Vaccine 31 (2013) 6239–6246

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A replication-incompetent influenza virus bearing the HN glycoprotein of human parainfluenza virus as a bivalent vaccine Hirofumi Kobayashi a , Kiyoko Iwatsuki-Horimoto a , Maki Kiso a , Ryuta Uraki a , Yurie Ichiko a , Toru Takimoto b , Yoshihiro Kawaoka a,c,d,e,∗ a

Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 672, Rochester, NY 14642, USA c Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA d International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan e ERATO Infection-Induced Host Responses Project, Saitama 332-0012, Japan b

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Article history: Received 1 May 2013 Received in revised form 23 September 2013 Accepted 8 October 2013 Available online 19 October 2013 Keywords: Influenza virus Vaccine Mucosal immunity Antibody Hamster

a b s t r a c t Influenza virus and human parainfluenza virus (HPIV) are major etiologic agents of acute respiratory illness in young children. Inactivated and live attenuated influenza vaccines are approved in several countries, yet no vaccine is licensed for HPIV. We previously showed that a replication-incompetent PB2knockout (PB2-KO) virus that possesses a reporter gene in the coding region of the PB2 segment can serve as a platform for a bivalent vaccine. To develop a bivalent vaccine against influenza and parainfluenza virus, here, we generated a PB2-KO virus possessing the hemagglutinin-neuraminidase (HN) glycoprotein of HPIV type 3 (HPIV3), a major surface antigen of HPIV, in its PB2 segment. We confirmed that this virus replicated only in PB2-expressing cells and expressed HN. We then examined the efficacy of this virus as a bivalent vaccine in a hamster model. High levels of virus-specific IgG antibodies in sera and IgA, IgG, and IgM antibodies in bronchoalveolar lavage fluids against both influenza virus and HPIV3 were detected from hamsters immunized with this virus. The neutralizing capability of these serum antibodies was also confirmed. Moreover, the immunized hamsters were completely protected from virus challenge with influenza virus or HPIV3. These results indicate that PB2-KO virus expressing the HN of HPIV3 has the potential to be a novel bivalent vaccine against influenza and human parainfluenza viruses. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Over the last 60 years, annual vaccination has been the most effective strategy for the prevention and control of influenza virus infection [1,2]. Inactivated influenza vaccines are licensed in many countries, and in some countries live attenuated influenza vaccines are also licensed. The former provide protection with minimal reactogenicity but a short duration of effect [1,3]. The latter, by contrast, can confer longer protection and higher efficacy particularly in young children but have the potential to cause minor symptoms of respiratory illness [4]. In the US, a live attenuated influenza vaccine is currently licensed only for healthy, non-pregnant persons ranging from 2 to 49 years of age [1,2]. Hence, a novel influenza

∗ Corresponding author at: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5549 5281; fax: +81 3 5449 5408. E-mail addresses: [email protected] (H. Kobayashi), [email protected] (K. Iwatsuki-Horimoto), [email protected] (M. Kiso), [email protected] (R. Uraki), [email protected] (Y. Ichiko), toru [email protected] (T. Takimoto), [email protected] (Y. Kawaoka). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.10.029

vaccine that has minimal reactogenicity with higher efficacy is desirable. Human parainfluenza virus (HPIV) causes serious lower respiratory tract diseases in young children. It is one of the most common causes of hospitalization for fever and/or acute respiratory illness in children under 5 years of age, and in particular, in infants aged 0–5 months [5,6]. Among the four types of HPIV, a vaccine for children against HPIV type 3 (HPIV3) is most desirable as HPIV3 contributes to more than half of all of the HPIV hospitalizations annually [6,7]. The quest for an HPIV vaccine began soon after HPIV was first isolated in the 1960s. Hemagglutinin-neuraminidase (HN) glycoprotein, one of the major surface antigens of HPIV, has been targeted for triggering immunity against HPIV because of its high immunogenicity [8]. Despite efforts to develop an effective HPIV vaccine, no such vaccine had yet been licensed [7]. Considering that the hospitalization rate for influenza virus is also highest among infants aged 0–5 months, a combined vaccine of HPIV and influenza virus would decrease the burden on this population [9]. Previously, we genetically engineered a replicationincompetent PB2-knockout (PB2-KO) influenza virus that harbors a foreign gene [10], and demonstrated its potential in a mouse model to serve as a platform for bivalent vaccines [11]. In the next

