Ebola Specific Immune Response Figure 2. Strong humoral IgM, IgA, IgG responses are induced in the IM, OR, and IN immunization routes. A ZEBOV VLP ELISA was utilized to determine the ZEBOV-specific IgA (a), IgG (b), IgM (c) titres in post-vaccination and post-challenge sera from cynomolgus macaques immunized either orally (OR; n = 4), intranasally (IN; n = 4) or intramuscularly (IM; n = 2). Titres are presented as endpoint dilutions of the average value per group. Table (d) illustrates fold increases in peak titres post-challenge compared to post-vaccination for IgA and IgG. IgM is not shown as there were no increases post-challenge. doi:10.1371/journal.pone.0005547.g002 Figure 3. Neutralizing antibodies generated by the various immunization routes. ZEBOV GP-specific neutralizing antibodies in the sera from NHPs vaccinated either (A) intramuscularly (IM), (B) orally (OR), or (C) intranasally (IN), were investigated for their ability to inhibit infection by ZEBOV-GFP. NHP sera from days 0 and 21 post-vaccination were incubated for 1 hour with ZEBOV-GFP before being added to a monolayer of Vero cells. The positive control was ZEBOV-GFP in DMEM without sera. The level of GFP fluorescence of ZEBOV-GFP infected cells was determined by flow cytometry. The percent reduction of infection by ZEBOV-GFP was calculated as follows: % ZEBOV-GFP reduc- tion = (12(Test samples/Positive control))6100). The day 21 results for each NHP are displayed and have been normalized by subtracting each NHP’s day 0 results from the day 21 results. The numbers in the legend represent the dilution of the NHP sera that was added to ZEBOV-GFP. doi:10.1371/journal.pone.0005547.g003 PLoS ONE | www.plosone.org 4 May 2009 | Volume 4 | Issue 5 | e5547