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Molecular analysis of varicella vaccines and varicella-zoster virus from vaccine-related 1

skin lesions 2

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Sonja Thiele#, Aljona Borschewski#, Judit Küchler, Marc Bieberbach, Sebastian Voigt and 4

Bernhard Ehlers 5

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Division of Viral Infections, Robert Koch Institute, D-13353 Berlin, Germany 7

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Corresponding author: 9

Dr. Bernhard Ehlers, Robert Koch Institute, Nordufer 20, D-13353 Berlin 10

Phone: ++4930187542347; Fax: ++4930187542598; email: [email protected]

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# These authors contributed equally to this work 13

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Running title: Molecular Analysis of VZV from vaccines and vaccinees 16

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05021-11 CVI Accepts, published online ahead of print on 11 May 2011

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ABSTRACT 18

To prevent complications that might follow an infection with varicella-zoster virus (VZV), 19

the live attenuated Oka strain (V-Oka) is administered to children in many developed 20

countries. Three vaccine brands (Varivax from Sanofi Pasteur MSD; Varilrix and Priorix-21

Tetra, both from Glaxo-Smith-Kline) are licensed in Germany that have been associated both 22

with different degrees of vaccine effectiveness and adverse effects. To identify genetic 23

variants in the vaccines that might contribute to rash-associated syndromes, single nucleotide 24

polymorphism (SNP) profiles of the three vaccines and rash-associated vaccine-type VZV 25

from German vaccinees were quantitatively compared by PCR-based pyrosequencing (PSQ). 26

The Varivax vaccine contained an estimated three-fold higher diversity of VZV variants with 27

20% more wt SNPs compared to Varilrix and Priorix-Tetra. These minor VZV variants in the 28

vaccines were identified by analysing cloned full length ORF 62 sequences by chain 29

termination sequencing and PSQ. Some of these sequences amplified from vaccine VZV were 30

very similar or identical to those of the rash-associated vaccine-type VZV from vaccinees and 31

were almost exclusively detected in Varivax. Therefore, minorities of rash-associated VZV 32

variants are present in varicella vaccine formulations and it can be concluded that the analysis 33

of a core set of four SNPs is required as a minimum for a firm diagnostic differentiation of 34

vaccine-type VZV from wt VZV. 35

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Key Words: Varicella-zoster virus, ORF62, vaccine, rash-associated syndrome, 39

pyrosequencing40


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INTRODUCTION41

The live attenuated Oka strain (V-Oka) of varicella-zoster virus (VZV) is licensed in 42

developed countries for vaccination against varicella. In Germany, routine vaccination of 43

healthy children >11 months of age is performed since 2004. Since then, varicella morbidity 44

declined (16). The mixtures of V-Oka strains with different single nucleotide polymorphism 45

(SNP) profiles present in vaccine preparations and the reported genomic variation of vaccine 46

VZV have prompted investigations of the linkage of certain genomic variants to episodes of 47

vaccine-induced rash (9, 10). 48

We aimed at comparing the SNP profiles of the varicella vaccines used in Germany and 49

of vaccine-type VZV from German vaccinees for several reasons. First, three vaccine brands 50

are distributed in Germany (Varivax from Sanofi Pasteur MSD; Varilrix and Priorix-Tetra, 51

both from GlaxoSmithKline) that have been associated with different degrees of vaccine 52

effectiveness (17). In addition, data from the German varicella sentinel (16) indicated that the 53

vaccines might be related to different frequencies of rash-associated syndromes, i.e., mild 54

forms of varicella or zoster (Anette Siedler and Bernhard Ehlers, unpublished data). Second, 55

vaccine-type VZV strains causing rash-associated clinical syndromes in vaccinees have been 56

reported to contain a number of wildtype (wt) SNPs. It has been hypothesized that these 57

vaccine-type, wt SNP-containing VZV strains are minor components of the vaccines (1, 9, 10, 58

13). To substantiate this hypothesis and to search for genetic correlates of the observed 59

differences among the vaccines, we compared the SNP profiles of (i) the three vaccine brands, 60

(ii) rash-associated vaccine-type VZV from German vaccinees and (iii) wt VZV from non-61

vaccinated and vaccinated individuals suffering from varicella by PCR, quantitative 62

pyrosequencing (PSQ) and chain termination sequencing (CTSQ). In addition, ORF 62 63

sequences were cloned in E. coli and analysed by the same methods. 64

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MATERIAL AND METHODS66

Sample collection, vaccines and DNA extraction. Vesicle material from vaccinees with 67

varicella-like rash or herpes zoster was collected as part of the varicella sentinel in Germany 68

(16). Three vaccines from two manufacturers were analysed: Varivax (monovalent, Lots: 69