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step toward the development of a bivalent vaccine for influenza and parainfluenza, here we generated a PB2-KO virus expressing the HN of HPIV3 (HPIV3-HN), and tested its efficacy as a bivalent vaccine in a hamster model.

2. Materials and methods 2.1. Cells Human embryonic kidney 293 (HEK293) and HEK293T (a derivative of the HEK293 cell line into which the gene for the simian virus 40T antigen has been inserted) cells were maintained in Dulbecco’s modified Eagle medium (Sigma–Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (Life Technologies, Carlsbad, CA). Madin–Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM) (Life Technologies) supplemented with 5% newborn calf serum (NCS) (Sigma–Aldrich). AX4 cells, which are an MDCK-derived cell line with enhanced expression of the human ␣-2,6-sialyltransferase, the human-type receptors for influenza virus [12], were maintained in 5% NCS–MEM supplemented with puromycin (2 ␮g/ml, Nacalai Tesque, Kyoto, Japan). AX4/PB2 cells (AX4 cells stably expressing the PB2 protein derived from A/Puerto Rico/8/34 [PR8]) were maintained in 5% NCS–MEM supplemented with puromycin (2 ␮g/ml, Nacalai Tesque) and blasticidin (10 ␮g/ml, Life Technologies) [10]. Rhesus monkey kidney epithelial (LLCMK2) cells were maintained in medium 199 (Life Technologies) supplemented with 1% horse serum (ATCC, Manassas, VA). All cells were maintained in a humidified incubator at 37 ◦ C in 5% CO2 .

2.2. Viruses and plasmid-driven reverse genetics The wild-type HPIV3 (strain C243) obtained from ATCC was propagated in LLC-MK2 cells. The wild-type influenza virus (PR8) and the PR8-based PB2-KO virus expressing HPIV3-HN (strain C243) from its PB2 gene (designated as PR8/PB2-HPIV3HN) used in this study were engineered by using reverse genetics as previously described [13]. We also engineered a PB2-KO virus expressing enhanced GFP (EGFP) from its PB2 gene (designated as PR8/PB2-EGFP) as a control for the PB2-KO virus vector. Briefly, for the expression of viral RNA (vRNA), plasmids containing the cloned cDNAs of the PR8 genes between the human RNA polymerase I promoter and the mouse RNA polymerase I terminator (referred to as PolI plasmids) were constructed. Plasmids [pPolIPB2(120)HPIV3HN(336)] and [pPolIPB2(120)EGFP(336)] were constructed to replace the PolI plasmid encoding the PB2 segment with a segment encoding the PR8-derived 3 PB2 noncoding region, 120 nucleotides that corresponded to the PB2-coding sequence at the 3 end of the vRNA followed by the HPIV3HN-coding or EGFP-coding sequence, 336 nucleotides that corresponded to the PB2-coding sequence at the 5 end of the vRNA, and finally the 5 PB2 noncoding region [14]. To generate the PR8/PB2-HPIV3HN or PR8/PB2-EGFP virus, pPolIPB2(120)HPIV3HN(336) or pPolIPB2(120)EGFP(336), respectively, and the remaining 7 PolI plasmids of PR8 were cotransfected into HEK293T cells along with eukaryotic protein expression plasmids for PB2, PB1, PA, and NP derived from PR8 by using the TransIT 293 transfection reagent (Mirus Bio Corp., Madison, WI), following the manufacturer’s instructions. At 48 h post-transfection, the supernatants containing the PR8/PB2-HPIV3HN or PR8/PB2-EGFP virus was harvested and inoculated into AX4/PB2 cells to make stock viruses.

