One- and Two-Dose Vaccinations With Modified Vaccinia Ankara-Bavarian Nordic Induce Durable B-Cell Memory Responses Comparable to Replicating Smallpox Vaccines

Abstract

Background

Although modified vaccinia Ankara-Bavarian Nordic (MVA-BN) vaccination is approved for smallpox and monkeypox prevention, immunological persistence and booster effects remain undescribed.

Methods

Participants naive to smallpox vaccination were randomized to 1 dose MVA-BN (1×MVA, n = 181), 2 doses MVA-BN (2×MVA, n = 183), or placebo (n = 181). Participants with previous smallpox vaccination received 1 MVA-BN booster (HSPX, n = 200). Subsets of the formerly naive groups (approximately 75 each) received an MVA-BN booster 2 years later.

Results

Neutralizing antibody (nAb) geometric mean titers (GMTs) increased from 1.1 (baseline, both naive groups) to 7.2 and 7.5 (week 4, 1×MVA and 2×MVA, respectively), and further to 45.6 (week 6, 2×MVA after second vaccination). In HSPX, nAb GMT rapidly increased from 21.6 (baseline) to 175.1 (week 2). At 2 years, GMTs for 1×MVA, 2×MVA, and HSPX were 1.1, 1.3, and 10.3, respectively. After boosting in the previously naive groups, nAb GMTs increased rapidly in 2 weeks to 80.7 (1×MVA) and 125.3 (2×MVA), higher than after primary vaccination and comparable to boosted HSPX subjects. Six months after boosting, GMTs were 25.6 (1×MVA) and 49.3 (2×MVA). No safety concerns were identified.

Conclusions

Anamnestic responses to boosting without sustained high nAb titers support presence of durable immunological memory following primary MVA-BN immunization.

Clinical Trials Registration. NCT00316524 and NCT00686582.

One or 2 primary MVA BN vaccinations induced similar durable B-cell memory responses as a booster administered 2 years after primary vaccination showed rapid and robust antibody response. No safety concerns were identified following the booster.

The discontinuation of smallpox vaccinations in the 1970s has left much of the world's population susceptible to variola, the causative agent of smallpox, and other orthopox viruses for which the vaccine provided cross-protection [1]. While the risk of smallpox from occupational exposure [2] or bioterrorism [3] remains concerning, the most prevalent human orthopox infection is currently monkeypox. In the 1970s and 1980s, monkeypox cases documented in Africa were suspected to result primarily from animal to human transmission with limited secondary spread between humans [4, 5]. However, an increase in human monkeypox has been reported in Africa [6, 7], and beginning in 2018, isolated travelers from Nigeria experienced cases of monkeypox upon arriving in Israel, Singapore, and the United Kingdom, with occasional secondary infection of close contacts [8]. Since May 2022, a geographically widespread monkeypox outbreak has occurred, primarily in men who identify as gay or bisexual, or men who have sex with men, leading to declaration of a Public Health Emergency of International Concern by the World Health Organization [9]. These outbreaks highlight the absence of population protection against orthopox viruses and the urgency to better understand immunogenicity of different vaccine schedules.

Although worldwide protection against orthopox viruses was achieved with replicating vaccinia-based vaccines, they have rare, but potentially serious, adverse effects including progressive vaccinia, encephalitis, and myopericarditis [10–12]. Risks of adverse reactions are higher in immunocompromised individuals and those with exfoliative skin conditions [13–16]. These concerns prompted use of the highly attenuated modified vaccinia Ankara (MVA) virus to develop the nonreplicating and substantially less reactogenic MVA-Bavarian Nordic (MVA-BN) vaccine [17–19]. MVA-BN induces immune responses comparable to traditional smallpox vaccines [18–20] and is well tolerated by healthy adults [21–28] and those with human immunodeficiency virus [29–31], hematopoietic stem cell transplantation [32], or exfoliative skin conditions [33, 34]. These data and nonhuman primate studies [35] supported approval of liquid frozen MVA-BN for prevention of smallpox and monkeypox under the trade names Jynneos (United States), Imvanex (European Union, United Kingdom), and Imvamune (Canada).

