A Herbal Mixture Formula of OCD20015-V009 Prophylactic Administration to Enhance Interferon-Mediated Antiviral Activity Against Influenza A Virus

OCD20015-V009 is an herbal mix of water-extracted Ginseng Radix, Poria (Hoelen), Rehmanniae Radix, Adenophorae Radix, Platycodi Radix, Crataegii Fructus, and Astragali Radix. In this study, its in vitro and in vivo antiviral activity and mechanisms against the influenza A virus were evaluated using a GFP-tagged influenza A virus (A/PR/8/34-GFP) to infect murine macrophages. We found that OCD20015-V009 pre-treatment substantially reduced A/PR/8/34-GFP replication. Also, OCD20015-V009 pre-treatment increased the phosphorylation of type-I IFN-related proteins TBK-1 and STAT1 and the secretion of pro-inflammatory cytokines TNF-α and IL-6 by murine macrophages. Moreover, OCD20015-V009 prophylactic administration increased IFN-stimulated genes-related 15, 20, and 56 and IFN-β mRNA in vitro. Thus, OCD20015-V009 likely modulates murine innate immune response via macrophages. This finding is potentially useful for developing prophylactics or therapeutics against the influenza A virus. Furthermore, pre-treatment with OCD20015-V009 decreased the mortality of the mice exposed to A/PR/8/34-GFP by 20% compared to that in the untreated animals. Thus, OCD20015-V009 stimulates the antiviral response in murine macrophages and mice to viral infections. Additionally, we identified chlorogenic acid and ginsenoside Rd as the antiviral components in OCD20015-V009. Further investigations are needed to elucidate the protective effects of active components of OCD20015-V009 against influenza A viruses.


INTRODUCTION
The genome of the influenza A virus (IAV) contains eight segments of negative-sense single-stranded RNA and remains a major threat to public health. IAV infection leads to enormous morbidity and economic loss (Lam et al., 2013); each year, seasonal influenza virus (IV) infects 5-15% of the global human population causing approximately 300,000-500,000 deaths (Clayville, 2011;Kim et al., 2020). Several epidemics and pandemics have occurred over the past century due to antigenic drift or shift in the IAV (Yin et al., 2018;Degoot et al., 2019). Antigenic shifts are substantial in the influenza A virus caused by genetic re-assortment, resulting in novel hemagglutinin (HA) and/or novel HA and neuraminidase (NA) from Avian Influenza into currently circulating human influenza viruses that infect humans (Yin et al., 2018;Degoot et al., 2019). However, predicting the next antigen shift or the resultant outbreak is challenging (Yin et al., 2018;Degoot et al., 2019). Moreover, influenza vaccines have become ineffective (Berlanda Scorza et al., 2016). Thus, antiviral agents are crucial in disease control (Berlanda Scorza et al., 2016;Pardi and Weissman, 2020). Anti-influenza medications such as matrix protein 2 (M2) ion channel blockers (Takeda et al., 2002) and neuraminidase (NA) inhibitors (Alymova et al., 2005) have been approved globally, while an RNA polymerase inhibitor (Hayden and Shindo, 2019), an antivirus medication, has been approved regionally (Zhang et al., 2019). Additionally, NA inhibitors such as oseltamivir and zanamivir are frequently administered. However, due to the emergence of resistant influenza strains, M2 ion channel blockers, such as amantadine and rimantadine, are rarely used. Furthermore, the resistance of influenza strains to NA inhibitors, such as oseltamivir and zanamivir, has increased (Hussain et al., 2017).
Conversely, the host initiates the innate immune system, the first line of defense against most pathogens, including the influenza virus, via the production of antiviral cytokines (Chen et al., 2018). For instance, activated interferon (IFN) is a critical component of innate immunity against the influenza virus (Chen et al., 2018;Wu and Metcalf, 2020). Type I IFN plays a crucial role in the innate immune response against many viruses and is also a component of the adaptive immune response to viral and nonviral pathogens (Chen et al., 2018;Wu and Metcalf, 2020). However, overproduction of IFNs and proinflammatory factors may cause a cytokine storm that aggravates a disease by disrupting the immune suppression of viral infections and causing tissue damage. Thus, IFN and proinflammatory cytokines, the first line of defense against the influenza virus, act as a double-edged sword (Gu et al., 2019).
After the influenza virus infects the lungs, type I IFNs are rapidly expressed in numerous myeloid and parenchymal cells (Jewell et al., 2007;Kumagai et al., 2007). Type I IFNs' proinflammatory activity allows for immunological modulation of this antiviral cytokine family (Kopitar-Jerala, 2017). Cytokine secretion from macrophages is tightly regulated at the transcriptional level. Post-transcriptional modulation of IFNs and proinflammatory cytokines also occurs (Kopitar-Jerala, 2017). The innate immune response is involved in the various inflammatory processes and is particularly vital for viral infections, which affect the cellular, tissue, and overall physiological functions (Teijaro, 2016;Kopitar-Jerala, 2017). Therefore, rapid IFN production is required during viral infection to inhibit virus spread in the host cells (Teijaro, 2016;Kopitar-Jerala, 2017).
Traditional herbal medicines or natural products such as Clove (Syzygium aromaticum (L.) Merr. & L.M. Perry) and Opuntia ficus-indica (L.) strengthen the antiviral properties against influenza or SARS-CoV-2 (Vicidomini et al., 2021a;Vicidomini et al., 2021b). Particularly, IFN-β and proinflammatory cytokines are essential in the defense against the IAV (Koerner et al., 2007). The activity of IFN-β and proinflammatory cytokines can be enhanced with traditional herbal medicines or herbal products that strengthen the host's antiviral defense response to influenza (Talactac et al., 2015;Mousa, 2017;Trinh et al., 2020). Therefore, researchers are examining herbal medications or natural compounds with immunomodulatory properties for influenza virus infection treatment (Choi et al., 2016;Choi et al., 2017a;Choi et al., 2017b). In this study, we investigated whether OCD20015-V009-induced signaling triggers antiviral mediators, such as type I interferons, proinflammatory cytokines, and interferonstimulatory genes responsible for the antiviral state in murine macrophage cells. Here, we investigated whether OCD20015-V009, a herbal complex containing the water extract of Panax ginseng C.