2.3. Detection of the HN glycoprotein expressed from PR8/PB2-HPIV3HN AX4 and AX4/PB2 cells grown in 12-well plates (Asahi Techno Glass, Shizuoka, Japan) were inoculated with PR8/PB2-HPIV3HN or PR8/PB2-EGFP virus, and incubated for 48 h prior to being subjected to the immunofluorescence assay (IFA). Cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde (Wako, Ltd., Osaka, Japan) and permeabilized with 0.1% Triton X-100 (Nacalai Tesque). They were then incubated with a goat antiHPIV3 polyclonal antibody (ab28584, Abcam, Cambridge, UK) and a mouse anti-influenza NP monoclonal antibody (clone 2S-347/3). All cells were then incubated with an Alexa Fluor 488-labeled donkey anti-goat secondary antibody (Life Technology), an Alexa Fluor 549-labeled chicken anti-mouse secondary antibody (Life Technology), and Hoechst 33342 (Life Technologies) for the detection of influenza PR8-NP, HPIV3-HN, and nuclei, respectively.

2.4. Growth kinetics and virus titration To determine virus growth rates, triplicate wells of confluent AX4 or AX4/PB2 cells were infected with PR8/HPIV3HN, PR8/PB2EGFP or wild-type PR8 at a multiplicity of infection (MOI) of 0.001. Supernatants were collected every 12 h for 3 days and subject to plaque assays in AX4 or AX4/PB2 cells.

2.5. Genetic stability of the HN gene in PR8/PB2-HPIV3HN virus PR8/PB2-HPIV3HN virus was passaged 5 times by inoculating virus into AX4/PB2 cells at an MOI of 0.001 and incubating for 3 days. Supernatant of the fifth passage was subject to a plaque assay and IFA as described above to determine the number of plaques that retain the HN gene.

2.6. Immunization and protection tests Four-week-old female Syrian hamsters (n = 3, Japan SLC, Inc., Shizuoka, Japan) were anesthetized with Ketamine and Xylazine via intraperitoneal injection, and then intranasally inoculated with 200 ␮l/hamster of PBS, PR8/PB2-EGFP virus (109 plaque-forming units [PFU]/hamster), or PR8/PB2-HPIV3HN virus (109 PFU/hamster) three times at 2-week intervals. The PB2-KO viruses used for these immunizations were prepared by ultracentrifugation of viral supernatants in a Type19 Beckman rotor (18,000 × g, 2 h, 4 ◦ C) through a 20% sucrose cushion. Two weeks after the final immunization, hamsters were intranasally challenged with 106 PFU/hamster of PR8 virus or 104 PFU/hamster of HPIV3 (strain C243), and body weight was monitored. We first tested two inoculation volumes of virus, 50 and 200 ␮l/animal, and found that the latter was better for robust infection. We then determined the optimum amount of virus to use for the inoculum; for A/PR/8/34 virus, preliminary experiments with 106 PFU/animal resulted in consistent infection. For HPIV3, among the three virus doses (104 , 105 and 106 PFU/anima) tested, we found that virus was present longer in the lungs of animals infected with 104 PFU/animal than in those infected with higher doses. We therefore used the virus doses of 106 PFU/200 ␮l/animal for influenza virus and 104 PFU/200 ␮l/animal for parainfluenza virus. On days 3, 6 (and 9 for the HPIV3-challenged group) postchallenge, nasal turbinates, tracheae, and lungs of hamsters (n = 3) were collected after euthanasia, and homogenized. Virus titers were determined by using plaque assays with AX4 cells for PR8 virus and LLC-MK2 cells for HPIV3.

