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Analysis of variola and vaccinia neutralization assays for smallpox vaccines 1 2 Christine M. Hughes*1, Frances K. Newman2, Whitni B. Davidson1, Victoria A. Olson1, 3

Scott K. Smith1, Robert C. Holman1, Lihan Yan3, Sharon E. Frey2, Robert B. Belshe2, 4

Kevin L. Karem1, Inger K. Damon1 5

Running title: Variola and vaccinia neutralization assay analysis 6 7

1Centers for Disease Control and Prevention 8

National Center for Emerging and Zoonotic Infectious Diseases 9

Division of High-Consequence Pathogens and Pathology 10

1600 Clifton Road NE, Atlanta, Georgia 30333 11

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2Saint Louis University School of Medicine 13

Division of Infectious Diseases and Immunology 14

Edward A. Doisy Research Center, 8th Floor 15

1100 S. Grand Blvd, Saint Louis, Mo 63104 16

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3The EMMES Corporation 18

401 N. Washington St., Rockville, MD 20850 19

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*Corresponding Author: 21

Christine Hughes, 1600 Clifton Rd. NE, MS G-06, Atlanta, GA 30333 22

Phone: 404-639-2764, Fax: 404-639-1060, Email: Christine.Hughes@cdc.hhs.gov 23

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Funding: N01-AI-25464 25

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00056-12 CVI Accepts, published online ahead of print on 16 May 2012

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Abstract 26

Possible smallpox re-emergence drives research for third-generation vaccines that 27

effectively neutralize variola virus. Comparison of neutralization assays using different 28

substrates, variola and vaccinia (Dryvax and MVA), showed significantly different 90% 29

neutralization titers; Dryvax underestimated while MVA overestimated variola 30

neutralization. Third-generation vaccines may rely upon neutralization as a correlate of 31

protection. 32

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Text 66 67 Previous studies (6) suggested the quantitative titer of human sera plaque reduction 68

neutralization tests (PRNTs) differed when variola or vaccinia virus were used as the 69

neutralization target. Similar observations were made during Acambis2000 vaccine 70

studies in post-immunization, non-human primate sera (10). This study was designed to 71

evaluate the correlation between PRNTs when different virus species or strains were used 72

as the neutralization target. This study is the first to look extensively at these differences 73

using the same set of well-defined human post-vaccination sera. If the ability to 74

neutralize variola is the desired outcome of smallpox vaccination, then understanding the 75

relative significance of variola and vaccinia neutralization titers will be critical surrogate 76

measures; especially if previous measures of vaccine efficacy (i.e. “the Jennerian 77

pustule” or “take”) are not available for third generation vaccine and if variola stocks are 78

destroyed. The comparable efficacy of vaccination regimen, using vaccinia (Dryvax or 79

modified vaccinia Ankara (MVA)) or variola virus as the neutralizing target, is presented 80

elsewhere (3, 4) 81

82

Sera (46 participants) from a National Institutes of Health funded smallpox vaccine trial 83

(DMID 02-017) were evaluated at Saint Louis University (SLU) and at the Centers for 84

Disease Control and Prevention (CDC). 20 participants received MVA (IMVAMUNE®) 85

subcutaneously (SC) (1 X108 TCID50, 2 doses, 1 month apart), 15 received MVA 86

intramuscularly (IM) (1 X108 TCID50, 2 doses, 1 month apart), and 11 received Dryvax 87

vaccination by scarification (1 dose) (4). MVA is a replication deficient, less 88

reactogenic third-generation smallpox vaccine (1, 7, 9). Sera at “peak” response times 89

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post-vaccination were evaluated using variola, Dryvax, and MVA PRNTs. Individuals 90

were evaluated either 28-30 days post-Dryvax vaccination or 14 days post-second MVA 91

dose. 92

93

At SLU, sera samples were tested in a qualified PRNT assay using an American Type 94

Culture Collection (ATCC) strain of MVA (Catalog # VR-1508) or Dryvax (2, 8) as the 95

neutralization reference virus. The Dryvax PRNT was modified by substituting MVA for 96

Dryvax as the neutralizing target and by identifying plaques through immunostain in 97

place of crystal violet staining. Sonicated MVA virus was diluted to ~30-50 plaque 98

forming units (pfu)/well. An equal volume of diluted MVA was mixed with each serial 2-99

fold dilution of heat-inactivated serum or media and incubated overnight at 37°C. Each 100

serum-virus mixture and virus-media mixture (virus-only control) was inoculated onto 101

BSC-40 cell monolayers, a one hour adsorption time was utilized, and then the 102

plateswere incubated for two days at 37°C to allow for plaque formation. Plates were 103

fixed with cold acetone/methanol (50/50) for 1 hour at 2-8°C. Plaques were then 104

elucidated by immunostaining using anti-vaccinia antibody (rabbit anti-vaccinia, 105

