JVI Accepted Manuscript Posted Online 8 July 2015 J. Virol. doi:10.1128/JVI.01373-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial

2

Virus Pre-fusion F Protein Expressed by a Vaccine Candidate

3 4 5

Bo Lianga, Sonja Surmana, Emerito Amaro-Carambota, Barbora Kabatovaa, Natalie

6

Mackowa, Matthias Lingemanna, Lijuan Yanga, Jason S. McLellanb*, Barney S.

7

Grahamb, Peter D. Kwongb, Anne Schaap-Nutta, Peter L. Collinsa, Shirin Munira#

8 9

RNA Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy

10

and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA a ;

11

Vaccine Research Center, National Institute of Allergy and Infectious Diseases,

12

National Institutes of Health, Bethesda, Maryland, USA b

13 14

Running title: Improved bivalent HPIV3/RSV live vaccine

15 16 17

#Address correspondence to Shirin Munir, [email protected]

18 19

*Present address: Department of Biochemistry, Geisel School of Medicine at

20

Dartmouth, 7200 Vail, Hanover, New Hampshire, United States of America

21 22

1

23

Abstract

24

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are

25

the first and second leading viral agents of severe respiratory tract disease in infants

26

and young children worldwide. Vaccines are not available, and an RSV vaccine is

27

particularly needed. A live attenuated chimeric bovine/human PIV3 (rB/HPIV3)

28

vector expressing the RSV fusion (F) glycoprotein from an added gene has been

29

under development as a bivalent vaccine against RSV and HPIV3. Previous clinical

30

evaluation of this vaccine candidate suggested that increased genetic stability and

31

immunogenicity of the RSV F insert were needed. This was investigated in the

32

present study. RSV F expression was enhanced 5-fold by codon-optimization and by

33

modifying the amino acid sequence to be identical to that of an early passage of the

34

original clinical isolate. This conferred a hypo-fusogenic phenotype that presumably

35

reflects the original isolate. We then compared vectors expressing stabilized pre-

36

and post-fusion versions of RSV F. In a hamster model, pre-fusion F induced

37

increased quantity and quality of RSV-neutralizing serum antibodies and increased

38

protection against wt RSV challenge. In contrast, vector expressing the post-fusion F

39

was less immunogenic and protective. The genetic stability of the RSV F insert was

40

high and was not affected by enhanced expression or the pre- or post-fusion

41

conformation of RSV F. These studies provide an improved version of the

42

previously-well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a

43

superior RSV-neutralizing serum antibody response.

44 45

2

46

Importance

47

Respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are two

48

major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector

49

expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV.

50

Previous clinical evaluation indicated the need to increase the immunogenicity and

51

genetic stability of the RSV F insert. Here, we increased RSV F expression by codon

52

optimization and by modifying the RSV F amino acid sequence to conform to that of

53

an early passage of the original isolate. This resulted in a hypo-fusogenic phenotype,

54

which likely represents the original phenotype before adaptation to cell culture. We

55

also included stabilized versions of pre- and post-fusion RSV F protein. Pre-fusion

56

RSV F induced a higher quantity and quality of RSV-neutralizing serum antibodies

57

and was highly protective. This provides an improved candidate for further clinical

58

evaluation.

59 60 61

3

62

Introduction

63

Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3

64

(HPIV3) are enveloped non-segmented negative stranded RNA viruses of the family

65

Paramyxoviridae. They are, respectively, the first and second leading viral causes of

66

severe acute lower respiratory tract infections in infants and children worldwide.

67

RSV alone is responsible for an estimated 34 million annual pediatric cases of lower

68

respiratory tract illness worldwide with >3.5 million hospitalizations and

69

66,000~199,000 pediatric deaths, which occur predominantly in the developing

70

world (1). Licensed vaccines or effective antiviral drugs are not available for either

71

RSV or HPIV3. Experimental inactivated (RSV, HPIV3) and subunit (RSV) vaccines

72

have been linked to vaccine-induced enhanced disease in young children

73

(inactivated RSV) and experimental animals (subunit RSV, inactivated HPIV3) (2-4).

74

In contrast, disease enhancement is not observed with live attenuated RSV strains

75

or vectors expressing RSV antigens (5-7), indicating that suitably attenuated

76

candidates are safe for immunization of infants and young children.

