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Safety and Immunogenicity of Modified Vaccinia Ankara (ACAM3000): Effect of Dose and Route of Administration

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. Author manuscript; available in PMC: 2011 May 1.

Published in final edited form as: J Infect Dis. 2010 May 1;201(9):1361–1370. doi: 10.1086/651561

Abstract

Background

We conducted a clinical trial of the safety and immunogenicity of Modified Vaccinia Ankara (ACAM3000 MVA) to examine the effects of dose and route of administration.

Methods

Seventy-two healthy, vaccinia-naïve subjects received one of six regimens of MVA or placebo in two administrations one month apart.

Results

MVA was generally well tolerated at all dose levels and by all routes. More pronounced local reactogenicity was seen by the intradermal (ID) and subcutaneous (SC) routes than by intramuscular (IM) administration. Binding antibodies to whole virus and neutralizing antibodies to IMV and EEV forms of vaccinia virus (VV) were elicited by all routes of MVA administration, and were greater for the higher dose by each route. Similar levels of neutralizing antibodies were seen at a ten-fold lower dose given ID (107 TCID50), compared to responses after 108 TCID50 given IM or SC. T-cell immune responses to VV were detected by IFN-γ ELISPOT, but had no clear relationship to dose or route.

Conclusion

These data suggest that ID immunization with MVA provides a dose sparing effect by eliciting antibody responses similar in magnitude and kinetics to those elicited by IM or SC routes, but at a 10-fold lower dose.

Keywords: Safety, Immunogenicity, Dose, Route, MVA

INTRODUCTION

An effective vaccination program led by the World Health Organization (WHO), eradicated smallpox in 1980 [1, 2]. Despite this extraordinary achievement, the immunologic basis for the efficacy of vaccination against smallpox remains incompletely understood, and correlates of protection are not fully defined. Furthermore, despite its effectiveness, the use of smallpox immunization with strains of vaccinia such as Dryvax can be associated with significant morbidity, particularly in subjects with certain host-defense defects and dermatopathologic conditions [3, 4]. Therefore, the development of safer, yet efficacious vaccines for future use against smallpox remains of considerable interest.

We studied an attenuated strain of vaccinia, Modified Vaccinia Ankara (MVA) [5–7] which has been reported to be less reactogenic than widely used vaccinia strains such as Dryvax, and yet possibly confers a degree of protection against orthopox virus infections, such as those caused by variola. MVA is severely host restricted, and is either unable to replicate in mammalian cell lines or replicates at a very low level (less than 2 pfu/cell) [8–10].

The optimal regimen of immunization with MVA is not known. Therefore, we conducted a clinical study of immunization with MVA to examine the effects of dose and route of administration on tolerability and immune responses. We were particularly interested in exploring the ID route of administration, because it has been associated with similar levels of immune responses to those elicited by higher doses administered SC or IM for several vaccines, thus resulting in a potential dose sparing effect [11–15].

MATERIALS AND METHODS

Vaccine

The MVA vaccine used in the study was ACAM3000 (lot number 460304KA, ACAMBIS, Inc., Cambridge, MA) formulated with 20mM Tris, 0.9% NaCl USP, and 0.01% neomycin USP with a titer of 2.54 × 108 TCID50/mL. The vaccine was reconstituted with 0.9% NaCl and diluted to the appropriate dose. Sterile saline, 0.9% NaCl, was used as the placebo. The dose of MVA administered was verified by back titration for each dose tier that was studied.

Study Design and Subjects

The study design was a dose escalation of MVA administered IM at doses of 107 or 108 TCID50, SC at 107 or 108 TCID50, ID at 106 or 107 TCID50, or placebo, given in a two-dose regimen at day 0 and day 28. (107 TCID50 was the maximum dose that could be given ID, because of the volume [0.1ml] which could be given by that route.) (Table 1). Study preparation or placebo was administered under a randomized, double-blind allocation. Subjects were healthy men or women, at least 18 years of age who were born after 1971 and had no history of smallpox vaccination. Good health was determined by history, physical examination, and laboratory tests.

Table 1.