The initial and follow-up clinical studies described herein demonstrate the duration and nature of immunological memory in both younger individuals never immunized against smallpox and older individuals vaccinated in the distant past using live-replicating vaccinia vaccines. These findings are of relevance when defining optimal vaccination strategies for outbreaks in a supply-constrained environment and for prophylaxis for those at risk of occupational orthopox exposure, such as health care providers, laboratory staff, and military personnel.

METHODS

Study Design

Two studies were conducted to assess safety, immunogenicity, and boost response with MVA-BN in healthy adults with and without prior smallpox vaccination. These studies were conducted from 2006 to 2009 at 1 European site.

The initial study (NCT00316524) was a phase 2, partially randomized, double blind, placebo-controlled, noninferiority trial. Participants without prior smallpox vaccination were randomized 1:1:1 to receive vaccinations 4 weeks apart with 1 dose of MVA-BN followed by 1 dose of placebo (1×MVA), 2 doses of MVA-BN (2×MVA), or 2 doses of Tris buffer placebo (PBO). Participants with prior smallpox vaccination (HSPX) were given a single booster dose of MVA-BN. The primary immunogenicity objective was to compare the humoral responses of the HSPX and 2×MVA groups to determine if a single booster dose of MVA-BN in a previously vaccinated population could induce a response comparable to 2 MVA-BN primary vaccinations in a population never vaccinated against smallpox. Secondary objectives included comparisons of immune response kinetics across groups. The safety results from the initial study were reported previously [25].

The follow-up study (NCT00686582) was a phase 2 open-label trial that assessed immunogenicity in the 1×MVA, 2×MVA, and HSPX groups 2 years following their last dose in the initial study. An MVA-BN booster dose (BD) was also administered to a subset of the participants without prior smallpox vaccination (1×MVA BD and 2×MVA BD groups) to evaluate postbooster immunogenicity and safety.

Participants

The study was conducted in accordance with the Declaration of Helsinki (2000/2008). All participants were informed verbally and in writing about the purpose, procedures, and potential risks and benefits, and provided consent before participation in any study-related assessments.

The initial study included healthy men and nonpregnant women, aged 18 to 55 years who had normal laboratory values at screening and agreed to use birth control. For the 1×MVA, 2×MVA, and PBO groups, participants had no known or suspected previous smallpox vaccination and no detectable vaccinia scar. For the HSPX group, participants had a prior smallpox vaccination that was documented or manifested as a typical vaccinia scar. The Supplementary Material contains full eligibility criteria.

Participants who completed the initial study were able to have the persistence of their humoral response evaluated 2 years later, at the beginning of the follow-up study. Consenting eligible participants from the 1×MVA and 2×MVA groups received a booster and immunogenicity assessments over 6 months.

Vaccine

MVA-BN is a highly attenuated, purified, live vaccine [17]. It and the Tris buffer placebo were produced at IDT Biologika GmbH (Dessau-Rosslau, Germany) according to current Good Manufacturing Practice standards. MVA-BN was provided in liquid frozen 0.5-mL aliquots of ≥ 0.5 × 10⁸ 50% tissue culture infectious dose (TCID₅₀) MVA titer. MVA-BN and PBO were shipped and stored at −20°C ± 5°C, avoiding direct light, and administered subcutaneously with a 24- or 25-gauge needle in the upper arm.

Immunogenicity Assessments

Total and neutralizing serum antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). In the initial study, samples were drawn at screening, 2, 4, 6, and 8 weeks, and 6 months after the last vaccination. In the follow-up study, samples were drawn 2 years (−2 to +3 months) after the initial vaccination from all available participants. Those who received the booster also had samples drawn at 1, 2, and 4 weeks, and 6 months postboosting. The Supplementary Material contains ELISA and PRNT methodologies.

