Infectious Diseases, National Institute of Allergy ... - Journal of Virology

8 downloads 489 Views 712KB Size Report
Nov 19, 2014 - A live attenuated equine H3N8 influenza vaccine is highly ...... staff at MedImmune's Animal Care Facility for technical support for animal studies ...
JVI Accepts, published online ahead of print on 19 November 2014 J. Virol. doi:10.1128/JVI.02449-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1 2 3 4 5 6 7 8 9 10 11

JVI02449-14

12 13 14 15 16 17 18 19 20

Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America 
 X

A live attenuated equine H3N8 influenza vaccine is highly immunogenic and efficacious in mice and ferrets

Mariana Baz1, Myeisha Paskel1, Yumiko Matsuoka1, James Zengel, Xing Cheng, John J. Treanor2, Hong Jin3 and Kanta Subbarao1#

1

Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, United States Department of Medicine, University of Rochester Medical Center, Rochester, New York, United States 3 MedImmune LLC, Mountain View, CA 94043, United States 2

21 22

Running title: Equine H3 influenza vaccine in mice and ferrets

23 24 25 26 27 28 29

Abstract count: 227 Text count: 4556 Inserts: 4 figures, 2 tables

30 31 32 33 34 35 36 37 38 39 40 41 42 43

#

Corresponding author: Kanta Subbarao, MD, MPH Emerging Respiratory Viruses Section Laboratory of Infectious Diseases, NIAID, NIH Bldg 33, Room 3E13C.1, 33 North Drive, MSC 3203 Bethesda, MD 20892-3203 Phone: 301-451-3839 Fax: 301-480-4749 Email: [email protected]

44 45 46 47

ABSTRACT

48

respiratory disease in horses. Although natural infection of humans with EIV have not

49

been reported, experimental inoculation of humans with these viruses can lead to a

50

productive infection and elicit a neutralizing antibody response. Moreover, EIV have

51

crossed the species barrier to infect dogs, pigs and camels and therefore may also pose a

52

threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different

53

lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a

54

live attenuated candidate vaccine by reverse genetics with the hemagglutinin and

55

neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein

56

genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (H2N2) vaccine donor virus, which

57

is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice

58

and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus

59

induced neutralizing antibodies and conferred complete protection from homologous wt

60

virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also

61

induced neutralizing antibodies and conferred complete protection in mice and nearly

62

complete protection in ferrets upon heterologous challenge with the H3N8

63

(eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca

64

vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus

65

to humans.

Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of

66 67 68

2

69 70 71 72 73

IMPORTANCE

Equine influenza viruses have crossed the species barrier to infect other mammals such as

74

dogs, pigs and camels and therefore may also pose a threat to humans. We believe that it

75

is important to develop vaccines against equine influenza viruses in the event that an EIV

76

evolves, adapts and spreads in humans causing disease. We generated a live attenuated

77

H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and

78

protected mice and ferrets against homologous and heterologous EIV.

79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107

3

108 109 110 111

INTRODUCTION

112

respiratory disease in horses for centuries. Influenza A viruses contain a single-stranded,

113

negative-sense RNA genome, consisting of 8 gene segments and are further classified

114

into subtypes on the basis of the antigenicity of the two major surface glycoproteins:

115

hemagglutinin (HA) and neuraminidase (NA) (1). Two subtypes of EIV have been

116

isolated from horses: H7N7 and H3N8 viruses. The prototype equine H7N7 virus

117

(A/equine/Prague/56) virus emerged in 1956 (2) but has not been isolated since the late

118

1970s (3), although serological evidence suggest that this virus subtype circulated among

119

horses in Europe and the Americas before 1956 (4, 5); its circulation in unvaccinated

120

horses was recorded in the 1980s in India (6) and the beginning of the 1990s in Europe

121

and USA (7, 8). Equine H3N8 viruses were first isolated during a major epidemic in

122

Miami in 1963 (A/eq/Miami/1/63) (9) and since then have circulated enzootically in

123

horses, causing significant disease and economic burden worldwide (10). These viruses

124

have continued to evolve and have diverged into two antigenically and genetically

125

distinct American and the European lineages since 1986. The American lineage further

126

evolved into Kentucky, South American and Florida sublineages. Subsequent evolution

127

within the Florida sublineage has resulted in the emergence of two distinct clades (clades

128

1 and 2) (11).

129

Influenza A viruses can transmit between species and this characteristic of interspecies

130

transmission allows the emergence of reassortant influenza viruses (12). The H3N8 EIV

131

has crossed the species barrier and transmitted to racing greyhounds that shared a racing

132

track with horses in Florida in January 2004 (13) although retrospective serological

Equine influenza viruses (EIV) have been responsible for rapidly spreading outbreaks of

4

133

analysis suggests that H3N8 influenza viruses were circulating in racing greyhounds

134

since 1999 (14). Subsequently, canine H3N8 influenza viruses spread to pet dogs and

135

became enzootic in the USA (15). Canine H3N8 infections have also been reported in the

136

United Kingdom, Australia and Algeria (16-19). Studies on the distribution of the

137

sialoreceptors in the respiratory tract of horses and dogs have shown that both horses and

138

dogs have a predominance of SAα2,3-gal receptors (13, 18, 20). Pecorano et al., have

139

recently shown by binding assays that canine and equine influenza isolates have a higher

140

affinity for SAα2,3-gal compared to SAα2,6-gal receptors (20). These data may explain

141

the natural transmission of equine influenza virus to dogs.

142

In addition, two H3N8 influenza viruses were isolated from pigs in central China during

143

surveillance for swine influenza in 2004-2006. Sequence and phylogenetic analyses of

144

the eight gene segments revealed that the two swine isolates were of equine origin and

145

were most closely related to European H3N8 EIV from the early 1990s (21). Recently, an

146

EIV (H3N8) was isolated from a Bactrian camel in Mongolia highlighting a novel

147

interspecies transmission (22).

148

While natural transmission of EIV to humans has not been documented, experimental

149

challenge studies done in the 1960s indicate that the influenza A/equi 2/Miami/1/63 virus

150

was able to infect 64% of 33 human volunteers who received an intranasal dose of

151

between 104.6 and 105.3 fifty percent tissue culture infectious doses (TCID50) of virus.

