PEGylation of vesicular stomatitis viru s (VSV) extends virus

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PEGylation of vesicular stomatitis virus (VSV) extends virus persistence in 1 blood circulation of passively immunized mice 2 3 Mulu Z. Tesfay1, Amber C. Kirk1, Elizabeth M. Hadac1, Guy E. Griesmann1, Mark J. 4 Federspiel1, Glen N. Barber2, Stephen M. Henry3, Kah-Whye Peng1, Stephen J. 5 Russell1# 6 7 1Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905; 2Department 8 of Medicine and Sylvester Comprehensive Cancer Center, 9 University of Miami School of Medicine, Miami, Florida 331362 10 3Biotechnology Research Institute, AUT University, Private Bag 92006, Auckland 1142, 11 New Zealand 12 13 14 15 16 17 18 19 Running Title: VSV PEGylation for systemic delivery 20 21 Key words: Oncolytic VSV, non-immune serum, PEGylation, Multiple myeloma 22 23 #Corresponding author: Stephen Russell 24 Email: [email protected] 25 26 Abstract word counts: 223 words 27 Text word counts: 4094 words 28 29 30 Abstract 31 32 We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of 33 multiple myeloma, an incurable malignancy of antibody secreting plasma cells that are 34 specifically localized in the bone marrow. One of the presumed advantages for using VSV as an 35 oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically 36 lacking in cancer patients, which is very important for clinical success. However, our studies 37 show that nonimmune human and mouse serum can neutralize clinical grade VSV, reducing the 38 titer by up to 4-log in 60 minutes. In addition, we show that neutralizing anti-VSV antibodies 39 negate the anti-tumor efficacy of VSV, a concern for repeat VSV administration. We have 40 investigated the potential use of covalent modification of VSV with poly-ethylene glycol (PEG) 41 or a Function-Spacer-Lipid construct (FSL)-PEG to inhibit serum neutralization and to limit 42 hepatosplenic sequestration of systemically delivered VSV. We report that in passively 43 immunized mice with neutralizing anti-VSV antibodies, PEGylation of VSV has improved the 44 persistence of VSV in blood circulation maintaining more than a 1-log increase in VSV genome 45 copies for up to 1hr as compared to the non-PEGylated virus, which was mostly cleared within 10 46 minutes after intravenous injection. We are currently investigating if this increase in PEGylated 47 VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse 48 models of multiple myeloma. 49

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.02832-12 JVI Accepts, published online ahead of print on 16 January 2013

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Introduction 50 51 Multiple myeloma (MM) is an incurable disseminated cancer of antibody secreting 52

plasma cells that localize primarily to the bone marrow, which is characterized by the 53

development of progressive and destructive osteolytic bone disease (9, 16, 21, 28, 29, 31, 54

45). MM is the second most prevalent blood cancer after non-Hodgkin’s lymphoma, 55

representing 1% of all cancers and 2% of all cancer deaths (9, 10). The American Cancer 56

Society estimates that in the United States about 50,000 patients have MM, 57

approximately 20,000 new cases are being diagnosed every year and it is responsible for 58

the death of about 10,000 annually (9, 28, 50). Significant progress has been made in the 59

understanding of disease pathogenesis and emerging therapies, however, MM remains an 60

incurable disease with a median survival of 5-6 years after conventional therapies (10, 21, 61

28, 29, 31, 54). This underscores the urgent need for the development of alternative 62

approaches to therapy. 63

64

Oncolytic viruses can selectively infect tumor cells and cause direct cell destruction 65

and/or elicit antitumor innate and cellular immune responses (8, 26, 41, 46, 48, 51, 52, 66

60). Oncolytic viruses have shown potential utility for the treatment of MM (19, 35, 39, 67

40, 55). We are particularly interested in utilizing vesicular stomatitis virus (VSV) for the 68

treatment of MM. VSV is a Vesiculovirus of the family Rhabdoviridae with a negative 69

sense RNA genome that encodes a nucleocapsid protein (N), a phosphoprotein (P), a 70

matrix protein (M), a glycoprotein (G), and an RNA-dependent RNA polymerase (L) 71

(62). VSV infects a wide variety of animals and different cells. VSV has a rapid 72

replication cycle (62) with ability to reach to high titers in infected tumor cells while 73


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normal cells are effectively protected by the antiviral activity of the host interferon 74

response (3, 5, 43, 56, 57). Because of these attributes, together with, the presumed lack 75

of pre-existing antibody response to VSV in humans, makes VSV a preferred candidate 76

platform for oncolytic virus development against a variety of cancers (32, 33, 39, 40, 43, 77

