Alloferon and Zanamivir Show Effective Antiviral Activity against Influenza A Virus (H1N1) Infection In Vitro and In Vivo

The use of vaccines is the most effective and reliable method for the prevention of viral infections. However, research on evaluation of effective therapeutic agents for use in treatment after infection is necessary. Zanamivir was administered through inhalation for treatment of pandemic influenza A/H1N1 in 2009. However, the emergence of drug-resistant strains can occur rapidly. Alloferon, an immunomodulatory drug developed as an NK cell activator, exerts antiviral effects against various viruses, particularly influenza viruses. Therefore, alloferon and zanamivir were administered in combination in an effort to improve the antiviral effect of zanamivir by reducing H1N1 resistance. First, we confirmed that administration of combined treatment would result in effective inhibition of viral proliferation in MDCK and A549 cells infected with H1N1. Production of IL-6 and MIP-1α in these cells and the activity of p38 MAPK and c-Jun that are increased by H1N1 were inhibited by combined treatment. Mice were then infected intranasally with H1N1, and examination of the antiviral efficacy of the alloferon/zanamivir combination was performed. The results showed that combined treatment after infection with H1N1 prevented weight loss, increased the survival rate, and improved lung fibrosis. Combined treatment also resulted in reduced infiltration of neutrophils and macrophages into the lungs. Combined treatment effectively inhibited the activity of p38 MAPK and c-Jun in lung tissue, which was increased by infection with H1N1. Therefore, the combination of alloferon/zanamivir effectively prevents the development of H1N1-mediated inflammation in the lungs by inhibiting the production of inflammatory mediators and migration of inflammatory cells into lung tissue.


Introduction
With an increased incidence of viral infections including SARS-CoV2 worldwide, the research and development of vaccines for the prevention of viral infection has also shown a recent increase [1,2]. Vaccines are known to be effective in the prevention of viral infection [3,4]; however, with regard to viral infectious diseases caused by avoidance of immune responses induced through vaccination, the development of therapies for the treatment of viral infection is important. Therefore, this study was conducted in order to

Alloferon Increases the Antiviral Effect of Zanamivir against H1N1 Infection
First, real-time RT-PCR was performed in order to assess the effect of alloferon and zanamivir on viral proliferation in H1N1-infected Madin-Darby canine kidney (MDCK), A549, adenocarcinomic human alveolar basal epithelial cell line and Calu-3, human lung adenocarcinoma cell line. As shown in Figure 1, when combined with zanamivir, alloferon showed more effective suppression of viral replication in both cell lines, A549 and MDCK, compared with control and a group treated with either drug alone. The suppressive effect was observed from 48 h post treatment and lasted for up to 72 h. A more efficient effect on the suppression of virus replication was observed in A549 cells compared with MDCK cells. In Calu-3 cells, the suppressive effect mediated by treatment with alloferon/zanamivir was also apparent at 48 h (see Supplementary Figure S1). increased in both subcutaneous injection and intranasal administration of alloferon, and that intranasal administration of alloferon was more effective than subcutaneous injection [28]. Therefore, in this study, we attempted to determine whether administration of a combination of zanamivir and alloferon can compensate for the weaknesses of the only zanamivir when used for treatment of influenza A infection.

Alloferon Increases the Antiviral Effect of Zanamivir against H1N1 Infection.
First, real-time RT-PCR was performed in order to assess the effect of alloferon and zanamivir on viral proliferation in H1N1-infected Madin-Darby canine kidney (MDCK), A549, adenocarcinomic human alveolar basal epithelial cell line and Calu-3, human lung adenocarcinoma cell line. As shown in Figure 1, when combined with zanamivir, alloferon showed more effective suppression of viral replication in both cell lines, A549 and MDCK, compared with control and a group treated with either drug alone. The suppressive effect was observed from 48 h post treatment and lasted for up to 72 h. A more efficient effect on the suppression of virus replication was observed in A549 cells compared with MDCK cells. In Calu-3 cells, the suppressive effect mediated by treatment with alloferon/zanamivir was also apparent at 48 h (see Supplementary Figure S1). Quantitative real-time PCR analysis of the levels of influenza virus (normalized to GAPDH) was performed in the presence or absence of alloferon (0.5 μg/mL) and zanamivir (35 μg/mL). Briefly, cells were infected with influenza A virus (MOI 0.01), extraction of total RNA was performed, and RT-PCR was performed using primers specific for H1N1. The amount of virus in treated cells is shown relative to that in control cells (cells infected with virus but not treated). Results are representative of more than three independent experiments. Control: Virus only (A) MDCK (B) A549 * p < 0.1, ** p < 0.01, *** p < 0.001, NS; not significant.

