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1 Supporting Information Sui et al..7/pnas.997 Pre-CLP CM9 LA9 SL Tat# Pol Vif % Tetramer + CD + CD + Vac+IL- +IL- Vac Fig. S. Frequencies of six different CD + CD + Mamu-A*-tetramer + cells were measured in the colonic lamina propria weeks after the second modified vaccinia virus Ankara (MVA)- simian immunodeficiency virus (SIV) boost (at week 9). Sui et al. of7

2 A % + CD + CD + pre-vaccine pre-boost *p=. *p=. post-boost *p=. B %IL- + CD + CD + pre-vaccine pre-boost IL- p=. *p=. post-boost *p=. C % + CD + CD + *p=. *p=. *p=. pre-vaccine pre-boost post-boost D MFI (log ) IL- Single *p<. *p=. *p=. IL- Triple E r=.7 F IL- r=.7 G r=. - - LOG(% + /CD + CD + ) - - LOG(%IL- + /CD + CD + ) - - LOG(% + /CD + CD + ) H I J r= -.9 *p= LOG(% + IL- + + /CD + CD + ) CD + r=-. p= LOG(% + CD + ) CD + r= LOG(% + CD + ) Fig. S. Cellular immune responses before viral challenge. (A C) Frequency of CD + CD + T-cell response (positive for γ, IL-, or α) were measured and comparisons among the groups were performed by two-tailed Wilcoxon s rank sum tests. Peripheral blood mononuclear cells (PBMCs) were measured at week (prevaccine), week (preboost), and week 9 (postboost). (D) Comparisons of geometric mean of fluorescent intensity (MFI) of γ, IL-, and α in triplecytokine-producing cells and single-cytokine-producing cells in the PBMCs of macaques receiving vaccine adjuvanted with Toll-like receptor (TLR) agonists and IL-. Comparisons among the groups were performed by repeated measures ANOVA. (E H) Correlations between prechallenge CD + CD + T-cell response frequency (positive for γ, IL-, or α) and set-point viral load (VL) was evaluated by three-decay dose-dependent inhibition curves using animals in groups to(e G) or by linear correlation using all five animals in group (H). (I and J) Correlation between prechallenge γ + CD + T-cell response frequency and set-point VL was evaluated by two-tailed Spearman s rank test (I), or evaluated by a three-decay dose-dependent inhibition curve (J). From D to J, PBMCs were measure weeks after the second rmva-siv boost (at week 9). Sui et al. of7

3 Relative mrna expression levels TRIM *p=. CXCL9 *p= IRF- IL *p=. *p=. *p=. IRF-7 GranzymeB %CD - CD + CD9a + *p=. *p=. *p= MxA %CD - CD + CD9a + Fig. S. Innate immune responses before viral challenge (mesenteric lymph node samples weeks after the second rmva-siv boost, at week 9). Natural killer (NK) cell population (CD - CD + CD9a + ) were measured by FACS, and relative mrna expression level of TRIMα, α, α, β, IRF-, IRF-7, MxA, α, CXCL9, IL-β, and GranzymeB was measured by Taqman real-time RT-PCR using macaque-specific primers/probe sets, which were either purchased from ABI as ready-made sets (TRIMα, α, α, β, IRF-, IRF-7, CXCL9, IL-β, AG, GranzymeB, and α) or kindly provided by Christopher J. Miller (California Primate Research Center, University of California, Davis) (MxA). Comparisons among the groups were performed by two-sided Wilcoxon s rank sum tests. Sui et al. of7

4 TRIM p=.9 p=.97 p=.99 p= LOG(Relative TRIM mrna level) LOG(Relative mrna level) LOG(Relative mrna level) LOG(Relative mrna level) p= LOG(Relative mrna level) CXCL9 p=.7 IRF LOG(Relative IRF- mrna level) IL- p=. IRF LOG(Relative IRF-7 mrna level) GranzymeB p=. MxA LOG(Relative MxA mrna level) NK p=. p=. p=. p= LOG(Relative CXCL9 mrna level) - - LOG(Relative IL- mrna level) - - LOG(Relative GranzymeB mrna level)..... %CD - CD + CD9a + Fig. S. Correlations between set-point plasma VL and the relative mrna expression level of TRIMα, α, α, β, IRF-, IRF-7, MxA, α, CXCL9, IL- β, and GranzymeB in the MLN for all of the animals were evaluated by a two-tailed Spearman s rank test. The animals in different groups were color-coded: group : in red, group : Vac+IL- in blue, group : +IL- in green, group : Vac-only in orange, and group : -adjuvant-only in purple. A % AG + CD+ pdc mdc CD+CD+CD9+ B AG pre-o pre-q post-o post-q pre-q post-q pre-o post-o -actin C Group : Group : Vac+IL- Group : Relative APOBECG mrna level Pre-Vac Post-Vac Relative APOBECG mrna level Pre-Vac Post-Vac Relative APOBECG mrna level Pre-Vac Post-Vac Fig. S. Comparison of AG expression levels in pre- and postvaccination PBMC samples (at week and week 9). (A) Flow cytometry analysis of AG + cell populations from two representative animals in groups and. AG + CD + CD9 + T cells, dendritic cells, and monocyte cell populations were shown. (B) Western blots of AG with lysates of pre- and postvaccination PBMC samples from animals O (group ) and Q (group ). (C) Relative mrna expression level of AG in pre- and postvaccination PBMC samples from animals in groups,, and. Sui et al. of7