Safety Assessments

The safety profile for the initial study was previously reported [25]. Thus, the safety assessments described herein are specific to the follow-up study. Safety and reactogenicity assessments included solicited local and systemic adverse events (AEs), unsolicited AEs, and serious adverse events (SAEs). Solicited AEs comprised a set of predefined, expected local and systemic reactions. See Supplementary Material for specifics. These AEs were listed on a memory aid where participants recorded them for 8 days postbooster. Unsolicited AEs (AEs that were not listed on the memory aid or occurred outside the 8-day postbooster period) were reported at any study visit. All solicited local AEs occurring during the 8-day period were considered related to study vaccination, while the investigator determined causality for all other reported events.

Safety tests were performed at screening and 2 weeks postbooster. Abnormal laboratory values assessed as clinically significant and grade 3 (severe) or grade 4 (potentially life threatening) abnormal values were documented as AEs.

AEs of special interest (AESIs) were clinically significant cardiac symptoms, electrocardiogram changes, or cardiac enzymes above normal limits. Participants developing an AESI returned for examinations and diagnostic tests and were followed until resolution.

Statistical Methods

Statistical analyses were performed using SAS 9.1 (SAS Institute). The safety and primary immunogenicity analyses were based on the full analysis set, comprising all participants given at least 1 vaccination. Immunogenicity was also summarized for the per protocol set, comprising those participants who adhered to all vaccinations and protocol conditions.

In the initial study, a randomization list was deposited in the electronic case report form; in the follow-up study, no randomization was required. In both studies, conduct was overseen by an independent data and safety monitoring board. Unsolicited AEs were coded with Medical Dictionary of Regulatory Activities terminology.

For participants seronegative at baseline, seroconversion was defined as development of antibody titers above the limit of detection (≥6 for PRNT and ≥50 for ELISA). For participants seropositive at baseline, seroconversion was defined as an at least 2-fold increase compared to baseline titer in the initial study and to the prebooster titer in the follow-up study. Geometric mean titer (GMT) was calculated as the antilogarithm of the mean of the log₁₀ titer transformations. Antibody titers below the detection limit were given an arbitrary value of 1; titers between the detection limit and quantification limit (20 for PRNT and 200 for ELISA) were analyzed as reported.

RESULTS

Clinical Participant Population and Conduct of the Study

The initial study included 753 participants. Of these, 204 participants vaccinated against smallpox in the distant past received an MVA-BN booster. The other 549 participants without prior smallpox vaccination were randomized among 1×MVA, 2×MVA, and PBO (Figure 1). Eight participants who did not meet eligibility criteria were not included. Primary MVA-BN vaccination was administered to 181 participants in the 1×MVA group and 183 participants in the 2×MVA group; placebo was administered to 181 participants. MVA-BN booster was administered to 200 participants in the HSPX group. In total, 745 participants received study vaccination and were included in the full analysis set.

Figure 1.

Participant disposition for initial and follow-up MVA-BN vaccination studies. ^aThe per protocol set comprised the subset of participants who received all vaccinations and adhered to all protocol conditions without major protocol deviations. Abbreviations: BD, booster dose; HSPX, history of smallpox vaccine positive; MVA, modified vaccinia Ankara; MVA-BN, modified vaccinia Ankara-Bavarian Nordic; n, total number of participants per group; PBO, placebo.

Approximately 2 years later, 304 participants (91 from the 1×MVA group, 92 from the 2×MVA group, and 121 from the HSPX group) provided blood samples to assess antibody persistence. A total of 77 and 75 participants initially given 1 or 2 MVA primary vaccinations, respectively, were boosted with MVA-BN.

Demographic characteristics were generally comparable across treatment groups at initial study onset (Table 1). Slightly more than half of participants were female (53.0% to 61.9%); most were white (97.2% to 99.0%). The HSPX group was older (mean age 41.5 years) than the other groups (25.3 and 26.0 years), as they were vaccinated prior to the declaration of worldwide smallpox eradication in 1980. At baseline, 64.4% of participants were on concomitant medication, most commonly over-the-counter pain medications and birth control. The follow-up study population had similar baseline characteristics.