Combination Alloferon/Zanamivir Suppresses Production of IL-6 and MIP-1α Induced by H1N1 Infection In Vitro.
Infection with influenza A virus induces the production of IL-6 and MIP-1α, which are involved in development of sepsis and induction of the cytokine storm in the lung [50][51][52][53]. Therefore, the question of whether combined treatment with alloferon and zanamivir can suppress production of IL-6 and MIP-1α after infection with H1N1 was examined. The results showed that the production of IL-6 in MDCK cells increased at 72 h post infection with H1N1, while increased production of MIP-1α was observed at 48 h post infection; however, effective suppression of both was achieved by combined treatment with alloferon and zanamivir (Figure 2A,C). Regarding A549, increased production of IL-6 was observed at 48 h after infection with H1N1, and an increased production of MIP-1α was observed at 72 h; combined treatment with alloferon and zanamivir resulted in suppression of both ( Figures 2B,D). Alloferon increased the antiviral efficacy of zanamivir after infection with H1N1. Quantitative real-time PCR analysis of the levels of influenza virus (normalized to GAPDH) was performed in the presence or absence of alloferon (0.5 µg/mL) and zanamivir (35 µg/mL). Briefly, cells were infected with influenza A virus (MOI 0.01), extraction of total RNA was performed, and RT-PCR was performed using primers specific for H1N1. The amount of virus in treated cells is shown relative to that in control cells (cells infected with virus but not treated). Results are representative of more than three independent experiments. Control: Virus only (A) MDCK (B) A549 * p < 0.1, ** p < 0.01, *** p < 0.001, NS; not significant.

Combination Alloferon/Zanamivir Suppresses Production of IL-6 and MIP-1α Induced by H1N1 Infection In Vitro
Infection with influenza A virus induces the production of IL-6 and MIP-1α, which are involved in development of sepsis and induction of the cytokine storm in the lung [50][51][52][53]. Therefore, the question of whether combined treatment with alloferon and zanamivir can suppress production of IL-6 and MIP-1α after infection with H1N1 was examined. The results showed that the production of IL-6 in MDCK cells increased at 72 h post infection with H1N1, while increased production of MIP-1α was observed at 48 h post infection; however, effective suppression of both was achieved by combined treatment with alloferon and zanamivir (Figure 2A,C). Regarding A549, increased production of IL-6 was observed at 48 h after infection with H1N1, and an increased production of MIP-1α was observed at 72 h; combined treatment with alloferon and zanamivir resulted in suppression of both ( Figure 2B,D).

The Antiviral Effects of Combined Treatment with Alloferon and Zanamivir are Mediated by Inhibition of the p38MAPK and JNK Signaling Pathways
Activation of p38 mitogen activated protein kinase (MAPK) and Jun-amino-terminal kinase (JNK) in virus infected cells in the presence or absence of alloferon (0.5 μg/mL) and/or zanamivir (35 μg/mL) was examined in order to confirm the signaling pathways involved in the antiviral effects of combined treatment with alloferon and zanamivir. As shown in Figures 3A,B, infection of MDCK and A549 cells with H1N1 resulted in increased phosphorylation of p38 MAPK and JNK, which was suppressed by both drugs individually and in combination, although combination treatment was more effective. Regarding P38MAPK, its phosphorylated form appeared to show a decrease in the group treated only with Zanamivir, however, a decrease in the total form of p38MAPK was also observed. Therefore, no difference in the ratio was observed when phosphorylated p38MAPK was divided by total p38MAPK. An analysis of all bands was performed using Image J software in order to clarify the results, as shown in Supplementary Figure S6. Combined treatment with alloferon and zanamivir suppressed production of IL-6 and MIP-1α in H1N1-infected cells MDCK and A549 in vitro. At 48 and 72 h post-H1N1 infection, culture supernatants were collected and measurement of the levels of IL-6 and MIP-1α in the presence or absence of alloferon (0.5 µg/mL) and/or zanamivir (35 µg/mL) was performed using an enzymelinked immunosorbent assay (ELISA) as described in the Section 4. Results are representative of more than three independent experiments. Control: Virus uninfected cells (A,C) MDCK (B,D) A549 * p < 0.1, ** p < 0.01, *** p < 0.001, NS; no significant.