52, 53, 57). Studies from our laboratory and others show that VSV and its recombinant 78

derivatives have some promise as antimyeloma agents (19, 33, 39, 40, 47, 52). 79

80

For cancers like MM that are disseminated from the onset, systemic delivery of oncolytic 81

viruses that can target both primary and metastatic deposits at the same time is the 82

preferred option for better efficacy (18, 52, 61). However, intravenous delivery presents 83

its own challenges such as potential failure of oncolytic virus extravasation from tumor 84

blood vessels, mislocalization in liver and spleen, and neutralization by antiviral 85

antibodies (27, 23, 35, 44, 52, 59). These potential challenges to systemic virus delivery 86

emphasize the difficulties of using free virus as an oncolytic agent in immuno-competent 87

hosts. In an effort to circumvent the neutralization of oncolytic viruses and to increase the 88

efficiency of virus delivery, different approaches have been employed, such as the use of 89

cell carriers, serotype switching and polymer shielding (11, 12, 30, 34, 38, 42, 47, 52). 90

We are testing covalent modification of VSV with poly-ethylene glycol (PEG) molecules 91

and non-covalent membrane-inserting Function-Spacer-Lipid (FSL)-PEG constructs, 92

termed PEGylation, to overcome the challenges for intravenous delivery of VSV. PEG is 93

an uncharged, hydrophilic, linear polymer that is nonimmunogenic and with low toxicity, 94

leading to its approval by the Food and Drug Adminstration (FDA) for human use (1, 95

11). PEGylation can potentially protect oncolytic viruses from serum neutralization and 96


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from non-specific localization in organs such as liver, leading to increased virus 97

circulation half-life (7, 11, 13, 15, 17, 30, 64). Accordingly, we hypothesized that 98

PEGylation of VSV will increase its stability in blood circulation and this will enhance 99

virus delivery into tumor sites and improve its anti-tumor efficacy. 100

101

In this study, we characterized the interaction of VSV with non-immune normal serum 102

from human and mouse. We found that clinical grade VSV is rapidly neutralized by non-103

immune human and mouse serum. Neutralization requires both IgM antibody and a heat 104

labile factor(s). In addition, we demonstrate that covalent modification of VSV with PEG 105

molecules or with membrane inserting FSL-PEG constructs (PEGylation) protects the 106

virus from serum neutralization in vitro. Using passively immunized mice we further 107

show that PEGylation extends the persistence of VSV in the circulation and increases 108

virus trafficking to tumor growth sites while reducing virus induced hepatotoxicity. We 109

are currently investigating if this increase in VSV circulating half-life in blood can 110

translate to better VSV efficacy in mouse models of multiple myeloma tumors. 111

112

Material and Methods 113 114 Cells: 115

Human 293T cells and African green monkey kidney Vero cells were cultured in 116

Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated 117

fetal calf serum (FCS) for 293T cells and mouse plasmacytoma (MPC-11) cells 118

(American Type cell culture [ATCC # CCL-167]) or 5% FCS for Vero cells, penicillin 119

and streptomycin (GIBCO). 120

121


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Virus: 122

We obtained VSV coding for mIFNβ or GFP (Indiana strain) from the laboratory of Dr. 123

Glen N. Barber (University of Miami School of Medicine, Miami, FL) (43) that was 124

constructed by insertion of IFNβ or GFP gene at XhoI/NheI restriction sites between the 125

G and L viral genes. Clinical grade oncolytic VSV was manufactured in the Mayo Clinic 126

Vector Manufacturing Facility. 127

128 Virus neutralization assays: 129

For neutralization assay, VSV-GFP virus (5µl volume with 1*10^8 50% Tissue Culture 130

Infective Dose [TCID50]) was incubated with 100µl undiluted non-immune human (a 131

pool of 3-5 volunteer healthy donors) or naïve mouse serum at 37°C for 1hr or for the 132

indicated times followed by virus titer (TCID50/ml) determination on Vero cells (1*10^4 133

cells/well) that were plated overnight prior to infection with VSV treated with serum. In 134

some cases, heat inactivation of serum was performed at 56°C for 30 min (2). The effect 135

of serum IgM was analyzed by pre-incubation of serum with anti-IgM antibody (equal 136

volume, for 30 minutes at RT). As control, virus was incubated with medium only and 137

plated on Vero cells. Neutralization assay result was read 48 hrs after infection with virus 138

that was treated with or without serum. All virus neutralization assay experiments were 139

conducted in triplicates. 140

141

In vivo mice experiments: 142

5*10^8 TCID50 VSVGFP was intravenously injected into immuno-competent Balb/c or 143

