2.7.1. For HIV Studies

Spleens were isolated from C57BL/6 mice either 10 days post final vaccination for acute time points or 50 days post final vaccination for memory timepoint. Single-cell suspensions of splenocytes were made by homogenizing and processing the spleens through a 40-μm cell strainer. Cells were then re-suspended in ACK Lysing buffer (GibcoTM) for 5 min to lyse red blood cells before two washes with PBS and final re-suspension in RPMI complete media (RPMI 1640 + 10% FBS + 1% penicillin–streptomycin). Two hundred thousand splenocytes were added to each well and stimulated overnight at 37 ◦C in 5% CO2 with R10 (negative control), concanavalin A (3 μg/mL; positive control), or 15-mer HIV envelope clade C peptides overlapping by 11 amino acids (NIH AIDS Research & Reference Reagent Program). Peptide pools consisted of 15-mer residues overlapping by 11 amino acids, representing the entire protein consensus sequence of HIV-1 clade C, were obtained from the NIH AIDS Research and Reference Reagent Program. The Env peptides were pooled at a concentration of 2 μg/mL/peptide into four pools, and three of the four pools were used as antigens for specific stimulation of IFN-γ release.