Cell Lines and Virus
Accoding to a prior publication, the replication and viral titer of the two strains were determined .

Cell Viability Assay
Cell viability was determined using the MTT assay. The RAW 264.7 and MDCK cells were seeded into 24-well plates at 1 × 10 5 cells/well, and OCD20015-V009 was added to the wells at a concentration of 0-400 μg/ml. MTT solutions were added to each well after 24 h, and the cells were incubated for another 30 min (D'Alessandro et al., 2019;Mosmann, 1983). Subsequently, 1 ml DMSO was added before measuring the absorbance at 540 nm using an Epoch Microplate Reader (BioTek, United States). The data were represented by the mean ± SEM of four independent experiments.

Cytokine Determination
For ELISA, RAW 264.7 cells were seeded and incubated for 18 h. The cells were treated with 20 ng/ml LPS or OCD20015-V009 at 50 or 100 μg/ml for 6 or 24 h. The levels of the inflammatory cytokine TNF-α and IL-6 in the culture medium were measured using the ELISA antibody set purchased from eBioscience (#88-7324-77 and #88-7064-77).

Total RNA Extraction and qRT-PCR
Total RNA extraction and cDNA synthesis were conducted using the Easy-BLUE ™ RNA extraction kits (iNtRON Biotech) and AccuPower ® CycleScript RT PreMix (Bioneer), respectively. A total of 1 μg RNA was reverse-transcribed into cDNA, and qPCR oligonucleotide primers for macrophage cell cDNA are indicated in Table 1 qPCR reactions were performed in triplicate 20 μL reactions with 1 μL of 0.3 μM of the forward and reverse primer each, 10 μL of the AccuPower ® 2× Greenstar qPCR master mix, 5 μL of template DNA, and 3 μL of RNase-free water. The PCR cycle was as follows: 95°C for 10 min, 40 cycles of 95°C for 20 s, 60°C (ISG15), 53°C (ISG20), 60°C (IFN-β), 56°C (ISG56), or 60°C (TNF-α) for 40 s, and at each experiment end, a melting curve analysis was conducted to confirm that a single product per primer pair was amplified. Amplification and analysis were performed using the QuantStudio 6 Flex Real-time PCR System (Thermo Fisher), and each sample was compared using the relative CT method. Fold changes in gene expression were determined relative to the blank control after normalization to GAPDH expression using the 2−ΔΔCt method.