The Antiviral Effects of Combined Treatment with Alloferon and Zanamivir Are Mediated by Inhibition of the p38MAPK and JNK Signaling Pathways
Activation of p38 mitogen activated protein kinase (MAPK) and Jun-amino-terminal kinase (JNK) in virus infected cells in the presence or absence of alloferon (0.5 µg/mL) and/or zanamivir (35 µg/mL) was examined in order to confirm the signaling pathways involved in the antiviral effects of combined treatment with alloferon and zanamivir. As shown in Figure 3A,B, infection of MDCK and A549 cells with H1N1 resulted in increased phosphorylation of p38 MAPK and JNK, which was suppressed by both drugs individually and in combination, although combination treatment was more effective. Regarding P38MAPK, its phosphorylated form appeared to show a decrease in the group treated only with Zanamivir, however, a decrease in the total form of p38MAPK was also observed. Therefore, no difference in the ratio was observed when phosphorylated p38MAPK was divided by total p38MAPK. An analysis of all bands was performed using Image J software in order to clarify the results, as shown in Supplementary Figure S6. h. Cell lysates were prepared. and western blot analysis was performed for detection of changes in activation of p38 MAPK and JNK, as described in the Materials and Methods Section 4. Results are representative of more than three independent experiments. (C,D) Calculation of the relative expression of the phospho form/total form was performed using Image J software. * p < 0.1, ** p < 0.01, *** p < 0.001, NS; no significant.
Based on the in vitro results from MDCK and A549 cells, the antiviral effects of alloferon and zanamivir in mice infected with H1N1 were then examined using the experimental scheme shown in Figure 4A. H1N1 was administered intranasally to mice after performance of an HA assay for measurement of the effective viral titer ( Figure 4A). On Day 4 post virus infection, a decrease in weight was observed in untreated virus inoculated mice, which was effectively rescued by combined treatment with alloferon and Figure 3. The antiviral effects of combined treatment with alloferon (0.5 µg/mL) and zanamivir (35 µg/mL) are mediated by inhibition of the p38 MAPK and JNK signaling pathways. MDCK (A) and A549 (B) cells were cultured in the presence or absence of alloferon and/or zanamivir for 12 and 24 h. Cell lysates were prepared. and western blot analysis was performed for detection of changes in activation of p38 MAPK and JNK, as described in the Materials and Methods Section 4. Results are representative of more than three independent experiments. (C,D) Calculation of the relative expression of the phospho form/total form was performed using Image J software. * p < 0.1, ** p < 0.01, *** p < 0.001, NS; no significant.

Combined Treatment with Alloferon and Zanamivir Inhibits Viral Replication and Increases Survival of Mice Infected with H1N1
An experiment was performed to determine the optimal amount of alloferon and zanamivir prior to conduct of an experiment to confirm the antiviral effect. According to our previous report on the effect of alloferon [21,[23][24][25]54,55], 0.5-2 µg/mL was used for examination of the antiviral efficacy or immune enhancement function. Determination of a dose of 35 µg/mL of zanamivir was based on conversion of the amount administered for the treatment of patients with influenza virus infection into a weight of mice. As a result, alloferon, 0.5 and 1 µg/mL and zanamivir 35 µg/mL and 70 µg/mL were established for study on the combined treatment of alloferon and zanamivir, and a pilot study was conducted, as shown in Supplementary Figure S3. According to the results shown in Supplementary Figure S3, the optimal concentration for the drug combination was determined as 0.5 µg alloferon and 35 µg zanamivir. The results confirmed that there was no cytotoxicity at that concentration (Supplementary Figure S4). Regarding in vivo toxicity and safety, there was no acute toxicity in mice (up to 6000 mg/kg), rat (up to 5000 mg/kg) and dog (up to 20 mg/kg) as well as no embryo-toxic or teratogenic effect in rat (up to 15 mg/kg). In addition, no special toxicity was observed in phase 1 clinical studies in which 0.1, 1, 5, and 10 mg of alloferon were administered subcutaneous in 40 healthy people. In a phase 2 clinical study of patients with recurrent genital herpes and acute hepatitis B virus infection, it was observed that it was effective in controlling diseases caused by viruses. In our previous report [27], 25-50 µg/mL was mainly used in experiments to confirm in vivo anti-cancer efficacy, but 0.5 µg/mL of alloferon were used in this experiment. It is quietly lower concentration than the concentration in the experiment for anti-cancer experiment.
Based on the in vitro results from MDCK and A549 cells, the antiviral effects of alloferon and zanamivir in mice infected with H1N1 were then examined using the experimental scheme shown in Figure 4A. H1N1 was administered intranasally to mice after performance of an HA assay for measurement of the effective viral titer ( Figure 4A). On Day 4 post virus infection, a decrease in weight was observed in untreated virus inoculated mice, which was effectively rescued by combined treatment with alloferon and zanamivir ( Figure 4B). In addition, the survival rate of infected mice began to show a decline at three days post inoculation with virus; however, the rates showed marked improvement after combined treatment with alloferon and zanamivir ( Figure 4C). Real-time RT-PCR was also performed in order to examine replication of H1N1 in the lungs of mice treated with (or without) alloferon and/or zanamivir ( Figure 4D), as described in the Section 4. As in MDCK and A549 cells, combined treatment with alloferon and zanamivir resulted in markedly inhibited proliferation of H1N1.

Combined Treatment with Alloferon and Zanamivir Suppresses Production of IL-6 and MIP-1α Induced by H1N1 Infection In Vivo
An examination of the levels of these molecules in serum and BALF from mice infected with H1N1 was performed. As described above, we demonstrated that production of IL-6 and MIP-1α in MDCK and A549 cells was suppressed by combination of alloferon/zanamivir ( Figure 2). Similar to the in vitro results, combined treatment with alloferon and zanamivir resulted in suppression of the H1N1-mediated increase in the amounts of IL-6 and MIP-1α in Broncho alveolar lavage fluid (BALF) ( Figure 5A,C); however, no change in the levels of IL-6 and MIP-1α in serum were observed ( Figure 5B,D). cline at three days post inoculation with virus; however, the rates showed marked improvement after combined treatment with alloferon and zanamivir ( Figure 4C). Real-time RT-PCR was also performed in order to examine replication of H1N1 in the lungs of mice treated with (or without) alloferon and/or zanamivir ( Figure 4D), as described in the Materials and Methods section. As in MDCK and A549 cells, combined treatment with alloferon and zanamivir resulted in markedly inhibited proliferation of H1N1.