Experimental Design of Clinical Trial of Immunization of Subjects with ACAM3000 MVA

Vaccination at Days 0 and 28
Group N Vaccine Dose Route
A 10 ACAM3000 MVA 106 TCID50 ID
2 Placebo — ID
B 10 ACAM3000 MVA 107 TCID50 IM
2 Placebo — IM
C 10 ACAM3000 MVA 107 TCID50 SC
2 Placebo — SC
D 10 ACAM3000 MVA 108 TCID50 SC
2 Placebo — SC
E 10 ACAM3000 MVA 107 TCID50 ID
2 Placebo — ID
F 10 ACAM3000 MVA 108 TCID50 IM
2 Placebo — IM
Total 72

Twelve subjects were enrolled sequentially into six groups each consisting of 10 vaccine and 2 placebo recipients, for a total of 72 subjects (Table 1). The study was approved by the Institutional Review Board, and written informed consent was obtained from all subjects.

Safety and Reactogenicity Evaluation

To assess reactogenicity after each immunization, subjects maintained a diary to record daily temperatures and reactions for at least 14 days or until any symptoms resolved if longer. Hematology and chemistry evaluations were then performed on days 4, 7, 14, 28, 32, 35, 42, 56, 84, and 180. Cardiac evaluations using standardized questions were performed at each visit. ECGs and troponin levels were performed on days 14, 28, 42, 56, and 180 after the first immunization. Non-serious adverse events were collected through day 28 after the last immunization, and serious adverse events (SAEs) were collected throughout the study period. Toxicity was graded according to standard NIAID-DMID Toxicity Tables.

Cell Lines and Vaccinia Viruses

HeLa, CV-1, and DF-1 cell lines, and vaccinia viruses [Western Reserve Strain (VV:WR); ACAM3000 MVA; recombinants containing a luciferase reporter gene (VV:Luc and MVA:Luc), and IHD-J strain (VV:IHD-J)] are described in the online supplemental material [17].

Immunogenicity Assays

Enzyme-Linked Immunosorbent Assays (ELISA)

ELISA assays were performed on serum samples obtained on days 0, 14, 28, 35, 42, 84, and 180 post immunization, as previously described [18]. (See online supplemental material).

Neutralization Assay

Neutralizing antibody responses against VV and MVA were measured on serum samples obtained on days 0, 7, 14, 28, 35, 42, 56, 84, and 180 post immunization, using a luciferase based assay in HeLa or DF-1 cells as previously described [18]. (See online supplemental material).

Comet Reduction Assay

Comet reduction assays were performed on serum samples obtained on day 42 post immunization with VV: IHD-J in CV-1 cells and analyzed using densitometry with Image J Software as previously described [19] (See online supplemental material).

T cell IFN-γ ELISPOT

ELISPOT assays were performed on PBMCs obtained on days 0, 14, 28, 35, 42, 56, 84, and 180 post immunization as described with minor modifications [20–23]. (See online supplemental material).

Statistical Analysis

Fisher’s exact test was used to test for association between categorical variables. The exact Wilcoxon rank-sum test was used to assess group differences in continuous measures. All tests were two-sided. All subjects received the first vaccine, and 69 (97.2%) received the second injection. Of the three subjects who did not receive the second injection, two were in the MVA SC 107 TCID50 group, and one was an ID placebo recipient. One subject was lost to follow-up, and two voluntarily withdrew due to personal reasons unrelated to the study. Thus, 69 subjects who received both injections were included in an intent-to-treat analysis of immunogenicity. To adjust for multiple comparisons of immunogenicity, P values were considered significant at the 0.01 level.

RESULTS

Subject Characteristics

Seventy-two subjects were enrolled in the study from October 2005 through March 2007. Forty-three participants (59.7%) were female. Fifty-eight subjects (80.5%) were white, 7 (9.7%) were Asian, and 7 (9.7%) were of other racial groups. Subjects ranged in age from 18 to 34 years, with a median age of 25 years.