C57BL/6 mice with or without passively transferred VSV-immune serum (1:100 diluted 144

serum that was harvested from actively immunized mice) injected intraperitoneally (IP) 145


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24 hrs prior to intravenous (IV) tail vein injection of VSV. Blood samples were drawn via 146

retro-orbital and/or submandibular method at indicated time points for virus titer 147

determination of TCID50 on Vero cells and viral RNA analysis by quantitative PCR. 148

149

For survival studies, BALB/c mice (4-to-6 week old) were immunized by IV challenge 150

with VSV-mIFNβ virus (1*10^8 TCID50/mouse). Two weeks after immunization, serum 151

titer was determined using neutralization assay. Mice with similar serum titers (Figure 152

2a) were selected and MPC11 subcutaneous tumor were established by injecting with 153

5*10^6 MPC11 cells subcutaneously, including control naive mice. Ones tumors were 154

established (~100mm3 tumor volume), mice were randomly divided into three groups: 155

Group 1 was immunized and IV injected with no virus (PBS only) (n=8), Group 2 was 156

immunized and IV injected with VSV-mIFNβ virus (2.5*10^8 TCID50/mouse) (n=9), 157

and Group 3 was non-immunized (naïve) and IV injected with VSV-mIFNβ virus (2.5 158

*10^8 TCID50/mouse) (n=5). Tumor size and survival data was collected as shown in the 159

Figure legend. All of the animal experimental procedures in this study were performed 160

following the guidelines of Mayo Clinic Institutional Animal Care and Use Committee 161

(IACUC). 162

163

VSV PEGylation: 164

VSV was covalently modified with activated monomethoxypoly (ethylene) glycol 165

(PEG5000)-succinimidyl succinate (Jenkem Technology, Beijing, China). A range of 166

concentrations of PEG (0 to 20mg) were added to 5*10^8 TCID50 of VSV with a final 167

volume of 500 µl in 100mM potassium phosphate-buffered saline (pH 7.4) for each 168


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preparation. PEG conjugation reactions were performed as described previously (4, 11, 169

42). Briefly, all conjugation reactions were performed at 25°C with gentle agitation. The 170

reactions were stopped by 15 minutes incubation at 4°C (42). Reaction products were 171

purified by buffer exchange over a Micro-Bio Spin P-30 chromatography column (Bio-172

Rad) equilibrated with 100 mM potassium phosphate-buffered saline (pH 7.4) following 173

the supplier’s protocol. A similar approach was used to modify VSV with FSL-PEG2000 174

(KODE Biotech, Auckland, New Zealand) but non-covalently used to modify VSV by 175

simple direct contact (4, 20). As a control, VSV was incubated with 100 mM potassium 176

phosphate-buffered saline (pH 7.4) without PEG or FSL-PEG under similar condition and 177

processed in similar manner as the PEG conjugated virus. PEGylation status was assessed 178

by in vitro infectivity assay on Vero cells. Serum neutralization assay was performed 179

after exposing viruses to different concentrations of PEG (0.31 mg to 20 mg) or FSL-180

PEG (5 to 200 µg) treated VSV. 181

182

Purification of RNA and cDNA synthesis for qPCR analysis: 183

Tumor tissue disruption and homogenization for RNA harvest was performed using 184

TissueLyser in combination with Qiagen sample purification kit (Qiagen). Total blood 185

RNA was harvested using an RNeasy protect animal blood kit (Qiagen). 160 ng of total 186

RNA was subjected to Reverse Transcription PCR (RT-PCR) assay for cDNA sysnthesis 187

using Supercript® III First-Strand Synthesis SuperMix kit (Invitrogen) with random 188

hexamers following the manufacturer’s instructions. 189

190

Quantitative PCR (QPCR) analysis: 191


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To measure the number of VSV genome copies in total RNA samples harvested from 192

blood or tumor, cDNA was subjected to QPCR analysis with Mx4000 Multiplex 193

Quantitative PCR systems (Stratagene) with specific primers to VSV N (VSVN-R: 5’-194

CCTTGCAGTGACATGACTGCTCTT-3’ and VSV-N-F: 5’-TGATAGTACCGGAGGA 195

TTGACGAC-3’) and as internal control specific primers for GAPDH (GAPDH-F: 5’-196

CCCACTCCTCCACCTTTGAC-3’ and GAPDH-R: 5’-CTAACCAGGAAATGAGCTT 197

GACCA-3’). Amplified sequence detection was performed by SYBR green-based real-198

time quantitative PCR assay (Roche Diagnostics, Indianapolis, USA). Data is presented 199

as VSV N genome copy number determined using standard curve of known quantities of 200