Combined Treatment with Alloferon and Zanamivir Suppresses Production of IL-6 and MIP-1α Induced by H1N1 Infection In Vivo
An examination of the levels of these molecules in serum and BALF from mice infected with H1N1 was performed. As described above, we demonstrated that production of IL-6 and MIP-1α in MDCK and A549 cells was suppressed by combination of alloferon/zanamivir ( Figure 2). Similar to the in vitro results, combined treatment with (B) Body weight and (C) survival were monitored for an additional eight days. (D) Total RNA was extracted from lung tissues and an analysis of viral replication was performed using real-time PCR as described in the Section 4. Results are representative of more than three independent experiments. * p < 0.1, ** p < 0.01, *** p < 0.001.

Pathological Changes in the Lungs of H1N1-Infected Mice Are Prevented by Combined Treatment with Alloferon and Zanamivir
Following the harvest of lung tissues from mice, histological examination was performed in order to evaluate both pathological changes and cytokine production, caused by infection with H1N1, and to determine whether combined treatment with alloferon and zanamivir can inhibit these pathological changes. As shown in Figure 6A, widespread inflammatory lesions were detected in the lungs of mice infected with H1N1. However, treatment with alloferon and zanamivir effectively prevented these pathological changes, particularly when used in combination ( Figure 6). Further analysis was performed using Masson's Trichrome staining in order to examine fibrotic changes in lung tissue caused by infection with H1N1. Severe fibrosis caused by H1N1 was detected in lungs, which was effectively prevented by combined treatment with alloferon and zanamivir ( Figure 6B).
The effect of combined treatment with alloferon and zanamivir on the number of immune cells, particularly neutrophils and macrophages, in the BALF of H1N1-infected mice was also examined. Combined treatment with alloferon and zanamivir resulted in a decrease of the total number of immune cells in BALF; however, the number of macrophages and neutrophils showed a particularly significant decline ( Figure 6C).  and serum (B,D) were collected, and ELISA was performed for measurement of the levels of IL-6 and MIP-1α as described in the Materials and Methods section. Reading of plates was performed using a microplate reader at 450 nm. Results are representative of more than three independent experiments. * p < 0.1, ** p < 0.01, *** p < 0.001, NS; no significant.

Pathological Changes in the Lungs of H1N1-Infected Mice are Prevented by Combined Treatment with Alloferon and Zanamivir.
Following the harvest of lung tissues from mice, histological examination was performed in order to evaluate both pathological changes and cytokine production, caused by infection with H1N1, and to determine whether combined treatment with alloferon and zanamivir can inhibit these pathological changes. As shown in Figure 6A, widespread inflammatory lesions were detected in the lungs of mice infected with H1N1. However, treatment with alloferon and zanamivir effectively prevented these pathological changes, particularly when used in combination ( Figure 6). Further analysis was performed using Masson's Trichrome staining in order to examine fibrotic changes in lung tissue caused  D) were collected, and ELISA was performed for measurement of the levels of IL-6 and MIP-1α as described in the Section 4. Reading of plates was performed using a microplate reader at 450 nm. Results are representative of more than three independent experiments. * p < 0.1, ** p < 0.01, *** p < 0.001, NS; no significant. was effectively prevented by combined treatment with alloferon and zanamivir ( Figure  6B). The effect of combined treatment with alloferon and zanamivir on the number of immune cells, particularly neutrophils and macrophages, in the BALF of H1N1-infected mice was also examined. Combined treatment with alloferon and zanamivir resulted in a decrease of the total number of immune cells in BALF; however, the number of macrophages and neutrophils showed a particularly significant decline ( Figure 6C).

Combined Treatment with Alloferon and Zanamivir Inhibits H1N1-Induced Activation of p38 MAPK and JNK Signaling Pathways in Lung Tissue.
As shown in Figure 3, combined treatment with alloferon and zanamivir inhibited H1N1-induced activation of the p38 MAPK and JNK signaling pathways in MDCK and A549 cells. Therefore, we attempted to determine whether the same result would be obtained in vivo. The results showed that combined treatment with alloferon and zanamivir resulted in significant suppression of H1N1-induced activation of p38 MAPK and JNK in

Combined Treatment with Alloferon and Zanamivir Inhibits H1N1-Induced Activation of p38 MAPK and JNK Signaling Pathways in Lung Tissue
As shown in Figure 3, combined treatment with alloferon and zanamivir inhibited H1N1-induced activation of the p38 MAPK and JNK signaling pathways in MDCK and A549 cells. Therefore, we attempted to determine whether the same result would be obtained in vivo. The results showed that combined treatment with alloferon and zanamivir resulted in significant suppression of H1N1-induced activation of p38 MAPK and JNK in lung tissue (Figure 7). Analysis of WB images was also performed using Image J software in order to clarify the results, as shown in Supplementary Figure S6. lung tissue (Figure 7). Analysis of WB images was also performed using Image J software in order to clarify the results, as shown in Supplementary Figure S6.