NA28970, HW08500) from Sanofi Pasteur MSD (SPMSD, Leimen, Germany), Varilrix 70

(monovalent, Lot: A70CA216B) and, with the same VZV component, Priorix-Tetra 71

(tetravalent; Lot: #A71CA024A) from GlaxoSmithKline (GSK, München, Germany). DNA 72

was extracted using the DNeasy Blood & Tissue Kit DNA (Qiagen, Hilden, D) according to 73

the manufacturer’s instructions.74

75

Long-Distance PCR and sequence analysis. Long-distance-PCR (LD-PCR) was performed 76

as follows: The reaction mixture was set up on ice and contained 50 μL of ExTaq buffer with 77

MgCl2, 400 μM of each dNTP, 400 nM of each primer, 2.5 units of TaKaRa ExTaq 78

polymerase (TaKaRa Bio Inc, Japan). Thirty cycles of amplification were performed, each 79

cycle consisting of a denaturing step at 98 °C for 20 sec, an annealing step at 63 °C for 30 sec 80

and an elongation step at 72 °C. The duration of the elongation step was 7 minutes in the first 81

15 cycles and 10 min plus 5 sec ramp time for each of the last 15 cycles. The final elongation 82

step was performed for 30 minutes at 72 °C. LD-PCR was performed in a nested format using 83

the primer pairs LD_a1 and LD_a2 in 1st and 2nd round (Table 2). When no PCR product was 84

obtained (because of very low copy number) the three alternative primer pairs LD_b1, LD_b2 85

and LD_b3 were used (Table 2). 86

PCR products were purified with the Invisorb Spin PCRapid Kit (Invitek, Berlin, D), 87

and directly sequenced by chain termination sequencing (CTSQ) using the BigDye-terminator 88

cycle sequencing kit and an ABI PRISM Genetic Analyzer (Applied Biosystems, USA) 89

according to the manufacturer’s instructions. The programs DNASTAR Lasergene 8 and 90


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MacVector 10.6 were used to examine the SNPs by comparing the obtained sequences with 91

those of reference strains. 92

When patient specimens were nearly exhausted, DNA was expanded in toto with the 93

GenomiPhi™ V2 DNA Amplification Kit (GE Healthcare Life Sciences, München, 94

Germany) according to the manufacturer’s instructions. The qualitatively and quantitatively 95

correct reamplification of VZV DNA and VZV variant mixtures from specimens with the 96

GenomiPhi kit had been previously controlled using quantitative real-time PCR, conventional 97

PCR, CTSQ and PSQ (Judit Küchler and Bernhard Ehlers, unpublished data). 98

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Cloning in E. coli. PCR products spanning the complete ORF62 (approximately 4 kb) and 100

short PCR products containing a single SNP (<0.3 kb) were transformed into chemically 101

competent E. coli cells using the TOPO-TA cloning kit (Invitrogen, Karlsruhe, D) according 102

to the manufacturer’s instructions. The plasmids containing the 4 kb inserts were extracted 103

with the QIAprep Spin Miniprep Kit (Qiagen, Hilden, D) and analysed by cycle sequencing 104

(see above). The plasmids containing the short inserts were tested with colony PCR and 105

pyrosequencing (see below). 106

107

PCR-based pyrosequencing. Pyrosequencing (PSQ) (12) was used for analysing the allele 108

frequency of VZV SNPs. 32 PSQ assays consisting each of a primer pair (one primer 109

biotinylated) for PCR (to generate a product containing the SNP of interest) and a separate 110

sequencing primer were designed by using the Pyrosequencing Assay Design Software 111

(Biotage, Uppsala, SE) (Table 1). Each PCR reaction mixture contained 35 μL of AmpliTaq 112

Gold buffer, 2,0 mM MgCl2, 200 μM of each dNTP, 1 μM of each primer, 1,5 units of 113

AmpliTaq Gold (Applied Biosystems, USA) and extracted DNA, either from vaccines 114

(adjusted to 105 VZV genome copies per reaction set-up) or from vaccinated individuals. 115


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Forty-five cycles of amplification were performed after enzyme activation at 95 °C for 12 116

minutes. Each cycle consisted of a denaturing step at 95 °C for 30 sec, an annealing step at an 117

appropriate temperature (see Table 1) for 30 sec and an elongation step at 72 °C for 60 sec. 118

The final elongation step was done for 10 minutes at 72 °C.119

For analysis of single E. coli clones, colony PCR was performed using the PSQ primer 120

pairs with the same reaction setup. Colonies were picked and transferred directly into the 121

pyrosequencing PCR reaction mixtures. Spare material of the colony was stripped on a grid 122

plate for later countercheck. 123

For PSQ, 30 μL of the PCR products were immobilized, denaturized and washed using 124

the PyroMark Vakuum Prep Workstation (Biotage, Uppsala, SE). The single-stranded DNA 125

was transferred into a 96 well optical reaction plate (PSQTM 96 Plate low Biotage, Uppsala, 126

