Purified protein derivative

Two hundred and seven Mtb antigens, selected based on their discriminatory potential for TB diagnosis and their immunogenicity (Franken et al., 2000 (link); Serra-Vidal et al., 2014 (link); Coppola and Ottenhoff, 2018 (link)) were obtained from Dr. Tom Ottenhoff and Kees Franken (Nziza et al., 2022 (link)), and prepared as previously described (Franken et al., 2000 (link); Commandeur et al., 2013 (link)). Purified LAM was obtained from BEI Resources and purified protein derivative (PPD) was received from the Statens Serum Institute. Influenza Virus A, pertussis and gp120 were purchased from ImmuneTech.

As antigen-specific interferon gamma release assay, an ELISpot (T-Spot.TB^® test, Oxford Immunotec, Abingdon, UK) was performed on PBMC as recommended for blood according to the manufacturer’s recommendation (30 ). 200,000 PBMCs were cultured in RPMI medium containing 1% penicillin–streptomycin and 5%-fetal-calf-serum (FCS) at 37°C on a precoated 96-well T-spot.TB^® test plate (Oxford Immunotec). Negative controls were left unstimulated, positive controls were stimulated with anti-CD3-antibody (10 ng/mL, Beckmann Coulter, Brea, CA, USA). Purified protein derivative (PPD) (10 µg/mL, Statens Serum Institute, Copenhagen, Denmark) was utilized to detect cellular immune-responses toward M. tuberculosis, M. bovis BCG, or non-tuberculous mycobacteria (31 (link), 32 (link)). Spot forming cells (SCF) were counted after 18–24 h incubation.
ELISpot assay results were considered conclusive if the number of SFCs in the positive control well was more than 20 SFCs after subtracting the number of spots in the negative control well and had less than twice the number of spots of the negative control well, positive if more than five spots were counted in the ESAT-6 or CFP-10 well after subtracting the number of the SFC in the negative control and if the total number of SFC was at least twice the number of the negative control, and negative in any other case.

HIV-1 Tat protein from IIIB-BH-10 (subtype B) strain was produced in Escherichia coli and prepared as previously reported [20 (link)]. The lipopolysaccharide content of this preparation was measured by a Lymulus amebocyte lysate test and shown to be <0.06 EU/μg of protein. The recombinant (r)Ag85B protein was prepared as previously reported [21 (link)]. The lipopolysaccharide content of this preparation was measured by a Lymulus amebocyte lysate test and shown to be below 4.3 EU/μg of protein. All these reagents were purchased from Diatheva, (Fano, PU, Italy). Purified protein derivative (PPD) was purchased from Statens Serum Institute, (Copenhagen, Denmark).

Four weeks after MTBVAC HK vaccination, mice were euthanized and splenocytes and lung cells collected. 10⁶ cells per experimental timepoint were stimulated with Purified Protein Derivative (PPD) (Statens Serum Institute, SSI) 5 μg/ml during 48 h for supernatants collection and cytokine detection by ELISA. IL17A or IFNγ concentrations were determined with specific ELISA commercial kits (MabTECH). For bronchoalveolar lavage (BAL) collection, trachea was cannulated and BAL was performed with 0.8 ml of ice-cold PBS. Supernatant was separated from cells by centrifugation and frozen at −80°C for further protein detection analysis. For antibodies or J chain determination in BAL, maxisorp ELISA plates (NUNC) were coated with 10 μg/ml of PPD and incubated overnight at 4°C. After a washing step with PBS-Tween20 0.05% (v/v) buffer, plate was blocked with Bovine Serum Albumin 1% (w/v) in washing buffer for 1 h at 37°C. Then, plates were incubated with 100 μl of BAL during 90 min at 37°C. Following washing, plates were incubated for 1 h at 37°C with the corresponding anti IgA, IgG, or IgM antibodies conjugated with Horseradish Peroxidase (HRP) (Sigma Aldrich) at a 1:10000 dilution. Finally, enzyme-substrate reaction was developed using 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma Aldrich) as substrate, and reaction was stopped with H₂SO₄ 0.1N.

