NIH Public Access Author Manuscript J Infect Dis. Author manuscript; available in PMC 2011 May 1.

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1 NIH Public Access Author Manuscript Published in final edited form as: J Infect Dis May 1; 201(9): doi: / Effect of Immunization with Modified Vaccinia Ankara (ACAM3000) on Subsequent Challenge with Dryvax Michael S. Seaman 1,5, Marissa B. Wilck 2,5, Lindsey R. Baden 2,5, Stephen R. Walsh 3,5, Lauren E. Grandpre 1, Colleen Devoy 1, Ayush Giri 1, Lizanne C. Noble 2, Jane A. Kleinjan 2, Kristen E. Stevenson 4, Haesook T. Kim 4, and Raphael Dolin 1,2,5 1 Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Boston, MA 02115, United States 2 Division of Infectious Diseases, Brigham and Women s Hospital, Boston, MA 02115, United States 3 Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114, United States 4 Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, United States 5 Department of Medicine, Harvard Medical School, Boston, MA 02115, United States Abstract Background Despite the success of smallpox vaccination, the immunological correlates of protection are not fully understood. To investigate this question, we examined the effect of immunization with Modified Vaccinia Ankara (MVA) upon challenge with replication competent vaccinia (Dryvax). Methods Dryvax challenge via scarification was conducted in 36 healthy subjects who had received MVA (29) or placebo (7) in a previous study of doses and routes of immunization. Subjects were followed for clinical takes, virus shedding and immune responses. Results MVA administration attenuated takes in 21/29 (72%) subjects compared to 0/7 placebo recipients (P=0.001). Attenuation was most significant in MVA groups that received 10 7 TCID 50 intradermally (P=0.0014) and 10 7 TCID 50 intramuscularly (P=0.0013). Both duration and peak titer of virus shedding were reduced in MVA recipients. Peak neutralizing antibody responses to vaccinia or MVA previously induced by MVA immunization were associated with attenuated takes (P=0.02) and reduced duration (P= and ) and titer (P=0.005) of virus shedding. Conclusions MVA immunization results in clinical and virologic protection against Dryvax challenge. Protection is associated with prior induction of neutralizing antibodies to MVA or vaccinia. MVA administered intradermally has protective and immunologic responses similar to a ten-fold higher dose given subcutaneously. Reprints or Correspondence: Dr. Raphael Dolin, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, E/CLS-1003, Boston, MA Potential Conflict of Interest: none Presented in part at: New England Regional Center of Excellence/Biodefense and Emerging Infectious Diseases Annual Meeting, Brentwood, New Hampshire, Nov , 2007 Trial Registration: Identifier: NCT

2 Seaman et al. Page 2 Keywords Protection; MVA; Dryvax challenge; clinical takes Introduction Despite the extraordinary success of immunization with vaccinia in the eradication of smallpox, the immunologic basis for the effectiveness of the smallpox vaccine is not fully understood [1]. To investigate this, we conducted two clinical studies. The first was a study of the safety and immunogenicity of a candidate smallpox vaccine, modified vaccinia Ankara (MVA), ACAM3000 [2]. The purpose of this first study was to investigate the humoral and cell-mediated immune responses to immunization with MVA, and to determine the effects of dose and route of administration. The second study, reported herein, examines the effect of previous immunization with MVA on subsequent challenge with a replication competent vaccinia virus, Dryvax. A candidate smallpox vaccine, such as MVA [3], is intended to provide protection against variola (smallpox virus), but clinical studies to directly examine the effect of MVA vaccination on infection with variola are inappropriate because of biosafety and ethical considerations. However, we believe that challenge with Dryvax can serve as an appropriate human model to investigate the protective effects of immunization against smallpox. For this purpose, we offered Dryvax challenge to all participants in our previously conducted MVA immunization study. Subjects who elected to participate were followed for the effects of Dryvax challenge, including clinical manifestations ( takes ), vaccinia virus shedding, and humoral and cellular immune responses. Materials and Methods Dryvax Vaccinia Virus The vaccinia virus used for challenge was Dryvax (Wyeth Laboratories, Marietta, PA; lot number ) provided by the Centers for Disease Control and Prevention. Dryvax was a lyophilized preparation derived from calf lymph and reconstituted with a diluent containing 50% glycerine and 0.25% phenol in sterile water (Chesapeake Biologics Lab., Inc.). The reconstituted vaccine contains 10 8 pfu/ml of vaccinia virus. Study Design and Subjects The subjects were healthy volunteers, whose entry and exclusion criteria are previously described [2]. The study was approved by the institutional IRB, and written informed consent was obtained from all subjects. Double-blind allocation to MVA or placebo was maintained [2]. The Dryvax challenge was to have been administered six months after MVA immunization, but in 2006, in discussion with NIH and FDA, the investigational use of Dryvax was delayed because of concern for possible cardiac adverse events [4 7]. Therefore, Dryvax challenges were delayed in some subjects, and the interval between MVA vaccination and Dryvax challenge was extended to 15 months. In 2007, the FDA prohibited the investigational use of Dryvax in clinical studies, so the group that had previously received the highest dose of MVA intramuscularly (10 8 TCID 50 ) was not able to be challenged with Dryvax. Administration of Vaccinia Vaccinia was administered by the standard scarification technique utilizing a bifurcated needle pressed 15 times to a 5 mm area of skin over the deltoid [8], after which a dressing