Viral Replication Inhibition Assay
A viral replication inhibition assay was performed using the A/PR/8/34 and A/PR/8/34-GFP viruses . We tested the antiviral effect of OCD20015-V009 on viruses Immunofluorescence Staining RAW 264.7 cells seeded onto cover slides at 1 × 10 5 cells/ml were cultured at 37°C with 5% CO 2 for 24 h. The cells were then pretreated with OCD20015-V009 or IFN-β and incubated at 37°C with 5% CO 2 for 18 h before infection with A/PuertoRico/8/34 at the MOI of 10 for 2 h. After viral infection, the virus and medium were removed, and the cells were rinsed with phosphate-buffered saline (PBS) thrice. Next, a complete medium was added, and the cells were incubated at 37°C with 5% CO 2 . After 24 h, the cells were rinsed with cold PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.1% Triton-X100 in PBS for 15 min. After blocking, the cells were incubated with a rabbit polyclonal antibody against M2 (1:250 in 3% BSA; GeneTex, United States) at 4°C overnight, rinsed with cold PBS thrice, and incubated with an Alexa Fluor 568 goat anti-rabbit IgG antibody (1:500 in 3% BSA; Thermo Fisher) for 1 h. The nuclei were visualized by staining with DAPI (0.5 μg/ml; Thermo Fisher) for 10 min. Then, the images were captured using a fluorescence microscope (Nikon).

Western Blot
The RAW 264.7 cells seeded in 6-well plates at 1 × 10 6 cells/well were incubated with OCD20015-V009 and LPS at 37°C with 5% CO 2 . Afterward, the cells were harvested and lysed in RIPA buffer (Millipore, United States) containing protease and phosphatase inhibitors. The total protein content in the samples was normalized using Bradford's reagents. The proteins were separated using SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). After blocking with 3% BSA, the blots were incubated with primary anti-STAT1, anti-TBK1, anti-phospho-STAT1, anti-phospho-TBK1, antiβ-actin, M1, NA, NP, and PA antibodies (1:1,000 dilution) at 4°C overnight. After the blots were washed in TBS-T thrice, they were incubated with an HRP-conjugated secondary antibody.
The proteins were quantified using a ChemiDoc ™ Touch Imaging System (Bio-Rad), and the relative intensities of protein bands were measured using ImageJ.

Animal Studies
This study was conducted following the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Center (LAC) of Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF). The animal studies were approved by the IACUC of the LAC of DGMIF under approval number DGMIF-18071602-00. Five-week-old female BALB/c mice (Orient Bio Inc., Seongnam, South Korea) were acclimated for at least 1 week under standard housing conditions at the LAC of DGMIF and provided with a standard rodent chow diet and water ad libitum. For the oral inoculation of OCD20015-V009 and the IAV challenge, the mice were separated into four experimental groups of ten mice in each group and administered with control, PBS, 100 or 300 mg/kg OCD20015-V009 with IAV, respectively. The group of mice without virus infection was used as a negative control. The mice in each experimental group were orally administered PBS, 100 and 300 mg/kg OCD20015-V009 at a total volume of 200 µL once daily for 7 days before infection, respectively (Choi et al., 2017a). The mice were infected intranasally with 20 µL of A/PR/8/34 in PBS at the 50% mouse lethal dose (LD50).
Survival was monitored for 10 days post-infection (dpi) at fixed time points. At 7 dpi, three mice from each group were randomly selected and sacrificed to measure lung histopathology. The lung tissue samples were immediately fixed in paraffinembedded neutral buffer containing 10% formalin and sliced to 4-6-µm sections using a microtome. The sections were mounted on a slide, stained with eosin, and examined under an optical microscope (Choi et al., 2017a). The remaining mice were used to measure survival at 10 dpi.

Statistical Analysis
The data were expressed as mean ± SEM. The significance of the differences in the mean values between the treatment and control groups was determined using one-way ANOVA. Additionally, Tukey's post-hoc test was utilized for multi-group comparisons.
Analyses were performed using GraphPad PRISM ® Version 5.02 (GraphPad, United States). A p-value less than 0.05 denotes statistical significance.

