Trial Design and Oversight

In this phase 3, open-label, randomized trial, MVA was compared with ACAM2000, and two indicators of efficacy for protection against smallpox were assessed. The two primary end points were immunogenicity (noninferiority of the MVA vaccine to ACAM2000 with respect to the peak geometric mean titer of serum neutralizing antibodies) and a surrogate of efficacy (attenuation of the major cutaneous reaction by previous vaccination with MVA, measured as the maximum lesion area, after receipt of ACAM2000). A total of 440 healthy participants who had not previously received any vaccinia-based vaccine were eligible for enrollment and randomization, in a 1:1 ratio, to one of the two vaccination groups. The MVA group received two doses of MVA, one each on weeks 0 and 4, followed by ACAM2000 on week 8. The ACAM2000-only group received ACAM2000 on week 0.

Safety and reactogenicity, including solicited local and general adverse events, as well as unsolicited adverse events, were assessed throughout the active trial phase. Cardiac monitoring was performed until 6 months after the last vaccination in both the MVA group and the ACAM2000-only group.

The trial was designed by Bavarian Nordic on the basis of comments and feedback received from the FDA. Trial data were collected and analyzed by the U.S. Army Medical Research Institute of Infectious Diseases, Bavarian Nordic, and Chiltern International, a contract research organization. All the authors vouch for the completeness and accuracy of the data and analyses and for the fidelity of the trial to the protocol (available with the full text of this article at NEJM.org). The manuscript was prepared by authors who are employees of Bavarian Nordic. The conduct of the trial was managed by Bavarian Nordic and Chiltern. The trial was conducted at a U.S. military garrison in South Korea led by the U.S. Army Medical Research Institute of Infectious Diseases in collaboration with the Defense Health Agency.

The trial was conducted in accordance with the Declaration of Helsinki and the Belmont Report and was approved by the institutional review boards of the U.S. Army Medical Research Institute of Infectious Diseases. All the participants provided written informed consent.

Participants

Participants were enrolled at the Brian Allgood Army Community Hospital at Yongsan U.S. Military Garrison and Camp Hovey, both in South Korea. They were recruited from Department of Defense personnel who were scheduled to receive an initial smallpox vaccination as part of their in-process orientation.

Healthy persons who were 18 to 42 years of age were eligible to participate if they had a body-mass index (the weight in kilograms divided by the square of the height in meters) between 18.5 and 34.9, a negative serum pregnancy test, a white-cell count of 2500 to 10,999 per cubic millimeter, normal hemoglobin and platelet levels, normal renal and hepatic function, a troponin I level that was less than 2 times the upper limit of the normal range, and echocardiographic (ECG) tests without clinically significant findings (normal laboratory ranges are provided in the Supplementary Appendix, available at NEJM.org).

Participants were excluded if they had a known or suspected history of smallpox-based or poxvirus-based vaccination, a history of or active immunodeficiency or immune suppression or close contact with a person with immunodeficiency, a history of certain heart conditions, a high risk of death from cardiovascular causes, or certain active skin conditions or a history of such conditions.

Vaccine

MVA was produced at Bavarian Nordic (Kvistgård, Denmark) and filled, formulated, and labeled at IDT Biologika (Dessau-Roßlau, Germany). A standard 0.5-ml dose of liquid frozen vaccine had a nominal titer of 1×108 TCID 50 (50% tissue-culture infectious dose). MVA was injected subcutaneously into the upper arm.

ACAM2000 vaccine was supplied by the Defense Health Agency. Department of Defense personnel administered the vaccine through scarification with a bifurcated needle (standard dose, 2.5×105 to 12.5×105 plaque-forming units of live vaccinia virus) into the upper arm.

Assessments of Immunogenicity

Serum antibody titers were measured with the use of a plaque-reduction neutralization test (PRNT) and an enzyme-linked immunosorbent assay (ELISA). The vaccinia-specific PRNT and ELISA used the vaccinia virus Western Reserve strain and MVA, respectively, as antigens; the detection limits were titers of 2 and 50, respectively.

Assessments of the Major Cutaneous Reaction

The Silhouette Connect camera system was used to photograph the major cutaneous reaction and calculate the lesion area (Fig. S1 in the Supplementary Appendix). The maximum lesion area was defined as the larger area measured once during days 6 through 8 and once during days 13 through 15 (relative to scarification). An independent review committee consisting of three assessors who were unaware of the trial-group assignments confirmed or updated the lesion area measurements (Fig. S2). Using photos of the lesions taken on one day during each of the two periods (days 6–8 and 13–15), the committee also evaluated, in blinded fashion, each lesion as full (major lesion diameter, ≥5 mm), partial, or without a major cutaneous reaction (at days 6–8 after scarification). The diameter of the major lesion, major erythema, and major induration were assessed by an investigator twice (at days 6–8 and 13–15). The committee also reviewed local symptom data from participants on printed diary cards through 14 days after scarification.

Statistical Analysis

We specified that statistical significance at a type I error of 0.05 would be needed for both primary end points. No adjustment was made for multiple comparisons, and the 95% confidence intervals were not adjusted. All tests and confidence intervals are two-sided. The sample size provided at least 80% power to assess both primary end points. Efficacy analyses were conducted in the per-protocol population, the full-analysis population, and in all participants who underwent randomization, with the per-protocol population being the primary analysis population. Safety analyses were based on the full-analysis population (participants who underwent randomization and were vaccinated).

The first primary end point was the noninferiority of MVA to ACAM2000 with respect to the geometric mean titer of neutralizing antibodies at peak visits (week 6 in the MVA group and week 4 in the ACAM2000-only group, according to published data).15,18,25 The noninferiority of MVA to ACAM2000 was shown if the 95% confidence interval of the geometric mean titer ratio as measured by PRNT (MVA:ACAM2000-only, obtained by calculating the antilog of the difference in the mean log 10 -transformed titers) was entirely above 0.5. Secondary immunogenicity end points included seroconversion and geometric mean titers at each visit determined with the use of PRNT and ELISA. Seroconversion was defined as seropositivity after baseline (in patients who were seronegative at baseline) or two or more times the baseline titer (in patients who were seropositive at baseline).

The second primary end point was the attenuation of a major cutaneous reaction when ACAM2000 was administered after previous MVA vaccination, as determined by the maximum lesion area confirmed by the independent review committee. The goal was an area attenuation ratio (1 – the maximum lesion area ratio [MVA:ACAM2000-only]) that was significantly above 40%. The maximum lesion area ratio and 95% confidence interval were based on the Hodges–Lehmann estimate of the shift for log-transformed maximum lesion areas. Secondary efficacy end points included evaluation of the maximum lesion diameter and the lesion diameter measured by the investigator twice (at days 6–8 and 13–15) as well as classification of the major cutaneous reaction as full, partial, or absent.

A safety analysis, including the number and percentage of participants who had adverse events, was performed during each vaccination period. There were three vaccination periods in the MVA group (period 1 [weeks 0–4], period 2 [weeks 4–8], and period 3 [weeks 8–12]) and one vaccination period in the ACAM2000-only group (period 1 [weeks 0–4]). Each period in the MVA group was compared with the period in the ACAM2000-only group by means of Fisher’s exact test.

Randomization was performed by a third-party organization using a validated interactive Web portal. This was an open-label trial, but the independent review committee and personnel responsible for immunogenicity testing were unaware of the trial-group assignments. Further specific details regarding the trial design are provided in the protocol.