3 Seaman et al. Page 3 was applied. Subjects were evaluated, and the vaccination site was photographed on days 4, 7, 10, 14, and 28 after vaccinia challenge. Subjects who had lesions after day 14 following Dryvax challenge had additional visits every 3 5 days until the site healed. Assessment of Dryvax Takes Following Dryvax challenge, the vaccination site reaction was categorized into the following take categories that have been derived from standard definitions [9,10]: category 0: no take no skin reaction at vaccination site; category 1: modified take, without vesicle a papule with or without surrounding erythema by the 3 rd day and without vesicle or pustule formation prior to resolution; category 2: modified take, with vesicle a papule by the 3 rd day that became vesicular by the 5 7 th day and dried shortly thereafter; and category 3: primary or full take a papule develops, becomes vesicular, umbilicated, and pustular by the 7 10 th day. The lesion becomes crusted and dries by the 3 rd week. Categories 1 and 2 were considered attenuated takes and category 3 represented a full take or no attenuation. The determination of take category was conducted by three physicians blinded to the volunteers vaccination status with respect to either MVA or placebo. Assessment of Virus Shedding Immunogenicity Assays Statistical Analysis Results Virus cultures of the inoculation sites were performed to assess virus shedding at each visit. (See online supplemental material) Humoral antibody assays (neutralization, comet-reduction, and ELISA) and T-cell IFN-γ ELISPOT assays were carried out as previously described [2]. Fisher s exact test was used to test for association between categorical values. The Wilcoxon rank-sum test or the Kruskal-Wallis test was used to assess group differences in continuous measures. All tests were two-sided. To investigate the effect of pre-dryvax immune responses on viral shedding (post-dryvax), a simple group comparison as well as a linear regression analysis was performed. The maximum values of pre-dryvax MVA and VV antibody titers, the maximum values of titer and duration of virus shed, and ELISPOT baseline adjusted counts for each subject were used. In the linear model, dose and route of MVA immunization were adjusted. Maximum pre-dryvax immune response measures and maximum post-dryvax virus titers were log 10 -transformed prior to modeling. The interaction between dose and route was also examined in each of these models. Locally weighted scatterplot smoothing (Lowess) [11,12] was used to describe the impact of the immune response to MVA vaccination on swab titer and viral shedding over time after Dryvax challenge (Figures 1 and 6). Characteristics of Subjects 36 subjects received Dryvax challenge at 6 15 months following two doses of MVA immunization. 29 of these subjects had received MVA vaccination as part of the previous study [2], and 7 had received placebo and were thus still vaccinia naïve prior to Dryvax challenge. For the 29 subjects who had previously received MVA immunization, the median day of challenge (scarification) with Dryvax was 190 (range ) after the first immunization. 18/36 subjects were male, 83% were white, 11% were Latino, and the median age was 27 years (range 20 34).

