protein family34
and functions as a molecular chaperone,25
in part by preventing heat-induced aggregation of proteins in M. tuberculosis
Although it is unclear how this chaperoning function may contribute to the hypoxic response of the bacilli, it has been proposed that HspX may help to stabilize cell structures in the thickening cell wall, thereby allowing the bacilli to survive low oxygen tension within the granuloma.18
In this regard, we hypothesized that protective immunity is generated against a molecule the native HspX protein chaperones, and that this chaperoned molecule is produced by M. tuberculosis
but not by E. coli
. If this hypothesis is correct, the reason rHspX protein failed to protect was because E. coli
did not produce the substrate molecule to which HspX binds, thereby preventing epitope exposure and subsequent generation of a protective immune response when rHspX alone is used as the vaccine. As shown in , the fact that the recombinant protein, when incubated with whole-cell lysate from ΔHspX M. tuberculosis
, was able to confer statistically significant protection against infection, suggested that this hypothesis could be true. Further investigation of potential molecular and functional differences among the native, recombinant and recombinant pulldown proteins is currently in progress.
In summary, the results of this study showed that native HspX subunit vaccination protected mice when used on its own and that protective immunity was long-lived. Perhaps more clinically relevant, though, is that native HspX boosted the protective effect of BCG vaccination, maintaining the positive aspects of BCG while improving upon its shortcomings, namely BCG's inability to stimulate strong T-cell responses against latency-associated antigens. Another important finding of this study was that the native HspX subunit vaccine was protective, while the recombinant form was not, most likely because it chaperoned an immunogenic, mycobacterial molecule produced by M. tuberculosis. Based on the results presented here, the native HspX proves to be a promising multistage vaccine for use in both prophylactic and therapeutic approaches and to boost BCG vaccination.