a chimeric vesiculo/alphavirus is an effective...

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A Chimeric Vesiculo/Alphavirus is an effective Alphavirus Vaccine 1

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Anasuya Chattopadhyay1, Eryu Wang2, Robert Seymour2, Scott C. Weaver2, and John K. Rose1, # 4

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Author Affiliation: 1Department of Pathology, Yale University School of Medicine, New 6

Haven, CT 06510; 2Institute for Human Infections and Immunity, Sealy Center for Vaccine 7

Development and Department of Pathology, University of Texas Medical Branch, Galveston, 8

TX. 9

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# Corresponding Author: John K. Rose, Department of Pathology, Yale University School of 11

Medicine, 310 Cedar Street (LH315), New Haven, CT 06510, U.S.A. Phone: 203-785-6794. Fax: 12

203-785-767. E-mail address: [email protected] 13

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Running Title: VSV∆G Chikungunya Vaccine 15

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Word count for Abstract: 164 17

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Word count for text: 4368 (excluding References, figure legends) 19

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.01860-12 JVI Accepts, published online ahead of print on 17 October 2012

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ABSTRACT 20

While a large number of mosquito-transmitted alphaviruses are known to cause serious human 21

diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus 22

chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This 23

virus has the potential to cause a worldwide epidemic and has generated strong interest in 24

development of a prophylactic CHIKV vaccine. We report here on the development of a potent 25

experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) 26

expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV 27

glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV 28

glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated 29

for growth in tissue culture, but could be propagated to high titers without VSV G 30

complementation. They also generated robust neutralizing antibody and cellular immune 31

responses to CHIKV in mice after a single dose, and protected mice against CHIKV infection. 32

VSV∆G-alphavirus chimeras could have general applicability as alphavirus vaccines. 33

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INTRODUCTION 43

Alphaviruses are found worldwide and many are associated with serious disease in 44

humans and other vertebrates. They are typically transmitted by mosquitoes to vertebrate hosts, 45

and can cause fever, arthritis, and lethal encephalitis (9). There are currently no licensed 46

vaccines that protect against alphavirus infection and disease. Chikungunya virus (CHIKV) is an 47

alphavirus that causes chikungunya fever. The virus was first isolated in 1953 in Tanzania and 48

spread across Africa and Southeast Asia. More recent outbreaks have spread to Europe, and 49

CHIKV has been diagnosed in the United States in travelers returning from endemic areas (25, 50

39). CHIKV has generated global public health concern in part because its spread has been 51

associated with a new mosquito vector, Aedes albopictus, which is widely distributed in Europe 52

and the Americas (25). 53

CHIKV infection in humans results in high fever, headache, vomiting, skin rash and 54

crippling arthritis. While most symptoms resolve by about 10 days, the crippling arthritis can 55

persist for months or even years (25). Recent CHIKV epidemics have shown even more severe 56

symptoms including encephalitis, hemorrhagic disease and mortality (34). 57

CHIKV has a single, positive sense, 11.8 kb RNA genome, which encodes four structural 58

proteins (nsP1-4) and five structural proteins (C, E3, E2, 6K, E1) that are cleaved from a 59

precursor to generate the capsid and envelope glycoproteins (35). Neutralizing antibodies 60

(nAbs) play dominant roles in resolving alphavirus infections and neutralizing epitopes have 61

been mapped within E1 (fusion protein) and E2 (attachment protein) glycoproteins in several 62

alphaviruses (38). However, cellular immunity is also capable of protecting against fatal 63

alphaviral disease (22). 64


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Recent CHIKV epidemics in the La Reunion Island and the Indian subcontinent, 65

southeast Asia and Europe have generated substantial interest in development of CHIKV 66

vaccines. A number of successful vaccine approaches based on expression of the E1 and E2 67

CHIKV glycoproteins have been described recently. These include chimeric alphavirus vectors 68

(42, 43), an adenovirus vector (41), VLP vaccine (1), DNA vaccine (18) and IRES based live-69

attenuated CHIKV vaccine (24). 70

Vesicular stomatitis virus (VSV), a negative-strand RNA virus in the Rhabdoviridae 71

family (Vesiculovirus genus), has been used extensively as an experimental vaccine vector 72

against several viral and bacterial pathogens (4, 12-15, 26, 27, 29, 33). VSV-based vaccine 73

vectors are currently being used in HIV vaccine clinical trials 74

(http://clinicaltrials.gov/ct2/show/NCT01438606). 75

In this study, we initially wanted to determine if we could construct VSV-based vaccine 76

vectors expressing the entire CHIKV E3-E2-6K-E1 precursor polyprotein. We did this in a full-77

length VSV construct as well as in a VSVΔG construct (lacking the VSV glycoprotein gene). 78

Interestingly, we found that the VSVΔG vector expressing CHIKV envelope proteins 79

incorporated the CHIKV glycoproteins efficiently into virus particles and propagated without 80

VSV G complementation. This chimeric VSV/alphavirus vector also induced more potent 81

CHIKV immune responses than the full-length vector with VSV G and protected mice from 82

CHIKV challenge after a single dose. Such chimeric viruses could be generally applicable as 83

alphavirus vaccines. 84

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MATERIALS AND METHODS 86

Cells. Baby Hamster Kidney (BHK)-21 cells were maintained in Dulbecco’s modified 87

Eagle’s medium (DMEM) supplemented with 5% Fetal Bovine Serum (FBS). Vero cells, 88

derived from African green monkey kidney cells, were maintained in DMEM containing 10% 89