Table 1.

Demographics of Study Participants at Initial and Follow-up Study Baselines

Characteristic	Initial Study				Follow-up Study
	2×MVAa	1×MVAa	HSPX+b	Placebo	2×MVA BDc	1×MVA BDc
No. of participants	183	181	200	181	75	77
Age, y, mean ± SD	25.3 ± 5.0	25.4 ± 4.4	41.5 ± 7.6	26.0 ± 5.1	27.3 ± 6.0	27.9 ± 4.5
Sex, No. (%)
ȃFemale	97 (53.0)	112 (61.9)	115 (57.5)	107 (59.1)	40 (53.3)	45 (58.4)
ȃMale	86 (47.0)	69 (38.1)	85 (42.5)	74 (40.9)	35 (46.7)	32 (41.6)
BMI, kg/m2, mean ± SD	23.6 ± 3.9	23.7 ± 3.4	24.6 ± 3.9	23.9 ± 4.3	23.7 ± 4.0	24.3 ± 3.6
Ethnicity, No. (%)
ȃWhite	178 (97.3)	176 (97.2)	198 (99.0)	177 (97.8)	74 (98.7)	74 (96.1)
ȃAsian	1 (0.5)	2 (1.1)	0	1 (0.6)	1 (1.3)	1 (1.3)
ȃBlack	0	1 (0.6)	1 (0.5)	0	0	0
ȃArabic	2 (1.1)	1 (0.6)	0	1 (0.6)	0	1 (1.3)
ȃOther	2 (1.1)	1 (0.6)	1 (0.5)	2 (1.1)	0	1 (1.3)

Percentages are based on the number of participants in the group.

Abbreviations: BD, booster dose; BMI, body mass index; HSPX, history of smallpox vaccination; MVA, modified vaccinia Ankara; MVA-BN, modified vaccinia Ankara-Bavarian Nordic.

Participants in these treatment groups had not been previously immunized against smallpox and received either 1 or 2 primary MVA-BN vaccinations in the initial study.

Participants in this treatment group had been immunized against smallpox in the distant past with replicating smallpox vaccines and received a booster MVA-BN vaccination in the initial study.

Participants in these treatment groups received a booster MVA-BN vaccination 2 years after receiving either 1 or 2 primary MVA-BN vaccinations in the initial study.

Immunogenicity

Early Responses to Vaccination, Initial Study

In the 1×MVA group, neutralizing antibody (nAb) GMTs rapidly increased from initial study baseline (1.1; 95% confidence interval [CI], 1.0–1.1) to week 2, then further increased by week 4 (7.2; 95% CI, 5.5–9.4) (Figure 2A). A similar increase in nAb occurred after first vaccination in the 2×MVA group from baseline (1.1; 95% CI, 1.0–1.2) to week 4 (7.5; 95% CI, 5.7–10.0). After the second MVA-BN vaccination, nAb GMT peaked at week 6 (45.6; 95% CI, 35.1–59.3), nearly 10 times higher than levels observed 2 weeks following the first primary vaccination. GMT then decreased slightly but remained elevated at week 8.

Figure 2.

GMTs over time in the initial (A and C) and follow-up (B and D) studies. Dashed lines connect postbooster data points. The 1-week postvaccination time point for the follow-up study (week 109), included in Supplementary Table 1, has been omitted from this figure to facilitate comparisons of immune response kinetics between studies. In A and C, the 2-year follow-up time point (at week 108) depicts immunogenicity data obtained from all 304 participants in the follow-up study who provided blood draws 2 years after MVA-BN and included 92 participants in the 2×MVA group, 91 participants in the 1×MVA group, and 121 participants in the HSPX group (for additional details see Figure 1). Abbreviations: BD, booster dose; ELISA, enzyme-linked immunosorbent assay; GMT, geometric mean titer; HSPX, history of smallpox vaccine positive; n, total number of participants per group; PBO, placebo; PRNT, plaque reduction neutralization test.