77 78

A chimeric bovine/human PIV3 (rB/HPIV3) virus (Fig. 1) has been under

79

development as a replication-competent intranasal pediatric vaccine vector. The

80

PIV3 genome is a negative-sense RNA of 15.5 kb that contains six genes in the order

81

3´-N (nucleoprotein)- P (phosphoprotein)- M (matrix protein) – F (fusion

82

glycoprotein)- HN (hemagglutinin-neuraminidase glycoprotein) – L (polymerase

83

protein)-5´ (Fig. 1). The rB/HPIV3 vector consists of the BPIV3 Kansas strain

84

backbone in which the F and HN genes have been replaced by those of the HPIV3 JS

4

85

strain (8). This combines the attenuation phenotype of BPIV3 in primates with the

86

HPIV3 F and HN viral neutralization antigens. Inclusion of sequence encoding the

87

RSV F protein, which is the major RSV neutralization and protective antigen, as a

88

supernumerary gene (Fig. 1) provides a bivalent HPIV3/RSV vaccine. In a previous

89

clinical study in children seronegative for both viruses, rB/HPIV3 expressing RSV F

90

from the second (N-P) gene position (MEDI-534) was found to be infectious,

91

attenuated and well tolerated. However, only 50% of the recipients developed

92

detectable RSV neutralizing serum antibodies (7), although the sensitivity of the

93

assay would have been reduced because it was done without added complement.

94

Analysis of the shed vaccine virus from vaccinees showed that ~50% of the

95

specimens contained vaccine virus with mutations predicted to perturb RSV F

96

expression, and 2.5% of the clinical trial material did not express RSV F (9). Our

97

goal was to enhance the immunogenicity of RSV F in the rB/HPIV3 vector and

98

evaluate its genetic stability during replication in vitro and in vivo.

99 100

Like F proteins of all paramyxoviruses, the RSV F protein is a type I fusion protein

101

that mediates the fusion of viral envelope and cellular membrane during entry. It

102

initially assembles into trimers in a metastable (unstable) pre-fusion conformation

103

on the surface of infected cells and virions. Pre-fusion F can be triggered, such as by

104

contact with an adjacent target cell membrane, to undergo a massive

105

conformational change that mediates membrane fusion, leaving the F protein in a

106

post-fusion conformation(10-14). Triggering of RSV F does not require the

107

engagement of attachment protein (G), unlike the triggering of parainfluenza virus F

5

108

proteins that is mediated by the binding of attachment protein to a cellular receptor.

109

Pre-fusion RSV F protein is thought to be susceptible to premature triggering. There

110

also is evidence that much of the RSV F protein expressed during infection is

111

conformationally heterogeneous, which may act as a decoy to reduce the induction

112

of virus-neutralizing antibodies (15). These problems might be obviated by

113

expression of the F protein in a stabilized conformation.

114 115

The pre-fusion form of RSV F appears to be a more effective neutralization antigen

116

than the post-fusion form when evaluated as a subunit vaccine in experimental

117

animals (16, 17), although the post-fusion form does induce a substantial

118

neutralizing antibody response and has the advantage of being highly stable (13).

119

The pre-fusion form of RSV F also can be substantially stabilized through structure-

120

based mutations, one of which involves the introduction of a disulfide bond between

121

S155 and S290 (called DS)(17). In the present study, we codon-optimized the RSV F

122

ORF, made its amino acid sequence identical to that of an early passage of the

123

original clinical isolate (18), and introduced the stabilized pre- and post-fusion F

124

protein. The resulting series of rB/HPIV3-RSV F constructs were evaluated for

125

replication, immunogenicity, and protective efficacy in a hamster model and for

126

stability of the RSV F gene in vitro and in vivo. Since the prototype rB/HPIV3-RSV F

127

construct has already been shown to be well-tolerated in seronegative children, as

128

noted, an improved version could advance expeditiously to clinical evaluation.