4 Seaman et al. Page 4 Clinical Responses to Dryvax Challenge All 36 subjects who were challenged with Dryvax experienced local reactions at the site of inoculation. Prior MVA vaccination was significantly associated with attenuation of takes in 21/29 (72%) MVA recipients. None (0/7) of the placebo recipients demonstrated attenuation of takes (P=0.001). Of note, 2/ ID recipients also experienced full takes, indicative of no attenuation. Each of the other groups experienced a significantly higher rate of attenuation compared to those who received placebo, except for 10 7 SC (Table 1). The most statistically significant differences in rates of attenuation occurred in the groups that received 10 7 ID (P=0.0014) and 10 7 IM (P=0.0013) compared to placebo recipients. Systemic reactions to Dryvax challenge occurred in 14/29 (48%) of MVA recipients and 6/7 (86%) of placebo recipients, were either mild or moderate in severity, and did not differ in characteristics among any of the MVA and placebo groups. Virus Shedding from Dryvax Lesions Virus shedding from Dryvax lesions was highly correlated with take categorization (Figure 1A). Prior immunization with MVA was significantly correlated with a lower peak titer of virus shedding in MVA recipients (median of pfu/ml) compared to placebo recipients (median of pfu/ml, P< 0.004), and with a shorter duration of viral shedding [median of 14 days (range 2 21) versus 21 days (range 20 29) in MVA and placebo recipients, respectively, P<0.0001] (Figure 1B). MVA recipients with category 3 takes had peak virus titers that were similar to placebo recipients, but had significantly shorter durations of shedding (median of 17 days versus 21 days respectively, P=0.003). Furthermore, MVA recipients with category 1 and 2 attenuated takes had lower peak titers of virus shed (median of and pfu/ml; P=0.001 and compared to placebo, respectively), and shorter duration of virus shedding (median of 7 days and 14 days, P=0.001 and compared to placebo, respectively). Neutralizing Antibody Responses Following Dryvax Challenge The development of neutralizing antibody responses to VV:WR in placebo recipients was consistent with a primary immune response (Figure 2). In these subjects, serum neutralizing antibody titers were first detected on day 14 after challenge, and continued to rise through day 28. In contrast, all MVA recipients had detectable anti-vv neutralizing antibody titers by day 7 post challenge. The responses in the 10 8 SC, 10 7 ID, 10 7 IM, and 10 7 SC groups peaked at day 14, and remained stable at day 28. The magnitude of the anti-vv neutralizing antibody responses in the two 10 6 ID recipients was lower than that of the other MVA vaccine groups at day 14, but was equivalent by day 28. At day 7 and 14, neutralizing antibody titers were significantly higher in MVA recipients (median ID 50 titers of 42 and 803, respectively) compared to placebo recipients (median ID 50 titers of < 10 and 58, respectively) (P=0.002 and for day 7 and 14, respectively). Sera were also tested for neutralizing activity against MVA virus, and the kinetics and magnitude of responses were similar to those observed against VV:WR (data not shown). We further assessed anti-eev neutralizing antibody activity in sera from MVA and placebo recipients by comet reduction assay 14 days after Dryvax challenge (Figure 3). Placebo recipients had detectable anti-eev neutralization activity by day 14 post-challenge (median 49% comet reduction), but responses were higher in the groups that received 10 8 SC, 10 7 ID, 10 7 IM, and 10 7 SC MVA (P=0.05, 0.04, 0.03, and 0.005, respectively). Only minimal comet reduction activity was observed in sera from the two 10 6 ID MVA recipients at this time point. Analysis of anti-eev neutralization at day 28 post-challenge showed similar levels of activity in all MVA and placebo groups (data not shown).

