Antiviral Activity of Recombinant Mouse β-defensin 3 against Influenza a Virus in vitro and in vivo

Abstract

Background:

Influenza causes significant morbidity and mortality. Mammalian β-defensins are small peptides of about 4.5–6 kDa in mass and are effectors of the innate immune response with potent antimicrobial activity. In this paper, we focused on the anti-influenza A activity of the recombinant mouse β-defensin 3 (rMBD-3) in vivo and in vitro.

Methods:

The rMBD-3 peptide was added to Madin-Darby canine kidney (MDCK) cells at different stages of influenza A virus (IAV) A/PR/8/34 (H1N1) infection and its virus inhibitory properties were determined. Mice were infected with IAV and treated with rMBD-3 peptide from 12 h post-infection. The effect of rMBD-3 peptide was determined by pulmonary viral load, pathology and mortality. In addition, the expression of interleukin (IL)-12, interferon (IFN)-γ and tumour necrosis factor (TNF)-α genes in mice with or without rMBD-3 treatment was determined by semi-quantitative reverse transcriptase PCR.

Results:

rMBD-3 was shown to protect MDCK cells against IAV infection and had a major role in inhibition of adsorption and uptake by cells infected with IAV. Following the addition of 100 μg/ml rMBD-3 to MDCK cells medium, approximately 80% of cells were protected from infection in vitro. rMBD-3 given by tail vein injection (10 mg/kg/day) was the most effective method to improve the survival rate of the mice. Treatment with rMBD-3 was found to up-regulate IFN-γ and IL-12 gene expression, but reduced expression of the TNF-α gene. Conclusions: These results demonstrate that rMBD-3 possesses anti-influenza virus activity both in vivo and in vitro that might be of therapeutic use.

Introduction

Influenza continues to present a serious threat to human health. Human influenza A viruses (IAV) is one of the major causes of morbidity and life-threatening respiratory tract diseases worldwide [1]. Globally, seasonal influenza causes between 250,000 and 500,000 deaths every year [2]. Influenza is not only a disease of great medical importance but also of economic importance. To date, an influenza pandemic seems to be inevitable as the nature of the virus means that there is currently no option of a generic vaccine. Furthermore, a rapid increase of resistance to several antiviral drugs has been highlighted [3]; therefore, new prevention and treatment methods are still needed.

The main site of IAV infection is the respiratory mucosa, including nasal sinuses, bronchiolar epithelium and alveolar cells. After the initial exposure to IAV, virus infection progresses rapidly, reaching peak virus titres in the upper airway within 48 h after infection [4]. This situation is readily demonstrated in experimentally infected people and in laboratory animals, such as mice, ferrets, cotton rats and guinea pigs [5 –8].

Defensins provide a central role in innate immunity in all species of plants and animals and are cationic peptides classified by their pattern of conserved cysteines that are primarily known for their antimicrobial properties. Mammalian defensins are organized into three classes: α-, β- and θ-defensins, based primarily on the spacing of cysteine residues and the topology of the disulfide bridges [9]. Defensins are antimicrobial peptides that are important for innate host defence and, in addition to their antimicrobial properties, they can modulate immune responses. The α-, β- or θ-defensins are known to protect against HIV [10,11], influenza virus [12,13], human adenovirus [14], severe acute respiratory syndrome coronavirus [15], papillomavirus [16] and herpes simplex viruses [17,18]. Human β-defensins 1 and 2 are chemotactic for memory T-cells and immature dendritic cells [19].

In our previous study [20], pcDNA 3.1(+)/MBD-3, a eukaryotic expression plasmid was expressed and purified, and transfected into Madin-Darby canine kidney (MDCK) cells. The plasmid was injected into mice to verify its effects against influenza virus infection. The results showed that recombinant mouse β-defensin 3 (rMBD-3) protected against influenza virus infection in mice and cells with an efficiency ranging between 60% and 70% in vivo and in vitro. Furthermore, we constructed an rMBD-3 using a prokaryotic expression system and verified its antibacterial and antifungal activity [20]. In this report, we examine the activities of rMBD-3 against IAV infection in mice in vivo and in MDCK cells in vitro.

Materials and methods

Recombinant mouse β-defensin 3 peptides

A prokaryotic expression plasmid pET32a(+) that contained MBD-3 was constructed and transformed into Escherichia coli Rosetta-gami(2)™ strain (Novagen, Schwalbach, Germany). Expression of the plasmid was induced and rMBD-3 was recovered by rounds of nickel (Ni) affinity chromatography and thrombosin digestion. The molecular weight and specificity of rMBD-3 were verified by mass chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Ni affinity purification of fusion MBD-3 accounted for 62.9% of the total protein of the bacterial cells. According to the mass spectrometry there were about 90% 4,632-Da peptides in the recombination peptides [21]. The mature peptide sequence of MBD-3 is KINNPVSCLRKGGRCWNRCIGNTRQIGSCGVPFLKCCKRK. Purified and filtered (with a membrane with a 0.22 μm pore size) rMBD-3 (lipopolysaccharide <0.001 EU/μg) was diluted in 10 mM phosphate-buffered saline (PBS; 8.2 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and tested for cytotoxicity and antiviral activity at concentrations of 2,000, 1,000, 500, 250, 125 and 62.5 μg/ml.