Discussion
Zanamivir is known to bind to the active site of the neuraminidase of the influenza virus to prevent the spread of the virus and to inhibit the replication of the virus by itself [20,56]. According to a paper published in PNAS by Sergey et al. in 2002, it was reported that the production of IFN was increased in both subcutaneous injection and intranasal administration, and that intranasal administration was more effective than subcutaneous injection [28]. In addition, we have already found that IFN production was increased by alloferon in our previous study [27]. Therefore, it is thought that it will be more effective if alloferon and interferon are used together, since alloferon indirectly shows it anti-viral effect through the increase of IFN production.
The study of infectious diseases and their causes has long been an important area of research in the medical field. The introduction and adoption of a variety of vaccines in different countries around the world has enabled control of viral infection. Two classes of antiviral drugs are currently approved for treatment of influenza in humans; adamantanes (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, zanamivir, and peramivir) [57][58][59]. Many studies have reported on resistance to these drugs [60][61][62], and development of chemical conjugates as treatments has been reported [63,64]. Conduct of research on new approaches such as this one is underway, however, no studies on the synergistic effects have been reported. Therefore, our study examined the effect of combination of zanamivir, a drug approved for treatment of influenza viruses, Figure 7. Combined treatment with alloferon (0.5 µg/mL) and zanamivir (35 µg/mL) inhibited H1N1-induced activation of the p38 MAPK and JNK signaling pathways in mouse lung tissue. (A) Lung tissues were collected after sacrifice, lysates were prepared in RIPA buffer, and western blot analysis was then performed for detection of p38 MAPK and JNK as described in the Section 4. Results are representative of more than three independent experiments. (B,C) Calculation of the relative intensity of the phospho form/total form was performed using Image J software. * p < 0.1 (The p < 0.1 statistical implications are results for determining tendency only), ** p < 0.01, *** p < 0.001, NS; no significant.