5 Seaman et al. Page 5 Binding Antibody Responses Post Dryvax Challenge Serum antibody binding titers to the intracellular mature virion (IMV)-associated protein antigens, L1R and A27L, and the extracellular enveloped virion (EEV)-associated protein antigens, A33R and B5R, were measured by ELISA on days 14 and 28 post-challenge (Figure 4). For these analyses, endpoint titers measured in all MVA recipients as a group were compared to those measured in placebo recipients. On day 14 post-challenge, serum endpoint titers against B5R, A33R, and L1R were higher in MVA recipients compared to placebo recipients (P=0.003, 0.02, 0.009, respectively), whereas endpoint titers against A27L were similar among these groups. Of note, we had previously observed only low level antibody responses against A27L following MVA prime/boost immunization [2]. Antibody titers against all 4 proteins were similar by day 28 post-challenge in MVA and placebo recipients. Together, these data demonstrate that prior MVA vaccination with doses of 10 7 and 10 8 resulted in rapid, anamnestic neutralizing and binding antibody responses against both IMV and EEV forms of vaccinia virus following Dryvax challenge. T-cell Responses Post Dryvax Challenge T-cell responses in MVA and placebo recipients following Dryvax challenge were assessed by IFN-γ ELISPOT assay (Figure 5). In the placebo group, T-cell responses peaked by day 14 after Dryvax challenge and declined by nearly 50% by day 28. Following Dryvax challenge, T-cell responses were found to be quite variable in MVA recipients. Of note, peak T-cell responses in the 10 8 SC and 10 7 ID groups were only slightly elevated compared to the pre-challenge baseline, and were significantly lower than peak responses measured in the placebo group (P= for both comparisons). A more rapid cellular immune response was observed in the 10 6 ID, 10 7 SC and 10 7 IM MVA groups compared to placebo recipients, with responses measurable by day 7 post-challenge. Responses in the 10 7 SC and 10 7 IM groups remained stable through day 28, whereas ELISPOT responses for the two individuals in the 10 6 ID group peaked on day 14 post-challenge. Together, these data suggest that anamnestic T-cell responses in MVA vaccinees after a Dryvax challenge are variable, and are possibly influenced by the dose and route of MVA utilized in the vaccination regimen, in which the lower dose groups demonstrate a more robust recall response. Effect of Prior MVA-Elicited Immune Responses on Subsequent Takes and Viral Shedding after Dryvax Challenge We examined the relationship between immune responses elicited by MVA immunization prior to Dryvax challenge, and the attenuated takes and decreased viral shedding that were subsequently observed after Dryvax challenge. The immune responses we examined included neutralizing antibody responses to MVA and VV, ELISA antibody responses to MVA and VV, protein specific antibody responses to IMV and EEV antigens, and anti-vv T-cell responses as measured by IFN-γ ELISPOT assay. The only predictor of an attenuated take among the immune responses prior to Dryvax challenge was the peak anti-mva neutralizing antibody responses following MVA immunization: median titers of 353, 82, and 139 for categories of attenuated takes 1, 2, and 3 respectively (P=0.02). We further investigated this association by categorizing peak pre-challenge anti-vv:wr neutralizing antibody titers as 20, , or >100. As shown in Figure 6, the maximum titer of virus shed was inversely correlated to increasing antibody category: , , and pfu/ml, for each increasing antibody category respectively (P=0.005). The magnitude of the pre-challenge anti-vv:wr neutralizing antibody category was also inversely correlated to duration of virus shedding: median duration of 21 (range 8 29), 14 (4 21) and 7.5 (2 18) days for each respectively (P=0.0007). A similar significant association was seen between