SE) containing an annealing buffer and a specific sequencing primer. The annealing of the 127

primer was done at 80 °C for 2 minutes. After cooling down of the reaction mixture, PSQ was 128

performed at room temperature on the PyroMark ID (Biotage, Uppsala, SE). The resulting 129

pyrograms allowed qualitative and quantitative SNP analysis. Quantification was conducted 130

by PSQ software and is based on the fact that PSQ peak heights are proportional to the 131

frequency of an allele in the sample. Therefore, PSQ peak heights provide an accurate 132

measure of the proportion of VZV wildtype and vaccine-type alleles in a sample. In addition, 133

the results of the analysis are presented in sequence context, confirming that the analysis was 134

made at the correct sites. All PCRs plus PSQ measurements of the vaccines and the patient 135

specimens with vaccine-type VZV were repeated two to four times. 136

137

Reference strains. As VZV reference sequences were used: Dumas, Acc. NC_001348; Oka-138

P, Acc. AB097933; Oka-V, Acc. AB097932; Varilrix, Acc. DQ008354; and Varivax Acc. 139

DQ008355.140


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RESULTS and DISCUSSION 143

As part of the German varicella sentinel (16), specimens from vaccinated individuals with 144

rashes were subjected to VZV PCR and PSQ for differentiation between wt VZV and 145

vaccine-type VZV by analysing five SNP positions (nt 105544, 105705, 106262, 107252, 146

108111; positions based on the genome sequence of the Dumas strain). Between January 2004 147

and December 2010, 505 specimens were diagnosed by PCR and sequencing as VZV wt and 148

20 as VZV vaccine-type. Of the latter, twelve had been clinically diagnosed as originating 149

from a varicella-like rash and eight from herpes zoster (HZ) [Bernhard Ehlers and Annette 150

Siedler, unpublished data]. To comprehensively determine the frequency of wt alleles and 151

their fraction in mixed alleles at SNP positions in these vaccine-type VZV isolates, SNPs 152

(n=32) present in the VZV genome were analysed with a panel of PCR reactions and 153

subsequent PSQ. The PCR and PSQ primers used are listed in Table 1. The specimens of nine 154

vaccinees (nos. 1 to 9; Figures 1, 3 and 5) contained enough VZV genome copies for this 155

genome-wide SNP analysis. Those with low copy numbers (nos. 1 to 6) had to be expanded in156

toto as described in the Methods section to perform all analyses of this study. The remaining 157

eleven specimens were of such low copy number that not all PCR amplifications were 158

successful. These were not included in the study. For comparison, the SNP profiles of wt 159

VZV from non-vaccinated (n=4) and vaccinated (n=5) individuals suffering from varicella 160

were analysed by the same PSQ approach. Before the patient specimens were analysed, the 161

SNP profiles of the three varicella vaccines distributed in Germany (Varivax from SPMSD, 162

Varilrix and Priorix-Tetra from GSK) were quantitatively evaluated. 163

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Comparison of the vaccines Varivax, Varilrix and Priorix-Tetra by PSQ. To get more 165

detailed information about the SNP distribution in Varivax, Varilrix and Priorix-Tetra, we 166


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examined 32 SNP positions by PSQ. All SNP profiles were tabulated and compared to the 167

previously published SNPs of Varivax and Varilrix (18), the original vaccine strain Oka-V as 168

well as the wt parent strain Oka-P (7) and the wt Dumas strain (3) (Figure 1). To firmly 169

discriminate pure wt and pure vaccine-type alleles from mixed alleles in the vaccines the 170

performance of all PSQ assays on specimens from nine patients infected with wt VZV was 171

assessed. Here, we expected PSQ measurements of 100% wt and 0% vaccine-type at all 32 172

SNP positions. In fact, only a part of the PSQ assays revealed 100% wt allele. The others 173

revealed background signals in a highly reproducible fashion falsely indicating the presence 174

of the vaccine-type allele (not shown). Therefore, we calculated individual limits of detection 175

for the minority allele for all PSQ assays. To achieve this, the median height of the 176

background signals obtained from the nine patients with wt VZV was calculated for 25/32 177

PSQ assays. For the remaining seven PSQ assays targeting the SNP positions nt 560, 105310, 178

105544, 105705, 106262, 107252, and 108111 cloned VZV sequences were available (see 179

below), and their pyrograms were used for calculation of the detection limits. The median of 180

the peak height of the background signal plus two-fold standard deviation was defined as the 181

individual limit of detection for each assay. The resulting 32 detection limits of up to 12% 182

partially exceeded those of a previous study aimed at detecting mutant minorities of human 183

cytomegalovirus. In that study, detection limits of 6% had been defined for four PSQ assays 184

