Accepted Manuscript Title: Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants Author: Katharina Klingbeil Elke Lange Ulrike Blohm Jens P. Teifke Thomas C. Mettenleiter Walter Fuchs PII: DOI: Reference:

S0168-1702(15)00015-5 http://dx.doi.org/doi:10.1016/j.virusres.2015.01.009 VIRUS 96511

To appear in:

Virus Research

Received date: Revised date: Accepted date:

9-10-2014 18-12-2014 10-1-2015

Please cite this article as: Klingbeil, K., Lange, E., Blohm, U., Teifke, J.P., Mettenleiter, T.C., Fuchs, W.,Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidaseexpressing attenuated pseudorabies virus recombinants, Virus Research (2015), http://dx.doi.org/10.1016/j.virusres.2015.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 

Revised manuscript of VIRUS-D-14-00536

2 

Protection of pigs against pandemic swine origin H1N1

3 

influenza

4 

neuraminidase-expressing attenuated pseudorabies virus

5 

recombinants

virus

infection

by

hemagglutinin-

cr

6 

Katharina Klingbeil,a Elke Lange,b Ulrike Blohm,c Jens P. Teifke,b Thomas C.

8 

Mettenleiter,a and Walter Fuchsa

us

7 

9 

12 

Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal

an

11 

a

Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany b

Department of Experimental Animal Facilities and Biorisk Management, Friedrich-

M

10 

or

ip t

A

13 

Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald-Insel

14 

Riems, Germany

Institute of Immunology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal

d

16 

c

Health, 17493 Greifswald-Insel Riems, Germany

te

15 

17 

Ac ce p

18  19  20  21  22 

Corresponding author:

Walter Fuchs

23 

Friedrich-Loeffler-Institut

24  25 

Südufer 10

26 

17493 Greifswald – Insel Riems

27 

Germany

28 

phone:

+49 38351 71258

29 

fax:

+49 38351 71151

30 

e-mail:

[email protected]

31  Page 1 of 47

2   

Abstract

32 

Influenza is an important respiratory disease of pigs, and may lead to novel human

33 

pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore,

34 

improved influenza vaccines for pigs are required. Recently, we demonstrated that

35 

single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus

36 

recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge

37 

(Klingbeil, K., Lange, E., Teifke, J.P., Mettenleiter, T.C., Fuchs, W., 2014. Immunization

38 

of

39 

haemagglutinin of pandemic swine origin H1N1 influenza A virus. J. Gen. Virol. 95, 948-

40 

959). Now we investigated enhancement of efficacy by prime-boost vaccination and/or

41 

intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing

42 

codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus

43 

was only slightly affected compared to parental virus. Unlike HA, the abundantly

44 

expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with

45 

the two PrV recombinants, either singly or in combination, induced B cell proliferation

46 

and the expected SoIV-specific antibodies, whose titers increased substantially after

47 

boost vaccination. Animals immunized with either PrV recombinant were protected from

48 

disease after challenge with H1N1 SoIV, and challenge virus replication was significantly

49 

reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-

50 

expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced

51 

by combination. Despite higher serum antibody titers obtained after intramuscular

52 

immunization, transmission of challenge virus to naïve contact animals was only

53 

prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba.

attenuated

pseudorabies

virus

cr

an

recombinant

us

with

expressing

the

te

d

M

an

pigs

Ac ce p

54 

ip t

31 

55  56  57  58 

Keywords: Pandemic H1N1 SoIV; hemagglutinin (HA); neuraminidase (NA); attenuated

59 

PrV strain Bartha; vectored vaccine

60 

Page 2 of 47

3    60 

1.

Introduction

61 

Influenza A viruses belong to the family Orthomyxoviridae (King et al., 2012) and cause

63 

mainly respiratory infections in nearly all mammalian species. Frequent mutations during

64 

replication of their eight-segmented single-stranded RNA genomes, and reassortment of

65 

entire genome segments from different viruses provide a high genetic variability which

66 

facilitates adaptation to novel host species. Furthermore, antigenic variation of the two

67 

envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) permits immune

68 

escape and frequent reinfections of the same individuals. Birds are considered the

69 

natural hosts of influenza viruses in which infections are mostly asymptomatic (Palese

70 

and Shaw, 2007). In pigs the course of disease is frequently mild, and they represent

71 

important reservoir hosts which may function as mixing vessels for the development of

72 

new influenza reassortants (Webster et al., 1993). At present three different subtypes of

73 

influenza viruses, H1N1, H3N2 and H1N2 are circulating in swine worldwide (Van Reeth,

74 

2007).

75 

In 2009 a novel pandemic H1N1 influenza A virus infected the human population starting

76 

from North America. It was identified as a swine-origin influenza virus (SoIV), which

77 

contained six genome segments encoding HA, the RNA polymerase complex (PB1,

78 

PB2, PA), nucleoprotein (NP) and nonstructural proteins (NS) from a triple reassortant

79 

previously isolated from North American pigs, and NA and matrix protein (M) genes from

80 

current Eurasian H1N1 swine influenza virus (Garten et al., 2009) (Smith et al., 2009). In

81 

pigs the new virus exhibited similar clinical signs of disease and respiratory tract

82 

pathology like classical porcine influenza A viruses (Brookes et al., 2009) (Lange et al.,

83 

2009) (Vincent et al., 2010). Crossing of the species barrier by, and rapid spread of the

84 

2009 pandemic H1N1 SoIV demonstrated that more effective control strategies for

85 

influenza virus infections of swine are required. To reduce economic losses in pig

86 

husbandry and to prevent the development of further human pathogens with pandemic

87 

capacity, transmission of influenza viruses within the swine population must be limited.

Ac ce p

te

d

M

an

us

cr

ip t

62 

Page 3 of 47

4   

Commercially available influenza virus vaccines for pigs containing mixtures of

89 

inactivated H1N1, H1N2 and H3N2 subtypes do not always prevent infection (Kobinger

90 

et al., 2010), presumably because they induce good humoral, but limited cell mediated

91 

and no mucosal immune responses which are suggested to be also important for

92 

protection (Ma and Richt, 2010) (Van Reeth, 2007). Higher efficacies might be achieved

93 

with engineered, live-attenuated influenza virus vaccines containing mutations in HA

94 

cleavage site or viral polymerase (Babiuk et al., 2011) (Kappes et al., 2012) (Pena et al.,

95 

2011) (Vincent et al., 2012) which, however, bear risks from the high mutation and

96 

recombination rates of this virus family. One alternative to overcome these problems are

97 

vectored live-virus vaccines which express major influenza virus antigens, and are

98 

capable to induce humoral and cellular immunity. Such vectored vaccines may also

99 

permit differentiation of infected from vaccinated animals (DIVA) (Capua et al., 2003)

100 

(van Oirschot, 1999) by testing for antibodies against influenza virus antigens absent

101 

from the vaccine. Efficacy of vectored vaccines against highly pathogenic avian

102 

influenza A viruses based on attenuated fowlpox virus, Newcastle disease virus, or

103 

infectious laryngotracheitis virus has been demonstrated (Pavlova et al., 2009) (Taylor et

104 

al., 1988) (Veits et al., 2006). For protection of pigs against H1N1 SoIV HA-expressing

105 

recombinants of equine herpesvirus 1 (Said et al., 2013), and pseudorabies virus (PrV)

106 

(Klingbeil et al., 2014) have been evaluated.

107 

PrV or Suid herpesvirus 1 (SuHV-1) is a member of the Alphaherpesvirinae subfamily of

108 

the Herpesviridae within the order Herpesvirales (King et al., 2012). Although PrV can

109 

infect many mammals except higher primates and humans, pigs are its natural host, in

110 

which it causes respiratory disease, abortions and high mortality rates particularly of

111 

piglets (Aujeszky´s disease). Attenuated live-virus vaccine strains like PrV Bartha (PrV-

112 

Ba) (Bartha, 1961) have been successfully used for control, and enabled eradication of

113 

Aujeszky´s disease of domestic pigs in several European and North American countries

114 

(Pomeranz et al., 2005). Attenuated PrV strains were also used as viral vectors for

115 

expression of foreign antigens and conferred protection against the respective

116 

pathogens (Jiang et al., 2007) (Thomsen et al., 1987b) (van Zijl et al., 1991). In a recent

117 

study we have cloned the genome of PrV-Ba as an infectious bacterial artificial

118 

chromosome (BAC), and used this construct for insertion of the codon-optimized HA

Ac ce p

te

d

M

an

us

cr

ip t

88 

Page 4 of 47

5   

gene of pandemic H1N1 SoIV. The obtained PrV recombinant (PrV-BaMI-synH1)

120 

showed abundant HA expression in infected cell cultures, and intranasal infection of pigs

121 

induced HA-specific serum antibodies. After challenge infection with a related H1N1

122 

SoIV isolate, the animals proved to be protected from disease, and challenge virus

123 

shedding was significantly reduced (Klingbeil et al., 2014). However, HA-specific

124 

antibody titers, and the degree of inhibition of challenge virus replication varied

125 

considerably between animals.

