on DNA and viral based vaccines, or virus like particles (VLP) have been more promising. These newer vaccines are more effective in several animal models however, to date only five EBOV vaccine platforms have demonstrated over 80% efficacy in the more stringent NHP model [18–26]. Previously, we described the generation and use of live attenuated recombinant Vesicular Stomatitis Virus (VSV) with the VSV glycoprotein replaced by the glycoprotein of ZEBOV (VSVDG/ZEBOVGP) or MARV (VSVDG/MARVGP) [27]. Furthermore, we demonstrated that these live attenuated recombinant VSV vaccines completely protect NHPs against lethal challenge with the corresponding filoviruses by intramuscular injection (IM) [25,26,28]. In this study we are investigating for the first time the protection provided by intranasal (IN) or oral (OR) immunization with the VSV-based vaccines against the standard systemic IM challenge with ZEBOV in NHPs. We investigated the humoral and cellular immune responses following vaccination as well as the long term memory B and T cell immune responses post-challenge. Results Clinical Observations We used 12 cynomolgus macaques, of which 10 were immunized with VSVDG/ZEBOVGP either orally (OR; n = 4), Ebola Specific Immune Response intranasally (IN; n = 4) or intramuscularly (IM; n = 2). The remaining 2 control animals were vaccinated intramuscularly with VSVDG/MARVGP. VSVDG/MARVGP does not provide any heterologous protection against ZEBOV, therefore these NHPs succumb to ZEBOV infection. All animals were challenged with 1000 PFU of ZEBOV 28 days later, and monitored daily for clinical symptoms. An IM challenge was chosen as we have previously demonstrated that it produces a rapid and lethal ZEBOV HF [25]. An aerosol mucosal delivery mechanism was not employed as the disease course progressed more slowly, and we wished to test the vaccine against the most severe disease [13]. In addition, we have already demonstrated that the VSVDG/ ZEBOVGP vaccine fully protects against an aerosolized lethal dose of ZEBOV [13]. In this current study, all of the VSVDG/ ZEBOVGP immunized animals were protected from the high dose challenge and showed no evidence of clinical illness after vaccination or ZEBOV challenge. However, the control animals demonstrated typical symptoms associated with ZEBOV HF, such as fever, macular rashes, lethargy, unresponsiveness and were euthanized on day 6 (Figure 1). We performed haematology analysis at each examination date, and other than increases in the platelet-crit in the OR and IN groups post-challenge, we saw no significant changes in any NHPs post-immunization or in the VSVDG/ZEBOVGP immunized NHPs post-challenge (Table 1). Figure 1. Survival of vaccinated cynomolgus macaques. A) Flow chart of experimental design. Arrows indicate vaccination and challenge date. Tick marks indicate sampling days. B) Kaplan-Meier survival curve for cynomolgus macaques immunized by different routes and challenged with Zaire ebolavirus. Cynomolgus macaques were immunized orally (OR; n = 4), intranasally (IN; n = 4) or intramuscularly (IM; n = 2) with 26107 PFU of VSVDG/ ZEBOVGP, or injected intramuscularly with VSVDG/MARVGP (control; n = 2). All animals were challenged 28 days later with 1000 PFU ZEBOV. The animals were scored daily for fever, macular rashes, lethargy, and unresponsiveness. doi:10.1371/journal.pone.0005547.g001 PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5547