Elsevier

Veterinary Microbiology

Volume 160, Issues 3–4, 7 December 2012, Pages 277-289
Veterinary Microbiology

Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate/chitosan nanoparticles induces protection against highly pathogenic influenza A virus

https://doi.org/10.1016/j.vetmic.2012.05.035Get rights and content

Abstract

Intranasal administration of recombinant influenza hemagglutinin (rHA) antigen or inactivated virus with nanoparticles (NPs) composed of poly-γ-glutamic acid (γ-PGA) and chitosan which are safe, natural materials, and able to target the mucosal membrane as a mucosal adjuvant, could induce a high degree of protective mucosal immunity in the respiratory tract. Intranasal immunization with mixture of rHA antigen or inactivated virus and γ-PGA/chitosan nanoparticles (PC NPs) induced not only a high anti-HA immunoglobulin A (IgA) response in lung and IgG response in serum, including anti-HA neutralizing antibodies, but also an influenza virus-specific cell-mediated immune response. Also, PC NPs could function as a potential mucosal adjuvant when it was compared with the well-known mucosal adjuvant, cholera toxin (CT). Intranasal administration of rHA antigen or inactivated virus with PC NPs protected mice against challenge with a lethal dose of the highly pathogenic influenza A H5N1 virus. These results suggested that application of PC NPs with a subunit antigen of influenza produced by prokaryotic expression system provides several solutions to the current problems of the influenza vaccines using inactivated influenza virus. Moreover, our finding about a sufficient function of PC NPs to elevate vaccine efficacy led us to consider that it can be useful in clinical applications as a potent mucosal adjuvant with safety.

Introduction

Influenza A viruses are a major cause of respiratory diseases in human (Wright et al., 2007, Korteweg and Gu, 2010, Zhao et al., 2010) and of serious economic losses in the poultry industry. Especially, a pandemic with the highly pathogenic avain influenza (HPAI) H5N1 virus is being continuously reported in avian species and human beings (WHO, 2011). There are numerous studies that have been conducted on influenza A viruses and efforts to develop defenses against the virus (Jindrich et al., 2007) and several currently available influenza vaccines have managed to prevent several influenza infections. However, the current parenteral vaccines using inactivated virus have been shown some defects in protection and preparation (Quan et al., 2008).

Concern of recombinant subunit vaccine has recently been increased. The production of subunit protein from prokaryotic expression system is able to bring lower cost and shorter period of preparation on influenza vaccine. However, most protein antigens of influenza virus produced as recombinant subunit vaccines are poor immunogens and are not able to induce a sufficient level of vaccine-specific immunoglobulin to disrupt viral infection (Okamoto et al., 2007). For the benefits of recombinant subunit vaccine, many researchers have tried to make up this defect and the insufficient level of immune responses could be reinforced with an adjuvant.

The respiratory mucosal surface is the main site of influenza virus infections as well as a major site of immune defense. Several recent studies have focused on development of mucosal vaccines capable of effectively inducing both mucosal and systemic immune responses (Yuki and Kiyono, 2003). In the respiratory tract, secreted mucosal IgA for the influenza virus is more effective against the virus infection than the serum IgG induced by systemic immunization (Liew et al., 1984, Greenbaum et al., 2001, Okamoto et al., 2009).

The coadministration of an adjuvant with vaccine is essential to induce the effective and protective mucosal immunity. Since 1980, the mucosal adjuvants that were toxin based, such as cholera toxin (CT) and Escherichia coli heat-labile toxin (HLT), have been extensively studied (Tamura et al., 1994, Gluck et al., 2000). However, these toxin-based adjuvants caused severe diarrhea and a threat to the central nervous system (van Ginkel et al., 2000). Therefore, development of a safer and more effective adjuvant is necessary for mucosal immunization.

The use of self-assembling biodegradable nanoparticles (NPs) as a vaccine delivery system has recently been explored and highlighted (Akagi et al., 2005, Uto et al., 2007). The NPs associate with the antigen and in turn enhance antigen uptake by antigen-presenting cells, such as macrophages and dendritic cells (DCs), resulting in enhanced antigen-specific acquired immune responses (Akagi et al., 2005). Furthermore, NP uptake by DCs results in the stimulation of cytokine production, upregulation of costimulatory molecules, and enhanced T-cell stimulatory capacity in the cells (Uto et al., 2007). Thus, NP delivery systems are considered as effective adjuvants for subunit vaccines. However, there is still one problem in application of NP for vaccine delivery that its non-biodegradable nature.