FBS. 90

Plasmid constructions. To generate pVSV-CHIKV, we first designed a codon 91

optimized synthetic E3-E2-6K-E1 gene (CHIKV S27 prototypic African strain) and had it 92

synthesized with flanking Xho I and Nhe I restriction sites (Genscript, Inc.). This gene was 93

inserted into Xho I-Nhe I digested pVSVXN2 vector (31). pVSVΔG-CHIKV was prepared by 94

deleting the VSV G gene from the pVSV-CHIKV by Mlu I-Xho I digestion, filling-in with T4 95

DNA polymerase (New England Biolabs) and re-ligation. pCAGGS-CHIKV was made by 96

inserting the Xho I-Nhe I digested synthetic E3-E2-6K-E1 fragment into corresponding sites of a 97

modified pCAGGS vector (20) containing these sites. 98

Recombinant virus recovery. Recombinant VSVs (rVSVs) were recovered from 99

pVSV-CHIKV and pVSVΔG-CHIKV as described previously (16, 32). In brief, BHK-21 cells 100

were infected at a multiplicity of infection (MOI) of 10 with vTF-7.3 (7), a vaccinia virus 101

recombinant expressing T7 RNA polymerase. The cells were then transfected with rVSV 102

plasmids (pVSV-) together with support plasmids, pBS-N, pBS-P, pBS-L and pBS-G, encoding 103

VSV proteins. VSV-CHIKV was recovered by transferring the transfected cell supernatants onto 104

fresh BHK-21 cells at 48 h post transfection, and collecting supernatant containing the virus after 105

another 48 h. Virus stock was prepared from individual plaques grown in BHK-21 cells and 106

stored at -70°C. To recover VSV G complemented VSVΔG-CHIKV, transfection supernatant 107

was transferred to cells that were transfected with pCAGGS-G (21) one-day prior, and 108


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supernatant containing the virus was collected after 48 h. The virus was further plaque purified 109

on BHK-G cells (32), and VSV G-complemented stock was prepared and stored at -70°C. A 110

part of VSVΔG-CHIKV recovery supernatant was also plaque-purified without VSV G 111

complementation on BHK-21 cells and further passaged on BHK-21 cells to generate a non-G 112

complemented VSVΔG-CHIKV stock. 113

pCAGGS transfection. 10 μg of the appropriate pCAGGS vector diluted in 0.6 ml of 114

OptiMEM (Invitrogen, CA) was mixed with 30 μl of Lipofectamine Reagent (Invitrogen, CA) 115

also diluted in 0.6 ml of OptiMEM, and incubated at room temperature for 30 min. Confluent 116

monolayers of BHK-21 cells in a 10 cm dishes were washed once with phosphate buffered saline 117

(PBS) and once with OptiMEM. OptiMEM (4.8 ml) was then added followed by addition of the 118

DNA-Lipofectamine mix and incubation for 5 h at 37°C. Subsequently, the transfection mix was 119

removed from the plate and replaced with DMEM containing 10% FBS and incubated overnight 120

at 37°C. 121

Indirect immunofluorescence microscopy. BHK-21 cells on coverslips were infected 122

with recombinant wild-type (rwt)-VSV, VSV-CHIKV or VSVΔG-CHIKV. At 4 h post 123

infection, cells were washed two times with PBS and fixed with 3% paraformaldehyde at room 124

temperature for 20 min. Cells were then washed with PBS containing 10 mM glycine (PBS-125

glycine) and incubated with a 1:200 dilution of either VSV G monoclonal antibodies (MAbs; I1 126

and I14) (17) or an anti-CHIKV antibody [4-week sera from CHIKV-La Reunion (LR) strain 127

infected mouse]. Following this, cells were washed with PBS-Glycine and incubated with 1:500 128

diluted goat anti-mouse AlexaFluor 488 IgG (Molecular probes, Eugene, OR). Cells were then 129

washed twice with PBS-Glycine, mounted on slides using ProLong Gold antifade reagent with 130


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DAPI (Molecular probes, Eugene, OR) and imaged with a Nikon Eclipse 80i fluorescence 131

microscope using a 40X objective. 132

Metabolic labeling and SDS-PAGE of purified viruses and cell lysates. To label 133

infected cells, BHK-21 cells in 3 cm dishes were infected with recombinant viruses or mock 134

infected at an MOI of 20. At 5 h post-infection, cells were washed with methionine-free DMEM 135

followed by labeling with 100 μCi of [35S]-methionine in 1 ml methionine-free DMEM for 30 136

min at 37°C. Cells were then washed twice with PBS and lysed with detergent lysis buffer (1% 137

Nonidet P-40, 0.4% deoxycholate, 50 mM Tris-HCl, pH 8.0, 62.5 mM EDTA) on ice for 5 min. 138

Lysates were collected and clarified at 16,000 x g for 2 min at 4°C. To label rVSVs, BHK-21 139

cells on 6 cm dishes were infected with rwt-VSV, VSV-CHIKV or VSVΔG-CHIKV at an MOI 140

of 100. After adsorption for 1 h, virus was removed and replaced with DMEM containing 5% 141

FBS. After 2 h, cells were washed two-times with pre-warmed methionine-free DMEM. 142

Subsequently, labeling media (90% methionine-free DMEM + 9% serum-free DMEM + 1% 143

FBS) containing 300 μCi of [35S]-methionine was added to each dish and incubated over night at 144

37°C. Virus supernatants were collected after 20 h, clarified at 16,000 x g for 5 min to remove 145

cell debris, layered onto a 20% sucrose gradient (in 10 mM Tris pH 7.4) and centrifuged for 1 h 146

at 38,000 rpm at 4°C. Virus pellet was dissolved in TE. 147

Labeled viruses and cell lysates were analyzed on a 4-12% Bis-Tris NuPAGE gel 148

(Invitrogen, CA) and protein bands were imaged using a Fujifilm BAS 1800 imaging system. 149

