key: cord-0887904-k4ebs2wj
authors: Kim, Eun-Ha; Kim, Young-il; Jang, Seung-Gyu; Im, Minju; Jeong, Kyeongsoo; Choi, Young Ki; Han, Hae-Jung
title: Antiviral effects of human placenta hydrolysate (Laennec(®)) against SARS-CoV-2 in vitro and in the ferret model
date: 2021-10-06
journal: J Microbiol
DOI: 10.1007/s12275-021-1367-2
sha: f889f48f70302fe50703f83dc1d17292fac9fda4
doc_id: 887904
cord_uid: k4ebs2wj

The COVID-19 pandemic has caused unprecedented health, social, and economic crises worldwide. However, to date, there is an only a limited effective treatment for this disease. Human placenta hydrolysate (hPH) has previously been shown to be safe and to improve the health condition in patients with hyperferritinemia and COVID-19. In this study, we aimed to determine the antiviral effects of hPH against SARS-CoV-2 in vitro and in vivo models and compared with Remdesivir, an FDA-approved drug for COVID-19 treatment. To assess whether hPH inhibited SARS-CoV-2 replication, we determined the CC(50), EC(50), and selective index (SI) in Vero cells by infection with a SARS-CoV-2 at an MOI of 0.01. Further, groups of ferrets infected with 10(5.8) TCID(50)/ml of SARS-CoV-2 and treated with hPH at 2, 4, 6 dpi, and compared their clinical manifestation and virus titers in respiratory tracts with PBS control-treated group. The mRNA expression of immune-related cytokines was determined by qRT-PCR. hPH treatment attenuated virus replication in a dose-dependent manner in vitro. In a ferret infection study, treatment with hPH resulted in minimal bodyweight loss and attenuated virus replication in the nasal wash, turbinates, and lungs of infected ferrets. In addition, qRT-PCR results revealed that the hPH treatment remarkably upregulated the gene expression of type I (IFN-α and IFN-β) and II (IFN-γ) IFNs in SARS-CoV-2 infected ferrets. Our data collectively suggest that hPH has antiviral efficacy against SARS-CoV-2 and might be a promising therapeutic agent for the treatment of SARS-CoV-2 infection.

In early December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the cause of severe pneumonia, named coronavirus disease 2019 , in human patients in Wuhan City, China . Since then, the number of SARS-CoV-2 cases has increased continuously, resulting in the COVID-19 pandemic. As of May 2021, SARS-CoV-2 has spread through 220 countries and caused more than 163 million confirmed cases and 3.3 million deaths worldwide (WHO, 2021) . SARS-CoV-2 infection causes clinical symptoms, such as nausea, vomiting, diarrhoea, cytokine storm, lymphopenia, as well as tissue damage to the lungs, liver, kidneys, heart, and other organs, eventually resulting in death in some severe cases . Notably, convergent evolution of SARS-CoV-2 has resulted in novel variants able to evade host immune responses (CDC, 2021) , making the virus difficult to control. Thus, there is an urgent need to develop novel vaccines and antiviral drugs for effective control of SARS-CoV-2 infections.