Discussion
Zanamivir is known to bind to the active site of the neuraminidase of the influenza virus to prevent the spread of the virus and to inhibit the replication of the virus by itself [20,56]. According to a paper published in PNAS by Sergey et al. in 2002, it was reported that the production of IFN was increased in both subcutaneous injection and intranasal administration, and that intranasal administration was more effective than subcutaneous injection [28]. In addition, we have already found that IFN production was increased by alloferon in our previous study [27]. Therefore, it is thought that it will be more effective if alloferon and interferon are used together, since alloferon indirectly shows it anti-viral effect through the increase of IFN production.
The study of infectious diseases and their causes has long been an important area of research in the medical field. The introduction and adoption of a variety of vaccines in different countries around the world has enabled control of viral infection. Two classes of antiviral drugs are currently approved for treatment of influenza in humans; adamantanes (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, zanamivir, and peramivir) [57][58][59]. Many studies have reported on resistance to these drugs [60][61][62], and development of chemical conjugates as treatments has been reported [63,64]. Conduct of research on new approaches such as this one is underway, however, no studies on the synergistic effects have been reported. Therefore, our study examined the effect of combination of zanamivir, a drug approved for treatment of influenza viruses, and alloferon, which has several immune effects and is much more effective than a single agent against influenza viruses. Alloferon was developed as an immunomodulatory agent; however, it also has anti-tumor [54], anti-inflammatory [23],and anti-viral [55] efficacy, particularly against influenza A virus [24]. Zanamivir was developed for the treatment of infection with influenza A virus. Compared with other antiviral agents, it has a higher safety profile, but less antiviral efficacy when used for treatment of infection with influenza A virus [65,66].
MDCK, A549, and Calu-3 cells were used in this study to assess the effects of combined treatment with alloferon and zanamivir against infection with influenza A virus. The results obtained from experiments using MDCK and A549 cells suggest that the extent of effective suppression of viral replication with combined treatment is significantly greater than that obtained with use of either alloferon or zanamivir alone. Although A549 and Calu-3 are known lung adenocarcinoma cell lines, we focused more attention on A549 because it is derived from type II alveolar epithelial cells, while Calu-3 is derived from the submucosal layers of bronchial airways. This is because in the case of animal experiments, the focus was on identifying changes in the lungs, rather than changes in the bronchial tubes. Therefore, data acquired from A549 cells was compared with data from MDCK cells. In addition to the in vitro studies using MDCK and A549 cells, our findings confirmed that combined treatment with alloferon and zanamivir (1) prevented weight loss in mice infected with H1N1; (2) increased survival rates post-infection; (3) inhibited viral replication in lung tissue; and (4) inhibited inflammatory responses and pulmonary fibrosis. Considering these findings, the data suggested the usefulness of alloferon as an adjuvant capable of enhancing the antiviral efficacy of zanamivir. In our experiments, alloferon along with zanamivir was administered directly into the nasal cavity of mice infected with H1N1. As a result, alloferon effectively induced an increase of the efficacy of zanamivir, which is safe, but less effective than other drugs due to its local action on respiratory tissues and the fact that it does not spread systemically. In general, a nasal spray is used for delivery of drugs administered into the nasal cavity, so that a smaller amount of drug is delivered to the target lesion compared with oral or intravenous administration. In an effort to compensate for this, we are also conducting research on formulations that involve combination of alloferon and zanamivir with drug carriers capable of more effective transfer of drugs to target tissues. These studies are expected to provide evidence that the combination of alloferon and other drugs is an effective option for treatment of respiratory diseases.
The Th1 immune response is suppressed and the Th2 immune response is boosted in humans and mice infected with influenza A virus [67]. This explains the symptoms that are observed with proliferation of the influenza A virus. Among the Th2 cytokines, IL-6 can trigger a "cytokine storm" (known as cytokine release syndrome), as well as macrophage activation syndrome [68,69]. As shown in Figure 2, increased production of IL-6 in MDCK and A549 cells was observed after infection with H1N1; however, production of IL-6 showed a marked decreased after treatment with combination alloferon/zanamivir that was significantly greater than that after treatment with alloferon or zanamivir alone. In addition, the amount of IL-6 in the BALF of virus-infected mice showed a significant decrease after treatment with combination alloferon/zanamivir ( Figure 5). This finding indicates that combination alloferon/zanamivir might be an effective treatment for H1N1-induced inflammation of lungs. Like IL-6, increased production of MIP-1α was observed in MDCK and A549 cells, as in H1N1-infected mice; however, levels of H1N1 infection showed a significant decrease after treatment with combination alloferon/zanamivir (Figures 2 and 5).
Increased levels of MIP-1α (also known as CCL3), a chemokine in inflammatory diseases such as rheumatoid arthritis, and in tumors such as multiple myeloma, have been reported [70,71]. It is produced primarily by macrophages, but also by other types of lymphocytes including T cells, B cells, and NK cells [72,73]. Synthesis of MIP-1α by nonhematopoietic cells such as osteoblasts and inflamed epithelial cells occurs upon stimulation with viruses [38,73]. During the inflammatory process, macrophages are recruited to sites where tissue damage has occurred, where they initiate the process of regeneration and repair [74,75]. An increase in the levels of MIP-1α occurs upon viral infection, particularly after infection with respiratory viruses such as Respiratory Syncytial Virus (RSV) and influenza virus. For example, Respiratory Syncytial Virus (RSV) causes an increase in the activity of neutrophils in lung tissue, leading to severe damage [76,77]. In addition, alleviation of pneumonia in MIP-1α-deficient mice infected with the influenza virus has been reported [78]. Increased levels of MIP-1α have also been reported in patients with asthma and pulmonary fibrosis, and in the alveolar fluid of patients with acute respiratory distress syndrome (ARDS) [78,79]. The results of this study showed that the production of IL-6 and MIP-1a in the BALF of mice infected with H1N1 was inhibited by combination alloferon/zanamivir ( Figure 5). Thus, combination alloferon/zanamivir might also be an effective treatment for inflammatory responses caused by infection with respiratory viruses, as well as for regulation of inflammatory responses in the lung mediated by IL-6 and MIP-1a.
Activation of p38 MAPK has a close association with replication of influenza viruses; suppression of the activity of p38 MAPK results in effective inhibition of replication of influenza virus [80][81][82]. JNK, another representative signaling molecule, promotes replication of viruses within host cells infected with influenza virus [83][84][85]. Many studies on attempts to treat influenza virus infection using inhibitors of p38 MAPK and JNK have been reported thus far [82]. However, effective suppression of activation of p38 MAPK and JNK by combination zanamivir/alloferon was confirmed in this study (Figures 3 and 7). Therefore, the replication of influenza A virus is controlled by combination alloferon/zanamivir through inhibition of the activity of p38 MAPK and JNK. This could be the basis for the development of effective therapeutic agents through combinations of other drugs with alloferon or zanamivir for treatment of infection with influenza A virus.
Considering the findings of this study, the data presented herein demonstrate that combination alloferon/zanamivir can (1) inhibit virus-mediated activation of p38 MAPK and JNK; (2) inhibit production of inflammatory cytokines; and (3) inhibit infiltration of inflammatory cells into lung tissue; therefore, it can be regarded as an effective treatment for influenza A virus infection. In addition, our data provide a rationale for the development of therapeutic agents based on the combination of alloferon and zanamivir to control of the development and progression of chronic inflammatory lung diseases, as well as infection with influenza A virus.

Cells and Preparation of Virus Stocks
The human lung epithelial cell line A549 was maintained in RPMI 1640 (Welgene, Daegu, Korea), and MDCK and Calu-3 were maintained in Dulbecco's modified Eagle's medium (DMEM) (Welgene). Both media contained 10% heat-inactivated fetal bovine serum (Gibco, Queensland, Australia) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; Welgene). Cells were cultured at 37 • C in a humidified atmosphere containing 5% CO 2 . Isolation and passage of influenza A viruses was performed using MDCK cells. The influenza A/Puerto Rico/8/1934 was generously provided by Nam Hyuk Cho (Department of Microbiology, Seoul National University College of Medicine, Seoul, Korea). All animal experiments using influenza A virus were performed in an Animal Biosafety Level 2 facility at Seoul National University.