129 130

Materials and Methods

6

131

Cells and viruses. Rhesus monkey LLC-MK2 (ATCC CCL-7) cells and African green

132

monkey Vero cells (ATCC CCL-81) were maintained in Opti-MEMI (1X)+GlutaMax-1

133

medium (Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine

134

serum (FBS) (HyClone, Thermo Scientific, Atlanta, GA). BSR T7/5 hamster kidney

135

cells that constitutively express T7 RNA polymerase were maintained as described

136

(19). Recombinant (r) wild-type (wt) RSV and rB/HPIV3 were previously described

137

(8, 20). All rB/HPIV3 vectors were propagated at 32°C in LLC-MK2 cells or Vero

138

cells (21). RSV and its gene sequences were derived from strain A2 (GenBank

139

accession: M74568); HPIV3 and BPIV3 sequences were from strains JS (Z11575)

140

and Kansas/15626/84 (AF178654), respectively.

141 142

Construction of antigenomic cDNA encoding rB/HPIV3 vectors expressing RSV

143

F

144 145

The full-length cDNA of rB/HPIV3 was previously modified to contain a unique AscI

146

restriction site at the 2nd genome position between the N and P genes (22) (Fig 1).

147

The DNA sequence encoding the RSV F ORF with HEK assignments was codon-

148

optimized (HEK/opt) (Fig 1) and synthetically derived (GeneArt, Life Technologies).

149

The HEK/opt and Ecto insert sequences were amplified by PCR, with Advantage HF

150

2 PCR Kit (Clontech, Mountain View, CA) using synthetic HEK/opt RSV F as

151

template. The shared forward primer for both HEK/opt and Ecto was

152

ATCATGGCGCGCCAAGTAAGAAAAACTTAGGATTAATGGACCTGCAGGATGGAACTGCT

153

GATCCTGAAGGC, the reverse primer for HEK/opt was

7

154

GAGATGGCGCGCCGCTAGCTATCAGTTGGAGAAGGCGATATTGTTG, and the reverse

155

primer for Ecto was

156

GAGATGGCGCGCCGCTAGCTATCATCACAGCAGCTCGTCGCTCTTTCTGATG

157

(Restriction site Asc I underlined; ORF translation initiation and termination codons

158

in boldface). PCR products of HEK/opt and Ecto inserts were cloned into the pCR4-

159

TOPO vector with TOPO TA Cloning kit (Life Technologies) and sequences were

160

confirmed by automated sequencing. The pCR4-TOPO vectors with Non-HEK/opt

161

and DS inserts were generated with QuickChange Lightning Multi Site-Directed

162

Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using pCR4-TOPO vector

163

DNA containing HEK/opt insert as template. The pCR4-TOPO vector with post-

164

fusion insert was generated by deleting the first 10 amino acids of FP in RSV F

165

(aa137-146) with QuickChange II Site-Directed Mutagenesis Kit (Agilent

166

Technologies) using pCR4-TOPO vector containing Ecto insert as template. To

167

mutate two residues in RSV F to Non-HEK assignments (P101Q and E66K), two

168

mutagenesis reactions were performed. The following primers were used for

169

mutagenesis: for the P101Q mutation the primers

170

TGATGCAGTCCACCCAAGCCACCAACAACCGG and

171

CCGGTTGTTGGTGGCTTGGGTGGACTGCATCA were used; for the E66K mutation, the

172

primers TCGAGCTGTCCAACATCAAGAAAAACAAGTGCAACGGCAC and

173

GTGCCGTTGCACTTGTTTTTCTTGATGTTGGACAGCTCGA were used. To stabilize RSV

174

F in pre-fusion conformation, two DS mutations (S155C and S290C) were

175

introduced in RSV F by mutagenesis. For the S155C mutation, the primers

176

GGCGTGGCCGTGTGCAAGGTGCTGCACC and GGTGCAGCACCTTGCACACGGCCACGCC

8

177

were used; for the S290C mutation, the primers

178

CAGAGCTACTCCATCATGTGCATCATCAAAGAAGAGG and

179

CCTCTTCTTTGATGATGCACATGATGGAGTAGCTCTG were used; for the 10aa FP

180

deletion in post-fusion form of RSV F, the primers

181

GCGGAAGCGGCGGGCCATTGCCTCTG and CAGAGGCAATGGCCCGCCGCTTCCGC were

182

used.

