Ctl imager

Mouse splenocytes were used to enumerate NP-specific antibody secreting cells 21 days post-vaccination. ELISPOT plates (Millipore Sigma cat. no. S2EM004M99) were coated with 10μg/ml recombinant NP (Sino Biological inc, cat. no 11675-V08B) in PBS and then incubated overnight at 4°C. Mouse splenocytes were processed as outlined above and then 3x10⁶ splenocytes in 100μl of media were plated in NP-coated plates in duplicate and then incubated overnight (~16hrs) at 37°C. The following day, the cells were discarded and the wells were washed 5 times with PBS. Following the final wash, 100μl of anti-mouse IgG-HRP, IgG₁-HRP, or IgG_2C–HRP diluted at 1:2000 in PBS were added to each well. Spots were developed using BD ELISPOT AEC substrate set (BD cat. no. 551951) and then visualized using a CTL imager (Cellular Technologies) and were enumerated using an ImageJ cell counter.

NHP T cell ELISPOT reagents were obtained from Mabtech (Cincinnati, OH). VEEV E1 and VEEV E2 specific IFN-γ⁺ T cells were quantified per manufacturer instructions. Positive control wells were stimulated with a 1:1000 dilution of anti-CD3 monoclonal antibody CD3-1 (Mabtech kit catalog no. 3421M-4HST-10). Pooled 15-me peptides with an 11-base overlap spanning the VEEV IAB E1 or E2 envelope glycoprotein (Pepscan, Lelystad, Netherlands) were added to the appropriate wells at 10 µg/mL. Cells were incubated in RPMI media (Gibco) containing 10% HI-FBS (Hyclone) and 5% penicillin and streptomycin (Gibco) for 20 h at 37 °C in 5% CO₂. Positive spots were visualized on a CTL Imager (Cellular Technology Ltd., Shaker Heights, OH) and counting was performed with Immunospot software (CTL).