Study Population

A total of 76 of the 123 volunteers who were screened for eligibility were vaccinated (Fig. S1 in the Supplementary Appendix). Of the 60 participants who were included in the original analysis of the ChAd3 vaccine, 59 completed at least 28 days of follow-up. One participant in group 1 withdrew on day 1 after vaccination owing to an aversion to venipuncture. Communication with the participant on day 10 after vaccination confirmed that the participant remained well, with no symptoms to report. Among the participants who were followed for 180 days were all 30 who received the MVA booster and 28 who did not receive the booster.

Safety

A complete list of the frequency and maximum severity of solicited, unsolicited, and laboratory adverse events, according to dose group, is provided in Tables S1 through S8 in the Supplementary Appendix. The majority of adverse events that were reported in all dose groups were mild in severity, with no unexpected serious adverse reactions or serious adverse events. Local reactogenicity appeared to be more pronounced after boosting vaccination than after priming vaccination, a finding that is consistent with the results of other studies of heterologous prime–boost vaccine schedules incorporating a ChAd prime and MVA booster. In contrast, fewer systemic adverse events were reported with boosting vaccination than with priming vaccination.

The majority of adverse events were self-limited and mild. Local pain was the most common local event (with one case reported as severe). Moderate systemic adverse events were fever, myalgia, arthralgia, headache, fatigue, nausea, and malaise. No severe systemic solicited adverse events were reported. Four episodes of mild fever (37.6 to 38.0°C in 4 participants) were reported. No fever persisted for more than 24 hours.

A prolonged activated partial-thromboplastin time was observed in four participants during the first 2 weeks after vaccination (three with a grade 1 elevation and one with a grade 2 elevation). None of the prolongations were associated with symptoms or clinical features of coagulopathy. The elevations fully resolved in all participants by 10 weeks after vaccination. No further abnormality was found on extended hematologic and coagulation evaluation.

A transient induction of an antiphospholipid antibody causing an in vitro artifact on the laboratory assay for activated partial-thromboplastin time after the administration of adenovirus vectors has been reported previously.15,16 Transient mild lymphocytopenia was noted on day 1 after vaccination in five participants in group 1, four in group 2, and eight in group 3; moderate lymphocytopenia was noted in two participants each in group 2 and group 3 on day 1. Transient mild or moderate elevations in bilirubin were recorded in three participants in group 2 and three in group 3. Transient hyperbilirubinemia in the severe range was recorded in two participants (one in group 2 and one in group 3) who had a prevaccination diagnosis of Gilbert’s syndrome.

Antibody Responses

Figure 1. Figure 1. Antibody Responses to the Zaire ebolavirus (ZEBOV) Glycoprotein. Panel A shows the geometric mean titer of antibody responses to increasing doses of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of ZEBOV, followed by a booster dose of a modified vaccinia Ankara (MVA) strain. Antibody responses are shown according to measurements on a standardized enzyme-linked immunosorbent assay (ELISA) for doses of 1×1010 viral particles (in 19 participants), 2.5×1010 viral particles (in 20 participants), and 5×1010 viral particles (in 20 participants). Solid symbols indicate that participants received only the ChAd3 vaccine, and open symbols, that participants received the ChAd3 vaccine followed by booster MVA. Antibody responses increased significantly by 7 days after the MVA dose and peaked at day 14 after boosting and then decreased slightly by day 28. There were no significant differences in responses among the dose groups in the cohort that received the MVA booster at any time point after vaccination. The days of the analysis are indicated by a plus sign after administration of the ChAd3 vaccine (A) and the MVA vaccine (M). Panel B shows the responses of participants after administration of the prime and booster vaccines, according to results on anti-ZEBOV glycoprotein (GP) IgG ELISA. The solid horizontal lines represent the geometric mean titer. Percentages of vaccinees with positive antibody responses at each time point are indicated below the graph. The horizontal dashed line represents the threshold for a positive result (arbitrary ELISA units, +0.561), calculated as the mean plus 3 standard deviations of the response on day 0 for all participants. Panel C shows antibody titers to inactivated whole ZEBOV virions (Makona strain) as measured on ELISA. The data show that immunogenicity at 4 weeks after priming with ChAd3 was similar to that measured after immunization with a recombinant vesicular stomatitis virus–based vaccine expressing ZEBOV glycoprotein (rVSV-ZEBOV) in 10 vaccinees in Hamburg, Germany. Panel D shows titers of neutralizing antibodies against live ZEBOV (Mayinga strain) from all participants who received the MVA booster, as measured at 28 days after the ChAd3 dose and 14 days after the MVA dose. Low levels of neutralizing antibodies that were detected in participants at 28 days were similar to levels reported after the administration of the rVSV-ZEBOV vaccine. By 14 days after MVA vaccination, the levels had increased by a factor of 9. In Panels C and D, the columns represent the geometric mean titer, the I bars represent 95% confidence intervals, and the horizontal dashed lines represent the positive threshold. In Panels B and D, the asterisk denotes P<0.001 by the two-tailed Mann–Whitney test.

