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Title Page

Title: Characterization of recombinant influenza A virus as a vector expressing

respiratory syncytial virus fusion protein epitopes

Running title: Influenza A virus expressing RSV F epitopes

The author’s information:

Peirui Zhang, 302 Military Hospital, Beijing 100039, China

Hongjing Gu, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and

Biosecurity, Beijing 100071, China

Chengrong Bian, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen

and Biosecurity, Beijing 100071, China;

Na Liu, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and

Biosecurity, Beijing 100071, China;

Zhiwei Li, 302 Military Hospital, Beijing 100039, China

Yueqiang Duan, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and

Biosecurity, Beijing 100071, China

Shaogeng Zhang, Department of Hepatobiliary, 302 Military Hospital, Beijing 100039, China

Xiliang Wang, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and

Biosecurity, Beijing 100071, China

Penghui Yang, Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and

Biosecurity, Beijing 100071, China (E-mail: ypenghuiamms@hotmail.com)

JGV Papers in Press. Published June 9, 2014 as doi:10.1099/vir.0.064105-0

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Corresponding authors: Fax: +86-10-66948678

E-mail addresses: ypenghuiamms@hotmail.com(Penghui Yang)

Subject Category: Animal Viruses- Negative-Strand RNA

Total number of words: 4202

The number of figures: 5

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Characterization of recombinant influenza A virus as a vector expressing respiratory

syncytial virus fusion protein epitopes

Peirui Zhanga§

, Hongjing Gub§

, Chengrong Biana,b§

, Na Liub,c

, Zhiwei Lia, Yueqiang Duan

b, Shaogeng

Zhanga, Xiliang Wang

b*, Penghui Yanga,b

*

a 302 Military Hospital, Beijing 100039, China

b Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity,

Beijing 100071, China

c Department of Pathogenic Biology and Medical Immunology, School of Basic Medical Sciences, Ningxia

Medical University, Yinchuan 750004, China

§ These authors contributed equally to this paper.

* Corresponding authors: E-mail addresses: ypenghuiamms@hotmail.com

xiliangw@126.com

SUMMARY

Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants

and the elderly, and no vaccine against this virus has yet been licensed. In this report, a

recombinant PR8 influenza virus with the RSV fusion protein epitopes of the subgroup A gene

inserted into the influenza virus nonstructural (NS) gene (rFlu/RSV/F) was generated as an

RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western

blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein

(NEP) of rFlu/RSV/F were also investigated using an immunofluorescent assay. The rescued

virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line, and

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the CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with

rFlu/RSV/F had specific hemagglutination inhibition antibody responses against the PR8

influenza virus and RSV NT proteins. Furthermore, intranasal immunization with rFlu/RSV/F

elicited Th1-dominant cytokine profiles against the RSV-A2 virus. Taken together, our

findings suggest that rFlu/RSV/F is immunogenic in vivo, and warrants further development as

a promising candidate vaccine.

Keywords: Influenza virus; RSV; RSV fusion protein epitopes; Viral vector

INTRODUCTION

Since the isolation of respiratory syncytial virus (RSV) in 1956, its significance as a

human pathogen in infants and the elderly has been established (Oshansky et al., 2009). RSV is

the primary cause of hospitalization for respiratory tract illness in young children, with

infection rates approaching 70% in the first year of life (Robinson et al., 2012). Despite this

substantial disease burden, no vaccines are currently available. Therefore, development of

effective RSV vaccines is urgently needed.

In the 1960s, a formalin-inactivated RSV vaccine was used to immunize children, which

elicited non-protective, pathogenic antibodies. Immunized infants experienced increased

morbidity after subsequent RSV exposure (Sawada et al., 2011). This enhanced respiratory

disease (SRD) was thought to be due to low-affinity, poorly neutralizing antibodies and a

biased TH2 immune response to the RSV fusion (F) protein, which correlated with enhanced

lung pathology when compared with live RSV infection (Collins & Melero, 2011; Kamphuis et

al., 2012). The only currently available, effective prophylaxis for RSV is a humanized

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monoclonal antibody Synagis® (palivizumab) (MedImmune, USA) which is specific for the

RSV F protein. However, use of palivizumab may produce anallergic reaction, which is

potentially life threatening (Fuller & Del Mar, 2006; Fuller & Del Mar, 2010). Various

strategies for the development of an RSV vaccine have been considered.

Influenza viruses are attractive candidate viral vaccine vectors because they elicit strong

humoral and cell-mediated immune responses and can be manipulated by reverse genetics.