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2.7. Anti-virus antibody detection by use of an ELISA Sera and nasal washes from hamsters (n = 3) were obtained prior to each immunization. Sera were obtained via saphenous vein bleeding and nasal washes were collected by passing 400 ␮l of PBS through the nasal cavity. Sera, nasal washes, and bronchoalveolar lavage (BAL) fluids were also obtained one day before challenge (n = 3). Hamsters for BAL collection were sacrificed by exsanguination via cardiocentesis, and BAL fluids were collected by perfusing lungs with 1 ml of PBS. IgG antibodies in the sera, and IgA, IgG and IgM (designated as IgA/G/M) antibodies in the nasal washes and BAL fluids were detected by using an enzymelinked immunosorbent assay (ELISA) as previously described [15]. Briefly, flat-bottom 96-well ELISA plates (Asahi Techno Glass) were coated with purified PR8 virus or HPIV3 disrupted with 0.05 M Tris(hydroxymethyl)aminomethanehydrochloride (Tris–HCl) (pH 8.0) containing 0.5% Triton X-100 and 0.6 M KCl. After incubation of the virus-coated plates with the test samples, the IgA/G/M antibodies in the nasal washes and BAL fluids, or the IgG antibodies in the sera, were detected by using rabbit anti-hamster IgA/G/M antibodies (Brookwood Biomedical, Birmingham, AL) or goat anti-hamster IgG antibodies (Rockland Immunochemicals, Inc., Gilbertsville, PA) conjugated to horseradish peroxidase, respectively. 2.8. Virus neutralization assay Virus neutralization assays were performed as described in the WHO Manual on Animal Influenza Diagnosis and Surveillance [16] with the following modifications. Briefly, sera were treated with receptor-destroying enzyme (RDE; Denka Seiken Co., Ltd., Tokyo, Japan) to remove nonspecific virus inhibitors. PR8 virus or HPIV3 (100 50% tissue culture infectious doses [TCID50 ]/ml for both viruses) was incubated with 2-fold serial dilutions of RDE-treated sera for 30 min at 37 ◦ C, and the mixtures of virus and sera were then added to confluent AX4 or LLC-MK2 cells on 96-well microplates to determine the neutralizing activity. 2.9. Statistical analysis Statistical analysis of body weight changes and antibody levels was done with a two-tailed, unpaired Student’s t-test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Characterization of the PR8/PB2-HPIV3HN virus To examine whether the PB2-KO virus vector can accommodate HPIV3-HN as a bivalent vaccine, we generated a PB2-KO virus possessing the HN gene of HPIV3 in its PB2 segment (PR8/PB2HPIV3HN). The HN gene was flanked by the packaging signals of the PB2 segment (120 and 336 nucleotides at the 3 and 5 ends, respectively), which help the recombinant PB2 segment to be effectively incorporated into the virions [14,17]. We then confirmed HN expression in PR8/PB2-HPIV3HN virus-infected AX4/PB2 cells by use of an IFA (Fig. 1A). The coexpression of PR8-NP and HPIV3-HN was observed only in PR8/PB2-HPIV3HN virus-infected cells. These results thus demonstrate the successful generation of a PB2-KO virus expressing HPIV3-HN. We next examined the growth properties of the PR8/PB2HPIV3HN virus in AX4/PB2 cells. PR8/PB2-HPIV3HN, PR8/PB2EGFP, or wild-type PR8 virus were inoculated into both AX4 and AX4/PB2 cells. With the exception of wild-type PR8 virus, no infectious virus was detected in AX4 cells, whereas in AX4/PB2 cells, both PR8/PB2-HPIV3HN and PR8/PB2-EGFP virus replication was comparable to that of wild-type PR8 virus (Fig. 1B). These results