Antibody responses as measured by means of standardized glycoprotein ELISA increased significantly by 7 days after the MVA dose and peaked at day 14 after boosting and then decreased slightly by day 28 (P<0.01 by the Friedman test for all comparisons) (Figure 1A). Responses remained significantly above pre-boost levels at 180 days after MVA vaccination and were four times as high as titers measured at 180 days after priming with the ChAd3 vaccine alone (P<0.001 by the Mann–Whitney test) (Figure 1B); in addition, 100% of the participants who received the MVA vaccine remained seropositive, as compared with less than half of those who received the priming vaccination alone.

Titers on whole-virion ELISA showed that immunogenicity at 4 weeks after priming with ChAd3 was similar to that measured after immunization with rVSV-ZEBOV in 10 vaccinees in Hamburg, Germany, at the dose administered in the ring vaccination study (geometric mean titer with ChAd3, 752.4; 95% confidence interval [CI], 541 to 1647; geometric mean titer with rVSV-ZEBOV, 920.7; 95% CI, 541 to 1566). In our study, after boosting with MVA, titers increased by a factor of 9 (geometric mean titer, 6625; 95% CI, 4748 to 9245) at 1 week and by a factor of 12 (geometric mean titer, 9007; 95% CI, 6909 to 11741) at 4 weeks (Figure 1C).

Six months after vaccination, titers in the group primed only with ChAd3 were similar to those detected 1 month after vaccination (geometric mean titer, 758; 95% CI, 561 to 1023; P=0.90 by the two-tailed Wilcoxon matched-pairs test). Titers remained significantly higher in the group that received the MVA booster (geometric mean titer, 1750; 95% CI, 1247 to 2456) than in the ChAd3 prime-only group (P<0.001 by the two-tailed Mann–Whitney test). At that time, 77% of vaccinees in the group that received the MVA booster remained seropositive, as compared with 25% of those in the prime-only group. (Summary data are provided in Tables S9 through S12 in the Supplementary Appendix.)

Neutralizing antibody titers to live ZEBOV (Mayinga strain) from all participants who received the MVA booster were measured at 28 days after the ChAd3 dose and at 14 days after the MVA dose (Figure 1D). Low levels of neutralizing antibodies were detected in participants at 28 days (geometric mean titer, 14.9; 95% CI, 12 to 18.5) — levels that were similar to those reported after the rVSV-ZEBOV vaccine4 (geometric mean titer, 22.2; 95% CI, 15.7 to 31.4); by 14 days after the MVA vaccine, the levels had increased by a factor of 9 (geometric mean titer, 139; 95% CI, 90 to 215) and all participants were seropositive (geometric mean titer, >8). Boosting with the high dose of MVA elicited neutralizing antibody titers that were higher than those with the low dose (geometric mean titer in the high-dose group, 243.9; 95% CI, 96 to 628; geometric mean titer in the low-dose group, 95.7; 95% CI, 65 to 142; P=0.03 by the two-tailed Mann–Whitney test) (Figure 1D). (Additional details regarding neutralizing antibodies and IgG antibodies are provided in Fig. S2 and S3 in the Supplementary Appendix.)