In October 2020 the FDA approved the use of remdesivir, an antiviral drug for RNA viruses, for use as a therapeutic for COVID-19 (FDA, 2020) . Remdesivir was originally developed by Gilead Sciences in 2009 to target hepatitis C and respiratory syncytial viruses (Mackman et al., 2021) . A recent study revealed that the Remdesvir inhibits the viral RNA-dependent RNA polymerase to terminate viral RNA synthesis (Eastman et al., 2020) . Further, pre-treatment with remdesivir effectively inhibits virus replication in vitro (Wang et al., 2020a) , and reduces clinical signs in SARS-CoV-2 infected rhesus macaques (Williamson et al., 2020) . Clinical trials have shown that remdesivir accelerates clinical improvement in COVID-19 patients (Singh et al., 2020; Wang et al., 2020b) . Nevertheless, a systematic review and network meta-analysis showed that remdesivir may have little or no effect on the length of hospital stay (Siemieniuk et al., 2020) . Thus, the need for therapeutics to effectively control SARS-CoV-2 and treat COVID-19 still exists. Human placenta hydrolysate (hPH; Laennec ® , GC Wellbeing, Co. Ltd.), a safe therapeutic agent for the improvement of hepatic function, was authorized by the PMDA (Pharmaceuticals and Medical Devices Agency) in Japan in 1974 and by MFDS (Ministry of Food and Drug Safety) in Korea in 1993 for hepatic function improvement. Preclinical studies showed that Laennec ® could stimulate liver regeneration, anti-stress mechanisms and hair growth in rats (Liu et al., 1998; Kwon et al., 2015; Park et al., 2018) , as well as promote ligament healing in rodents (Shin et al., 2019) and antioxidant effects in mice (Bak et al., 2019) . In clinical studies, hPH has been reported to improve the health status of elderly Koreans (Kong and Park, 2012) and to alleviate menopausal symptoms and fatigue in middle-aged Korean women (Kong et al., 2008; Lee et al., 2009) . Moreover, it was also approved for use as an i) hepatoprotector, ii) immune-modulating agent for the treatment of atopic dermatitis, psoriasis and acne, and iii) for the treatment of recurring herpes (varicella-zoster virus) infection by the Russian Ministry of Health in 2003. Recently, Maksimov et al. (2020) demonstrated that Laennec ® administration improved the health of hyperferritinemia patients with COVID-19 by decreasing ferritin levels, reducing lung damage, and increasing blood oxygenation. Given the various functions of hPH, in this study we determined the CC 50 , EC 50 , and selective index (SI) of hPH in Vero cells to investigate the antiviral effects of hPH following infection with SARS-CoV-2 at an MOI of 0.01. Further, we used a ferret infection model, which has high natural susceptibility in the respiratory tract , to evaluate the antiviral effect of this therapeutic and compared the results with those of a remdesivir-treated group.

Vero cells (CCL-81) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) (Gibco). A SARS-CoV-2 virus (NMC-nCoV02) was isolated from a patient with confirmed COVID-19 in South Korea . The virus was propagated in Vero cells in DMEM supplemented with 1% PS and 0.5 μg/ml L-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (Worthington Biochemical) at 37°C for 72 h. Propagated viruses were harvested and stored at -80°C until further use. The 50% tissue culture infective dose (TCID 50 ) of the virus was measured through fixation and crystal violet staining.

Male and female ferrets, 10-12 months old, and seronegative for influenza A viruses, MERS-CoV and SARS-CoV, were maintained in isolators (Woori IB Corporation, Daejeon, South Korea) in a biosafety level 3 laboratory at Chungbuk National University, South Korea. All ferrets were housed under a 12/12 h light/dark cycle and allowed access to food and water ad libitum. Body weight and temperature were measured every two days, before and after virus infection, during all experimental periods. Animal handling and experiments using SARS-CoV-2 were carried out following protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University.

Human placenta hydrolysate (Laennec ® ) was manufactured by GC Wellbeing Co., Ltd. (Seongnam, South Korea), as previously described (Kong and Park, 2012) . Briefly, hPH, an aqueous placenta extract, is known for abundant source of various bioactive substances, such as peptides, amino acids, nucleic acids, and minerals. It is prepared by the hydrolysis of human placenta with HCl and pepsin. The final product is aliquoted 2 ml in ampule as liquid. Each ampule of hPH possesses various amino acids including arginine (0.08%), lysine (0.1%), phenylalanine (0.08%), tyrosine (0.03%), leucine (0.12%), methionine (0.03%), valine (0.04%), alanine (0.08%), seine (0.07%), and threonine (0.06%). hPH has been investigated to possess various therapeutic properties ranging from anti-inflammation to immunomodulation.

To evaluate the antiviral efficacy of hPH, Vero cells were cultured overnight in 96-well plates at a density of 2 × 10 4 cells/well. The cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. The virus was then removed, and cells were further cultured with fresh medium containing different concentrations of hPH. After 72 h, the cells were fixed with formaldehyde and stained with 0.1% crystal violet. The CellTiter 96 ® Non-Radioactive Cell Proliferation Assay Kit (Promega) was used to determine the cytotoxic concentration (CC 50 ) and effective concentration (EC 50 ). The values were calculated from the percentage of cells whose viability was inhibited by various concentrations of hPH.

Ferrets (n = 10 per group) were anesthetized with isoflurane and then infected with 30 μl of 10 5.8 TCID 50 /ml of SARS-CoV-2 through the intranasal route. The infected ferrets were injected with either hPH (4 ml/animal by intravenous injection) on days 0, 2, 4, and 6 post-infection (dpi), or remdesivir (17.6 mg/kg on day 1 as a loading dose and 8.8 mg/kg daily on days 2-4 post infection as maintenance doses). A group of ferrets injected daily with PBS from days 0-4 post infection was used as the PBS-control group. The body weight and temperatures of the ferrets were measured every 2 days until 14 dpi.