One-step growth curve. BHK-21 cells were infected with rwt-VSV, VSV-CHIKV or 150

VSVΔG-CHIKV at a MOI of 10. After virus adsorption for 30 min, cells were washed twice 151

with PBS and DMEM containing 5% FBS was added. Supernatant samples were collected at 0, 152

2, 4, 6, 8 and 24 h post adsorption and titered by plaque assay on BHK-21 cells. 153


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CHIKV pseudotype generation. VSVΔG-eGFP1 pseudotyped with CHIKV envelope 154

proteins was generated by transfecting BHK cells with pCAGGS-CHIKV, followed by infection 155

with VSV G complemented VSVΔG-eGFP1 (15). At 1 h post infection input virus was 156

removed, cells were washed twice with PBS and DMEM containing 5% FBS was added. 157

Supernatant containing VSVΔG-eGFP1 pseudotyped with CHIKV envelope proteins was 158

collected at 24 h and virus titered on BHK-21 cells by counting the number of enhanced green 159

fluorescent protein (eGFP) expressing cells. 160

Animal experiments. Mice, housed under BSL-2 conditions in microisolator cages, 161

were inoculated with recombinant VSV vectors using 106 plaque forming units (PFU) per mouse 162

by the intramuscular (I.M.) route in a volume of 50 μl of serum-free DMEM administered into 163

the right hind leg muscle. For the challenge study, 3 week old female C57Bl/6 mice were 164

obtained from Jackson Laboratories, ME and acclimatized for 4 days before immunization. 165

Blood was collected at 30 days post immunization (dpi) from retro-orbital sinus. These animals 166

were challenged with the wild-type CHIKV-LR strain derived from a cDNA clone (39) using 104 167

PFU per mouse by the subcutaneous (S.C.) route in the left rear footpad. Foot swelling 168

measurements were taken on the day of challenge and for 10 days following using a caliper to 169

measure the height of the foot at the ball. Animals from each immunization group were divided 170

into two subgroups and were bled on alternate days post challenge to measure viral titers by 171

plaque assay on Vero cells. Two animals from each group were sacrificed at 4 days post 172

challenge (38 dpi) for histopathology analysis. UV inactivation of virus was performed using 173

two exposures of virus to 5 x 105 μJoules in a UV Stratalinker 1800 (Stratagene). 174


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To look at cellular immune responses, 6-8-week-old female C57Bl/6 mice were 175

inoculated with virus after 1-week acclimatization, and sacrificed by anesthesia overdose at 7 dpi 176

for isolating splenocytes. 177

Yale University and/or UTMB Institutional Animal Care and Use Committee approved 178

all animal experiments. 179

Virus neutralization assays. The VSVΔG-eGFP1/CHIKV pseudotype neutralization 180

assay was similar to that described previously for Nipah virus pseudotypes (5). Briefly, 181

approximately 50 infectious pseudotype particles were mixed with pooled and serially diluted 182

serum samples from each immunization group, added to monolayer of BHK-21 cells in a 96-well 183

plate and incubated for 20-24 h at 37°C. Infection was determined by visualizing the number of 184

eGFP expressing cells using an Olympus CK-40 microscope equipped with epifluorescence. 185

Absence of infection in duplicate wells for each sample was scored as 100% neutralization. 186

VSV neutralization assay was performed as described previously (29). The VSV 187

neutralization titers were defined as the highest dilution of serum that could completely 188

neutralize infectivity of 100 PFU of VSV on BHK-21 cells 189

CHIKV plaque reduction neutralization tests were performed using standard methods (2) 190

and the CHIKV-LR strain. 191

ELISPOT assay. A gamma interferon (IFN-γ) enzyme linked immunospot assay 192

(ELISPOT) kit (BD Biosciences) was used to quantify T cell activation following immunization. 193

Splenocytes were isolated from immunized mice at 7 dpi by disrupting spleen between frosted 194

ends of two microscope slides. The red blood cells were removed by using red blood cell lysis 195

buffer (eBioscience Inc., San Diego, CA) and spenocytes were collected in RPMI 1640 196

containing 10% FBS after passing through strainer. 2 x 105 splenocytes per well were then 197


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added to a 96-well plate pre-coated with mouse IFN-γantibody and incubated with or without 198

CHIKV E1 (HSMTNAVTI) or E2 (IILYYYELY) peptides at a concentration of 20 μg/ml for 24 199

h at 37°C. Thereafter, cells were first washed with water and then with wash buffer (kit) 200

followed by incubation with biotinylated anti-mouse IFN-γ antibody at 1:2000 dilution for 2 h at 201

room temperature. Following this, cells were again washed with wash buffer; streptavidin-202

horseradish peroxidase (HRP) conjugate (kit) was added at 1:100 dilution and incubated at room 203

temperature for 1 h. The plate was developed according to manufacturer’s protocol using 3-204

amino-9-ethyl-carbazole (AEC) chromogen diluted in AEC substrate buffer. Reaction was 205

stopped by adding water and the plate was air-dried before counting spot forming cells (sfc). 206

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RESULTS 208

Construction and characterization of recombinant VSVs expressing Chikungunya 209

virus envelope proteins. To generate a VSV recombinant expressing CHIKV surface 210

glycoproteins, a synthetic gene encoding the CHIKV E3-E2-6K-E1 polyprotein was inserted 211

between the G and L genes of VSV (Indiana) in the plasmid vector pVSVXN2 (31) and the 212

resulting plasmid pVSV-CHIKV was used to recover VSV-CHIKV (Fig. 1A) using established 213

procedures (16). A VSV recombinant expressing CHIKV envelope proteins in the absence of 214

VSV G (Fig. 1A) was also recovered by complementation with VSV G (32) and was designated 215