Safety and Reactogenicity

MVA immunization was well tolerated at all dose levels and by all routes of administration. Local reactogenicity was common in all regimens and consisted of discomfort, erythema, or induration at the inoculation site, which generally resolved within 4 to 7 days, with either no treatment or with over the counter analgesics (Figure 1A). Severe local reactogenicity which consisted of erythema and induration of 31 to 70 mm in size, was more frequent in ID (8/20) and SC (5/20) vaccine recipients than in IM (0/20) vaccinees (P=0.003 and 0.047, respectively). Local reactogenicity was correlated with immune responses on days 14, 35 and 42 (binding antibody responses: P≤0.0005; neutralizing antibody responses: P≤0.0008). Thirteen (65%), 17 (85%) and 10 (50%) subjects who received ID, IM and SC MVA respectively, experienced systemic reactogenicity [P=0.02, 0.0005, and 0.13 respectively compared to placebo (2/12)], (Figure 1B). Systemic reactogenicity, graded as mild or moderate, occurred in 33 subjects and consisted of fever (100.1–101.0 F°) for one to two days in two subjects, and malaise, myalgia, arthralgia, headache, or nausea which lasted from one to seven days. Systemic reactogenicity, graded as severe, occurred in five subjects and consisted of malaise, headache or chills, which resolved within 24 hours. There were no differences in frequency or severity of systemic reactions between the higher and lower doses of MVA or among the different routes. There were no differences in reactogenicity noted after the first vaccination compared to the second vaccination. All systemic reactogenicities were self-limited and resolved without sequelae. Systemic reactogenicities were not correlated with immune responses.

Figure 1.

Figure 1

Proportion of vaccinees experiencing (A) local or (B) systemic symptoms after first or second MVA vaccination, by dose and route. Severity of symptoms were graded by NIAID-DMID toxicity tables.

Adverse Events

Two non-serious adverse events were considered to be associated with the vaccine. One was a subcutaneous lump (15 mm in diameter) proximal to the vaccine site, which resolved over seven days without therapy in a subject who received MVA at 107 SC. The other adverse event was skin pigmentation at the vaccination site in the form of a reddish brown macule, which also resolved without therapy in a subject who received MVA at 107 ID. No serious adverse events were related to vaccination.

Because of the reports of myopericarditis in recipients of vaccinia, subjects were examined closely for possible cardiac effects of immunization. Four subjects experienced mild chest pain or discomfort within 24 hours to three weeks after the second immunization. These were found to be related to musculoskeletal or gastrointestinal disorders, resolved, and were deemed not related to the vaccine. No subject had clinical evidence of myopericarditis or ECG findings or troponin levels suggestive of myopericarditis.

Binding Antibody Responses Detected by ELISA

Binding antibody responses developed to MVA in 93% (54/58) and to VV in 88% (51/58) of subjects who received two immunizations. Following the first vaccination, elevated anti-MVA titers compared to placebo were first seen at day 14 in groups receiving 108 SC and 108 IM (P≤ 0.001 for both groups) and by day 28 in groups receiving 107 SC, 108 SC, 107 ID and 108 IM (P=0.02, < 0.001, 0.01, and <0.001, respectively) (Figure 2A). A significant increase in anti-MVA ELISA titers was observed following the second immunization (day 42) in all MVA groups compared to the placebo group (P=0.01 for 106 ID, and P< 0.001 for the other 5 groups). At day 42, the higher dose groups for ID and SC routes had greater anti-MVA ELISA titers compared to the lower dose groups (P=0.005 and 0.002, respectively). Importantly, MVA recipients demonstrated serum ELISA antibody binding titers to VV:WR antigen that were similar in time course and magnitude to the responses measured against MVA, suggesting a high degree of antibody cross-reactivity (Figure 2B).

Figure 2.

Figure 2

Binding antibody responses elicited by MVA prime/boost immunizations. Serum samples were obtained at days 0, 14, 28, 35, 42, 84, and 180 following MVA immunization. Serial dilutions were tested for antibody binding activity against (A) ACAM3000 MVA or (B) VV:WR by ELISA. Data are presented as median serum endpoint with the interquartile ranges for each dose and route-of-immunization group. The dashed line represents the limit of detection (serum endpoint titer=30), and arrows indicate days of vaccination.