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indicate that the replication of PR8/PB2-HPIV3HN virus is restricted to PB2-expressing cells. The stability of HN gene incorporation into PR8/PB2-HPIV3HN virus was assessed by serially passaging the virus five times in AX4/PB2 cells. We found that after five passages 97% of the plaques expressed HN (data not shown), thus demonstrating that the HN gene is stably maintained in PR8/PB2-HPIV3HN virus. 3.2. Virus-specific antibody production in immunized hamsters To examine the antibody responses elicited upon PR8/PB2HPIV3HN virus infection, hamsters were intranasally inoculated with PR8/PB2-HPIV3HN virus, PR8/PB2-EGFP virus, or PBS three times at 2-week intervals. Sera, nasal washes, and BAL fluids were collected prior to each immunization and virus challenge, and then subject to a neutralization assay and an ELISA. The IgG antibody response to PR8 virus reached a high titer after a single immunization with PR8/PB2-EGFP or PR8/PB2-HPIV3HN virus and did not increase with additional immunizations, whereas the IgG antibody response to HPIV3 exhibited a dose-dependent increase in animals immunized with PR8/PB2-HPIV3HN virus (Fig. 2A). In nasal washes, we wanted to measure IgA and IgM responses; however, because secondary antibodies specific for hamster IgA, IgG, and IgM were not available, we could only measure the combined total IgA, IgG, and IgM responses (IgA/G/M). Although no significant differences were detected in IgA/G/M antibody responses against HPIV3, significant increases were detected in the antibody responses against PR8 after the second immunization with PR8/PB2-EGFP or PR8/PB2-HPIV3HN virus (Fig. 2B). In BAL fluids, substantial increases were observed in IgA/G/M antibody responses against both PR8 and HPIV3, although only one sample demonstrated a substantial increase against HPIV3 (Fig. 2C). No antibodies were detected in any samples from the hamsters inoculated with PBS (Fig. 2). These results demonstrate that PB2-KO virus induced antibodies against PR8 virus and that the HN expressed from the PB2 segment induced antibodies against HPIV3 in this animal model. Virus neutralizing assays were also performed to determine whether the antibodies detected by ELISA had virus neutralizing activity. All but one of the hamsters singly immunized with PR8/PB2-EGFP or PR8/PB2-HPIV3HN virus showed neutralizing capability against PR8 virus (Fig. 2D). With the additional immunizations, the virus neutralizing titers of all of the immunized animals increased to high levels. By contrast, only a few animals developed relatively low titers of neutralizing antibodies with activity against HPIV3 after two immunization and even with three immunization, not all animals developed high-neutralizing antibodies (Fig. 2E). These data suggest that three immunizations, two weeks apart, with PR8/PB2-HPIV3HN virus are appropriate for immunization against HPIV3. 3.3. Vaccine efficacy of the PR8/PB2-HPIV3HN virus To assess the vaccine efficacy of the PR8/PB2-HPIV3HN virus, hamsters immunized with the PR8/PB2-HPIV3HN, PR8/PB2-EGFP virus or PBS three times at 2-week intervals were challenged with 106 PFU/hamster of PR8 virus or 104 PFU/hamster of HPIV3 two weeks after the final immunization. Body weight changes and virus replication in the respiratory organs of challenged hamsters were evaluated as described below. 3.3.1. Body weight changes To evaluate the efficacy of vaccination, body weight changes of hamsters upon virus challenge were monitored. Upon PR8 virus challenge, hamsters mock-immunized with PBS, but not those immunized with PR8/PB2-HPIV3HN or PR8/PB2-EGFP virus,

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Fig. 1. Characterization of PR8/PB2-HPIV3HN virus. (A) Expression of HPIV3-HN from PR8/PB2-HPIV3HN virus. Confluent AX4/PB2 cells were mock-infected or infected with PR8 or PR8/PB2-HPIV3HN virus at an MOI of 1. At 12 h post-infection, cells were fixed and incubated with an anti-HPIV3 polyclonal antibody, an anti-NP monoclonal antibody, and Hoechst 33342. Scale bar, 40 ␮m. (B) Growth kinetics of PR8/PB2-HPIV3HN virus monitored over 72 h. AX4/PB2 cells were infected with wild-type PR8, PR8/PB2-EGFP, or PR8/PB2-HPIV3HN virus at an MOI of 0.001. Supernatants collected at the indicated time points were assayed for infectious virus in plaque assays with AX4/PB2 cells.