VSVΔG-CHIKV. Initially this recombinant was grown in cells expressing VSV G because we 216

assumed that CHIKV glycoproteins would not be incorporated efficiently into VSV particles or 217

promote efficient infection. As we show below, the VSVΔG-CHIKV recombinant does 218

propagate in the absence of VSV G complementation. 219


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Expression of CHIKV proteins in VSV recombinants. To determine if CHIKV 220

proteins were expressed in cells infected with the VSV/CHIKV recombinants, we initially used 221

indirect immunofluorescence microscopy. BHK-21 cells were infected with VSV-CHIKV, 222

VSVΔG-CHIKV or rwt-VSV (control). Fixed, non-permeablized cells were incubated with 223

MAbs to VSV G or with serum from CHIKV-infected mice, followed by 2° Ab (anti-mouse 224

AlexaFlour-488 conjugate) treatment. As shown in Figure 1B, cells infected with both 225

recombinants showed surface expression of CHIKV proteins, while rwt-VSV infected cells 226

showed no CHIKV protein expression. The CHIKV antiserum recognizes both E1 and E2. VSV 227

G protein was expressed on the cell surface of rwt-VSV and VSV-CHIKV infected cells, while 228

VSVΔG-CHIKV infected cells did not express VSV G. 229

To visualize the expression levels of both E1 and E2 proteins, we performed a 30 min 230

metabolic labeling ([35S]-methionine) of BHK-21 cells infected with VSV-CHIKV, VSVΔG-231

CHIKV, or rwt-VSV. Lysates from infected and mock-infected cells were separated by SDS-232

PAGE and analyzed on a Fujifilm imaging system (Fig. 1C). Because VSV infection shuts off 233

host protein synthesis, the cell lysate from rwt-VSV infected cells shows mainly the viral 234

proteins L, G, N, P and M (Fig 1C, lane 2). VSV-CHIKV infected cells expressed an additional 235

protein with the mobility expected for CHIKV E1 protein (~ 52 kDa) (Fig. 1C, lane 3). Because 236

this was only a 30 minute labeling, the CHIKV E2 would still be in its precursor form of E3-E2 237

(p62). The p62 protein is evident in the VSVΔG-CHIKV infected cells along with E1 (Fig. 1C, 238

lane 4). However, in the VSV-CHIKV infected cells, the p62 protein runs just ahead of and 239

overlapping VSV G, and cannot be discerned clearly. 240

CHIKV glycoproteins are incorporated into VSV recombinants. To determine if the 241

CHIKV glycoproteins were incorporated into VSV particles, BHK-21 cells were infected with 242


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rwt-VSV or either VSV/CHIKV recombinants and labeled for sixteen hours with [35S]-243

methionine. Virus particles purified from the medium were then analyzed by SDS-PAGE (Fig. 244

1D). rwt-VSV and VSV-CHIKV particles contained the five indicated VSV proteins, while 245

VSVΔG-CHIKV particles lacked VSV G. Both VSV-CHIKV and VSVΔG-CHIKV particles 246

contained a single extra protein band with the mobility of a 52 kDa protein, the mobility 247

expected for both E1 and mature E2. The precursor protein p62 present in cells was absent from 248

the VSVΔG-CHIKV virions as expected, because it is cleaved prior to transport to the cell 249

surface. 250

VSVΔG-CHIKV recombinants grow in the absence of VSV G complementation. 251

Although VSV is promiscuous in terms of accepting foreign proteins into its envelope (30, 46), 252

VSVΔG recombinants with foreign viral envelopes often grow to titers 100-fold lower than VSV 253

itself in the absence of complementation with VSV G (3). However, we found that the VSVΔG-254

CHIKV recombinant typically grew to titers only 2-3 fold lower than rwt-VSV and did not 255

require VSV G complementation. To examine the growth of VSVΔG-CHIKV and VSV-CHIKV 256

in more detail we performed a one-step growth curve as shown in Figure 2A. VSV and VSV-257

CHIKV grew at similar rates and reached equivalent titers, while the VSVΔG-CHIKV appeared 258

to lag initially but eventually reached similar titer as VSV by 24 h. VSVΔG-CHIKV also 259

generated smaller plaques in BHK-21 cells compared to VSV-CHIKV (Fig. 2B). The plaques 260

generated by VSV-CHIKV are indistinguishable from VSV plaques (not shown). 261

VSV/CHIKV recombinants induce CHIKV nAbs in C57Bl/6 mice. To determine if 262

the VSV recombinants expressing CHIKV glycoproteins were immunogenic, we initially 263

performed a pilot study to test the immunogenicity of VSV-CHIKV, VSVΔG-CHIKV (G-264

complemented) or VSVΔG-CHIKV (non-complemented) in C57Bl/6 mice. A single I.M. 265


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immunization (106 PFU) with these VSV/CHIKV recombinants induced antibodies that 266

neutralized a VSVΔG-eGFP1/CHIKV pseudotype (not shown). Moreover, the non-267

complemented VSVΔG-CHIKV elicited a CHIKV nAb titer that was at least as good as that of 268

the G-complemented virus or VSV-CHIKV. 269

The immune response requires viral RNA synthesis. To determine if the nAb 270

response to VSVΔG-CHIKV was dependent on viral replication, we examined immune 271

responses to UV inactivated virus particles. As shown in Fig. 3, UV inactivation completely 272

eliminated the antibody response to G-complemented or non-complemented VSVΔG-CHIKV. 273

This result indicates that proteins on incoming virions are not sufficient to generate the immune 274

response and that viral transcription and presumably replication are required to generate 275

sufficient mRNA and protein to induce the immune response. Because the non-complemented 276