152

However, illness only occurred in 12% of the volunteers, suggesting that the virus was

153

more virulent for horses than for humans (23-25). Human birth cohorts from the late 19th

154

century, particularly individuals born before 1890, demonstrated serologic reactivity with

155

equine H3N8 viruses many decades later (26). However, a recent study reported by

5

156

Burnell et al., showed sparse evidence for H3N8 infection in 100 subjects enrolled during

157

equine events in Australia (27). In this study, only nine subjects showed serologic

158

reactivity against EIV antigens and although eight of the subjects reported horse

159

exposure, antibody titers were low except for one subject who had a titer of 1:80 by

160

microneutralization assay. Another study done by Khurelbaatar et al., also showed sparse

161

evidence for EIV infection in 439 subjects ≥18 years of age (28) though 76% of the

162

participants reported exposure to horses.

163

The social and economic impact of widespread disease caused by EIV in humans could

164

be devastating since people in different regions of the world still rely heavily upon horses

165

for recreation, communication, military or general transport, and EIV has already crossed

166

the species barrier to dogs. We believe it is important to develop vaccines against animal

167

influenza viruses of the H3 subtype. The ongoing circulation of seasonal H3N2 viruses

168

does not preclude the possibility of a pandemic caused by an antigenically distinct animal

169

H3 virus as demonstrated by the unexpected emergence of a swine-origin H1N1

170

influenza virus as a pandemic strain in 2009 despite the ongoing circulation of seasonal

171

H1N1 viruses.

172

Seasonal live attenuated influenza vaccines (LAIV) have been licensed in the United

173

States since 2003 and they elicit both systemic and local mucosal immunity (29, 30). Our

174

laboratory has previously generated live attenuated H1N1, H2N2, H5N1, H6N1, H7N3,

175

H7N7, H7N9 and H9N2 viruses and found that these candidate vaccines were safe and

176

efficacious in conferring protection against wild-type (wt) viruses in mice and ferrets (31-

177

36) and several of these vaccines have been evaluated in phase 1 clinical trials (33, 37,

178

38).

6

179

We analyzed the antigenic relatedness and replicative capacity of H3N8 EIV from the

180

pre-divergent and American lineage (sublineage Florida Clade 1 and 2) viruses using

181

post-infection mouse and ferret sera (39). We selected A/equine/Georgia/1/1981

182

(eq/GA/81) for vaccine development because it induced the most broadly cross-

183

neutralizing antibodies (NtAb) and replicated to a high titer in the upper respiratory tract

184

of mice and ferrets (39). We used reverse genetics to generate a live attenuated cold-

185

adapted (ca) H3N8 virus bearing wild-type (wt) HA and NA genes from the eq/GA/81wt

186

virus, and the six internal protein gene segments from the ca influenza A vaccine donor

187

strain, A/Ann Arbor/6/60 ca (AA ca) (H2N2). The immunogenicity and protective

188

efficacy against challenge with the homologous wt eq/GA/81 and heterologous

189

A/equine/Newmarket/5/2003 (eq/Newm/03) viruses were evaluated in mice and ferrets.

190 191

MATERIALS AND METHODS

192 193

Viruses.

194

H3N8 EIV isolates were provided by Richard Webby, St. Jude Children’s Research

195

Hospital, Memphis, TN (A/equine/Georgia/1/1981 [H3N8]) and Debra Elton, Animal

196

Health Trust, Newmarket, UK (A/equine/Newmarket/5/2003 [H3N8]). The HA amino

197

acid sequence identity between eq/GA/81 and eq/Newm/03 is 97.3 % (39). Eq/GA/81

198

belongs to the “Florida Clade 1” sub-lineage and eq/Newm/03 belongs to the “American

199

lineage” “Florida Clade 2” (40). We have previously reported (39) that sera from ferrets

200

infected with eq/GA/81 showed cross-reactivity against the homologous virus (NtAb titer

201

≥1280) as well as eq/Newm/03 (NtAb titer ≥640). Virus stocks were propagated in the

7

202

allantoic cavity of 9- to 11-day-old embryonated specific-pathogen-free hen’s eggs

203

(Charles River Laboratories, North Franklin, CT) at 35°C. The allantoic fluid was

204

harvested at 72 h postinoculation (p.i.), tested for hemagglutinating activity using 0.5%

205

turkey red blood cells (Lampire Biological Laboratories, Pipersville, PA), pooled,

206

aliquoted, and stored at -80°C until use. Virus titers were determined in Madin-Darby

207

canine kidney (MDCK) cells (ATCC, Manassas, VA) and calculated using the Reed and

208

Muench method (41).

209 210

Generation of reassortant eq/GA/81 ca vaccine virus by reverse genetics.

211

The HA and NA gene segments of eq/GA/81 (H3N8) were amplified from vRNA by

212

reverse transcription-polymerase chain reaction (RT-PCR) using primers that are

213

universal to the HA and NA genes, sequenced and cloned into the plasmid vector

214

pAD3000 (42). The 6:2 reassortant vaccine virus was generated by co-transfecting eight

215

plasmids encoding the HA and NA of the eq/GA/81 virus and the 6 internal protein gene

216

segments of the AA ca virus into co-cultured 293T and MDCK cells. At 3 to 5 days post-

217

transfection, the transfected cell supernatant was inoculated into the allantoic cavity of 10

218

to 11 day old embryonated chicken eggs (Charles River Laboratories, Franklin, CT) and

219

incubated at 33°C for 2 days. Virus titer was determined by immunostaining plaques

220

using an anti-NP monoclonal antibody and expressed as log10 PFU (plaque-forming

221

units)/ml as previously described (43). The HA and NA sequences of the rescued virus

222

were verified by sequencing the genes amplified from viral RNA by RT-PCR.

223 224

8

225 226

Serologic assays.

227

Anti-influenza antibody titers in serum samples were measured by hemagglutination

228

inhibition (HAI) according to standard protocols (44) or microneutralization (MN) assay

229

as previously described (45). For the HAI assay, nonspecific inhibitors were removed

230

from serum by overnight treatment with receptor-destroying enzyme (Denka Seiken,

231

Tokyo, Japan). Sera were 2-fold serially diluted in 96-well V-bottom plates starting at a

232

dilution of 1:10, and 4 HA units of virus was added. Control wells received phosphate-

233

buffered saline (PBS) alone. Virus and sera were incubated together for 30 min at room

234

temperature and 50 μl of a 0.5% (vol/vol) suspension of turkey erythrocytes was added.