Cells, virus and mice

MDCK cells, A549 (human lung carcinoma) cells and IAV A/PR/8/34 (H1N1) were maintained in the laboratory (Sichuan University, Chengdu, China).

MDCK cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. The DMEM medium used for the antiviral test was serum-free. A549 cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. The RPMI 1640 medium used for the antiviral test was serum-free. IAV titre was determined in MDCK cells or in A549 cells and expressed as the 50% tissue culture infectious dose (TCID₅₀) using the method described by Reed and Muench [22].

Specific-pathogen-free male BALB/c mice (4-week-old) were obtained from the Laboratory Animal Center of Sichuan University (Sichuan, China).

Cytotoxicity assays

MDCK and A549 cells were grown in DMEM or RPMI 1640 medium, seeded into 96-well tissue culture plates and incubated at 37°C until a confluent monolayer were formed. Different concentrations of rMBD-3 were added to the medium (final concentrations were 200, 100, 50 or 25 μg/ml) for 48 h. Cell proliferation and viability were determined using a cell reproduction quantitative detection kit (GMS10039; Genmed Scientifics Inc., Shanghai, China; MTT method).

Anti-IAV activity

Anti-IAV activity was detected by protocols described by Hazrati and colleagues [17]. There were four different assays performed, and the steps for the synchronized infectivity assays are shown in Figure 1. IAV was incubated with rMBD-3 at 4°C for 2 h and MDCK cells were cooled to 4°C and then pre-incubated IAV (100xTCID₅₀/well) was added and cells were kept at 4°C for a further 2 h. Unbound viruses were removed by washing and the cell cultures were incubated at 37°C for 60 min to permit virus uptake. Viruses that had bound to the cell, but which had not been taken up were inactivated by treating the monolayer with a buffer of pH 3.0. The cells were then overlaid with fresh serum-free medium. This was the first pre-incubation step. IAV (100×TCID₅₀/well) was added and cells were kept at 4°C for a further 2 h and the next steps were as described previously. rMBD-3 (6.25–100 μg/ml) and control buffer was added at each step (step 2, binding; step 3, entry; and step 4, post-entry) as indicated in Figure 1. The IAV titre was determined 2 days after infection by titration in MDCK cells.

Figure 1.

Schematic of synchronized infectivity assays that demonstrate that rMBD-3 is active at multiple steps in the virus life cycle

Therapeutic efficacy in mice

Mice were anaesthetized with ether and infected intranasally with 20 μl of mouse-adapted IAV (10×50% lethal dose [LD₅₀]/mouse). The mice were divided into eight groups of nine mice per group: (1) PBS control group, 10 mM/l PBS 0.2 ml/mouse/day, once daily, intraperitoneal injection; (2) low-dose rMBD-3, 5 mg/kg/day, once daily, intranasal infection; (3) high-dose rMBD-3, 10 mg/kg/day, once daily, intranasal infection; (4) low-dose rMBD-3, 5 mg/kg/day, once daily, intraperitoneal injection; (5) high-dose rMBD-3, 10 mg/kg/day, once daily, intraperitoneal injection; (6) low-dose rMBD-3, 5 mg/kg/day, once daily, tail intravenous injection; (7) high-dose rMBD-3, 10 mg/kg/day, once daily, tail intravenous injection; (8) ribavirin group, 75–100 mg/kg/day, once daily, intraperitoneal injection. A no-treatment group was also included. Treatment began 12 h after infection and was performed once a day for 3 weeks. After 3 days of treatment, three mice per group were sacrificed and the IAV titre of bronchoalveolar lavage fluids, lung pathology and lung index of these mice were determined. In addition, the decreasing rates of the IAV titre were used to determine the effects of treatment of rMBD-3 (the decreasing rates = [the IAV titre of PBS control group - the IAV titre of each treatment of rMBD-3 group]/the IAV titre of PBS control group). The remaining six mice per group were fed and treated, and change of weight or mortality were measured. Lung indices were calculated as described by Wilson et al. [23]: lung index = ([lung weight/body weight]× test animal)/([lung weight/body weight] × control animal).

