Intranasal administration of poly-gamma glutamate induced antiviral activity and protective immune responses against H1N1 influenza A virus infection
© Kim et al. 2015
Received: 17 February 2015
Accepted: 17 September 2015
Published: 6 October 2015
The global outbreak of a novel swine-origin strain of the 2009 H1N1 influenza A virus and the sudden, worldwide increase in oseltamivir-resistant H1N1 influenza A viruses highlight the urgent need for novel antiviral therapy.
Here, we investigated the antiviral efficacy of poly-gamma glutamate (γ-PGA), a safe and edible biomaterial that is naturally synthesized by Bacillus subtilis, against A/Puerto Rico/8/1934 (PR8) and A/California/04/2009 (CA04) H1N1 influenza A virus infections in C57BL/6 mice.
Intranasal administration of γ-PGA for 5 days post-infection improved survival, increased production of antiviral cytokines including interferon-beta (IFN-β) and interleukin-12 (IL-12), and enhanced activation of natural killer (NK) cells and influenza antigen-specific cytotoxic T lymphocytes (CTL) activity.
These results suggest that γ-PGA protects mice against H1N1 influenza A virus by enhancing antiviral immune responses.
Influenza A virus is an important human pathogen that continues to affect global health and cause pandemics sporadically, including the 2009 H1N1 influenza outbreak. Vaccination is the most economic and effective strategy to control influenza pandemics. However, because influenza vaccines target the virus’s surface antigen, they do not provide effective protection against influenza virus strains having variant antigens. Moreover, current anti-influenza drugs have several limitations and recent studies show that some human isolates of influenza A virus (H1N1) are resistant to antiviral drugs [1–3]. Because we cannot presage which influenza strain will cause the next epidemic or pandemic to ensure that vaccines target the most troublesome strain, we need to develop antiviral drugs that broadly control influenza by enhancing effective host immune responses.
During infection, viruses begin counteracting the host immune response comprising not only the innate but also the adaptive immune systems. For instance, infection of influenza virus blocks many features of antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, including production of the antiviral cytokine type I IFN . Moreover, influenza virus-infected DCs fail to maturate costimulatory molecules and upregulate MHC class II molecules that are pivotal for antigen-specific, T-cell activation . García-Satre et al. suggested that such modulation of the host immune response is a major mechanism whereby viruses create a favorable environment for their replication and persistence . The hypothesis that prophylactic immunomodulators can be used to decrease influenza virus-induced mortality has been tested: The results suggest that immunomodulators, including CpG-oligonucleotide (CpG-ODN), can indeed initiate a rapid immune response characterized by antiviral and inflammatory cytokine production [7–9]. In fact, CpG-ODN have such strong immunostimulatory properties that pre-treatment with a single CpG-ODN dose of 1.5 or 2.5 μg provides partial protection to mice against lethal seasonal influenza virus.
Unfortunately, potent immunomodulator action is often correlated with increased toxicity. However, the immunomodulator poly-gamma glutamate (γ-PGA), which is produced by Bacillus subtilis chungkookjang, isolated from Chungkookjang, a traditional Korean fermented soybean food, is safe and edible . Unlike other pathogen-associated molecular patterns, γ-PGA retains its water-soluble, biodegradable, and non-toxic properties [11, 12]. Previously, we identified this molecule as a potent inducer of the host immune response in in vivo tumor models [13, 14]. Recently, treatment with γ-PGA enhanced the type I interferon and protected them against highly pathogenic influenza A virus (H5N2) in the B6.A2G-Mx1 mouse model . A biodegradable nanoparticles also have been demonstrated to be excellent immune stimulators with the potential to protect against viral infections . Taken together, these investigations suggest that γ-PGA may be effective therapy for tumor and infectious diseases. On the basis of these results, we hypothesized that γ-PGA might be effective as an antiviral agent against influenza virus.