VSVΔG-CHIKV would have the major advantage of not inducing anti-G antibodies, we 277

performed a more detailed analysis of this virus as well as VSV-CHIKV to assess induction of 278

immune responses and protective efficacy in a CHIKV challenge model. 279

Immunization and challenge study. Fig. 4A shows the timeline of the 280

immunization/challenge study using 3.5 week-old C57Bl/6 mice. Two groups of eight mice each 281

were immunized I.M. with VSV-CHIKV or VSVΔG-CHIKV and a control group (n=8) received 282

rwt-VSV. Blood was collected from the animals at day 30-post immunization and used to 283

determine the CHIKV nAb response from each group. As shown in Fig. 4B, while both 284

VSV/CHIKV recombinants elicited 100% neutralization of VSV/CHIKV pseudotypes 285

(homologous S27 strain) at low dilution, VSVΔG-CHIKV induced a superior response at 286

dilutions greater than 1:160. However, the differences between the two vaccine groups did not 287

reach statistical significance. Control rwt-VSV immunized mice had no CHIKV nAb. To assess 288


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the nAb responses to the heterologous CHIKV-LR strain (challenge virus), plaque reduction 289

neutralization tests (PRNT) were also performed using these serum samples. As shown in Table 290

1, all rVSV/CHIKV immunized animals had PRNT80 titers of ≥80 with the VSVΔG-CHIKV 291

group showing a higher nAb titer on average (ranging from 160 to >640) compared to VSV-292

CHIKV group (titer ranging from 80 to 320). Control mice that received rwt-VSV showed titers 293

below the detection limit (<20). Anti-vector nAb titers directed against VSV G were also 294

measured using pooled serum samples from each immunization group (not shown). Mice 295

immunized with rwt-VSV and VSV-CHIKV had VSV nAb titers of 1: 2,560, while mice 296

immunized with VSVΔG-CHIKV did not generate detectable VSV nAb (<1:20). 297

rVSV/CHIKV immunized mice are protected against CHIKV infection. To 298

determine if the VSV/CHIKV recombinants can protect against CHIKV-induced viremia and 299

disease, the immunized C57Bl/6 mice were subjected to S.C. challenge in their rear left footpad 300

with 104 PFU of CHIKV-LR strain at 34 days post immunization and followed for signs of 301

disease over 10 subsequent days. Four mice from each immunization group were bled on 302

alternate days post challenge to assess viremia. Control animals that received rwt-VSV showed 303

viremia during the first two days after challenge while none of the rVSV/CHIKV-vaccinated 304

mice had detectible viremia (Table 2). CHIKV induced disease was also monitored for 10 days 305

post challenge by following signs of local inflammation as measured by swelling of the 306

inoculated foot. As shown in Fig. 5A, rwt-VSV immunized control animals displayed 307

substantial footpad swelling that was sustained for 10 days after challenge. In contrast, mice in 308

both the rVSV/CHIKV-vaccinated groups separately showed significantly less (p<0.001, 309

ANOVA) foot swelling, which returned to pre-challenge levels by day 8 or 9 post challenge (Fig. 310

5A). While the control animals showed a transient weight loss or failed to gain weight after 311


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challenge, VSV/CHIKV-immunized mice did not show any weight loss and gained weight (Fig. 312

5B). However, the differences in weight loss did not reach statistical significance because of the 313

high degree of animal-to-animal variability. 314

Two animals from each group were sacrificed at day 4-post challenge for histopathologic 315

evaluation (not shown). The brain, lung, heart, kidney, bowel, spleen, and muscle, demonstrated 316

no significant histopatholgic differences among the groups. It is possible that we might have 317

noted more significant histopathologic change at later times after infection. This has been noted 318

by others in the muscle at day 7 following challenge (8). We did however note microgranulomas 319

in the liver of all groups, and the vaccine groups showed a 75% reduction in the number of 320

microgranulomas compared to the rwt-VSV control group. These microgranulomas most likely 321

represent a non-specific reaction to hepatic injury. 322

VSV/CHIKV recombinants induce cellular immune responses in C57Bl/6 mice. 323

Because VSV based vaccine vectors are known to be potent inducers of both humoral and 324

cellular immune responses (28, 47), we also looked at the CHIKV specific cellular immune 325

response to the VSV/CHIKV vectors. A previous study, using a multi-dose DNA vaccine 326

expressing consensus CHIKV E1 or E2 sequences, reported induction of CHIKV specific CD8+ 327

CTL responses using peptide pools, and predicted two dominant CD8+ epitopes one in E1 328

protein and one in E2 protein (19). We used synthetic peptides corresponding to these predicted 329

epitopes (HSMTNAVTI on E1 and IILYYYELY on E2) in an IFN-γ ELISPOT assay. Our 330

studies (Fig. 6) showed that the VSV/CHIKV vectors induced CHIKV cellular immune 331

responses by day 7 after a single immunization. Although the response to the E1 epitope was 332

higher than the response to the E2 epitope in both vaccinated groups, VSVΔG-CHIKV induced a 333


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significantly stronger (p<0.01, ANOVA) response to the E2 epitope compared to that of VSV-334

CHIKV. 335

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DISCUSSION 337

We report here the development of experimental CHIKV vaccines based on VSV vectors 338

expressing the envelope proteins of CHIKV. These were either a full-length VSV-CHIKV 339

vector or a VSVΔG-CHIKV vector that lacks the VSV G gene. Both of these vectors generated 340

robust nAb responses to CHIKV in mice after a single dose, and protected mice from CHIKV 341

challenge. Most interestingly, we found that the VSVΔG-CHIKV recombinant incorporated 342

functional CHIKV envelope proteins into the viral envelope and propagated in the absence of 343

VSV G complementation. Although the VSVΔG-CHIKV recombinant grew more slowly than 344

the full-length vector and made only small plaques, it generated a stronger nAb response to 345

CHIKV than the full-length VSV-CHIKV vector. The stronger antibody responses probably 346

resulted from a combination of greater expression of CHIKV glycoproteins in the absence of 347

