Study Population

Figure 1. Figure 1. Screening, Enrollment, Vaccinations, and Follow-up. Groups 1 and 2 were enrolled sequentially according to a dose-escalation protocol. All available study data and samples were used for study analyses. Overall, 100% of the participants completed the study-injection regimen and 4 weeks of follow-up. One participant was lost to follow-up at 48 weeks. For assessment of the durability of response at 48 weeks, an additional 17 participants who received the 2×1011 particle-unit dose were evaluated. The abbreviation cAd3 denotes chimpanzee adenovirus type 3.

Table 1. Table 1. Characteristics of the Participants at Enrollment.

A total of 20 participants were enrolled and vaccinated from September 2 through September 23, 2014 (Figure 1). The study population comprised 11 women and 9 men; the mean age was 37 years (range, 25 to 50) (Table 1). For assessment of the durability of response, an additional 17 participants who received the 2×1011 particle-unit dose were evaluated.

Vaccine Safety

Information on local and systemic reactogenicity was solicited from participants at each scheduled visit. When present, reactogenicity was usually mild to moderate. There was evidence of a dose effect with respect to the use of medication for symptom relief (e.g., acetaminophen or nonsteroidal antiinflammatory drugs) and lowering of body temperature (Table S1 in the Supplementary Appendix, available at NEJM.org). No fever was reported after vaccination in the group that received the 2×1010 particle-unit dose; in the group that received the 2×1011 particle-unit dose, 2 of 10 vaccine recipients reported fever: one case of grade 1 fever (temperature of 38.1°C) and one of grade 3 fever (temperature of 39.9°C). Fever developed 8 to 24 hours after vaccination, responded to antipyretic medication, and resolved within 1 day. There were no serious adverse events.

Asymptomatic prolonged aPTT was observed 2 weeks after vaccination in one recipient of the 2×1010 particle-unit dose (aPTT, 70.5 seconds; grade 4) and in two recipients of the 2×1011 particle-unit dose (aPTT, 48.4 seconds and 58.4 seconds; grade 2 and grade 4, respectively). Further evaluation (Table S2 in the Supplementary Appendix) showed that all three instances of prolonged aPTT were consistent with the induction of an antiphospholipid antibody, which causes an in vitro effect in the laboratory assay for aPTT and does not indicate a coagulopathy. The aPTT measurement in these participants decreased by the next visit and returned to almost baseline levels before the end of the study. Asymptomatic neutropenia or leukopenia of grade 1 or 2 was observed 3 to 4 days after vaccination in one recipient of the 2×1010 particle-unit dose and in three recipients of the 2×1011 particle-unit dose.

Ebola-Specific Antibody Responses

Table 2. Table 2. Geometric Mean Antibody Titers and Positive T-Cell Responses.

Figure 2. Figure 2. Glycoprotein Antibody Titers in Groups 1 and 2 as Assessed by an Enzyme-Linked Immunosorbent Assay (ELISA), According to Antigen. Individual antibody titers as assessed by glycoprotein ELISA at 2 weeks and 4 weeks after vaccination, as well as at 8 weeks after vaccination for Zaire–Mayinga, are shown according to antigen and vaccine-dose group. Geometric mean titers (horizontal lines) are shown for each group and time point; I bars indicate 95% confidence intervals. Vaccine-induced antibodies against Zaire–Mayinga glycoprotein (Panel A) were higher at week 4 than at week 2 in both group 1 and group 2 (group 1, P=0.001, and group 2, P<0.001), as well as higher at week 4 than at week 8 (group 1, P=0.003, and group 2, P<0.001). The difference between the groups reached significance at weeks 2, 4, and 8 (P=0.03, P=0.001, and P=0.004, respectively). Vaccine-induced antibodies against Sudan glycoprotein (Panel B) were higher at week 4 than at week 2 in group 2 (P=0.004), and the difference between group 1 and group 2 reached significance at week 4 (P=0.01). Vaccine-induced antibodies against Zaire–Guinea glycoprotein (Panel C) were higher in group 2 than in group 1 at week 4 (P=0.02).

At 4 weeks after vaccination, vaccine-induced anti-glycoprotein antibodies to Zaire species were detected in 90% of the participants in group 1 and in 100% of the participants in group 2, anti-glycoprotein antibodies to Sudan species were detected in 70% and 80% of the participants, respectively, and anti-glycoprotein antibodies to Zaire–Guinea species were detected in 90% and 90% of the participants, respectively. Anti-glycoprotein antibody responses (by ELISA) to at least one species or strain were detected in all 20 participants. At 8 weeks after vaccination, vaccine-induced anti-glycoprotein antibodies to Zaire–Mayinga species were detected in 90% of the participants in group 1 and in 80% of the participants in group 2. In both groups, the EC90 geometric mean titer against the Zaire–Mayinga antigen was higher at week 4 than at week 2 (331 vs. 106 in group 1 [P=0.001] and 2037 vs. 376 in group 2 [P<0.001]), as well as higher at week 4 than at week 8 (331 vs. 85 in group 1 [P=0.003], and 2037 vs. 464 in group 2 [P<0.001]) (Table 2 and Figure 2). The EC90 geometric mean titer against the Sudan antigen was also higher at week 4 than at week 2 (279 vs. 161 in group 1 and 936 vs. 400 in group 2), and the difference reached significance in group 2 (P=0.004) (Table 2 and Figure 2). The EC90 geometric mean titer against the Zaire–Guinea glycoprotein antigen was higher in group 2 than in group 1 at week 4 (623 vs. 177, P=0.02).