(15). In the present study, the detection limits of 19 assays were <6%, but those of 10 assays 185

were >6% and three >10%. The 13 high detection limits were probably due to the high GC 186

content of the VZV genome which occasionally hampered the design of optimal PSQ assays. 187

All PSQ values determined in our study which exceeded the detection limit of the respective 188

PSQ assay were considered as representing true alleles (Figure 1 and Figures 3 to 5). 189

The qualitative SNP profile of Varivax determined here matched only at 20 SNP 190

positions with the Varivax profile published by Tillieux et al. (18). Similarly, the present SNP 191

profiles of Varilrix and Priorix Tetra matched only at 18 positions with the Varilrix profile 192


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determined by these authors (18). Using CTSQ methodology, they detected (with reference to 193

the panel of 32 SNPs investigated in this study) eleven pure wt SNPs in Varivax and four in 194

Varilrix. Here, we found only four pure wt SNPs in Varivax and none in Varilrix or Priorix-195

Tetra. In addition, nine pure vaccine-type SNPs in Varivax and 14 in Varilrix had been 196

detected by CTSQ. We identified only five pure vaccine-type SNPs in Varivax, six in Varilrix 197

and seven in Priorix-Tetra (Figure 1). In addition, some mixed SNPs revealing minority allele 198

fractions of >20% in our study (Varivax: nt 703, 763, 58595, 107797; Varilrix/Priorix-Tetra: 199

nt 763, 19431; 31732; 71252; 87306; 89734; 105310; 107797) had not been identified as 200

mixed in earlier reports using CTSQ (7, 10). Most likely, this indicates differences between 201

vaccine batches (Figure 1). It has already been noted that Varivax V-Oka has undergone some 202

changes in variant content, compared with the original Biken vaccine (9) (compare also 203

Varivax Acc.-no. DQ008355 and V-Oka Acc.-no. AB097932 in Figure 1). This is underlined 204

by the SNP profile of the Varivax lot analysed in this study which differs from both published 205

vaccine profiles (Figure 1) and therefore indicates additional changes over time. Finally, we 206

also identified mixed SNPs with wt allele fractions below 20%. These minor wt fractions had 207

been missed by CTSQ (7, 10), most likely because the PSQ method is more sensitive and 208

quantifies more accurately than CTSQ (12). 209

A quantitative examination of all vaccine PSQ measurements was performed and the 210

percentages of wt alleles are plotted in Figure 2. The data show nearly identical wt allele 211

fractions in Varilrix and Priorix-Tetra. This was theoretically expected for all 32 SNPs since 212

the VZV component in Priorix-Tetra is the same as in Varilrix. In addition, the wt allele 213

percentages of Varivax measured by PSQ in this study were very similar to the PSQ values 214

reported previously for Varivax (10) (Figure S1 of the supplement). These data convincingly 215

demonstrate the suitability of the PSQ technique for quantitative molecular typing of VZV. 216

By comparing the three vaccines through quantification of the PSQ signals, we detected on 217


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average 54% wt alleles in Varivax which exceeded the wt allele fractions in Varilrix and 218

Priorix-Tetra (both 35% wt alleles) by about 20% (Figure 2). 219

PSQ revealed 100% vaccine markers at positions 560, 105705, 106262, 107252 and 220

108111 in all three vaccines. All other loci revealed mixed markers for Varilrix and Priorix-221

Tetra (percentage wt alleles: 5-90%) and Varivax (percentage wt alleles: 11-90%) except four 222

loci in Varivax where only wt markers were detected (nt 12779, 31732, 89734 and 107599) 223

(Figure 2). 224

SNP positions containing 100% vaccine markers in vaccines as well as in vaccine-type 225

VZV from vaccinees are suitable positions for differentiation between vaccine-type VZV and 226

wt VZV in routine diagnostics. 100% vaccine markers have been detected previously by PSQ 227

at positions 560, 105705, 106262, 107252 and 108111 in the Varivax vaccine as well as in 228

patient isolates (except SNP 560) (10). In accordance, vaccine-type alleles were also detected 229

in our PSQ analysis, albeit with weak wt signals at positions 560 (5-9%) and 107252 (5-7%), 230

just below the detection limits of 10% and 8%, respectively (Figure 2). To assay these SNPs 231

in more depth, we analysed all five positions by cloning the respective five PCR fragments 232

from Varivax in E. coli K12 and screened 73-125 E. coli clones per SNP position for inserts 233

with wt alleles. 1/100 clones (1%) revealed the wt allele at position 560, 0/96 at position 234

105705, 7/125 (6%) at position 106262, 0/99 at position 107252 and 4/73 (5%) at position 235