ViroStat, Portland, ME) as the primary antibody followed by goat anti-rabbit IgG 106

conjugated to horseradish peroxidase (Kirkegaard and Perry, Gaithersburg, MD). The 107

substrate used was the enhanced orange system (Kirkegaard and Perry, Gaithersburg, 108

MD). Immunostained plaques were counted using a dissecting microscope. 109

110

Variola PRNT assays were performed at CDC using a method adapted from that 111

previously described (3, 4). Duplicate 2-fold dilutions of sera were prepared in RPMI-112

1640 supplemented with 2% fetal bovine serum, mixed with variola virus strain Solaimen 113

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(final serum dilutions 1:10 to 1:40 for prebleeds; 1:40-1:1280 for post-vaccination sera), 114

and incubated at 35ºC overnight. Media alone was used to quantitate the “virus only” 115

control. Positive (sera from previously vaccinated persons) and negative serum controls 116

were used to confirm that the assay performed within predetermined parameters.(5). The 117

positive control, Vaccinia Immune Globulin Intravenous (Human) (Cangene Corporation, 118

Winnipeg, Canada), was used at dilutions of 1:1000 to 1:32,000 based on prior 119

knowledge of vaccinia neutralizing capacity. After overnight incubation, one milliliter of 120

serum-virus or control-virus mixture was added to BSC40 cell monolayers, adsorbed for 121

one hour, and an additional milliliter of media was applied. Plaques developed over 72 122

hours and were counted following crystal violet staining of cell monolayers. 123

124

Linear regression analysis was applied to a log transformation of each individual’s serum 125

dilutions to facilitate linear interpolation of actual 90% PRNTs titers at “peak” post-126

vaccination response. The medians with their quartiles at 90% neutralization were 127

calculated for each neutralization target overall and by vaccine treatment group; the 128

geometric mean titers (GMTs) were also calculated (Table 1). The overall 90% PRNT 129

titers for the different orthopoxvirus neutralization targets (Dryvax, MVA, and variola) 130

were compared using the Wilcoxon signed rank test; non-parametric statistics were used 131

as the data are not normally distributed. A p-value < 0.05 was considered statistically 132

significant. The 90% PRNT titers for the different orthopoxvirus neutralization targets 133

were displayed graphically (Figure 1). 134

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Median 90% PRNT titers for each neutralization target were compared and GMTs 136

presented (Table 2). Statistically significant differences were noted between the overall 137

90% PRNTs titers for Dryvax and variola (1:32.5 vs 1:63.41; p=0.008), MVA and variola 138

(1:110.5 vs. 1:63.41; p=0.0007), and Dryvax and MVA (1:32.5 vs. 1:110.5; p<0.0001) 139

(Table 2). Graphical analyses did not reveal visual relationships between the 90% PRNT 140

titers for the comparison of the three different neutralization targets. Graphs of the MVA 141

90% PRNT titers with the Dryvax and variola 90% PRNT titers show a clustering of 142

titers <500 with the MVA PRNT titer higher than the corresponding Dryvax or variola 143

PRNT titer for some individuals (Figures 1a & 1b). The graph of the Dryvax 90% PRNT 144

with the variola 90% PRNT showed a clustering of titers < 200 with the variola 90% 145

PRNT higher than the corresponding Dryvax 90% PRNT for some individuals (Figure 146

1c). 147

148

Previous studies (6) suggesting that PRNTs differed when variola or vaccinia virus were 149

used as the neutralization target are largely anecdotal, with evaluation of human 150

convalescent and post vaccination sera, as well as some hyperimmune sera raised in other 151

species. In addition, the results were not truly comparable as different sets of sera were 152

used for the variola and vaccinia neutralization tests. This manuscript is the first to look 153

extensively at the differences in PRNT results for the same set of well-defined post-154

vaccination sera using different viruses (variola and vaccinia) and different strains (MVA 155

and Dryvax) as the substrate. . Significant differences in PRNT titers were observed 156

using different viruses as the substrate for neutralization when all subjects are combined 157

regardless of vaccination regimen. Using Dryvax as the neutralization antigen results in 158

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significantly lower 90% PRNT titers than using variola as the neutralization antigen. 159

Using MVA as the neutralization antigen results in significantly higher 90% PRNT titers 160

than using variola as the neutralization antigen. The mechanisms for this observation are 161

uncertain and likely include subtle antigenic differences between viruses and between the 162

vaccine regimens. PRNT titer differences were not due to variances in viral preparation 163

quality, since the amount of virus used is limited and calculations of genomes/pfu for 164

both variola and Dryvax were similar (variola ~147 genomes/pfu; vaccinia ~100 165

genomes/pfu). Although the Dryvax PRNT protocol had to be modified to include IHC 166

staining in order to elucidate plaques formed by MVA, staining techniques were the same 167

for both Dryvax and variola virus PRNTs. Therefore, it is unlikely that differences in 168