In the HSPX group, nAb GMT increased from baseline (21.6; 95% CI, 16.3–28.5) to week 2 (175.1; 95% CI, 140.0–219.1) and was approximately 4 times greater than that of the 2×MVA group 2 weeks after the second primary vaccination. The nAb GMT remained elevated 4 weeks following MVA-BN booster (144.3; 95% CI, 117.9–176.5; Figure 2A).

Approximately half of participants in the 1×MVA and 2×MVA groups seroconverted for nAb at week 2, which increased by week 4 to 62.1% and 56.7%, respectively (Figure 3A). The second primary vaccination in the 2×MVA group further elevated seroconversion by week 6 (89.2%).

Figure 3.

A and B, Seroconversion rates over time in the initial and follow-up studies. The 2-year follow-up time point (at week 108) depicts immunogenicity data obtained from participants who provided blood draws 2 years following primary immunization with MVA-BN and included 92 participants in the 2×MVA group and 91 participants in the 1×MVA group (for additional details see Figure 1). Abbreviations: BD, booster dose; ELISA, enzyme-linked immunosorbent assay; n, total number of participants per group; PB, postbooster; PBO, placebo; PRNT, plaque reduction neutralization test; SC, seroconversion.

The HSPX group had a wide range of neutralizing antibodies at initial study baseline and therefore higher titers were needed to achieve seroconversion for individuals with high baseline titers. Nonetheless, 78.5% of the participants were seroconverted at week 2.

The same general postvaccination trends were also observed for total antibodies (Figure 2C and Figure 3B). However, while the booster in the HSPX⁺ group induced higher levels of nAb compared to primary vaccination in the 2×MVA group, peak total antibody titers were more comparable (568.8; 95% CI, 473.3–683.6 vs 495.8; 95% CI, 431.8–569.4, respectively). As expected, antibody GMTs and seroconversion rates in the PBO group were low at all time points, with negligible changes over the course of the initial study.

Immunogenicity at 6 Months and 2 Years

Six months after the last vaccination (week 30), nAb GMT remained higher than at baseline in the HSPX group (106.5; 95% CI, 89.1–127.2). Sustained detectable nAb levels above baseline were also observed, although at lower levels, in the 1×MVA (1.9; 95% CI, 1.6–2.2) and 2×MVA groups (7.2; 95% CI, 5.6–9.4) (Figure 2A). Seroconversion based on nAb levels continued to be observed in 23.6% of the 1×MVA group and 65.2% in the 2×MVA group (Figure 3A). At 2 years, nAb GMTs were comparable to initial study prevaccination levels (Figure 2A).

Total antibody GMTs at the 2-year time point remained above initial study baseline for all groups, with the most notable sustained titers in the HSPX group (134.7; 95% CI, 111.9–162.0 vs 38.8; 95% CI, 29.4–51.3) and more modest sustained titers in the 2×MVA (23.3; 95% CI, 15.2–35.9 vs 1.4; 95% CI, 1.2–1.7) and 1×MVA (6.2; 95% CI, 4.0–9.7 vs 1.3; 95% CI, 1.1–1.5) groups (Figure 2C). The durability of total antibody responses was also reflected by sustained levels of seroconversion from 6 months to 2 years in the 1×MVA (37.9% and 42.9%) and 2×MVA (73.0% and 71.7%) groups (Figure 3B).