VSV N plasmid. 201

202

Statistical analysis: 203

Data analysis was carried out using GraphPad Prism software (GraphPad Software, CA, 204

USA). A two-tailed Student’s t test was used to compare mean values. A p-value<0.05 205

was considered statistically significant (p values: *p<0.05, **p<0.01, ***p<0.001). Error 206

bars represent standard deviation. For survival studies comparison was performed using 207

Log rank test from Kaplan Meier survival curves. 208

209

Results 210 211 VSV is rapidly neutralized by non-immune normal serum 212

One of the presumed advantages for using VSV as an oncolytic virus was that human 213

infections are rare and that pre-existing anti-VSV immunity is lacking, which is very 214

important for clinical success (52). To test this assumption we performed neutralization 215


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assays by exposing clinical grade VSV to normal human or mouse serum for one hour, or 216

at earlier and later time points, at 37 ºC and virus titer was determined on Vero cells. Our 217

data show that normal human serum quickly neutralizes VSV resulting in up to 4-log 218

virus titer reduction compared to control virus incubated with medium only. Normal 219

mouse serum similarly neutralized VSV, albeit to a lesser extent than observed with 220

human serum (Figures 1a, 1b and 1d). We also show that the anti-VSV activity of normal 221

human serum can be largely abrogated by anti-IgM antibody pre-treatment (Figure 1b) or 222

by heat inactivation of the serum (Figure 1a) prior to incubation with virus, suggesting 223

the involvement of the antibody and heat-labile factor(s) of normal non-immune serum in 224

the anti-VSV activity, which is in agreement with findings from previous studies (2). 225

226

VSV is rapidly cleared from blood circulation in vivo 227

We assessed the stability of VSV after intravenous injection into immuno-competent 228

C57BL/KaLwRij mice, using the 5TGM1 multiple myeloma mouse model (49). Our in 229

vivo VSV stability studies show that VSV is cleared rapidly from the bloodstream with 230

up to 3-log reduction in VSV titer within 10 minutes after virus injection into non-231

immunized mice (Figure 1c). In addition, using actively immunized Balb/c mice, bearing 232

MPC11 subcutaneous tumors, we show that immune anti-VSV antibody completely 233

negates the antitumor efficacy of VSV (Figures 2b and 2c), underscoring a challenge for 234

repeat VSV administration. 235

236

Our in vitro and in vivo data show that non-immune serum can effectively neutralize 237

VSV and the antitumor efficacy of VSV is negated by VSV-specific immune serum. 238


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These results, together with, the potential failure of oncolytic VSV to extravasate from 239

tumor blood vessels and its sequestration in liver and spleen (27, 35, 44, 52, 59), 240

emphasize the difficulties in using free VSV as an oncolytic agent in an immuno-241

competent host. Because of this, we are focused on finding ways to circumvent serum 242

neutralization of VSV in the blood circulation and to increase the efficiency of VSV 243

delivery into tumor growth sites. One of the innovative approaches being tested is 244

covalent and non-covalent modification of VSV with PEG, termed PEGylation. 245

246

PEGylation protects VSV from neutralization with VSV-specific immune serum 247

PEGylation, the covalent modification of virus with PEG molecules, is one of the ways 248

being tested to protect non-enveloped viruses from serum neutralization and is showing 249

some promise (7, 11, 13, 15, 17, 30). We therefore tested if PEGylation can render VSV, 250

an enveloped virus, resistant to serum neutralization. To our knowledge, this is the first 251

report to modify an enveloped animal virus with PEG for protection of virus from serum 252

neutralization. We used two approaches to bind PEG molecules to the virus surface, 253

either by directly PEGylating the VSV glycoprotein G (VSV G), which is required for 254

virus attachment and membrane fusion during VSV entry into susceptible cells and is the 255

target for host neutralizing antibody (62), or alternatively using FSL-PEG conjugates that 256

insert into the VSV lipid membrane rather than covalently bind to VSV G (4, 20). We 257

proposed that PEGylation can protect VSV from serum neutralization and limit non-258

specific hepatosplenic sequestration, extending virus persistence in blood and increasing 259

its trafficking to sites of multiple myeloma tumor growth. While the direct VSV G 260

covalent modification with PEG may result in reduced virus infectivity, FSL-PEG, which 261


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inserts into the virus lipid membrane leaves VSV G available for receptor binding (4,20) 262

may be less likely to block virus infectivity. To test our hypothesis, we first identified 263

PEGylation conditions that can provide both a good level of virus infectivity and 264

protection from serum neutralization. 265

266

We assessed level of PEGylation using FACS analysis of FSL-Flouro membrane 267

incorporation (data not shown, 20) and inhibition of VSV infectivity due to increased 268

concentrations of PEG or FSL-PEG (Figure 3 and 4a). We show that while low levels of 269

