Interestingly, the incidence of ground glass opacities (GGO) on high resolution computed tomography seemed to be higher for virus-positive hospitalized patients than for virus-negative patients, but it did not reach statistical significance. For example, **Figure 2A** shows a patchy GGO with thickening of interlobular septa in a 28-year-old woman who was admitted during an asthma attack induced by HRV-A. **Figure 2B** also shows GGO in a 62-year-old man with an asthma attack caused by HRV-C infection. These GGO in both patients could only be detected in HRCT, not in chest X-ray.

These results suggested that HRV was the major cause of virusinduced asthma, and was possibly involved in lower airway or lung parenchyma features, appearing as GGO. Viral infection significantly exaggerated the respiratory status (low SpO2 and hypercapnea) when compared to that of virus-negative asthma exacerbative patients at the time of admission. Indeed, in recent years, HRV has been recognized as a common cause of hospital admission, both as an agent of bronchopneumonia and through exacerbation of chronic pulmonary conditions, even in the elderly over 65 years of age (Pierangeli et al., 2011).

Curiously, after initiation of treatment with intravenous steroid, both the virus-positive and -negative groups had no significant difference in duration of respiratory failure, wheezing, days in hospital, and even in the time required for steroid treatment.

#### **TREATMENT**

No established treatment for prevention of HRV-induced asthma is available, and we describe the exploratory interventions as follows.

#### **INHALED CORTICOSTEROID**

Inhaled corticosteroid (ICS) is the main drug for regular asthma therapy. ICS treatment improved airway hyperresponsiveness in asthmatic patients experimentally challenged with HRV, however, ICS treatment did not reduce accumulation of inflammatory cells, except for eosinophils in bronchial epithelium (Grunberg et al., 2001). Double-stranded RNA (dsRNA), a viral product and a ligand for the Toll-like receptor-3 (TLR3), upregulates the expression of inflammatory chemokines in airway epithelial cells. Matsukura et al. (2013) reported that treatment of BEAS-2B cells with fluticasone propionate significantly and dose-dependently inhibited dsRNA-induced expression of CCL5, CXCL8, and CXCL10 protein

**Table 4 | Clinical characterization of hospitalized patients with asthma attack based on the presence of virus infection.**

\*p < 0.05, \*\*p < 0.01. All data are presented as (mean ± SD).

and mRNA. To confirm the effect on ssRNA, such as that of HRV, would need further studies.

#### **LEUKOTRIENE RECEPTOR ANTAGONIST**

Leukotriene receptor antagonist was prescribed in asthmatic patients with or without ICS. Montelukast treatment did not improve asthma control or cold symptom scores when HRV were experimentally inoculated into mild asthmatics, or healthy subjects (Kloepfer et al., 2011). It is uncertain whether leukotriene receptor antagonist treatment is effective in the reduction of asthma symptoms associated with HRV infection.

#### **ANTI-IgE THERAPY**

Zambrano et al. (2003) reported that high serum IgE levels in mildly asthmatic children with experimental HRV infection may be associated with enhanced lower respiratory symptoms and elevation of inflammatory markers, such as nasal eosinophil cationic protein and expired nitric oxide, than those of healthy subjects and/or low IgE asthmatic patients. The prevalence of asthma was closely associated with the serum IgE levels standardized for age and sex (Burrows et al., 1989), and airway hyperresponsiveness appears to be closely linked to the allergic diathesis, as reflected by the serum total IgE level (Sears et al., 1991). Omalizumab, an anti-IgE monoclonal antibody, was indicated in inadequately controlled moderate-to-severe persistent allergic asthma patients who were treated with high dose ICS. Durrani et al. (2012) showed that the IgE receptor FcεRI is inversely associated with IFN-α and IFN-λ1 secretion when plasmacytoid dendritic cells derived from allergic asthmatic children were challenged with HRV. Omalizumab downregulates FcεRI expression on dendritic cells (Prussin et al., 2003), which may reduce exacerbation of asthma associated with increased production of IFNs, through FcεRI.

#### **ANTI-VIRAL TREATMENT**

No drugs are clinically used in HRV infection, although several drugs have been tried for treatment and prevention of HRV infection. These drugs are summarized in a review (Jacobs et al., 2013). IFNs had a potential protective role in viral induced asthma (Cakebread et al., 2011; Gaajetaan et al., 2013). Becker et al. (2013)showed that exogenous IFN-α, IFN-β, IFN-λ1, and IFN-λ2 inhibited HRV replication in BECs from healthy donors.

#### **MACROLIDE THERAPY**

Macrolides are known to possess anti-inflammatory and immunomodulatory actions extending beyond their antibacterial activity in pulmonary inflammatory disorders (Takizawa et al., 1995; Min and Jang, 2012). Erythromycin inhibits HRV infection by reducing ICAM-1 expression on the surface of human tracheal epithelial cells, and modulates inflammation by suppressing the production of proinflammatory cytokines (Suzuki et al., 2002).

#### **OTHER AGENTS**

Yamaya et al. (2014) reported that the mucolytic drug ambroxol hydrochloride, antibiotic drug of levofloxacin (Yamaya et al., 2012b), and bronchodilators (Tiotropium, Tulobuterol, and Procaterol) for asthma or COPD (Yamaya et al., 2011, 2012a, 2013) may have a beneficial effect in HRV infection, by inhibiting HRV replication and partly reducing ICAM-1 expression and acidic endosome production, via the inhibition of NF-kappaB activation (Yamaya, 2012).

#### **SUMMARY**

We reviewed the previous reports regarding HRV-induced asthma exacerbations, together with our results from an institutional prospective study. HRV is a major pathogen for asthma exacerbations, and certainly associated with more serious clinical conditions such as hypoxemia or hypercapnea in hospitalized patients. Further accumulation of evidence of virus-induced asthma for multidisciplinary assessment would be helpful for physicians in recognizing the condition or understanding the pathogenic mechanisms.

#### **REFERENCES**

Andries, K., Dewindt, B., Snoeks, J., Wouters, L., Moereels, H., Lewi, P. J., et al. (1990). Two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity. *J. Virol.* 64, 1117– 1123.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 March 2014; accepted: 28 April 2014; published online: 26 May 2014. Citation: Saraya T, Kurai D, Ishii H, Ito A, Sasaki Y, Niwa S, Kiyota N, Tsukagoshi H, Kozawa K, Goto H and Takizawa H (2014) Epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus. Front. Microbiol. 5:226. doi: 10.3389/fmicb.2014.00226*

*This article was submitted to Virology, a section of the journal Frontiers in Microbiology. Copyright © 2014 Saraya, Kurai, Ishii, Ito, Sasaki, Niwa, Kiyota, Tsukagoshi, Kozawa, Goto and Takizawa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Influenza A(H1N1)pdm09 virus and asthma

## *Masatsugu Obuchi 1\*, Yuichi Adachi 2 , Takenori Takizawa1 and Tetsutaro Sata1*

1 Department of Virology, Toyama Institute of Health, Toyama, Japan

2 Department of Pediatrics, Faculty of Medicine, University of Toyama, Toyama, Japan

#### *Edited by:*

Hirokazu Kimura, National Institute of Infectious Diseases, Japan

#### *Reviewed by:*

Jianwei Wang, Chinese Academy of Medical Sciences, Peking Union Medical College, China Wei Zhang, Yele University, USA

#### *\*Correspondence:*

Masatsugu Obuchi, Department of Virology, Toyama Institute of Health, 17-1 Nakataikoyama, Imizu-shi, Toyama 939-0363, Japan e-mail: masatsugu.obuchi@pref. toyama.lg.jp

Respiratory viral infection is a major cause of asthma exacerbations in both children and adults. Among the respiratory viruses, influenza virus is a particularly important pathogen due to its enormous morbidity and mortality in annual epidemics.The swine-origin influenza A virus, designated as A(H1N1)pdm09, emerged in the spring of 2009 and caused the first influenza pandemic in the 21st century. With the emergence of the novel A(H1N1)pdm09 virus, numerous epidemiologic studies detected asthma as a frequent comorbid condition in patients infected with this virus. Here we review recent reports regarding asthma in patients infected with influenza A(H1N1)pdm09 virus, and we discuss the utility of influenza vaccines and antivirals.

**Keywords: asthma, pandemic influenza, influenza A(H1N1)pdm09 virus, vaccine, antiviral drug**

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### **INTRODUCTION**

Asthma is a chronic airway disease with the symptoms of repetitive cough, wheezing and dyspnea, with reversible airway narrowing accompanied by airway hyper-responsiveness (Ohta et al., 2011). It is estimated that worldwide, approximately 300 million people including both children and adults have asthma (Masoli et al., 2004). Inhaled irritants, inhaled allergens, and microorganism infections of the respiratory tract are common causes of asthma exacerbations. Respiratory viral infection is closely associated with asthma (Jacoby, 2002; Papadopoulos et al., 2011). Human rhinovirus (HRV) is the most common virus in asthmatics of all ages (Papadopoulos et al., 2011). Respiratory syncytial virus and enterovirus are also frequently detected in infants, whereas influenza virus seems to induce severe exacerbations, mostly in adults (Papadopoulos et al., 2011).

Influenza virus causes influenza characterized by a sudden onset of high fever and respiratory symptoms such as cough, sore throat and coryza, as well as systemic symptoms such as headache, muscle ache and fatigue. Influenza epidemics occur yearly during the autumn and winter in temperate regions, whereas the disease patterns in tropical and subtropical regions are less well established (World Health Organization, 2009). Annual epidemics result in approximately three to five million cases of severe illness and approximately 250,000 to 500,000 deaths, which occur mostly among people age 65 or older (World Health Organization, 2009).