The animals were anesthetized using 150 μl Zoletil/xylazine mixture (Zoletil 50 ® , 80 mg/kg, Virbac; Rompun ® , 20 mg/kg, Bayer HealthCare) through intramuscular injection. Nasal washes were then collected in 1 ml PBS containing antibiotics at 0, 2, 4, 6, 8, and 10 dpi. Lung tissue and nasal turbinates were collected at 3 and 6 dpi and homogenized in a medium containing penicillin G (2 × 10 6 U/L), polymyxin B (2 × 10 6 U/L), ofloxacin HCl (60 mg/L), and sulfamethoxazole (0.2 g/L). After centrifugation at 12,000 × g for 10 min at 4°C the supernatants were collected. All samples were stored at -80°C until further use.

To determine infectious SARS-CoV-2 titers, 10-fold serial dilutions of the lung, nasal wash, and nasal turbinate homogenates were incubated with confluent Vero cells in 96-well flat-bottom plates for 1 h at 37°C. The cells were washed with PBS and incubated with DMEM containing 1% PS and 0.5 μg/ml TPCK-trypsin for 4 days at 37°C. The cytopathic effect (CPE) was recorded daily, and the TCID 50 was calculated according to the Reed and Muench method (Leed and Muench, 1938) and expressed as log 10 TCID 50 /ml.

Briefly, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), or an RNeasy Kit (Qiagen), and cDNA was generated with an oligo (dT) primer by reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR was performed using the SYBR Green Supermix (Bio-Rad) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with specific primer sets (IFN-α: Forward: 5′-AACATCATCCCTGCTTC-3′, Reverse: 5′-AGGCCAT GCCAGTGAGCT-3′, IFN-β: Forward: 5′-GGTGTATCCT CCAAACTG-3′, Reverse: 5′-ACTCCACACTGCTGCTGC-3′, and IFN-γ: Forward: 5′-CCATCAAGGAAGACATGC-3′, Reverse: 5′-GAAACACACTGTGACT-3′). Finally, the target mRNA was quantified, normalized to GAPDH (endogenous housekeeping gene) and relative to the calibrator, using the 2 -ΔΔCT method. Thus, all experimental samples are expressed as a fold-change relative to the calibrator.

Statistical significance between groups was determined by two-way analysis of variance (ANOVA) and a subsequent Dunnett test using GraphPad Prism version 8.20 (GraphPad Software). Statistical significance was set at p < 0.05.

To assess the antiviral efficacy and cell cytotoxicity of hPH, the CC 50 , EC 50 and selective index (SI) of this extract were measured in Vero cells. Briefly, SARS-CoV-2 infected cells were treated with different concentrations of hPH, and inhibition of cytopathic effect (CPE) was visually observed using crystal violet staining. The CPE inhibition assay revealed that hPH treatment reduces SARS-CoV-2 induced CPE in Vero cells at both a 50% dilution and its original concentration (Fig. 1A) . Additionally, cell viability and antiviral activity were measured using the CellTiter 96 ® Non-Radioactive Cell Proliferation Assay and hPH did not show any cytotoxicity in Vero cells at up to 100% with the EC 50 values of hPH (EC 50 = 19.42%). Therefore, we concluded that the SI (CC 50 /EC 50 ) value of hPH was 5.1 (Fig. 1B) . These results suggest that hPH has antiviral effects against SARS-CoV-2 in vitro and with low cytotoxicity.

To investigate the antiviral effects of hPH against SARS-CoV-2 in vivo groups of ferrets (n = 10/group) infected with 10 5.8 TCID 50 /ml of SARS-CoV-2 were treated with hPH from 24 h post-infection (hpi) as described in the Methods. All SARS-CoV-2 infected fqerrets showed a gradual decrease in body weight (~6%) until 4 dpi, but recovered to their initial weight by 10-14 dpi. Compared with the PBS-treated control group, the ferrets treated with hPH or remdesivir showed significantly reduced body weight loss and earlier recovery to their initial weight (Remdesivir, p < 0.05). There was no difference in weight loss between ferrets treated with hPH or remdesivir ( Fig. 2A) . However, there were no significant differences between groups in body temperature, as all groups of animals had slightly elevated body temperature at 4 dpi and recovered after 10-14 dpi (Fig. 2B) . To evaluate the antiviral activity of hPH, we measured infectious virus titers in nasal washes and tissues from each ferret group. Infectious SARS-CoV-2 was detected in nasal washes from all groups until 6 dpi. However, the hPH and remdesivirtreated groups showed significantly lower virus titers than

