37uC before adding 8 ml PBS, 4% paraformaldehyde (PFA) overnight. The cells were acquired (10,000 events) and analyzed with CellQuest Pro v3.3 on a Becton Dickinson FACSCalibur flow cytometer. The percent reduction of green fluorescent protein (GFP) fluorescence was calculated as follows: Percent GFP reduction = (12(Test sample/Positive control))6100). The positive control was ZEBOV-GFP and the negative control was DMEM. The day 21 samples were normalized by subtracting each NHPs day 0 value, therefore the limits of detection for this assay are at or below the zero v alue of the y-axis. ELISPOT Assay Human IFN-c and IL-2 BD ELISPOT assays (BD Bioscience) were performed as per manufacturer’s instructions. Briefly, 26105 PBMCs/well were incubated with 1.5 mg/ml of each ZE- BOVGP1,2 peptide. Spots were quantified by an automated ELISPOT reader (CTL, Cleveland, OH). The ELISPOT response was considered positive when the number of specific spots/million PBMCs was over 5 times the number detected in the dimethylsulfoxide (DMSO) control wells. Immunophenotyping Whole blood (100 ml) was stained with a 100 ml mastermix of antibodies (CD4 PerCP Cy5.5, CD14 PeCy7, CD3 APC Cy7). CD8+ T cells were identified as the CD3+42 population. After 30 minutes, 4uC, red blood cells (RBCs) were lysed with 1.5 ml of 16 FACS lysing solution (Becton Dickinson), vortexed, incubated at room temperature for 10 minutes before centrifugation at 300 g, 10 minutes. The pellet was washed with 1 ml of wash buffer (Dulbecco’s PBS, Invitrogen). Cells were pelleted as before and resuspended in 900 ml of PBS, 4% PFA. Samples were acquired (10,000 events) and analyzed on a BD LSRII flow cytometer using FACS Diva software v5.0.2 (Becton Dickinson). Prior to running, 100 ml of Flowcount fluorospheres (Beckman Coulter) were added for determination of absolute counts. T lymphocyte ZEBOVGP-specific Memory Response For the memory response assays, 56105 PBMCs in 500 ml RPMI supplemented with 10% FBS, 2 mM L-glutamine and 100 U/ml penicillin, 100 mg/ml streptomycin (RPMI-10) were incubated with 1.5 mg/ml ZEBOVGP1,2 peptides, or media, at 37uC, 5% CO2 and measured for their ability to produce IFN-c References 1. Feldmann H, Jones S, Klenk HD, Schnittler HJ (2003) Ebola virus: From discovery to vaccine. Nat Rev Immunol 3(8): 677–685. 2. Geisbert TW, Jahrling PB (2004) Exotic emerging viral diseases: Progress and challenges. Nat Med 10(12 Suppl): S110–21. 3. Mason C (2008) The strains of ebola. CMAJ 178(10): 1266–1267. 4. [Anonymous] (2008) Outbreak news. ebola haemorrhagic fever, uganda–end of the outbreak. Wkly Epidemiol Rec 83(10): 89–90. 5. Towner JS, Sealy TK, Khristova ML, Albarino CG, Conlan S, et al. (2008) Newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda. PLoS Pathog 4(11): e1000212. 6. Leroy EM, Rouquet P, Formenty P, Souquiere S, Kilbourne A, et al. (2004) Multiple ebola virus transmission events and rapid decline of central african wildlife. Science 303(5656): 387–390. 7. Walsh PD, Abernethy KA, Bermejo M, Beyers R, De Wachter P, et al. (2003) Catastrophic ape decline in western equatorial africa. Nature 422(6932): 611–614. 8. Formenty P, Boesch C, Wyers M, Steiner C, Donati F, et al. (1999) Ebola virus outbreak among wild chimpanzees living in a rain forest of cote d’ivoire. J Infect Dis 179 Suppl 1: S120–6. 9. Groseth A, Feldmann H, Strong JE (2007) The ecology of ebola virus. Trends Microbiol 15(9): 408–416. 