To further examine MVA-elicited antibodies, we assessed binding antibody responses to antigens associated with the vaccinia intracellular mature virions (IMV: A27L and L1R) or extracellular enveloped virions (EEV: A33R and B5R). Data for the higher dose groups by each route and for the placebo group are shown in Figure 3. By day 14 after the first immunization with MVA, 19/20 individuals in the 108 SC and 108 IM groups had ELISA antibody titers to A33R, and 20/20 subjects had titers to B5R and L1R. By day 42, the 107 SC (data not shown), 107 ID, 108 SC, and 108 IM groups had titers to all three antigens significantly greater than placebo (P<0.007), and no significant differences in responses were observed among the high doses by each route. Responses to A27L were generally quite low and did not consistently increase following the second immunization when compared with responses to the other 3 antigens.

Figure 3.

Figure 3

Antibody responses to IMV and EEV associated antigens following MVA prime/boost immunizations. Serum samples were obtained two weeks following primary immunization (d.14) and two weeks following boost immunization (d.42). Serial dilutions were tested for antibody binding activity against two IMV (A27L and L1R) and two EEV (A33R and B5R) associated protein antigens by ELISA. Data are presented as individual endpoint titers with bars indicating median titer per group. PL indicates the placebo group.

Neutralizing Antibody Responses to MVA and VV:WR

Neutralizing antibody responses to MVA were detected in 91% (53/58) of vaccinees who received two immunizations (Figure 4A). Following primary immunization, elevated titers were observed on day 14 in the higher dose group compared to the corresponding lower dose group (P=0.04 for ID, 0.004 for IM, and 0.002 for SC). These responses were increased following the second immunization, and the higher dose groups continued to exhibit higher titers than the lower dose groups through day 180 (P<0.01). Peak neutralizing antibody titers typically occurred on days 35–42 (7–14 days after the second immunization), and median titers were greater than placebo for all six vaccination groups (P=0.03 for the 106 ID group, and P<0.001 for the other five groups). By day 180, only titers in the higher dose groups by each route remained significantly increased compared to placebo (P<0.001 for all). No differences were observed in the time course or magnitude of the anti-MVA neutralizing antibody responses among the higher dose groups (107 ID, 108 SC, and 108 IM). These data demonstrate that the 107 ID group, despite receiving a 10-fold lower dose of MVA, elicited similar neutralizing antibody responses to those observed in individuals who received a 108 dose via IM or SC routes.

Figure 4.

Figure 4

Neutralizing antibody responses elicited by MVA prime/boost immunizations. Serum samples were obtained at days 0, 7, 14, 28, 35, 42, 56, 84, and 180 following MVA immunization. Serial dilutions were tested for neutralizing activity against (A) MVA:Luc or (B) VV:Luc. Data are presented as median ID50 titers with interquartile ranges for each dose and route-of-immunization group. The dashed line represents the limit of detection (serum ID50 titer=10), and arrows indicate days of immunization.

The cross-reactivity of MVA-elicited neutralizing antibody responses to VV:WR virus was also assessed. Neutralizing antibodies to VV:WR were seen in 81% (47/58 of subjects). Overall, the kinetics of anti-VV neutralizing antibody responses for each group were similar to the anti-MVA neutralizing antibody responses, though the magnitude was diminished (Figure 4B). By day 42, the 107 ID, 107 SC, 108 SC, and 108 IM groups all had neutralizing antibody titers that were significantly increased compared to placebo (P=0.007 for 107 SC, and P<0.001 for the others), and each higher dose given by a particular route had significantly increased responses compared to the corresponding lower dose (P<0.001 for all). No significant differences were observed in the magnitude of anti-VV neutralizing antibody titers among the 3 higher dose groups by each route through day 180. However, differences were found among the three 107 groups. The ID route had higher anti-VV neutralizing titers on day 14 (P=0.05 for 107 ID vs. 107 IM, P=0.02 for 107 ID vs. 107 SC), which peaked by day 42 (P=0.008 107 ID vs. 107 IM and P=0.009 for 107 ID vs. 107 SC). No significant differences were observed among these groups by day 180.