Booster Immunogenicity, Follow-up Study

In response to revaccination with a single MVA-BN booster 2 years after the start of the initial study, nAb GMTs rapidly increased after 1 week and peaked 2 weeks after boosting (week 110) in both the 1×MVA BD and 2×MVA BD groups (80.7; 95% CI, 54.4–119.7 and 125.3; 95% CI, 89.5–175.3, respectively; Figure 2B). These booster responses were similar in magnitude and kinetics between treatment groups and exceeded peak responses following primary vaccination. Four weeks postbooster (week 112), nAb GMTs decreased by nearly half in both groups and declined further by 6 months postbooster, although nAb levels remained elevated (25.6; 95% CI, 15.8–41.4 for 1×MVA BD and 49.3; 95% CI, 32.4–75.0 for 2×MVA BD) relative to prebooster baseline, and were notably higher than those observed 6 months following primary vaccinations.

Consistent with these results, nearly all participants rapidly underwent nAb seroconversion as early as 1 week postbooster, peaking at 2 weeks postbooster (96.1% for 1×MVA BD and 98.7% for 2×MVA BD). Seroconversion rates remained high 6 months later (76.6% and 88.7%) (Figure 3A).

Total antibodies also showed rapid postbooster titer increases (Figure 2D and Figure 3B). Total antibody GMTs were higher following the booster compared with either 1 or 2 primary vaccinations and nearly 3 times higher in the 2×MVA BD group (1688.2; 95% CI, 1381.5–2062.9) compared to the HSPX group in the initial study (568.8; 95% CI, 473.3–683.6).

All observed GMTs for nAbs and total antibodies are provided in Supplementary Table 1.

Across all time points, immunogenicity results based on the per protocol set were consistent with those of the full analysis set (data not shown).

Overall Safety Assessment

The safety results of the initial study have been previously described [25]. As such, only the safety results for the follow-up study are described herein.

Overall, for solicited AEs, the most common local events were injection site erythema (82.2%) and pain (80.3%), while the most common systemic events were fatigue (32.2%), myalgia (23.7%), and headache (28.9%) (Table 2). Most solicited AEs were mild or moderate in intensity. However, 1 subject in the 1×MVA group and 3 subjects in the 2×MVA group experienced grade 3 (severe) events of headache, myalgia, and/or fatigue. No subjects experienced any grade 4 (life-threatening or disabling) events.

Table 2.

Overview of Solicited and Unsolicited Adverse Events in the Follow-up Study

Type of Adverse Event	2×MVA BDa (n = 75)	1×MVA BDa (n = 77)	All Participants (n = 152)
Solicited local adverse events
ȃInjection site erythema	60 (80.0)	65 (84.4)	125 (82.2)
ȃInjection site pain	58 (77.3)	64 (83.1)	122 (80.3)
ȃInjection site swelling	51 (68.0)	48 (62.3)	99 (65.1)
ȃInjection site induration	58 (77.3)	58 (75.3)	116 (76.3)
ȃInjection site pruritus	30 (40.0)	36 (46.8)	66 (43.4)
Solicited systemic adverse events
ȃFatigue	22 (29.3)	27 (35.1)	49 (32.2)
ȃMyalgia	17 (22.7)	19 (24.7)	36 (23.7)
ȃHeadache	19 (25.3)	25 (32.5)	44 (28.9)
ȃNausea	6 (8.0)	12 (15.6)	18 (11.8)
ȃBody temperature increased	3 (4.0)	4 (5.2)	7 (4.6)
Unsolicited adverse events
ȃAdverse event	40 (53.3)	38 (49.4)	78 (51.3)
ȃRelated adverse eventb	11 (14.7)	9 (11.7)	20 (13.2)
ȃSerious adverse event	2 (2.7)	0	2 (1.3)
ȃRelated serious adverse eventb	0	0	0
ȃAdverse event of special interest	2 (2.7)	3 (3.9)	5 (3.3)
ȃRelated adverse event of special interestb	0	0	0

Data are No. (%). Percentages are based on the number of participants in the group.

Abbreviations: BD, booster dose; MVA, modified vaccinia Ankara; MVA-BN, modified vaccinia Ankara-Bavarian Nordic.

Participants in these treatment groups received a booster MVA-BN vaccination 2 years after receiving either 1 or 2 primary MVA-BN vaccinations in the initial study.