IL-12, IFN-γ and TNF-α genes identification by semi-quantitative reverse transcriptase PCR

Twelve mice were infected intranasally with IAV 0.2×LD₅₀. The mice were divided into four groups each containing three mice: (1) high-dose rMBD-3, 10 mg/kg/day, tail vein injection; (2) low-dose rMBD-3, 5 mg/kg/day, tail vein injection; (3) ribavirin group, 75–100 mg/kg/day, intraperitoneal injection; and (4) PBS control group, 10 mM/l PBS 0.2 ml/mouse/day, tail vein injection. Treatment started 12 h after infection. After 5 days of treatment, the mice were sacrificed and their spleens recovered and stored in liquid nitrogen. Interleukin (IL)-12, interferon (IFN)-γ and tumour necrosis factor (TNF)-α gene sequences were obtained from GenBank, accession numbers AL607030, AC153498, and CR974444, respectively. RNA was isolated from the spleen tissue and reverse transcribed using the First Strand cDNA Synthesis Kit (Fermentas, Hanover, MD, USA). The resulting complementary DNAs (cDNAs) were subjected to PCR amplification with 20 pM each of the forward and reverse primers (Table 1) specific for mouse IL-12, IFN-γ, TNF-α or β-actin. PCR products were analysed by gel electrophoresis using a 2% agarose gel. Gels were scanned and yield of each band was quantitated by densimetric analysis of the image using Bandscan 4.3 software (Glyko, Hayward, CA, USA).

Table 1.

Primers used for the amplification and product sizes of IL-12, IFN-γ, TNF-α, Mbd-3 and ACTB

Target mRNA	Forward primer 5′-3′	Reverse primer 5′-3′	Size of product, bp
IL-12	TTATGTTGTAGAGGTGGACTGG	TTTCTTTGCACCAGCCATGAGC	734
IFN-γ	TGAACGCTACACACTGCATCTTGG	CGACTCCTTTTCCGCTTCCTGAG	460
TNF-α	AGCCCACGTCGTAGCAAACCACCAA	ACACCCATTCCCTTCACAGAGCAAT	446
ACTB	CCATGGATGACGATATCGCTGC	GCTTCTCTTTGATGTCACGCACG	669

ACTB, β-actin; IFN, interferon; IL, interleukin; Mbd-3, mouse β-defensin 3; mRNA, messenger RNA; TNF, tumour necrosis factor.

Statistics

SPSS 11.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Data were obtained from three independent experiments, each performed in triplicate and reported as mean ±sd. Comparison between groups in the antiviral assays was performed using the one-way analysis of variance (ANOVA), and results were deemed significance if the P-value was <0.05.

Results

Cytotoxicities of rMBD-3 peptides

Doses of 25–200 μg/ml rMBD-3 were not cytotoxic to either MDCK or A549 cells, and cell proliferation was comparable to that of the control groups without rMBD-3 (data not shown). These doses were used for subsequent antiviral studies.

rMBD-3 inhibits IAV infection

This study examined the ability of rMBD-3 to protect MDCK cells from infection by IAV. Cells incubated with rMBD-3 for 2 h before IAV infection had lower levels of virus production. With this regime, rMBD-3 was effective at decreasing IAV titre in a dose-dependent manner (Figure 2). An assay of the confluence of the cell monolayer showed that at a higher virus titre (100× TCID₅₀) of infection, rMBD-3 when applied during the entry or binding period decreased the IAV titre from infected cells; however, rMBD-3 when applied after the post-binding step had almost no antiviral effect compared with the virus control (Figure 2).

Figure 2.

The inhibition IAV infection in MDCK cells by rMBD-3

Therapeutic efficacy of rMBD-3 in mice

Mice administered 10 mg/kg/day rMBD-3 alone exhibited no toxic effects in vivo, as measured by clinical signs and weight loss (data not shown). On the third day after infection, the IAV titre of bronchoalveolar lavage fluids was significantly lower in all the rMBD-3 treated groups compared with the control group (P<0.05). Interestingly, the IAV titre in the high-dose rMBD-3 intranasally infected group was decreased by approximately 99.9% (Figure 3). Concurrently, the lung index and lung inhibitory rate of all groups were measured. The lung index was lower in all groups treated with rMBD-3, as shown in Figure 4.

Figure 3.

Virus titre in bronchoalveolar lavage fluids of different treatment groups

Figure 4.

Changes in lung index of different treatment groups

The therapeutic efficacy of rMBD-3 was evaluated on the basis of the survival rate at 3 weeks post-infection. Low- or high-dose intranasal infection, intraperitoneal injection or tail intravenous injection of rMBD-3 had a protective effect against lethal virus challenge. High-doses of rMBD-3 injected intravenously had an especially significant therapeutic effect, and the mouse survival rate was 83.3% (Figure 5).

Figure 5.