6 Seaman et al. Page 6 Discussion decreased duration and titer of virus shedding and the maximum vaccine elicited neutralizing antibody responses to MVA. For peak anti-mva neutralizing antibody categories of 20, and >100, virus was shed for a median of 21 (18 29), 14 (8 19), and 10.5 (2 21) days respectively (P=0.0001), and was shed with titers of , , and pfu/ml, respectively (P=0.005). Thus, elicitation of a neutralizing antibody response to MVA or VV prior to Dryvax challenge is associated with a decrease in duration of vaccinia shedding by 50 67%, and a reduction in maximum titer of virus shed by 10 to 100-fold. When the above analyses were adjusted for dose and route in a linear model, the relationship between decreased titer of virus shed and maximum neutralizing antibody response to MVA or VV pre-dryvax challenge was maintained (P=0.02 and 0.04 respectively), as was the relationship between decreased duration of virus shedding and maximum neutralizing antibody response to MVA or VV prior to Dryvax challenge (P=0.01 and 0.04 respectively). Immunization with ACAM3000 MVA conferred protection against challenge with Dryvax administered by scarification as reflected by attenuation of Dryvax takes and decreased titer and duration of virus shedding. Attenuation of takes was observed with MVA immunization by each of the three routes (ID, SC, and IM), and was most significantly associated with the highest dose administered by each route. Of particular note, the protective effects were obtained with ID administration of MVA at a dose of 10 7 TCID 50, which was a ten-fold lower dose than the highest dose given SC (10 8 TCID 50 ). Studies of other MVA candidate vaccines in humans have also reported an attenuation of takes and decreased virus shedding after Dryvax challenge. Frey et al administered MVA- BN (Bavarian Nordic A/S, Kristgard, Denmark) to 90 vaccinia naïve volunteers, SC at doses of to TCID 50 or IM at TCID 50, and challenged them with Dryvax by scarification 112 days later [13]. MVA immunization was associated with decreased lesion size and decreased titers of virus shed after Dryvax challenge, but without a dose or route relationship. Parrino et al studied 76 vaccinia-naïve and 68 vaccinia-immune volunteers immunized with TBC-MVA (Therion Biologics, Cambridge, MA) IM in multiple dose regimens [10]. A somewhat lower dose of TBC-MVA than that originally intended was administered (10 6 pfu), and subjects were challenged with Dryvax by scarification three months after MVA immunization. MVA immunization reduced severity of lesion formation and systemic reactogenicity induced by Dryvax in both vaccinia-naive and immune subjects, and two or three doses of MVA reduced the duration of virus shedding. Protective effects were seen in both vaccinia naive and immune subjects, but were more profound in the former [10]. MVA has also afforded protection against high dose challenge with monkeypox virus, administered either intravenously or intratracheally, to non-human primates. In a study by Earl et al, two inoculations of MVA at 10 8 pfu IM significantly reduced viral load and poxvirus lesions following challenge. MVA vaccinated animals remained clinically well compared to non-vaccinated animals which became extremely ill [14]. Anamnestic binding and neutralizing antibody responses were observed following challenge, although no significant correlation could be established because of the small number of animals. Postchallenge T-cell responses were not measured in this study because of biocontainment issues. In another report, monkeys immunized with two inoculations of an MVA-based candidate HIV vaccine (10 8 pfu IM) similarly demonstrated protection against viral load, the number of poxvirus lesions, and death following monkeypox challenge three years later [15]. The magnitude of the post-challenge anti-monkeypox neutralizing antibody response