Definite, probable, possible, or unknown relationship to vaccination.

Approximately half (51.3%) of all follow-up study participants experienced at least 1 unsolicited AE, and 13.2% of participants had AEs considered vaccine related (Table 2). Injection site warmth was most commonly reported (3.9%) (Table 3). Pain in extremity, reported by 1 participant in the 2×MVA BD group, was the only severe unsolicited AE considered possibly related to study treatment. This event was not serious, and the participant recovered the same day.

Table 3.

Overview of Related Unsolicited Adverse Events Experienced by >1 Participant in the Follow-up Study

Preferred Term	2×MVA BDa (n = 75)	1×MVA BDa (n = 77)	All Participants (n = 152)
Injection site warmth	3 (4.0)	3 (3.9)	6 (3.9)
Lymphadenopathy	3 (3.9)	0	3 (2.0)
Chills	0	2 (2.6)	2 (1.3)
Dizziness	1 (1.3)	1 (1.3)	2 (1.3)
Injection site irritation	2 (2.7)	0	2 (1.3)
Nasopharyngitis	2 (2.7)	0	2 (1.3)

Data are No. (%). Percentages are based on the number of participants in the group.

Abbreviations: BD, booster dose; MVA, modified vaccinia Ankara; MVA-BN, modified vaccinia Ankara-Bavarian Nordic.

Participants in these treatment groups received a booster MVA-BN vaccination 2 years after receiving either 1 or 2 primary MVA-BN vaccinations in the initial study.

Two participants (1.3%), both in the 2×MVA group, experienced SAEs (gastroenteritis and concussion) that resolved without sequelae; neither was considered related to study vaccination. Five participants (3.3%) experienced AESIs: 2 participants in the 2×MVA BD group experienced palpitations, and in the 1×MVA BD group, 1 participant each experienced musculoskeletal chest pain, palpitations, and noncardiac chest pain. All AESIs were mild or moderate in intensity and considered unlikely related to study medication. No deaths occurred during the follow-up study and no participants withdrew or discontinued due to an AE.

DISCUSSION

In this study, an MVA-BN vaccine booster dose induced a robust immune response regardless of smallpox vaccination history and use of 1 or 2 doses of MVA-BN for priming. Two years after priming in naive individuals, nAb titers rapidly increased, were highest at 2 weeks, and remained elevated relative to baseline at 6 months after boosting. A single MVA-BN booster administered to those vaccinated with older-generation replicating smallpox vaccines in the distant past also evoked the same pattern of rapid increase in nAb titers. The anamnestic nAb response was similar in magnitude in all groups, although slightly lower in both MVA-BN–primed groups. Booster responses greater than responses to primary vaccination are consistent with what has been previously observed following revaccination with other smallpox vaccines [36].

These data suggest that priming with 1 or 2 doses of MVA-BN can induce B-cell immune memory similarly to that of older-generation replicating smallpox vaccines, which have conferred long-term protection and are believed to have prevented orthopox outbreaks (including monkeypox transmission) until recently [6, 7].

While nAbs in those immunized long ago were detectable decades later, a protective threshold has not been defined for smallpox, monkeypox, or other orthopox viruses [37]. At booster study baseline, those who received primary MVA-BN immunizations 2 years earlier exhibited nAb levels that had declined to near baseline levels. Even so, a single MVA-BN booster induced robust anamnestic responses, regardless of the primary vaccination schedule administered. The rapid kinetics mirrored those observed after MVA-BN boosting in participants immunized long ago with older-generation replicating smallpox vaccines. This demonstration of long-lived B-cell memory strongly supports protection in the absence of persisting nAbs, particularly when considering the mean incubation periods for smallpox and monkeypox are both over 1 week (12.5 and 8.5 days, respectively) [38, 39], with minimum latencies of 7 and 3 days [38, 40].

