Ebola Specific Immune Response Figure 4. PBMCs from immunized animals produce IFN-c or IL-2 in response to ZEBOVGP peptides. ZEBOVGP-specific IFN-c or IL-2 secreting cells from PBMCs were detected by either an (A) IFN-c or (B) IL-2 ELISPOT assay. PBMCs were obtained from cynomolgus macaques immunized with VSVDG/ZEBOVGP either orally (OR; n = 4), intranasally (IN; n = 4), or intramuscularly (IM; n = 2). The control NHPs (n = 2) were immunized IM with the heterologous VSVDG/MARVGP. PBMCs were incubated with 1.5 mg/ml of peptides spanning the entire ZEBOV glycoprotein. Bars represent the average number of ZEBOVGP-specific IFN-c or IL-2 secreting cells detected in each immunized group. doi:10.1371/journal.pone.0005547.g004 (Figure 4). IFN-c is produced by activated T lymphocytes and NK cells. Prior to challenge on days 10 to 14 post-vaccination there was a detectable ZEBOVGP-specific IFN-c response in all immunized animals (Figure 4a). The IM route was the most potent, inducing approximately 2 fold more IFN-c secreting cells than OR (p,0.001) or IN (p = 0.043) routes. However, post- challenge a strong secondary IFN-c response was induced in all VSVDG/ZEBOVGP immunized animals with the IM route producing the most IFN-c cells at day 6 but by day 10 was overtaken by a stronger response in the OR group. The IFN-c in the IN group rose steadily, peaking at day 26 post-challenge with 4.3 and 2 fold more ZEBOVGP specific IFN-c secreting cells than the IM (p = 0.003) and OR (p = 0.075) group, respectively. All three routes produced strong ZEBOVGP-specific IFN-c respons- es, with the IM route dominant early after challenge but surpassed by the OR and IN routes at later time points. Another T-cell cytokine, IL-2, plays a key role during infection by inducing proliferation/activation of CD4+ and CD8+ T cells, potentiates the cytotoxicity of CD8+ T lymphocytes and Natural Killer (NK) cells, and stimulates B lymphocyte function. Post- vaccination, the IM group had more ZEBOVGP-specific IL-2 secreting cells than either of the mucosally immunized groups (Figure 4b). This difference was significant for the IN route on day 10 (p,0.001) and 14 (p = 0.022). Post-challenge the IM route continued to dominate early after challenge peaking on day 10. This difference shows a trend when compared to the IN group (p = 0.067) and is significant when compared to the OR group (p,0.001). Additionally, the IN group had more IL-2 producing PLoS ONE | www.plosone.org 5 May 2009 | Volume 4 | Issue 5 | e5547