PEG or FSL-PEG modification maintain VSV infectivity, increasing the concentration of 270

PEG molecules leads to loss of VSV infectivity in vitro (Figures 3 and 4a). We found that 271

in our hands, depending on the freshness of the stock used, we needed 10-200X increased 272

amount of PEG as compared to FSL-PEG to inhibit infectivity of VSV in vitro (Figure 3 273

and Figure 4a medium treated). We speculate the need of increased amount of PEG as 274

compared to that of FSL-PEG to result in loss of VSV infectivity is due to either 275

increased efficiency of binding of FSL-PEG as it inserts to the virus membrane envelope 276

versus PEG that covalently binds to VSVG, or the presence of FSL that increases the 277

length of the polymer as compared to PEG alone may lead to increased inhibition of VSV 278

infectivity with less amount of FSL-PEG molecules per virion. In addition, we 279

determined the level of modification needed for protection of VSV from in vitro 280

neutralization with VSV-specific immune serum, a very stringent condition compared to 281

non-immune serum (Figure 4a). 282

283

PEGylation of VSV extends virus RNA persistence in blood 284


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Our in vitro data show that serum from naïve mice can inactivate VSV at a rate similar to 285

that of non-immune human serum (Figures 1a, 1b and 1d) and that the virus is cleared 286

rapidly from the circulation in vivo (Figure 1c). Interestingly, we show that VSV can be 287

conjugated with PEG (Figure 3a) or modified with FSL-PEG (Figure 3b) and that 288

PEGylation can protect VSV from VSV-specific immune serum (Figure 4a). These 289

findings led us to hypothesize that modification of VSV with PEG will increase VSV 290

stability in the blood circulation by protecting VSV from serum neutralization and 291

limiting non-specific sequestration in tissues. To assess the effect of VSV PEGylation in 292

vivo, C57BL/6 mice were either injected with VSV-specific immune serum (1:100 293

diluted serum that was empirically determined to block VSV infectivity in vivo as shown 294

in Figure 4b) or PBS prior to a single intravenous dose of either PEGylated or 295

unmodified VSV (with a dose of 5*10^8 TCID50 per mouse). In order to determine the 296

kinetics of VSV disappearance from the circulation, we collected whole blood following 297

VSV injection at 10 minutes, 1hr, 24hrs and 72hrs post-VSV injection and, total RNA 298

was isolated and subjected to qPCR analysis with specific primers to the VSV N gene to 299

assess virus genome copy number. Our data show that in the immunized mice, the 300

unmodified VSV was cleared within 10 minutes from the circulation (Figure 4c) in 301

contrast to the virus levels in non-immunized mice which was cleared more slowly (data 302

not shown). However, PEGylation significantly extended VSV virus RNA persistence 303

leading to a 1-log increase in virus genome copies present in the circulation at 10 minutes 304

and residual virus still detectable 1hr after virus injection in VSV neutralizing antibody 305

treated mice (Figure 4c). This suggests PEGylation may have protected VSV from serum 306

neutralization in vivo, supporting our in vitro results (Figure 4a). There was no detectable 307


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VSV RNA at later time points (data not shown). We anticipate that the extended 308

persistence of PEGylated virus in the circulation will lead to an increase in the efficiency 309

of virus delivery to tumor growth sites and will improve virus efficacy. 310

311

PEGylation increases virus genome accumulation in tumor and lowers virus 312

induced hepatotoxicity 313

Considering the improved persistence of PEGylated VSV in blood (Figure 4c), we 314

hypothesized that this increase in plasma half-life of PEGylated VSV may lead to 315

increase in virus trafficking to tumor sites. To test our hypothesis we compared the 316

accumulation of unmodified VSV to that of PEGylated VSV following IV injection of 317

VSV into four subcutaneous MPC11 tumor bearing Balb/c mice in the presence of 318

passively transferred VSV-specific immune serum. Control mice were treated with 200µl 319

of potassium phosphate-buffered saline (PBS). Four days after virus injection mice were 320

euthanized and total RNA was isolated from tumor, and then analyzed for VSV N 321

genome copy number using qPCR. Animals treated with PEGylated VSV showed a 2- to 322

3-log higher VSV N genome copy number compared to animals that were treated with 323

unmodified VSV (Figure 5). When liver, Lung, kidney, and spleens were assayed for 324