The dose of ChAd3 or MVA vaccine had no significant effect on post-boost IgG titers, nor did the interval between priming and boosting vaccinations affect the magnitude of the antibody response (r=0.20, P=0.30) (Fig. S3C in the Supplementary Appendix). However, there was a significant positive correlation between the prime–boost interval and the neutralizing antibody titer, regardless of the MVA dose (r=0.72, P<0.001) (Fig. S3D in the Supplementary Appendix). Antibody induction to the Sudan Ebola virus glycoprotein was assessed, but as expected in the absence of priming with this antigen, antibody titers against the Sudan virus glycoprotein were not detected after administration of the prime vaccine, which suggests a lack of cross-reactivity to the Sudan strain with antibodies raised against the ZEBOV glycoprotein. However, after boosting with the MVA vaccine (which expresses a Sudan Ebola virus glycoprotein), IgG titers increased significantly (geometric mean titer before boosting, 0.1; 95% CI, 0.07 to 0.2; geometric mean titer 14 days after boosting, 1.5; 95% CI, 1.1 to 2.0; P<0.001 by the Friedman test) (Fig. S3F in the Supplementary Appendix).

Cell-Mediated Immunity Induced by Vaccination

Figure 2. Figure 2. T-Cell Responses and Induction of Cytokines after Boosting with MVA. Panel A shows the median T-cell responses to ChAd3 vaccination and MVA boosting on enzyme-linked immunosorbent spot (ELISPOT) assay at all time points, as measured in spot-forming cells (SFCs) per million peripheral-blood mononuclear cells (PBMCs). The dose of MVA is indicated in plaque-forming units (PFU). Panel B shows the relationship between the prime–boost interval and the peak ELISPOT response 7 days after MVA vaccination, as calculated by means of a two-tailed Spearman’s test. Panel C shows the total cytokine response on flow cytometry with intracellular cytokine staining at 28 days after priming (post-prime) or 7 days after boosting, according to the MVA dose. The secretion of interferon-γ, interleukin-2, and tumor necrosis factor α (TNF-α) by CD4+ and CD8+ T cells was quantified for each booster-dose group and expressed as the frequency of cells expressing any one of the three cytokines. Panel D shows the expression of the degranulation marker CD107a 28 days after priming or 7 days after boosting. In Panels C and D, the solid horizontal lines indicate median values, and the dashed horizontal lines indicate the positive threshold. Panel E shows the proportions of CD4+ and CD8+ T cells that secreted any combination of interferon-γ, interleukin-2, and TNF-α after stimulation with two different MVA doses.

ELISPOT responses peaked 7 days after boosting with MVA at a median of 2068 spot-forming cells (SFCs) (interquartile range, 1197 to 3447) per million PBMCs and were significantly higher than peak responses after prime vaccination at 14 days (SFCs, 633; interquartile range, 274 to 820; P<0.001 by the two-tailed Wilcoxon matched-pairs test). Responses were maintained at 180 days after boosting (SFCs, 498; interquartile range, 207 to 905) and were significantly higher than non-boosted responses (SFCs, 84; interquartile range, 50 to 192; P<0.001 by the Mann–Whitney test) (Figure 2A). There was a modest negative correlation between the prime–boost interval and the peak ELISPOT response (r=−0.42, P=0.03 by two-tailed Pearson’s correlation coefficient) (Figure 2B). However, there was no significant relationship between the magnitude of the antibody response and the T-cell response (Spearman’s correlation coefficient, 0.17; P=0.39).