OCD20015-V009 Inhibited IAV Infection in RAW 264.7 Cells
The antiviral activity of OCD20015-V009 was examined by detecting GFP levels in RAW 264.7 cells after suppressed A/PR/ 8/34-GFP replication. The untreated cells had high GFP levels upon infection by A/PR/8/34-GFP. Conversely, the GFP level of RAW 264.7 cells pre-treated with OCD20015-V009 was considerably lower ( Figure 1B). The replication of A/PR/8/34-GFP in RAW 264.7 cells was significantly decreased by 81.5 and 91.1% with OCD20015-V009 pre-treatment at 50 and 100 μg/ml, respectively, compared to the vehicle (the virus treatment) group ( Figure 1C). Furthermore, we observed that dose-dependent OCD20015-V009 decreased plaque formation in MDCK cells ( Figure 1D).

Immunofluorescence (IF) and
Western blots were performed to validate the production of IAV proteins. Cells pre-treated with OCD20015-V009 produced significantly less M2 ( Figure 1E). Additionally, the production of HA, PA, and NP was significantly inhibited in RAW 264.7 cells pre-treated with 100 μg/ml OCD20015-V009 before infection with A/PR/8/34(H1N1)-GFP ( Figure 1F).
These data imply that OCD20015-V009 pre-treatment significantly inhibits IAV infection and viral protein production in RAW 264.7 cells. Thus, OCD20015-V009 pretreatment likely reduces influenza H1N1 viral protein production and inhibits infection.

OCD20015-V009 Inhibited IAV Infection in vivo
We investigated the protective effects of OCD20015-V009 on IAV infection in BALB/c mice. The mice treated once daily with 100 or 300 mg/kg OCD20015-V009 maintained a relatively stable body weight with no significant clinical symptoms in this study (data not shown). All untreated A/PR/8/34-infected mice were dead within 7 dpi (Figure 2). Contrarily, the mortality of the mice pre-treated with OCD20015-V009 after A/PR/8/34 infection was reduced (Figure 2A). The lungs from the mice were sampled at 7 dpi for hematoxylin and eosin staining to investigate  histopathological changes caused by viral infection. Lung inflammation or pathological changes were not found in the normal control group ( Figure 2B). However, bronchial epithelial cells were necrotized with thickened alveolar walls in the mice in the vehicle group; in addition, severe pulmonary congestion and lesions were observed. Also, the alveolar space was occupied with moderate inflammatory infiltrates of neutrophils, macrophages, and lymphocytes. However, lung samples from the mice pretreated with 300 mg/kg OCD20015-V009 revealed pulmonary congestion and lesion alleviation, indicating lower lung inflammation compared to untreated mice ( Figure 2B).
Effects of OCD20015-V009 on Pro-Inflammatory Cytokine Production and Type-I IFN Signaling Pathway Activation in RAW 264.7 Murine Macrophages Pro-inflammatory cytokines and type-I IFN are significant in inducing immunoregulatory activities and antiviral responses.
Herbal medicine's immunomodulatory effect for treating IAV infection has been extensively studied (Choi et al., 2017a). Additionally, innate immune responses through the production of pro-inflammatory cytokines and type-I IFN may be responsible for OCD20015-V009s antiviral action. Using ELISA, we evaluated the effect of OCD20015-V009 on TNF-α and IL-6 the secretion.
Additionally, we studied TBK1 and STAT1 phosphorylation in RAW 264.7 cells pre-treated with OCD20015-V009 using Western blots to determine the OCD20015-V009 effect on the activation of type-I IFN signaling molecules. The results show that OCD20015-V009 treatment upregulates STAT1 and TBK1 phosphorylation, and they are key molecules in the type-I IFN signaling pathway ( Figures 3E,F).
We further analyzed the interaction between OCD20015-V009 and IFN-stimulated genes in RAW 264.7 cells. The expression of the IFN-stimulated gene (ISG)-15, ISG-20, and ISG-56, and TNF-α and IFN-β genes time-dependently increased in the OCD20015-V009-treated RAW 264.7 cells compared with that in the untreated cells ( Figures 4A-E). Additionally, the upregulation of the TNF-α, IFN-β, and ISG was notable. The observed pattern was similar to that of LPS-treated positive control ( Figure 4); the transcription of the ISG-15, ISG-20, and ISG-56 genes increased by 132.9 ± 5.2, 114.2 ± 6.7, and 98.1 ± 7.5-fold, respectively, in the cells pre-treated with 100 μg/ ml OCD20015-V009 for 6 h ( Figures 4A-E). Overall, the results indicate that OCD20015-V009 can induce an antiviral state by modulating the IFN signaling pathway and ISG expression in macrophages, thus inhibiting viral infection.

