been absent or low (1:20 to 1:180) [41,42]. Therefore a non-NAb response appears to be the mechanism involved in the humoral response to filovirus infections. In addition, all mice immunized with VSVDG/ZEBOVGP survived challenge with MA-EBOV even when depleted of CD8+ lymphocytes, suggesting that the cytotoxic T lymphocyte response may not be required [40]. As further confirmation, PBMCs from NHPs that had been immunized with an EBOV GP plus NP DNA prime then Adenovirus ZEBOV GP boost were examined for their ability to proliferate to ZEBOV peptides. When the CD4+ population had been depleted the ability to proliferate in response to the peptides was reduced to background levels, but when the CD8+ population was depleted there was no drop in proliferation [22]. In our system, total Ab response, and the pre-challenge IgG titres achieved from all routes of immunization with the VSVDG/ ZEBOVGP vaccine were high (1:6,400 to 1:57,600). Although there is no evidence for the importance of IgA, it is vital for mucosal immunity especially in the event of an aerosol delivery of ZEBOV [43]. All routes were able to produce high IgA titres although the IN and OR delivery was most effective. In addition, the OR route was able to induce a detectable CD4+ CMIR which also appears to be vital to survival. Development of a vaccine for OR or IN delivery would be desirable because they can be self administered significantly reducing the requirement for trained personnel especially in areas where the virus is endemic; they stimulate mucosal immune responses, which play an essential role in protecting the lungs from aerosol exposure; and they are more accepted by patients as there is no needle. We have shown conclusively that mucosal delivery of the vaccine can protect from IM systemic challenge and very surprisingly mucosal delivery particularly IN immunization seems to be more potent than IM injection in many immune parameters. Mucosal immunization would be huge benefit in any emergency mass vaccination campaign for natural outbreaks or following intentional release. Furthermore, mucosal immunization may be of great significance if immunization of great apes is attempted in the wild. Materials and Methods Ethics Statement Animal studies were performed in biosafety level 4 biocontain- ment at Public Health Agency of Canada and approved by the Canadian Science Centre for Human and Animal Health Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Animals were acclimatized for 14 days prior to infection. Animals were fed and monitored twice daily (pre- and post-infection) and fed commercial monkey chow, treats and fruit. Husbandry enrichment consisted of commercial toys and visual stimulation. Vaccine vectors, viruses, and peptides The recombinant VSVDG/ZEBOVGP and VSVDG/ MARVGP vaccine expressing the glycoproteins of ZEBOV (strain Mayinga) or Lake victoria marburgvirus (MARV) (strain Musoke) were generated using VSV (Indiana serotype) as described previously [27,44]. The ZEBOV (strain Kikwit) challenge virus was passaged in Vero E6 cells prior to challenge, as described previously [25,45]. A ZEBOVGP1,2 peptide pool consisting of 15mers with 11 amino acid overlaps (Sigma-Genosys) spanning the entire 676 amino acids of the ZEBOV, strain Mayinga 1976 GP was used. Animals Twelve 5 to 19 year old healthy cynomologus macaques (macaca fascicularis) were from a Health Canada non-human primate Ebola Specific Immune Response colony (Health Canada Animal Resources Division, Tunney’s Pasture, Ottawa, Ontario). Animal studies were performed in biosafety level 4 biocontainment at Public Health Agency of Canada and approved by the Canadian Science Centre for Human and Animal Health Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Animals were acclimatized for 14 days prior to infection. Animals were fed and monitored twice daily (pre- and post-infection) and fed commercial monkey chow, treats and fruit. Husbandry enrich- ment consisted of commercial toys and visual stimulation. Vaccination and Challenge Experiment Twelve filovirus na¨ıve cynomolgus monkeys randomized into four groups received 2 ml of 16107 PFU/ml of vaccine in Dulbecco’s modified Eagle’s medium (DMEM). Animals in the three experimental groups were vaccinated with either: 1) 2 ml orally (OR) (n = 4); 2) 1 ml dripped into each nostril, intranasally (IN) (n = 4); or 3) 1 ml each into two sites intramuscularly (IM) (n = 2). The two controls were injected intramuscularly with 2 ml of 16107 PFU/ml of VSVDG/MARVGP. All animals were challenged intramuscularly 28 days later with 1,000 PFU of ZEBOV (strain kikwit). Routine examination was conducted on 0, 2, 4, 6, 10, 14 and 21 days post-vaccination, then 0, 3, 6, 10, 14, 19, 26 days, 6 and 9 months after the ZEBOV challenge. For the examinations animals were anaesthetized by intramuscular injection with 10 mg/kg of ketaset (Ayerst). Examinations included haematological analysis, monitoring temperature (rectal), respiration rate, lymph nodes, weight, hydration, discharges and mucous membranes. Also, swabs (throat, oral, nasal, rectal, vaginal) and blood samples were collected (4 ml from femoral vein, 1 ml in EDTA vacutainer tube; 3 ml in serum separator vacutainer tube). Cynomolgus monkey PBMCs were isolated using BD CPT sodium citrate Vacutainers (Becton Dickinson) as per manufacturer’s protocol. ELISA Generation of ZEBOV VLPs were described previously [46]. High binding polystyrene microtitre plates (Thermo) were coated with 60 ml 5 mg/ml ZEBOV VLPs in phosphate buffered saline (PBS) for I hour, 37uC. Plates were washed 46 with PBS, 0.1% Tween-20, then blocked with 200 ml PBS-2% skim milk overnight, 4uC, before sera diluted in blocking solution (60 ml) was added, in triplicate, for 1 hour, 37uC. After washing 46, 60 ml of detection antibodies (Peroxidase-conjugated goat-anti-monkey, IgG, IgM, or IgA) diluted 1:2000 in blocking solution were added for 1 hour, 37uC. After washing 46, 100 ml of substrate (ABTS+H2O2) was added for 30 minutes at room temperature, before reading on a Versamax microplate reader at 405 nm. A sample was positive when the absorbance was higher than the mean plus 4 standard deviations of the pre-vaccination negative control from each monkey. ZEBOV-GFP Flow Cytometric Neutralization Assay Serum samples from days 0 and 21 post-vaccination were assessed, in duplicate, for their ability to neutralize an infection with ZEBOV-GFP in VeroE6 cells. Serially diluted serum samples were incubated with an equal volume of ZEBOV-GFP [29] in DMEM, at 37uC, 5% CO2 for 1 hr before 150 ml was added per well of a confluent 12 well plate of VeroE6 cells (MOI = 0.0005). After 2 hours at 37uC, 5% CO2, 1 ml of DMEM, 2% fetal bovine serym (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin was added per well and incubated for 5 days. Cells were harvested by removing the culture supernatant, washing with 1 ml PBS, 0.04% EDTA, then adding 800 ml of PBS 0.04% EDTA for 5 minutes at PLoS ONE | www.plosone.org 9 May 2009 | Volume 4 | Issue 5 | e5547