memory responses yet conducted with ZEBOV infected NHPs. This data forms a foundation for future critical studies on the immune correlates of protection, the potential for rapid mass vaccination programs in humans, and the immunization of endangered great ape populations in Africa. We demonstrated previously that a single IM dose of VSVDG/ ZEBOVGP protects cynomolgus macaques from a high dose challenge with ZEBOV [25]. In this study, we explored potential routes of immunization to compare the protection conferred as well as the immune response each route would elicit. All immunization routes were equivalently efficacious as no NHPs died or displayed symptoms of HF. In all cases both ZEBOVGP- specific humoral and cell mediated immune responses (CMIR) were generated, although some routes generated more potent humoral responses. All animals demonstrated a high IgM response post-vaccination, which was not exceeded following challenge. This response is normal since IgM is produced upon initial exposure to a new antigen during the primary but not the secondary immune response. In general the post-vaccination IgM and IgA titres were the highest in the IN group followed closely by the OR group, but 2.4 and 6.8 fold lower, respectively, in the IM route. This suggests that IN immunization was effective in eliciting not only a primary immune response but also IgA antibodies important for mucosal immunity. Importantly, no adverse effects were seen in any of the immunized animals. The most crucial immunoglobulin is IgG since it provides the majority of the systemic antibody, which is important for a haematological infection of ZEBOV. Once again the IN route was most effective with post-vaccination titres 8–9 fold higher than with OR and IM routes. With ZEBOV challenge, the secondary immune response IgG titres increased 43, 16 and 2 fold for OR, IM and IN, respectively. Although IN titres only increased 2 fold, the final titres were similar to the OR route as the IN pre-challenge titres were high initially, and the OR immunized animals had a very strong secondary IgG immune response after challenge. Very significantly the IM route consistently resulted in lower IgG titres before and after challenge when compared to the mucosally immunized animals. However, the IM animals all survived and this may indicate a minimum threshold of antibody required for protection, with all three routes achieving this minimum. In contrast to the total IgG titres, the NAb titres were systemically very low (titres = 1:40 to 1:320) and seem unlikely to be important as a protective mechanism, which is consistent with previous reports. In murine studies and other NHP studies, anti-ZEBOV NAb were low or absent yet the animals were resistant to a ZEBOV challenge [35–37]. The CMIR is a vital protective mechanism against viral pathogens and in this study was induced by all immunization routes. ZEBOV infections result in the loss of peripheral blood lymphocytes, presumably due to apoptosis [30–32,38]. In this study, absolute lymphocyte counts rather than percentages were used to determine whether the VSVDG/ZEBOVGP vaccine was able to prevent their decline. This is very important since changes in percentages can be caused by increases or losses in cell populations other than the one under observation. Absolute counts in contrast measure a true value for the number of circulating cells under investigation and other population changes do not effect the measurement. While the controls demonstrated the typical loss in lymphocytes, after challenge, all vaccination routes displayed a relatively stable or mild increase of CD3+, CD4+ and CD8+ numbers. An increase in lymphocyte numbers is typical of the expansion seen during a secondary immune response, and was most pronounced for the OR administration. Further evidence to support the induction of CMIR is the production of IFN-c and IL-2 cytokines measured by ELISPOT. Ebola Specific Immune Response Previous studies have investigated the production of TNFa by T cells rather than IL-2 however, TNFa does not have a specific role in the induction or maintenance of the CMIR and was therefore not included in this study. IFN-c secretion by T lymphocytes, NKs, and dendritic cells, occurs early in the immune response to a pathogen, and promotes the activation of antigen presenting cells as well as the induction of a Th1 response. IL-2 which is produced by T lymphocytes during an immune response, stimulates the growth, differentiation and survival of antigen-specific cytotoxic cells. All three routes induced the production of both IL-2 and IFN-c. Post-vaccination and very early post-challenge the IM route induced the most IFN-c and IL-2 secreting cells. However, cytokine production in the OR and IN routes increased steadily post-challenge and by day 26 post-challenge the OR and IN cytokine secreting cells surpassed the IM group. The production of IFN-c post-vaccination contrasts a previous study were we had demonstrated that IFN-c was not produced in peripheral blood mononuclear cells (PBMCs) post-vaccination [25]. The discrep- ancy could be due to the different assays and tissues used, as intracellular cytokine staining of PBMCs by flow cytometry was performed in the earlier study versus the ELISPOT assay on the peripheral blood cells in this paper. Overall, in this paper, the strong production of IL-2 and IFN-c by IM injection early on suggests the ability to induce a strong Th1 CMIR. The induction of long term immunity is a key requirement of any vaccine. In this study we examined the immune response induced by the vaccines up to 28 days, as well as the long term immunity that occurred after vaccination plus challenge. The immune responses directly attributable to the vaccine demonstrate that the OR and IN vaccination produce stronger humoral responses while the IM route provides a stronger CMIR. After challenge the strength, type and duration of the immune responses were also measured. The ZEBOVGP-specific T lymphocyte memory response and antibody titre was examined at 6 and 9 months post-vaccination, respectively. The OR and IN adminis- tration produced long lasting CD4+ and CD8+ memory lymphocytes that were able to proliferate and produce IFN-c in response to ZEBOVGP peptides. On the other hand the IgA and IgG serum titres were high for all three routes with a ranking of IN.OR.IM. Based on this data it suggests that different routes of immunization prime the memory responses differently. These differences are reflected in the memory responses seen after challenge in regards to the strength and type of response. The IM route is more likely to produce a strong Th1 CMIR while the IN route induced a predominantly Th1 humoral response. The potent CMIR seen in the IM group may be a product of the high IFN-c and IL-2 production detected post-vaccination, and early on post-challenge. IFN-c has immunoregulatory activities during an immune response to viral pathogens, and in addition it has antiviral effects that may inhibit early viral replication and thereby slow the infection [39]. Although the memory responses elicited at 6 and 9 months do not represent the effectiveness of the vaccine alone, this measurement indicates the route of immunization has an impact on the strength and type of immune response that is initiated upon a subsequent infection. In addition it demonstrates that long term protection to filoviruses is achievable, and provides us with clues as to the correlates of immune protection. The immune correlates of protection have not been defined for the protection of NHPs against filoviruses. Previously, we demonstrated an 80% survival in mouse adapted ZEBOV (MA- ZEBOV) infected mice that had received VSVDG/ZEBOVGP immune sera, indicating that the humoral response is partially responsible for protection [40]. NAb titres in vaccinated animals, or humans that have survived EBOV infections have consistently PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5547