VSV deposition at day 4, PEGylation increased virus genome levels in these tissues (data 325

not shown). The PEGylation related increase in virus level in tissues could be due to 326

initial virus protection from serum neutralization in the circulation followed by virus 327

amplification in tissues. 328

329


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In an independent experiment, we assessed virus induced hepatotoxicity by performing 330

blood chemistry to determine serum alanine aminotransferase (ALT) and alkaline 331

phosphatase (ALP) levels at day-3 post-virus injection in BALB/c mice. ALP and ALT 332

levels were significantly increased in animals treated with unmodified VSV as compared 333

to control animals (p=0.0003 and p=0.00045, respectively) or those treated with 334

PEGylated VSV (p=0.0092 and p=0.0003, respectively). Conversely, animals treated 335

with PEGylated VSV had lower levels of ALP and ALT that do not differ significantly 336

from that levels in control animals treated with PBS (p=0.3449 and p=0.4922, 337

respectively) (Figures 6a and 6b). 338

339

Discussion 340

341

We are developing oncolytic VSV, a negative stranded RNA virus, for treatment of 342

multiple myeloma, an incurable cancer of antibody secreting plasma cells primarily 343

localized to bone marrow (9, 16, 28, 29, 31). VSV is a promising oncolytic virus that 344

specifically infects and kills tumor cells and has shown promise in preclinical studies for 345

treatment of various tumors (3, 5, 32, 33, 39, 40, 43, 52, 53, 56, 57). In this work, we 346

show that non-immune normal human serum rapidly neutralizes clinical grade VSV, a 347

process that is dependent on a heat-labile factor(s)- and IgM antibody, and which reduces 348

viral titer by about 4-log within 1hr in vitro (Figures 1a, 1b and 1d). In support to our 349

findings, inactivation of serum antiviral activity by complement depletion with heat 350

treatment has been previously reported for influenza (25), VSV (2), and adenovirus 351

vector (24). In agreement with our results, Ikeda, et al, 1999 (23) have also shown that 352

non-immune serum inactivates HSV with IgM playing a contributing role. However, in 353


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contrast to our results that show complement inactivation by heat treatment abrogates the 354

antiviral activity of non-immune mouse and human serum against VSV, Ikeda, et al, 1999 355

(23) reported complement-depleted plasma only partially reverses antiviral activity 356

against HSV, suggesting complement-independent virus inactivation (23). Variation in 357

serum inactivation of viruses related to virus- and/or cell species-specific has been 358

reported (14, 37, 58, 63) that may explain the above differences. 359

360

Virus clearance was much faster in vivo with about 3-log virus titer reduction within 10 361

minutes following IV injection of VSV into naïve mice (Figure 1c). Furthermore, we 362

demonstrated that VSV-immune serum very rapidly neutralizes VSV and negates its anti-363

tumor efficacy in the MPC11 multiple myeloma Balb/c mouse model (Figures 2b and 364

2c). Together, these data underscore the challenges of using VSV as an oncolytic agent in 365

an immuno-competent subject. 366

367

We are investigating different approaches that can protect VSV from serum 368

neutralization, increase virus trafficking to tumor growth sites and lead to increased virus 369

efficacy. One of the innovative ways to protect virus from serum neutralization is virus 370

PEGylation, a covalent conjugation of PEG molecules to the virus surface (7, 11, 13, 15, 371

17, 30). We have developed PEGylation conditions for VSV. Our in vitro data show that 372

low concentrations of PEG don not compromise VSV infectivity. However, higher 373

concentrations of PEG led to loss of virus infectivity (Figures 3a and 3b). Our in vitro 374

studies further demonstrate that PEGylation of VSV with PEG or FSL-PEG protects VSV 375

from neutralization with VSV-specific immune serum, a very stringent condition as 376


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compared to that of non-immune serum (Figure 4a). Using Balb/c mice bearing 377

subcutaneous MPC11 multiple myeloma tumors, we show that virus RNA persisted for a 378

longer time in the circulation of mice treated with PEGylated VSV than in mice treated 379

with unmodified VSV in the presence or absence of passively transferred VSV-specific 380

neutralizing antibodies (Figure 4c and data not shown). 381

382

More importantly, we show that VSV RNA was enriched in tumor tissues harvested on 383

day 4 from mice treated with PEGylated as compared to mice that received unmodified 384

VSV (Figure 5). FSL-PEG-VSV levels tend to be higher in blood (Figure 4c), while it is 385

lower in tumor tissues as compared to PEG-VSV levels. We speculate this may be related 386

to differences in sequestration levels by other tissues, particularly lung and liver, and 387

binding to and/or infection of blood cells. The larger molecular size of FSL-PEG may 388

reduce VSV sequestration by liver than that of PEG bound VSV, leading to increased 389

level of FSL-PEG-VSV in blood (Figure 4c) (22). However, FSL-PEG that inserts to 390