183 184

All RSV F sequences in pCR4-TOPO vectors were confirmed by automated

185

sequencing analysis. The RSV F inserts were digested and ligated into full-length

186

cDNA of rB/HPIV3 at the 2nd genome position using AscI restriction site (Fig. 1) , and

187

the sequences were confirmed by automated sequencing. Full-length cDNA of

188

rB/HPIV3 with Non-HEK/non-opt RSV F in the 2nd genome position was generated

189

previously (23). The genome nucleotide length for each construct was a multiple of

190

six (24). The phasing of each gene was maintained as the native phasing (25). The

191

phasing of the inserted RSV F gene at the second gene position was identical to the

192

native phasing of the P gene, which normally is in the second gene position in PIV3

193

(25).

194 195

Recovery of rB/HPIV3-RSV F viruses from cDNA. The rB/HPIV3-RSV F viruses

196

were recovered by reverse genetics (8) in BSR-T7/5 cells constitutively expressing

197

T7 RNA polymerase (26). Recovered virus was amplified by two passages in LLC-

198

MK2 cells at 32°C. Viral RNA was isolated from the virus stocks using the Qiagen Viral

199

Amp kit (Qiagen, Valencia, CA) followed by DNase I treatment to remove plasmid

9

200

DNA used for rescue. Virus sequences were confirmed in their entirety (except for

201

the last 30 and 120 nucleotides at the 3’Iand 5’nterminal ends, respectively) by

202

sequence analysis of un-cloned reverse transcription (RT)-PCR products amplified

203

from viral RNA. Control RT-PCR reactions lacking reverse transcriptase indicated

204

that RT-PCR products were not derived from the input antigenomic cDNAs (data not

205

shown). The percentage of vector virions expressing RSV F in each working stock of

206

inoculum was determined by fluorescent double staining plaque assay (described

207

below) to ensure that each stock had 99-100% of virions expressing RSV F and that

208

plaques were homogeneous in size and shape.

209 210

Fluorescent double-staining plaque assay. Vero cell monolayers in 24-well plates

211

were infected with 10-fold serially diluted samples. Infected monolayers were

212

overlaid with culture medium containing 0.8% methylcellulose, incubated at 32°C

213

for 6 days, and fixed by two overnight incubations in 80% methanol at 4lC. The

214

monolayers were washed in 1xPBS, followed by incubation in Odyssey Blocking

215

Buffer (LiCor) for 1h at room temperature (R.T.). Monolayers were then incubated

216

with Odyssey Blocking Buffer containing RSV F-specific monoclonal antibodies

217

diluted at 1:2000 (19) and HPIV3 hyperimmune serum diluted 1:1000 for 1h at R.T.

218

The HPIV3 hyperimmune serum was generated by immunizing rabbits with sucrose

219

purified HPIV3 virions as described (23). After washing three times in 1xPBS,

220

monolayers were then incubated for 1h at R.T. with Odyssey Blocking Buffer

221

containing IRDye800CW conjugated goat anti-rabbit antibody and IRDye680LT

222

conjugated goat anti-mouse antibody each diluted at 1:800. The plaques were

10

223

scanned and visualized with the Odyssey Infrared Imaging System (LiCor) under

224

800nm and 680nm channels. Fluorescent plaques showing staining for HPIV3

225

proteins and RSV F were detected as green (800 nm) and red (680 nm),

226

respectively, and were yellow when merged (800 and 680 nm).

227 228

Analysis of RSV F expression by Western blot. Vero cells (2 x 105) were infected

229

with rB/HPIV3-RSV F viruses at MOI of 10 TCID50 or wt RSV at 10 PFU, and

230

incubated at 32CC or 37oC as indicated. At 48 h post-infection (p.i.), medium

231

supernatants were removed and monolayers were washed twice with ice-cold PBS

232

and lysed with 200 μe of ice-cold RIPA buffer containing 1x Complete Cocktail

233

Protease Inhibitor (Roche, Indianapolis, IN). For Western blot analysis under

234

reducing and denaturing conditions, supernatants or lysates were mixed with 1x

235

LDS buffer (Life Technologies), 1x reducing reagent (Life Technologies) and boiled

236

at 95°C for 5 min. 30 μ of reduced, denatured lysate was loaded onto 4-12%

237

NuPAGE gels (Novex-Life Technologies) with NuPage antioxidant reagent (Novex-

238

Life Technologies) added in the running buffer in the cathode chamber. For Western

239

blot analysis under non-reducing conditions, lysates were mixed with 1x LDS buffer