126 

To achieve more robust protection, and reliable prevention of influenza virus

127 

transmission, we have now evaluated the effect of boost immunizations performed three

128 

weeks after initial vaccination of seven-week old piglets, and also compared the

129 

efficacies

130 

Furthermore, we constructed a second PrV-Ba recombinant expressing NA of pandemic

131 

H1N1 SoIV A/Regensburg/D6/09 (Lange et al., 2009). To enhance protein expression

132 

under control of the murine cytomegalovirus immediate early promoter (P-MCMV), a

133 

fully synthetic neuraminidase gene with PrV-adapted codon usage was generated, like

134 

previously done for the hemagglutinin gene (Klingbeil et al., 2014). In vitro replication

135 

properties of the NA-expressing PrV recombinant (PrV-BaMI-synN1) were analyzed, and

136 

in animal experiments PrV-BaMI-synN1 was applied either singly, or in a 1:1 mixture

137 

with PrV-BaMI-synH1. The H1N1 SoIV isolate A/California/7/09 (Garten et al., 2009),

138 

which is closely related to the Ha and NA gene donor isolate, was used for challenge

139 

three weeks after boost vaccination. Sera collected weekly after both immunizations and

140 

challenge infection were tested for HA- and NA-specific antibodies. Furthermore, the

141 

effects on B lymphocyte populations were investigated by flow cytometry and

142 

restimulation studies. Besides observation for clinical symptoms, challenge virus

143 

replication was quantified by real-time RT PCR (Hoffmann et al., 2010). Furthermore,

144 

shedding of infectious challenge virus was monitored using naïve contact animals.

intranasal

and

intramuscular

administration

of

PrV-BaMI-synH1.

Ac ce p

te

d

M

of

an

us

cr

ip t

119 

145 

Page 5 of 47

6   

2.

Materials and Methods

147 

2.1.

Viruses and cells

148 

Rabbit (RK13) and canine (MDCK) kidney cells were cultivated at 37°C in minimum

149 

essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and

150 

maintained in MEM containing 5% FBS and antibiotics (penicillin 100 U/ml and

151 

Streptomycin 0.1 mg/ml) after infection. For plaque assays infected cells were overlaid

152 

with semisolid medium containing 6 g/l methylcellulose. PrV-Ba (Bartha, 1961), PrV-

153 

BaMI-synH1 (Klingbeil et al., 2014), and the NA-expressing virus recombinants were

154 

grown in RK13 cells. The pandemic H1N1 SoIV isolates A/Regensburg/D6/09 (Lange et

155 

al., 2009), and A/California/7/09 (Garten et al., 2009), were propagated in MDCK cells

156 

with serum-free medium containing 2 µg/ml trypsin.

145 

M

an

us

cr

ip t

146 

157 

2.2.

Generation of PrV recombinants

159 

The neuramindase open reading frame (ORF) of H1N1 SoIV A/Regensburg/D6/2009

160 

was reverse transcribed and amplified by PCR using primers SIRN1-R and SIRN1-F

161 

(Fig. 1b). The PCR primers, as well as the synthetic, PrV-adapted NA ORF (purchased

162 

from Eurofins Genomics, Fig. 1b) contained engineered XbaI and EcoRI restriction sites

163 

for cloning into the correspondingly digested transfer vector pUC-BaKJPMI (Klingbeil et

164 

al., 2014). The resulting plasmids, together with EcoRI-digested DNA of pPrV-Ba∆gGG

165 

were used for cotransfections of RK13 cells as described (Klingbeil et al., 2014). The

166 

desired mutants PrV-BaMI-N1 and PrV-BaMI-synN1 (Fig. 1a) were isolated from

167 

transfection progenies by screening for non-fluorescent virus plaques.

Ac ce p

te

d

158 

168  169 

2.3

Western Blot and indirect immunofluorescence (IIF) analyses

170 

For Western Blot analyses RK13 cells were infected with PrV-Ba or the N1-expressing

171 

PrV recombinants PrV-BaMI-N1 and PrV-BaMI-synN1 at a multiplicity (m.o.i.) of 2 and

Page 6 of 47

7   

harvested 20 h after infection. PrV and H1N1 SoIV A/California/7/09 particles were

173 

prepared as described (Klupp et al., 2000) (Klingbeil et al., 2014). Proteins of approx.

174 

104 cells or 3 µg of virion proteins were separated by SDS-PAGE, transferred to

175 

nitrocellulose membranes, and incubated with monospecific rabbit antisera raised

176 

against bacterial fusion proteins with an avian influenza virus N1 neuraminidase (α-GST-

177 

N1) (Pavlova et al., 2009), or PrV pUL34 (α-pUL34) (Klupp et al., 2000), or against

178 

affinity-purified PrV gB (α-gB) (Kopp et al., 2003) at dilutions of 1:100,000.

179 

Chemiluminescence reactions of the peroxidase-conjugated secondary antibodies were

180 

detected as described (Klingbeil et al., 2014). For IIF tests PrV-infected RK13 cells were

181 

incubated under plaque assay conditions and fixed after 48 h with methanol and

182 

acetone (1:1) for 30 min at -20°C. The cells were blocked with 10% FBS in PBS,

183 

incubated with a 1:500 diluted antiserum from a rabbit which had been infected with a

184 

vaccinia virus recombinant expressing N1 neuraminidase of an avian influenza virus

185 

(Pavlova et al., 2009), and an Alexa Fluor 400-conjugated secondary antibody (Life

186 

Technologies) for 1 h each. After each step the cells were repeatedly washed with PBS,

187 

and analyzed by fluorescence microscopy (Eclipse Ti, Nikon).

188 

te

d

M

an

us

cr

ip t

172 

2.4.

One-step replication kinetics and determination of plaque sizes

190 

RK13 cells were infected with PrV-Ba, PrV BaMI-synH1 or PrV-BaMI-synN1 at an m.o.i.

191 

of 5. One hour after infection the inoculum was removed, non-penetrated virus was

192 

inactivated by low pH treatment (Mettenleiter, 1989), and incubation at 37°C was

193 

continued. After 0, 4, 8, 12, 24 and 48 h cells were harvested, lysed by freeze-thawing

194 

and progeny virus titers were determined by plaque assays on RK13 cells. After 3 d cells

195 

were fixed with 2% formaldehyde, and stained with crystal violet. Virus titers were

196 

determined and plaque diameters were measured microscopically.

Ac ce p

189 

197  198 

2.5.

Animal experiment and challenge virus detection

Page 7 of 47

8   

Three groups of five seven-week old pigs were vaccinated intramuscularly with 2x107

200 

plaque forming units (p.f.u.) of either PrV-PrV-BaMI-synH1, PrV-BaMI-synN1 or a 1:1

201 

mixture of both PrV recombinants. A fourth group was immunized intranasally with the

202 

same dose of PrV-BaMI-synH1. A fifth group of four animals was vaccinated

203 

intramuscularly with the same dose of PrV-Ba, and three pigs remained unvaccinated.

204 

Three weeks after primary immunization, the animals were vaccinated again (boost) with

205 

the same PrV mutant and virus dose, and in the same way like before. Another three

206 

weeks later all animals were challenged intranasally with 2x106 infectious doses

207 

(TCID50) of pandemic H1N1 SOIV A/California/7/09, and two days later two naïve

208 

contact pigs were added to each group. During the whole trial the animals were

209 

observed daily for clinical signs, and rectal temperatures were measured. Serum

210 

samples were prepared immediately before vaccination, as well as 7, 14 and 21 days

211 

after each immunization, and after challenge infection, respectively. Additional blood

212 

samples for flow cytometry were collected on days 1, 3 and 10 after challenge. Nasal

213 

swabs were taken before and 1 to 10 days after challenge infection or contact to infected

214 

animals to analyze the amount of influenza virus RNA by real-time RT-PCR as

215 

described previously (Hoffmann et al., 2010) (Klingbeil et al., 2014).