7 Seaman et al. Page 7 significantly correlated with reduced peak viral load, whereas no significant correlations were observed with post-challenge T-cell responses. In our study, rapid anamnestic neutralizing antibody responses to both MVA and VV:WR were seen in all previously MVA immunized groups after challenge with Dryvax, and the time course was consistent with a recall response. In contrast, placebo recipients who were challenged with Dryvax had a pattern more consistent with a primary response. Importantly, we demonstrated a rapid antibody response in MVA vaccinees to key protein antigens associated with protection against IMV and EEV forms of infectious vaccinia [16 20], and also demonstrated effective in vitro neutralization against these viruses. The titers of neutralizing antibodies after Dryvax challenge were ultimately similar by day 28 at all the doses and routes of MVA vaccination studied. However, the group that received 10 7 ID had anamnestic antibody responses which were similar in magnitude and kinetics to those who had received a 10-fold higher dose of MVA in the SC group. Our studies of the effect of MVA immunization on Dryvax challenge showed a strong correlation between the presence of neutralizing antibodies to MVA or VV:WR prior to Dryvax challenge and attenuation of takes and decreased virus shedding. The relationship between neutralizing antibody titers and reduction of virus shedding was maintained even when dose and route were adjusted in linear models. Consistent with these observations, other studies have found antibody responses to be important in protection against orthopox infection and disease [21 23], and high titers of neutralizing antibodies against VV have been correlated with protection against smallpox [24 26]. Neutralizing antibody protected non-human primates against lethal intravenous challenge with monkeypox, and protection afforded by immunization with vaccinia was abrogated by B cell depletion [27]. Other clinical and experimental animal studies have implicated T cell, as well as B cell immunity in protection against orthopoxvirus infection. Individuals with either genetic T cell or B cell deficiencies have increased complications with smallpox vaccination, although patients with T cell abnormalities appear to be more at risk than those with agammaglobulinemia [28,29]. Adoptive transfer of lymphocytes from vaccinia-immunized donors was associated with a resolution of vaccinia necrosum that was unresponsive to vaccinia immune globulin [29], and protection against vaccinia infection has been achieved with adoptive transfer with virus specific T cells [30,31]. In mice, protection against orthopox infection has been associated with both B and T cell responses [32,33]. In our studies, T cell responses measured by IFN-γ ELISPOT using autologous VV:WR infected target cells were seen after Dryvax challenge, and showed varied patterns with respect to dose and route of MVA administration. Interestingly, the lowest T-cell responses measured following Dryvax challenge were in groups receiving high-dose MVA immunizations (10 8 SC and 10 7 ID), which also had the highest titer of neutralizing antibody responses prior to challenge [2]. It is possible that pre-existing neutralizing antibodies or the rapid expansion of such responses subsequently blunted cellular immune responses that require replication of virus for optimal stimulation. While our studies did not show a correlation between T cell responses and protection, additional studies entailing more detailed analyses of T cell responses in terms of epitope specificity, and phenotypic and functional characteristics will be required to adequately assess the relationship of such responses to protection against Dryvax challenge. In summary, MVA immunization by ID, SC, and IM routes resulted in attenuation of takes and decreased virus shedding after Dryvax challenge. The protective effects of ID administration were seen at a 10-fold lower dose of MVA than that administered SC, and suggest that ID administration may thus offer a dose sparing effect in vaccination regimens. Among the immune responses elicited by MVA immunization, the presence of neutralizing

8 Seaman et al. Page 8 antibodies against MVA or VV was highly associated with decreased viral shedding following challenge. Supplementary Material Acknowledgments References Refer to Web version on PubMed Central for supplementary material. Funding: National Institute of Allergy and Infectious Diseases (U.S. Public Health Service Grants U54 AI 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. 1. Fenner, F.; Henderson, D.; Arita, I.; Jezek, Z. Smallpox and Its Eradication. World Health Organization; p Wilck MB, Seaman MS, Baden LR, Walsh SR, Grandpre LE, Devoy C, Giri A, Kleinjan J, Noble L, Stevenson KE, Kim H, Dolin R. Safety and Immunogenicity of Modified Vaccinia Ankara (ACAM3000): Effect of Dose and Route of Administration. submitted to J Infect Dis McCurdy LH, Larkin BD, Martin JE, Graham BS. Modified vaccinia Ankara: potential as an alternative smallpox vaccine. Clin Infect Dis 2004;38: [PubMed: ] 4. Halsell JS, Riddle JR, Atwood JE, et al. Myopericarditis following smallpox vaccination among vaccinia-naive US military personnel. JAMA 2003;289: [PubMed: ] 5. Eckart RE, Love SS, Atwood JE, et al. Incidence and follow-up of inflammatory cardiac complications after smallpox vaccination. J Am Coll Cardiol 2004;44: [PubMed: ] 6. Cassimatis DC, Atwood JE, Engler RM, Linz PE, Grabenstein JD, Vernalis MN. Smallpox vaccination and myopericarditis: a clinical review. J Am Coll Cardiol 2004;43: [PubMed: ] 7. Neff J, Modlin J, Birkhead GS, et al. Monitoring the safety of a smallpox vaccination program in the United States: report of the joint Smallpox Vaccine Safety Working Group of the Advisory Committee on Immunization Practices and the Armed Forces Epidemiological Board. Clin Infect Dis 2008;46 (Suppl 3):S [PubMed: ] 8. Fulginiti VA, Papier A, Lane JM, Neff JM, Henderson DA. Smallpox vaccination: a review, part I. Background, vaccination technique, normal vaccination and revaccination, and expected normal reactions. Clin Infect Dis 2003;37: [PubMed: ] 9. Centers for Disease Control and Prevention. Evaluation of Takes and Non-Takes. [Accessed 22 July 2009]. Available at Parrino J, McCurdy LH, Larkin BD, et al. Safety, immunogenicity and efficacy of modified vaccinia Ankara (MVA) against Dryvax challenge in vaccinia-naive and vaccinia-immune individuals. Vaccine 2007;25: [PubMed: ] 11. Cleveland WS. Robust locally weighted regression and smoothing scatterplots. J Amer Statist Assoc 1979;74: Fan J. Design-adaptive nonparametric regression. J Amer Statist Assoc 1992;87: Frey SE, Newman FK, Kennedy JS, et al. Clinical and immunologic responses to multiple doses of IMVAMUNE (Modified Vaccinia Ankara) followed by Dryvax challenge. Vaccine 2007;25: [PubMed: ] 14. Earl PL, Americo JL, Wyatt LS, et al. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature 2004;428: [PubMed: ] 15. Nigam P, Earl PL, Americo JL, et al. DNA/MVA HIV-1/AIDS vaccine elicits long-lived vaccinia virus-specific immunity and confers protection against a lethal monkeypox challenge. Virology 2007;366: [PubMed: ]