Poly-gamma glutamate (γ-PGA) is a capsular polymer secreted by Bacillus subtilis, which is generally regarded as a safe organism (Poo et al., 2010). γ-PGA is a safe, edible substance and is naturally degraded by γ-glutamyl transpeptidase, which is widely distributed throughout the body. γ-PGA has a self-assembling ability and can form biodegradable NPs. Recently, properties of NPs composed of γ-PGA have been reported, such as antitumor, antiviral, and especially adjuvant activities (Lin et al., 2005, Lin et al., 2007, Lee et al., 2009). According to the report by Okamota et al., γ-PGA NPs were highly effective in enhancing the efficacy of an inactivated influenza HA vaccine, with stimulation of influenza-specific cell-mediated responses and the induction of humoral immune responses (Okamoto et al., 2007, Okamoto et al., 2009, Akagi et al., 2005, Akagi et al., 2006).

However, different from the low-molecular-weight γ-PGA used in that study, which was obtained from B. subtilis natto (low molecular weight, LMW: 10–1000 kDa), high-molecular-weight γ-PGA from B. subtilis chungkookjang (high molecular weight, HMW: >2000 kDa) showed higher levels of induction of natural killer cell-mediated cytotoxicity, interferon (IFN)-γ secretion, and DCs than LMW γ-PGA (Kim et al., 2007, Lee et al., 2009, Poo et al., 2010).

Chitosan is a cationic polysaccharide, derived from chitin by alkaline deacetylation. It is also a safe and biocompatible polymer (Vila et al., 2004). Additionally, it is known that chitosan has a special feature of adhering to mucosal surfaces for the adsorption of protein drugs (Lin et al., 2005). Lin et al. reported that when LMW chitosan (polycationic) was mixed with HMW γ-PGA (anionic) by a mild ionic-gelation method, the resulting γ-PGA/chitosan nanoparticles (PC NPs) showed effective delivery of peptides and protein drugs and other large hydrophilic molecules (Lin et al., 2005). Therefore, such NPs composed of LMW chitosan and HMW γ-PGA may address the need for a strong immune response along with safety, especially at mucosal surfaces, not only against influenza, but also for other viruses.

In this study, we examined whether PC NPs composed of HMW γ-PGA and chitosan can function as an effective mucosal adjuvant for an influenza vaccine. Intranasal inoculation of PC NPs with the expressed recombinant influenza hemagglutinin (HA) protein based on HPAI H5N1 virus through prokaryotic expression system or intranasal inoculation of PC NPs with inactivated virus as a representative current influenza vaccine into mice was able to induce effective mucosal, antigen-specific humoral, cell-mediated immune responses, and provided a protective immune response against HPAI virus infection. Thus, the present study reports that biological functions of PC NPs as an effective mucosal adjuvants and also a protective ability of the recombinant subunit vaccine with PC NPs as a potential influenza vaccine means.

Section snippets

Virus for challenge test

The HPAI H5N1 virus (A/EM/Korea/W149/06) used in this study was isolated from fecal specimens collected from wild bird habitats (Lee et al., 2008). Viruses were serially diluted 10-fold and inoculated in eggs; titers were calculated as log EID50/mL (Reed and Muench, 1938). Stock viruses were kept at −80 °C before use. The mouse 50% lethal dose (MLD50) was defined as the EID50 resulting in 50% mortality, calculated by the method of Reed and Muench (1938) with some modifications. All use of the

Confirmation of the target protein and rHA-loaded NPs

Expression of the purified rHA (globular head domain of HA1) protein was confirmed by SDS-PAGE and immunoblotting. The electrophoresed proteins on the SDS-PAGE gel were stained with Coomassie brilliant blue, and a protein band was observed at the expected molecular weight (26.3 kDa) after destaining (Fig. 1A). Moreover, reactions between rHA protein and anti-His (C-term) mouse antibody and rHA and anti-A/EM/Korea/W149/06 virus mouse antibody were confirmed by immunoblotting at 26.3 kDa (Fig. 1B

Discussion

It has been reported that induction of mucosal immune responses in the respiratory tract, which is a major route of virus entry, is effective immunization strategies for protection against influenza virus infection. For more effective protection against influenza virus infection at the mucosa, several mucosal adjuvants (e.g., CTB, LT) have been studied. However, intranasal administration of influenza vaccine together with the mucosal adjuvant LT reportedly led to some cases of Bell's palsy (

Conflicts of interest

There are no potential conflicts of interest.

Acknowledgments

This study was supported by National Agenda Project grant from the Korea Research Council of Fundamental Science and Technology and the KRIBB Initiative program (Grant no. KGM0821113), and the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant no. 110057-03, 111106-02). The study was also financially supported by the research fund of Chungnam National University in 2011.

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    These authors contributed equally to this study.

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