Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

The study population consists of 45 individuals from Belarus (vaccinated three times with BCG) and 17 individuals from Sweden (who did not receive BCG vaccination). The following individuals were enrolled from Belarus: i) individuals with active pulmonary TB (n = 15) were recruited from the Research Institute for Pulmonology and Tuberculosis in Minsk. TB diagnosis was based on patient history, chest X-ray and TST, confirmed by positive acid fast staining (AFS) and subsequent culture (MGIT system). The detailed characteristics of the study population are provided in the Additional file 1: Table S1. The mean age was 39 years, 60% males and 40% females ii) individuals with previous pulmonary TB (n = 15), mean age 40 years, 0% males, 100% females, all had drug-susceptible TB) and iii) clinically healthy individuals (n = 15) with no symptoms of TB at the time of sample collection (mean age 42 years, 20% males, 80% females). These 'healthy individuals' were health care workers and laboratory staff, and many of them had most likely been exposed to M.tb. All subjects included in this study were HIV negative. The current health care policies, diagnostic steps and TB treatment protocols have been recently reviewed and reported for Belarus [2]. Informed consent was obtained from all the subjects and the study was approved by the Local Research Ethics Committee of Belarus (No. 3, 22.07.2008). Individuals from Sweden provided informed consent; their mean age was 20 years (29% males, 71% females, immune cells analysis for M.tb -associated target antigens was approved by the Ethics Committee in Stockholm, Sweden; diary number 2011/863-31/2), all individuals from Sweden tested negative in the QFT-GIT test.

The Mantoux skin test (TST) (Biolek, Kharkov, Ukraine) was used as a standard method for the diagnosis of TB/LTBI. The skin reaction was read after 3 days by measuring the transverse diameter of induration in mm. The classification of tuberculin reaction was: negative result = no infiltration, prick reaction 0-1 mm; doubtful result = any size of hyperemia or papula up to 5 mm; positive reaction = papule or infiltrate from 5 to 21 mm. This readout is according to the Belarus national standard criteria. Sputum was obtained from each TB-patient and acid -fast bacilli were detected by direct microscopy. The positive results in acid-fast-staining (AFS) were confirmed by culture using the BACTEC MGIT 960 (Becton Dickinson, Franklin Lakes, NJ, USA).

The QFT-GIT test was performed according to the manufacturer's instructions (Cellestis, Copenhagen, Denmark). In brief, 1 ml of blood was drawn directly into vacutainer tubes, either pre-coated with saline (negative control), peptides from ESAT-6, CFP-10 and TB-7.7 or PHA (mitogen control). Tubes were incubated for 18-20 hours at 37°C in CO₂ 5% and supernatants were harvested and the amount of IFN-γ produced was measured by ELISA.

Synthetic 15-mer peptide pools were generated (JPT Peptide Technologies, Berlin, Germany) which have been found to bind to MHC class II molecules. The respective protein and peptide targets are listed in Table 1 and the individual peptide sequences are provided in the Additional file 2: Table S2. The recombinant proteins Rv3804c (Ag85A), Rv1886c (Ag85B) and Rv0288 (TB10.4) and peptide pools from Rv0959 (Ag85A/B) and Rv0288 (TB10.4) were kindly provided by the AERAS Global TB Foundation (AERAS, Washington DC, US). Recombinant ESAT-6 (Rv3875) was purchased from Statens Serum Institute (SSI, Copenhagen, Denmark). Recombinant PPE-proteins Rv0754, Rv0978c and Rv1917c [18‐21] were provided by K. N. Balaji, Bangalore, India. PHA (Sigma Aldrich, Stockholm, Sweden) was used as a positive control for IFN-γ production.