235

The virus-serum mixture and erythrocytes were gently mixed, and the results were

236

recorded after incubation for 45 min at room temperature. HAI titers were recorded as the

237

inverse of the highest antibody dilution that inhibited hemagglutination. A cross-reactive

238

antibody response was defined as a ≤4-fold difference between the homologous HAI titer

239

and the titer generated against the heterologous virus. For the MN assay, serial 2-fold

240

dilutions of heat-inactivated serum were prepared starting from a 1:20 dilution. Equal

241

volumes of serum and virus were mixed and incubated for 60 minutes at room

242

temperature. The residual infectivity of the virus-serum mixture was determined in

243

MDCK cells in 4 replicates for each dilution of serum. The neutralizing antibody (NtAb)

244

titer was defined as the reciprocal of the serum dilution that completely neutralized the

245

infectivity of 100 TCID50 of the virus as determined by the absence of cytopathic effect

246

on MDCK cells at day 4. A cross-reactive antibody response was defined as a ≤4-fold

247

difference between the homologous NtAb titer and the titer generated against the

9

248

heterologous virus.

249

Immunogenicity and protective efficacy of the H3N8 ca virus in mice.

250

Six- to 8-week-old female BALB/c mice (Taconic Farms, Inc., Germantown, NY) were

251

used in all mouse experiments. Animal studies were conducted in biosafety level 2

252

laboratories (BSL-2) at the National Institutes of Health (NIH) and protocols were

253

approved by the National Institutes of Health Animal Care and Use Committee.

254

Groups of eight mice were lightly anesthetized and inoculated intranasally (i.n.) with 50

255

µl containing 106 PFU of the H3N8 ca vaccine virus in one or two doses. Mock-

256

inoculated controls received Leibovitz-15 (L15) medium alone. Neutralizing antibody

257

responses to homologous (eq/GA/81) and heterologous (eq/Newm/03) H3N8 wt viruses

258

were determined from sera collected prior to inoculation (prebleed) and at 38 days after

259

the first or second immunization by MN assay (45).

260

On day 38 after the first or second dose of vaccine, groups of eight mice were challenged

261

i.n. with 105 TCID50 of the H3N8 equine wt viruses, eq/GA/81 or eq/Newm/03. Four

262

mice per challenge virus were sacrificed on days 2 and 4 post-challenge (p.c.), and lungs

263

and nasal turbinates (NTs) were harvested and stored at -80°C. We chose these time

264

points based on previous observations in our laboratory that equine wt viruses replicate to

265

high titers in the NTs and lungs of mice from days 2 to 4 post-infection (39). Organs were

266

weighed and homogenized in L15 medium containing 2X antibiotic-antimycotic

267

(penicillin, streptomycin, and amphotericin B) (Invitrogen-GIBCO) to make 10% and 5%

268

(wt/vol) tissue homogenates of lung and NT, respectively. Tissue homogenates clarified

269

by centrifugation at 1,500 rpm for 10 min were titered in 24 and 96-well tissue culture

270

plates containing MDCK cell monolayers The virus titer for each organ was determined

10

271

by Reed and Muench method (41) and was expressed as log10 TCID50/gram of tissue.

272

Replication of H3N8 equine wt and ca viruses in the respiratory tract.

273

Ten- to 12-week-old ferrets (Triple F Farms, Sayre, PA) were used in these ferret

274

experiments. Animals were seronegative for antibodies to circulating human H3N2,

275

H1N1 and B influenza viruses. Ferret studies were conducted in a BSL-2 at MedImmune

276

and NIH and protocols were approved by the MedImmune and NIH Animal Care and

277

Use Committees.

278 279

Groups of ferrets were lightly anesthetized with isoflurane and inoculated i.n. with 500 μl

280

containing 107 PFU of wt or ca viruses. At 3 and 5 dpi, ferrets were euthanized, and right

281

middle and the caudal portion of the left cranial lobe of the lungs and the NTs were

282

harvested and stored at -80°C. Organs were thawed, weighed and homogenized in L-15

283

medium as described above to make a 10% (wt/vol) suspension and titers were

284

determined by plaque assay on MDCK cells using an anti-NP monoclonal antibody and

285

expressed as log10 PFU/gram of tissue.

286 287

Immunogenicity and protective efficacy of the H3N8 ca virus.

288

Groups of ferrets were inoculated i.n. with one or two doses 28 days apart of 500 μl

289

containing 107 PFU of eq/GA/81 ca or L15 medium (mock immunized) and serum

290

samples were collected on days 0 (pre-immunization), 28 or 56 p.i. Antibody titers in pre

291

and post-infection ferret sera were determined by MN and HAI assays, as described

292

above.

293

On day 28 or 56 p.i. ferrets were challenged i.n. with 107 PFU of each of the H3N8

11

294

equine wt viruses, eq/GA/81 or eq/Newm/03. Three ferrets per challenge virus were

295

euthanized on days 3 and 5 p.c., and lungs and NTs were harvested and stored at -80°C.

296

We chose these time points based on previous observations in our laboratory that wt EIV

297

are detected at high titers in the NTs of ferrets from days 1 to 5 p.i. (39). Challenge virus

298

titers were determined in MDCK cells and expressed as log10TCID50 per gram of NT or

299

lung tissue as described above.

300 301

Human sera.

302

Sera collected during a study in 2009 from healthy adult men and nonpregnant women

303

before vaccination with the monovalent inactivated 2009 pH1N1 vaccine were provided

304

by John Treanor (University of Rochester) (46). Subjects were enrolled in 3 age cohorts:

305

18 to 32 years (n = 19), 60 to 69 years (n = 19), and ≥70 years (n = 18). The study was

306

conducted under a protocol approved by the University of Rochester Research Subjects

307

Review Board. Informed written consent was obtained from each participant.

308 309

RESULTS

310 311 312 313 314

Immunogenicity of the eq/GA/81 ca virus in mice.

A single dose of eq/GA/81 ca virus induced a robust neutralizing antibody response

315

against the homologous virus, with a geometric mean titer (GMT) of 418 (range 57 to

316

1016) (Table 1). The NtAb titers against the heterologous virus, eq/Newm/03, were

317

similar (GMT of 490; range 160 to 1613). When two doses of the eq/GA/81 ca vaccine

318

were administered to mice, the GMT achieved was 264 (range 57 to 905) following the

319

first dose, with a further increase (GMT 2426; range 640 to 7241) 38 days after the

12

320

second dose (Table 1). A similar profile was observed against the heterologous virus

321

(Table 1). As expected, mock-immunized mice did not develop detectable NtAb

322

antibodies. These results demonstrate that the eq/GA/81 ca vaccine candidate is highly

323

immunogenic in mice and serum antibodies cross-reacted well with the heterologous

324

eq/Newm/03 wt virus.

325 326 327 328 329

Efficacy of the eq/GA/81 ca virus in mice.