240

without boiling. 30 μ0 of lysate with 1x LDS buffer was loaded onto 4-12% NuPAGE

241

gels (Novex-Life Technologies) without adding NuPage antioxidant reagent (Novex-

242

Life Technologies) in the running buffer. Membranes were probed with murine

243

monoclonal anti-RSV F antibody (Ab43812, Abcam, Cambridge, MA), rabbit

244

polyclonal antibodies generated by immunizing rabbits with sucrose-purified wt

245

RSV, goat polyclonal anti-GAPDH antibody (G8795, Sigma, St Louis, MO), and anti-

11

246

HPIV3 HN antibodies generated as described (23). The following fluorescent dye-

247

conjugated secondary antibodies were used: donkey anti-rabbit IRDye680, and

248

donkey anti-mouse IRDye 800CW (LiCor, Lincoln, NE). Membranes were scanned

249

and the fluorescence intensities of protein bands were quantified using the Odyssey

250

Infrared Imaging System (Image Studio, LiCor).

251 252

Analysis of cell surface RSV F expression by flow cytometry. Vero cells were

253

infected with rB/HPIV3 vectors at an MOI of 5 TCID50 or wt RSV at an MOI of 5 PFU.

254

After incubation at 32fC for 48h, infected cells were harvested by incubation in PBS

255

containing 1mM EDTA at 37°C for 5 min, then washed twice in ice-cold FACS buffer

256

(1x PBS, 2% FBS), and incubated with an optimized dilution of Alexa Fluor 488

257

(AF488)-conjugated RSV F MAb 1129 (27) or unconjugated D25 human monoclonal

258

antibody. The AF488-conjugated RSV F MAb was prepared using the Alexa Fluor

259

488 Antibody Labeling Kit (Life Technologies) following the kit instructions. Cells

260

stained with D25 were washed twice with ice-cold FACS buffer and stained with

261

Alexa Fluor 488 conjugated Goat F(abab2 Anti-human IgG(γ) (Life Technologies).

262

Stained cells were washed twice in ice-cold FACS buffer and incubated in Near-IR

263

LIVE/DEAD dye (Life Technologies) to discriminate dead cells. Stained cells were

264

analyzed using a BD FACSCanto II flow cytometer.

265 266

Hamster studies. All animal studies were approved by the NIH Institutional

267

Animal Care and Use Committee (IACUC). Six-week old Golden Syrian hamsters

268

(Harlan Laboratories, Frederick, MD), which were confirmed to be seronegative for

12

269

HPIV3 and RSV by hemagglutination-inhibition (28) assay and RSV-specific virus

270

neutralization assay, respectively (29, 30), were anesthetized and inoculated

271

intranasally (IN) with 0.1 ml containing 105 TCID50 of rB/HPIV3 vectors or 106

272

plaque forming units (PFU) of wt RSV (A2 strain). Six hamsters per group were

273

inoculated with each virus. To evaluate vector replication, nasal turbinate and lung

274

tissues were collected separately on day 3 and 5 p.i. for virus quantification by serial

275

dilution on LLC-MK2 cells (rB/HPIV3 vectors) or plaque assay on Vero cells (wt

276

RSV) (19, 31). For determining vector immunogenicity, sera were collected from

277

hamsters 3 days prior to and 28 days after inoculation. Titers of RSV- and HPIV3-

278

specific NAbs were determined by 60% plaque reduction neutralization assays

279

(PRNT60) on Vero cells and LLC-MK2 cells respectively, and performed in the

280

presence or absence of guinea pig complement (Lonza) (32). (Note that all sera

281

were heated at 56°C for 30 min to destroy endogenous complement prior to assay

282

and the addition of guinea pig complement.) Protective efficacy was determined by

283

challenge infection performed by intranasal infection with 106 PFU of wt RSV in 0.1

284

ml at 30 days after immunization. Viral load of challenge RSV in the nasal turbinates

285

and lungs was determined 3 days after challenge by plaque titration of tissue

286

homogenates on Vero cells as described (33).

287 288

Results

289

Codon optimization and an early-passage form of RSV F protein.