9 Seaman et al. Page Kaufman DR, Goudsmit J, Holterman L, et al. Differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice. J Virol 2008;82: [PubMed: ] 17. Heraud JM, Edghill-Smith Y, Ayala V, et al. Subunit recombinant vaccine protects against monkeypox. J Immunol 2006;177: [PubMed: ] 18. Hooper JW, Thompson E, Wilhelmsen C, et al. Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J Virol 2004;78: [PubMed: ] 19. Berhanu A, Wilson RL, Kirkwood-Watts DL, et al. Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge. J Virol 2008;82: [PubMed: ] 20. Lustig S, Fogg C, Whitbeck JC, Eisenberg RJ, Cohen GH, Moss B. Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge. J Virol 2005;79: [PubMed: ] 21. Czerny CP, Mahnel H. Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies. J Gen Virol 1990;71 (Pt 10): [PubMed: ] 22. Galmiche MC, Goenaga J, Wittek R, Rindisbacher L. Neutralizing and protective antibodies directed against vaccinia virus envelope antigens. Virology 1999;254: [PubMed: ] 23. Ramirez JC, Tapia E, Esteban M. Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions. J Gen Virol 2002;83: [PubMed: ] 24. Mack TM, Noble J Jr, Thomas DB. A prospective study of serum antibody and protection against smallpox. Am J Trop Med Hyg 1972;21: [PubMed: ] 25. Sarkar JK, Mitra AC, Mukherjee MK. The minimum protective level of antibodies in smallpox. Bull World Health Organ 1975;52: [PubMed: ] 26. Downie AW, Mc CK. The antibody response in man following infection with viruses of the pox group. III. Antibody response in smallpox. J Hyg (Lond) 1958;56: [PubMed: ] 27. Edghill-Smith Y, Golding H, Manischewitz J, et al. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat Med 2005;11: [PubMed: ] 28. Hammarlund E, Lewis MW, Hansen SG, et al. Duration of antiviral immunity after smallpox vaccination. Nat Med 2003;9: [PubMed: ] 29. Kempe CH. Studies smallpox and complications of smallpox vaccination. Pediatrics 1960;26: [PubMed: ] 30. Zinkernagel RM, Althage A. Antiviral protection by virus-immune cytotoxic T cells: infected target cells are lysed before infectious virus progeny is assembled. J Exp Med 1977;145: [PubMed: ] 31. Derby M, Alexander-Miller M, Tse R, Berzofsky J. High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL. J Immunol 2001;166: [PubMed: ] 32. McCurdy LH, Rutigliano JA, Johnson TR, Chen M, Graham BS. Modified vaccinia virus Ankara immunization protects against lethal challenge with recombinant vaccinia virus expressing murine interleukin-4. J Virol 2004;78: [PubMed: ] 33. Belyakov IM, Earl P, Dzutsev A, et al. Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proc Natl Acad Sci U S A 2003;100: [PubMed: ]