Additionally, several groups have reported the use of recombinant influenza viruses as viral

vectors for chlamydia, tuberculosis, malaria, and cancer vaccines (He et al., 2007; Miyahira et

al., 1998; Sereinig et al., 2006; Strobel et al., 2000). Furthermore, many exogenous genes, such

as bacterial chloramphenicol acetyltransferase (CAT) (Percy et al., 1994), the HIV-1 antigens

p17Gag

and Rev (de Goede et al., 2009), the Mycobacterium tuberculosis early secretory

antigenic target (ESAT-6)(Stukova et al., 2006), biologically active human interleukin-2(Kittel

et al., 2005), various influenza virus genes(Horimoto et al., 2004; Li et al., 2005), and GFP

(Kittel et al., 2004), have been tested using a recombinant influenza virus as a vector. The RSV

F protein is one of the major antigens expressed on the virion surface and contains many

neutralizing antibody epitopes and several T-cell epitopes (Singh et al., 2007). Therefore, we

hypothesized that a recombinant influenza virus containing a F protein or F protein epitopes

would induce strong RSV-specific immune responses and immunity. In the present study, we

describe the construction and characterization of rFlu/RSV/F, a recombinant influenza virus

vector expressing F protein epitopes of RSV. Using reverse genetics, a recombinant influenza

virus that contains the influenza virus PR8 backbone and RSV F protein epitopes was

generated and inserted into the nonstructural NS1 protein, encoded by the NS gene. The

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immune responses against influenza and RSV were investigated in BALB/c mice immunized

with rFlu/RSV/F.

RESULTS AND DISCUSSION

In the present study, we describe the construction and characterization of a recombinant

influenza virus (rFlu/RSV/F) containing RSV F protein epitopes produced using reverse

genetics. As shown in Fig. 1A, the NS gene segment of the recombinant influenza viruses

contains the RSV F protein epitopes. The incorporation of RSV F protein epitopes was

confirmed by Western blot with RSV polyclonal antibodies (Fig. 1B). The rFlu/RSV/F virus

had a typical lipid bilayer membrane and spherical shape with surface spikes (Fig. 1C). The

rescued virus had a peak size distribution of 80–120 nm (Fig. 1D). Thus, the rFlu/RSV/F virus

was similar to the influenza virus in both morphology and size. Additionally, the antigenic

properties of the rFlu/RSV/F virus were stable for at least 1 year at −80°C (data not shown).

Next, the expression of NS1 and NEP by the rFlu/RSV/F virus was determined with an

immunofluorescent assay. As shown in Fig. 2, the rFlu/RSV/F virus was able to express NS1

and NEP in MDCK cells 24h post-infection. NS1 and NEP staining was detected in the

cytoplasm, and no staining was detected following RSV infection. To further evaluate the

relationship between cytopathicity and viral replication in the tested cell lines, the growth

kinetics of rFlu/RSV/F and PR8 viruses were compared. MDCK, A549, and CNE-2Z cells

infected with the rFlu/RSV/F virus or the PR8 influenza virus at a MOI of 0.002 were

incubated at 35°C in 5% CO2, and virus titers were determined by PFU assay at 12, 24, 48, and

72 h post-infection. rFlu/RSV/F virus reached a peak titer of 107.2

PFU/ml 48 h after MDCK

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cell infection (Fig. 3A). The rescued viruses could also effectively replicate in A549 (Fig. 3B)

and CNE-2Z cells (Fig. 3C), similar to wild-type PR8 virus.

A specific antibody response is an important functional component of immune responses

induced by vaccination. De Baets et al. determined that PR8/NA –F (85-93), a recombinant

influenza virus that contained RSV F 85-93 epitopes in its neuraminidase stalk, provided a

significant reduction in the lung RSV viral load upon subsequent challenge with RSV. (De

Baets et al., 2013). Herein, we described the rFlu/RSV/F virus, which contains RSV F 205-223;

255-278 epitopes in its NS1 fragment. The two major distinctions between these two studies are: 1)

different RSV epitopes and 2) the different targets for RSV eptiope insertion within the

influenza virus, NA and NS. Following vaccination with rFlu/RSV/F, the antibody response to

the influenza virus and RSV F protein epitopes were detected by HI (Fig. 4A) and NT (Fig. 4B)

assays, respectively. HI and NT titers increased gradually with increasing doses of rFlu/RSV/F

after prime and boost immunizations. After two intranasal immunizations with the rFlu/RSV/F

virus, influenza virus-specific HI antibodies were induced in the serum of immunized mice.