(A) Nasal wash samples were collected at 0, 2, 4, 6, 8, and 10 dpi (* p < 0.05 and ** p < 0.01). (B) Virus titres in lung samples were quantified at 3 and 6 dpi. (C) Nasal turbinate tissue collected from ferrets at 3 and 6 dpi. The limit of detection is 0.75 log10 TCID50/g as indicated by the dotted line. those of the PBS control group at 6 dpi (hPH, p < 0.05; remdesivir, p < 0.01) (Fig. 3A) . Further, SARS-CoV-2 titers in the lung tissue of hPH and remdesivir-treated groups were significantly lower than that of the control group at 3 dpi (Fig.  3B ). In addition, hPH and remdesivir-treated groups showed attenuated viral titers in the nasal turbinates compared to PBS-treated ferrets at 6 dpi (Fig. 3C) . Collectively, these data demonstrate that hPH exerts antiviral activity which leads to a reduction of the viral burden in the nasal wash, lungs, and nasal turbinates of SARS-CoV-2 infected ferrets with efficacy comparable with that of remdesivir.

To identify potential host factors contributing to the antiviral activity of hPH treatment, lung tissues from each group of ferrets were collected at 3 and 6 dpi, and the mRNA expression levels of immune-related cytokines were determined by qRT-PCR. Interestingly, hPH-treated SARS-CoV-2 infected ferrets showed a significant increase in expression of type I IFN cytokines, including IFN-α and IFN-β at 3 and 6 dpi, compared with that of the PBS-treated control ( Fig. 4A and B). Further, although IFN-γ expression was lower in hPHtreated ferrets compared to remdesivir-treated ferrets at 3 dpi, the IFN-γ expression was higher in the hPH-treated group than in the PBS control group. The expression of IFN-γ at 6 dpi was similar in all groups (Fig. 4C) . Taken together, our results suggest that administration of hPH reduces the viral burden and clinical symptoms in SARS-CoV-2 infected ferrets by enhancing the expression of type I and II IFNs.

Due to the uncontrolled nature of the COVID-19 pandemic, remdesivir was given emergency use approval for the treatment of COVID-19. However, its effect on the disease is not yet fully understood (Liang et al., 2020) , and multiple clinical trials are ongoing to evaluate its safety and efficacy as a treatment for COVID-19 (Eastman et al., 2020) . Thus, there is still a need to explore other potential antiviral drugs for this disease. Recently, hyperferritinemia contributes to a cytokine storm and is considered an indicator of severe infection in COVID-19 patients (Maksimov et al., 2020) . hPH is a safe and approved drug for the improvement of hepatic function (Kong and Park, 2012) and for treatment of hyperferritinemia patients with COVID-19 (Maksimov et al., 2020) , making it a potential candidate drug for the treatment of COVID-19. Recently, Maksimov et al. (2020) reported that the short peptide fragment in hPH was deduced to improve the serum ferritinemia through inactivation of the pro-inflammatory factor NF-κB and, consequently, to a decrease in the level of chronic inflammation. Further, hPH is already approved by the PMDA in Japan and by the MFDS in Korea for therapeutic treatment in humans, hPH did not show any cytotoxicity in vitro while it showed strong antiviral activity against SARS-CoV-2.

Regarding the antiviral activity of remdesivir, many studies have reported that therapeutic treatment results in a reduction in clinical signs in rhesus macaques infected with MERS-CoV (de Wit et al., 2020) and SARS-CoV-2 (Kim et al., 2021) and clinical improvement was observed in 68% of COVID-19 patients treated with remdesivir in a clinical trial (Abdolvahab et al., 2021) . Further, a recent study demonstrated that remdesivir-treated ferrets showed reduced virus titers in the nasal wash and lung tissues compared with the control group (Kim et al., 2021) . Thus, for the systemic evaluation of antiviral efficacy of hPH, we adapted the ferret as an in vivo evaluation model for SARS-CoV-2 infection and compared antiviral effects of hPH with those of remdesivir, an FDA-approved drug for COVID-19.