Comet Reduction Assay

The ability of MVA vaccination regimens to elicit neutralizing antibodies against the EEV form of VV was assessed by the comet reduction assay. Two weeks following the second immunization (day 42), serum comet reduction activities in the 108 IM and 108 SC groups were higher than in the placebo group (P=0.0003 and 0.005, respectively) (Figure 5). In contrast, no significant comet reduction was detected in sera from each of the lower dose groups. Importantly, comet inhibition activity in the 107 ID group was higher than in the 107 IM and 107 SC groups, and was similar to levels observed in the 108 IM and 108 SC groups (Figure 5).

Figure 5.

Figure 5

Assessment of anti-EEV neutralizing antibody responses by comet reduction assay. Serum samples were obtained two weeks following boost immunization (d. 42) and tested in a comet reduction assay at a 1:50 dilution. Data are presented as the percent comet reduction observed from individual subjects in each dose and route-of-immunization group, with bars indicating the median response. PL indicates the placebo group.

T-cell Responses by IFN-γ ELISPOT

The magnitude and kinetics of anti-VV T-cell responses were assessed by IFN-γ ELISPOT assay (Figure 6). At day 14 after the first immunization, only MVA administration by the IM route elicited significantly higher responses compared to the placebo group (P=0.005 and <0.001 for the 107 and 108 dose groups, respectively). Two weeks following the boost immunization (day 42), both the 107 and 108 dose groups for the IM and SC routes of administration had significantly higher responses than the placebo group (P≤ 0.007 for all four groups). T-cell responses elicited by the ID route were consistently lower than responses measured in groups receiving MVA via the IM or SC routes.

Figure 6.

Figure 6

Cellular immune responses elicited by MVA prime/boost immunizations. PBMC were obtained at days 0, 14, 28, 35, 42, 56, 84, and 180 following MVA immunization and tested by the IFN-γ ELISPOT assay against autologous VV:WR-infected target cells. Data are presented as median SFC per 106 effector PBMC with interquartile ranges for each dose and route-of-immunization group following subtraction of responses to medium alone and pre-immune background values.

Discussion

ACAM3000 was safe and generally well tolerated at all doses and by all three routes, and self-limited local discomfort was the most frequent reactogenicity. More pronounced local reactogenicity was more common in the ID and SC groups compared to the IM group. Self-limited systemic reactogenicities were encountered in half of the vaccinees and were not significantly different among the various regimens. No serious adverse events were associated with vaccination. Phase I studies of other MVA candidate vaccines have also shown that they are well tolerated [24–26].

We extensively characterized antibody responses elicited by MVA vaccination by various routes and doses. Both binding and neutralizing antibody responses against MVA, VV:WR and three individual IMV and EEV associated antigens were clearly generated by the 107 and 108 doses for the IM and SC routes, and by the 107 dose for the ID route, and the higher dose groups for each route elicited generally greater responses. Importantly, a single administration of MVA at the higher dose groups (107 ID, 108 SC, and 108 IM) elicited detectable anti-MVA binding and neutralizing antibody titers in the majority of subjects by day 14. These titers substantially increased by two weeks after the second immunization, and all subjects in the higher dose groups had detectable responses. Furthermore, antibody responses in the higher dose groups by each route remained detectable through day 180. While antibody responses in the lower dose groups for each route also significantly increased after the second immunization, peak titers were significantly lower than those in the higher dose groups, and a few individuals in the 106 ID and 107 IM group still lacked detectable responses.

Of note, at the maximum doses that were administered, there were no significant differences in antibody responses according to route of administration. However, similar responses were obtained with a 10-fold lower dose of vaccine (107) administered ID, compared to 108 given IM or SC. This dose sparing effect is consistent with that observed with several other vaccines given ID [11–15], and suggests that ID administration may be a particularly efficient route for administration of certain immunogens. Immune responses after the first ID dose were not as robust as after the first dose given SC or IM, but were equivalent after the second ID dose, reflecting the impact of a prime/boost regimen. The dose sparing effect of ID administration may offer a significant advantage in terms of availability and cost.

Serum antibody responses to MVA and Dryvax have been reported in the studies of immunization with MVA-BN (IMVAMMUNE™), and appeared to be highly dose dependent when vaccine was given SC [24, 25]. In the study of TBC-MVA (Therion Biologics Corporation, Cambridge, MA), a lower dose of vaccine was utilized (106 pfu IM), and neutralizing antibody responses were not elicited [26]. This finding is consistent with our observation that MVA given at 106 ID generated lower levels of immune responses, and suggests that this dose may represent a lower threshold for stimulating immune responses to MVA in humans.