ELISpot assays were performed on freshly isolated PBMC, from all volunteers at screening or on day of vaccination and on days 7, 14, 28, 84 and 168 post-vaccination, as previously described [13] (link). Ag85A single pool of 66-peptides (Peptide Protein Research (PPR), UK), IMX313 peptides and C4bp (IMAXIO, France) were all used at a final concentration of 2 μg/ml. Purified protein derivative (PPD), (Statens Serum Institute, Denmark) was used at 20 μg/ml.
ELISpot plates (Millipore) were coated with capture mAb then incubated overnight at 4 °C. 3 × 10⁵ PBMC were incubated for 18–20 h at 37 °C/5% CO₂ with Ag85A (single pool of 66 15mer peptides, overlapping by 10 amino acids) (PPR, UK), purified protein derivative (PPD) (Staten Serum Institute, Denmark), MVA CD4 and CD8T cell epitopes (PPR, UK), IMX313 (IMAXIO, France) and hC4bp (provided by Anna Blom, Sweden). Staphylococcal enterotoxin B (Sigma) was used as a positive control and unstimulated PBMC were used as negative controls. Antigen-specific background subtracted results are presented as spot-forming cells (SFC) per million PBMC.

For T cell assays, overlapping peptide pools targeting ESAT-6 (BEI Resources Cat#NR-50711), CFP-10 (BEI Resources Cat#NR-50712), Ag85A (BEI Resources Cat#NR-34827), Ag85B (BEI Resources Cat#NR-34828), and TB10.4 (BEI Resources Cat#NR-34826) as well as H37Rv M.tb whole cell lysate (BEI Resources Cat#NR-14822) were used. Staphylococcal Enterotoxin Type B (SEB) (List Biological Laboratories Cat#122) was a positive assay control, and dimethyl sulfoxide (DMSO) (Sigma-Aldrich Cat#D8418) was a negative assay control.
For antibody assays, M.tb antigens tested were: purified protein derivative (PPD) (Statens Serum Institute), Ag85A and B in a 1:1 ratio (BEI Resources Cat#NR-49427 and #NR-53526), recombinant ESAT-6 (BEI Resources Cat#NR-49424) and CFP-10 (BEI Resources Cat#NR-49425) in a 1:1 ratio, HspX (BEI Resources Cat#NR-49428), 1-tuberculosyladenosine (1-TbAd) (provided by Dr. Branch Moody), and lipoarabinomannan (LAM) (BEI Resources Cat#NR-14848). An equal mixture of influenza antigens from HA1(B/Brisbane/60/2008) and HA1(H1N1) (A/New Caledonia/20/99) (Immune Technology Corp ITIT-003-001p and IT-003-B3p) was used as a positive assay control.

IFN-γ ex-vivo ELISpot was performed on freshly isolated Peripheral Blood Mononuclear Cells (PBMC) from volunteers in the Starter Group and Group A at: D0, D14, D28, D56, D84 and D168, Group B at: D0, D14, D28, D56, D63, D84, D140 and D224 and Group C at: D0, D14, D28, D42, D56, D119, D126, D147, D203 and D287, as previously described [14] (link) using the Human IFN-γ ELISpot (ALP) kit (Mabtech).
Ag85A-specific responses were measured using a single pool of Ag85A peptides (66 15mer peptides, overlapping by 10 amino acids). Anti-vector responses were measured using ChAdOx1 (2 IU:1 PBMC) (Vector Core Facility, The Jenner Institute, Oxford, UK) and responses to MVA were measured using separate pools of CD4 (27 14-21mer peptides) and CD8 (36 9mer peptides) epitopes from Vaccinia and MVA (Peptide Protein research, UK) (final concentration 2 μg/ml) and Purified Protein Derivative (PPD) (Statens Serum Institute, Denmark) (20 μg/ml). Staphylococcal enterotoxin B (SEB) (Sigma) (10 μg/ml) was used as a positive control and unstimulated PBMC as a negative control.