Our results show that remdesivir treatment following SARS-CoV-2 infection in ferrets resulted in minimal body weight loss and attenuated virus titers in the respiratory tract, which are well within agreement with the previous study. Further, notably, hPH-treated ferrets showed a significantly reduced SARS-CoV-2 viral load in the nasal wash and nasal turbinates at 6 dpi and in the lung at 3 dpi compared to PBS treatment. Importantly, virus titers in hPH-treated ferrets were comparable to those of the remdesivir treatment group indicating that Laennec ® has comparable antiviral activity with remdesivir in ferrets.

It is well known that type I IFNs, including IFN-α and IFNβ, are involved in innate antiviral defense. Once IFNs are induced and bind their receptors, STAT (signal transducer and activator of transcription) signaling is activated, which triggers the transcription of numerous IFN-stimulated genes, ultimately eliciting an effective antiviral response (Platanias, 2005; Schoggins et al., 2011) . Further, recent studies reported that treatment with IFN-α or IFN-β reduces SARS-CoV-2 virus titers in Vero cells (Mantlo et al., 2020) . Moreover, a phase II clinical trial revealed that adminiatration of IFN-β-1b in combination with lopinavir-ritonavir and ribavirin can shorten the duration of virus shedding and reduce clinical symptoms in COVID-19 patients (Mantlo et al., 2020) .

In this study, we observed significantly enhanced IFN-α and IFN-β mRNA expression following hPH treatment compared to treatment with remdesivir or PBS. Notably, administration of hPH induced the production of IFN-α and IFN-β at an earlier time point (3 dpi) than was seen following remdesivir treatment. Thus, hPH treatment appears to induce an early and strong type I IFN antiviral response compared to the control group. Unlike virus-induced type I and III IFNs, IFN-γ (known as type II IFN) is mostly produced by immune cells and initiates antiviral activity through expansion of the cytotoxic T lymphocyte population and through activation of monocytes and macrophages to express cytokines. Therefore, IFN-γ is also listed as a potential treatment for COVID-19 (Abdolvahab et al., 2021) . Herein, an increase in IFN-γ mRNA expression at an early point (3 dpi) was found in the lungs of SARS-CoV-2 infected ferrets following hPH treatment. Thus, this suggests that the hPH treatment may enhance IFN-γ cytokine expression in lungs and contribute to the inhibition of SARS-CoV-2 replication. Although Vero cells could not secret functional IFN-alpha or beta due to the deletion of the type I interferon gene cluster set, it still have type I IFN receptors and other interferon-inducible genes (ISG) set. Actually, hPH is a human placenta extract which is a cocktail of biologically active compunds including various peptides, growth factors, amino acids, antioxidants, vitamins, minerals etc. Thus such multiple component could exert multiple tar-get mode of action for antiviral activity as well as induction of type I IFNs. Further, although type I IFN response is important for antiviral activity, there are many other alternative antiviral pathways (Sa Ribero et al., 2020; Mdkhana et al., 2021) that the other biologically active compounds found in hPH may exert a broad antiviral effects. This study encompass the multiple component, multiple target (MCMT) concept which has been favored to identify therapeutic agents with a broad spectrum antiviral activity using Vero cells. Although a recent study (Maksimov et al., 2020) demonstrated that hPH contains numerous compounds that can inhibit virus activation, fusion, replication and budding, however, exact antiviral pathway in vitro against SARS-CoV-2 remains to be elucidated. Therefore, to understand the detailed antiviral mechanisms of hPH, further expanded molecular studies are needed. Collectively, our results demonstrate that hPH has antiviral effects against SARS-CoV-2 and is as effective as remdesivir at reducing the clinical features of virus-infected animals. Moreover, these effects may be associated with enhanced type I and II IFN expression. Although studies of the safety of hPH and elucidation of its detailed antiviral mechanisms are needed, hPH could be a promising candidate for the treatment of COVID-19.

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The materials and budget were sponsored by GREENCROSS WellBeing Co. Ltd.

The author(s) have declared that no conflicts of interest exists.

All animal experiments were approved by the Medical Research Institute, a member of the Laboratory Animal Research Center of Chungbuk National University (approval number CBNUA-1352-20-02), and all animal experiments were conducted in strict accordance and adherence to relevant policies regarding animal handling as mandated under the Guidelines for Animal Use and Care of the Korea Centers for Disease Control. Viruses were handled in an enhanced biosafety level 3 (BSL3) containment laboratory, as approved by the Korean Centers for Disease Control (KCDC-14-3-07). The materials and budget were sponsored by GC Wellbeing Co. Ltd.