Because influenza viruses enter through a mucosal surface, preventing infection at the viral entry site by inducing mucosal immunity via intranasal immunization is an attractive strategy for influenza protection. In this study, we intranasally administered γ-PGA to C57BL/6 mice in an attempt to protect them from lung pathogenicity during infection with lethal doses of PR8 or CA04 H1N1 influenza A virus, strains known to induce severe immune pathogenicity, cause high mortality, and localize to pulmonary tissue in mice. We found that γ-PGA-treated mice had reduced mortality and pathogenicity during influenza infection. Moreover, intranasal administration of γ-PGA was found to effectively induce protective innate and CTL immune responses against both viruses. These findings suggest that γ-PGA may be an effective novel immunomodulator against influenza virus-induced pulmonary pathogenicity.
γ-PGA induces antiviral activity against influenza A virus infection
γ-PGA induces viral clearance in mice with lethal influenza virus infection
Next, we evaluated the extent of the pathologic changes in the lungs of influenza virus-infected mice treated with immunomodulators. Typically, influenza A virus replication is accompanied by infiltration of immune cells in the lung tissue of the infected host . Consistent with published data, we observed induction of widespread inflammatory processes in influenza virus-infected mice not treated with immunomodulators. As expected, no histologic alterations indicative of severe inflammation or disruption of the membrane barrier were observed in the lung tissue of virus-infected mice given γ-PGA (Fig. 2c). Furthermore, only mild histopathologic changes were detected in the lungs of a PR8-infected mouse given CpG-ODN. However, lungs of CA04 virus-infected mice given CpG-ODN had considerably more bronchial cell infiltrates and peribronchial inflammation. Collectively, these results show that intranasal administration of γ-PGA could prevent histopathologic alterations in the lungs and protect mice against lethal influenza virus infection.
γ-PGA enhances activation of innate immune cells in influenza-infected mice
Next, we assessed γ-PGA’s ability to induce maturation of DCs in influenza virus-infected mice by using FACS analysis to measure the expression of maturation marker CD86 in lung DCs. Both γ-PGA- and CpG-ODN-treated mice had higher CD86 expression than untreated mice did (Fig. 3b). In addition, we confirmed that other DC maturation markers such as CD40 and CD80 are expressed in higher amounts in lung DCs from γ-PGA-treated mice than in those from untreated mice (data not shown). Taken together, these results show that intranasal γ-PGA promotes an innate immune response in the lung after activating NK cells’ cytotoxicity and DCs’ maturation during influenza virus infection.
γ-PGA induces cytokine production in mice infected with influenza virus
γ-PGA promotes influenza virus-specific CTL activity in mice infected with influenza virus
The antiviral effects of γ-PGA are dependent on CD8+ T cells in CD4+ or CD8+ T cell depleted mice models
The emergence of highly contagious influenza virus strains, such as the new pandemic H1N1 influenza, represents a serious threat to global human health, and influenza virus vaccines do not protect against antigenically drifted strains that frequently appear to cause seasonal influenza outbreaks. Given the emergence of drug-resistant strains and limited protection capability, a great need exists to develop strategies to protect against the next pandemic influenza. The current study evaluated the antiviral efficacy of intranasal administration of γ-PGA to prevent lethality in mice infected with H1N1 influenza A viruses.
Norton et al. reported that intranasal immunomodulators induce a local immune response that results in decreased mortality upon influenza A virus infection . In the present study, we showed that intranasal administration of γ-PGA shortly after viral infection could inhibit influenza virus replication, leading to a significant reduction in pulmonary viral titers and an improved survival rate of infected mice. Recently, Okamoto et al. reported that intranasal vaccination with γ-PGA nanoparticles containing influenza HA protein enhances protection against influenza virus infection . Taken together, although therapeutic evaluation in humans is required, these findings indicate a therapeutic utility for γ-PGA when used as an anti-influenza virus drug.