330

To determine whether immunization with the eq/GA/81 ca virus induced protection, we

331

inoculated mice with either a single dose or two doses of the vaccine candidate. Thirty-

332

eight days after the final vaccination, mice were challenged with 106 PFU/50μl of the

333

homologous (eq/GA/81) or the heterologous (eq/Newm/03) wt viruses. In mock-

334

immunized mice, the mean titers on days 2 and 4 p.c. in the NTs after challenge with the

335

eq/GA/81 wt virus were 105.5 and 105.25 TCID50/g, respectively, and the mean titers in the

336

lungs were 106.1 and 104.6 TCID50/g, respectively. In mock-immunized mice, the mean

337

titers on days 2 and 4 p.c. in the NTs after challenge with the heterologous eq/Newm/03

338

wt virus were 106.8 and 104.9 TCID50/g, respectively, and the mean titers in the lungs were

339

106.4 and 104.95 TCID50/g on days 2 and 4 p.c., respectively (Fig. 1). A single dose of the

340

eq/GA/81 ca virus provided complete protection against challenge with homologous and

341

heterologous wt viruses in both the upper and lower respiratory tract (Fig.1). A similar

342

pattern was observed for mice vaccinated with two doses of the eq/GA/81 ca virus (Fig.

343

1). We had previously observed that the eq/GA/81 and eq/Newm/03 wt viruses did not

344

cause weight loss or mortality in mice, so we did not assess protection from clinical

13

345

illness in mice (39). Thus, the eq/GA/81 ca vaccine candidate offered complete

346

protection against homologous and heterologous H3N8 wt virus challenge in mice.

347 348 349

Level of replication of the eq/GA/81 wt and ca viruses in ferrets.

350

To determine whether the eq/GA/81 ca vaccine virus was attenuated in ferrets, the level

351

of replication in the NT and lungs 3 and 5 days following i.n. administration were

352

compared with those of the eq/GA/81 wt virus. The mean titers on days 3 and 5 in the

353

NTs of ferrets inoculated with the eq/GA/81 wt virus were 107.2 and 106.9 PFU/g,

354

respectively, and 104.8 and 105.7 PFU/g in ferrets inoculated with the eq/GA/81 ca virus.

355

Thus, the replication of the eq/GA/81 ca virus was 16 to 250-fold lower than the

356

corresponding wt virus in the URT of ferrets. The eq/GA/81 wt virus did not replicate

357

well in the lower respiratory tract of ferrets, as previously reported (39) and replication of

358

the vaccine candidate was not detected in the lungs of ferrets (Fig. 2). No notable signs of

359

disease were observed in ferrets infected with the equine ca or wt viruses. These data

360

indicate that the eq/GA/81 ca virus was attenuated in ferrets.

361 362 363

Immunogenicity of the eq/GA/81 ca virus in ferrets.

364

Ferrets that received a single dose of eq/GA/81 ca virus developed neutralizing and HAI

365

antibodies to the homologous wt virus at titers that ranged from 320 to 905 (GMT=538)

366

and from 40 to 160 (GMT=85), respectively (Table 2). One dose of the eq/GA/81 ca

367

virus elicited cross-reactive neutralizing and HAI antibodies to the heterologous

368

eq/Newm/03 wt virus at titers that ranged from 113 to 453 (GMT=196) and from 160 to

369

640 (GMT=226), respectively. In animals that received two doses of the vaccine, the first

14

370

dose of the eq/GA/81 ca vaccine induced homologous NtAb and HAI response at titers

371

that ranged from 226 to 1280 (GMT=559) and 40 to 160 (GMT=90), respectively. Titers

372

increased after the second dose and ranged from 453 to 3620 (GMT=932) and 80 to 1280

373

(GMT=302), respectively. One dose of the eq/GA/81 ca virus elicited cross-reactive

374

neutralizing and HAI antibodies to the heterologous wt virus at titers that ranged from

375

320 to 1280 (GMT=512) and 80 to 640 (GMT=151), respectively. Again, titers increased

376

after the second dose and ranged from 320 to 5120 (GMT=1243) and 160 to 2560

377

(GMT=678), respectively (Table 2). Consistent with findings from the study in mice,

378

these data indicate that a single dose of the eq/GA/81 ca virus was immunogenic in

379

ferrets and that serum antibodies cross-reacted with a heterologous H3N8 virus. Sera

380

from vaccinated ferrets failed to neutralize an older (A/Port Chalmers/1973) and a recent

381

(A/Texas/50/2012) human H3N2 virus (data not shown).

382 383 384

Efficacy of the eq/GA/81 ca virus in ferrets.

385

To determine whether immunization with the eq/GA/81 ca virus induced protection in

386

ferrets, we inoculated animals intranasally with either a single dose or two doses of the

387

vaccine candidate and challenged them 28 days later with 107 PFU of the homologous

388

(eq/GA/81) or the heterologous (eq/Newm/03) wt viruses. In mock-immunized ferrets the

389

titers of eq/GA/81 wt challenge virus on days 3 and 5 p.c. in the NTs were 107.1 and 105.0

390

TCID50/g, respectively, and the mean virus titers in the lungs were 102.3 and 102.5

391

TCID50/g on days 3 and 5 p.c. In mock-immunized ferrets the mean titers of the

392

heterologous eq/Newm/03 wt virus on days 3 and 5 p.c. in the NTs were 108.1 and 107.2

393

TCID50/g, respectively, and the mean virus titers in the lungs were 102.5 and 101.7

15

394

TCID50/g on days 3 and 5 p.c. (Fig. 3). A single dose of the eq/GA/81 ca virus provided

395

complete protection against challenge (no detectable replication) with homologous wt

396

virus in the upper respiratory tract of the ferrets and restricted replication and early

397

clearance of the heterologous wt challenge virus (only one ferret out of 3 had 102

398

TCID50/g on day 5 p.c.) (Fig.3). Similar results were observed in ferrets that received a

399

second dose of the vaccine. Because the eq/GA/81 and the eq/Newm/03 wt viruses did

400

not replicate well in the lower respiratory tract of ferrets, the protection conferred by the

401

eq/GA/81 ca virus in the lungs could not be evaluated (Fig.3).

402 403 404 405 406 407

Testing for presence of cross-reactive antibodies in human sera representing three age cohorts.

408

whether prior exposure to seasonal H3N2 viruses induced cross-reactive Ab against EIV.

409

We assessed the presence of antibodies that cross-reacted with eq/GA/81 (H3N8) virus in

410

human sera collected in 2009. As a control we assayed the levels of antibodies against the

411

seasonal influenza virus A/Wisconsin/67/2005 (H3N2) that was circulating at the time

412

the sera were collected. Subjects from three age groups, 18-32 years old (n=19), 60-69

413

years old (n=19), and 70 years or older (n=18), were enrolled in a clinical trial of a

414

monovalent 2009 H1N1pdm vaccine that has been reported previously (46).

