cells than the OR group (p = 0.090) on day 10 post-challenge. By day 26 post-challenge all three routes continued to produce a ZEBOVGP-specific IL-2 response, however the IN group response was strongest with a ranking of IN.IM>OR (IN vs. IM p = 0.051; IM vs. OR p = 0.838). At day 26 post-challenge the IN group had the most potent IFN-c and IL-2 responses, as well as the highest IgA and IgG antibody titre, indicating this immuni- zation route, followed by a ZEBOV challenge, results in the development of potent and sustained effector responses. Absolute white blood cell counts following ZEBOV challenge In immunized animals a secondary immune response should occur upon ZEBOV challenge resulting in a rapid amplification of lymphocytes. However, previous reports on ZEBOV infections in unvaccinated animals demonstrate a decrease in lymphocytes possibly due to apoptosis [30–32]. In order to compare the changes in cell numbers between the immunization routes, and also confirm that lymphocytes were not lost in vaccinated animals following ZEBOV challenge, absolute lymphocyte numbers for CD3+, CD4+, and CD8+ (CD3+4 2 ) T cell populations were determined by flow cytometry (Figure 5). As expected, in the VSVDG/MARVGP (control) vaccinated animals who are not protected from ZEBOV, the lymphocyte numbers decreased 28– 57% (Figure 5a: CD3+:day 3 Q0.586, p = 0.001; day 6 Q0.726, p = 0.015. Figure 5b:CD4+:day 3 Q0.586, p = 0.003; day 6 Ebola Specific Immune Response Q0.436, p = 0.002. Figure 5c:CD8+:day 3 Q0.576, p = 0.003, day 6 q1.046, p = 0.642) on days 3–6 compared to day 0 post- challenge data. In contrast, there was no decrease in the lymphocyte populations for any of the VSVDG/ZEBOVGP vaccinated NHPs (Figures 5a–c). Comparison within VSVDG/ ZEBOVGP inoculation routes demonstrated no significant changes from day 0 in either the IM or IN groups, although numbers of both CD4+ (Figure 5b) and CD 8+ (Figure 5c) T cells did increase on day 3 post-challenge in the IN group (CD4+: day 3 IN q1.226, p = 0.433; CD8+: day 3 IN q1.396, p = 0.053). The most substantial increases in total CD3+, CD4+, and CD8+ numbers were on day 3 (Figure 5a:CD3+: day 3 q1.686, p = 0.032; Figure 5b:CD4+: day 3 q1.566, p = 0.093; Figure 5c:CD8+: day 3 q1.806, p = 0.025) in the OR group, that then returned to normal levels by day 6. VSVDG/ ZEBOVGP vaccination prevented the loss of lymphocytes seen in non-protected NHPs. In fact the OR and IN routes showed increased numbers of lymphocytes on day 3 post-challenge probably indicating a proliferation of ZEBOVGP-specific T-cells. As antigen presenting cells, the macrophages have a major role in the initiation of the immune response to foreign antigens. However, they are also the primary cells infected by ZEBOV early during infection [33,34]. Therefore, the number of CD14+ macrophage/monocytes was examined post-challenge (Figure 5d). The number of CD14+ cells increased 2.26(p = 0.190) on day 3 in the control group, but the numbers were greater in the VSVDG/ Figure 5. Absolute white blood cell numbers do not decrease in immunized animals after Zaire ebolavirus challenge. Whole blood from cynomolgus macaques immunized either orally (OR; n = 4), intranasally (IN; n = 4) or intramuscularly (IM; n = 2) was stained for lympocytes (CD3+, CD4+, CD8+), monocytes (CD14+). Control animals (n = 2) received non protective VSVDG/MARVGP. The absolute numbers in blood was determined for the each of the monkeys by flow cytometry on days 0, 3 and 6 post-challenge with ZEBOV. The average for each inoculation route is represented. doi:10.1371/journal.pone.0005547.g005 PLoS ONE | www.plosone.org 6 May 2009 | Volume 4 | Issue 5 | e5547