216 

Ac ce p

te

d

M

an

us

cr

ip t

199 

217 

2.6.

Immunological studies

218 

Hemagglutination inhibition (HI) assays using H1N1 SoIV A/California/7/09 as antigen

219 

were performed according to standard procedures (Said et al., 2013) (Vincent et al.,

220 

2010). Mean reciprocal values of maximum log2 serum dilutions that inhibited

221 

hemagglutination were specified as HI titers. NA-specific serum antibodies were

222 

detected using a commercial N1-specific NA-ELISA (id screen® influenza N1 Antibody

223 

Competition, IDvet) according to the manufacturer’s instructions.

224 

For analysis of B cell subpopulations, peripheral blood mononuclear cells (PBMC) were

225 

subjected to multicolor immunostaining, and analyzed by flow cytometry (BD

226 

FACSCanto™ flow cytometer, BD Biosciences). Briefly, cells collected from 50 µl of

227 

heparinized blood were suspended in FACS-buffer (0.1% BSA, 0.035% NaHCO3, 0.02%

Page 8 of 47

9   

NaN3 in Hank's balanced salt solution) and stained with fluorochrome-labeled antibodies

229 

specific for porcine lymphocyte markers for 15 min at 4°C in the dark. After washing

230 

erythrocytes were lysed by addition of 150 mM NH4Cl, and the remaining lymphocytes

231 

were separated into CD3-positive and CD3-negative subpopulations. The CD3-negative

232 

B cells were further differentiated into activated B cells (CD3-CD2-CD21+), and antibody-

233 

forming plasma cells plus memory B cells (CD3-CD2+CD21-).

234 

For In vitro restimulation of memory B cells PBMC prepared three weeks after SoIV

235 

infection were cultivated and incubated with UV-inactivated H1N1 SoIV A/California/7/09

236 

(formerly 106 TCID50/ml) for 1 h. One, 3, 5 and 7 days after after restimulation 100 µl of

237 

the culture supernatants were analyzed for HA-specific antibodies by HI assays.

an

us

cr

ip t

228 

238 

2.7.

240 

The significance of differences between virus titers, plaque sizes, Ct values, ELISA and

241 

HI titers, as well as B cell subpopulations was evaluated using Student’s t-tests.

d

te Ac ce p

242 

Statistical analyses

M

239 

Page 9 of 47

10   

3.

Results

244 

3.1.

Generation of NA-expressing PrV mutants

245 

We have previously shown that expression of the HA gene of H1N1 SoIV

246 

A/Regensburg/D6/09 in PrV-Ba could be substantially enhanced by adaptation of codon

247 

usage to that of the vector (Klingbeil et al., 2014). To investigate, whether similar effects

248 

might occur for the NA gene, we generated two PrV-Ba recombinants containing either

249 

the native, or a fully synthetic, codon-optimized, neuraminidase ORF under control of P-

250 

MCMV (PrV-BaMI-N1, PrV-BaMIsynN1; Fig. 1). In both constructs, the NA gene was

251 

preceded by an expression-promoting synthetic intron in the 5’-nontranslated part of the

252 

transcription unit (Pavlova et al., 2009), and the nonessential glycoprotein G (gG) gene

253 

locus of PrV (Thomsen et al., 1987a) was chosen as insertion site. Mutagenesis of the

254 

BAC pPrV-BaΔgGG was done as described (Klingbeil et al., 2014), and led to

255 

substitution of the bacterial vector and a GFP reporter gene by the neuramindase

256 

expression cassettes (Fig. 1). Correct insertion was verified by restriction and Southern

257 

blot analyses of genomic DNA, as well as by amplification and sequencing of the

258 

mutated genome region (data not shown).

259 

Protein expression of the virus recombinants was investigated by IIF tests and Western

260 

blot analyses (Fig. 2) with monospecific rabbit antisera raised against the NA of an avian

261 

H5N1 influenza virus (Pavlova et al., 2009). Although the overall identity of this protein to

262 

the NA of H1N1 SoIV was only 84%, the sera showed good reactivities in both assays.

263 

IIF tests of cells infected with either of the two PrV recombinants revealed a specific

264 

cytoplasmic fluorescence which was not found in PrV-Ba-infected or uninfected RK13

265 

cells, and the signals of PrV-BaMIsynN1-infected cells were significantly stronger than of

266 

cells infected with PrV-BaMI-N1 (Fig. 2a). Enhanced expression of the codon-usage

267 

adapted NA was confirmed by Western blot analyses in which the specifically detected

268 

PrV- as well as SoIV-expressed proteins exhibited an apparent mass of approx. 70 kDa

269 

(Fig. 2b, upper panel). The additional unspecific reaction of the serum with an ubiquitous

270 

nonviral protein of 68 kDa has been observed previously (Pavlova et al., 2009). Unlike

242 

Ac ce p

te

d

M

an

us

cr

ip t

243 

Page 10 of 47

11   

the influenza virus HA in PrV-BaMI-synH1 (Klingbeil et al., 2014), the NA of H1N1 SoIV

272 

was abundantly detected in purified particles of PrV-BaMI-synN1 (Fig. 2b, upper panel).

273 

A control Western blot confirmed the presence of comparable amounts of the different

274 

uncleaved and cleaved forms of PrV glycoprotein B (gB) (Whealy et al., 1990) in the

275 

analyzed infected cell lysates and virion preparations (Fig. 2b, middle panel). The purity

276 

of virion preparations was demonstrated by the absence of PrV pUL34 (Fig. 2b, lower

277 

panel), which is present in primary enveloped immature, but not in mature virus particles

278 

(Klupp et al., 2000). In view of the significantly enhanced expression of the codon-

279 

optimized compared to the authentic NA gene of SoIV A/Regensburg/D6/09, only PrV-

280 

BaMI-synN1 was used in subsequent experiments.

an

us

cr

ip t

271 

281 

3.2.

In vitro replication of NA- and HA-expressing PrV

283 

To investigate the effect of the NA transgene insertion on replication, one-step growth

284 

kinetics and plaque sizes were determined in RK13 cells (Fig. 3). As previously

285 

demonstrated for PrV-BaMI-synH1 (Klingbeil et al., 2014), PrV-BaMI-synN1 also showed

286 

lower progeny virus titers than parental PrV-Ba between 8 and 12 h after high m.o.i.

287 

infection, but final titers of all three viruses were similar (Fig. 3a). Plaque sizes of both

288 

transgene-expressing PrV recombinants were also significantly reduced by approx. 20%

289 

compared to those of PrV-Ba (Fig. 3b). No significant differences were observed

290 

between the two mutants. Since the minor in vitro growth defects did not compromise

291 

the usability of PrV-BaMI-synH1 as potential live-virus vaccine in pigs (Klingbeil et al.,

292 

2014), the new recombinant PrV-BaMI-synN1 should be also applicable in vivo.

Ac ce p

te

d

M

282 

293  294 

3.3.

B cell differentiation and antibody induction after prime-boost Immunization

295 

of pigs with HA- and NA-expressing PrV

296 

Animal experiments were performed to evaluate the suitability of PrV-BaMI-synN1 as a

297 

vectored vaccine. In these studies efficacy of the NA-expressing mutant was compared

298 

to that of HA-expressing PrV-BaMI-synH1, and a mixture of both PrV-mutants as well as

Page 11 of 47

12   

different application protocols were tested. To ensure uptake of equal virus doses by all

300 

individuals, four groups of seven-week old pigs were vaccinated intramuscularly with

301 

PrV-Ba, PrV-BaMI-synN1, PrV-BaMI-synH1, or PrV-BaMI-synN1 and PrV-BaMI-synH1

302 

(1:1), and a fifth group was vaccinated intranasally with PrV-BaMI-synH1. All animals

303 

were immunized twice at three-week intervals with 2x107 p.f.u of PrV each. Three weeks

304 

after boosting all vaccinated, as well as naïve control pigs were challenged intranasally

305 

with 2x106 TCID50 per animal of pandemic H1N1 SoIV A/California/7/09.