108111. This indicated very low wt allele minorities at positions 560, 106262 and 108111, but 236

not at 105705 and 107252. Likewise, the minor, mostly doubtful wt signals in Varilrix at 237

positions 105310, 105544 and 107136 were evaluated by cloning. Inserts with wt alleles were 238

identified in 3%, 0% and 4%, respectively, of the clones. This confirmed very low wt allele 239

minorities at positions 105310 and 107136 in Varilrix (Figure 1). 240

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SNP analysis of (i) vaccine-type VZV from vaccinees and (ii) wildtype VZV from non-242

vaccinated individuals and vaccinees with PSQ. In the nine specimens from vaccinees with 243


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vaccine-type VZV, five SNPs in ORF62 homogeneously displayed the vaccine-type allele (nt 244

560, 105705, 106262, 107252 and 108111), being in accordance (except nt 560) with 245

previous analyses of vaccinees with rashes (9) (11) (10) (6). All specimens displayed also 246

mixed alleles and therefore contained mixtures of variants (see below) (Figure 1). The high 247

variability of the mixed allele percentage (6.2-40.6%) may be in part due to the fact that the 248

specimens contained highly different total VZV copy numbers (100-107) and that the firm 249

determination of a minority allele is limited to specimens with a sufficient copy number. In 250

addition, the samples were most likely derived from different numbers of lesions which may 251

have influenced the numbers of variants detected. 252

At four positions (nt 12779, 31732, 107599 and 107797), all specimens revealed 100% 253

wt allele. This is in accordance with previous reports that observed 90-100% wt alleles at 254

these positions (9, 10). Furthermore, it largely matched the Varivax profile (100% wt allele at 255

positions 12779, 31732, 107599; nt 107797: 52%) rather than those of the GSK vaccines 256

which uniformly revealed mixed markers (Figure 1). 257

The remaining 23 SNPs of the specimens displayed vaccine, wt or mixed markers, 258

apparently in a random fashion. There was no specimen that revealed wt alleles in all 23 259

variable positions (maximum: 18/23; specimen no. 8) (Figure 1). The percentage of wt 260

markers in the different specimens varied between 34% and 69% and that of mixed markers 261

between 6% and 41% (Figure 3). This indicated the presence of more than one vaccine variant 262

in each specimen. Likewise, Loparev and coauthors reported on the presence of more than 263

one vaccine variant in specimens from vaccinees. In accordance with our results (on average 264

54% vaccine, 34% wt and 12% mixed markers), these authors observed 59% vaccine, 33% wt 265

and 8% mixed markers in vaccine-related rashes (9). 266

Finally, PSQ of wt VZV from non-vaccinated (n=4) and vaccinated (n=5) individuals 267

uniformly revealed alleles at all 32 SNP positions that had been defined as wt on the basis of 268


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the wt strains Oka-P and Dumas (Figure 1). This showed that there is most likely no variation 269

among wt strains in the 32 SNPs analysed in this study (Figure 1). 270

271

Comparison of ORFs 62 from vaccines with those from vaccinees suffering from rashes. 272

To determine whether vaccine-type VZV strains in patient specimens are genetically identical 273

to vaccine-type VZV strains present as minorities in vaccines, we examined ORF62 that 274

encodes a transactivator protein thought to contribute to the attenuation of the vaccine (4, 5). 275

ORF62 encompasses more than one third of the SNPs analysed here and therefore is a highly 276

representative locus in the genome. To analyse SNP profiles of single molecules, we cloned 277

the entire ORF62 in E. coli. Clones from the three vaccines were completely sequenced by 278

CTSQ, and eleven SNP positions were analysed. It appeared that Varivax contains a larger 279

diversity of VZV variant strains than Varilrix and Priorix-Tetra. Thirty different SNP profiles 280

were observed for Varivax and only ten for Varilrix and Priorix-Tetra (Figures 4a and 4b, 281

respectively). Furthermore, the Varivax variant strains contained on average more wt SNPs 282

(37%; Figure 4a) than the variant strains of Varilrix and Priorix-Tetra (17%; Figure 4b). In 283

addition, the SNP profiles of the cloned vaccine variant ORF62 sequences were compared 284

with the profiles of the vaccine-type VZV in the nine patient specimens with vaccine-type 285

VZV. Importantly, one or more vaccine clone profiles were identical to one of the ORF62 286

profiles originating from the specimens and were almost exclusively detected in Varivax 287

(Figure 5). It has previously been speculated (but not formally demonstrated) that the vaccine 288

VZV variants detected in patient specimens pre-exist in the administered vaccine preparations 289

