Extended Data Fig. 1

(A) Representative FACS plots (NHP 4 and NHP 10) and pie charts for each population (% of total CD8⁺ or % of CD8⁺IFNγ⁺, mean for each group (n = 6 per group), week 13 post prime (one week post samRNA boost). Analysis of antigen-specific (IFNγ⁺) memory T cell populations, using CCR7 and CD45RA (Naïve: CCR7⁺CD45RA⁺, purple; Central Memory: CCR7⁺CD45RA^-, blue, Effector memory: CCR7^-CD45RA^-, green; Effector memory RA⁺: CCR7⁺CD45RA⁺, orange). Pie chart represents mean percent of total tetramer population. (B) Analysis of memory T cell populations at 80 weeks post prime (1 year post last boost). Antigen-specific T cells were identified by combinatorial tetramer staining for six different antigens. Memory analysis was performed on total tetramer+ population, using CCR7 and CD45RA (Naïve: CCR7⁺CD45RA⁺, purple; Central Memory: CCR7⁺CD45RA^-, blue, Effector memory: CCR7^-CD45RA^-, green; Effector memory RA⁺: CCR7⁺CD45RA⁺, orange). Pie chart represents mean percent of total tetramer population (n = 6). (C) Antigen-specific T cell response in each individual animal (n=6 animals) 2 years post prime (week 106) and one week post samRNA boost (week 107), for six SIV antigens, as measured by IFNγ ELISpot. SFU/10⁶ to each antigen (mean of three technical replicates per antigen with SD; negative control value subtracted) stacked to provide sum total response. Red asterisk represents values that were too numerous to count and were set to maximum measured value (11000 for TL8, plated at 2.5x10⁴ cells/well, and 3100 for TL9, plated at 1x10⁵ cells/well). (D) Killing of antigen loaded target cells by CD8 T cells (collected 2 weeks post-boost, week 108), relative viability of SIV-loaded PBMCs (as determined by 7-AAD staining), normalized to negative control peptide-loaded PBMCs for each sample, at specified effector to target ratios.

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