306 

B cell development after vaccination of pigs with PrV mutants and challenge infection

307 

with H1N1 SoIV was analyzed by flow cytometry of peripheral blood mononuclear cells

308 

(PBMC), which were stained with antibodies against pig-specific lymphocyte surface

309 

markers CD3, CD2 and CD21 (Fig. 4). CD3 is present on T- but not on B-cells, and

310 

immature B cells show a CD2+CD21+ phenotype. After activation of naïve B cells by an

311 

antigen stimulus, CD2 is down-regulated, resulting in a CD2-CD21+ cell population.

312 

During further maturation of activated B cells CD2 is re-expressed on the cell surface,

313 

and CD21 is down-regulated. Thus, antibody-forming plasma cells and memory B cells

314 

exhibit a CD2+CD21- phenotype (Sinkora and Butler, 2009).

315 

In all groups vaccinated with PrV-BaMIsynH1 the mean percentage of activated B cells

316 

(CD2-CD21+) among CD3-negative cells increased on day 14 or 21 after the first

317 

immunization (dpv) beyond the level detected in naïve control animals and remained

318 

higher until the end of the trial (Fig. 4a). An increased proportion of activated B cells was

319 

also found in pigs immunized with PrV-BaMI-synN1 alone, but not before two weeks

320 

after boost vaccination (i.e. from 35 dpv). Surprisingly, vaccination with PrV-Ba did not

321 

induce a lasting increase of activated B cells (Fig. 4a), indicating that the effects

322 

observed in the other groups were mainly caused by the overexpressed influenza virus

323 

proteins HA or NA. Challenge with H1N1 SoIV led to a temporary decrease of circulating

324 

CD3-CD2-CD21+ lymphocytes, but until two weeks after challenge (56 dpv) all animal

325 

groups which had been immunized with PrV-BaMI-synH1 and/or PrV-BaMI-synN1,

326 

exhibited higher proportions of activated B cells than non-vaccinated pigs (Fig. 4a).

327 

Maturation of activated B cells to antibody-forming plasma cells or memory B cells leads

328 

to a CD2+CD21- phenotype. In animals vaccinated with HA- and/or NA-expressing PrV

Ac ce p

te

d

M

an

us

cr

ip t

299 

Page 12 of 47

13   

recombinants the percentage of these subpopulations increased beyond the level

330 

observed in naïve pigs after the second immunization (35 dpv), and the differences

331 

became even more pronounced after challenge infection (Fig. 4b). At the end of the trial,

332 

percentages of CD2+CD-, as well as of CD2-CD21+ B cells had decreased to similar

333 

levels in all groups (Fig 4a, b).

334 

To investigate the development of HA specific antibodies serum samples were analyzed

335 

by HI assays (Fig. 5). As expected, all sera of naïve animals and of pigs immunized with

336 

PrV-Ba or PrV-BaMIsynN1 were negative until challenge. In contrast, two weeks after

337 

the first intranasal or intramuscular immunization (14 dpv) with PrV-BaMI-synH1, or with

338 

a mixture of the HA- and NA-expressing viruses (PrV-BaMI-synH1 + PrV-BaMI-synN1)

339 

most animals had developed comparable titers of HA-specific antibodies, and sera of

340 

one or two of the intramuscularly vaccinated piglets were positive as early as one week

341 

after immunization (Fig. 5a). After the second intramuscular immunization (28 dpv) an

342 

approx. 8-fold increase of HA-specific antibody titers was observed, whereas in animals

343 

immunized intranasally this boost effect was significantly less pronounced and delayed

344 

(Fig. 5a).

345 

Challenge infection three weeks after the second immunization induced similar levels of

346 

HA-specific serum antibodies in naïve and PrV-Ba immunized control animals like in

347 

pigs which had been intranasally vaccinated with PrV-BaMI-synH1 (Fig. 5b, 14dpi). In

348 

animals immunized intramuscularly with PrV-BaMI-synH1 alone or together with PrV-

349 

BaMI-synN1, HI titers re-increased to significantly higher levels (Fig. 5b). However, the

350 

maximum titers of HA-specific antibodies monitored two weeks after challenge were only

351 

slightly increased compared to those obtained after second vaccination. Interestingly,

352 

the piglets vaccinated with PrV-BaMIsyn-N1 alone developed lower amounts of HA-

353 

specific antibodies after challenge than control animals (Fig. 5b), indicating an inhibition

354 

of influenza virus replication.

355 

To investigate the effect of immunization with recombinant PrV vaccines on

356 

development of HA-specific memory B cells, peripheral blood lymphocytes isolated three

357 

weeks

358 

A/California/7/09. At different times after restimulation culture supernatants were tested

Ac ce p

te

d

M

an

us

cr

ip t

329 

after

challenge

infection

were

restimulated

with

inactivated

SoIV

Page 13 of 47

14   

for HA-specific antibodies by HI assays (Fig. 6). No reactions were detectable in

360 

lymphocyte cultures from animals which had not been vaccinated, or immunized with

361 

PrV-Ba, indicating that the period of three weeks after the first expossure to HA by the

362 

H1N1 SoIV challenge was too short for establishment of specific memory B cells.

363 

Nevertheless, the supernatant of stimulated B cells of one of five animals immunized

364 

with NA-expressing PrV-BaMI-synN1 was weakly HI-positive after 7 days (Fig. 6).

365 

Significantly higher amounts of HA-specific antibodies, increasing from day 5 to 7 after in

366 

vitro restimulation, were produced by B cells of 3 animals vaccinated intramuscularly

367 

with PrV-BaMI-synH1 and of all 5 animals vaccinated with PrV-BaMI-synH1 and PrV-

368 

BaMI-synN1 (Fig. 6). In contrast, restimulated lymphocytes of pigs which had been

369 

vaccinated intranasally with PrV-BaMI-synH1 did not produce detectable amounts of

370 

HA-specific antibodies (Fig. 6), which was in line with the relatively low HI titers in serum

371 

samples of these animals.

372 

To evaluate the development of NA-specific antibodies serum samples were analyzed

373 

using a commercial N1-specific ELISA. After the first immunization with NA-expressing

374 

PrV-BaMI-synN1, either singly or in combination with PrV-BaMI-synH1, only few piglets

375 

exhibited NA-specific antibodies at very low titers. However, one week after the second

376 

immunization (28 dpv), antibody titers had increased substantially, and all animals of

377 

both groups were positive (Fig. 7a). These antibody titers remained nearly constant after

378 

challenge infection. In contrast, the other groups, including PrV-Ba-vaccinated and naïve

379 

controls, developed almost no detectable NA-specific antibodies within 3 weeks after

380 

H1N1 SoIV challenge (Fig. 7b). Thus, our results indicate that NA-specific antibodies

381 

were induced at later times than HA-specific antibodies. On the other hand, duration of

382 

NA-specific antibody expression was obviously prolonged.

Ac ce p

te

d

M

an

us

cr

ip t

359 

383  384 

3.4.

Vaccination of pigs with NA- and HA-expressing PrV reduces H1N1 SoIV

385 

challenge virus shedding

386 

As expected, vaccination of pigs with PrV-Ba, PrV-BaMI-synH1 or PrV-BaMI-synN1 did

387 

not cause any clinical signs. After challenge infection with pandemic SoIV

Page 14 of 47

15   

A/California/7/09 vaccinated as well as most control animals also remained clinically

389 

inconspicuous, and no increase of body temperature was observed (data not shown). To

390 

investigate the effects of vaccination on influenza virus replication and shedding, nasal

391 

swabs of all pigs were taken before, and daily until 10 days after challenge infection.

392 

Total RNA was isolated from swabs and examined by real-time RT-PCR for the

393 

influenza virus M gene (Hoffmann et al., 2010). Throughout the experiment, all animal

394 

groups previously vaccinated with HA- or NA-expressing PrV recombinants, or with PrV-

395 

BaMI-synH1 and PrV-BaMIsyn-N1 showed significantly lower amounts of viral RNA,

396 

indicated by higher Ct values, than the control groups of naïve animals and PrV-Ba

397 

immunized pigs (Fig. 8). Most of these differences proved to be statistically significant.

398 

Inhibition of challenge virus replication with respect to both amount and duration was

399 

more pronounced in pigs immunized with PrV-BaMI-synH1, or PrV-BaMI-synH1 and

400 

PrV-BaMI-synN1, than in animals vaccinated with PrV-BaMI-synN1 only (Fig. 8).