Figure 3. Figure 3. Durability of Glycoprotein Antibody Titers, as Assessed by an Enzyme-Linked Immunosorbent Assay (ELISA). Individual vaccine-induced antibody titers against Zaire–Mayinga glycoprotein, as assessed by ELISA, at 1, 2, 4, 8, 16, 24, and 48 weeks after vaccination are shown according to vaccine-dose group. Geometric mean titers are shown for each dose group and time point. Peak titers were reached by week 4 in both dose groups, and within the high-dose group (2x1011 particle units), week 4 titers were significantly higher than both week 1 titers (P<0.001) and week 48 titers (P<0.001).

Table 3. Table 3. Durability of Vaccine-Induced Zaire–Mayinga Antibody Response

The durability of the vaccine-induced antibody response was assessed by ELISA. At 48 weeks after immunization, the latest time point sampled in this study, anti-glycoprotein antibodies to Zaire–Mayinga species remained elevated and similar to those at early time points. The high-dose group had consistently higher EC90 geometric mean titer values than the low-dose group. Across the time points sampled, both groups displayed the highest antibody responses at week 4 (Figure 3 and Table 3), and titers remained high at week 48, which highlights the durability of the vaccine-induced antibody response.

Ebola-Specific T-Cell Responses

Figure 4. Figure 4. Vaccine-Induced Memory CD4 and CD8 T-Cell Responses, According to Dose. Panels A through D show the individual responses for increases from baseline to week 2 and week 4 in glycoprotein Zaire–specific and glycoprotein Sudan–specific CD4 and CD8 memory T cells. A Wilcoxon test was used to calculate the P values for the comparison of the magnitude of the T-cell response; all comparisons are with week 0. Horizontal lines indicate medians, and shaded areas interquartile ranges. The proportions, at week 4, of glycoprotein-specific CD4 and CD8 T cells (i.e., cells secreting at least one cytokine) that make any given combination of the three cytokines are shown in Panel E. The proportion of vaccine-specific CD8 T cells capable of simultaneously secreting interferon-γ (interferon-γ+) and tumor necrosis factor (TNF+) is labeled; this level of cells with this functional profile was shown to be associated with protection in a preclinical nonhuman primate model. The kinetics of glycoprotein-specific CD4 and CD8 T cells making any given combination of the same three cytokines are shown in Panel F for group 2 at baseline, week 1, week 2, week 4, and week 8. Gray lines indicate profiles associated with protection (thin line) and durability (thick line) in a preclinical nonhuman primate model.

Vaccine-induced CD4 and CD8 T-cell responses, as determined by expression of cytokines (interferon-γ, interleukin-2, and TNF) in response to stimulation with Zaire or Sudan glycoprotein peptides, were assessed by means of intracellular cytokine staining at weeks 2 and 4, and the expression of the cytokines (as a percentage of total CD4 and CD8 T cells) was compared with the baseline expression in all participants. Glycoprotein-specific CD8 responses to at least one antigen (Zaire or Sudan) were detected by week 4 in 20% of the participants in group 1 and in 70% of those in group 2 (P=0.07). Glycoprotein-specific CD8 responses to Zaire–Mayinga antigen were detected by week 8 in 10% of the participants in group 1 and in 40% of the participants in group 2. Glycoprotein-specific CD4 responses to at least one antigen (Zaire or Sudan) were detected by week 4 in 30% of the participants in group 1 and in 100% of those in group 2 (P=0.004). Glycoprotein-specific CD4 responses to Zaire–Mayinga antigen were detected by week 8 in 20% of the participants in group 1 and in 50% of the participants in group 2 (Table 2). Glycoprotein-specific memory CD4 and CD8 T-cell responses were greater in magnitude at week 4 than at week 2 and greater in group 2 than in group 1 (Figure 4). The majority of the memory CD4 and CD8 glycoprotein-specific T-cell responses were polyfunctional, expressing two or three cytokines (Figure 4); the vaccine elicited high proportions of CD8 cells coproducing interferon-γ and TNF, which are known to be associated with protection in nonhuman primates.16

cAd3 and Ad5 Serologic Assessment

Background immunity may affect the response to virus-vectored vaccines. We assessed cAd3 neutralizing antibody titers in all the participants at baseline and 4 weeks after vaccination and compared them with antibody and T-cell responses (Fig. S1 in the Supplementary Appendix). At baseline, reciprocal titers of anti-cAd3 neutralizing activity ranged from undetectable (<12) to 911. After vaccination, cAd3 antibody titers increased by a factor of at least 1.9 in all participants (Table S3 in the Supplementary Appendix). As assessed with the use of the Spearman rank-correlation method, preexisting antibodies to cAd3 did not significantly correlate with glycoprotein-specific antibody responses at week 4 for Zaire species (correlation, −0.277; P=0.24) or Sudan species (correlation, −0.333; P=0.15). In addition, preexisting antibodies to cAd3 did not significantly correlate with the magnitude of vaccine-induced memory CD4 T-cell response (correlation, −0.232; P=0.33). There was a moderate association between the titer of preexisting antibodies to cAd3 and the memory CD8 T-cell response (correlation, −0.511; P=0.02) (Fig. S1 in the Supplementary Appendix).

Owing to the genetic relatedness of Ad5 to cAd3, anti-Ad5 neutralizing antibody titers were also assessed in all the participants at baseline and 4 weeks after vaccination. At baseline, reciprocal titers of anti-Ad5 neutralizing activity ranged from undetectable (<12) to higher than 8748 (Table S3 in the Supplementary Appendix). As assessed with the use of the Spearman rank-correlation method, preexisting antibodies to Ad5 did not significantly correlate with glycoprotein-specific antibody responses at week 4 for Zaire species (correlation, 0.194; P=0.41) or Sudan species (correlation, 0.162; P=0.50) or with vaccine-induced glycoprotein-specific memory T-cell responses for CD4 (correlation, −0.014; P=0.95) or CD8 (correlation, −0.144; P=0.55).