90% PRNTs observed between the neutralization viral antigens are due to differences in 169

PRNT techniques. This analysis, in combination with data from other vaccine trials, may 170

assist in determining if neutralization titers with vaccinia virus as the target can be 171

bridged to variola virus neutralizing titers. Furthermore, better understanding of 172

neutralizing capacity is invaluable for third generation vaccines which do not produce the 173

historic correlate of protection (the “take”). 174

175

Acknowledgements 176

The authors would like to acknowledge the assistance of Mark Challberg and Robert 177

Johnson of DMID/NIAID for their assistance in facilitating this study. 178

179

The findings and conclusions in this report are those of the authors and do not necessarily 180

represent the views of the Centers for Disease Control and Prevention181

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3. Damon, I. K., W. B. Davidson, C. M. Hughes, V. A. Olson, S. K. Smith, R. C. 188 Holman, S. E. Frey, F. Newman, R. B. Belshe, L. Yan, and K. Karem. 2009. 189 Evaluation of smallpox vaccines using variola neutralization. J Gen Virol 90:1962-6. 190

4. Frey, S. E., F. K. Newman, J. S. Kennedy, V. Sobek, F. A. Ennis, H. Hill, L. K. Yan, 191 P. Chaplin, J. Vollmar, B. R. Chaitman, and R. B. Belshe. 2007. Clinical and 192 immunologic responses to multiple doses of IMVAMUNE (Modified Vaccinia Ankara) 193 followed by Dryvax challenge. Vaccine 25:8562-73. 194

5. Karem, K. L., M. Reynolds, Z. Braden, G. Lou, N. Bernard, J. Patton, and I. K. 195 Damon. 2005. Characterization of acute-phase humoral immunity to monkeypox: use of 196 immunoglobulin M enzyme-linked immunosorbent assay for detection of monkeypox 197 infection during the 2003 North American outbreak. Clin Diagn Lab Immunol 12:867-72. 198

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8. Newman, F. K., S. E. Frey, T. P. Blevins, M. Mandava, A. Bonifacio, Jr., L. Yan, 205 and R. B. Belshe. 2003. Improved assay to detect neutralizing antibody following 206 vaccination with diluted or undiluted vaccinia (Dryvax) vaccine. J Clin Microbiol 207 41:3154-7. 208

9. Vollmar, J., N. Arndtz, K. M. Eckl, T. Thomsen, B. Petzold, L. Mateo, B. Schlereth, 209 A. Handley, L. King, V. Hulsemann, M. Tzatzaris, K. Merkl, N. Wulff, and P. 210 Chaplin. 2006. Safety and immunogenicity of IMVAMUNE, a promising candidate as a 211 third generation smallpox vaccine. Vaccine 24:2065-70. 212

10. Weltzin, R., J. Liu, K. V. Pugachev, G. A. Myers, B. Coughlin, P. S. Blum, R. 213 Nichols, C. Johnson, J. Cruz, J. S. Kennedy, F. A. Ennis, and T. P. Monath. 2003. 214 Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 9:1125-215 30. 216

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Table 1. 90% PRNT median and geometric mean titers (GMTs) for different orthopoxvirus neutralization targets by vaccination 219

regimen 220

Orthopoxvirus Neutralization Target Vaccination Regimen

All Vaccinees (n=46) MVA SC

(n=20) MVA IM (n=15)

Dryvax (n=11)

Dryvax Median

(25-75th percentile) 35.0 (13.0-95.5) 14.0 (11.0-33.0) 71.0 (29.0-167.0) 32.5 (12.0-93.0)

GMT

30.9 18.0 47.5 28.7

MVA Median

(25-75th percentile) 245.0 (61.5-673.0) 117.0 (92.0-247.0) 10.0 (4.0-59.0) 110.5 (18.0-345.0)

GMT

152.9 156.0 15.5 89.3

Variola Median

(25-75th percentile) 63.4 (49.9-130.2) 78.1 (42.6-104.8) 41.7 (14.1-64.1) 63.4 (41.7-108.0)

GMT

83.9 73.7 35.9 65.6 221

Table 2. Comparison of 90% PRNT titers for orthopoxvirus neutralization targets

Target Comparison Medians p-value

Dryvax vs. Variola 32.5 vs. 63.4 .008

MVA vs. Variola 110.5 vs. 63.4 .0007

Dryvax vs. MVA 32.5 vs. 110.5 <.0001

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222

Figure 1. 90% PRNT titer by vaccination regimen for Dryvax vs. MVA (A.), MVA vs. Variola 223

(B.), Dryvax vs. Variola (inset graph with reduced x and y-axis scale) (C.) 224

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