401 

Furthermore, in only two to three of the pigs in groups vaccinated with PrV-BaMI-synH1

402 

could influenza virus be detected at any time, whereas all animals vaccinated

403 

exclusively with PrV-BaMI-synN1 were positive. The additive effects observed after

404 

combined intramuscular administration of both vaccine candidates were marginal, and

405 

manifested in reduction of duration of challenge virus detection to four days (2 to 5 dpi)

406 

compared to 5 days in piglets immunized with PrV-BaMI-synH1 alone, and 9 days in

407 

PrV-BaMI-synN1 vaccinated and control animals. Remarkably, the best efficacy in the

408 

present trial was achieved by intranasal prime-boost vaccination of animals with PrV-

409 

BaMI-synH1, which showed the lowest viral loads for the shortest periods of time (Fig.

410 

8). Viral RNA could be detected in swab samples of only two animals from this group for

411 

one or three days, respectively. These results confirmed the relevance of local mucosal

412 

immunity for protection against swine influenza.

Ac ce p

te

d

M

an

us

cr

ip t

388 

413  414 

3.5.

Vaccination of pigs with HA- and NA-expressing PrV affects transmission of

415 

H1N1 SoIV challenge virus to contact animals

416 

Two naïve pigs were brought into contact to each vaccinated or unvaccinated animal

417 

group two days after challenge infection with H1N1 SoIV. To investigate challenge virus Page 15 of 47

16   

transmission nasal swabs were taken before, and from day 1 to 10 after contact (dpc).

419 

Total RNA was isolated and analyzed by real-time RT-PCR as described above (Fig. 9).

420 

Challenge virus was efficiently transmitted from non-vaccinated and PrV-Ba immunized

421 

pigs, and the respective contact animals shed H1N1 SoIV over a period of nine days (1

422 

to 9 dpc) at similar amounts as directly infected, unprotected pigs (Fig. 8, Fig. 9). Swabs

423 

taken from pigs brought into contact to PrV-BaMI-synN1-vaccinated animals also

424 

contained considerable amounts of challenge virus RNA for eight days (2 to 9 dpc).

425 

Transmission of H1N1 SoIV from pigs vaccinated intramuscularly with PrV-BaMI-synH1

426 

or PrV-BaMI-synH1 and PrV-BaMI-synN1 was significantly delayed, and challenge virus

427 

was not detectable before day 6 after contact (Fig. 9). The respective contact animals

428 

also showed reduced amounts of influenza virus RNA, indicating transmission of lower

429 

virus doses than to contact animals of control- or PrV-BaMI-synN1-vaccinated pigs. One

430 

contact animal of the group immunized with HA- and NA-expressing PrV shed challenge

431 

virus for only one day, but in the other one, as well as in one contact pig of the group

432 

vaccinated intramuscularly with PrV-BaMI-synH1, could influenza virus RNA be detected

433 

until the end of the monitoring period (10 dpc). In contrast, in the two animals brought

434 

into contact to pigs vaccinated intranasally with PrV-BaMI-synH1 influenza virus RNA

435 

could not be detected at any time (Fig. 9). This finding was in line with the most

436 

pronounced inhibition of challenge virus RNA replication in this group (Fig. 8), and

437 

indicated that the amount of infectious H1N1 SoIV in nasal discharge, if present at all,

438 

were too low for successful transmission to contact animals.

cr

us

an

M

d

te

Ac ce p

439 

ip t

418 

Page 16 of 47

17    439 

4.

Discussion

440 

Recently, we used the pseudorabies virus vaccine strain Bartha as vector for expression

442 

of the HA of H1N1 SoIV, and performed initial in vivo evaluation studies of the obtained

443 

recombinant PrV-BaMI-synH1 in pigs (Klingbeil et al., 2014). In this study, we aimed at

444 

optimization of vaccination protocols. Furthermore, PrV-Ba recombinants expressing

445 

NA, of H1N1 SoIV were constructed, characterized in vitro, and included in the in vivo

446 

studies. Like previously observed for the HA gene of H1N1 SoIV (Klingbeil et al., 2014),

447 

the native NA gene under control of the strong murine cytomegalovirus immediate-early

448 

promoter/enhancer (Dorsch-Hasler et al., 1985) followed by an expression-promoting

449 

synthetic intron (Pavlova et al., 2009) was only inefficiently translated in cells infected

450 

with the corresponding recombinant PrV-BaMI-N1. However, expression could be

451 

substantially enhanced by insertion of a synthetic NA gene with PrV-adapted codon

452 

usage, which exhibits a pronounced preference for C or G nucleotides in the third

453 

position (Klupp et al., 2004). Possibly, these modifications protected the HA- and NA

454 

mRNAs from selective degradation by the herpesvirus host-shutoff RNase (Shu et al.,

455 

2013) (Taddeo et al., 2013). Furthermore, it should be noted that besides codon usage

456 

also the environments of the HA- and NA start codons were modified in the synthetic

457 

genes according to the rules for efficient translation initiation in vertebrates (Kozak,

458 

1987). Interestingly, NA was not only abundantly expressed in cells infected with PrV-

459 

BaMI-synN1, but, unlike HA in PrV-BaMI-synH1, also detected at high amounts in

460 

mature virions. Thus, our previous speculations that incorporation of influenza virus

461 

proteins into herpesvirus particles might be impeded by protein targeting to the different

462 

budding sites of the two virus families at the plasma membrane or in the trans-Golgi

463 

network, respectively (Klingbeil et al., 2014) (Pavlova et al., 2009), cannot be

464 

generalized.

465 

Compared to the parental strain PrV-Ba, the new mutant PrV-BaMI-synN1 exhibited

466 

moderately delayed in vitro replication and cell-to-cell spread which, however, did not

467 

result in a significant reduction of maximum virus titers. Similar effects have been also

468 

observed with PrV-BaMI-synH1 (Klingbeil et al., 2014). In both virus recombinants the

Ac ce p

te

d

M

an

us

cr

ip t

441 

Page 17 of 47

18   

transgenes replaced the PrV gene encoding the secreted glycoprotein gG. However,

470 

previous studies have shown that gG deletion does not affect in vitro replication of PrV

471 

(Thomsen et al., 1987a), and, thus, the observed minor replication defects were more

472 

likely due to competition of the overexpressed transgenes with functionally relevant virus

473 

genes for enzymes or components of mRNA or protein synthesis.

474 

Like PrV-Ba, PrV-BaMI-synH1 and PrV-MIsyn-N1 were completely avirulent for the

475 

seven-weeks old piglets used in our studies. Nevertheless, a single intranasal

476 

vaccination with PrV-BaMI-synH1 induced HA-specific antibodies, and conferred

477 

protection against disease after challenge with pandemic H1N1 SoIV (Klingbeil et al.,

478 

2014). However, challenge virus replication was only incompletely inhibited and sterile

479 

immunity was not achieved. Our present studies revealed that prime-boost vaccination

480 

with PrV-BaMI-synH1 induced enhanced HA-specific serum antibody titers, in particular,

481 

if the vaccine was applied intramuscularly. Flow cytometry analyses of peripheral blood

482 

lymphocytes also indicated enhanced activation and proliferation of B cells to antibody-

483 

forming plasma cells (Sinkora and Butler, 2009) after boost vaccination.

484 

Remarkably, the HA-specific serum antibody titers of piglets intramuscularly immunized

485 

with PrV-BaMIsynH1 were significantly higher than those observed after H1N1 SoIV

486 

infection of naïve animals. Similarly, intramuscular prime-boost vaccination of pigs with

487 

the new mutant PrV-BaMI-synN1 induced high titers of NA-specific antibodies, although

488 

they appeared at later times than the HA-specific ones. A slower humoral immune

489 

response against NA was also indicated by flow cytometry of B cell populations. This

490 

might explain why specific serum antibodies remained almost undetectable in control

491 

animals within three weeks after H1N1 SoIV infection. As desired, combined

492 

intramuscular immunization of pigs with PrV-BaMI-synN1 and PrV-BaMI-synH1 induced

493 

HA-, as well as NA-specific antibodies at comparable levels like monovalent

494 

vaccinations with either of the PrV recombinants.