VSV G, greater display of the CHIKV proteins on the surface of the viral particles, and/or lack 348

of antigenic competition from VSV G. 349

Chimeric alphaviruses expressing structural protein genes from CHIKV and non-350

structural protein genes of either Venezuelan (VEEV) or eastern equine encephalitis virus 351

(EEEV) have been successfully used as experimental vaccines for CHIKV (43). These viruses 352

also generated potent CHIKV nAbs in mice and protection from challenge after a single dose. It 353

is interesting to note that the titers generated following a single dose VSVΔG-CHIKV vector 354

were comparable to those reported for the VEEV/EEEV-CHIKV chimeras (43). 355


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Passive antibody transfer studies have shown that antibody alone can confer immunity to 356

CHIKV challenge (1, 6, 24). The role of cellular immunity in clearance of CHIKV infection is 357

not well studied. However, recent studies have shown that strong CHIKV-specific T-cell 358

activation occurs early in infection and likely plays a role in early control of viral replication 359

prior to generation of nAb (8, 10, 44). We showed here that a single dose of VSV/CHIKV 360

recombinants induced potent CD8 T-cell responses to dominant epitopes in CHIKV E1 and E2 361

proteins. Recall of memory T-cells generated by vaccination with VSV/CHIKV vectors could 362

therefore complement the robust B-cell responses and aid in viral clearance and protection from 363

CHIKV-induced disease. 364

A major advantage of the VSVΔG vector system is that it does not induce any anti-vector 365

nAb since it lacks the VSV G protein. Hence it could be used in multiple vaccine applications. 366

Based on our experience here with CHIKV, it is likely that VSV∆G vectors could be a general 367

chimeric vaccine platform for other medically important alphaviruses such as VEEV, EEEV and 368

western equine encephalitis virus (WEEV), which are widespread in the Americas. Although the 369

VSVΔG-CHIKV chimeric virus grew more slowly than wild-type VSV, it eventually reached 370

titers nearly equivalent to VSV and thus could easily be scaled up for large-scale vaccine 371

production. We have also found that the VSVΔG-CHIKV recombinant grows in Vero cells to 372

titers equivalent to those obtained in BHK-21 cells. This is important because Vero cells are an 373

approved cell line for vaccine production. 374

VSV-based vaccine vectors given by the I.M. route typically cause no measurable 375

pathogenesis in mice. They are trafficked to draining lymph nodes and rapidly eliminated 376

without causing systemic infection (11, 36). We also observed that the VSV-CHIKV and 377

VSV∆G-CHIKV viruses caused no pathogenesis when given intramuscularly. Interestingly, our 378


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preliminary studies have shown that the VSV∆G-CHIKV vectors are non-pathogenic but 379

immunogenic when given by the intranasal route, while the VSV-CHIKV vector or the VSV 380

vector alone induce substantial pathogenesis (weight loss). A more extensive analysis of 381

pathogenesis and vector spread using multiple routes of inoculation will of course be required 382

before VSV∆G-CHIKV vectors could be moved into clinical trials. 383

Our studies have demonstrated that a VSV-vectored vaccine provides protection against a 384

CHIKV strain (La Reunion) that is about 2% divergent in sequence (37) compared to the vaccine 385

strain (S27). Based on these studies and others showing vaccine cross-protection among more 386

distantly related arthrogenic alphaviruses (8, 23), the VSV-vectored vaccine is likely to provide 387

protection against all three CHIKV clades. Vaccine cross-protection within the encephalitic 388

alphaviruses has also been reported (40, 45). Given the potent immune responses generated by 389

the VSVΔG-CHIKV recombinant, it will be important in the future to examine possible cross 390

protection against other arthrogenic viruses as well as the general applicability of the platform 391

for other alphaviruses. 392

393

ACKNOWLEDGEMENTS 394

This work was supported by NIH grant AI057158. We thank Drs. John Schell and Kapil Bahl 395

for advice on the T-cell studies, and Dr. Alex Ryder for assistance with statistical analysis. 396

397

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FIGURE LEGENDS 557

Fig. 1. Characterization of recombinant VSVs encoding the CHIKV E3-E2-6K-E1 polyprotein. 558

(A) Diagram of rVSV genomes with the insertion of CHIKV envelope polyprotein in full-length 559

VSV and VSVΔG vectors. The gene order is shown in 3’ to 5’ direction on the negative sense 560

RNA genome. (B) Expression of CHIKV envelope proteins detected by indirect 561

immunofluorescence microscopy using anti-VSV G MAb or anti-CHIKV mouse sera in infected 562

(as indicated) BHK-21 cells. Photomicrographs show cell nuclei stained with DAPI in all fields. 563

(C) [35S]-methionine labeled crude cell lysates of infected BHK-21 cells analyzed by SDS-PAGE 564

showing VSV proteins and CHIKV envelope proteins. VSV proteins are indicated to the left, 565

and CHIKV envelope proteins and molecular mass of VSV proteins are indicated to the right. 566

(D) [35S]-methionine labeled and purified virions analyzed by SDS-PAGE. Proteins 567

incorporated into the virions are indicated. 568

569

Fig. 2. Growth of recombinant VSVs and plaque morphology. (A) BHK-21 cells were infected 570

(MOI=10) for 30 min with rwt-VSV, VSV-CHIKV or VSVΔG-CHIKV and unadsorbed virus 571

was removed by washing cells twice with PBS. Complete growth medium was added and 572

supernatants were collected at indicated times post-adsorption. Virus titers were determined by 573

plaque assay on BHK-21 cells. (B) Plaque morphology of VSV-CHIKV and VSVΔG-CHIKV at 574