415

The 18-32 year old subjects failed to show detectable NtAb against the equine H3N8

416

virus (Fig.4A). Interestingly, subjects from the other two cohorts, 60-69 and ≥70 years

417

old, had a GMT of 22 (range 10 to 57) and 20 (range 10 to 113), respectively. In subjects

418

older than 60 years of age (n=37), seven had NtAb titers of 40, one of 50, three of 57 and

419

one of 113. Nine subjects showed lower levels of NtAb (between 20 and 28). In total, 22

Because human H3N2 viruses have circulated since 1968, we sought to determine

16

420

out of 37 individuals had NtAb titers ≥20 against the equine H3N8 virus. Unfortunately,

421

we did not enroll subjects who were between 33 and 59 years of age in the study, so we

422

cannot comment on the level of cross-reactive antibody in this age group. However, the

423

study was conducted in 2009-2010 and therefore subjects born after 1968, when H3N2

424

viruses emerged and became established in humans, would have been 42 years of age or

425

younger. Therefore, we would expect that people over 42 years of age would have been

426

exposed to H3N2 viruses and could have some cross reactive antibody. The GMT in the

427

18-32 year old, 60-69 year old and ≥70 year old subjects against the

428

A/Wisconsin/67/2005 (H3N2) virus was 248 (range 10 to 3620), 232 (range 10 to 3620)

429

and 134.5 (range 10 to 1613), respectively (Fig.4A).

430

Interestingly, none of the subjects had detectable HAI antibody against the equine H3N8

431

virus (Fig. 4B). The GMT of HAI antibodies in the 18-32 year old, 60-69 year old and

432

≥70 year old subjects against the A/Wisconsin/67/2005 (H3N2) virus was 46 (range 10 to

433

1280), 67 (range 10 to 640) and 27 (range 10 to 640), respectively (Fig.4B). The

434

detection of cross-reactive NtAb in the absence of HAI antibodies in 59% of subjects

435

over 60 years of age suggests that the antibody was induced by prior or repeated exposure

436

by infection or vaccination with older seasonal H3N2 viruses and that the Abs could be

437

directed at the HA stalk.

438 439 440 441

DISCUSSION

442

Although direct transmission of EIV to humans has not been reported, experimental

443

infection of humans with EIV can lead to a productive infection and elicit a significant

444

NtAb response (24, 25). The fact that EIV can infect humans and these viruses have

17

445

crossed the species barrier and infected dogs (13, 16, 18), pigs (21) and camels (21)

446

underscores the potential threat posed to human health by viruses of this subtype. The

447

emergence and pandemic spread of the swine-origin H1N1 influenza virus in 2009,

448

despite the ongoing circulation of human H1N1 viruses suggests that an antigenically

449

distant animal origin H3 virus may pose a pandemic threat despite the circulation of

450

H3N2 viruses in humans since 1968.

451

The purpose of our study was to generate and evaluate a vaccine candidate to be used in

452

humans in the event that an EIV evolves, adapts and spreads in humans causing disease.

453

To this end, we previously evaluated three H3N8 equine influenza viruses from different

454

lineages and selected the eq/GA/81 virus for vaccine development because it elicited

455

cross-reactive antibodies against heterologous EIV (39). We generated an eq/GA/81

456

candidate LAIV by plasmid-based reverse genetics on the backbone of the AA ca donor

457

virus that is used to produce the licensed seasonal live attenuated influenza vaccine.

458

In mice, a single dose of the eq/GA/81 ca vaccine virus induced robust neutralizing

459

antibody titers against the homologous and heterologous wt challenge viruses and

460

conferred full protection against homologous and heterologous virus challenge in both

461

the upper and lower respiratory tract. In ferrets, as in mice, one dose of the vaccine was

462

highly immunogenic and conferred complete protection against homologous challenge

463

virus and near complete protection against the heterologous challenge virus. We observed

464

a direct correlation between serum antibody response and protection against challenge in

465

mice and ferrets that received one dose of the eq/GA/81 ca vaccine virus, although

466

contributions from other arms of the immune system such as the cellular or mucosal

467

immune response cannot be excluded.

18

468

In 2009, when the novel H1N1pdm virus emerged, it was assumed that two doses of

469

vaccine would be needed to immunize the human population against the pandemic virus

470

because studies in 1977 had demonstrated the need for two doses of vaccine in a naïve

471

population; preclinical evaluation of 2009 H1N1pdm vaccines in influenza-naïve animal

472

models supported this conclusion. However, when clinical trials of the inactivated 2009

473

H1N1pdm vaccine were undertaken, a single dose of vaccine was sufficient in all except

474

children younger than 3 years of age, indicating that most of the population had been

475

primed by prior exposure or vaccination with seasonal H1N1 viruses. We evaluated this

476

phenomenon in a mouse model and demonstrated that priming was achieved by infection

477

with seasonal H1N1 influenza virus or seasonal LAIV but not by seasonal inactivated

478

influenza vaccine (47). In the present study, we evaluated sera from 56 subjects from

479

three age groups, 18-32 years old, 60-69 years old, and 70 years and older, who were

480

enrolled in a previous study for cross-reactive H3 antibodies. Most subjects in each

481

cohort reported receiving the 2009-2010 seasonal trivalent influenza vaccine 2 to 4

482

months before their blood sample was collected (46). We do not know if the participants

483

had been exposed to horses. We observed reactivity against the eq/GA/81 virus in

484

individuals over 60 years of age and speculate that the detectable cross-reactive NtAb

485

titers may be explained by cross-reactivity due to previously circulating human influenza

486

A H3N2 viruses. In the event of a pandemic caused by a related virus, a large portion of

487

the human population may be immunologically primed because of previous exposure to

488

seasonal H3N2 influenza viruses and therefore one dose of the H3N8 vaccine may be

489

sufficient to confer protection.

490

In summary, we generated a candidate LAIV against an EIV and demonstrated that a

19

491

single dose of the vaccine was highly immunogenic and efficacious in protecting mice

492

and ferrets from challenge with the homologous and an antigenically distinct

493

heterologous H3N8 virus from a different sub-lineage. Based on these promising

494

preclinical data, careful clinical evaluation of the eq/GA/81 (H3N8) ca vaccine is

495

warranted as part of pandemic preparedness efforts. We found evidence of cross-reactive

496

antibodies in subjects >60 years of age that could be directed at the stalk domain of the

497

HA protein. Although data from persons between 32-60 years of age are lacking, it

498

appears that a proportion of the human population may be previously primed for a robust

499

response to an equine influenza H3N8 vaccine.