Intracellular cytokine staining revealed that all participants had positive CD4+ and CD8+ interferon-γ T-cell responses after boosting. The median frequency of CD4+ T cells secreting interferon-γ, interleukin-2, or tumor necrosis factor α (TNF-α) increased from 0.13% (interquartile range, 0.004 to 0.19) at 14 days after prime vaccination to 0.20% (interquartile range, 0.15 to 0.31) at 7 days after MVA boosting (P<0.001 by the Kruskal–Wallis test) (Figure 2C). The increase in the median frequency of cytokine-secreting CD8+ T cells was even more pronounced, from 0.004% (interquartile range, 0.004 to 0.09) at 14 days after prime vaccination to 0.25% (interquartile range, 0.10 to 0.65) at 7 days after MVA boosting (P<0.001 by the Kruskal–Wallis test).

The expression of the degranulation marker CD107a on CD8+ T cells increased by a factor of 5 after boosting (P=0.003 by the Kruskal–Wallis test) (Figure 2D). The MVA dose had no significant effect on the magnitude of the T-cell response as measured by means of ELISPOT (Figure 2A) or intracellular cytokine staining (Figure 2C and 2D). Analysis of polyfunctionality confirmed the dominance of TNF-α–secreting CD4+ T cells over cells secreting either interferon-γ or interleukin-2 (Figure 2E). Cells that were positive only for interferon-γ and double-positive cells secreting interferon-γ and TNF-α (with the latter being associated with protection in macaques17) were the largest subgroups in the CD8+ T-cell response (Figure 2E).

Short-Interval Boosting

Figure 3. Figure 3. Effect of Reduced Prime–Boost Intervals on Cellular Immunogenicity. Panel A shows the results of reducing the interval between prime vaccination with ChAd3 and booster vaccination with MVA to either 1 week or 2 weeks (as compared with 3 to 10 weeks) in two groups of eight participants each. The results are shown as median T-cell responses on ELISPOT assay, as measured in SFCs per million PBMCs. D0 indicates the beginning of the prime–boost interval for each group. Responses in all three groups peaked at 7 days after boosting, regardless of the prime–boost interval. Panel B shows the relationship between the prime–boost interval and the peak ELISPOT response 7 days after boosting, with a modest negative correlation between the prime–boost interval and peak T-cell immunogenicity. Also shown are individual ELISPOT responses to summed glycoprotein peptide pools at 7 days (Panel C) and 28 days (Panel D) after boosting; no significant differences between the groups were seen at either 7 days or 28 days. The black horizontal lines indicate median values. In these analyses, all the participants received 2.5×1010 viral particles of the ChAd3 vaccine and a booster dose of 1.5×108 PFU of MVA.

Given the pivotal role that has been shown for T cells in preclinical efficacy studies in macaques and the finding of high T-cell and antibody immunogenicity even with the shortest prime–boost intervals, we assessed the effect of reducing the prime–boost interval further to either 1 week or 2 weeks in two groups of eight participants each. ELISPOT responses in the two groups still peaked at 7 days after boosting (Figure 3A). We observed a modest negative correlation between the prime–boost interval and peak T-cell immunogenicity among all participants (r=−0.30, P=0.04 by two-tailed Spearman’s correlation coefficient) (Figure 3B). In a comparison of the median ELISPOT response in groups that received the MVA booster dose at an interval of 1 week, 2 weeks, or 3 to 10 weeks after the priming vaccination, there were no significant between-group differences at either 7 days or 28 days after the MVA dose (Figure 3C and 3D).

An analysis of antibody responses showed that reducing the prime–boost interval resulted in a decrease in the peak IgG titer after the MVA dose (P<0.05 for all comparisons) (Fig. S4A in the Supplementary Appendix). Additional cellular and humoral immunologic analyses are described in the Supplementary Appendix.