#### **INFLUENZA A(H1N1)pdm09 VIRUS**

Influenza A viruses can be subtyped according to their two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Currently, there are 16 subtypes of HA (H1–H16) and nine subtypes of NA (N1–N9), and all have been found in wild aquatic birds, which are the natural reservoir of influenza A viruses. Only two subtypes of these viruses (H1N1 and H3N2) are currently circulating in humans, as seasonal influenza. Influenza A viruses have negative-sense, single-stranded, and eight-segmented RNAs as the genome (Lamb and Choppin, 1983). It is known that simultaneous infection of a single cell by two distinct influenza A viruses can lead to gene reassortment (Hause et al., 2012), which results in the generation of a novel influenza virus strain. It is believed that most human pandemic influenza A viruses arose in this manner.

In March and early April 2009, a novel swine-origin influenza A(H1N1) virus, designated as A(H1N1)pdm09, emerged in Mexico and the United States (Centers for Disease Control and Prevention, 2009) and rapidly spread worldwide. Genetic and evolutionary analyses revealed that this pandemic virus contains a combination of gene segments which had not been reported previously in swine or human influenza viruses in any part of the world. In the late 1990s, reassortment among North American avian (unknown subtype), human A(H3N2), and classical swine A(H1N1) viruses resulted in triple reassortant swine A(H3N2) and A(H1N2) viruses.

 The polymerase basic 2 (PB2) and polymerase acidic (PA) gene segments were derived from the avian virus lineage, whereas the polymerase basic 1 (PB1) gene segment was from human A(H3N2) virus. The HA, nucleoprotein (NP), and nonstructural protein (NS) gene segments were from classical swine A(H1N1) virus. The NA and matrix (M) gene segments were from the Eurasian avian-like swine A(H1N1) virus.

A(H1N1)pdm09 virus has none of the known hallmarks of virulent influenza viruses such as highly pathogenic avian A(H5) and A(H7) viruses, except for an amino acid substitution of aspartic acid by glycine at position 222 (D222G) in the HA, which was observed in severe and fatal cases with high frequency. The D222G substitution changes the receptor binding specificity of the virus from α2–6 (mammalian type) to α2–3 (avian type) sialylated

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glycans (Puzelli et al., 2010; World Health Organization, 2010; Belser et al., 2011). This amino acid substitution may result in a more efficient infection of human alveolar type II pneumocytes, which express avian type receptors, reducing the availability of progenitor cells for essential lung functions and thus leading to severe pulmonary impairment.

We recently reported that A(H1N1)pdm09 viral isolates derived fromfatal cases manifested sporadic amino acid changes in the PB2 and PA proteins (which are subunits of viral RNA polymerase) more frequently than those derived from mild cases (Obuchi et al., 2012). More recently, reassortant viruses generated by reverse genetics have shown that lysine or isoleucine at position 340 or 649 of the PB2, respectively, and threonine at position 667 of the PB2 also contribute to virulence in a mouse model (Uraki et al., 2013). Further studies are needed to elucidate the role of the viral RNA polymerase of A(H1N1)pdm09 virus as a virulence factor.

## **A(H1N1)pdm09 VIRAL INFECTION AND ASTHMA**

Widespread activity of pandemic A(H1N1) 2009 occurred and reached its peak a couple of months earlier than the usual seasonal influenza in the northern hemisphere, from April 2009 to January 2010 (Amato-Gauci et al., 2011; Jhung et al., 2011). The A(H1N1)pdm09 viral infection was considered a mild disease, similar to seasonal influenza. However, many severe and fatal cases were observed not only in the high-risk groups, but also among healthy children and young adults during the pandemic waves (Athanasiou et al., 2010; Reichert et al., 2010).

Asthma was one of the most common underlying medical conditions among patients hospitalized with A(H1N1)pdm09 viral infection in 2009 worldwide (Jain et al., 2009; Van Kerkhove et al., 2011). Kloepfer et al. (2012) reported that children with asthma had increased susceptibility to A(H1N1)pdm09 viral infection. They collected weekly nasal samples from 161 children (95 with asthma and 66 without asthma) between September 5 and October 24, 2009, and a total of 346 viral infections were detected. The majority were HRV (62%), followed by enterovirus (12%), A(H1N1)pdm09 virus (10%), adenovirus (2%), and others. Overall, 34% of the children were infected with A(H1N1)pdm09 virus during the study period. The incidence of A(H1N1)pdm09 viral infection was significantly higher in the children with asthma (41%) than in the children without asthma (24%), whereas the incidences of HRV (95% each) and the other viral infections (47% vs. 41%) were similar.

A Canadian group reported that children admitted to a hospital with A(H1N1)pdm09 viral infection tended to have pre-existing asthma to a greater extent compared to the children with seasonal influenza A viral infection (15% vs. 5%), although there was no significant difference in the severity of pre-existing asthma between the groups of children with these infections (Morris et al., 2012). An age-matched control study in Hong Kong demonstrated that hospitalized children with A(H1N1)pdm09 viral infection were more susceptible to asthma exacerbations compared to seasonal A(H1N1) (8.1 vs. 1.0%) or A(H3N2) (8.1 vs. 1.0%) viral infection (Chiu et al., 2011). A Japanese group reported similar findings (Hasegawa et al., 2011). It seems likely that A(H1N1)pdm09 viral infection rather than A(H1N1) or A(H3N2) viral infection may enhance the already elevated inflammatory response and worsen the symptoms in asthma. The underlying mechanisms of increased susceptibility to A(H1N1)pdm09 viral infection and the asthma exacerbation remain to be explored.

In contrast, it is not clear whether A(H1N1)pdm09 viral infection can frequently cause the development of asthma compared to seasonal A(H1N1) or A(H3N3) viral infection. The study by Hasegawa et al. (2011) mentioned above showed that 7 (31.8%) of 22 asthmatic children with A(H1N1)pdm09 viral infection admitted to a hospital between October and December 2009 were not previously diagnosed with asthma. The sample size of that study is small, and thus a larger patient population must be studied.

Influenza A viral infection induces the production of interleukin 1 beta (IL-1β), IL-6, IL-8, tumor necrosis factor-alpha (TNF)-α, histamine, protease, interferon (IFN)-α, and IFNgamma (IFN-γ) from airway epithelial cells and other cells including peripheral blood basophils (Yamaya,2012). These proinflammatory cytokines, monokines, and inflammatory substances may contribute to the development of airway inflammation, damaging the barrier function and leading to a subsequent asthma attack (**Figure 1**).

Camp et al. (2013) examined the phenotypic differences in virulence and immune response in A(H1N1)pdm09 virus isolates obtained from hospitalized patients with severe pneumonia. In that study, all viral isolates showed high similarity in nucleic acid sequences in viral gene and replication levels in nasal turbinates and lung, but the isolates' virulence and host responses in mice varied. Proinflammatory cytokines such as IL-1β, TNF-α and a keratinocyte-derived chemokine (KC) were expressed early in mice infected with virulent isolates compared to avirulent isolates, including a vaccine strain of A(H1N1) virus in the 2008–2009 season, A/Brisbane/59/2007. In vitro experiments demonstrated that a virulent isolate – but not an avirulent isolate – was able to replicate productively in macrophages, suggesting that viral susceptibility to macrophages may be one of the key determinants of their pathogenicity (Camp et al., 2013).

## **UTILITY OF INFLUENZA VACCINES AND ANTIVIRAL DRUGS IN PATIENTS WITH ASTHMA**

Many respiratory viruses are associated with asthma exacerbations, among which the influenza virus is the only virus for which both vaccines and antiviral drugs are available (**Figure 1**). Two types of influenza vaccines are currently available; inactivated vaccine and live, attenuated vaccine. The live, attenuated nasal-spray influenza vaccine has been approved for use in the United States since 2003. However, it has not been recommended in high-risk groups including asthmatics because its safety is not fully demonstrated. The widespread use of inactivated influenza vaccines contain a trivalent mixture of strains of A(H1N1)pdm09, A(H3N2), and type B viruses likely to circulate during the next influenza season. Many studies indicated that no increase in asthma exacerbations was reported for both vaccinated children and adults (American Lung Association Asthma Clinical Research Centers, 2001; Kramarz et al., 2001; Hak et al., 2005).

A randomized, open-label study to investigate the safety and immunogenicity of two administrations of an unadjuvanted, inactivated A(H1N1)pdm09 virus vaccine was conducted in the United States (Busse et al., 2011). The results indicated that both the 15 μg (standard dose) and 30-μg vaccine doses generally provided excellent seroprotection against viral antigen 21 days after a single immunization in patients (12 to 79 years of age) with mild-tomoderate asthma. In patients with severe asthma, the response to the 15-μg dose was lower than that to the 30-μg dose. The authors of that study did not identify any safety concerns with the A(H1N1)pdm09 vaccine. Collectively, the findings described above indicate that inactivated influenza vaccines are well tolerated in patients with asthma.

Specific antiviral drugs against influenza viruses could be used for the treatment and prophylaxis for influenza. Based on their chemical properties and spectra of activity against influenza viruses, the drugs can be classified into two categories: the M2 ion channel inhibitors, i.e., adamantanes (amantadine and rimantadine) and the NA inhibitors, i.e., zanamivir, oseltamivir, peramivir, and laninamivir. Currently, the NA inhibitors are exclusively used for the treatment and prophylaxis of influenza because the circulating strains of A(H1N1)pdm09 and A(H3N2) viruses have a known amino acid substitution of serine by asparagines at position 31 in the M2 protein, which confers resistance to the adamantanes.

Few studies have examined the safety of NA inhibitors in patients with asthma. A double-blind, placebo-controlled crossover study indicated that zanamivir inhaled as a dry powder did not significantly affect the pulmonary function and airway responsiveness of subjects (19 to 49 years of age) with mild or moderate asthma (Cass et al., 2000). However, a number of studies suggested that the use of NA inhibitors was beneficial in hospitalized patients with A(H1N1)pdm09 viral infection, particularly when they are started within 48 h after the onset of illness (Domínguez-Cherit et al., 2009; Jain et al., 2009; Louie et al., 2010). Accordingly, WHO recommended that for patients at increased risk for severe or complicated illness, treatment with oseltamivir or zanamivir should be started as soon as possible after the onset of illness (Writing Committee of the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 influenza, 2010). Although A(H1N1)pdm09 virus resistance to NA inhibitors has been detected at very low frequency among circulating viral strains (World Health Organization, 2013b), there is concern about the recent report that oseltamivir-resistant A(H1N1)pdm09 viral mutants were detected in untreated patients and from a few clusters in some countries (Samson et al., 2013).