To develop optimal drugs against the influenza viruses, it is important to understand their mechanisms of action on the immune system . Host innate immune response is the first line of protection against infection by viruses and is essential in local control of invading microbes. During influenza infection, DCs are necessary to induce an immune response that clears the virus from the lungs [29, 30]. In addition, influenza virus is able to evade host immunity by attenuating NK cell lysis of influenza virus-infected cells [30, 31]. Österlund et al. reported that pandemic H1N1 (A/Finland/553/09) virus and seasonal H1N1 virus (A/Brisbane/59/07) induce a relatively weak innate immune response in macrophages and DCs, as evidenced by a poor expression of antiviral and inflammatory cytokine genes . IFN-β can inhibit the replication of a wide variety of viruses, which contributes to key effector molecules of the immune response to viruses . IL-12 and RANTES are also important cytokines involved in cellular immunity, which can potently stimulate cytotoxic T cells as well as NK cells’ activation [33–35]. Therefore, cytokines are promising of antiviral drugs for enhancing the host immune response against influenza virus infection. We found that cytokine production levels and activation of innate immune cells in lung samples from γ-PGA-treated mice were higher than those from untreated controls. In addition, we also observed a significantly higher number of activated NK1.1+ cells in total lung cells from γ-PGA-treated mice than in those from untreated mice (data not shown). Thus, our findings suggest that intranasal γ-PGA promotes the innate immune response during virus infection, protecting against influenza.
Cell-mediated immune responses play a crucial role in protecting the host from invading pathogens [36, 37]. Virus clearance requires strong Th1-polarized immune responses characterized by IFN- γ production and CTL responses in the systemic compartment . Moreover, earlier studies show that both CD4+ and CD8+ T cells contribute equally to protection against pandemic H1N1 influenza virus [24, 25]. In accordance with these reports, we observed that in vivo depletion of CD4+ and CD8+ T cells completely abrogated antiviral activity in γ-PGA-treated mice, with γ-PGA having no antiviral effects in CD8+ T cell-deficient mice. Our data clearly showed that γ-PGA increases the cytotoxic activity of CD8+ T cells in the lung against H1N1 influenza A viruses. Hanna et al. reported the precise contribution of NK cells to induction of adaptive immunity during influenza infection of the lung . Moreover, other studies have shown that depletion of NK1.1+ cells decreases the induction of virus-specific CTL activity and increases influenza disease severity during influenza virus infection in mice . The major function of DCs during an antiviral immune response is to process and present viral antigenic peptides in the draining lymph nodes to rare, antigen-specific T cells . Additionally, cytokines such as IL-12 are key molecules that trigger the adaptive immune responses (including of DCs maturation, differentiation of Th1 and Th2 cells, and induction of cytotoxic NK cells and CTLs), resulting in protective layers against virus infection [42, 43]. Taken together, our results indicate that γ-PGA enhances influenza-specific CD8+ T cell immunity, increasing cytokine production and activation of antigen-presenting cells. However, the molecular mechanism by which γ-PGA provided complete protection against influenza virus remains unknown.
In our study, we confirmed that γ-PGA enhances influenza-specific CD8+ T cell immunity, increasing cytokine production and activation of antigen-presenting cells. We also showed that administration of γ-PGA significantly improved the survival rate and virus inhibition of mice from infections of PR8 and CA04 viruses. Although further mechanism studies are needed, we showed that intranasal γ-PGA induced a higher influenza-specific CTL response and innate immune response than did CpG-ODN after influenza H1N1 virus infection. In light of this evidence, we suggest that γ-PGA may be a more potent immunomodulator against H1N1 influenza A virus infection than CpG-ODN.
In summary, we report for the first time that intranasal administration of γ-PGA generated significant antiviral activity in H1N1 influenza A virus-infected mice. These novel findings indicate that γ-PGA mediates anti-viral immunity via the induction of antigen-specific CTL activity, suggesting that this molecule could be a good candidate for development as a novel immunomodulator for anti-influenza treatment.
Mice, cells, and viruses
Six-week-old female C57BL/6 (H-2b) mice were purchased from KOATECH (Pyeongtaek, Korea) and housed in specific pathogen-free conditions at Korea Institute of Bioscience and Biotechnology (KRIBB) in Daejeon, Korea. Mouse experiments were approved by KRIBB’s Institutional Review Board. The mouse lymphoma cell line EL-4 (H-2b) was obtained from the Korea Cell Line Bank (Seoul, Korea) and maintained in complete Dulbecco’s modified Eagle’s medium (DMEM; Hyclone; UT, USA) supplemented with 10 % fetal bovine serum (FBS; Hyclone). Both A/Puerto Rico/8/1934 (PR8) and A/California/04/2009 (CA04) H1N1 influenza A viruses were grown for 2 days at 37 °C in the allantoic cavities of 10-day-old fertile chicken eggs. Virus stocks were propagated in the allantoic cavity of 10-day-old, embryonated, and specific pathogen-free hen’s eggs at 37 °C. Clarified allantoic fluid was aliquoted and immediately frozen at −80 °C.