Plaque Forming Unit Assay
MDCK (5.0 × 10 5 /well) and A549 (4.0 × 10 5 /well) cells were seeded onto six well plates at 37 • C in a 5% CO 2 atmosphere. After overnight culture, washing of the confluent monolayer of MDCK cells was performed twice using 1× PBS buffer, followed by treatment with a 10-fold serial dilution of H1N1 influenza virus in serum-free medium containing only 1% penicillin streptomycin (Welgene). The plates were incubated for 1 h at 37 • C with constant agitation to induce viral infection. Washing of the monolayers with 1 × PBS buffer was performed for removal of virus, followed by application of 2 mL of agar overlay medium composed of 2× DMEM (Welgene), 2 µg/mL TPCK-treated trypsin, and 1.6% agar (Lonza Bioscience, Morrisville, NC, USA). The incubation of the plates was then performed for three days at 37 • C under 5% CO 2 and 95% relative humidity. The agar-covered monolayers were fixed with 4% para-formalin (Daejung, Siheung-si, Korea). Following removal of agar, staining of the fixed cells was performed using 0.5% crystal violet solution (Sigma-Aldrich, St. Loise, MO, USA) and plaques were counted.

Titration in a HA (Hemagglutination) Assay
Measurement of virus propagation was performed using an HA assay in viruscontaining culture medium. Serial dilution of virus supernatants in saline in 96-well U-bottom plates was performed. Each well was then filled with 50 µL of a 0.5% suspension of human red blood cells (RBC). The plates were then allowed to sit at room temperature for 30 min. Following completion of the incubation period, an analysis of the assay was performed to determine RBC agglutination in each well. Across a row, agglutinated wells containing high concentrations of virus and exhibiting a diffuse reddish appearance progressed to wells with low concentrations of virus, which contained a dark red pellet. The results from the low concentration wells were nearly identical to those observed in the no-virus negative control wells. RBCs settled to the lowest point of the U-bottom well, resulting in a button-like appearance. Therefore, based on the experiments above, we measured the effective viral titer of HA (1:32 diluted in saline) before intranasally infecting mice (Supplementary Figure S5).

Animal Experiments
C57BL/6 wild-type mice were housed under pathogen-free conditions at the Seoul National University College of Medicine. The experiments were conducted using eightweek-old males. All experiments using animals were reviewed and approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC: SNU-200319-2). In this experiment, total five group, 1. Control (No infection) group, 2. H1N1 infection group, 3. H1N1 + Alloferon 0.5 µg/mL group, 4. H1N1 + Zanamivir 35 µg/mL, 5. H1N1 + Alloferon 0.5 µg/mL + Zanamivir 35 µg/mL group, (n = 30/group) for in vivo experiments. Therefore, results represent more than three independent experiments. Intranasal administration of influenza A/H1N1 virus in mice was performed under isoflurane anesthesia. Drugs of alloferon (0.5 µg/mL) and zanamivir (35 µg/mL) were administered daily following intranasal inoculation of H1N1. Monitoring of symptoms and mortality was performed daily for eight days. Over the course of eight days, measurements of body weight, clinical signs: 1. Changes in activity/mobility (Decreased mobility may be localized to area of pain) 2. Behavior changes during handling (placid) 3. Separation from group (Mice are social and normally rest in close proximity to one another)], and survival were performed daily. Mice with weight loss exceeding 30%, or mice that were clearly terminal were euthanized in accordance with guidelines established by the animal ethics committee.

Serum and BALF Collection
Blood samples were collected from the retro-orbital plexus of each animal using heparinized capillaries and immediately centrifuged at 3000 rpm for 10 min at 4 • C to obtain serum. For BALF samples, PBS containing 1% bovine serum albumin and 0.1% NaN 3 (blocking solution) was used to lavage the trachea and lungs. After centrifuging the lavage fluid for 10 min at 300× g, the supernatant was stored at -80 • C [87]. The levels of TNF-α, IL-6, IL-1β, IFN-λ, MIP-1α, and IL-10 in serum and BALF were measured using ELISA kits (BioLegend, San Diego, CA, USA).