## **CLOSING REMARKS**

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Epidemiological studies as described above demonstrated that A(H1N1)pdm09 viral infection is closely associated with asthma in both children and adults. Although A(H1N1)pdm09 virus has not shown a high mortality rate similar to that of the highly pathogenic avian influenza virus of the H5N1 subtype, patients with A(H1N1)pdm09 viral infection were more susceptible to asthma exacerbation compared to A(H1N1) or A(H3N2) viral infection. Detailed analyses of virus-host interactions are needed to elucidate the mechanism underlying A(H1N1)pdm09 viral infection-induced asthma.

Since March 31, 2013 when the public health authorities of China reported three cases of human infection with an avian influenza A(H7N9) virus, a total of 135 human cases including 44 fatal cases have been reported in China and Taiwan as of August 12, 2013 (World Health Organization, 2013a). The current avian influenza viral infections in humans present considerable pathogenic potential with high mortality rates, suggesting that the pandemic viruses, if they emerge in human beings, could also present high pathogenicity and result in an excessive number of

### **REFERENCES**

A (H1N1) infection in two children – southern California, March-April 2009. *Morb. Mortal Wkly. Rep.* 58, 400–402.

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deaths in high-risk groups, including asthmatics. It will therefore be important to make preparations for drugs and vaccines for anti-influenza treatments and the prophylaxis of influenza.

#### **ACKNOWLEDGMENTS**

This work was supported in part by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases, Labour and Welfare Programs from the Ministry of Health, Labour and Welfare of Japan (H24-Shinko-Ippan-014).

Louie, J., et al. (2009). Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009. *N. Engl. J. Med.* 361, 1935–1944. doi: 10.1056/NEJMoa0906695

of pandemic A(H1N1)2009 influenza virus in a mouse model. *J. Virol.* 87, 2226–2233. doi: 10.1128/JVI. 01565-12

(H1N1) 2009 viruses. *Wkly. Epidemiol. Rec.* 85, 21–22.

(2010). Clinical aspects of pandemic 2009 influenza A (H1N1) virus infection. *N. Engl. J. Med.* 362, 1708–1719. doi: 10.1056/NEJMra 1000449

Yamaya, M. (2012). Virus infectioninduced bronchial asthma exacerbation. *Pulm. Med.* 2012, 834826. doi: 10.1155/2012/834826

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 28 June 2013; accepted: 24 September 2013; published online: 14 October 2013.*

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*Citation: Obuchi M, Adachi Y, Takizawa T and Sata T (2013) Influenza A(H1N1)pdm09 virus and asthma. Front. Microbiol. 4:307. doi: 10.3389/ fmicb.2013.00307.*

*This article was submitted to Virology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2013 Obuchi, Adachi, Takizawa and Sata. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, providedthe original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Development of oligomannose-coated liposome-based nasal vaccine against human parainfluenza virus type 3

## *Kyosuke Senchi 1, Satoko Matsunaga1, Hideki Hasegawa2 , Hirokazu Kimura3 and Akihide Ryo1\**

1 Department of Microbiology, Yokohama City University School of Medicine, Yokohama, Kanagawa, Japan

2 Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan

3 Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan

#### *Edited by:*

Hironori Sato, National Institute of Infectious Diseases, Japan

#### *Reviewed by:*

Komei Shirabe, Yamaguchi Prefectural Institute of Public Health and Environment, Japan Ichiro Aoki, Yokohama City University School of Medicine, Japan

#### *\*Correspondence:*

Akihide Ryo, Department of Microbiology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan e-mail<EMAIL_ADDRESS>
Human parainfluenza viruses (HPIVs) are the etiologic agents of lower respiratory infections and pneumonia in infants, young children and immunocompromised hosts.The overarching goal for the prevention of HPIV infection is the development of an effective vaccine against HPIVs. In the present study, we investigated the effectiveness of oligomannose-coated liposomes (OMLs) as an antigen-delivery system in combination with a synthetic doublestranded RNA analog for the induction of mucosal and systematic immunity against HPIV3. Full-length hemagglutinin-neuraminidase (HN) protein was synthesized using the wheat germ cell-free protein production system and then encapsulated into OML to serve as the antigen. Intranasal administration of the HN-filling OML (OML-HN) with the synthetic double-stranded RNA adjuvant, polyriboinosinic-polyribocytidylic acid [poly(I:C)] generated significant viral-specific systemic and mucosal immune responses as evidenced by the prominent induction of serum IgG and nasal wash IgA, respectively. On the other hand, no significant immune responses were observed in mice immunized with OML-HN without the adjuvant. Furthermore, serum from mice immunized with OML-HN plus poly(I:C) significantly suppressed viral infection in cell culture model. Our results provide the first evidence that intranasal co-administration of OML-encapsulated HN with the poly(I:C) adjuvant augments the viral-specific immunity against HPIV3.

**Keywords: HPIV3, HN, vaccine, oligomannose-coated liposome, adjuvant**

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## **INTRODUCTION**

Human parainfluenza viruses (HPIVs) belong to the Paramyxoviridae family and are one of the major causes of acute respiratory infections (ARIs) and asthma in infants and young children (<5 years old). HPIVs were classified into four serotypes including HPIV1-4 (Henrickson, 2003; Mizuta et al., 2011). In particular, human parainfluenza virus type 3 (HPIV3) is an important infectious agent, second only to respiratory syncytial virus (RSV), that causes bronchiolitis and pneumonia in infants (Glezen et al., 1984; Counihan et al., 2001; Belshe et al., 2004; Schmidt, 2011). Therefore, the development of a practical vaccine that can prohibit HPIV3 infection in infants is urgently needed.

Currently, there is no prophylactic human vaccine against HPIV3 infection. Several previous studies employed attenuated viruses or recombinant viruses for vaccination by intranasal administration (Haller et al., 2000; Karron et al., 2011; Schmidt et al., 2011; Mason et al., 2013). The HPIV3 cp45 is a practical nasal vaccine that is derived from the JS wild-type strain of HPIV3 through 45 passages in African green monkey cells at a low temperature. This vaccine has been evaluated in clinical human trials and is known to induce the hemagglutinationinhibiting (HAI) antibody in seronegative children (Skiadopoulos et al., 1999; Karron et al., 2003; Belshe et al., 2004). The rB/HPIV3b vaccine is a cDNA-derived chimeric HPIV3 in which the genomic cDNA is partially recombined with bovine PIV3 (BPIV3); the hemagglutinin-neuraminidase (HN) and F genes from HPIV3 fused with BPIV3 whole genome (Schmidt et al., 2001; Karron et al., 2012). The rB/HPIV3 vaccine was shown to induce significantly higher titers of HAI antibodies against HPIV3 in seronegative children. A major limitation of these vaccines is their potential to cause actual infection diseases in children or immunocompromised hosts because they are live attenuated vaccines. Therefore, it is necessary to develop a safer HPIV3 vaccine with lower risks for infection that will be useful for infants and young children in clinics. In this regard, component vaccines are desirable because they use non-infectious viral subunit proteins as antigens. A previous report demonstrated the efficacy of subunit vaccines that target the HPIV3 HN and F proteins in an animal model (Ray et al., 1988). Other reports also demonstrated the induction of protective antibodies that prohibit HPIV3 infection in response to subunit vaccines that target HPIV3 antigens (Morein et al., 1983; Ray et al., 1985; Ambrose et al., 1991; Brideau et al., 1993). A caveat of subunit-based vaccination strategies is their requirement for large amounts of antigens, thus rendering them costly to produce. Therefore, it is important to develop an effective subunit vaccine that utilizes lower quantities of antigen.

To circumvent the aforementioned problems, oligomannosecoated liposome (OML) was used as a natural and non-toxic antigen-delivery system. OML efficiently targets proteins to antigen presenting cells (APCs), such as macrophages or dendritic cells (Shimizu et al., 2007; Nishimura et al., 2013). Furthermore, previous reports showed that antigens incorporated into OML were efficiently delivered toAPCs by intranasal administration (de Haan et al., 1995; Ishii and Kojima, 2010; Giddam et al., 2012). The effect of OML was shown to be relatively ineffective at inducing humoral immunity, while it preferentially activated cell-mediated immunity via cytotoxic T lymphocytes (CTLs). Therefore, for optimal induction of both humoral and mucosal immunity it is necessary to use vaccination strategies that combine OML with other adjuvant systems.

Herein, we sought to utilize OML in combination with an adjuvant double-stranded RNA polyinosinic-polycytidylic acid [Poly(I:C)] for the induction of effective humoral and mucosal immunity against HPIV3. The overarching goal was to establish a vaccination strategy that required a small amount of antigen and a few doses. Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4+ T cell responses against viral infection. Poly(I:C) was shown to be an effective mucosal adjuvant for the development of antigen-specific immunity even when hosts were immunized with a relatively small quantity of antigen (Ichinohe et al., 2005; Hasegawa et al., 2009). In addition, we also took advantage of the wheat germ cell-free protein production system to synthesize our antigen, full-length HPIV3-HN protein (Takai et al., 2010). Our results highlight the utility of combining sophisticated systems in the development of a novel vaccine against HPIV3.

## **MATERIALS AND METHODS**

#### **CONSTRUCTION OF WHEAT CELL-FREE EXPRESSION VECTOR**

HPIV3 (C243) cDNA was kindly provided by Dr. Tsukakoshi. The HN fragment was amplified by PCR using the primers BamHI-HN F (5- -GAGAGGATCCCATGGAATACTGGAAGCAT) and NotI-HN R (5- -GAGAGCGGCCGCTTAACTGCAGCTTTTTGGA). The amplified fragment was digested with BamHI and NotI and cloned into either pEU-His or pEU-GST vectors that were previously digested with the same enzymes. GST-tagged HPIV3-HN (GST-HN) construct was mutated using the reagents of a PrimeS-TAR Mutagenesis Basal Kit (TakaraBio, Otsu, Japan) according to the manufacturer's instructions.