Influenza virus infection
Mice were lightly anaesthetized and challenged intranasally with 50 % of the mouse lethal dose (MLD50) of PR8 or CA04 influenza A viruses in a total volume of 30 μl. After infection, mice were monitored daily for morbidity (i.e., body weight loss) and survival for up to 14 days post-infection (dpi). Individual body weights were recorded for each group on various dpi. The mice having more than 20 % of body weight lost were considered to have reached the experimental endpoint and were humanely killed.
To determine the titer of infectious virus, lung samples of 3 mice per group were collected at 3, 5, and 7 dpi. Lung samples were homogenized in 1 ml of sterile PBS containing penicillin G (200,000 U/ml), streptomycin (40,000 U/ml), polymyxin B (20,000 U/ml), and gentamicin (5 mg/ml). The titers of clarified lung tissue homogenates were determined by calculating the standard 50 % egg infectious dose (EID50); the detection limit was 0.75 log10 EID50/ml.
Because CpG-ODN has been demonstrated to be an excellent immunomodulator with the potential to protect against influenza virus infection , it was used as the positive control for our experiments to test the anti-influenza efficacy of γ-PGA immunomodulator treatment. CpG-ODN was purchased from InvivoGen (SD, USA), and γ-PGA was kindly provided by Bioleaders Corporation (Daejeon, Korea). Mice were infected with either CA04 or PR8 (1 MLD50), and the immunomodulators were used as follows: γ-PGA (100 μg in 20 μl endotoxin-free PBS [Sigma; MO, USA]) was intranasally administered to mice for 7 days starting 24 h after virus infection or CpG-ODN (5 μg in endotoxin-free PBS) was intranasally administered once 24 h prior to virus infection. Control mice were administered an equal volume of endotoxin-free PBS.
At 5 dpi, fresh lung tissues were collected from PBS-treated, CpG-ODN-treated, and γ-PGA-treated mice (n = 3 per group), fixed in 10 % neutral buffered formalin and then dehydrated, embedded in paraffin, and cut into 4 μm-thick sections. The sections were stained with hematoxylin and eosin (H&E) and examined by using an Olympus TH4-200 System Microscope attached to a DP72 Microscope Digital Camera. The captured images were analyzed by using DP2-BSW software (Ver.2.1, OLYMPUS).
Flow cytometry analysis
Single-cell suspensions were prepared from mouse lung by using type II collagenase and DNase treatment. In brief, whole lung tissue was removed, minced in collagenase digestion solution (10 ml PBS containing 20 μg/ml type II collagenase [Roche, NJ, USA] and 100 μg/ml DNase I [Roche]), and incubated at 37 °C for 30 min. Lung tissue pieces were meshed through a 40-μm cell strainer (Falcon) before red blood cells in the supernatant were lysed by using red blood cell-lysis buffer (Sigma). To measure the population of lung immune cells, approximately 1 × 106 cells were stained in 100 μl FACS buffer (PBS containing 2 % FBS [v/v] and 0.02 % sodium azide [v/v]) with FITC-conjugated anti-mouse CD40, CD80, and CD86 antibodies. To avoid nonspecific counting, cells were gated with either anti-mouse CD49b or anti-mouse CD11c+ antibody. All antibodies were purchased from BD Biosciences (CA, USA). Data were acquired on a BD FACSCalibur (BD Biosciences) and analyzed by using the CELLQuest Pro software (BD Biosciences).
Quantification of cytokines
To count cytokines, mouse lung tissue was harvested and homogenized in 2 ml of tissue protein extraction reagent (Pierce: IL, USA) containing protease inhibitor cocktail (Roche). The homogenate was centrifuged at 16,000 g for 20 min at 4 °C before the supernatant was harvested. The amounts of the following cytokines were analyzed: IL-1β, IL-4, IL-6, IL-12p40, IL-15, IFN-γ, tumor necrosis factor-α (TNF-α), and regulated on activation normal T-cell expressed and secreted (RANTES). The concentration of IFN-β in the homogenate was determined by using an IFN-β ELISA kit (PBL Laboratories; NJ, USA).