10 Seaman et al. Page 10 Figure 1. A, Magnitude and duration of viral shedding in relation to Dryvax take category* in subjects who had received MVA vaccination. Swabs of Dryvax-induced lesions were obtained at the indicated timepoints following challenge, and viral shedding was determined by tissue culture pfu assay. Data are presented as the median pfu/ml with interquartile ranges per group, and the dashed line represents the limit of detection (pfu =10/ml). Subjects who received placebo all had full takes* and are plotted separately. * Take categories defined in text [9,10] B, Prior vaccination with MVA correlates with reduced titer and duration of viral shedding following Dryvax challenge. Data are presented as individual swab titers (pfu/ml) from individuals grouped as MVA or placebo recipients. A lowess fit line is plotted for each group. The assay limit of detection was 10 pfu/ml.

11 Seaman et al. Page 11 Figure 2. Neutralizing antibody responses following Dryvax challenge. Serum samples were obtained at days 0, 4, 7, 14, and 28 following Dryvax challenge. Serial dilutions were tested for neutralizing activity against VV:Luc. Data are presented as median ID 50 titers with interquartile ranges for each dose and route-of-immunization group, with the number of individuals in each group indicated. The dashed line represents the assay limit of detection (serum ID 50 titer =10).

12 Seaman et al. Page 12 Figure 3. Anti-EEV neutralizing antibody activity. Serum samples were obtained from vaccinated subjects two weeks following Dryvax challenge and tested in a comet reduction assay at a 1:50 dilution. Anti-vaccinia hyperimmune serum and matched pre-immune sera were used as positive and negative controls, respectively. Data are presented as the percent comet reduction observed from individual volunteers in each dose and route-of-immunization group, with bars indicating the median response. PL indicates the placebo group.

13 Seaman et al. Page 13 Figure 4. Antibody responses to vaccinia IMV- and EEV-associated antigens following Dryvax challenge. Serum samples were obtained on days 14 and 28 following Dryvax challenge. Serial dilutions were tested for antibody binding activity against two IMV (A27L and LIR) 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.

14 Seaman et al. Page 14 Figure 5. Cellular immune responses elicited by Dryvax challenge. PBMC were isolated at days 0, 4, 7, 14, and 28 following Dryvax scarification and tested in an IFN-γ ELISPOT assay against autologous VV:WR-infected target cells isolated at day 4 following the first MVA (or placebo) inoculation. Data are presented as median SFC per 10 6 effector PBMC with interquartile ranges for each dose and route-of-immunization group following subtraction of responses to medium alone. SFC at baseline are subtracted from each data point.

15 Seaman et al. Page 15 Figure 6. Magnitude of vaccine-elicited anti-vv:wr neutralizing antibody responses prior to challenge correlate with reduced titer and duration of viral shedding following Dryvax challenge. Swabs of Dryvax lesions were obtained at the indicated timepoints following challenge, and viral shedding was determined by pfu assay. Data are presented as individual swab titers (pfu/ml) from individuals grouped according to peak anti-vv:wr neutralizing antibody titers measured following MVA or placebo vaccination as follows: ID (black circles), ID (red triangles), or ID 50 >100 (blue squares). A lowess fit line is plotted for each group. The assay limit of detection is 10 pfu/ml.

16 Seaman et al. Page 16 Table 1 Cutaneous Responses to Dryvax Challenge in MVA Immunized Groups Group MVA Regimen * Number (Total) Number with Indicated Finding P Value + 36 Attenuated Response Unattenuated Response A 10 6 ID N/A B 10 7 IM C 10 7 SC D 10 8 SC E 10 7 ID Placebo * Dose in TCID 50 given at day 0 and day 28; Dryvax challenge by scarification at median day 194 including both MVA and placebo recipients (range of ); ID: intradermal; IM: intramuscular; SC: subcutaneous + Comparison of rate in MVA group vs. placebo by Fisher s Exact Test N/A: Not Applicable