Figure 1.

Production of conserved elements (CE) antigen-specific nonhuman primate T-cells (NHP CE-XTCs) from vaccinated macaques. (A) Macaques received a CE DNA vaccine on vaccination weeks 0 and 4 (open triangles) followed by two additional doses of the CE DNA vaccine co-delivered with a second plasmid expressing full length (FL) SIV Gag and Env on weeks 8 and 20 (closed purple triangles). NHP CE-XTC expansion began 2 weeks after the final CE vaccine dose with 30-50 mL of peripheral blood. Schedule is also shown relative to weeks post-SHIV infection (“SHIV Week”) and post-antiretroviral therapy (ART) initiation (“ART Week”). (B) Following Ficoll separation of week 22 blood draws and a plastic adherence step, the non-adherent fraction was cryopreserved, and an 8-day dendritic cell (DC) culture was initiated (blue bar). NHP CE-XTCs (red bar) were induced by mixing with autologous, CE peptide-pulsed, irradiated DC. DC manufacturing spanned Days -8 through 0, and were added to CE-XTCs on Day 0. Non-adherent PBMC stimulated with phytohemagglutinin (PHA) and IL2 (PHA blasts, green bar) were similarly pulsed with CE peptides and irradiated on Day 11. Finally, GM-K56 cells, typically expanded over a 2-week time course (orange bar), were irradiated and added to the culture along with PHA blasts on Day 11. Day 11 CE-XTC cultures were mixed with irradiated, peptide-pulsed PHA blasts and irradiated GM-K562 feeder cells at a 1:1:4 ratio. This mixed culture was rapidly expanded in G-Rex 100 flasks for at least one week prior to infusion.