VSV membrane envelope is likely to retain VSV G free to effect binding and infection of 391

blood cells that may reduce its level in tumor and other tissues (6, 36) as compared to 392

PEG-VSV, where binding of PEG directly on the VSV G may reduce its infectivity of 393

blood cells, leading to increased level in tumor (Figure 5). 394

395

Interestingly, virus induced hepatoxicity assessed using blood levels of ALP and ALT 396

show that mice treated with unmodified VSV had significantly elevated enzyme levels as 397

compared to animals treated with PEGylated VSV or PBS treated mice (Figure 6a and 398

6b). 399


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400

Overall, our data identified barriers for systemic VSV delivery in immuno-competent 401

mice and demonstrated the feasibility of using covalent modification of VSV with poly-402

ethylene glycol (PEG) molecules for virus protection from serum neutralization. We 403

further established that PEGylation extended the persistence of VSV in the circulation in 404

the presence of neutralizing anti-VSV antibodies and improved virus trafficking to tumor 405

sites with no noticeable virus induced hepatotoxicity. Our studies are in line with 406

published preclinical studies on PEGylation of non-eneveloped viruses that demonstrated 407

PEGylation provided virus protection from serum neutralization and reduced virus-408

induced toxicity (7, 11, 13, 15, 17, 30, 64). To our knowledge, this work is the first study 409

to report the feasibility of PEGylation of an enveloped animal virus and to demonstrate 410

PEGylation protecting VSV from serum neutralization. We are currently investigating if 411

the increased stability of PEGylated VSV in blood will improve the efficacy of the virus 412

as an oncolytic agent following intravenous delivery into immuno-competent multiple 413

myeloma mouse models in the presence of neutralizing antibodies. 414

415

Acknowledgements 416

417

This work was supported by NIH grant CA129966-01A1-05 to SJR. 418

We thank Rebecca Nace for excellent technical support in animal work. We also thank 419

Mayo Clinic core lab for providing us with clinical grade VSV. 420

421

Conflict of Interest Disclosures 422


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423

SMH is an employee and stock holder of KODE Biotech the supplier of the FSL-PEG. 424

425

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612

Figure Legends 613

Figure 1: VSV is rapidly neutralized by normal human serum. VSV-GFP was 614

incubated with normal human serum (Figures 1a and 1d) or anti-IgM antibody-treated 615

human serum (Figure 1b) or heat-treated (56 °C for 30 minutes) human serum (Figure 1a) 616

or as control with medium and then it was used to infect Vero cells. Figure 1d shows time 617

course experiments at which virus was incubated with serum for 5min, 10min, 15min, 618

30min, 60min, 120min or 240min followed by virus titer determination on Vero cells. In 619

vivo stability of VSV in naïve mice after IV injection is shown in Figure 1c. 620

621

Figure 2: VSV-immune serum negates the oncolytic efficacy of intravenous VSV. (a) 622

BALB/c mice (4-to-6 week old) were immunized by intravenous (IV) injection of VSV 623

(1*10^8 TCID50/mouse). Two weeks after immunization, serum titer was determined 624

using neutralization assay. Mice with similar serum titer were selected and MPC11 625

subcutaneous tumors were implanted by injecting 5*10^6 MPC11 cells subcutaneously, 626

including to control naive mice. When tumors reached an average volume of 100mm3, 627


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mice were randomly divided into three groups: Group 1 was immunized and IV injected 628

with no virus (PBS only) (n=8), Group 2 was immunized and IV injected with VSV-629

mIFNβ virus (2.5*10^8 TCID50/mouse) (n=9), and Group 3 was non-immunized (naïve) 630

and IV injected with VSV-mIFNβ virus (2.5 *10^8 TCID50/mouse) (n=5). Tumor size 631

(b) and survival data (c) were collected. In Figure 2b, at day 12 in PBS group there is no 632

error bar as only one mouse was left for measurement. 633

634

Figure 3: PEGylation of VSV. Different amounts of (a) monomethoxypoly (ethylene) 635

glycol (PEG5000) activated by succinimidyl (Jenkem Technology, Beijing, China) or (b) 636

FSL-PEG2000 (KODE Biotech, Auckland, New Zealand) were added for 5*10^8 TCID50 637

of VSV. All conjugation reactions were performed at 25°C with gentle agitation. The 638

reactions were stopped by 15 minutes incubation at 4°C as in O’riordan, et al., 1999 (42). 639

Unreacted PEG and reaction by-products were eliminated by buffer exchange over a 640