More importantly, BALB/c mice developed RSV-specific NT antibody responses. Mice

immunized with the influenza PR8 virus were used as a negative control and neutralizing

(anti-RSV) antibodies were not detected in their sera, in contrast to mice who received the

rFlu/RSV/F virus (data not shown). These results confirm that recombinant influenza viruses

are immunogenic in mice and may be used as viral vectors. Of course, the protective efficacy of

the rFlu/RSV/F virus, including data on body weight, viral load, and pathology of rFlu/RSV/F

immunized mice post wild type RSV challenge, requires further studies. Although not

demonstrated, we anticipate that the rFlu/RSV/F virus would also induce protective functional

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antibodies to influenza and RSV in vivo and in vitro.

A Th2-biased immune response is thought to be an important factor in RSV disease. Here,

recombinant rFlu/RSV/F immunization induced Th1-type cytokines, which might play an

important role in immune regulation in anti-infection immune responses. The RSV F-specific

immune response in splenocytes was examined using IL-2, IL-4, IL-5, IFN-γ, and TNF-α

ELISA kits 2 weeks after boost immunization. As shown in Fig. 5, the levels of the Th1

cytokines IL-2, IFN-γ, and TNF-α were significantly higher than the Th2 cytokines IL-4 and

IL-5 when splenic lymphocytes were restimulated with RSV strain A2. However, these

cytokines were undetectable when culture medium was used for restimulation. Thus, our

rFlu/RSV/F candidate vaccine induced high-level Th1 responses but relatively low-level Th2

responses. This is encouraging because enhanced disease after RSV challenge is reportedly

related to elevated Th2-associated responses (Schmidt et al., 2012). Also, our data reveal that

the observed immune responses are dependent upon the rFlu/RSV/F vector, since these

responses do not occur in splenocytes isolated from mice immunized with the PR8 influenza

virus alone (data not shown).

In conclusion, recombinant influenza viruses are promising vaccine vector candidates that

may be used for the induction of antibody and cell-mediated immune responses. Of course,

additional research into the induction of humoral, cellular, and mucosal immune responses is

required to further develop recombinant influenza viruses as vaccine candidates. Additionally,

the most effective rFlu/RSV/F vaccination strategy, including the optimum route and schedule

of immunization, should be further investigated. Overall, our report demonstrates that a

recombinant influenza virus containing RSV proteins can trigger a robust immune response.

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We therefore conclude that the rFlu/RSV/F virus is a promising vaccine candidate, and its

protective effects should be confirmed in the cotton rat and monkey models.

MATERIALS AND METHODS

Viruses and cells

The wild-type influenza virus A/PR/8/34 (PR8) was inoculated into the allantoic cavity of

9-day-old specific pathogen-free (SPF) chicken eggs (Laboratory Animal Center, Beijing,

China). Three days later, allantoic fluids were harvested and stored at −70°C until use. The

RSV strain A2 (Subgroup A) was obtained from the American Type Culture Collection (ATCC)

and cultured in human laryngeal epithelial (HEp-2) cells (ATCC, Manassas, Virginia, USA)

with Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1:1) medium (GIBCO) containing

10% fetal bovine serum (FBS) at 37°C with 5% CO2. RSV virus supernatants were collected on

day 5 post-infection with centrifugation at 6000 rpm for 30 min at 4°C and stored at −70°C.

COS-1 cells (African green monkey kidney cells transformed by SV40) and Madin–Darby

canine kidney (MDCK) cells were obtained from ATCC and maintained in essential medium

(DMEM; Sigma, USA) containing 10% FBS at 37°C in 5% CO2. The medium was

supplemented with 10,000 IU/ml penicillin and 10,000 g/ml streptomycin.

Construction of recombinant rFlu/RSV/F

RSV F protein epitopes (F205-223: PIVNKQSCRI SNIETVIEF; F255-278: SELLSLIN

DMPITNDQKK LMSNNV) were inserted into the influenza virus NS gene. Notably, an

overlapping stop-start pentanucleotide cassette (TAATG) was introduced. The coding

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sequence of the RSV F epitopes was blunt end cloned downstream of the stop-start cassette to

create the pNS1-F plasmid. The sequence of the recombinant pNS1-F plasmid was synthesized

by the Shanghai Sangon Company. The cDNA of recombinant pNS1-F plasmid was cloned

into the BsmBI site of pHW2000 [origins of this plasmid], and the resulting clone was

confirmed by DNA sequencing (Chen et al., 2010). Positive plasmids carrying eight gene

segments of the PR8 virus (pHW191 to pHW198) were as described previously (Hoffmann et

al., 2001).