(9) (10). Our data are the first to indicate that this is most likely the case (Figure 5). However, 290

our analysis was restricted to ORF62 which covers only one third of the SNPs differentiating 291

vaccine strains from the parent wt strain Oka-P. Further analysis of complete genomes are 292

required to settle this issue. 293

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SNPs for diagnostic differentiation of wildtype versus vaccine-type VZV. Different 295

commercial preparations of varicella vaccine (Varilrix and Priorix-Tetra, both GSK; Varivax 296

from SPMSD) were compared by PSQ for the first time. Due to the higher sensitivity of PSQ 297

compared to CTSQ, an unprecedented high percentage of mixed SNPs was found in all three 298

vaccine brands. However, even SNPs that were found by PSQ to be pure vaccine-type and 299

had been reported previously as pure (7, 9, 10) revealed very low levels of wt SNPs when 300

larger numbers of E. coli clones were analysed (Figure 1). Four SNPs (nt 105705, 106262, 301

107252 and 108111) have been formerly reported to be pure vaccine-type in vaccines and 302

individuals with vaccine-related rashes (7, 9, 10). One of them, nt 106262, has been used in 303

real-time PCR to firmly discriminate wt strains from vaccine strains (8). In this study, we 304

detected 5-6% wt SNPs at two of the four positions (nt 106262 and 108111) by PSQ of a large 305

number of E. coli clones of the vaccine Varivax (Figure 1). In addition, a complete ORF62 of 306

Varivax was cloned in E. coli that revealed the wt allele at position 108111 (Figure 4a). Of 307

note, wt alleles were detected previously by CTSQ at positions 560, 105705, 107252 and 308

108111 in some older lots of Varilrix. In contrast, Varivax lots revealed pure vaccine types at 309

these positions (13, 14). These data and those presented here show that the mixtures of variant 310

strains in the vaccines as well as the relative strain proportions change with time in different 311

vaccine batches. It is therefore reasonable to assume that the SNP profiles of vaccine-type 312

VZV strains causing rash-like illnesses in vaccinees may change over time as well. Based on 313

this assumption, the firm discrimination of vaccine-type VZV from wt VZV strains should 314

include more than one SNP. Rather, the four-position core set (nt 105705, 106262, 107252 315

and 108111) should be used for diagnostic differentiation of wildtype versus vaccine-type 316

VZV. 317

318

Conclusions. In this study of VZV variants mixed alleles with small wt minorities were 319

detected with a high sensitivity by PSQ. While CTSQ usually does not detect allele or mutant 320


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minorities below 20% in mixed alleles (2, 15), most PSQ assays detect them with a detection 321

limit well below 10% ((15); this study). Furthermore, alleles can be quantified by PSQ, in 322

contrast to CTSQ. An additional advantage is that PSQ is much faster than CTSQ, enabling 323

the analysis of 96 PCR products in 10 minutes. By PSQ, we determined an estimated three-324

fold higher diversity of VZV variants with 20% more wt SNPs in Varivax, compared to 325

Varilrix and Priorix-Tetra. Furthermore, by analysing the complete ORF62, we observed 326

VZV variants that represented minor components of the Varivax vaccine in vaccinated 327

individuals suffering from mild rash-associated syndromes. Therefore, Varivax resembles a 328

vaccine that appears to be more “wildtype-like”. This might be the reason why Varivax on the 329

one hand seems to be related to comparatively higher frequencies of rash-associated 330

syndromes, i.e., mild forms of varicella or zoster and on the other hand protects against 331

varicella more efficiently. In the future, the ongoing German varicella sentinel will provide 332

further insight into this issue. Finally, from our PSQ data on vaccine virus cloned in E. coli it 333

is concluded that the analysis of a core set of four SNPs is required as a minimum for a firm 334

diagnostic differentiation of vaccine-type VZV from wt VZV. 335

336

337

Acknowledgements338

The technical assistance of Cornelia Walter and Sonja Liebmann is kindly acknowledged. 339

This work was funded by the Robert Koch Institute. The authors declare that they have no 340

competing interests. 341

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FIGURES 346

Figure 1 VZV SNPs determined by PCR and PSQ. SNPs (n=32) from nine patients with 347

vaccine-type VZV, four non-vaccinated and five vaccinated patients with wildtype VZV and 348

the three vaccine brands are shown. The corresponding SNPs of Varivax and Varilrix, the 349

original vaccine strain Oka-V and the wt strains Oka-P and Dumas are included for 350

comparison. The last two lines show the percentages of wt markers (positions 560, 105310, 351

106262, 107136 and 108111) and of vaccine-type markers (positions nt 105544, 105705 and 352

107252) that were revealed by PSQ of E. coli clones. Red: wt marker; blue: vaccine marker; 353

yellow: mixed marker; NCR: non coding region. 354

355

356

Figure 2 Percentage of wildtype markers determined by PSQ for 32 SNPs of vaccine 357

VZV genomes from three varicella vaccines. The vertical bars indicate the individual 358

detection limits. They were defined as described in the text. Black bars: Varivax; white bars: 359