495 

Since our present experiments confirmed that in swine clinical symptoms of infections

496 

with pandemic H1N1 SoIV are generally moderate (Lange et al., 2009) protective

497 

efficacy of vaccination had to be mainly evaluated on the basis of inhibition of challenge

498 

virus replication and spread. The analysis of nasal swabs by real-time RT-PCR revealed

Ac ce p

te

d

M

an

us

cr

ip t

469 

Page 18 of 47

19   

reduced amounts of influenza virus RNA, and shortened duration of replication in all

500 

animals immunized with HA- and/or NA-expressing PrV recombinants compared to PrV-

501 

Ba vaccinated or naïve control pigs. Remarkably, despite high NA-specific serum

502 

antibody titers, inhibition of H1N1 SoIV replication was least pronounced in animals

503 

vaccinated with PrV-BaMI-synN1. Incomplete protection was unlikely due to differences

504 

between the NA sequences of vaccine and challenge virus since they exhibited 99.8%

505 

identity (Garten et al., 2009) (Lange et al., 2009). However, earlier investigations also

506 

indicated that recombinant vaccines providing only NA do often not confer sufficient

507 

protection against influenza virus infections, but that NA-specific immune responses

508 

nevertheless can improve and broaden the efficacy of vaccination (Bodewes et al.,

509 

2010) (Chen et al., 1999) (Eichelberger and Wan, 2014) (Pavlova et al., 2009). In the

510 

present study, no unambiguous effect on reduction of challenge virus replication or

511 

spread to naïve contact pigs could be achieved by intramuscular double-vaccination with

512 

HA- and NA-expressing PrV compared to immunization with PrV-BaMI-synH1 only.

513 

Although intramuscular prime-boost vaccination of pigs with PrV-BaMI-synH1 plus PrV-

514 

BaMI-synN1, as well as with PrV-BaMI-synH1 alone reduced the amounts of H1N1 SoIV

515 

challenge virus RNA in nasal swabs much more significantly than immunization with

516 

PrV-BaMI-synN1, influenza virus transmission to naïve contact pigs was considerably

517 

delayed, but not prevented. In contrast, transmission was prevented by intranasal prime-

518 

boost vaccination of pigs with PrV-BaMI-synH1, although the titers of HA-specific serum

519 

antibodies were much lower in intranasally than in intramuscularly immunized animals.

520 

In line with the inhibition of transmission, the intranasally vaccinated pigs also showed

521 

the lowest viral loads after challenge. Compared to a single intranasal vaccination of

522 

piglets of the same age with the same dose of PrV-BaMI-synH1 (Klingbeil et al., 2014),

523 

the additional boost vaccination performed in the present study reduced duration of

524 

H1N1 SoIV replication from 7 to maximally 3 days, and the lowest mean CT values were

525 

increased from approx. 31 to 38. In previous experiments we could demonstrate a good

526 

correlation between influenza virus RNA detection in and virus re-isolation from nasal

527 

swabs, and that swab samples with CT values > 35 were usually negative in inoculated

528 

cell-cultures (Klingbeil et al., 2014). Thus, intranasal prime-boost vaccination of pigs with

529 

PrV-BaMI-synH1 expressing the HA of pandemic H1N1 SoIV A/Regensburg/D6/09 is

Ac ce p

te

d

M

an

us

cr

ip t

499 

Page 19 of 47

20   

obviously able to preclude productive replication leading to infectious progeny of a

531 

related influenza challenge virus. This result indicates once more the relevance of local

532 

mucosal antibody responses for rapid clearance of influenza virus infections in swine

533 

and other species (Cox et al., 2004) (Larsen et al., 2000) (Ma and Richt, 2010).

534 

Several previous studies revealed that pigs can be widely protected against respiratory

535 

disease caused by the 2009 pandemic H1N1 SoIV by immunization with inactivated

536 

swine influenza viruses, as well as with HA-expressing DNA- or vectored vaccines

537 

(Gorres et al., 2011) (Klingbeil et al., 2014) (Said et al., 2013) (Vincent et al., 2010).

538 

However, so far only prime-boost vaccination with an adjuvanted homologous

539 

inactivated SoIV vaccine completely prevented shedding of infectious challenge virus

540 

(Vincent et al., 2010). We have now achieved a similar efficacy by intranasal prime-

541 

boost live-virus vaccination of pigs with our described attenuated PrV recombinant PrV-

542 

BaMI-synH1 (Klingbeil et al., 2014) expressing only the HA of H1N1. Thus, this vectored

543 

vaccine should support DIVA diagnostics (van Oirschot, 1999) to control naturally

544 

occurring influenza virus infections in pigs. Since our studies also revealed that induction

545 

of NA-specific antibodies is dispensable for protective immunity against H1N1 SoIV,

546 

their presence or absence could be used for differentiation of vaccinated from infected

547 

animals. Furthermore we have shown that PrV-Ba and its derivatives can be propagated

548 

to high titers in many permanent mammalian cell lines, which would permit cost-efficient

549 

production of vectored vaccines.

550 

Although our novel NA-expressing virus recombinant PrV-Ba-synN1 conferred only

551 

limited protection and did not significantly enhance the efficacy of PrV-BaMI-synH1

552 

against homologous H1N1 SoIV challenge, it is conceivable that, due to the somewhat

553 

lower variability of NA, double-vaccination with PrV vectors expressing both influenza

554 

virus envelope proteins may improve protection against a heterologous challenge. It also

555 

remains to be tested whether intranasal vaccination with PrV-BaMI-synN1 is more

556 

efficacious than intramuscular administration, as shown for PrV-BaMI-synH1. Although

557 

intramuscular vaccination is obviously not optimal for rapid clearance of respiratory tract

558 

infections like influenza, the pronounced transgene-specific serum immune responses

559 

induced by this kind of administration of PrV-BaMI-synH1 and PrV-BaMI-synN1 might

Ac ce p

te

d

M

an

us

cr

ip t

530 

Page 20 of 47

21   

contribute to long term protection, as indicated by the detection of HA-specific memory

561 

B-cells in correspondingly immunized pigs. In addition, intramuscular vaccination of pigs

562 

or small laboratory animals with PrV-BaMI-synH1 and PrV-BaMI-synN1 might facilitate

563 

further investigations of the antigenic potential of HA and NA at cellular and molecular

564 

levels.

ip t

560 

cr

565 

Acknowledgments

567 

This study was supported by the FLUPIG project within the Seventh Framework

568 

Programme of the European Commission. The authors thank G. M. Keil for providing the

569 

MCMV promoter, O. and J. Stech for the cloned HA- and NA-genes, and B. G. Klupp for

570 

PrV-specific antisera. The technical assistance of C. Ehrlich, S. Knöfel, S. Sander, and