The whole-blood assay was performed as described previously [22, 23]. In brief, heparin whole blood was diluted 1:2.5 with serum-free medium (RPMI-1640 supplemented with 2 mM L-glutamine and penicillin-Streptomycin, (Invitrogen, Stockholm, Sweden). 100 μl of diluted blood was plated in duplicates in 96-well, round-bottom tissue culture plates (Nunc, Roskilde, Denmark) pre-coated with 100 μl of the diluted antigens resulting in the final volume of 200 μl in each well and the final whole blood dilution of 1:5. Peptides and proteins were used at the final concentration of 1 μg/ml and 5 μg/ml, respectively. PHA (5 μg/ml; Sigma Aldrich, Stockholm, Sweden) served as a positive control and medium diluted whole blood as a negative control. Cell cultures were incubated at 37°C with 5% CO₂. Supernatants were harvested on day 7 and stored at -80°C until analysis. IFN-γ in cell culture supernatants was determined by a commercially available ELISA assay following the manufactures instructions (IFN-γ Eli-pair, Biosite, Stockholm, Sweden). The detection limit of the assay was 6.5 pg/ml.

PBMCs from non-human primates (NHP) included in our earlier vaccine studies [24] were stained as described for ICS [25]. Peripheral blood samples were obtained from female rhesus macaques (Macaca mulatta) of Chinese origin with an age range between 3 and 4 years. Animals were captive bred for research purposes and housed in the Astrid Fagraeus laboratory at the contained experimental facilities at the Biomedical Primate Research Centre. The local Ethical Committees approved all procedures (protocol DNR238/2006-54 and DEC#551, respectively). PBMCs were isolated from freshly obtained, heparinized peripheral blood by Ficoll-Hypaque density gradient centrifugation and processed. In brief, frozen NHP PBMCs obtained before as well as after BCG/rBCG vaccination were rested overnight and stimulated 6 h with PMA/Ionomycin (Sigma-Aldrich, Stockholm, Sweden) and peptide pools Rv0447c (putative cyclopropane fatty-acid synthase) and mixture of Rv2957 and Rv2958c (possible glycosyl transferase) in a presence of Brefeldin-A (Sigma-Aldrich, Stockholm, Sweden). Cells were stained for cell surface markers using anti-CD3-Pacific Blue (SP34-2), CD4-PerCpCy5.5 (L200) and CD8-APCCy7 (SK1). Subsequently, cells were fixed, permeabilized (IntraPrep Fix/Perm Kit, Beckman Coulter, Marseille, France) and stained with anti-IFN-γ-PECy7 (B27), TNF-α-APC and IL-2-PE (MQ1-17H12) followed by flow cytometric analysis using FACSAria flow cytometer (BD Biosciences, Stockholm, Sweden) and FlowJo software (Tree Star Inc., Ashland, OR).

Results of TST, AFS and culture are shown in Tables 2 and 3, the detailed clinical and microbiological data for the study population with tuberculosis is provided in the Additional file 1: Table S1. A total of 43/45 (96%) of individuals tested were TST positive. Fourteen (93%) of TB patients exhibited a positive TST, including individuals with negative AFS and/or culture (Table 2), yet with typical X-ray signs. 11/15 (73%) of patients with TB tested positive for M.tb culture and 4/15 (27%) were positive in AFS. TST results of the individuals recovered from TB and of healthy participants are shown in Table 3. All participants from Sweden were TST negative (data not shown).

The results of the INF-γ production in response to ESAT-6, CFP-10 and TB7.7 are shown in Tables 2 and 3. 11/15 (73%) of TB+ patients, 9/15 (60%) of the individuals recovered from TB and 6/15 (40%) of healthy donors showed a positive response in a QTF-GIT test. In two TB cases, the QTF-GIT exhibited negative results, although M.tb could be detected by AFS and/or culture (ID 011-5 and 021-1). The cut-off for positive results was 0.35 IU/ml above the Nil (negative) control (set by the manufactures). Blood from the Swedish participants were not tested with QTF-GIT.

Diluted whole-blood cultures were stimulated with the panel of M.tb antigens, followed by detection of IFN-γ production in culture supernatants. We identified differences in the number of responders to specific peptide pools between the groups (Figure 1). Peptides from Rv2957 elicited T cell responses in 7/15 (47%) of healthy individuals, 9/15 (60%) of individuals recovered from TB and in 4/15 (27%) of patients with TB (adjusted p-value = 0.39). Rv2958c was recognized by T cells in 8/15 (53%) healthy individuals followed by those recovered from TB (4/15 = 27%) and TB-patients (3/15 = 20%) (adjusted p-value = 0.39). 5/15 (33%) of healthy participants, 6/15 (40%) of TB-recovered and 3/15 (20%) TB-patients responded to Rv2962c (p-value = 0.71) (Figure 1). The non-adjusted and FDR-adjusted p-values are summarized in the Additional file 3: Table S3. Rv0447c (Cyclopropane fatty acyl phospholipid synthase) induced IFN-γ production in 11/15 (73%) individuals recovered from TB and in 7/15 (47%) TB-patients, in contrast to healthy individuals (4/15 = 27%, p value 0.046, after FDR adjustment: p-value = 0.18) (Figure 1).