290

The RSV F sequence (strain A2) was codon-optimized (GeneArt, Life Technologies)

291

for human expression and, in addition, was modified by two amino acid

13

292

substitutions (K66E and Q101P) so as to be identical in amino acid sequence to an

293

early passage of strain A2 that originally had been propagated in human embryonic

294

kidney cells (HEK F protein)(18). Three versions of the RSV F gene - (i) non-

295

optimized with non-HEK assignments (Non-HEK/non-opt), a construct similar to

296

MEDI-534, (ii) codon-optimized with non-HEK assignments (Non-HEK/opt), and

297

(iii) codon-optimized with HEK assignments (HEK/opt) – were inserted at the 2nd

298

position between the N and P genes of the rB/HPIV3 vector (Fig. 1). Infectious virus

299

was recovered by reverse genetics as described in Material and Methods. Multi-

300

cycle replication growth curves in Vero (Fig. 2A) and LLC-MK2 cells (not shown)

301

showed that all three constructs replicated with similar kinetics and to high peak

302

titers (~108 TCID50/mL), although they were all slightly more attenuated than the

303

empty vector (Fig. 2A). Virus stocks of these constructs contained only trace

304

amounts of RSV F, indicating that it was not efficiently packaged into the vector

305

particles (not shown).

306 307

Codon-optimization and the HEK substitutions conferred increases in RSV F protein

308

expression of 2.1- and 2.4-fold, respectively, with an aggregate 5-fold increase (Fig.

309

2B, C). Also, following the HEK substitutions the F protein trimer migrated

310

somewhat more slowly than the non-HEK form under non-reducing polyacrylamide

311

gel electrophoresis (Fig. 2D). The non-HEK form of RSV F induced abundant syncytia

312

on Vero monolayers (Fig. 2F); and increasing its expression by codon-optimization

313

resulted in more extensive syncytia (Fig. 2G). Interestingly, the HEK form of RSV F

14

314

conferred a hypo-fusogenic phenotype (Fig. 2H), despite having the highest level of

315

expression (Figs. 2B and C, lane 4).

316 317

Expression of stabilized pre-and post-fusion forms of RSV F.

318

The full-length RSV F protein, (i.e., including the C-terminal transmembrane [TM]

319

and cytoplasmic [CT] domains that anchor it in the membrane) was stabilized in the

320

pre-fusion form (Fig. 1, “DS”) by introducing two mutations (S155C, S290C) that

321

generate a new disulfide bond, as previously described (17). Stabilized post-fusion

322

RSV F protein was generated as previously described by expressing the ectodomain

323

(aa 1-513, lacking the C-terminal TM and CT domains) with further deletion of the

324

first 10 amino acids of the fusion peptide (FP; aa 137-146) (Fig. 1., “Post-

325

fusion”)(12, 13). Another construct consisting of the entire RSV F ectodomain (aa 1-

326

513) was included for comparison (Fig. 1., “Ecto”). All three forms of RSV F were

327

codon-optimized and had HEK assignments.

328 329

rB/HPIV3 expressing the pre-fusion DS, post-fusion, and Ecto forms of RSV F protein

330

replicated in Vero cells (Fig. 3A) and LLC-MK2 cells (not shown) at rates and to final

331

titers similar to those of vector expressing native RSV F protein (HEK/opt). None of

332

these constructs induced visible syncytium formation in vitro (not shown),

333

consistent with the idea that F protein that is not anchored in the membrane, or that

334

is stabilized in the pre-fusion form, should not be efficient in directing fusion. The

335

pre-fusion DS form was expressed at a similar level as HEK/opt (Fig. 3B). Cleavage

336

of the pre-fusion DS F0 precursor was somewhat less efficient than that of HEK/opt.

15

337

As expected, the post-fusion and Ecto constructs were expressed primarily as

338

secreted forms (Fig. 3C), and only the cleaved protein was detected in the medium

339

supernatant. None of the forms was efficiently packaged into vector particles (not

340

shown).

341 342

Analysis of cell surface expression by flow cytometry using monoclonal antibody

343

1129 (the murine precursor of palivizumab, which recognizes both pre- and post-

344

fusion F) demonstrated very efficient surface expression of the HEK/opt and pre-

345

fusion DS forms of RSV F (Fig. 3D). Consistent with its lower total expression versus

346

HEK/opt (Fig. 2B, C), the non-HEK/non-opt form had relatively lower surface

347

expression (Fig. 3D). The Ecto form was detected at the cell surface at a reduced but

348

still substantial level, whereas the post-fusion form was not detected above

349

background (Fig. 3D). The difference in cell surface accumulation between these

350

two secreted forms might reflect the presence of the hydrophobic FP domain in

351

Ecto, which might mediate association with the plasma membrane. Except for the

352

post-fusion and Ecto forms, the cell-surface expression of RSV F protein by the

353

rB/HPIV3 vectors was more efficient than by wt RSV (Fig. 3D).