Viral Replication Inhibitory Effect of the Components Identified in OCD20015-V009
Next, we investigated whether the eight major compounds in OCD20015-V009, chlorogenic acid, ginsenoside Rg1, calycosin, ginsenoside Rb1, ginsenoside Rd, astragaloside II, astragaloside I, and polygalacin D, inhibited H1NA influenza virus replication in RAW 264.7 cells by suppressing the production of the viral The GFP levels and reduction in viral replication in RAW 264.7 cells pre-treated with the eight major components of OCD20015-V009 24 h after viral infection were assessed using flow cytometry. The bar graphs are plotted with the data from three experiments. ***p < 0.001 compared with the control, # p < 0.05 and ### p < 0.001 compared with the vehicle (IAV-infection). (D) The level of the IAV proteins in RAW 264.7 cells as assayed by Western blots using antibodies against various IAV proteins. RAW 264.7 cells were pre-treated with 10 and 20 μM chlorogenic acid or ginsenoside Rd, 1000 U/mL recombinant mouse interferon (IFN)-β, or the medium only (negative control) after viral adsorption. The levels of IAV proteins PA, PB1, PB2, and NA in the cell lysates were analyzed with Western blots; the level of tubulin was used as the internal control. Western blotting of viral expressions and then quantification using the ImageJ software. The data are presented as the mean ± SD (error bars) of three independent experiments. **p < 0.01 and ***p < 0.001 indicate a significant difference between control groups, #p < 0.05, ##p < 0.01 and ###p < 0.001 indicate a significant difference between the vehicle (IAV-infection).
Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 764297 9 proteins. The GFP-expressing influenza virus A/PR/8/34-GFP was used to infect the RAW 264.7 cells. The level of GFP was lower in cells pre-treated with chlorogenic acid or ginsenoside Rd than that in the untreated cells ( Figures 6A-C). Western blots showed that the levels of IAV proteins were suppressed in the RAW 264.7 cells pre-treated with chlorogenic acid or ginsenoside Rd compared to those in the untreated cells ( Figure 6D).

CONCLUSION
We additionally experimented on the co-and post-treatment antiviral activity of OCD20015-V009. However, the results demonstrated that OCD20015-V009 had no anti-influenza virus activity co-and post-treatment (Supplementary Figure  S1). Therefore, these data indicated that OCD20015-V009 has anti-influenza A virus activity in the pre-treatment assays, but not in co-and post-treatment assays.
Here, we have discovered that OCD20015-V009 pretreatment substantially reduces viral infection, based on the analysis of the infection of RAW 264.7 cells by A/PR/8/34-GFP. We have demonstrated that OCD20015-V009 pre-treatment increases the phosphorylation of type-I IFNrelated proteins STAT1 and TBK-1 and the secretion of proinflammatory cytokines TNF-α and IL-6 in RAW 264.7 cells. Additionally, OCD20015-V009 pre-treatment increased the mRNA of the ISGs-related ISG 15, 20 and 56 and the IFN-β gene in vitro. Thus, these findings show that OCD20015-V009 has immunomodulatory and antiviral properties. This finding shows potential to develop prophylactic or therapeutic treatments against the IAV. In mice exposed to the H1N1 IAV, an OCD20015-V009 pre-treatment at 300 mg/kg decreased mortality by 20% compared with that in untreated animals. These data suggest that OCD20015-V009 stimulates an antiviral response in murine macrophages and mice, thus protecting against IAV infection. Additionally, we identified active compounds in OCD20015-V009, chlorogenic acid, or ginsenoside Rd, using UPLC-MS/MS and demonstrated that they exhibited the most significant antiviral effects.
Further investigations are warranted to elucidate the molecular mechanisms for the protective effects of OCD20015-V009 and its components, chlorogenic acid, and ginsenoside Rd, against influenza virus A infection. Based on the results, we propose that OCD20015-V009 and its components could be effective antiviral agents or vaccine adjuvants for influenza virus infection.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by this study was carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Center (LAC) of Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF). The animal studies were approved by the IACUC of the LAC of DGMIF under approval number DGMIF-18071602-00.

AUTHOR CONTRIBUTIONS
E-BK, Y-CO, Y-HH, WL, and YK designed the study, conducted the experiments, and wrote the manuscript. S-MP and RK performed OCD20015-V009 preparation. J-GC supervised the research, designed the study, conducted the experiments and wrote the manuscript. All authors reviewed the manuscript.