#### **CELL-FREE PROTEIN SYNTHESIS AND PURIFICATION**

In vitro transcription and cell-free protein synthesis were performed as described (Takai et al., 2010). The bilayer translation method was used to synthesize His-tagged HPIV3-HN (His-HN) protein using wheat germ extract that was optimizedfor Ni-affinity purification (WEPRO 7240H; Cellfree Sciences,Yokohama, Japan) and a robotic synthesizer (Protemist XE; Cellfree Sciences) according to the manufacturer's instructions. The cell-free translation reaction (15 ml) was separated into soluble and insoluble fractions by centrifuged at 15000 rpm for 15 min. The insoluble fraction was lysed using 8M Urea at room temperature for 6 h and then mixed with Ni-sepharose High Permormance beads (GE Helthcare, Hino, Japan) in the presence of 20 mM imidazole. The beads were washed three times with washing buffer (20 mM Tris–HCl, pH 7.5, 500 mM NaCl) containing 40 mM imidazole. The His-HN protein was then eluted using washing buffer containing 8M Urea, 500 mM imidazole. Purified His-HN proteins were concentrated approximately 10–20-fold using Amicon Ultra Centrifugal Filters (Merck Millipore, Billerica, MA, USA). Fulllength GST-HN protein and GST-HN deletion mutant proteins synthesized using wheat germ extract optimized for GST-affinity purification (WEPRO 1240G; Cellfree Science) according to the manufacturer's instructions. Quantification of synthesized proteins was performed by densitometric scanning of the Coomassie Brilliant Blue® (CBB)-stained bands.

#### **PREPARATION OF LIPOSOMES**

Liposomes were prepared as described previously (Giddam et al., 2012; Nishimura et al., 2013). Briefly, a chloroform:methanol (2:1, *v*/*v*) solution containing 1.5 μmol of DPPC, 1.5 μmol of cholesterol and varying amounts of Man3-DPPE (0.15–0.0015 μmol) was added to a flask and evaporated to prepare a lipid film. PBS containing 1 or 0.5 mg/ml of full-length HPIV3-HN protein was added to the dried lipid film and multi-lamellar vesicles were prepared by intense vortex dispersion. The vesicles were extruded 10 times through a 1 μm pore polycarbonate membrane (Nucleopore, Pleasanton, CA, USA). The amount of entrapped protein was measured using a Modified Lowry Protein Assay Kit (Pierce, Rockford, IL, USA) in the presence of 0.3% (*w*/*v*) sodium dodecyl sulfate using HN as the standard. The particle size of the liposomes was determined using a dynamic light scattering particle size analyzer (BioMedCore Inc., Yokohama, Japan).

#### **IMMUNIZATION OF MICE**

Female BALB/c mice (Japan SLC Inc., Hamamatsu, Japan), age 6–8 weeks at the time of immunization, were used in all of the experiments. All animal experiments were carried out in accordance with the Guides for Animal Experiments Performed at Yokohama City University (YCU) and approved by the International Animal Care and Use Committee of YCU. Three to six mice for each experimental group were anesthetized with isoflurane prior to being immunized. For the primary immunization, 13 μl of single-shot mixtures were prepared as containing OML-HN (0.1 or 1.0 μg) and/or poly(I:C) (10 μg), and administered 6.5 μl of mixtures into each nostril. Three weeks later, the secondary immunization was administered in the same manner. Two weeks after the secondary immunization, as tertiary immunization, 16 μl single-shot mixtures were prepared as containing OML-HN (0.2 or 2.0 μg) and/or Poly(I:C) (10 μg), and administered 8.0 μl of mixtures into each nostril.

#### **ENZYME-LINKED IMMUNOSORBENT ASSAY**

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Serum was collected on days 7 and 14 after the secondary immunization and on days 7, 14, 21, and 28 after the tertiary immunization. On day 28 after the third immunization, all of the mice were sacrificed and nasal wash fluid was collected by washing the nasal cavity of the excised head with 1 ml of PBS(-) containing 0.1% BSA. The levels of IgG and IgA antibodies against HPIV3-HN in the serum and nasal wash fluid were determined by enzyme-linked immunosorbent assay (ELISA) as described previously. Briefly, ELISA was conducted sequentially from the solid phase (Anti-GST coated 96-well plate; Thermo, Waltham, USA) with a ladder of reagents consisting of the following: (1) GST-HN protein and GST protein as a control; (2) serum or nasal wash fluid; (3) either anti-mouse IgG antibody-conjugated HRP (1:10000, Thermo) or anti-mouse IgA antibody-conjugated HRP (1:10000, BETHYL, Montgomery, TX, USA); (4) TMB substrate buffer (Thermo); and (5) 2M sulfuric acid. The chromogen produced was measured by determining the absorbance at 450 nm with an ELISA reader. The relative levels of IgG and IgA antibodies against HN were determined relative values calculated as follows; Relative values = mean value in immunized vaccine group/mean value in immunized OML-empty group. Each values were normalized with the optic values to GST protein.

#### **IMMUNOBLOTTING**

Using standard immunoblotting methods, the presence of HNspecific IgG was detected using pooled serum from each group of mice and incubated with anti-mouse IgG HRP-conjugated secondary Ab (Thrmo) at a dilution of 1:10000 in TBST. Immobilon was used for detection (Merck Millipore).

#### **QUANTITATIVE REAL-TIME PCR**

We performed an infection inhibitor assay by mouse serum using immortalized MRC5 cells (pNifty cells). pNifty cells were seeded in 24-well plates at a concentration of 2.5 × 105 cells per well, and after 12 h the cells were infected with preincubated HPIV3 virus (107TCID50) with or without 5 μl mouse serum in 200 μl DMEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin and streptomycin (PS). At 4 h post-infection, the cells were washed and replaced in 200 μl of DMEM containing 10% FBS and 1% PS. At 48 h after medium change, the cells were washed with PBS and total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. cDNA was synthesized using a cDNA synthesis kit (Toyobo, Osaka, Japan) and subjected to RT-PCR analysis with the SYBR Premix Ex gent Kit TaqII (Takara Bio) using an Applied Biosystems 7300 real-time PCR System. The primer sets used were as follows: HPIV3, 5- -CTCGAGGTTGTCAGGATATAG-3 and 5- -CTTTGGGAGTTGAACACAGTT-3- ; mGAPDH, 5- - CCATGGAGAAGGCTGGGG-3 and 5- -CAAAGTTGTCATGGAT GACC-3- .

#### **RESULTS**

#### **GENERATION OF OML VACCINE AGAINST HPIV3-HN**

To produce the full-length HPIV3-HN antigen, we subcloned full-length HN cDNA into two different cell-free expression vectors, pEU-His and pEU-GST for the expression of Nterminally His-tagged or GST-tagged fusion proteins, respectively (**Figure 1A**). We found that both His-HN and GST-HN proteins were efficiently synthesized using the wheat germ cell-free system (**Figures 1B,C**). His-HN proteins precipitated into the insoluble fraction (**Figure 1B**) were purified using Ni-sepharose beads in the presence of 8M Urea. GST-HN proteins were purified using glutathione sepharose beads in a regular buffer (**Figure 1C**).

After the large scale preparation of His-HN, the protein was incorporated into OML (**Figure 1D**). The particle diameter of HNfilling OML (OML-HN) and empty OML were 882 and 519 nm, respectively. The amount of carrier HN protein was approximately 32 mg per 1 mg cholesterol (**Figure 1E**) and the molar ratio of enclosed-OML to non-enclosed OML was found to be approximately 7:3 (data not shown).

#### **IMMUNIZATION OF MICE WITH OML-HN**

We investigated whether intranasal administration of OML-HN could induce a humoral immune response against the HN. **Figure 2A** depicts the time course for the immunizations and blood collection from the immunized mice. Mice were immunized intranasally with OML-HN (1 or 0.1 μg) with or without Poly(I:C), OML with or without Poly(I:C), Poly(I:C) only, or PBS. One week after the third immunization, HN-specific serum IgG was detected in mice immunized with OML-HN (1 μg) plus Poly(I:C). The serum IgG levels were increased between days 7 and 14 and reached the peak at 21 days after the final immunization. Mice immunized with the lower amount of OML-HN (0.1 μg) plus Poly(I:C) also produced HN-specific serum IgG at 28 days after the last immunization (**Figure 2B**). In contrast, there was no significant HN-specific serum IgG in mice immunized with OML-HN without Poly(I:C) or the other negative control groups (**Figure 2B**).

We next measured the levels of serum IgG and nasal wash IgA in each individual mouse by ELISA and immunoblotting (**Figures 3A–C**). Mice immunized with OML-HN (1 μg) plus Poly(I:C) exhibited prominent induction of HN-specific IgG (**Figures 3A,C**). HN-specific IgA in nasal wash fluid was most prominently induced in mice with OML-HN (1μg) plus Poly(I:C) compared to other groups (**Figure 3B**). Interestingly, the induction of the HN-specific IgA was higher in mice that were immunized with the lower amount of antigens, OML-HN (0.1 μg) plus Poly(I:C) (**Figure 3B**).

#### **EPITOPE MAPPING OF INDUCED ANTIBODIES**

We next determined the region of HN that was recognized by the HN-specific serum IgG produced by the mice that were immunized with OML-HN (1 μg) plus Poly(I:C). Three domain mutants of HPIV3-HN, the N-terminal region (1-190), the middle region (168-408) and C-terminal region (400-572) were synthesized using the wheat germ cell-free system (**Figure 4A** left) and protein production was confirmed by SDS-PAGE (**Figure 4A** right). Based on ELISA analysis, all of the serum samples contained HN-specific antibodies that had high reactivity to the N-terminal region (**Figure 4B**).