5 A B AG + DC fold changes C DC AG + CD+ cells fold changes D CD + monocyte AG + pdc fold changes AG + CD+ T cells fold changes E CD + T pdc AG + CD+CD+CD9+ T cells fold changes AG + mdc fold changes CD+CD+CD9+ F Memory CD + T mdc AG + CD + fold changes G CD + monocyte AG + CD+CD+CD9+ T cells fold changes H Memory CD + T Fig. S. Comparison of AG expression levels in animals from different immunization groups (at week 9). Flow cytometry analysis of AG + cell populations were presented as AG + CD + T cells, CD + CD + CD9 + T cells, dendritic cells, and monocyte cell populations in MLN (A D) or colon intraepithelial lymphocyte (E H) compartments. Sui et al. of7

6 Colon Tissue VL r=-. *p= LOG(AG + %) Fig. S7. Correlation between colon tissue VL and AG expression level in the colon intraepithelial lymphocyte (week 9) was evaluated by a two-tailed Spearman s rank test. Table S. Peptide/MVA-SIV included in vaccine Peptide/MVA-SIV CD + helper and CD + CTL epitopes MHC Peptide. HIV gp VSTVQCTHGIRPVVSTQLLL DRB* Peptide. HIV gp 97 ELYKYKVVKIEPLGVA DRB*w Peptide. SIV Gag 7 GNIYRRWIQLGLQKC DRB*w Peptide. SIV Rev RKRLRLIHLLHQT DRB*w Peptide. SIV Gag -pc GNIYRRWIQLGLQKCCTPYDINQML A* Peptide. SIV Gag 7-Tat GNIYRRWIQLGLQKCSTPESANL A* PCLUS-CL (HIV-env+SIV-Gag): KQIINMWQEVGKAMYAPPISGQIRCTPYDINQML A* PCLUS.-CL (HIV-env+SIV-Gag): DRVIEVVQGAYRAIRHIPRRIRQGLERCTPYTDINQML A* PCLUS-Pol (HIV-env+SIV-Pol): KQIINMWQEVGKAMYAPPISGQIRLGPHYTPKIV A* PCLUS-Gag7 (HIV-env+SIV-Gag7 KQIINMWQEVGKAMYAPPISGQIRLAPVPIPFA A* PCLUS-Tat (HIV-env+HIV-Tat): KQIINMWQEVGKAMYAPPISGQIRKHPGSQPKTA A* PCLUS-Tat (HIV-env+HIV-Tat): KQIINMWQEVGKAMYAPPISGQIRVDPRLEPW A* PLCUS-Vif (HIV-env+SIV-Vif): KQIINMWQEVGKAMYAPPISGQIRQVPSLQYLA A* MVA Gag, Pol, Env MVA-Rev, Tat, Nef CD + CTL epitopes denoted by bold. Sequence based on Belyakov et al () and Dzuris et al ().. Belyakov IM, et al. () Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques. Nat Med 7:.. Dzuris JL, et al. () Molecular determinants of peptide binding to two common Rhesus macaque major histocompatibility complex class II molecules. J Virol 7:9 9. Sui et al. of7

7 Table S. SIV gp antibody titers ( week after SIVmac challenge) OD reading with different dilution factors Group # Animal name : : : : A <. <. <. <. F* K <. <. <. <. P..9. <. U <. <. <. <. B <. <. <. <. H <. <. <. <. L*..9.. Q <. <. <. <. V*....7 E <. <. <. <. G.9. <. <. M <. <. <. <. T <. <. <. <. W.... C <. <. <. <. I <. <. <. <. N <. <. <. <. R <. <. <. <. X <. <. <. <. D <. <. <. <. J <. <. <. <. O <. <. <. <. S <. <. <. <. Y <. <. <. <. *Animals with significant antibody titers to gp. Table S. MHC types of the Rhesus macaques included in the study () Group Animal ID Weight (kg) Sex A A A A B B B B B7 B9 A.9 M F. M + + K.9 M + P. M U. M + + B. M H. M + + L. M + + Q. M + + V. M + + E M G. M + + M. M + W. M + + T. M + C.7 M I.9 M N. M + R M + X.7 M D. M J. M + O. M + + S.7 M + Y. M +. Knapp LA, Lehmann E, Piekarczyk MS, Urvater JA, Watkins DI (997) A high frequency of Mamu-A* in the rhesus macaque detected by PCR with sequence-specific primers and direct sequencing. Tissue Antigens :7. Sui et al. 7of7