Rescue of the infectious rFlu/RSV/F viruses

COS-1 and MDCK cells were co-cultured in culture dishes at a ratio of 1:1. Transfection of

plasmid cDNA was performed using the Effectene transfection reagent (QIAGEN, Shanghai,

China,) by mixing 0.2 µg of each plasmid with 8 µl of the Effectene reagent diluted in 100-µl

DMEM. rFlu/RSV/F was generated by reverse genetics based on the gene segments of

influenza A/PR/8/34 as described previously (Yang et al., 2011). These viruses were amplified

in SPF chicken embryos. Allantoic fluid was collected on day 3 post-infection by

centrifugation at 3000 rpm for 20 min at 4°C. Viruses were concentrated using an

ultra-filtration membrane package (PALL, USA) and purified on a 20-30-60% discontinuous

sucrose gradient at 30000 rpm for 3 h at 4°C. Bands between 30% and 60% were collected, and

then diluted with phosphate-buffered saline (PBS). The hemagglutination titer was recorded,

and the 50% tissue culture infective dose (TCID50) was determined by serial titration of

rFlu/RSV/F virus in MDCK cells and calculated with the Reed and Muench method (Neumann

& Kawaoka, 2001).

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Western blotting and electron microscopy

Recombinant rFlu/RSV/F was characterized by Western blotting and electron microscopy.

Expression of NS1-F protein in concentrated and purified rFlu/RSV/F was evaluated by

Western blotting, as described by Sawada et al. (Sawada et al., 2011). A polyclonal goat

anti-RSV fusion protein (diluted 1:1000, ab20745, abcam) was used to probe RSV-F protein

epitopes. For size determinations, negative staining of rFlu/RSV/F was performed, followed by

transmission electron microscopy.

Virus growth

To assess viral replication, MDCK, A549, and CNE-2Z cells were infected with rFlu/RSV/F

and wild-type PR8 (multiplicity of infection (MOI) = 0.002) viruses, and the plates were

incubated at 35°C in 5% CO2. Cell culture supernatants were harvested every 12 h, and virus

titers were assayed and expressed as PFU/ml.

Immunofluorescent assay

MDCK cells were infected with rFlu/RSV/F virus at an MOI of 0.01 and harvested at 24 h.

Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100

for 5 min, blocked with 1 % BSA in PBS for 1.5 h. NS1-23-1 monoclonal antibody (SANTA

CRUZ) were used and the cells incubated for 1h at 37℃. The Cells were washed extensively

with wash buffer (PBS containing 0.2% BSA and 0.1% Triton X-100), and stained with

Fluorescein (FITC)-conjugated (Jackson ImmunoResearch Laboratories, INC) AffiniPure

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Goat Anti-Mouse second antibodies, and thereafter, NEP polyclonal antibody (GeneTex) was

used and followed by DyLight 594 (EarthOx, LLC) AffiniPure Goat Anti-Rabbit second

antibodies. Images were captured with a laser-scanning microscope.

Immunogenicity in vivo

Female BALB/c mice (Animal Experimental Center) aged 6–8 weeks were used in this study.

Groups of mice (12 per group) were immunized intranasally twice with 1×104 or 1×10

5 TCID50

of the rFlu/RSV/F virus in a 20-µL volume at 4-week intervals. Blood samples were collected

before immunization, 4 weeks after prime immunization, and 2 weeks after boost

immunization. Splenic lymphocyte (SPL) suspension samples were also collected 2 weeks

after boost immunization. All animal experiments were conducted under the guidelines of the

Academy of Military Medical Sciences Institutional Animal Care and Use Committee.

To detect rFlu/RSV/F-specific antibodies in the serum, a hemagglutination inhibition (HI)

assay was performed by standard methods using 4 hemagglutination (HA) units of PR8

influenza virus in V-bottom, 96-well microtiter plates with 0.5% turkey erythrocytes (Webster

et al., 1991). The inhibition of HA at the highest serum dilution was defined as the HI titer for

the sample. All experiments were performed three times.