#### **EFFECT OF OML-HN VACCINE ON HPIV3 INFECTION** *IN VITRO*

We asked whether sera from the immunized mice could inhibit the HPIV3 infection of fibroblast cells. Infectious HPIV3 virions were pre-incubated with mouse serum harvested from three mice that were immunized with OML-HN plus Poly(I:C) and then used to infect MRC5 cells. Cellular HPIV3 mRNA was measured using quantitative reverse transcription PCR (qRT-PCR). The levels of HPIV3 mRNA were significantly reduced in cells pre-incubated with the immunized mouse serum compared to control serum that immunized with OML-empty (**Figure 4C**). These results indicate that the immunization with OML-HN plus Poly(I:C) induced serum antibodies that protect HPIV3 infection.

## **DISCUSSION**

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Herein, we developed a novel subunit vaccine against HPIV3-HN using OML and a mucosal adjuvant Poly(I:C). Consequently we

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gation followed by the separation of the soluble supernatant (S) and insoluble precipitate (P) fractions. These samples were subjected to SDS-PAGE and visualized with Coomassie Brilliant Blue (CBB) staining. The single asterisk indicates the band of His-HN. **(C)** Purification of recombinant GST-tagged HPIV3-HN proteins that were synthesized using OML-encapsulated His-HN. The single asterisk indicates the band of His-HN. **(E)** The amount of encapsulated HN was quantitated by SDS-PAGE (right panel). The diameter of OML-HN or control OML were measured by dynamic light scattering particle size analyzer (left panel). The arrow indicates the band of His-HN.

successfully induced antigen-specific immunoglobulin G and A with the immunization of lower quantities of HN antigen via the nasal route. Furthermore, the immunized mouse serum exhibited the ability to suppress the virus infection in cell culture model. These results indicate that our newly developed OML vaccine could offer a powerful means to protect HPIV3 infection.

In our current study, we used the wheat germ cell-free protein production system to synthesize full-length HN protein as

an antigen (Takai et al., 2010). In comparison to cell-mediated procedures such as *Escherichia coli* and baculovirus systems, the wheat germ cell-free system is beneficial for the rapid and efficient preparation of high-quality proteins (Endo and Sawasaki, 2006). Moreover, this cell-free system is suitable for the generation of toxic viral proteins for immunization and beneficial for the purification of naturally folded proteins, as well as scalability. This system, however, may not be cost-effective for preparing large

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amounts of viral antigens for vaccine development. Therefore, efforts were made to reduce the amount of antigen needed vaccination. Herein, we utilized a OML and Poly(I:C) vaccination strategy in an attempt to reduce the amount of antigen required. OML is a lipid vesicle that has mannose on its surface, which aids in efficient targeting to APCs (Shimizu et al., 2007; Nishimura et al., 2013). In a previous report, antigenic proteins incorporated into OML were efficiently delivered to APCs via intranasal administration (Ishii and Kojima, 2010). In that report, intranasal administration of 5 μg ovalbumin (OVA) incorporated into OML four times effectively induced immune responses in mice (Ishii and Kojima, 2010). Poly(I:C) is a synthetic double-stranded RNA (dsRNA) molecule that induces effective mucosal immune responses by stimulating Toll-like receptor 3 (TLR3) as a molecular mimic of dsRNA, which is a byproduct of viral replication (Ichinohe

et al., 2005; Hasegawa et al., 2009). The efficacy of nasal vaccines made of subunit proteins in the combination with mucosal adjuvants was demonstrated for influenza virus and RSV (Ichinohe et al., 2005; Hasegawa et al., 2009; Ainai et al., 2010; Kamphuis et al., 2013). We utilized a mucosal adjuvant Poly(I:C) to induce HN-specific antibodies in serum and nasal wash fluid through intranasal immunization with OML-HN. Using our vaccination strategy, we were able to decrease the amount of antigen required to 20% relative to previous reports (Mader et al., 2000; Ishii and Kojima, 2010).

The mucosa of respiratory tracts is the site of defense against virus infection since respiratory viruses attack and infect the respiratory mucosal tissues and cells (Tamura and Kurata, 2004). Mucosa is generally protected by mucin and defensin produced from goblet cells and Paneth cells. The TLR family members,

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TLR3, TLR7, TLR8, and TLR9 can recognize viral nucleotides and induces type I interferon (IFN-I) production if viruses intrude into tissues beyond the barrier. IFN-I activates the defense mechanism against virus by promoting the maturation of DCs and the induction of NK cells (Takeda et al., 2003; Akira et al., 2006). On the other hand, it is known that Microfold cells (M cells) promotes adherence and transport of antigens to APCs (Sato and Kiyono, 2012). M cells reside in the follicle-associated epithelium of Peyer's patches in the intestinal tract or nasal lymphoid tissue (NALT) of rodents in the upper respiratory tract, and plays a pivotal role in the induction of antigen-specific immunity (Nochi and Kiyono, 2006). The APCs promote adaptive immune responses by presenting antigens to naïve B cells and activate it to differentiate into antigen specific B cells. In the mucosa, secretory IgA is transported to mucosal surface by polymeric Ig receptor (pIgR) and the secreted IgA plays an important role in the protection of viral infection in the respiratory tract (Mostov and Deitcher, 1986). It is known that the intranasal immunization can activate mucosal immunity thereby enhancing the induction of mucosal IgA in addition to the generation of systemic IgG against viral antigen. Our current study employed OML as an effective tool to deliver the antigen to APCs and M cells in respiratory mucosa. A recent report demonstrated that OML-mediated intranasal immunization can efficiently induce Th2 cytokines such as IL-5 and IL-6 that eventually help produce secretory IgA in mucosal system in mouse model (Ishii and Kojima, 2010). We further combined OML with a mucosal adjuvant Poly(I:C) to facilitate the specific mucosal immunity against HPIV3-HN. Poly(I:C) has been shown to be an effective mucosal adjuvant stimulating TLR3 as a molecular mimic. A previous report indicated that a nasal influenza virus vaccine combined with Poly(I:C) synergistically induced IFN-1 and Th2 cytokine leading to an effective humoral immunity including secretory IgA in mucosa (Ichinohe et al., 2005). Our current study also demonstrated that the combinatory use of nasal vaccine with Poly(I:C) has a profound effect in inducing mucosal immunity against viral antigen.

Our newly developed OML-HN vaccine has several advantages as compared with previously developed vaccine methods including live attenuated vaccines. Although there is no practical prophylactic vaccine against HPIV3 infection, several previous studies have indicated that attenuated vaccines created by reducing the virulence of HPIV3 can indeed effectively induce the mucosal immunity when treated by intranasal administration (Karron et al., 2003). However, a major problem of these vaccines is their potential to cause a live infection in infants and immunocompromised hosts. Furthermore, there is a small risk of reversion to

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virulence by genetic mutations that results in the onset of severe disease. Therefore, it is desirable to develop a safer HPIV3 vaccine with lower risks of infection. On the other hand, intranasal subunit vaccines against HPIV3 have been demonstrated to be effective in animal models without the risk of viral replication and live infection. According to a report by Ray et al., the intranasal administration of HN and F proteins extracted from virions could induce significant anti-viral immunity in hamsters (Ray et al., 1988). However a drawback of subunit vaccines is their requirements for large amounts of antigens and concomitant high cost. Therefore, it is important to develop a cost-effective subunit vaccine that dispenses with substantial quantity of antigens. In order

to overcome this problem, we used OML and Poly (I:C) aiming for efficient vaccine delivery and immune response, respectively. Indeed, our current study demonstrated that a combination of OML-HN with poly(I:C) induced antigen-specific IgA and IgG by three times more than the immunization without poly(I:C). The safety in the use of either OML or poly(I:C) has been reported in previous studies. For OML-based vaccine, Fukasawa et al. have reported that OML has indeed no obvious toxicity and immunogenicity by itself (Fukasawa et al., 1998). Furthermore, Poly(I:C) has shown to be non-toxic as compared with conventional vaccine adjuvant such as cholera toxin subunit B (Ichinohe et al., 2005). However, further careful analysis should be necessary to validate the effectiveness and feasibility of our newly-developed vaccine strategy using virus infection models with multiple genotypes of HPIV3.

In our current study, we demonstrated that the OML-based vaccine incorporated with full-length HN protein induced IgG that targets the N-terminal region of HN protein. The N-terminal region of HN contains the stalk region while the C-terminal region contains the globular head domain. The stalk region of HN is known to play a crucial role in virion-host cell fusion via an interaction with F protein while the globular head binds sialic acid and neuraminidase (Moscona, 2005; Porotto et al., 2012). Although effective antigenic epitopes for HPIV vaccine remain elusive (Henrickson, 2003), a monoclonal antibody targeting the stalk region of HPIV2-HN has been shown to have a profound inhibitory activity against viral infection (Yuasa et al., 1995). Based on the observation, it seems that the antibodies induced by our vaccine system could also target the stalk region since they effectively blocked the viral infection in cell culture model. Further careful analysis will be required for the mapping of the epitope affecting virus infection in our current model.

In this study, we did not investigate other routes of antigen administration besides the intranasal route. However, previous studies have indicated that non-nasal immunization of HPIV3 components failed to prohibit the infection of HPIV3 in a cotton rat model. Indeed, the intramuscular immunization with HN and F recombinant proteins could not protect virus infection in upper respiratory tract although it had some effects on the protection of pneumonia and lower respiratory tract infection (Ambrose et al., 1991). It is generally believed that intranasal immunization has a great benefit for protecting virus infection itself by inducing antigen-specific secretory IgA in respiratory mucosa (Hirabayashi et al., 1990; Durrer et al., 2003). Our current study also confirmed this advantage of nasal vaccination where the OML-based nasal vaccine provides high performance for the induction of antigenspecific secretory IgA in nasal wash fluids. Therefore, intranasal administration of OML-based vaccine with poly(I:C) adjuvant could be an effective way of vaccination against respiratory viruses including HPIV3.