Varilrix; shaded bars: Priorix-Tetra. 360

361

Figure 3 Percentage of markers detected in the nine specimens from vaccine-associated 362

rashes. For each specimen 32 SNP positions were analysed by PSQ. Red bars: wt markers; 363

blue bars: vaccine markers; yellow bars: mixed markers. 364

365

Figure 4 SNPs in the ORFs 62 of individual vaccine VZV genomes from three varicella 366

vaccines. SNPs were determined from individual E. coli clones of complete ORFs 62 from 367

Varivax (A) and Varilrix and Priorix-Tetra (B) with CTSQ. Red boxes: wt markers; blue 368

boxes: vaccine markers. Note the wt allele at position 108111 in clone 27 of Varivax.369

370


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Figure 5 SNPs in the ORFs 62 of nine vaccine-type VZV from vaccinees with rashes. The371

ORFs 62 were amplified with LD-PCR, and completely sequenced (chain termination 372

sequencing). Twelve SNP positions are compared with ORF62 clone sequences from Varivax 373

(n=16) and Priorix-Tetra (n=1). Red boxes: wt marker; blue boxes: vaccine marker; yellow 374

boxes: mixed marker. 375

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Table 1 PCR plus PSQ primer sets1

2

3

4

ORF SNP position in genome*

SNP position in

ORF

primer name annealing temp. (°C)

product size (bp)

sense primer (5'-->3')

antisense primer (5'-->3')

pyrosequencing primer (5'-->3')

PCRsense

PCR antisense

PSQ

5' noncoding 560 560 5'NCR-s 5'NCR-as seq_5'NCR-s 62 210 CACCCAACAgTCTTACgATTgC 5'bio-TCCCCTTCCggACAgTAgTTT ggAACCTCCCAACTC 1 703 115 orf1.1-s orf1.1-as seq_orf1.1-s 58 126 TCgCCATCTggAgTACTACACCC 5'bio-CACCggCggTTCTTATTCC CAgTACgTTgCATAACCT 1 763 175 orf1.2-s orf1.2-as seq_orf1.2-s 59 79 ATCgTggCCTTgggACATC 5'bio-AACAACgCgTCgCCACTC gCCTTgggACATCAAC 6 5745 420 orf6-s orf6-as seq_orf6-s 62 157 AgTTACCCCgCgCTCTTTTAg 5'bio-TTCAAgACCgCAgTCgAAT TTAAAgATATTTgCggg

9A 10900 233 orf9A-s orf9A-as seq_orf9A-s 58 99 gTgTgTAgCCCTTATTTCgTTAgC 5'bio-CCTTTTATAggCAAACgggTTTA TgTATTACgCAgCACg 10 12779 620 orf10-s orf10-as seq_orf10-s 58 121 CggCgAAAAggACgACAATAg 5'bio-AgAAgACgCgCCAAACTTg CAAAACCCTgACCAgA 14 19431 1 orf14-s orf14-as seqorf14-s 58 191 TgggACgATTTTCAgCTTgAT 5'bio-TCgCAgTTATCgCAACCCTATg CgTTTggTTggTTTCT 21 31732 974 orf21-s orf21-as seq_orf21-as 62 166 5'bio-CCgggAATgCTTgCTAggA CACgggTgTgTTCTgCTACATTAC gCgCCTCAATATgAAT 31 58595 1588 orf31-s orf31-as seq_orf31-s 60 121 TgTCCAgAACTgggATCAgAT 5'bio-TCCCggACCCATTTAAACTAACTA TgggATCAgATACACg 39 71252 620 orf39-s orf39-as seq_orf39-s 60 80 CTgTgTCgTggggTCCAAgT 5'bio-CgTgTCCACCAAACAgTCCATAg gTCCAgCCgTgTTACT 50 87306 732 orf50-s orf50-as seq_orf50-as 60 144 5'bio-TTTCCgCgTCCACAAAAATA TTTTTggggTCTTTTgTCATTgT CTTggACATTTTgCg 51 89734 1854 orf51-s orf51-as seq_orf51-s 62 251 CTACgCTTCCggTTggTTTC 5'bio-CgTTACAgCgCATAACCTCAAA gCCCCAAATTTAACCA 52 90535 43 orf52-s orf52-as seq_orf52-s 62 77 gCAACgCAgATTACCTTggTTAg 5'bio-CCggACTgTgTCCAggATgT CTTggTTAgAgAAAgCg 54 94167 493 orf54-s orf54-as seq_orf54-as 58 81 5'bio-TATgTACCTCgCTTTgAgTTACCA CATTACTAgTTgCgCCgTATTTT TACgggCCACCCgAT 55 97748 1753 orf55.1-s orf55.1-as seq_orf55.1-as 60 245 5'bio-ATCgAATATgCTTACCggTTTCT CAgAAggCTTTATCggCAgAAgT ACCgCTgCTTCgTgT 55 97796 1801 orf55.2-s orf55.2-as seq_orf55.2-s 58 89 ACAAAgCCCCCTTACACgAA 5'bio-gAAggCTTTATCggCAgAAgT gggCgggTgCCgggA 59 101089 788 orf59-s orf59-as seq_orf59-s 58 251 AATACCTTTCgCgTACggCTCTTT 5'bio-AggACATTTATgTTTTggCCCATC TCTAgCTTAACCCCCA