2 days post infection of BHK-21 cells. 575

576

Fig. 3. UV inactivation prevents induction of CHIKV nAbs. Mice were immunized with non-577

complemented (NC, filled square) or G-complemented (GC, filled triangle) VSVΔG-CHIKV 578

without or with UV inactivation [NC-UV (open square) and GC-UV (open triangle)] and pooled 579


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serum from day 30-post immunization were analyzed for induction of VSV∆G-eGFP/CHIKV 580

pseudotype nAb. The representative curve shows percent neutralization at indicated serum 581

dilutions. All assays were performed in duplicate. 582

583

Fig. 4. VSV-CHIKV and VSVΔG-CHIKV induce VSVΔG-eGFP/CHIKV pseudotype nAbs in 584

mice. (A) Timeline for vaccination-challenge study for testing efficacy of rVSV/CHIKV 585

vectors. Mice were immunized (day 0) I.M. with 106 PFU of indicated virus and challenged at 586

about 5 weeks post-immunization (day 34) with 104 PFU CHIKV-LR strain by the S.C. route in 587

hind-leg footpad. rwt-VSV immunization was used as control. (B) Percent neutralization of 588

VSV/CHIKV pseudotype using pooled serum samples from day 30-post immunization with 589

rwtVSV (open circle), VSV-CHIKV (open square) or VSVΔG-CHIKV (open triangle). All 590

assays were performed in duplicate. 591

592

Fig. 5. Footpad inflammation and body weight loss profiles in immunized mice following 593

CHIKV challenge. (A) Foot swelling, measured as the vertical height of the hind feet at the ball, 594

was monitored in rwt-VSV (open circle), VSV-CHIKV (open square) or VSVΔG-CHIKV (open 595

triangle) immunized mice, for 10 days post S.C. challenge with 104 PFU CHIKV-LR isolate. 596

Error bars represent standard deviation. (B) Body weight of mice in control rwt-VSV (white 597

bar), VSV-CHIKV (black bar) and VSVΔG-CHIKV (shaded bar) immunized groups were 598

measured on the day of challenge and monitored daily for 9 days post challenge. Graph 599

represents average percent pre-challenge body weight in each group over time. Error bars 600

represent standard deviation. 601

602


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Fig. 6. Induction of cellular immune response in rVSV/CHIKV immunized mice. Splenocytes, 603

harvested from uninfected naïve mice (n=2) or mice immunized with 106 PFU of VSV-CHIKV 604

(n=5) or VSVΔG-CHIKV (n=5), at day 7 post I.M. immunization were stimulated with peptides 605

corresponding to CHIKV E1 (HSMTNAVTI; open bar) or E2 (IILYYYELY; filled bar) CD8+ 606

epitopes for 24 h followed by detection of IFN-γ spot forming cells (SFC) using an Elispot assay. 607

The numbers of SFC from unstimulated control samples have been subtracted. Error bars 608

represent standard deviation. * Indicates a significant difference (p<0.01, ANOVA) between 609

VSV-CHIKV and VSVΔG-CHIKV groups in response to E2 peptide. 610

611

612

613

614

615


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TABLE 1. Titers of CHIKV specific neutralizing antibodies at day 30 post immunization with

different VSV recombinants

a Groups of 3.5 weeks old C57Bl/6 mice were immunized I.M. with 106 PFU of indicated

viruses. Neutralizing antibody titers were determined in day 30 post-immunization serum

samples using PRNT80.

Mouse Number

PRNT80a

rwt-VSV VSV-CHIKV VSVΔG-CHIKV

1 <20 320 640

2 <20 320 >640

3 <20 160 >640

4 <20 320 320

5 <20 320 160

6 <20 80 320

7 <20 80 160

8 <20 320 320


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TABLE 2. Viremia in vaccinated and control mice after challenge with CHIKVa

Immunization Group

Virus titer (log10 PFU/ml ± S.D.) (Fraction positive) Day 1b Day 2c Day 3b Day 4c

rwt-VSV 2.55±0.56 (2/4) 2.85±0.47 (4/4) <0.9 (0/4) 2.0±0.0 (1/4)

VSV-CHIKV <0.9 (0/4) <0.9 (0/4) <0.9 (0/4) <0.9 (0/4)

VSVΔG-CHIKV <0.9 (0/4) <0.9 (0/4) <0.9 (0/4) <0.9 (0/4) a S.C. challenge in rear left footpad with 104 PFU of CHIKV La Reunion strain.

b Four mice in each group were bled on day 1 and 3 post challenge.

c Remaining four mice in each group were bled on day 2 and 4 post challenge.


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VSV-CHIKV(E3-E2-6K-E1)5’3’ LGMPN E1E3 E2 6K

VSVΔG-CHIKV(E3-E2-6K-E1)5’LE1E3 E2 6K3’ MPN

A

rwt-VSV VSV-CHIKV VSVΔG-CHIKV

α-V

SV

GB

α-C

HIK

Vα

C D

p62

240

63

L

G

L

Gp62E1

47

26

GNPM

NPM

E1/E2

Fig. 1. Characterization of recombinant VSVs encoding the CHIKV E3-E2-6K-E1 polyprotein. (A) Diagram of rVSV genomes with insertion of CHIKV envelope polyprotein in full-length VSV and VSVΔG vectors. The gene order is shown in 3’ to 5’ direction on the negative sense RNA genome. (B) Expression of CHIKV envelope proteins detected by indirect immunofluorescence microscopy using anti-VSV G MAb or anti-CHIKV mouse sera in infected (as indicated) BHK-21 cells. Photomicrographs show cell nuclei stained with DAPI in all fields. (C) [35S]-methionine labeled crude cell lysates of infected BHK-21 cells analyzed by SDS-PAGE h i VSV i d CHIKV l i VSV i i di d h l f dshowing VSV proteins and CHIKV envelope proteins. VSV proteins are indicated to the left, and

CHIKV envelope proteins and molecular mass of VSV proteins are indicated to the right. (D) [35S]-methionine labeled and purified virions analyzed by SDS-PAGE. Proteins incorporated into the virions are indicated.