500 501

ACKNOWLEDGMENTS

502

This research was supported by the Intramural Research Program of the NIAID, NIH and

503

was performed as part of a Cooperative Research and Development Agreement between

504

the Laboratory of Infectious Diseases, NIAID, and MedImmune, LLC.

505

We thank Dr. Ian Moore, the staff of the Comparative Medicine Branch, NIAID, and the

506

staff at MedImmune’s Animal Care Facility for technical support for animal studies. We

507

are grateful to Drs. Richard Webby and Debra Elton for providing the viruses used in this

508

study and Dr. JoAnn Suzich for reviewing the manuscript.

509 510 511 512 513 514 515 516 517 518

20

519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564

REFERENCES

1.

2. 3. 4. 5. 6. 7.

8.

9. 10. 11.

12. 13.

14.

15.

Wright P, Neumann, G., Kawaoka, Y. 2007. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA. Orthomyxoviruses, p. 1691–1740. Sovinova O, Tumova B, Pouska F, Nemec J. 1958. Isolation of a virus causing respiratory disease in horses. Acta virologica 2:52-61. Webster RG. 1993. Are equine 1 influenza viruses still present in horses? Equine veterinary journal 25:537-538. Beveridge WI, Mahaffey LW, Rose MA. 1965. Influenza in Horses. The Veterinary record 77:57-59. Doll ER. 1961. Influenza of horses. The American review of respiratory disease 83(2)Pt 2:48-53. Singh G. 1994. Characterization of A/eq-1 virus isolated during the equine influenza epidemic in India. Acta virologica 38:25-26. Madic J, Martinovic S, Naglic T, Hajsig D, Cvetnic S. 1996. Serological evidence for the presence of A/equine-1 influenza virus in unvaccinated horses in Croatia. The Veterinary record 138:68. Mumford J, Wood J. 1993. WHO/OIE meeting: consultation on newly emerging strains of equine influenza. 18-19 May 1992, Animal Health Trust, Newmarket, Suffolk, UK. Vaccine 11:1172-1175. Waddell GH, Teigland MB, Sigel MM. 1963. A New Influenza Virus Associated with Equine Respiratory Disease. J Am Vet Med Assoc 143:587-590. Paillot R, Hannant D, Kydd JH, Daly JM. 2006. Vaccination against equine influenza: quid novi? Vaccine 24:4047-4061. Lai AC, Chambers TM, Holland RE, Jr., Morley PS, Haines DM, Townsend HG, Barrandeguy M. 2001. Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere. Archives of virology 146:1063-1074. Webster RG, Sharp GB, Claas EC. 1995. Interspecies transmission of influenza viruses. American journal of respiratory and critical care medicine 152:S25-30. Crawford PC, Dubovi EJ, Castleman WL, Stephenson I, Gibbs EP, Chen L, Smith C, Hill RC, Ferro P, Pompey J, Bright RA, Medina MJ, Johnson CM, Olsen CW, Cox NJ, Klimov AI, Katz JM, Donis RO. 2005. Transmission of equine influenza virus to dogs. Science 310:482-485. Anderson TC, Bromfield CR, Crawford PC, Dodds WJ, Gibbs EP, Hernandez JA. 2012. Serological evidence of H3N8 canine influenza-like virus circulation in USA dogs prior to 2004. Vet J 191:312-316. Payungporn S, Crawford PC, Kouo TS, Chen LM, Pompey J, Castleman WL, Dubovi EJ, Katz JM, Donis RO. 2008. Influenza A virus (H3N8) in dogs with respiratory disease, Florida. Emerging infectious diseases 14:902-908.

21

565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609

16.

17.

18.

19.

20.

21.

22.

23.

24. 25. 26.

27.

28.

29.

Crispe E, Finlaison DS, Hurt AC, Kirkland PD. 2011. Infection of dogs with equine influenza virus: evidence for transmission from horses during the Australian outbreak. Aust Vet J 89 Suppl 1:27-28. Kirkland PD, Finlaison DS, Crispe E, Hurt AC. 2010. Influenza virus transmission from horses to dogs, Australia. Emerging infectious diseases 16:699702. Daly JM, Blunden AS, Macrae S, Miller J, Bowman SJ, Kolodziejek J, Nowotny N, Smith KC. 2008. Transmission of equine influenza virus to English foxhounds. Emerging infectious diseases 14:461-464. Laabassi F, Lecouturier F, Amelot G, Gaudaire D, Mamache B, Laugier C, Legrand L, Zientara S, Hans A. 2014. Epidemiology and Genetic Characterization of H3N8 Equine Influenza Virus Responsible for Clinical Disease in Algeria in 2011. Transboundary and emerging diseases. Pecoraro HL, Bennett S, Garretson K, Quintana AM, Lunn KF, Landolt GA. 2013. Comparison of the Infectivity and Transmission of Contemporary Canine and Equine H3N8 Influenza Viruses in Dogs. Veterinary medicine international 2013:874521. Tu J, Zhou H, Jiang T, Li C, Zhang A, Guo X, Zou W, Chen H, Jin M. 2009. Isolation and molecular characterization of equine H3N8 influenza viruses from pigs in China. Archives of virology 154:887-890. Yondon M, Heil GL, Burks JP, Zayat B, Waltzek TB, Jamiyan BO, McKenzie PP, Krueger WS, Friary JA, Gray GC. 2013. Isolation and characterization of H3N8 equine influenza A virus associated with the 2011 epizootic in Mongolia. Influenza and other respiratory viruses 7:659-665. Alford RH, Kasel JA, Lehrich JR, Knight V. 1967. Human responses to experimental infection with influenza A/Equi 2 virus. Am J Epidemiol 86:185192. Kasel JA, Couch RB. 1969. Experimental infection in man and horses with influenza A viruses. Bull World Health Organ 41:447-452. Kasel JA, Alford RH, Knight V, Waddell GH, Sigel MM. 1965. Experimental Infection of Human Volunteers with Equine Influenza Virus. Nature 206:41-43. Minuse E, McQueen JL, Davenport FM, Francis T, Jr. 1965. Studies of Antibodies to 1956 and 1963 Equine Influenza Viruses in Horses and Man. Journal of immunology 94:563-566. Burnell FJ, Holmes MA, Roiko AH, Lowe JB, Heil GL, White SK, Gray GC. 2014. Little evidence of human infection with equine influenza during the 2007 epizootic, Queensland, Australia. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 59:100-103. Khurelbaatar N, Krueger WS, Heil GL, Darmaa B, Ulziimaa D, Tserennorov D, Baterdene A, Anderson BD, Gray GC. 2014. Little Evidence of Avian or Equine Influenza Virus Infection among a Cohort of Mongolian Adults with Animal Exposures, 2010-2011. PloS one 9:e85616. Belshe R, Lee MS, Walker RE, Stoddard J, Mendelman PM. 2004. Safety, immunogenicity and efficacy of intranasal, live attenuated influenza vaccine. Expert review of vaccines 3:643-654.