exhibited significant weight loss; however, all groups challenged with HPIV3 showed no differences in body weight (Fig. 3). 3.3.2. Virus replication in the respiratory organs We next evaluated vaccine efficacy by determining virus titers in nasal turbinates, tracheae, and lungs from vaccinated hamsters on days 3 and 6 post-challenge with PR8 virus. PR8 virus replicated to high titers in almost all organs of all mock-immunized hamsters. In contrast, no virus was detected in any organs of the hamsters immunized with PR8/PB2-EGFP or PR8/PB2-HPIV3HN virus, indicating that both viruses provided sterile immunity against PR8 virus (Table 1). We then examined whether the PR8/PB2-HPIV3HN virus could protect hamsters from HPIV3, by examining the replication of HPIV3 in the nasal turbinates, tracheae, and lungs of hamsters on days 3, 6, and 9 post-challenge with HPIV3 (Table 2). High titers of HPIV3 were detected in all organs of hamsters immunized with PR8/PB2-EGFP virus or mock-immunized until 6 days post-challenge. The hamsters immunized with PR8/PB2HPIV3HN virus, however, were completely protected from HPIV3 with no virus detected in any organs tested, indicating that protection against HPIV3 was conferred by the HN expressed from the PB2 segment. Taken together, these results demonstrate that the PR8/PB2-HPIV3HN virus can provide protective immunity against both PR8 virus and HPIV3 in the hamster model.

4. Discussion Here, we generated PR8/PB2-HPIV3HN virus, which is a PB2-KO influenza virus carrying the HN gene of HIPV3 in the coding region of its PB2 segment. We demonstrated that PR8/PB2-HPIV3HN virus elicited high antibody responses and protected hamsters from both PR8 virus and HPIV3 challenge. Using influenza virus as a vaccine vector is attractive. Earlier studies showed that neither subcutaneous nor intranasal immunization with HN glycoprotein alone completely protected hamsters from HPIV3 challenge [18]. In fact, four intranasal immunizations of both HN and F glycoproteins were required for complete protection [19,20]. Thus, PR8/PB2-HPIV3HN virus confers better protection than subunit vaccines. Another advantage of this approach is that the influenza virus vector itself can effectively elicit immunity against influenza virus. Attenuated virus strains are already approved for vaccine use, which is also advantageous [21]. However, the poor stability of the foreign gene introduced into the influenza virus has been a major problem [21]. In this study, however, we confirmed that the HN gene was stably maintained in the progeny PR8/PB2-HPIV3HN viruses. Furthermore, we found that hamsters immunized only once with PR8/PB2-HPIV3HN exhibited high antibody levels against PR8 virus in their sera, and that the second and third immunization were not compromised by preexisting immunity against PR8 virus because anti-HPIV3 antibodies increased in proportion to the number of immunizations (Fig. 2A).

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Fig. 2. Virus-specific antibody responses in immunized hamsters. Purified PR8 virus and HPIV3 were used as antigens to analyze IgG antibody levels in the sera and IgA/G/M antibody levels in the nasal washes and BAL fluids. Sera and nasal wash samples were obtained at different time points. Pre: preimmunization, 1: before the second immunization, 2: before the third immunization, 3: before challenge. BAL fluids were obtained 1 day before challenge. (A and B) Values are expressed as the mean absorbance ± standard deviation (SD) (n = 3 per group). Sera were diluted 20-fold; nasal wash samples were used undiluted. (C) Each dot represents the BAL fluid data from each hamster; horizontal bars represent the mean for each group. *p < 0.013. (D and E) Neutralizing antibody titers against PR8 virus and HPIV3, respectively. Each dot represents the neutralization titer for each hamster. N = 3 per group.

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A replication-incompetent influenza virus bearing the HN glycoprotein of human parainfluenza virus as a bivalent vaccine.

Influenza virus and human parainfluenza virus (HPIV) are major etiologic agents of acute respiratory illness in young children. Inactivated and live a...
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