We used pools of several MHC class II-binding peptides from M.tb for their ability to stimulate IFN-γ production in vitro using whole blood from Belarusian individuals with a history of previous TB, patients with active pulmonary TB and healthy individuals (most likely TB exposed). In addition, 17 non-BCG vaccinated healthy individuals from Sweden were also included in the study. The selected peptides have shown to be expressed in replicating bacteria (Rv0477c, Rv2940c, Rv1690, Rv1085c, Rv1886, Rv2962c, Rv2958c, Rv2957c), non-replicating bacteria (Rv2453c, yet also Rv0477c, Rv2940c) and are suggested to be able to differentiate between M.tb, MOTT and BCG (Rv3019, Rv0066c, Rv3347c). Some peptide pools (glycosyl transferase) are more frequently recognized by T cells from healthy individuals as compared to TB+ patients suggesting that these were sensitized to mycobacteria. In addition, individuals recovered from TB recognized a broader panel of peptide pools compared to the other two groups (healthy or TB+)which indicates that the responses to M.tb specific antigens apparently persist in individuals who had recovered from pulmonary TB (i.e. long-term memory cellular responses). Of note, the analysis presented in the current report is based on IFN-γ production in PBMCs. The simultaneous measurement of additional chemokines/cytokines, including CXCL10 and IL-10 will most likely increase the discriminatory power and biological value of immunological readouts to M.tb antigens .
One of the strategies in developing new diagnostic methods improving TB vaccine design involves the identification of biomarkers for protection and novel T cell targets. Of particular interest is glycosyltransferase, involved in cellular metabolism and lipid formation. It has been shown that glycosyltransferase genes contribute to M.tb survival in macrophages . Interestingly, the peptide pools from Rv2957c, Rv2958 and Rv2962c were more commonly recognized by T cells from healthy (most likely TB exposed) individuals as compared to TB-patients (Figure 1, see Additional file 3: Table S3), yet larger clinically well defined cohorts are needed in order to provide biologically relevant biomarkers to study M.tb immune responses and to test for novel M.tb vaccine targets.
Other peptide pools from M.tb proteins involved in lipid formation and synthesis of long fatty acids, Rv0477c (Cyclopropane fatty acyl phospholipid synthase) induced IFN-γ production in the majority of PBMCs from individuals suffering from TB (73%) (Figure 1). Since mycolic acids represent a major constituent of the mycobacterial cell wall complex, they provide the first line of defense against potentially lethal environmental conditions. Slow growing pathogenic mycobacteria such as M.tb modify their mycolic acids by cyclopropanation which is a common membrane modification in bacteria and plants [28, 29] and essential for viability, drug resistance and cell wall integrity  playing an important role in M.tb pathogenesis [31, 32].
Chemical inhibition of mycolic acid methyltransferases has shown to be lethal to M.tb and causes alterations in cell envelope structure and drug susceptibility . Thus, Rv0477c may present an interesting target for development of new antibiotics against M.tb. Detectable cellular responses to Rv0477c, although limited, were also found in PBMCs from healthy Swedish controls. It is possible that immune recognition of this antigen is partially primed by exposure to or infection with MOTT since mycolic fatty acids are also present in MOTT. Cyclopropane fatty acids are also present in many other bacterial species expressed in stationary phase cells, including E.coli ; structural homologies may be responsible for cross-reactive T-cell responses. Of interest, a recent report showed that mutations in the rpoS-regulated genes in E.coli (i.e. cfa, cyclopropane fatty acid synthetase and osmB, outer membrane lipoprotein) are significantly higher sensitive to (oxidative) stress and impaired in membrane repair mechanism .
This notion is supported by our observation that constitutive and antigen-specific IFN-γ, IL-2 and TNFα production can be detected by ICS in CD4+ and CD8+ T-cells from NHPs prior to BCG vaccination, yet that BCG lead to a strong expansion of polyfunctional T cells. Future experiments, using T cells from animals housed under sterile conditions may show the contribution of MOTT and their role in providing an immunological matrix which can be boosted by TB vaccination strategies.
While there are no precisely defined correlates of protection against mycobacterial infection at present, IFN-γ producing Th1 cells are believed to be essential in protection against TB [34, 35]. In this study we focused on INF-γ and showed that there is a difference in IFN-γ production between the study groups. At this point, we cannot determine if the IFN-γ production observed in this study was due several BCG vaccinations, due to contact with M.tb in Belarus, or - mutually not exclusive - to related target structures present in other bacterial species.
Rv3804c, Rv1886c and Rv0288 present in M.tb and BCG have been extensively used in different vaccine trials. In general, Rv3804c was most commonly recognized in each group tested. Also several Swedish subjects responded to this antigen. Furthermore, IFN-γ production was higher in response to proteins compared to the peptides (Figures 1, 2, 3, 4). Differences may be due to i) peptide instability, ii) differential antigen processing and presentation and iii) the fact that the peptide pools did not cover the entire protein antigen. These differences may also, in part, explain the strong recognition of the recombinant proteins from the PPE family members Rv0978, Rv0754 and Rv1971c in patients with TB [18–21] in PBMCs from individuals who recovered from TB and also in healthy individuals with no record with TB (yet most likely exposed, since these individuals are health care workers) (Figure 2). The same was true for target recognition from T cells obtained from individuals (TST-, non-BCG vaccinated) from Sweden (Figure 4). These antigens have been reported to provide epitopes differentiating humoral immune responses in individuals with TB. Yet more detailed experiments showed that Rv0978 and Rv0754 recognize TLR2 and induce maturation and activation of human DCs . They enhance the ability of DCs to stimulate CD4+ T-cell responses and activate the ERK1/2, p38 MAPK, and NF-kappaB signaling pathways in DCs and may be instrumental to shape the quality of the innate cellular immune response in exposure to mycobacterial species. This may, in part, explain the strong IFN-γ production in response to the M.tb proteins; corresponding peptide cocktails may not provide these signals leading to DC maturation.
Differences in M.tb antigen recognition may be associated with i) the 'genetic makeup' of the test population, ii) alternate M.tb target molecules in circulating strains, iii) extent of TB disease, previous exposures to M.tb and iv) cross-reactive T-cell responses directed against closely related targets from other bacterial species: only a clinically well defined study population, along with genetic M.tb strain [36, 37] and host immunological marker analysis will aid to visualize a more realistic pattern of anti-M.tb directed immune responses.
We also compared in the current study the cellular immune responses to M.tb-antigens in healthy individuals from Sweden and from Belarus. The number of responders to each antigen was higher and the IFN-γ production was stronger in the group of individuals from Belarus. The difference seen between the groups is most likely a reflection of the environment since Sweden is a low TB endemic country in contrast to Belarus where TB burden is high . Furthermore, individuals from Belarus received two to three BCG vaccinations, which may alter the immune responses to M.tb proteins. None of the Swedish controls were BCG vaccinated and had no identified risk of exposure to TB.
TST is the primary method for diagnosis of latent TB infection in many countries but is not able to distinguish between BCG vaccination and reactions caused by M.tb infection itself. In the present study, viable M.tb bacilli could be detected in some patients with a positive AFS, while the immunological assays used (TST and QFT-GIT) provided negative results (Table 2). In three culture confirmed TB cases, the IGRA tested negative. All the TST positive individuals were positive for QFT-GIT within TB-positive cases. The discrepancy seen between the immunological and bacteriological assays is most likely due to not fully functional T cells from patients with TB. We did not assess the role of different HLA backgrounds in response to selected peptides in this study. Although most of the people in Belarus are Caucasian, it is still likely that some individuals may not have responded to selected target peptide pools.