To isolate the CD8+ T cells and NK cells, total lung cells were first digested in collagenase digestion solution. Both cell types were magnetically separated via negative selection with a CD8+ T-cell isolation kit (Miltenyi Biotec; CA, USA) and positive selection with a CD49b (DX5) kit (Miltenyi Biotec). The cell-mediated cytotoxicity assay was performed using the EL-4 cells as target cells  and quantified by using the lactate dehydrogenase (LDH)-release assay kit according to the instructions (Promega; CA, USA). In brief, lung NK cells and CD8+ T cells were mixed with target cells at an effector cell to target cell ratio of 50:1 and incubated at 37 °C for 4 h. To determine the CD8+ T cells’ cytotoxicity, target cells were stimulated with the 50 % tissue culture-infective dose (TCID50) of UV-inactivated virus for 24 h. Each sample was incubated with 50 μl of substrate at room temperature for 30 min, and optical density (OD) values were measured at 490 nm wavelength by using the VICTOR3 1420 multilabel counter plate reader (Perkin-Elmer; MA, USA). The cytotoxicity percentage was calculated by using the following formula: (mean experimental OD value–mean spontaneous OD value)/(mean maximal OD value–mean spontaneous OD value) × 100.
IFN-γ ELISPOT assay
Ninety-six-well filtration plates (Millipore, MA, USA) were pre-coated with anti-mouse IFN-γ antibody (5 μg/ml; BD Biosciences) overnight at 4 °C. After the plates were washed four times with PBS and then blocked with RPMI 1640 medium (Gibco), CD8+ T cells (1 × 105 cells in 100 μl of medium) were added to each well of the filtration plates along with UV-inactivated influenza virus (1 TCID50)  in 100 μl of medium). The plates were incubated for 72 h, washed with PBS containing 0.5 % Tween-20 (PBS-T), and incubated with biotin-conjugated anti-mouse IFN-γ antibody (1 μg/ml) for 2 h at room temperature. After 5 washes with PBS-T, diluted horseradish peroxidase (HRP)-conjugated streptavidin antibody (1:200; BD Biosciences) was added to the each well and incubated at room temperature for 1 h. The cells were washed 5 times with PBS-T; ELISPOT AEC substrate (100 μl; BD Biosciences) was added to develop the reaction, and then the reaction was stopped by washing the cells with double-distilled water. The spots were counted automatically by using the ELISPOT CTL-ImmunoSpot S5 UV Analyzer (Cellular Technology; OH, USA).
CD4+ and CD8+ T cell depletion
For T-cell depletion studies, mice were immunized with 100 μg of γ-PGA 5 days after influenza virus inoculation. Groups of mice were then injected intraperitoneally with 100 μg anti-CD4 antibody (clone GK1.5; BioLegend), 100 μg anti-CD8 antibody (clone 53.6.72; BioLegend), and 100 μg of both IgG2b isotype control antibodies (BioLegend). Antibodies were injected every other day three times before and twice at 3 days and 7 days after inoculated with a MLD50 of CA04 virus (5.5 EID50/ml per mouse). The positive control group was not injected with antibodies but was γ-PGA-treated after challenge. After virus infection, mice were monitored daily for weight loss and survival.
The analyses were done by using GraphPad Prism version 5.00 for Windows (GraphPad Software, CA, USA). P values of less than 0.05 (p < 0.05) were considered to be statistically significant.
All animal experiments were approved by the Institutional Animal Use and Care Committee of the Korea Research Institute of Bioscience and Biotechnology and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
We thank Sun-Woo Yoon and C. Guess (St. Jude Children’s Research Hospital, USA) for scientific editing the manuscript. This work was supported by grants of National R&D Program for Cancer Control (0720510) from Ministry of Health & Welfare and a National Research Foundation of Korea grant ( 2014R1A2A1A10049622) to H. Poo.
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