61/62 105169 684 orf61/62-s orf61/62-as seq_orf61/62-as 60 299 5'bio-CTgggATAgggTAATgCAAgTC TgACggAgTCCCCTCCTTT TCCTTTTCTCgTgAgC 62 108838 296 orf62.1-s orf62.1-as seq_orf62.1-s 62 165 TTCAACCAgAACCCAgAACg 5'bio-gATCggCTCCTgTTggTTCTC ggACggTCAggATCT 62 108111 1023 orf62.2-s orf62.2-as seq_orf62.2-s 62 138 AgACCCgATTgAggATgACA 5'bio-gACCTgCTgCCTgTAgTTTCACT TgAggATgACAgCCC 62 107797 1337 orf62.3-s orf62.3-as seq_orf62.3-as 62 165 5'bio-CgCCAgAgACAgAAATCATTTTCCT TAAAgCggCACgggTTCAgT ggggTTCTggATCgC 62 107599 1535 orf62.4-s orf62.4-as seq_orf62.4-as 62 141 5'bio-AACAgggTgCggTTTggAC TCTAgCCggATCTCCCAACTC CggCACgTAAAgCgg 62 107252 1882 orf62.5-s orf62.5-as seq_orf62.5-as 62 104 5'bio-CAgAgTCTCCgCAgAgCCTT TgAgggTCgggAgCCTgT CCTCggggTATgCCA 62 107136 1998 orf62.6-s orf62.6-as seq_orf62.6-as 62 84 5'bio-ACAgACTCCCgACCCTCAgC TTgCgCggAgTTCgTAAAC CggTggACACACAgAAA 62 106262 2872 orf62.7-s orf62.7-as seq_orf62.7-as 62 171 5'bio-CggggCCgTCgAgTATCT TTCTgTgACCgCCgAgTCT ACAAAgCgggTCCAT 62 105705 3429 orf62.8-s orf62.8-as seq_orf62.8-as 62 227 5'bio-ACCTgggCgAgggTgTTT CCCgCCTgggTTTCTgAC ggAAgCACgAgTggT 62 105544 3590 orf62.9-s orf62.9-as seq_orf62.9-as 62 227 5'bio-ACCTgggCgAgggTgTTT CCCgCCTgggTTTCTgAC CCCgTggTgTCCgAA 62 105356 3778 orf62.10-s orf62.10-as seq_orf62.10-s 62 290 AggggAgCgACggAACAC 5'bio-ggACAACAgCTCCACCTTgA ACCggggTCATCCTT 62 105310 3824 orf62.11-s orf62.11-as seq_orf62.11-as 62 114 5'bio-gggTCATCCTTTggggTgAg AggACAACAgCTCCACCTTgA CTCCACCTTgACCgC

62/63 109137 657 orf62/63.1-s orf62/63.1-as seq_orf62/63.1-s 62 148 gTgTAgAgCgCTgCATCgg 5'bio-gggggCACAACACgTTTTAAgTAC gCATCggCggCgTAT 62/63 109200 720 orf62/63.2-s orf62/63.2-as seq_orf62/63.2-as 59 100 5'bio-AgACgTACCCgAgTTTTCCA gggggCACAACACgTTTT CTCCCTCACCAACCg

64 111650 86 orf64-s orf64-as seq_orf64-s 58 148 CTTTACTgCACACgACACgATACC 5'bio-ACAAACTCgCgCACggTCT ATACCCCCgCgCACC


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Table 2 Long distance-PCR primer sets 5

ORF primer name annealing temp. (°C)

product size (bp)

sense primer (5' - 3')

antisense primer (5' - 3')

sense antisense 62 LD_a1-s LD_a1-as 63 3818 gCggggTCgCCTgATACTT gAggACAACAgCTCCACCTTgAC 62 LD_a2-s LD_a2-as 63 3686 CgCCTgATACTTTggAgTTAATgg CCCCTCCTCgCTgTCCCA

ORF62+NCR LD_b1-s LD_b1-as 60 4565 AgTCgggTgTATTgggACAg AgTCgggTgTATTgggACAg ORF62+NCR LD_b2-s LD_b2-as 60 4516 gggACAgTTACTCCATTAgAggC gggACAgTTACTCCATTAgAggC ORF62+NCR LD_b3-s LD_b3-as 60 4436 CTgggATAgggTAATgCAAg CCTCgAgTCTCgTCCAATCA

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