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109A

1.00E+07

1.00E+08

1.00E+09pf

u/m

l)108

107

109A

1.00E+04

1.00E+05

1.00E+06

Viru

s tit

er (p

rwtVSV

VSV-CHIKV

VSVdelG-CHIKV

106

105

104

1.00E+03

0 2 4 6 8 10 12 14 16 18 20 22 24Hours post adsorption

103

B

VSV-CHIKV VSVΔG-CHIKV

Fig 2 Growth of recombinant VSVs and plaque morphology (A) BHK 21 cellsFig. 2. Growth of recombinant VSVs and plaque morphology. (A) BHK-21 cells were infected (MOI=10) for 30 min with rwt-VSV, VSV-CHIKV or VSVΔG-CHIKV and unadsorbed virus was removed by washing cells twice with PBS. Complete growth medium was added and supernatant samples were collected at indicated times post-adsorption. Virus titers were determined by plaque assay on BHK-21 cells. (B) Plaque morphology of VSV-CHIKV and VSVΔG-CHIKV at 2 days post infection of BHK-21 cells.ce s.


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80

100

120

zatio

n

NCNC-UVGCGC-UV

40

60

rcen

t neu

tral

iz

0

20

20 40 80 160 320 640 1280 2560 5120

Pe

Fig. 3. UV inactivation prevents induction of CHIKV nAbs. Mice were immunized with non-complemented (NC, filled square) or G-complemented (GC, filled triangle) VSVΔG-CHIKV without or with UV inactivation [NC-UV (open square) and GC-UV (open triangle)] and pooled serum from day 30-post immunization were analyzed for induction of VSV∆G-

Reciprocal serum dilution

eGFP/CHIKV pseudotype neutralizing antibodies. The representative curve shows percent neutralization at indicated serum dilutions. All assays were performed in duplicate.


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Serum sampling

Viremiaand

footpad swelling

A

Immunization: rwt-VSV

Day 0 30 34 38 44

Challengewith CHIKV

End of challenge

p g

rwt VSV VSV-CHIKV VSVΔG-CHIKV

Histopathology sample collection

B

100

120

n

rwtVSVVSV-CHIKV

60

80

neut

raliz

atio

n

VSVdelG-CHIKV

0

20

40

Perc

ent

Fig. 4. VSV-CHIKV and VSVΔG-CHIKV induce VSVΔG-eGFP/CHIKV pseudotype nAbsin mice. (A) Timeline for vaccination-challenge study for testing efficacy of rVSV/CHIKV

Reciprocal serum dilution

vectors. Mice were immunized (day 0) I.M. with 106 PFU of indicated virus and challenged at about 5 weeks post-immunization (day 34) with 104 PFU CHIKV-LR strain by the S.C. route in hind-leg footpad. rwt-VSV immunization was used as control. (B) Percent neutralization of VSV/CHIKV pseudotype using pooled serum samples from day 30-post immunization with rwtVSV (open circle), VSV-CHIKV (open square) or VSVΔG-CHIKV (open triangle). All assays were performed in duplicate.


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2.62.83

m)

rwt-VSVVSV-CHIKVVSVdelG-CHIKV

A

1.82

2.22.4

ht o

f foo

t (m

m

1.21.41.6

0 1 2 3 4 5 6 7 8 9 10

Hei

gh

Days post challengeDays post challenge

115

120

y w

eigh

t

rwt-VSVVSV-CHIKVVSVdelG CHIKV

B

105

110

chal

leng

e bo

dy VSVdelG-CHIKV

95

100

0 1 2 3 4 5 6 7 8 9

% p

re-c

Day post challengeFig. 5. Footpad inflammation and body weight loss profiles in immunized mice following CHIKV challenge. (A) Foot swelling, measured as the vertical height of the hind feet at the ball, was monitored in rwt-VSV (open circle), VSV-CHIKV (open square) or VSVΔG-CHIKV (open triangle) immunized mice, for 10 days post S.C. challenge with 104 PFU CHIKV-LR isolate. Error bars represent standard deviation. (B) Body weight of mice in control rwt VSV (white bar) VSV CHIKV (black bar)

y p g

(B) Body weight of mice in control rwt-VSV (white bar), VSV-CHIKV (black bar) and VSVΔG-CHIKV (shaded bar) immunized groups were measured on the day of challenge and monitored daily for 9 days post challenge. Graph represents average percent pre-challenge body weight on each day. Error bars represent standard deviation.


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800

900

1000

E1

*

500

600

700

FC/106cells E2

100

200

300

400S

0

100

Naïve VSV-CHIKV VSVdelG-CHIKVNaive VSV-CHIKV VSVΔG-CHIKV

Fig. 6. Induction of cellular immune response in rVSV/CHIKV immunized mice. Splenocytes, harvested from uninfected naïve mice (n=2) or mice immunized with 106 PFU of VSV-CHIKV (n=5) or VSVΔG-CHIKV (n=5), at day 7 post I.M. immunization were stimulated with peptides corresponding to CHIKV E1 (HSMTNAVTI; open bar) or E2 (IILYYYELY; filled bar) CD8+ epitopes for 24 h followed by detection of IFN-γ spot forming cells (SFC) using an Elispot assay. The numbers of SFC from unstimulated control samples have been subtracted. Error b t t d d d i ti * I di t i ifi t diff ( <0 01bars represent standard deviation. * Indicates a significant difference (p<0.01, ANOVA) between VSV-CHIKV and VSVΔG-CHIKV groups in response to E2 peptide.


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