IM NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC Ebola Specific Immune Response Controls VSVG/MARVGP q day 10* q day 3 & 6* Q day 3* NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC WBC % Lymphocytes % Monocytes %Neutrophils %Eosinophils %Basophils HCT RBC HGB Reticulocytes % Reticulocytes MCV % RDW MCHC MCH Platelets MPV PCT % PDW OR Post Vac q day 3 (2/4)* NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC IN NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC Table 1. Blood Hematology Results for Cynomolgus Monkeys after vaccination and challenge with Ebola Virus. Parameter VSVG/ZEBOVGP Post Chall Post Vac Post Chall Post Vac Post Chall Post Vac Post Chall q day 6–26 (3/4)* NC q day 10,14,19 (3/4)* Pre-vaccination test results for each animal (n = 12) were used to establish an average for each parameter. Post-vaccination and -challenge values were compared to the pre-vaccination average. Significant changes in comparison to this average are indicated (*). When values for each animal were compared to its’ own pre-vaccination values, no changes (NC) were observed in any of the parameters. HCT = hematocrit, RBC = red blood cells, HGB = hemaglobin, MCV = mean corpuscular volume, % RDW = red cell distribution width, MCHC = mean corpuscular hemoglobin concentration, MCH = mean corpuscular hemoglobin, MPV = mean platelet volume, PCT = platelet-crit, % PDW = platelet distribution width. Post Vac = post vaccination, Post Chall = post challenge, OR = oral, IN = intranasal, IM = intramuscular. doi:10.1371/journal.pone.0005547.t001 Due to the high variability across the 2.5 months of sampling it was difficult to determine any significance within or between groups. However, there was a trend towards decreased lympho- cytes and platelets that does correspond to previous reports. Evaluation of the antibody response To characterize the humoral immune response, a virus like particle (VLP) based ELISA was used to determine the levels of ZEBOVGP-specific antibodies. The VSVDG/MARVGP immu- nized animals never developed a detectable antibody response to ZEBOVGP. In contrast, potent antibody responses were detected in all VSVDG/ZEBOVGP immunized animals independent of immunization route (Figure 2). Between days 14 and 21 post- vaccination, all VSVDG/ZEBOVGP immunized NHPs devel- oped high levels of IgA (IM = 1:600; OR = 1:2,900; IN = 1:4,100), IgM (IM = 1:3,400; OR = 1:6,000; IN = 1:8,000), and IgG (IM = 1:6,400; OR = 1:7,200; IN = 1:57,600) against ZEBOVGP. After challenge the IgM titres did not exceed the post-vaccination levels, however, IgG and IgA antibody titres were significantly increased peaking 14 days post-challenge then slowly decreasing before maintaining a relatively high antibody titre up to 9 months. Overall in the post-vaccination response, the IgM titres ranked IN>OR.IM, with an IN titre 2.4 times higher than IM, while IgA and IgG responses ranked IN.OR.IM (IN 6.8 fold .IM) and IN.OR>IM (IN 9.0 fold .IM), respectively. In order to determine the level of neutralizing antibodies (NAb) present in the sera 21 days post-vaccination, a ZEBOV-GFP neutralization assay was performed (Figure 3). The ZEBOV-GFP virus contains the GFP between the NP and VP35 gene [29]. Although the ZEBOV-GFP virus is slightly attenuated in in vivo NHP studies, the growth kinetics in the Vero E6 cell line is virtually indistinguishable from the wild type ZEBOV, suggesting similar levels of infectivity [29,30] The IM immunization produced low but detectable levels of NAb (Figure 3a). In comparison, 3/4 NHPs in the OR group (Figure 3b) demonstrated a 50% reduction in ZEBOV-GFP positive cells at a titre of 1:40 (mean = 66.7%, range:42.3–81.1%). One of the OR immunized NHPs had a 55.5% reduction at the 1:320 dilution. Similarly, the IN route (Figure 3c) resulted in a reduction of ZEBOV GFP positive cells at the 1:40 dilution (mean = 51.4%, range:39.1– 74.8%). Similar results were obtained using the standard plaque neutralization assay (data not shown). Overall, while the OR and IN routes produced ZEBOVGP-specific Nabs, the NAb titres are relatively low, with the OR route producing the highest titres post- vaccination Evaluation of the cellular immune response To evaluate the ZEBOVGP-specific effector cellular immune responses, IL-2 and IFN-c ELISPOT assays were conducted to determine the number of IL-2 and IFN-c secreting lymphocytes PLoS ONE | www.plosone.org 3 May 2009 | Volume 4 | Issue 5 | e5547