Immunogenicity assays
tetanus
VIRUS, CYTOMEGALOVIRUS, TETANUS, INFLUENZA
All immunogenicity assessments on serum and PBMC samples were performed by ABL. Serum ELISAs were performed using high-binding 384-well ELISA plates coated for 2 h at ambient temperature with either purified ID93 or subunit proteins (RV1813, RV2608, RV3619, or RV3620), each at 1 µg per mL. Plates were then washed three times using Wash Buffer A (Teknova, Hollister, CA) and blocked for 4 h at ambient temperature with blocking buffer (1% w/v bovine serum albumin [BSA] and 0.05% w/v polysorbate 20 in PBS). The blocking buffer was then discarded, and samples were added to each well. Control serum was diluted fourfold, beginning with an initial 1:100 dilution in 0.1% BSA with 0.05% polysorbate 20 in PBS, added to appropriate wells, and incubated overnight at 4 Secretory immunoglobulin A (sIgA) ELISAs were performed with a nasal swab and tear samples collected on Study Days 0, 70, and 224ICS was performed according to the following procedures. PBMCs from study participants were thawed and rested in R10 media (RPMI 1640 with 10% FBS, 1% A short-term B cell ELISpot assay was performed to detect the frequency of circulating antibody-secreting cells. Plates were activated with 35% alcohol, coating appropriate wells with either ID93 capture antigen or IgG/IgA capture antibody (Clone MT91/145, Cat# 3850-3-1000, or Clone MT57, Cat# 3860-3-1000, respectively, MabTech) at 100-fold dilution, and incubated overnight at 4 °C. Plates were then blocked using R10 media. PBMCs were thawed, incubated for 2 h at 37 °C and 5% COA long-term B cell ELISpot assay to detect memory B cells was conducted following the same procedures described above, except that PBMCs were first cultured for 3 days with R10 media supplemented with Human B-Poly-S polyclonal B cell resiquimod/IL-2 stimulator (Cellular Technology Limited) and then plated (2 × 10IFN-γ and IL-10 T cell ELISpot assays were conducted according to the following procedures. Plates were activated with 35% alcohol, coating plates with capture antibodies to either IFN-γ or IL-10 (Clone 1-D1K, Cat# 3420-3-250, or Clone 9D7, Cat# 3430-3-250, respectively, MabTech) at 100-fold dilution, and incubated overnight at 4 °C. Study sample PBMCs and cytomegalovirus, Epstein-Barr virus, influenza virus, and tetanus toxin (CEFT) donor control PBMCs were thawed, counted, and incubated overnight at 37 °C and 5% COThe mycobactericidal activity was studied in whole blood cultures from Day 0, 7, and 224 as previously described