22

610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40. 41. 42.

Murphy BR, Coelingh K. 2002. Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines. Viral immunology 15:295-323. Chen GL, Lamirande EW, Cheng X, Torres-Velez F, Orandle M, Jin H, Kemble G, Subbarao K. 2014. Evaluation of three live attenuated h2 pandemic influenza vaccine candidates in mice and ferrets. Journal of virology 88:28672876. Min JY, Vogel L, Matsuoka Y, Lu B, Swayne D, Jin H, Kemble G, Subbarao K. 2010. A live attenuated H7N7 candidate vaccine virus induces neutralizing antibody that confers protection from challenge in mice, ferrets, and monkeys. Journal of virology 84:11950-11960. Karron RA, Talaat K, Luke C, Callahan K, Thumar B, Dilorenzo S, McAuliffe J, Schappell E, Suguitan A, Mills K, Chen G, Lamirande E, Coelingh K, Jin H, Murphy BR, Kemble G, Subbarao K. 2009. Evaluation of two live attenuated cold-adapted H5N1 influenza virus vaccines in healthy adults. Vaccine 27:4953-4960. Chen Z, Santos C, Aspelund A, Gillim-Ross L, Jin H, Kemble G, Subbarao K. 2009. Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets. Journal of virology 83:65-72. Suguitan AL, Jr., McAuliffe J, Mills KL, Jin H, Duke G, Lu B, Luke CJ, Murphy B, Swayne DE, Kemble G, Subbarao K. 2006. Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets. PLoS medicine 3:e360. Chen Z, Baz M, Lu J, Paskel M, Santos C, Subbarao K, Jin H, Matsuoka Y. 2014. Development of a High-Yield Live Attenuated H7N9 Influenza Virus Vaccine That Provides Protection against Homologous and Heterologous H7 Wild-Type Viruses in Ferrets. Journal of virology 88:7016-7023. Talaat KR, Karron RA, Callahan KA, Luke CJ, DiLorenzo SC, Chen GL, Lamirande EW, Jin H, Coelingh KL, Murphy BR, Kemble G, Subbarao K. 2009. A live attenuated H7N3 influenza virus vaccine is well tolerated and immunogenic in a Phase I trial in healthy adults. Vaccine 27:3744-3753. Karron RA, Callahan K, Luke C, Thumar B, McAuliffe J, Schappell E, Joseph T, Coelingh K, Jin H, Kemble G, Murphy BR, Subbarao K. 2009. A live attenuated H9N2 influenza vaccine is well tolerated and immunogenic in healthy adults. The Journal of infectious diseases 199:711-716. Baz M, Paskel M, Matsuoka Y, Zengel J, Cheng X, Jin H, Subbarao K. 2013. Replication and immunogenicity of swine, equine, and avian h3 subtype influenza viruses in mice and ferrets. Journal of virology 87:6901-6910. Daly JM, MacRae S, Newton JR, Wattrang E, Elton DM. 2011. Equine influenza: a review of an unpredictable virus. Vet J 189:7-14. Reed LJ, and H. Muench. . 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27:493–497. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. 2000. A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences of the United States of America 97:6108-6113.

23

656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703

43.

44.

45.

46.

47.

Chen GL, Lamirande EW, Yang CF, Jin H, Kemble G, Subbarao K. 2010. Evaluation of replication and cross-reactive antibody responses of H2 subtype influenza viruses in mice and ferrets. Journal of virology 84:7695-7702. WHO. 2002. WHO manual on animal influenza diagnosis and surveillance. http://www.who.int/vaccine_research/diseases/influenza/WHO_manual_on_anim al-diagnosis_and_surveillance_2002_5.pdf. Joseph T, McAuliffe J, Lu B, Jin H, Kemble G, Subbarao K. 2007. Evaluation of replication and pathogenicity of avian influenza a H7 subtype viruses in a mouse model. Journal of virology 81:10558-10566. Sangster MY, Baer J, Santiago FW, Fitzgerald T, Ilyushina NA, Sundararajan A, Henn AD, Krammer F, Yang H, Luke CJ, Zand MS, Wright PF, Treanor JJ, Topham DJ, Subbarao K. 2013. B cell response and hemagglutinin stalk-reactive antibody production in different age cohorts following 2009 H1N1 influenza virus vaccination. Clinical and vaccine immunology : CVI 20:867-876. Chen GL, Lau YF, Lamirande EW, McCall AW, Subbarao K. 2011. Seasonal influenza infection and live vaccine prime for a response to the 2009 pandemic H1N1 vaccine. Proceedings of the National Academy of Sciences of the United States of America 108:1140-1145.

24

704 705 706 707

FIGURE LEGENDS

708

heterologous challenge in the upper (A) or lower (B) respiratory tract of mice. Mice were

709

intranasally inoculated with either L-15 (mock) or 1 or 2 doses of 106 PFU/mouse of

710

eq/GA/81 ca vaccine and challenged 38 days following the last vaccine dose with 106

711

PFU/mouse of the indicated challenge virus. Virus titers were determined on days 2 and 4

712

postchallenge. The dashed horizontal line represents the lower limit of detection.

Fig 1. Protection conferred by the eq/GA/81 ca vaccine against homologous and

713 714

Fig 2. Level of replication of the eq/GA/81 ca vaccine virus compared with the

715

corresponding wt virus in the upper (A) and lower (B) respiratory tracts of ferrets. Lightly

716

anesthetized ferrets were inoculated intranasally with 107 PFU/ferret and tissues were

717

harvested on days 3 and 5 postinfection. The dashed horizontal line represents the lower

718

limit of detection.

719 720

Fig 3. Protection conferred by the eq/GA/81 ca vaccines against homologous and

721

heterologous challenge in ferrets. Animals were intranasally inoculated with either L-15

722

(mock) or 1 or 2 doses of 107 PFU/ferret of eq/GA/81 ca vaccine and challenged 28 days

723

following the last vaccine administration with 107 PFU/ferrets of the indicated challenge

724

virus. Virus titers were determined on days 3 and 5 postchallenge. Levels of replication

725

of the indicated challenge viruses in the upper (A) or lower (B) respiratory tract of ferrets

726

that were challenged following 1 dose of the ca vaccine. The dashed horizontal line

727

represents the lower limit of detection.