Western Blot Analysis
Western blotting was performed in order to evaluate the expression of proteins involved in signaling pathways. Protein lysate was obtained from lung tissue of H1N1 infected mice and MDCK and A549 cells treated with alloferon and zanamivir for 12 and 24 h after infection with H1N1 using lysis buffer containing 50 mM Tris-HCL (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktails (Sigma-Aldrich). The BCA assay was performed for quantification of the protein concentration by BCA kit (Sigma-Aldrich). Then, measurement of the relative absorbance at 562 nm was performed using a SpectraMax iD3 microplate reader (Molecular Devices, San Jose, CA, USA). The proteins (20 µg/sample) were resolved in a 10% polyacrylamide-SDS gel at 100 V for 2 h, followed by transfer to nitrocellulose membranes. Blocking was performed for 1 h at room temperature with 5% nonfat milk in PBS. Incubation of the blocked membrane was performed at 4 • C overnight with antibodies specific for p-p38 (1:1000; Cell Signaling; Boston, MA, USA), p-c-Jun (1:1000; Cell Signaling), p38 (1:1000; Cell Signaling), c-Jun (1:1000; Cell Signaling) or anti-β-actin Ab (1:5000; Santa Cruz Biotechnology; Santa Cruz, CA, USA). The membrane was washed three times (5 min each) with 0.1% PBST, the membrane was followed by incubation for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (1:5000; Cell Signaling Technology) for detection of p-p38, p-c-Jun, p38, and c-Jun, or with HRP-conjugated anti-mouse IgG antibody (1:10,000; Cell Signaling) for detection of β-actin. An electrochemical luminescence detection system (Thermo Scientific, Wilmington, DE, USA) was used for the visualization of the immunoreactive proteins after washing three times (5 min each). Analysis of the density of the bands was performed using Image J software (NIH), and the phospho form was normalized to the total form.

Quantification of Virus Replication by Real-Time RT-PCR Analysis
Extraction of total RNA from A549 and MDCK cells at 12 and 24 h post-infection and lung specimens of H1N1-infected mice was performed using Trizol Reagent (Invitrogen, Grand Island, NY, USA). Reverse transcription of RNA samples into cDNAs was performed using oligo (dT) primers and AMV reverse transcriptase (iNtRON; Daejeon, Korea). Briefly, 1 µg of total RNA was combined with 5 mM MgCl 2 , 1 mM of each dNTP, 1 µL of RT buffer, 1 U/mL RNasin, 15 U/mL reverse transcriptase, and DEPC treated water up to a total volume of 20 µL. The samples were incubated for 10 min at room temperature, followed by incubation for 1 h at 42 • C, 5 min at 72 • C, followed by another 5 min on ice. Following addition of 180 µL of DEPC treated water, samples were stored at −70 • C. Each reaction was run in a quadrant for each sample, and an analysis of the results was performed using the Rotor-Gene SYBR Green PCR kit (Qiagen, Hilden, Germany) and Rotor-Gene Q 2plex-real-time RT PCR instrument (Qiagen). The reaction mixture (20 µL) consisted of 2 µL of a cDNA template and 1.5 µL each of primers. Amplification was performed using SYBR Green master mix (Qiagen). Each cycle of PCR was monitored by measuring the fluorescence produced by binding of SYBR Green dye to dsDNA. The following primers were used: H1; Forward 5 -AGCAAAAGCAGGGGAAAATAAAA-3 ; Reverse 5 -CACGAGGACTTCTTTCCCTTTATCAT-3 ; and GAPDH; Forward 5 -GGTGGTCCAGGG-TTTCTTA-3 ; Reverse 5 -GTTGTCTCCTGCGACTTCA-3 . Calculation of differences in the amount of H1N1 was performed using the using the ∆∆CT method [88].

Histopathology
Lung tissues were harvested and fixed overnight in 4% Paraformaldehyde (PFA) at 4 • C. Fixed tissues were embedded in paraffin prior to cutting of sections with 4 µm thickness. Following deparaffinization and hydration, the sections were stained with hematoxylin and eosin, followed by viewing under a light microscope. Masson's Trichrome staining was performed using a VitroView TM Masson's Trichrome Stain Kit (VitroView TM , Hanam, Korea). Briefly, Weigert's iron hematoxylin, Biebrich Scarlet-Acid Fuchsin, and Aniline Blue Stain solutions were applied to deparaffinized sections pretreated with Bouin's fluid at 56 • C for 1 h. Finally, removal of non specific staining was performed using 1% acetic acid solution. The nuclei were then stained black, the cytoplasm and muscle tissue were stained red, and collagen was stained blue. Alveoli in the lungs did not show an even distribution. Although scanning of whole-mount lung sections was not performed for measurement of collagen in this study, Trichrome staining of lung tissue enabled an estimation of differences in the amount of collagen. All analyses were performed using Celleste (Thermo Scientific).

Statistical Analysis
Data are presented as the mean ± SD. The comparison of data between groups was performed using unpaired t-tests. Statistical analysis was performed using GraphPad Software Prism version 6.01 (GraphPad Software, La Jolla, CA, USA).

Conclusions
The combination of alloferon and zanamivir can be regarded as an effective treatment for influenza A virus infection by (1) inhibiting the activity of p38 MAPK and JNK; (2) inhibiting production of inflammatory cytokine in the lungs; and (3) inhibiting the infiltration of inflammatory cells into lung tissue. In addition, there is the potential for the use of alloferon/zanamivir in the development of other therapeutic agents capable of regulating the progression of chronic inflammatory lung diseases and influenza A/H1N1 infection.

Conflicts of Interest:
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.