#### **ACKNOWLEDGMENTS**

We thank Drs. A Kudoh, A Ainai, and Y Sato for discussion and comments. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Respiratory Infection Disease Research Grants from The Ministry of Health Labour and Welfare of Japan to Akihide Ryo.

### **REFERENCES**

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**Conflict of Interest Statement:** The authors, editor, and chief editor declare that while the authors Kyosuke Senchi, Satoko Matsunaga, Akihide Ryo, and the review editor Ichiro Aoki are currently employed by the same institution (Yokohama City University School of Medicine, Japan) and while Hirokazu Kimura and Hironori Sato are currently employed by the same institution (National Institute of Infectious Diseases, Japan) there has been no conflict of interest during the review and handling of this manuscript.

*Received: 09 August 2013; paper pending published: 26 August 2013; accepted: 30 October 2013; published online: 26 November 2013.*

*Citation: Senchi K, Matsunaga S, Hasegawa H, Kimura H and Ryo A (2013) Development of oligomannose-coated liposome-based nasal vaccine against human parainfluenza virus type 3. Front. Microbiol. 4:346. doi: 10.3389/fmicb.2013.00346 This article was submitted to Virology, a section of the journal Frontiers in Microbiology. Copyright © 2013 Senchi, Matsunaga, Hasegawa, Kimura and Ryo. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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## Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody

*Satoko Matsunaga1, Shiho Kawakami 1, Izumi Matsuo1, Akiko Okayama2 , Hiroyuki Tsukagoshi 3 , Ayumi Kudoh1,Yuki Matsushima4 , Hideaki Shimizu4 , Nobuhiko Okabe4 , Hisashi Hirano2 , Naoki Yamamoto5 , Hirokazu Kimura6 and Akihide Ryo1\**

1 Department of Microbiology, Yokohama City University School of Medicine, Kanagawa, Japan

2 Proteome Analysis Center, Yokohama City University School of Medicine, Kanagawa, Japan

3 Gunma Prefectural Institute of Public Health and Environmental Sciences, Gunma, Japan

4 Kawasaki City Health and Safety Research Center, Kanagawa, Japan

5 Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

6 Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan

#### *Edited by:*

Yasuko Tsunetsugu Yokota, National Institute of Infectious Diseases, Japan

#### *Reviewed by:*

Yoshimichi Okayama, Nihon University School of Medicine, Japan Masatsugu Obuchi, Toyama Institute of Health, Japan Takeshi Saraya, Kyorin University School of Medicine, Japan

#### *\*Correspondence:*

Akihide Ryo, Department of Microbiology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan e-mail<EMAIL_ADDRESS>
Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis.The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

**Keywords: human parainfluenza virus 3, monoclonal antibody, cell-free protein synthesis, proteomics**

#### **INTRODUCTION**

Human parainfluenza viruses (HPIVs) are major causes of lower respiratory infections in infants, young children, the immunocompromised, the chronically ill, and the elderly (Glezen et al., 1984; Counihan et al., 2001; Weinberg et al., 2009). HPIVs belong to the Paramyxoviridae family of medium-sized enveloped viruses and their genomes are organized on a single negative-sense strand of RNA. Of the four predominant serotypes of HPIV, the HPIV3 is the most frequently detected in viral infections in respiratory tracts. In fact, HPIV3 is second only to respiratory syncytial virus (RSV) as a cause of pneumonia and bronchiolitis in infants and young children. The HPIV3 genome contains approximately 15,000 nucleotides encoding at least six common structural proteins (3- -N-P-C-M-F-HN-L-5- ; Storey et al., 1984; Tashiro and Homma, 1985; Wechsler et al., 1985).

The HN protein is found on the lipid envelope of HPIVs, where it likely exists as a tetramer. HN is important for HPIV3 infection of host cells because it functions in virus-host cell attachment via sialic acid receptors and in virus release from cells with its neuraminidase activity (Huberman et al., 1995; Porotto et al., 2001; Chu et al., 2013). HN can be recognized by the host immune system and antibodies against epitopes within HN can neutralize its activity through the inhibition of the function and/or activity of either hemagglutinin or neuraminidase. Therefore, characterization of the protein structure and function of HN is of great importance for the understanding of HPIV3 infection and the host immunity against this virus.

Polyclonal antibodies and monoclonal antibodies (MAbs) against HPIV3 antigens have previously been generated, and animal antiserum to HPIV3 HN is also commercially available. However, the antibodies demonstrated cross-reactivity to other HPIV family viruses and exhibited relatively high non-specific background staining in immunoassays (Goswami and Russell, 1983; Waner et al., 1985). MAbs have been also produced to several components of HPIV3 (van Wyke Coelingh et al., 1985; Rydbeck et al., 1986). Although these commercially available MAbs have been shown to be specific, the utility of these antibodies for several immunological and/or proteomic analyses for

understanding the nature of HPIV3 infection have not been fully delineated.

In our current study, we utilized the innovative wheat germ cellfree protein production system to generate the antigen protein. The main advantage of the cell-free protein system is the synthesis of proteins that are properly folded and that possess biological activity because the proteins are expressed in a eukaryotic cell system. Moreover, this system is capable of producing toxic proteins, such as viral antigens, that cause severe cytotoxicity or interference with host cellular physiology. By utilizing HPIV3-HN protein synthesized by the wheat cell-free system, we established multiple hybridoma clones producing MAbs that specifically targeted the viral antigen and were applicable for several immunoassays. Furthermore, we used the MAbs in proteomic analyses for identifying host proteins that potentially act as HN binding partners.

#### **MATERIALS AND METHODS**

#### **CONSTRUCTION OF WHEAT GERM CELL-FREE EXPRESSION VECTOR**

Amplification of HN fragment from HPIV3 (Strain C243) genome was performed with the following primers: forward (5- -AGGAGTAAAGTTACGCAATCCAA) and reverse (5- - ATATTTCCCTTTTGTCTATTGTCTG). For producing the expression vector of wheat germ cell-free system, The HN openreading frame was amplified by PCR using the forward primer (5- -GAGAGGATCCCATGGAATACTGGAAGCAT) and reverse primer (5- -GAGAGCGGCCGCTTAACTGCAGCTTTTTGGA). The amplified fragment was subcloned using BamH I and Not I into pEU-His or pEU-bls-S1 (bls; biotin ligase site) vectors. Biotinylated HN mutants were generated using the PrimeSTAR Mutagenesis Basal kit (TakaraBio, Otsu, Japan) according to the manufacturer's instructions.

HN protein fragments comprising CT, TM, and stalk regions were generated by the wheat cell-free system based on the template cDNA amplified from clinical isolates of HPIV1 (GeneBank No. JQ901977; Beck et al., 2012), HPIV2 (GeneBank No. AF533010; Skiadopoulos et al., 2003), and Mumps (GeneBank No. AB699704; Momoki,2013) usingfollowing primer sets. HPIV1: (5- -ATGCTTATACTCTGGAGTCAAGA) and (5- - TCTAGCAAAACRTGAAGTTGAG); HPIV2: (5- -AAAAACCTA-AAATAAGCACGAA); and (5- -CCATTCTGGCCTATATYATAAT), Mumps: (5- -TTACTTATAAGACTGCGGTGC) and (5- -CTTGCA-ATGAGTTCTACTCTGA). Synthetic cDNA encoding the CT, TM and stalk regions of Sendai virus (UniProtKB entry no. P04853; Miura et al., 1985) was generated by Operon Biotechnologies (Huntsville, WI, USA). The amplified fragments were subcloned into pEU-bls-S1 vectors using In-fusion cloning system (TakaraBio) according to the manufacturer's instructions.

#### **CELL-FREE PROTEIN SYNTHESIS AND PURIFICATION**

*In vitro* transcription and cell-free protein synthesis were performed as described (Takai and Endo, 2010; Takai et al., 2010). For cell-free protein synthesis, the ENDEXT Wheat Germ Expression S Kit (CellFree Sciences, Yokohama, Japan) was used according to the manufacturer's instructions for the bilayer translation method. GST fusion and Biotinylated proteins were produced as previously described (Sawasaki et al., 2008; Takahashi et al., 2012). His-HPIV3-HN protein, used for the generation of hybridoma, was synthesized using the robotic synthesizer (Protemist XE; Cell-Free Sciences) according to the manufacturer's instructions. The cell-free translation reaction mixture (15 ml) was separated into soluble and insoluble fractions by centrifugation at 15,000 rpm for 15 min. The insoluble fraction was lysed by the addition of 8 M Urea at room temperature for 6 h, then mixed with Nisepharose High Performance beads (GE Healthcare, Waukesha, WI, USA) in the presence of 20 mM imidazole. The beads were washed three times with washing buffer (20 mM Tris–HCl pH 7.5, 500 mM NaCl) containing 40 mM imidazole. His-HN proteins were then eluted with washing buffer containing 8 M Urea, 500 mM imidazole. Amicon Ultra centrifugal filters (Millipore, Bedford, MA, USA) were used to concentrate the purified His-HN proteins by approximately 10- to 20-fold. The protein concentration was determined using the Bradford method with bovine serum albumin (BSA) as a protein standard.

#### **IMMUNIZATIONS AND GENERATION OF HYBRIDOMAS**

Monoclonal antibodies specific for HPIV3-HN were generated using the previously described hybridoma technology (Kimura et al., 1994). In brief, 300 μg of N-terminal, His-tagged full-length HPIV3-HN protein was injected into the footpad of Balb/c mice using keyhole limpet hemocyanin as an adjuvant. Four weeks later, spleen cells were isolated and fused to the myeloma cell line SP2/O using polyethylene glycol 1500 (PEG 1500) as previously described (Kimura et al., 1996). Isotype determination was performed with the mouse MAb isotyping test kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol.

#### **ELISA**

Microtiter plates coated with HPIV3-HN were incubated with threefold serial dilutions of each antibody (starting from 1:300 dilution of a hybridoma culture supernatant). After incubation with a peroxidase-conjugated secondary antibody and washing with PBS, the colorimetric signal of tetramethylbenzidine was detected by measuring the absorbance at 405 nm (Abs) using a plate reader.