354 355

The relative quantity of RSV F protein in the pre-fusion conformation on the cell

356

surface was measured with human monoclonal antibody D25, which recognizes

357

antigenic site Ø that is unique to the pre-fusion conformation and some

358

intermediate forms (11). The greatest cell-surface expression of F protein in the pre-

359

fusion conformation was observed with the DS construct; a lesser amount was

16

360

observed with HEK/opt (Fig. 3E), even though the latter was more efficiently

361

expressed on the surface as detected by antibody 1129 (Fig. 3D). This was

362

illustrated by calculating the ratio of the MFI values for D25 to 1129 (Fig. 3E). This

363

suggested that HEK/opt, i.e. a native form of RSV F protein, was only partially

364

displayed in pre-fusion conformation on the cell surface. The presence of the HEK

365

assignments (i.e., HEK/opt versus Non-HEK/non-opt) resulted in a marginal

366

increase in the D25:1129 ratio. There was essentially no secretory post-fusion form

367

detected at the cell surface with either antibody 1129 (Fig. 3D) or D25 (Fig. 3E).

368

There also was essentially no Ecto form detected with antibody D25 (Fig. 3E),

369

suggesting that this form was highly susceptible to triggering. F expressed by wt

370

RSV (strain A2) also was detected in the pre-fusion conformation (Fig. 3E), albeit at

371

a lower amount than DS and HEK/opt, likely because the total expression of F by wt

372

RSV was lower (Fig. 3D).

373 374

Replication of rB/HPIV3 vectors in the respiratory tract of hamsters.

375

Hamsters were infected intranasally (IN) with 105 TCID50 of each vector or with 106

376

plaque forming units (PFU) of wt RSV (A2). Replication in the upper and lower

377

respiratory tracts was determined by titrating the viral load in nasal turbinates and

378

lungs on days 3 and 5 (only day 3 for RSV), and samples of the input inocula also

379

were analyzed to confirm input titer. Compared with the empty vector, vectors

380

bearing an RSV F insert replicated slower (i.e., lower titers on day 3 versus day 5)

381

and were moderately more attenuated in the nasal turbinates (Fig. 4A) and

382

substantially more attenuated in the lungs (Fig. 4B) on both days 3 and 5. The

17

383

difference between the empty vector and vector bearing an RSV F insert was the

384

greatest with the DS construct on day 3, where the reduction was more than 3.0

385

log10 in the nasal turbinates and 4.0 log10 in the lungs. There was a single time point

386

at which a vector bearing RSV F was not more attenuated than the empty vector,

387

and that was for the construct expressing post-fusion F on day 5 in the nasal

388

turbinates. Among the various vectors, there was some variability in titer, but

389

differences were significant only in the case of vector expressing post-fusion F,

390

which replicated to higher titer in both the nasal turbinates and lungs. It may be that

391

this insert was more easily tolerated by the vector because it was secreted and had

392

minimal association with the infected cells, as was shown in Fig. 3. Wt RSV

393

replicated to 100 to 1000-fold higher titers than the various vectors expressing RSV

394

F protein, particularly in the lungs, which would be relevant to its relative

395

immunogenicity (see below).

396 397

Pre-fusion RSV F induced highly potent RSV neutralizing serum antibodies.

398

Hamsters were infected with the various vectors or with wt RSV, and sera were

399

collected 28 days later and analyzed for RSV-neutralizing antibodies by a 60%

400

plaque reduction neutralization test (PRNT60) performed in the (i) presence (Fig.

401

5A) and (ii) absence (Fig. 5B) of added complement. The presence of added

402

complement provides for more sensitive detection of virus-specific antibodies, since

403

complement potentially confers viral-lysis and steric-hindrance capabilities (34) to

404

both neutralizing and non-neutralizing antibodies, whereas the complement-

405

independent assay would detect only antibodies that directly neutralize the virus.

