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. 2018 Feb 26;9:308. doi: 10.3389/fimmu.2018.00308

Figure 2.

Figure 2

IFN-γ secretion after Ag85-ESAT immunization. C57BL/6j and HLA-A2tg mice B6.Cg-Tg (117) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed under specific pathogen-free conditions. Recombinant proteins were overexpressed in E. coli BL21 (DE3) and purified to remove any traces of endotoxin as described in Ref. (116, 118). For the production of the antigen 85B (Ag85B)-early secretory antigenic target (ESAT6), hybrid recombinant hybrid protein, the Ag85B and ESAT6 genes were fused together by PCR with a linker coding for the amino acids NVA. C57BL/6j mice [(A); 13–14 animals per group] and HLA-A2tg mice [(B); 5 animals per group] were immunized three times subcutaneously with Mycobacterium tuberculosis (Mtb) Ag85-ESAT or Mycobacterium leprae Ag85-ESAT recombinant protein (25 µg) adjuvanted with GLA-SE [glucopyranosyl lipid adjuvant-stable emulsion (23) kindly provided by Infectious Disease Research Institute; Seattle, WA, USA; TLR4 agonist; 20 µg]; or CpG (ODN1826 5′-TCC ATG ACG TTC CTG ACG TT -3′; InvivoGen, San Diego, CA, USA; TLR9 agonist; 50 µg) (119). Splenocytes were harvested 4 weeks after final injections and restimulated in vitro with Mtb or M. leprae Ag85-ESAT hybrid recombinant proteins or the single Ag85B and ESAT6 recombinant proteins (all 10 µg/ml). IFN-γ secretion was analyzed by ELISA after 5 days. All mice were analyzed separately. Data shown indicate the mean and SE value of five mice per group.