571 

S. Schuparis is greatly appreciated.

M

Ac ce p

te

d

572 

an

us

566 

Page 21 of 47

22    572 

References

573 

te

d

M

an

us

cr

ip t

Babiuk, S., Masic, A., Graham, J., Neufeld, J., van der Loop, M., Copps, J., Berhane, Y., Pasick, J., Potter,  A., Babiuk, L.A., Weingartl, H., Zhou, Y., 2011. An elastase‐dependent attenuated heterologous  swine  influenza  virus  protects  against  pandemic  H1N1  2009  influenza  challenge  in  swine.  Vaccine 29(17), 3118‐3123.  Bartha, A., 1961. Experimental reduction of virulence of Aujeszky’s disease virus. Magy Allatorv Lapja  16, 42‐45.  Bodewes,  R.,  Osterhaus,  A.D.,  Rimmelzwaan,  G.F.,  2010.  Targets  for  the  induction  of  protective  immunity against influenza a viruses. Viruses 2(1), 166‐188.  Brookes, S.M., Irvine, R.M., Nunez, A., Clifford, D., Essen, S., Brown, I.H., Van Reeth, K., Kuntz‐Simon,  G.,  Loeffen,  W.,  Foni,  E.,  Larsen,  L.,  Matrosovich,  M.,  Bublot,  M.,  Maldonado,  J.,  Beer,  M.,  Cattoli, G., 2009. Influenza A (H1N1) infection in pigs. Vet Rec 164(24), 760‐761.  Capua,  I.,  Terregino,  C.,  Cattoli,  G.,  Mutinelli,  F.,  Rodriguez,  J.F.,  2003.  Development  of  a  DIVA  (Differentiating  Infected  from  Vaccinated  Animals)  strategy  using  a  vaccine  containing  a  heterologous neuraminidase for the control of avian influenza. Avian Pathol 32(1), 47‐55.  Chen, Z., Matsuo, K., Asanuma, H., Takahashi, H., Iwasaki, T., Suzuki, Y., Aizawa, C., Kurata, T., Tamura,  S., 1999. Enhanced protection against a lethal influenza virus challenge by immunization with  both hemagglutinin‐ and neuraminidase‐expressing DNAs. Vaccine 17(7‐8), 653‐659.  Cox,  R.J.,  Brokstad,  K.A.,  Ogra,  P.,  2004.  Influenza  virus:  immunity  and  vaccination  strategies.  Comparison  of  the  immune  response  to  inactivated  and  live,  attenuated  influenza  vaccines.  Scand J Immunol 59(1), 1‐15.  Dorsch‐Hasler, K., Keil, G.M., Weber, F., Jasin, M., Schaffner, W., Koszinowski, U.H., 1985. A long and  complex  enhancer  activates  transcription  of  the  gene  coding  for  the  highly  abundant  immediate early mRNA in murine cytomegalovirus. Proc Natl Acad Sci U S A 82(24), 8325‐8329.  Eichelberger, M.C., Wan, H., 2014. Influenza Neuraminidase as a Vaccine Antigen. Curr Top Microbiol  Immunol.  Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., Sessions, W.M., Xu, X., Skepner,  E., Deyde, V., Okomo‐Adhiambo, M., Gubareva, L., Barnes, J., Smith, C.B., Emery, S.L., Hillman,  M.J.,  Rivailler,  P.,  Smagala,  J.,  de  Graaf,  M.,  Burke,  D.F.,  Fouchier,  R.A.,  Pappas,  C.,  Alpuche‐ Aranda,  C.M.,  Lopez‐Gatell,  H.,  Olivera,  H.,  Lopez,  I.,  Myers,  C.A.,  Faix,  D.,  Blair,  P.J.,  Yu,  C.,  Keene, K.M., Dotson, P.D., Jr., Boxrud, D., Sambol, A.R., Abid, S.H., St George, K., Bannerman,  T.,  Moore,  A.L.,  Stringer,  D.J.,  Blevins,  P.,  Demmler‐Harrison,  G.J.,  Ginsberg,  M.,  Kriner,  P.,  Waterman,  S.,  Smole,  S.,  Guevara,  H.F.,  Belongia,  E.A.,  Clark,  P.A.,  Beatrice,  S.T.,  Donis,  R.,  Katz, J., Finelli, L., Bridges, C.B., Shaw, M., Jernigan, D.B., Uyeki, T.M., Smith, D.J., Klimov, A.I.,  Cox,  N.J.,  2009.  Antigenic  and  genetic  characteristics  of  swine‐origin  2009  A(H1N1)  influenza  viruses circulating in humans. Science 325(5937), 197‐201.  Gorres,  J.P.,  Lager,  K.M.,  Kong,  W.P.,  Royals,  M.,  Todd,  J.P.,  Vincent,  A.L.,  Wei,  C.J.,  Loving,  C.L.,  Zanella,  E.L.,  Janke,  B.,  Kehrli,  M.E.,  Jr.,  Nabel,  G.J.,  Rao,  S.S.,  2011.  DNA  vaccination  elicits  protective immune responses against pandemic and classic swine influenza viruses in pigs. Clin  Vaccine Immunol 18(11), 1987‐1995.  Hoffmann, B., Harder, T., Lange, E., Kalthoff, D., Reimann, I., Grund, C., Oehme, R., Vahlenkamp, T.W.,  Beer,  M.,  2010.  New  real‐time  reverse  transcriptase  polymerase  chain  reactions  facilitate  detection and differentiation of novel A/H1N1 influenza virus in porcine and human samples.  Berl Munch Tierarztl Wochenschr 123(7‐8), 286‐292. 

Ac ce p

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  610  611  612  613  614  615  616 

Page 22 of 47

23   

te

d

M

an

us

cr

ip t

Jiang,  Y.,  Fang,  L.,  Xiao,  S.,  Zhang,  H.,  Pan,  Y.,  Luo,  R.,  Li,  B.,  Chen,  H.,  2007.  Immunogenicity  and  protective  efficacy  of  recombinant  pseudorabies  virus  expressing  the  two  major  membrane‐ associated  proteins  of  porcine  reproductive  and  respiratory  syndrome  virus.  Vaccine  25(3),  547‐560.  Kappes, M.A., Sandbulte, M.R., Platt, R., Wang, C., Lager, K.M., Henningson, J.N., Lorusso, A., Vincent,  A.L., Loving, C.L., Roth, J.A., Kehrli, M.E., Jr., 2012. Vaccination with NS1‐truncated H3N2 swine  influenza  virus  primes  T  cells  and  confers  cross‐protection  against  an  H1N1  heterosubtypic  challenge in pigs. Vaccine 30(2), 280‐288.  King,  A.M.Q.,  Adams,  M.J.,  Carstens,  E.B.,  Lefkowitz,  E.,  2012.  Virus  Taxonomy:  Ninth  Report  of  the  International Committee on Taxonomy of Viruses. Academic Press.  Klingbeil,  K.,  Lange,  E.,  Teifke,  J.P.,  Mettenleiter,  T.C.,  Fuchs,  W., 2014. Immunization of pigs with an  attenuated pseudorabies virus recombinant expressing the haemagglutinin of pandemic swine  origin H1N1 influenza A virus. J Gen Virol 95(Pt 4), 948‐959.  Klupp, B.G., Granzow, H., Mettenleiter, T.C., 2000. Primary envelopment of pseudorabies virus at the  nuclear membrane requires the UL34 gene product. J Virol 74(21), 10063‐10073.  Klupp, B.G., Hengartner, C.J., Mettenleiter, T.C., Enquist, L.W., 2004. Complete, annotated sequence of  the pseudorabies virus genome. J Virol 78(1), 424‐440.  Kobinger, G.P., Meunier, I., Patel, A., Pillet, S., Gren, J., Stebner, S., Leung, A., Neufeld, J.L., Kobasa, D.,  von  Messling,  V.,  2010.  Assessment  of  the  efficacy  of  commercially  available  and  candidate  vaccines against a pandemic H1N1 2009 virus. J Infect Dis 201(7), 1000‐1006.  Kopp,  M.,  Granzow,  H.,  Fuchs,  W.,  Klupp,  B.G.,  Mundt,  E.,  Karger,  A.,  Mettenleiter,  T.C.,  2003.  The  pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in  the cytoplasm. J Virol 77(9), 5339‐5351.  Kozak, M., 1987. An analysis of 5'‐noncoding sequences from 699 vertebrate messenger RNAs. Nucleic  Acids Res 15(20), 8125‐8148.  Lange,  E.,  Kalthoff,  D.,  Blohm,  U.,  Teifke,  J.P.,  Breithaupt,  A.,  Maresch,  C.,  Starick,  E.,  Fereidouni,  S.,  Hoffmann,  B.,  Mettenleiter,  T.C.,  Beer,  M.,  Vahlenkamp,  T.W.,  2009.  Pathogenesis  and  transmission of the novel swine‐origin influenza virus A/H1N1 after experimental infection of  pigs. J Gen Virol 90(Pt 9), 2119‐2123.  Larsen, D.L., Karasin, A., Zuckermann, F., Olsen, C.W., 2000. Systemic and mucosal immune responses  to H1N1 influenza virus infection in pigs. Vet Microbiol 74(1‐2), 117‐131.  Ma,  W.,  Richt,  J.A.,  2010.  Swine  influenza  vaccines:  current  status  and  future  perspectives.  Anim  Health Res Rev 11(1), 81‐96.  Mettenleiter, T.C., 1989. Glycoprotein gIII deletion mutants of pseudorabies virus are impaired in virus  entry. Virology 171(2), 623‐625.  Palese,  P.,  Shaw,  M.L.,  2007.  Orthomyxoviridae:  The  viruses  and  their  replication.  Fields  virology 5th  ed., 1647‐1689.  Pavlova,  S.P.,  Veits,  J.,  Keil,  G.M.,  Mettenleiter,  T.C.,  Fuchs,  W.,  2009.  Protection  of  chickens  against  H5N1  highly  pathogenic  avian  influenza  virus  infection  by  live  vaccination  with  infectious  laryngotracheitis  virus  recombinants  expressing  H5  hemagglutinin  and  N1  neuraminidase.  Vaccine 27(5), 773‐785.  Pena,  L.,  Vincent,  A.L.,  Ye,  J.,  Ciacci‐Zanella,  J.R.,  Angel,  M.,  Lorusso,  A.,  Gauger,  P.C.,  Janke,  B.H.,  Loving,  C.L.,  Perez,  D.R.,  2011.  Modifications  in  the  polymerase  genes  of  a  swine‐like  triple‐ reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1  viruses. J Virol 85(1), 456‐469.  Pomeranz, L.E., Reynolds, A.E., Hengartner, C.J., 2005. Molecular biology of pseudorabies virus: impact  on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 69(3), 462‐500. 