Most of the individuals in each group responded to Rv3019 (ESAT-6 like protein) with the strongest IFN-γ production in the group who recovered from TB. In addition, Rv3347c peptides (PPE-family-protein) were most commonly recognized by individuals recovered from TB 8/15 (53%) (Figure 1).

Rv2453c (probable molybdopterin-guanine dinucleotide biosynthesis), associated with non-replicating bacteria, was recognized by PBMCs in 7/15 (47%) healthy individuals and in 10/15 with previous TB (67%). Only 4/15 (27%) of patients with active TB responded to this antigen (adjusted p-value = 0.29). The peptide antigens Rv0288 (TB10.4) and Rv0959 were recognized by most of the Belarusian participants included in the study (Figure 1).

For comparison, we tested blood from 17 healthy, non-BCG vaccinated individuals from Sweden for T-cell reactivity directed against the identical target panel. The pattern of peptide recognition appears to be broader among participants from Belarus compared to the Swedish subjects (Figure 2). Yet, T cells from one of the Swedish participants responded to all antigens tested. Of note, several subjects from Sweden responded to Rv0447c peptides (Figure 2). There was a statistical significant difference in IFN-γ production and in the number of individuals responding to ESAT-6-like protein Rv3019 (p = 0.0267) from Belarus as compared to participants from Sweden.

A panel of M.tb proteins was used to gauge the cellular immune responses in blood from the cohorts in Belarus (healthy, previous TB or TB ± individuals) as well as in blood from the swedish control individuals (Figures 3 and 4). T cells from most of the individuals in all three groups from Belarus responded to the entire protein panel (Rv3804c, Rv1886c, Rv3875, Rv0288, Rv0754, Rv0978c and Rv1917) (Figure 3). There was a significant difference in INF-γ production and specific protein recognition between healthy individuals from Sweden and in healthy individuals from Belarus (Figure 4). Cells obtained from individuals from Belarus exhibited higher IFN-γ production and responded more frequently to antigens Rv3804c (p = 0.0170), Rv1886c (p = 0.0015), Rv3875 (p = 0.0170), Rv0754c (0.0447) and Rv1917c (0.0447) as compared to participants from Sweden. Several proteins used in this study were recognized by participants from Sweden, although the IFN-γ production was lower compared to the healthy individuals from Belarus (Figure 4).

Most the test antigens listed in Table 1 are also expressed by BCG and MOTT. At this point, we did not have access to PBMCs from individuals (i.e. infants) prior to BCG vaccination from Minsk, or as longitudinally sampled PBMCs reflecting the sequence of multiple BCG vaccinations. Although we could not control the exposure to MOTT in non-human primates (NHPs), we resorted to the analysis of more detailed cellular immune responses in PBMCs from NHP prior to and after BCG vaccination as a paradigm for testing polyfunctional T cells responding to M.tb test antigens. In an attempt to understand the immune responses to selected antigens seen in the study population, we performed measurements of intracellular IFN-γ, TNF-α and IL-2 production in CD4+ and CD8+ T cells from NHP in response to peptides from Rv0447c, and a cocktail from Rv2957/Rv2958c peptides using cryo-preserved PBMCs, previously obtained from NHPs included in a vaccine study [24]. As shown in Figure 5, PBMCs from NHPs vaccinated with BCG exhibited both CD4+ T cells and CD8+ T cells producing IL-2, IFN-γ and TNF-α in response to stimulation with Rv0447c (Cyclopropane fatty-acyl phospholipid synthase) and to Rv2957/Rv2958c (Glycosyltransferase). Cytokine production was higher after BCG vaccination in both CD4+ and CD8+ T cells (Additional file 4: Figure S1).