#### **IMMUNOBLOTTING**

Recombinant HPIV3-HN proteins (equivalent to ∼100 ng) or HPIV3-infected HeLa cell lysates were separated by 10% SDS-Gel and transferred onto a PVDF membrane (Millipore). The membrane was then soaked in Tris-buffered saline (TBS) containing 5% (w/v) skim milk for 1 h and incubated with a MAb (hybridoma supernatant, 1:10 dilution) in TBS containing 0.1% (v/v) Tween-20 (TBST) overnight at 4◦C. After washing three times with TBST, the membrane was incubated for 1 h in TBST containing goat-anti mouse IgG-HRP antibody (1:10000; GE Healthcare, Buckinghamshire, UK). After washing three times in TBST, the blot was detected with ImmobilonWestern Chemiluminescent HRP Substrate (Millipore) using FluorChem FC2 (Alpha Innotech, Santa Clara, CA, USA) in accordance to the manufacturer's protocol.

#### **IMMUNOPRECIPITATION**

HeLa cells were infected with HPIV3 (Strain C243) at multiplicity of infection (MOI) of 100 for 48 h. The cells were lysed with immunoprecipitation buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5% NP-40, 200μM PMSF, 50μMVO4, 2μg/ml Aprotinin, 5 μg/ml Leupeptin, 1 μg/ml Pepstatin A). For immunoprecipitation assay, cell lysate or wheat germ cell extract generating full-length HPIV3-HN was incubated with the individual MAb (hybridoma supernatant), preclearing overnight using proteinA/G sepharose beads, for 2 h at 4◦C. After washing three times with immunoprecipitation buffer, immunocomplexes were eluted from the beads with 2x SDS sample buffer. Then, the bound protein was analyzed by immnoblotting.

#### **IMMUNOFLUORESCENCE**

HeLa cells were grown on coverslips for 24 h and the cells were infected with HPIV3 (MOI = 100); mock-infected cells served as the control. At 48 h post-infection, the cells were washed with PBS before fixation with 3% formalin in PBS at room temperature for 15 min. Cells were washed twice with PBS for 5 min followed by 100% methanol for 10 min at −20◦C. Cells were permeabilized with PBS containing 0.05% Triton X-100 for 10 min at room temperature. The cells were incubated with individual primary antibodies for 1 h at room temperature. After being washed twice with PBS, the cells were incubated with secondary antibodies for 1 h at room temperature. The nucleus was counterstained with 4- ,6-diamidino-2-phenylindole (DAPI). Microscopic imaging was performed with an FV1000-D confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 60x oil-immersion objective.

#### **FLOW CYTOMETRY**

HeLa cells were seeded in 6-well plates at a concentration of 2×105 per well 24 h before infection, and the cells were washed with medium containing 0.0001% trypsin and infected with HPIV3 (MOI = 100); mock-infected cells were used as a control. At 4 days post-infection, cells were harvested in PBS containing 5 mM EDTA and washed twice with PBS. The cells were then incubated with either MAbs (hybridoma supernatant; 5X dilution) or non-immunized hybridoma supernatant in PBS containing 2% Blocking One (NACALAI TESQUE, INC., Kyoto, Japan) for 1 h at room temperature. After being washed twice with PBS, the cells were incubated with phycoerythrin (PE)-conjugated anti-mouse IgG antibodies (Beckman Coulter, Fullerton, CA, USA) for 1 h at room temperature. Flow cytometric analysis of the cells (10,000 cells per sample) was performed on a FACScanto II instrument (BD Biosciences, San Jose, CA, USA).

#### **EPITOPE MAPPING AND SPECIFICITY OF MAbs USING ALPHASCREEN ASSAY**

The AlphaScreen assay was performed using 384-well ProxiPlates (PerkinElmer, Boston, MA, USA). Biotinylated virus proteins or GST (negative control) were incubated with a 40-fold dilution of MAbs (hybridoma supernatant) in 15 μl of binding mixture containing reaction buffer (100 mM Tris-HCl, pH 7.5, 1 mg/ml BSA, 0.01% Tween-20) at 26◦C for 30 min. Then, combine 10 μl of the detection mixture containing 0.1 μl protein G-conjugated acceptor beads and 0.1 μl streptavidin-coated donor beads (AlphaScreen IgG detection kit, PerkinElmer) in reaction buffer were incubated at 26◦C for 1 h. Antigen-antibody interactions were analyzed using an Envision microplate reader (PerkinElmer).

#### **PROTEOMIC ANALYSIS**

Cell lysate from HPIV3-infected or mock-infected HeLa cells were immunoprecipitated with #21 MAb. The binding proteins were separated by SDS-PAGE and transferred to PVDF membranes. For LC-MS/MS analysis, the membranes digested with trypsin. LC-MS/MS analysis was performed using a TripleTOF MS (TripleTOF 5600 system, AB SCIEX, Foster City, CA, USA) and the Analyst version 1.6 TF (AB SCIEX) coupled to an DiNa-AP (KYA Technologies, Tokyo, Japan). Prior to injection into the mass spectrometer, the tryptic digests were filtered through a Ultrafree-MC, GV 0.22 μm filter (Millipore), then loaded onto a reverse phase pre-column (HiQ sil C18W-3, 500 μm id × 1 mm, KYA Technologies) and resolved on a nanoscale HiQ sil C18W-3 (100 μm id × 10 cm; KYA Technologies) at a flow rate of 200 nL/min with a gradient of acetonitrile/0.1% (v/v) formic acid. Peptides were separated using a 30 min gradient from 5 to 100% solvent B [0.1% (v/v) formic acid/80% (v/v) acetonitrile]. Solvent A was 0.1 % (v/v) formic acid/2% (v/v) acetonitrile. The obtained MS and tandem-MS data were searched against the human protein sequences in the Swiss-Prot database (version Jan 2013, 20233sequences) using the Protein Pilot software 4.5 (AB SCIEX).

## **RESULTS**

#### **PRODUCTION OF MAbs**

In our current study, we attempted to produce the full-length HPIV3-HN by the wheat cell-free production system (**Figure 1A**). Complementary DNA encoding HPIV3-HN open-reading frame was sub-cloned into pEU-His, the expression vector designed specifically for the wheat germ cell-free system for expressing His-tagged protein. Consequently, His-tagged HPIV3-HN protein was synthesized by this procedure in a large scale. Since HPIV3-HN exhibited high insolubility in regular buffer, the protein was suspended in the buffer including 8 M urea. This suspended His-tagged HPIV3-HN was further purified using Nisepharose followed by the elusion with imidazole. Balb/c mice were then immunized with purified full-length HPIV3-HN protein. After 4 weeks, splenocytes were isolated and hybridomas were created (**Figure 1A**). Finally, 36 stable hybridomas were obtained and designated #1 to #36. The resulting hybridomas were screened by ELISA with plates coated with HPIV3-HN protein conjugated with BSA. Of the 36 hybridoma clones established, seven clones (#4, #5, #7, #10, #14, #21, #23) exhibited relatively high absorbances (**Figure 1B**). Titration analyses with diluted hybridoma supernatants or antigenic HN protein revealed that these seven antibodies had higher specificities and intensities than control hybridoma supernatants (**Figure 1C**). These seven hybridomas were processed for further characterization.

#### **IMMUNOBLOTTING ANALYSES OF MAbs**

We next tested the MAbs in immunoblotting analysis. First, recombinant HPIV3-HN protein was separated by SDS-PAGE followed by immunoblotting with MAbs isolated from the seven

**FIGURE 1 | Production of hybridoma cells generating anti-HPIV3-HN antibodies. (A)** Schematic diagram of hybridoma cells production generating anti-HPIV3-HN monoclonal antibody (MAb). The recombinant Histidine-tagged recombinant HPIV3-HN (His-HN) protein was produced by wheat germ cell-free system and then purified by nickel-chelated sepharose beads in the presence of 8 M urea. The purified protein was separated by SDS-PAGE and visualized by CBB-staining. Purified His-HN protein was injected into the footpad of Balb/c mice. After 4 weeks, immunized mouse splenocytes were

fused with myeloma cells and then 36 hybridoma cells were established. SP6, SP6 promoter sequence; E01, translation enhancer sequence; His, Histidine-tagged sequence; TEV; TEV protease recognized sequence. **(B,C)** The specificity of MAbs (hybridoma supernatant) evaluated by ELISA. The specificity of 36 MAbs in 2700-fold dilution was determined **(B)**. The black arrows indicate the selected MAbs while the white arrow depicts a selected clone as a negative control (clone no. #30). The selected eight MAbs were diluted at serial points and analyzed by ELISA **(C)**.

hybridomas. As shown in **Figure 2A**, all seven MAbs recognized a single band that corresponded to the recombinant protein. Next, immunoblotting analysis was performed with cell lysates from HeLa cells either infected- or mock-infected with HPIV3. The all seven MAbs detected a 63 kDa protein band that was consistent with the molecular mass of the HN protein (**Figure 2B**). No other bands were detected by the MAbs indicating that they specifically recognized HN.

#### **IMMUNOFLUORESCENT ANALYSIS**

We next performed an immunofluorescent (IF) analysis of HPIV3 infected HeLa cells with seven MAbs (**Figure 2C**). Three of the MAbs (#7, #21, and #23) exhibited prominent IF staining of the infected cells, while the other four MAbs had no detectable IF staining. The IF staining with the antibodies revealed that HN protein exhibited granular staining pattern throughout cytoplasm and plasma membrane in HPIV3-infected cells (**Figure 2D**), which

was consistent with previous studies (Ali and Nayak, 2000; Stone and Takimoto, 2013). The control mock-infected cells did not show any signals when stained with the antibodies (**Figure 2C**). We thus selected the three MAbs (#7, #21, and #23) for further characterization.