10. Geisbert TW, Pushko P, Anderson K, Smith J, Davis KJ, et al. (2002) Evaluation in nonhuman primates of vaccines against ebola virus. Emerg Infect Dis 8(5): 503–507. Ebola Specific Immune Response on day 3, or proliferate on day 6. To observe the IFN-c response cells were washed twice with PBS, 2% FBS before blocking with 10 ul 1 mg/ml human c-globulin for 10 minutes at room temperature. Then a 100 ml mastermix of CD45RA PECY5, CD4 APC and CD8 PECy7 was added for 30 minutes, 4uC. Cells were stained for IFN-c using the intracellular staining protocol of BD’s cytofix/cytoperm Plus (with GolgiStop) Kit. Cells were then washed and the pellet resuspended in 1 ml of PBS, 4% PFA. Samples were acquired (100,000 events) and analyzed on the LSRII (Becton Dickinson) using the FACS Diva software v5.0.2. Cell proliferation on day 6 was monitored by a decrease in carboxyfluorsecein diacetate succinimidyl ester (CFSE) staining. On day 0 of the assay, 26106 PBMCs in 200 ml PBS were stained with 200 ul of 3 mM CFSE (Invitrogen) for 8 minutes, 37uC. After adding 400 ul of cold FBS for 1 minute, cells were washed 26 with 10 ml of PBS and resuspended at 16106 cells/ml in RPMI- 10. Then 500 ml of cells were combined with either media (negative control) or 1.5 mg/ml ZEBOV GP1,2 peptides. On the sixth day samples were subjected to the same blocking and surface staining as the day 3 samples before washing cells, then resuspending in 1 ml of PBS, 4% PFA. Samples were acquired (30,000 events) and analyzed as above. Statistics The T test was used to determine significant responses for the Elispot and flow cytometry data. A significant result was when p#0.050. Acknowledgments We wish to thank Shannon Paille for her assistance with the animals, as well as John Copps and Jocelyn Fournier for their veterinary support. We also wish to thank John Rose (Yale University) for kindly providing us with the vesicular stomatitis virus reverse genetics system; Yoshihiro Kawaoka for his ZEBOV GP Monoclonal antibody 12/1.1. Author Contributions Conceived and designed the experiments: XQ LF PLM HF SMJ. Performed the experiments: XQ LF JBA PLM FF DD HF SMJ. Analyzed the data: XQ LF JBA PLM HF SMJ. Contributed reagents/materials/ analysis tools: XQ LF JBA PLM FF DD US HF SMJ. Wrote the paper: XQ LF JBA PLM US HF SMJ. 11. Jaax NK, Davis KJ, Geisbert TJ, Vogel P, Jaax GP, et al. (1996) Lethal experimental infection of rhesus monkeys with ebola-zaire (mayinga) virus by the oral and conjunctival route of exposure. Arch Pathol Lab Med 120(2): 140–155. 12. Johnson E, Jaax N, White J, Jahrling P (1995) Lethal experimental infections of rhesus monkeys by aerosolized ebola virus. Int J Exp Pathol 76(4): 227–236. 13. Geisbert TW, Daddario-Dicaprio KM, Geisbert JB, Reed DS, Feldmann F, et al. (2008) Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with ebola and marburg viruses. Vaccine 26(52): 6894–6900. 14. Jaax N, Jahrling P, Geisbert T, Geisbert J, Steele K, et al. (1995) Transmission of ebola virus (zaire strain) to uninfected control monkeys in a biocontainment laboratory. Lancet 346(8991–8992): 1669–1671. 15. Borio L, Inglesby T, Peters CJ, Schmaljohn AL, Hughes JM, et al. (2002) Hemorrhagic fever viruses as biological weapons: Medical and public health management. JAMA 287(18): 2391–2405. 16. Ignatyev GM, Agafonov AP, Streltsova MA, Kashentseva EA (1996) Inactivated marburg virus elicits a nonprotective immune response in rhesus monkeys. J Biotechnol 44(1–3): 111–118. 17. Chepurnov AA, Chuev I, P’iankov OV, Efimova IV (1995) The effect of some physical and chemical factors on inactivation of the ebola virus. Vopr Virusol 40(2): 74–76. 18. Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, et al. (2007) Ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge. J Infect Dis 196 Suppl 2: S430–7. PLoS ONE | www.plosone.org 10 May 2009 | Volume 4 | Issue 5 | e5547