Micro-Bio Spin P-30 chromatography column (Bio-Rad) equilibrated with 100 mM 641

potassium phosphate-buffered saline (pH 7.4). As a control VSV with the same PBS 642

buffer but without PEG or FSL-PEG was incubated in a similar condition and processed 643

in the same manner as the conjugated virus. PEGylation status was assessed by in vitro 644

infectivity on Vero cells. The fluorescent (upper panels) and phase-contrast (lower 645

panels) images taken 24 hrs post infection are shown for each condition. 646

647

Figure 4: PEGylation provides protection of VSV from serum neutralization both in 648

vitro and in vivo. For in vitro experiments (a) 150 µl of VSV-immune serum diluted 649

1:15000 was incubated with 1*10^7 TCID50 PEGylated (with indicated concentrations 650


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of PEG or FSL-PEG) or unmodified GFP-expressing VSV for 1hr at 37°C. As a control 651

virus was incubated with medium. The mix was plated on Vero cells to determine virus 652

infectivity. Figure shows pictures taken at 18 hrs post infection that indicate PEGylation 653

confers some level of virus protection from serum neutralization. Both PEG and FSL-654

PEG experiments were performed at the same time. In vivo studies (b) shows recovered 655

anti-VSV titers in serum harvested from mice that were immunized by passive transfer of 656

VSV-specific antibody. Two-fold dilutions of serum were injected to different mice 657

groups and 24 hrs post injection serum harvested and titered on Vero cells. c) Shows 658

VSV genome copy numbers after intravenous injection of VSV with or without 659

PEGylation in the presence of passively transferred neutralizing antibodies (1:100 660

dilutions). Blood RNA samples harvested at 10 and 60 minutes were subjected to qRT-661

PCR with primers specific to VSV N gene to determine virus genome copies. Results 662

show PEGylation improved persistence of virus RNA in blood circulation significantly at 663

10 minutes in the presence of neutralizing antibodies (VSV vs. VSV PEG, p=0.0330; 664

VSV vs. FSL-PEG, p=0.0001), whereas the difference is not significant at 60 minutes 665

post injection (VSV vs. VSV-PEG, p=0.9610; VSV vs. VSV-FSL-PEG, p=0.0617). 666

667

Figure 5: PEGylation of VSV increases virus accumulation in tumor. Four 668

subcutaneous MPC11 tumor bearing Balb/c mice per group in the presence of passively 669

transferred VSV-specific immune serum were IV injected with 1*10^8 TCID50/mouse 670

unmodified VSV, PEG or FSL-PEG PEGylated. Control mice were treated with 200 µl of 671

potassium phosphate-buffered saline (PBS). Four days after virus injection mice were 672


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sacrificed and total RNA was isolated from tumor and analyzed for VSV N genome copy 673

number using qPCR. 674

675

Figure 6: PEGylation of VSV reduces virus induced toxicity. Hepatotoxicity 676

assessment was performed by blood chemistry analysis to determine serum alkaline 677

phosphatase (ALP) and alanine aminotransferase (ALT) levels at day-3 post-virus 678

injection in BALB/c mice. ALP (a) and ALT (b) levels were significantly increased in 679

animals treated with unmodified VSV as compared to control animals or PEGylated 680

VSV, whereas animals treated with PEGylated VSV have lower levels of ALP and ALT, 681

which is not significantly different from that of control animals treated with PBS. 682


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Erratum for Tesfay et al., PEGylation of Vesicular Stomatitis VirusExtends Virus Persistence in Blood Circulation of PassivelyImmunized Mice

Mulu Z. Tesfay,a Amber C. Kirk,a Elizabeth M. Hadac,a Guy E. Griesmann,a Mark J. Federspiel,a Glen N. Barber,b Stephen M. Henry,c

Kah-Whye Peng,a Stephen J. Russella

Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USAa; Department of Medicine and Sylvester Comprehensive Cancer Center, University of MiamiSchool of Medicine, Miami, Florida, USAb; Biotechnology Research Institute, AUT University, Auckland, New Zealandc

Volume 87, no. 7, p. 3752–3759, 2013. Page 3754, Fig. 1b: “P � 0.004” should read “P � 0.0004” and “P � 0.012” should read “P �0.0174.”

Citation Tesfay MZ, Kirk AC, Hadac EM, Griesmann GE, Federspiel MJ, Barber GN,Henry SM, Peng KW, SJ Russell. 2015. Erratum for Tesfay et al., PEGylation ofvesicular stomatitis virus extends virus persistence in blood circulation of passivelyimmunized mice. J Virol 89:2453. doi:10.1128/JVI.03446-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.03446-14

ERRATUM

February 2015 Volume 89 Number 4 jvi.asm.org 2453Journal of Virology

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