Ebola Specific Immune Response ZEBOVGP vaccinated groups with the IM route showing the most significant increases (IM q10.96, p = 0.001; OR q2.86, p = 0.062; IN q4.96, p = 0.009). ZEBOVGP Specific Memory Responses In order to determine the long term immune response after challenge, ZEBOVGP-specific CD4+ (Figure 6a) and CD8+ (Figure 6b) memory T lymphocytes were examined for their 2 ability to proliferate (CFSE ) or produce IFN-c in response to ZEBOVGP peptides at 6 months post-vaccination. The T lymphocyte marker CD45RA was used to determine the memory population (CD45RA2), and the na¨ıve plus effector populations (CD45RA+). In the IM immunized group, there was a very low level of proliferation in the CD8+ memory population accompa- nied by low levels of IFN-c production in the na¨ıve/effector CD8+ populations, as well as the CD4+ memory population. In comparison, the OR and IN immunized groups saw higher levels of proliferation and IFN-c production in their CD4+ and CD8+ memory cells. The memory cells in both the OR and IN groups proliferated equally well however the OR group had a higher percentage of memory cells producing IFN-c than the IN group. The CD45RA+ na¨ıve/effector populations also demonstrated low levels of proliferation as well as produced IFN-c in response to the ZEBOVGP peptides in the OR and IN groups. This is presumed to be primarily due to the effector population and not the na¨ıve cells. Overall, the ZEBOVGP-specific memory responses that occur as a result of vaccination followed by a ZEBOV challenge persist for at least 6 months indicating that long term immunity is possible in filovirus infections. The fact that the memory populations in OR and IN inoculation routes demonstrate the greatest potential for proliferation and IFN-c production post- challenge suggests that different routes of immunization prime different memory cell populations. Discussion In this study we have completed the only long term immune response study, and the most extensive investigation of the T-cell Figure 6. ZEBOVGP-specific CD4+ and CD8+ functional memory responses in PBMCs. Flow cytometry was utilized to evaluate the long term CD4+ (A) and CD8+ (B) ZEBOVGP-specific functional memory responses of freshly isolated PBMCs incubated with media or ZEBOVGP peptides at 6 months post-vaccination. The average for each inoculation route (IM = intramuscular n = 2, OR = orally n = 4, IN = intranasally n = 4) is represented. The CD45RA2 population denotes the memory lymphocytes, while CD45RA+ represents the na¨ıve and effector lymphocytes. The IFN-c response was evaluated after 3 days by intracellular cytokine staining. The background from the media sample was subtracted from the peptide stimulated sample, and the results are shown for each monkey with the average for each group being represented with a bar. The ability of the PBMCs to proliferate in response to the GP peptide was determined by staining the PBMCs with CFSE and then looking for loss of CFSE 6 days later as a measure of proliferation. The percent of CFSE2 cells for each memory population is shown for each monkey after subtracting the media sample, and the average for each group is represented by a bar. doi:10.1371/journal.pone.0005547.g006 PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5547