25

728

Fig 4. Serum neutralizing antibody (A) and hemagglutination inhibiting antibody (B)

729

titers in individuals of different age groups. The serum antibody titer against eq/GA/81

730

(circles) and A/WI/67/05 (triangles) are shown for individual subjects. Bars identify

731

geometric mean titer of the group. The dashed horizontal line represents the lower limit

732

of detection.

733 734 735

26

736 737 738 739

Table 1. Serum neutralizing antibody response to the eq/GA/81 ca vaccine in micea.

Test antigen

eq/GA/81 wt eq/Newm/03 wt 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778

Geometric mean titer of serum NtAb achieved at indicated days post-immunization in mice: 1 dose (D38)b 2 doses (D28/D66b) 418 264/2426 490 225/1540

a

Groups of eight mice were inoculated i.n. with 106 PFU of the eq/GA/81 ca vaccine. Serum was collected at the indicated days after the first immunization. Homologous antibody titers are in bold. b Mice were bled on day 38 after the first or second immunization because of technical reasons.

27

779 780 781 782

Table 2. Serum antibody responses to the eq/GA/81 ca vaccine in ferretsa. Test antigen

eq/GA/81 wt eq/Newm/03 wt 783 784 785 786 787 788 789 790 791 792 793 794 795 796

Geometric mean serum HAI or NtAb titers achieved at indicated days post-immunization in ferrets: Assay 1 dose (D28) 2 doses (D28/D56) MN 538 559/932 HAI 85 90/302 MN 196 512/1243 HAI 226 151/678

a

Groups of twelve ferrets were inoculated i.n. with 107 PFU of the eq/GA/81 ca vaccine. Serum was collected at the indicated days after immunization. Homologous antibody titers are in bold.

28

Fig. 1

Virus Titer (log10 TCID50/g)

A: NTs 8 7 6 5 4 3 2 1

1 dose

Challenged with:

1 dose

2 doses

eq/GA/81 wt

d4

d2

ca

/8 1

G A

eq /

eq /

G A

/8 1

ca

d4

d2

ca

/8 1

G A

eq /

eq /

G A

/8 1

ca

L1 5d L1 2 5d4

d4

d2

ca

/8 1

G A

eq /

eq /

G A

/8 1

ca

d4

ca

/8 1

G A

eq /

eq /

G A

/8 1

ca

L1 5d L1 2 5d4

Immunized with:

d2

0

2 doses

eq/Newm/03 wt

Virus Titer (log10 TCID50/g)

B: Lungs 8 7 6 5 4 3 2 1

1 dose

Challenged with:

2 doses

eq/GA/81 wt

d4 A eq /81 /G A ca d2 /8 1 ca d4 eq /G A eq /81 /G A ca d /8 2 1 ca d4

d2 5-

L1

L1

5-

/G

eq

eq

/G

A eq /81 /G A ca d /8 2 1 ca d4 eq /G A eq /81 /G A ca d2 /8 1 ca d4

d2

5-

L1

5L1

d4

0

Immunized with:

1 dose

2 doses

eq/Newm/03 wt

Fig 1. Protection conferred by the eq/GA/81 ca vaccines against homologous and heterologous challenge in the upper (A) or lower (B) respiratory tract of mice. Mice were intranasally inoculated with either L-15 (mock) or 1 or 2 doses of 106 PFU/mouse of eq/GA/81 ca vaccine and challenged 38 days following the last vaccine dose with 106 PFU/mouse of the indicated challenge virus. Virus titers were determined on days 2 and 4 postchallenge. The dashed horizontal line represents the lower limit of detection.

Fig. 2

Virus Titer (log10 PFU/g)

eq/GA/81 wt 8

eq/GA/81 ca

A: NTs

7 6 5 4 3 2 1 5

3

Days post-inoculation

B: Lungs

Virus Titer (log10 PFU/g)

8

5

3

0

7 6 5 4 3 2 1 5

3

5

3

0 Days post-inoculation

Fig 2. Level of replication of the eq/GA/81 ca vaccine virus compared with the corresponding wt virus in the upper (A) and lower (B) respiratory tract of ferrets. Lightly anesthetized ferrets were inoculated intranasally with 107 PFU/ferret and tissues were harvested on days 3 and 5 postinfection. The dashed horizontal line represents the lower limit of detection.

Virus Titer (log10 TCID50/g)

Fig. 3 9

A: NTs

8 7 6 5 4 3 2 1

/G eq A / 8 /G 1 A ca /8 1 d3 ca eq d5 /G eq A / 8 /G 1 A ca /8 1 d3 ca d5

5L1 d3 5d5

1 dose

eq

L1

L1 d3 5d5 eq

L1

5-

Immunized with:

/G eq A / 8 /G 1 A ca /8 1 d3 ca eq d5 /G eq A / 8 /G 1 A ca /8 1 d3 ca d5

0

1 dose

2 doses

eq/Newm/03 wt

eq/GA/81 wt

Challenged with:

2 doses

B: Lungs Virus Titer (log10 TCID50/g)

9 8 7 6 5 4 3 2 1

Challenged with:

2 doses

eq/GA/81 wt

eq A/8 /G 1 A ca /8 1 d3 ca eq d5 /G A eq /8 /G 1 A ca /8 1 d3 ca d5

d5

5-

G

L1

5-

d3

eq /

G eq /

1 dose

L1

L1 d3 5d5

5L1

eq A/8 /G 1 A ca /8 1 d3 ca eq d5 /G eq A/8 /G 1 A ca /8 1 d3 ca d5

0 Immunized with:

1 dose

2 doses

eq/Newm/03 wt

Fig 3. Protection conferred by the eq/GA/81 ca vaccines against homologous and heterologous challenge in the upper (A) or lower (B) respiratory tract of ferrets. Ferrets were intranasally inoculated with either L-15 (mock) or 1 or 2 doses of 107 PFU/ferret of eq/GA/81 ca vaccine and challenged 28 days following the last vaccine administration with 107 PFU/ferrets of the indicated challenge virus. Virus titers were determined on days 3 and 5 postchallenge. The dashed horizontal line represents the lower limit of detection.

Fig. 4

A 4096

NtAb titers

1024 256 64 16 4

18-32 yr

60-69 yr

³70 yr

n=19

n=19

n=18

Age groups

B

HAI antibody titers

4096 1024 256 64 16 4

18-32 yr

60-69 yr

³70 yr

n=19

n=19

n=18

Age groups

Fig 4. Serum neutralizing antibody (A) and hemagglutination inhibiting antibody (B) titers in individuals of different age groups. The serum antibody titer against eq/GA/81 (circles) and A/WI/67/05 (triangles) are shown for individual subjects. Bars identify geometric mean titer of the group. The dashed horizontal line represents the lower limit of detection.