We also assessed the efficiency of antigen cross-recognition by MVA-elicited antibodies by utilizing ELISA and neutralization assays that incorporated VV:WR as target antigens. The magnitude and kinetics of antibody responses recognizing whole virus VV, as determined by ELISA, were similar to responses observed against MVA, suggesting the presence of a high degree of antibody cross recognition. We also observed efficient cross neutralization of VV:WR in sera from MVA vaccinees, although titers were generally lower than those against MVA. Of interest, a recent study has reported that MVA vaccinees mounted serum neutralizing antibody responses against variola, demonstrating the ability of MVA immunization to elicit cross-reactive immunity against smallpox [27].

Data suggests that optimal protection against orthopox virus infection is achieved when antibody responses target two structurally and antigenically distinct forms of infectious poxviruses, IMV and EEV [28]. Since the ELISA and neutralization assays described above used the IMV form of virus, we further assessed the ability of MVA immunization to elicit antibody responses against EEV by comet reduction assay and protein-specific ELISA. Sera from all three of the higher dose groups demonstrated neutralizing activity against EEV following the second immunization. We also assessed the generation of antibodies against two IMV and two EEV protein antigens which have been implicated in protection against VV infection [29–33] and are expressed by MVA. ELISA responses to the EEV-associated antigens, A33R and B5R, and the IMV antigen, L1R, were detected with each route, and higher responses were generated by the higher doses for each route. Of interest, only low level antibody responses were detected against the IMV antigen A27L. We have previously observed a similar lack of anti-A27L antibody responses in rhesus macaques immunized with high doses of MVA in contrast to immunization with VV [18], and others have also described a lack of anti-A27L responses following NYVAC administration [34]. It may be that responses to this IMV protein are lacking in certain attenuated vaccine strains. Recent reports have described additional antigens against which antibody responses have been reported to convey protection against VV infection, including H3L, D8L, and A28L [32, 35, 36]. It will be informative to further characterize the nature of antibody responses elicited by MVA against a broader panel of vaccinia encoded proteins at the doses and routes described herein.

We conducted only limited studies of T-cell immune responses, using an IFN-γ ELISPOT assay with VV:WR infected target cells. T-cell responses were observed, but without a clear effect of dose or route of administration. Boosting with the second dose of MVA appeared to increase the T-cell responses for some doses and routes. A previous study of MVA-BN reported T-cell responses to Dryvax after MVA immunization, but also did not find a dose response relationship [25]. CD4+ and CD8+ T-cell responses to MVA or Dryvax were reported in subjects vaccinated with TBC-MVA, and responses were augmented after three doses of MVA compared to one dose [26]. Interestingly, we observed that the recall response of T-cells following the second MVA immunization was higher in the 107 IM group compared to the 108 IM group, despite a higher response in the 108 group after the first immunization. Whether this reflects functionally better T-cell priming at the lower dose, or whether the high level of neutralizing antibodies generated in the higher dose IM group impaired the ability of the second MVA immunization to boost cellular immunity, remains to be determined. Additional studies of T-cell responses to define epitope specificity, breadth, phenotype and functional characteristics are needed to further characterize the effect of dose and route of administration of MVA on T-cell responses.

Supplementary Material

1

Acknowledgments

Funding:

National Institute of Allergy and Infectious Diseases (U.S. Public Health Service Grants U54 AI057159 and U19 AI057330)

We wish to thank Robert Johnson, Stephen Heyse, and Carol Ostrye of NIAID, Heather Hill and Dewei She of the EMMES Corporation, and John Jarcho of Brigham and Women’s Hospital for their assistance in this study.

Footnotes

Potential Conflict of Interest: none

Presented in part at:

New England Regional Center of Excellence/Biodefense and Emerging Infectious Diseases Annual Meeting, Brentwood, Lake George, New York, October 29-31, 2006 And AIDS Vaccine Annual Meeting, Cape Town, South Africa, October 13-15, 2008

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