Neutralization tests (NTs) against RSV were performed using the 50% plaque reduction assay,

with the RSV strain A2. Mice sera were complement inactivated, diluted serially by 1:2 each

step, and mixed with an equal volume of RSV (100 PFU). The mixtures were inoculated onto

HEp-2 cell monolayers in six-well plates. The protocol was that of Jones et al. (Jones et al.,

2012). Plaques were counted, and NT antibody titers were calculated as the reciprocal of the

serum dilution that demonstrated a 50% reduction in plaque number. All experiments were

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performed three times.

Cytokine assay

SPL suspensions were plated in 96-well plates at 1×107 cells per well, followed by

restimulation with RSV or medium as a negative control. The viral antigen concentration used

was 5 µg/ml. Cells were incubated at 37°C for 3 days, and supernatants were collected for

detection of cytokines. ELISA kits (Dakewe Biotech Company) for Th cytokines (IL-2, IL-4,

IL-5, IFN-γ and TNF-α) were used to assay cytokine production.

Statistics

GraphPad Prism 5 software was used to analyze the data (GraphPad Software Inc., San Diego,

CA). Analysis of variance (ANOVA) was used, and differences were deemed statistically

significant at p <0.05. Antibody titers for each group and error bars extending to the upper 95%

confidence limit were plotted.

ACKNOWLEDGMENTS

This work was carried out in part with funding from the National Natural Scientific Foundation

(30800977), the Ministry of Science and Technology of China (2012CB518905,

2013ZX10004003 and SS2012AA020905). P.H.Y was supported by Beijing Nova Program of

Science and Technology (No.Z141107001814054). No other potential conflict of interest

relevant to this article was reported.

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FIGURE LEGENDS

Fig. 1. Construction of influenza vaccine vectors encoding the ecto domains of the RSV

F-protein and its expression in purified rFLU/RSV/F. Virus particles containing RSV F

epitopes were visualized and their size distributions determined. (A) Schematic representation

of the NS gene segment of the recombinant influenza viruses. The RSV F protein epitopes were

inserted into the NS1 coding region. Hatched boxes represent non-translated parts. (B) RSV F

protein epitopes were purified from rFlu/RSV/F, RSV and PR8 influenza viruses by sucrose

gradient ultracentrifugation and visualized by Western blot. (C) rFlu/RSV/F virus particles

containing RSV F-protein epitopes were visible by electron microscopy after negative staining.

(D) Seventy-eight percent of 200 rFLU/RSV/F virus particles were distributed between 80 and

120 nm.

Fig. 2. The expression of NS1 and NEP proteins. MDCK cells were infected with wild type

RSV, PR8 or rFlu/RSV/F virus at an MOI of 0.01 in 24-well plate and fixed at 24 h.

Mock-infected cells were also prepared as controls. The expression of NS1 and NEP proteins

are shown. Panels 3 are merged images.

Fig. 3. Replication of rFlu/RSV/F and PR8 influenza virus in various cell lines. (A) MDCK ,

(B) A549, and (C) CNE-2Z cells were infected with rFlu/RSV/F and PR8 influenza virus at a

MOI of 0.002. Culture supernatants were obtained every 12 h post-infection. Infectivity values

are shown as mean PFU/ml in MDCK, A549, and CNE-2Z cells. These experiments were

repeated at least three times.

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Fig. 4. HI and NT antibody titers following rFlu/RSV/F immunization.(A) HI antibody titers

for PR8 influenza virus and (B) NT antibody titers for RSV strain A2 after intranasal

vaccination of BALB/c mice (ten per group) with 104 TCID50 or 10

5 TCID50 of the rFlu/RSV/F

virus in a 20 µL volume or PBS as a negative control. Sera were collected 4 weeks after prime

and boost immunizations. HI antibody titers (A) were detected using 0.5% turkey erythrocytes.

NT antibodies (B) were determined using RSV strain A2 (Subgroup A). NT titers that resulted

in a 50% reduction in plaques are expressed as 10n. * p<0.01, ** p<0.001.

Fig. 5. Splenic lymphocyte cytokine production after boost immunization. (A) IL-2 , (B) IL-4,

(C) IL-5, (D) IFN-γ, and (E) TNF-α levels in splenic lymphocyte suspensions after intranasal

boost immunization of BALB/c mice with 104 TCID50 or 10

5 TCID50 of rFlu/RSV/F. Cytokine

levels were determined by ELISA. Values are mean±SD of samples from ten mice. * p<0.01,

** p<0.001.

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Figure 2

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Figure 3

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Figure 4

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Figure 5