Sustained MVA-BN–induced protection has been observed in nonhuman primates challenged with monkeypox virus [41, 42]. Although postimmunization nAb titers fell to prevaccination levels after about 3 years, they quickly rose upon exposure to the virus, and the animals were protected against disease. Thus, even a single MVA-BN vaccination, as administered in this study, may offer protection against future exposure.

This type of long-term immunological memory, observed in the absence of sustained nAbs, has been demonstrated with vaccines for hepatitis B virus (HBV) and measles, mumps, and rubella (MMR). In HBV studies, 90% to 100% of previously vaccinated participants who were seronegative exhibited rapid anamnestic responses when given a booster vaccination [43, 44]. Neutralizing mumps antibodies were found to decrease over time [45], as nearly half of young adults given MMR vaccinations 20 years earlier as infants were seronegative. However, over 70% of seronegative individuals had an anamnestic response upon revaccination [46].

In this study, MVA-BN booster vaccination induced robust total antibody responses in all groups. However, in contrast to nAb titers, total antibody titers were substantially higher upon booster vaccination in those primed with 1 or 2 doses of MVA-BN than in those primed with older-generation replicating vaccines in the distant past. Although the difference in time between priming and boosting in the different groups may confound this finding, increasing evidence suggests nonneutralizing antibodies may play significant roles in protecting against some viral infections [47, 48]. These non-nAbs may elicit effector pathways such as antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and antibody-mediated complement-dependent cytotoxicity. Furthermore, although nAbs are primarily effective against the targeted protein, less specific antibodies can target less variable, cross-serotype viral proteins. Thus, the very robust total antibody response induced by MVA-BN may be important in providing cross-protection against other orthopox viruses such as monkeypox [41, 42].

No safety concerns were identified in the follow-up study. This is consistent with the initial study [25]. Because reports of myopericarditis were reported in military personnel administered replicating smallpox vaccines [10, 12, 49, 50], cardiac events have been intensively monitored throughout the MVA-BN clinical development program. The cardiac AESIs observed in this study were considered unrelated to study vaccination. Therefore, the safety findings further support the existing safety profile of MVA-BN in adults, as no cardiac safety concerns or cases of myo-/pericarditis have been identified [23, 25, 26]. The immunological and safety findings described in this study are expected to translate to the recently developed freeze-dried formulation, as previous trials have demonstrated comparable safety profiles and noninferior immune responses between the 2 formulations.

The study was conducted in a single European site, and lack of racial or ethnic diversity is one of the limitations. However, other MVA-BN studies [19] that enrolled a more diverse population have not revealed any racial difference in immunogenicity or safety. Another limitation is that the antigens used in the immune assays were based on vaccinia, and not on Monkeypox. This is consistent with the antigen used for evaluation of other smallpox vaccines that have been demonstrated to confer protection against smallpox.

CONCLUSION

Priming with either 1 or 2 doses of MVA-BN induced durable immune memory as boosting 2 years later elicited a rapid and sizable anamnestic response comparable to boosting following immunization in the distant past with older-generation replicating smallpox vaccines.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Author contributions. H. I. contributed conceptualization, supervision, and investigation (trial site). N. S. contributed conceptualization, and supervision. D. R. performed project administration. D. S. and R. N. performed supervision, investigation (laboratory), and validation. J. D. P. prepared the initial draft. T. P. H. M. performed supervision, investigation (laboratory), validation, and visualization. G. S. performed formal analysis, validation, and software. L. D. M. contributed supervision. L. C. performed the formal analysis. H. W. contributed conceptualization, methodology, and supervision. P. C. contributed conceptualization, methodology, supervision, and funding acquisition. All authors reviewed and edited the manuscript.

Acknowledgments. The investigators thank all study participants. We acknowledge in memoriam the contributions of Frank von Sonnenburg and Stephan de la Motte. We also thank Josef Weigl, Alfred v. Krempelhuber, Siegfried Rösch, and Garth Virgin for their efforts.

Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases (grant number N01-AI40072); and was sponsored by Bavarian Nordic.