#### **IMMUNOPRECIPITATION**

We next examined whether these selected antibodies were useful in immunoprecipitation analysis. The wheat germ extract containing full-length HPIV3-HN with glutathione-*S*-transferase (GST) tag was incubated with protein A/G-coated sepharose beads (GE Helthcare) together with the three selected antibodies or non-immunized mouse IgG antibody. The precipitated samples were subjected to immunoblotting analysis with anti-GST antibody. The GST tagged HN protein was precipitated by all selected antibody (#7, #21, and #23) but not non-immunized IgG (**Figure 3A**).

#### **FLOW CYTOMETRY ANALYSIS**

We next addressed the usability of the antibodies #7, #21, and #23 in flow cytometry analysis. HeLa cells infected with HPIV3 were stained with the antibodies and then subjected to FACS analysis.

**FIGURE 3 | Immunoprecipitation and flow cytometry assay. (A)** Recombinant GST-HPIV3-HN or GST protein was immnoprecipitated with either #7, #21, #23 MAbs, or IgG (negative control), respectively. Then bound proteins were analyzed with immunoblotting using anti-GST antibody. **(B)** HPIV3-infected or uninfected HeLa cells were harvested at 4 days post-infection, followed by incubation with indicated MAbs. The cells were then fixed and stained with anti-mouse secondary antibody. The population of stained cells was calculated by flow cytometry. The shaded histogram shows negative hybridoma supernatant and the bold line shows specific MAbs.

Our results demonstrated that only #21 MAbs could specifically detect virus-infected cell populations, and distinguished between viral-infected and uninfected cell population with flow cytometory (**Figure 3B**).

#### **EPITOPE MAPPING OF MAbs**

To determine the binding domain of the MAbs within the HPIV3-HN protein, we synthesized six different deletion mutants of HPIV3-HN as depicted in **Figure 4A**. All of the deletion mutants contained N-terminal biotin tag were incubated with the antibodies (#7, #21, or #23), followed by the addition of AlphaScreen streptavidin donor and protein A acceptor beads, as depicted in **Figure 4B**. The reactivity was measured and calculated by the level of the AlphaScreen luminescent signal. The results showed that two MAbs (#7 and #23) reacted against the cytoplasmic tail (CT) of HPIV3-HN whereas #21 MAb detected extracellular stalk region of the protein (**Figures 4C–E**). This result is fully consistent with the result of flow cytometry analysis (**Figure 3B**).

#### **SPECIFICITY OF MAb**

We next investigate the specificity of MAbs using HN protein frangments derived from HPIV1, Sendai virus (SeV), HPIV2, and Mumpus virus (MuV). Partial HN protein fragments containing cytoplasmic tail (CT), transmembrane domain (TM), and stalk region were produced with a N-terminal biotin tag by wheat cellfree system, and then incubated with the antibodies (#7, #21, or #23) followed by the AlphaScreen (**Figure 5A**). The antigen reactivity was measured based on the level of the AlphaScreen luminescent signal (**Figure 5B**). Notably, there was no crossreactivity to HN proteins derived from other Paramyxoviruses except for HPIV3. This was also confirmed by immunoblotting analysis (**Figure 5C**). These results indicate the specificity of the antibodies for HPIV3-HN.

#### **PROTEOMIC ANALYSIS**

We next utilized our newly developed #21 MAb for the identification of host proteins that bind to HPIV3-HN during HPIV3 infection. Cell lysate from HPIV3-infected or mockinfected cells were immunoprecipitated with #21 MAb. Precipitated samples were collected and then digested with trypsin followed by LC-MS/MS analysis (**Figure 6A**). Annotation analysis using the Swiss-Prot database revealed that 10 proteins were putative HN binding proteins (**Figure 6B**). Based on the number of corrected peptides, top four proteins (HSP70, HSP90, tubulin, alpha 1c, and SERPINA3) were selected as the most likely candidates for association with HN and subjected to further binding analysis. The pull-down analysis of the host proteins with recombinant HPIV3-HN was performed. The subsequent immunoblotting analysis demonstrated that HPIV3-HN could indeed interact with these four proteins (**Figure 6C**). These results demonstrate the availability of our newly-developed antibody in comprehensive proteomic analysis.

#### **DISCUSSION**

Herein we produced HPIV3-HN proteins by the wheat cell-free system, and created MAbs that selectively target the HPIV3-HN protein. Characterization of the most potent MAb confirmed the antigen-specificity and usability in various applications including immunoblotting, immnofluorescent, flow cytometry, and immunoprecipitation analyses. Furthermore, the MAb could capture the endogenous HN protein from HPIV3-infected cells to identify HN-binding host proteins via mass spectrometry-based proteomic analysis. Our current results demonstrated the generation of useful antibody against HPIV3-HN and also shed new light

AlphaScreen with protein A-conjugated acceptor beads and streptavidin-coated donor. Upon excitation at 680 nm, singlet oxygen molecules were produced from the donor beads, which reacted with the

on the unexplored molecular link between the PIV3-HN and host proteins.

Currently, cell-based production (e.g., *E. coli* system or baculovirus-insect cell system) of recombinant virus proteins has been widely used. However, it is often difficult to produce sufficient quantities of viral antigens in the conventional cell-based system because many viral antigens are usually insoluble, cytotoxic, and are expressed in the inclusion body fraction. In contrast, the cell-fee protein production system enables the

(right panel). **(E)** Summary of properties of selected MAbs. Immunoglobulin isotyping was carried out with mouse monoclonal antibody isotyping test kit. synthesis of toxic proteins that are otherwise excluded from production in live cells. Among the cell-free approaches, the wheat germ cell-free system employs a eukaryotic translation system that warrants the synthesis of properly folded and bio-

logically active proteins similar to proteins that are expressed in living mammalian cells (Endo and Sawasaki, 2005, 2006). These advantages underscore the suitability and availability of the wheat germ cell-free system for the generation of antigenic proteins that can be used for animal immunizations to generate

MAbs. Our current study clearly demonstrated the benefit of using viral proteins synthesized by the wheat germ cell-free system to efficiently produce the MAbs against the viral antigen. Using this approach, we have created MAbs against HPIV3-HN that detected both denatured and native forms of the antigen. These MAbs were useful in various immunological assays including ELISA, IF, immunoblotting, and immunoprecipitation. Further careful studies of structural aspects are needed to determine whether the MAb can affect the virus infectivity.

We identified Hsp70 as a putative HN binding protein. Several previous studies demonstrated that Hsp70 was involved in the regulation of other RNA viruses. Hsp70 is known to associate with viral PB1 and PB2 subunits of influenza A virus, and it negatively regulated the expression of viral proteins in infected cells (Li et al., 2011). In another study, Hantavirus infection induced the expression of HSP70 that interacted with nucleocapsid protein and its overexpression suppressed viral infection in Vero E6 cells (Yu et al., 2009). In contrast, Hsp70 was

found to positively regulate rabies viral infection. Indeed, rabies infection induced the cellular expression of Hsp70 and accumulation in Negri body-like structures, which are the site of viral transcription and replication. Inhibition of Hsp70 resulted in a significant decrease of viral mRNAs, viral proteins, and virus particles (Lahaye et al., 2009, 2012). Taken together these results indicated a pivotal role of Hsp70 in viral replication and the pathogenicity of viral infection. Hsp70 binds and regulates many cellular proteins, as well as viral proteins (Pratt and Toft, 2003; Mayer, 2005; Silver and Noble, 2012), and the effects of Hsp70 on viral infection are diverse and unique between different viral species or cell systems. Further studies are required to investigate the precise molecular mechanism by which the association of Hsp70 and HN proteins mediate HPIV3 replication.

We also found that PIV3-HN can interact with Serpin3a. Serpin3a, as also known as alpha-1-antichymotrypsin, is a member of serpin proteins involved in the inhibition of serine and other types of proteases (Bauman et al., 2002). In humans, the majority

of serpins regulates the functions of proteases involved in the response against body's injuries such as coagulation, fibrinolysis, inflammation, wound healing and tissue repair. Serpins have been also implicated in various pathologies in respiratory system such as airway hyperresponsiveness (AHR) and asthma (Sivaprasad et al., 2011). The physical interaction of HPIV3- HN with serpin3a may be involved in hindering the function of Serpin3a toward respiratory disorders. Further analysis may shed new light on understanding the etiology of HPIV3-induced asthma.

In summary, we utilized the wheat cell-free production system to create and characterize MAbs that may be useful in various immunological applications. Our newly-developed MAbs could thus provide a valuable means to explore HPIV3 infection in human cells.

## **ACKNOWLEDGMENTS**

We thank Tomoe Suzuki for technical assistance, and Dr. Kei Miyakawa for FACS analysis. This work was partially supported by the fund for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems from the Ministry of Education, Culture, Sports, Science and Technology, and Research Grants from The Ministry of Health Labour and Welfare of Japan to Akihide Ryo.

#### **REFERENCES**

Ali, A., and Nayak, D. P. (2000). Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein. *Virology* 276, 289–303. doi: 10.1006/viro.2000.0556

Bauman, S. J., Whinna, H. C., and Church, F. C. (2002). Serpins (serine protease inhibitors). *Curr. Protoc. Protein Sci.* Chap. 21, Unit 21.7. doi: 10.1002/0471140864.ps2107s26

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 February 2014; accepted: 18 April 2014; published online: 13 May 2014. Citation: Matsunaga S, Kawakami S, Matsuo I, Okayama A, Tsukagoshi H, kudoh A, Matsushima Y, Shimizu H, Okabe N, Hirano H, Yamamoto N, Kimura H and Ryo A (2014)Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody. Front. Microbiol. 5:208. doi: 10.3389/fmicb.2014.00208*

*This article was submitted to Virology, a section of the journal Frontiers in Microbiology.*

*Copyright © 2014 Matsunaga, Kawakami, Matsuo, Okayama, Tsukagoshi, kudoh, Matsushima, Shimizu, Okabe, Hirano, Yamamoto, Kimura and Ryo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*