Survival analysis of mice after lethal challenge of 10xLD₅₀ IAV and with different treatment

Influence of rMBD-3 on the expression of IL-12, IFN-γ and TNF-α genes in mice

After 5 days of treatment, spleen cell cytokine expression in influenza-infected mice treated with rMBD-3 differed from that of the control. Treatment with rMBD-3 regulated IFN-γ and IL-12 gene expression, but TNF-α gene expression was reduced especially in the high-dose group (Figure 6) compared with the untreated group.

Figure 6.

Expressions of IL-12, IFN-γ and TNF-α in spleen tissues of mice of different treatment groups

Discussion

In the present study, rMBD-3 was examined for its ability to protect MDCK cells from infection by IAV. We showed that rMBD-3 prevented IAV infection by directly blocking virus binding to cells and cell entry. The lethal effects of IAV on MDCK cells were greatly reduced after incubation with rMBD-3 at 4°C for 2 h. The results showed that rMBD-3 can directly affect IAV infection. The direct effect of defensins on the microorganisms is mainly the destruction and function of membrane structure [24]. Leikina and colleagues [25] have shown that the synthetic primate θ-defensins (RC2) and human β-defensin 3 (HBD3) can block virus infection by a mechanism directly related to their ability to cross-link cell membrane glycoproteins into a fusion-resisting barricade. At the binding and entry stages of IAV infection, the addition of rMBD-3 can decrease the IAV titre. Similarly, rMBD-2 inhibits influenza virus infection by blocking entry into cells [12]. The effect of defensins on virus binding and entry steps has been widely reported [17,25,26]. Interestingly, when rMBD-3 was added after the entry step (post-entry), the effects were comparable to those obtained in the PBS control group. Therefore, these data indicate that rMBD-3 prevents influenza virus infection by blocking virus binding and entry rather than by inhibiting the subsequent stages of an ongoing infection.

To test this regime on animals, we selected different doses of rMBD-3 and different routes of administration. The results showed that the 10 mg/kg/day dose of rMBD-3 peptides given intranasally can reduce IAV titre in the respiratory tract of mice early after challenge, and that the 10 mg/kg/day dose of rMBD-3 peptides given intravenously was the most effective regime for improving the survival rate of mice challenged with influenza infection. Natural antiviral defences are mediated by the innate and adaptive immune systems. MBD-3 expression is induced after infection, and it can be expressed at low levels in some organs without any inducing factors [20]. In our study [20], the Mbd-3 gene was highly expressed in the upper respiratory tract (trachea) and lower respiratory tract (lung tissue) of mice following an influenza infection. This finding suggests that MBD-3 has an important role in natural immunity to influenza virus infection.

IL-12 and IFN-γ could promote the differentiation of Th0 cells to Th1 cells, activate cytotoxic T-lymphocytes and natural killer cells, and enhance their cytotoxic activity. Monteiro et al. [27] confirmed by experiments in mice that IL-12 is conducive to the early control of influenza. TNF-a, which was produced mainly by mononuclear macrophages and T-lymphocytes, could stimulate a variety of cells to produce inflammatory cytokines, and cause necrosis, protein damage and interstitial oedema. Defensins can activate the antiviral immune response indirectly. Niyonsaba and colleagues [28] reported that human β-defensin 2 acts as a chemotaxin for mast cells, and also enables the activation and degranulation of mast cells to release histamine and prostaglandin D₂. Furthermore, Pazgier and colleagues [19] reported that human β-defensins 1 and 2 were chemotactic for memory T-cells and immature dendritic cells. Similarly, we found that rMBD-3 could influence the gene expression of some cytokines (IL-12, IFN-γ and TNF-α) in mice infected with IAV. The data presented herein are consistent with the multiple effects of defensins reported by ourselves and others and provide evidence of both direct antiviral activity of rMBD-3. It is not clear whether the dose-dependent regulation of murine cytokines that are known to play a role in influenza pathogenesis was a direct effect or was indirectly due to antiviral activity, since the antiviral drug ribavirin showed a similar effect in reducing cytokine levels.

In summary, we have identified a novel antiviral peptide, rMBD-3, that inhibited IAV infection by blocking virus attachment and entry into cells and that provided protection against IAV infection in experimental mice. Although the rMBD-3 peptide has biological activity consistent with that of the authentic MBD-3 peptide and is therefore expected to be structurally related, we have yet to rigorously demonstrate it is structurally identical. Therefore, rMBD-3 may be considered a good candidate as an agent to prevent anti-influenza virus infection.

Footnotes

Acknowledgements

The authors appreciate the editorial input provided by Elixigen Corporation (Huntington Beach, CA, USA).

This work was supported by grant from the Natural Science Foundation of China (project number 30671964) and the Science and Technology Department of Guizhou Province of China (number [2010]3143). The authors declare no competing interests.

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