Ac ce p

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  656  657  658  659  660  661  662  663 

Page 23 of 47

24   

te

d

M

an

us

cr

ip t

Said,  A.,  Lange,  E.,  Beer,  M.,  Damiani,  A.,  Osterrieder,  N.,  2013.  Recombinant  equine  herpesvirus  1  (EHV‐1)  vaccine  protects  pigs  against  challenge  with  influenza  A(H1N1)pmd09.  Virus  Res  173(2), 371‐376.  Shu,  M.,  Taddeo,  B.,  Zhang,  W.,  Roizman, B., 2013. Selective degradation of mRNAs by the HSV host  shutoff  RNase  is  regulated  by  the  UL47  tegument  protein.  Proc  Natl  Acad  Sci  U  S  A  110(18),  E1669‐1675.  Sinkora, M., Butler, J.E., 2009. The ontogeny of the porcine immune system. Dev Comp Immunol 33(3),  273‐283.  Smith,  G.J.,  Vijaykrishna,  D.,  Bahl,  J.,  Lycett,  S.J.,  Worobey,  M.,  Pybus,  O.G.,  Ma,  S.K.,  Cheung,  C.L.,  Raghwani,  J.,  Bhatt,  S.,  Peiris,  J.S.,  Guan,  Y.,  Rambaut,  A.,  2009.  Origins  and  evolutionary  genomics of the 2009 swine‐origin H1N1 influenza A epidemic. Nature 459(7250), 1122‐1125.  Taddeo,  B.,  Zhang,  W.,  Roizman,  B.,  2013.  The  herpes  simplex  virus  host  shutoff  RNase  degrades  cellular  and  viral  mRNAs  made  before  infection  but  not  viral  mRNA  made  after  infection.  J  Virol 87(8), 4516‐4522.  Taylor,  J.,  Weinberg,  R.,  Kawaoka,  Y.,  Webster,  R.G.,  Paoletti,  E.,  1988.  Protective  immunity  against  avian influenza induced by a fowlpox virus recombinant. Vaccine 6(6), 504‐508.  Thomsen,  D.R.,  Marchioli,  C.C.,  Yancey,  R.J.,  Jr.,  Post,  L.E.,  1987a.  Replication  and  virulence  of  pseudorabies virus mutants lacking glycoprotein gX. J Virol 61(1), 229‐232.  Thomsen, D.R., Marotti, K.R., Palermo, D.P., Post, L.E., 1987b. Pseudorabies virus as a live virus vector  for expression of foreign genes. Gene 57(2‐3), 261‐265.  van Oirschot, J.T., 1999. Diva vaccines that reduce virus transmission. J Biotechnol 73(2‐3), 195‐205.  Van Reeth, K., 2007. Avian and swine influenza viruses: our current understanding of the zoonotic risk.  Vet Res 38(2), 243‐260.  van  Zijl,  M.,  Wensvoort,  G.,  de  Kluyver,  E.,  Hulst,  M.,  van  der  Gulden,  H.,  Gielkens,  A.,  Berns,  A.,  Moormann, R., 1991. Live attenuated pseudorabies virus expressing envelope glycoprotein E1  of hog cholera virus protects swine against both pseudorabies and hog cholera. J Virol 65(5),  2761‐2765.  Veits,  J.,  Wiesner,  D.,  Fuchs,  W.,  Hoffmann,  B.,  Granzow,  H.,  Starick,  E.,  Mundt,  E.,  Schirrmeier,  H.,  Mebatsion,  T.,  Mettenleiter,  T.C.,  Romer‐Oberdorfer,  A.,  2006.  Newcastle  disease  virus  expressing  H5  hemagglutinin  gene  protects  chickens  against  Newcastle  disease  and  avian  influenza. Proc Natl Acad Sci U S A 103(21), 8197‐8202.  Vincent,  A.L.,  Ciacci‐Zanella,  J.R.,  Lorusso,  A.,  Gauger,  P.C.,  Zanella,  E.L.,  Kehrli,  M.E.,  Jr.,  Janke,  B.H.,  Lager,  K.M.,  2010.  Efficacy  of  inactivated  swine  influenza  virus  vaccines  against  the  2009  A/H1N1 influenza virus in pigs. Vaccine 28(15), 2782‐2787.  Vincent, A.L., Ma, W., Lager, K.M., Richt, J.A., Janke, B.H., Sandbulte, M.R., Gauger, P.C., Loving, C.L.,  Webby,  R.J.,  Garcia‐Sastre,  A.,  2012.  Live  attenuated  influenza  vaccine  provides  superior  protection  from  heterologous  infection  in  pigs  with  maternal  antibodies  without  inducing  vaccine‐associated enhanced respiratory disease. J Virol 86(19), 10597‐10605.  Webster, R.G., Wright, S.M., Castrucci, M.R., Bean, W.J., Kawaoka, Y., 1993. Influenza‐‐a model of an  emerging virus disease. Intervirology 35(1‐4), 16‐25.  Whealy,  M.E.,  Robbins,  A.K.,  Enquist,  L.W.,  1990.  The  export  pathway  of  the  pseudorabies  virus  gB  homolog  gII  involves  oligomer  formation  in  the  endoplasmic  reticulum  and  protease  processing in the Golgi apparatus. J Virol 64(5), 1946‐1955.   

Ac ce p

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  704  705  706  707  708  709 

Page 24 of 47

25    709 

Figure legends

710 

Fig. 1. Construction of virus mutants. (a) A cloned genome fragment of PrV-Ba (pUC-

712 

BaKJHXAE) was used for generation of an infectious BAC (pPrV-BaΔgGG) containing

713 

an EGFP reporter cassette and the bacterial vector pMBO131 at the gG gene locus

714 

(Klingbeil et al., 2014). Transfer vector pUC-BaKJPMI permitted substitutive insertion of

715 

the authentic or codon-optimized NA gene of H1N1 SoIV A/Regensburg/D6/2009 in PrV-

716 

BaMI-(syn)N1. Relevant restriction sites, ORFs (pointed rectangles), promoters (P-

717 

MCMV), polyadenylation signals (A+), introns (IVS), and multiple cloning sites (MCS) are

718 

indicated. (b) The oligonucleotide primers used for PCR-amplification of the native NA

719 

gene (SIRN1-F/R), as well as the codon-optimized gene (synN1) contained artificial

720 

EcoRI and XbaI sites (printed in bold italics) for cloning. HA start and stop codons are

721 

underlined.

M

an

us

cr

ip t

711 

722 

Fig. 2. NA-expression of PrV recombinants. (a) For IIF analyses RK13 cells were

724 

infected with PrV-Ba, PrV-BaMI-N1, or PrV-BaMI-synN1 under plaque assay conditions.

725 

After 2 days cells were fixed and incubated with a NA-specific rabbit serum (α-vaccinia-

726 

N1) serum, and Alexa Fluor 488-conjugated secondary antibodies. (b) For Western

727 

blotting lysates of PrV-infected (c) and uninfected RK13 cells, as well as purified PrV

728 

and H1N1 SoIV particles (v) were separated by SDS-PAGE. Blots were probed with

729 

NA-, PrV gB-, and PrV pUL34-specific rabbit antisera (α-GST-N1, α-gB, α-pUL34).

730 

Molecular masses of marker proteins are indicated.

te

Ac ce p

731 

d

723 

732 

Fig. 3. In vitro replication of PrV-BaMI-synN1 and PrV-Ba. (a) For one-step growth

733 

analyses RK13 cells were infected at an m.o.i. of 5. After indicated times at 37°C the

734 

cells were harvested together with the supernatants, and progeny virus titers were

735 

determined by plaque assays. Shown are the mean results of three experiments. (b) For

736 

determination of plaque sizes infected RK13 cells were incubated 3 days under

737 

semisolid medium. Mean diameters of 50 plaques per virus as well as standard Page 25 of 47

26    738 

deviations are indicated. Statistical significance of the differences between PrV-Ba and

739 

the two mutants was calculated (*p