18

406 407

For the complement-containing assay (Fig. 5A), the most interesting observations

408

were that vector expressing post-fusion F was relatively less immunogenic than the

409

other vectors even though it replicated to the highest titers in hamsters (Fig. 4),

410

while the vector expressing the pre-fusion DS form of F induced the highest titer of

411

RSV neutralizing antibodies among the vectors (Fig. 5A). In the complement-

412

independent assay (Fig. 5B), only two viruses induced high, similar titers of potent

413

RSV-neutralizing serum antibodies: namely vector expressing pre-fusion DS RSV F,

414

and wt RSV. The observation that the RSV-neutralizing antibodies induced by these

415

two viruses were similar in magnitude is noteworthy because the vector replicated

416

100 to 1000 times less efficiently than wt RSV (Fig. 4), and in addition the

417

neutralizing activity conferred by wt RSV would have the additional contribution

418

from the RSV G protein. In this regard, it is clear that RSV G is an important

419

independent neutralization and protective antigen in this model, because a previous

420

study comparing rB/HPIV3 expressing RSV F versus RSV G showed that they were

421

statistically indistinguishable with regard to the induction of RSV-neutralizing

422

serum antibodies and protection against RSV challenge(35). Thus, the vector

423

expressing the pre-fusion DS form of RSV F induced an RSV-specific antibody

424

response that was both quantitatively and qualitatively greater than that of the

425

other vectors, and was very similar to that of wt RSV.

426

The induction of HPIV3-neutralizing serum antibodies was evaluated by a

427

PRNT60 in the presence of complement. This showed that all of the vectors induced

428

high antibody titers. The titer was greatest for the empty vector, and generally was

19

429

somewhat lower for vectors bearing the RSV F gene (Fig. 5C), likely reflecting the

430

reduced replication of the constructs (Fig. 4).

431 432

Protection against RSV challenge.

433

The immunized hamsters from the experiment in Fig. 5 were challenged 30 days

434

post-immunization with 106 PFU of wt RSV administered IN. Viral loads in the nasal

435

turbinates and lungs on day 3 after challenge were determined by plaque titration of

436

tissue homogenates. In the nasal turbinates (Fig. 6A), the construct expressing the

437

pre-fusion DS form of RSV F was the most protective of the vectors, although the

438

difference was not significant. In the lungs (Fig. 6B), each of the vectors expressing

439

RSV F completely restricted RSV replication in 5/6 of the animals in each group with

440

the exception of the post-fusion construct, which was less restrictive. Wt RSV

441

conferred nearly complete resistance in the nasal turbinates and complete

442

resistance in the lungs, but this high level of protection was aided by three factors:

443

(i) wt RSV replicated up to 1000-fold more efficiently than the attenuated

444

rB/HPIV3-RSV-F vectors, (ii) wt RSV expressed both RSV neutralization antigens, G

445

and F, and (iii) wt RSV expressed all of the other RSV proteins as potential antigens

446

for cellular immunity, which is known to be effective in protection in short term RSV

447

challenge studies (36).

448 449

Stability of RSV F protein expression during replication in vivo.

450

The stability of expression of RSV F protein during replication in vitro and in vivo

451

was evaluated by examining the virus stocks used for immunization as well as the

20

452

homogenates of hamster nasal turbinates and lungs from days 3 and 5, from the

453

study shown in Fig. 4. This was done using a fluorescence double-staining plaque

454

assay to simultaneously detect RSV F and HPIV3 proteins. The results are

455

summarized in Table 1, and fluorographs of plaque staining for two representative

456

in vivo specimens are shown in Fig. 7 and 8. In general, the expression of unmodified

457

RSV F protein (Non-HEK/non-opt) was substantially stable; and the stability of RSV

458

F expression was not affected by modifications (DS or post-fusion or Ecto) affecting

459

its structure or level of expression. For the viral stocks, all of the virus plaques

460

present in wells in which individual plaques could be enumerated were positive for

461

expression of RSV F and were scored as 100% (Table 1). The same was true for the

462

majority of tissue homogenate samples (108 out of 120)(Table 1). Note that in some

463

of the lower dilutions that contained too many plaques to clearly distinguish and

464

enumerate, occasional green plaques could be distinguished, suggesting a very low

465

background of virus that did not express RSV F (e.g., Fig. 7). In the other specimens

466

(11 out of 120), there was a greater loss of RSV F expression, usually