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Abstract The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyze blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyze their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) is achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against virus variant immune escape.

Background/Aims We used next-generation sequencing (NGS) to analyze resistance-associated substitutions (RASs) and retreatment outcomes in patients with chronic hepatitis C virus (HCV) infection who failed direct-acting antiviral agent (DAA) treatment in South Korea. Methods Using prospectively collected data from the Korean HCV cohort study, we recruited 36 patients who failed DAA treatment in 10 centers between 2007 and 2020; 29 blood samples were available from 24 patients. RASs were analyzed using NGS. Results RASs were analyzed for 13 patients with genotype 1b, 10 with genotype 2, and one with genotype 3a. The unsuccessful DAA regimens were daclatasvir+asunaprevir (n=11), sofosbuvir+ribavirin (n=9), ledipasvir/sofosbuvir (n=3), and glecaprevir/pibrentasvir (n=1). In the patients with genotype 1b, NS3, NS5A, and NS5B RASs were detected in eight, seven, and seven of 10 patients at baseline and in four, six, and two of six patients after DAA failure, respectively. Among the 10 patients with genotype 2, the only baseline RAS was NS3 Y56F, which was detected in one patient. NS5A F28C was detected after DAA failure in a patient with genotype 2 infection who was erroneously treated with daclatasvir+asunaprevir. After retreatment, 16 patients had a 100% sustained virological response rate. Conclusions NS3 and NS5A RASs were commonly present at baseline, and there was an increasing trend of NS5A RASs after failed DAA treatment in genotype 1b. However, RASs were rarely present in patients with genotype 2 who were treated with sofosbuvir+ribavirin. Despite baseline or treatment-emergent RASs, retreatment with pan-genotypic DAA was highly successful in Korea, so we encourage active retreatment after unsuccessful DAA treatment.

Summary: Here, we describe detailed steps to constitute an in vitro assay for monitoring papain-like protease of coronavirus and a cell-based immunofluorescence infection assay. These assays can be adapted for high-throughput screening to determine the efficacy of novel protease inhibitors of coronaviruses and other viruses. In addition, cell-based immunofluorescence infection assay can be used to visually analyze antiviral efficacy of any novel compounds.For complete details on the use and execution of this protocol, please refer to Jeong et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The molecular manifestations of host cells responding to SARS-CoV-2 and its evolving variants of infection are vastly different across the studied models and conditions, imposing challenges for host-based antiviral drug discovery. Based on the postulation that antiviral drugs tend to reverse the global host gene expression induced by viral infection, we retrospectively evaluated hundreds of signatures derived from 1,700 published host transcriptomic profiles of SARS/MERS/SARS-CoV-2 infection using an iterative data-driven approach. A few of these signatures could be reversed by known anti-SARS-CoV-2 inhibitors, suggesting the potential of extrapolating the biology for new variant research. We discovered IMD-0354 as a promising candidate to reverse the signatures globally with nanomolar IC50 against SARS-CoV-2 and its five variants. IMD-0354 stimulated type I interferon antiviral response, inhibited viral entry, and down-regulated hijacked proteins. This study demonstrates that the conserved coronavirus signatures and the transcriptomic reversal approach that leverages polypharmacological effects could guide new variant therapeutic discovery.

Summary: The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors. Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent of the coronavirus disease 2019 (COVID-19) pandemic, and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development, and this effort was partially successful. Since the beginning of the COVID-19 pandemic, the emergence of SARS-CoV-2 variants has been reported in many parts of the world, and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. Viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development; therefore, the inhibitory effects of transmembrane serine protease 2 (TMPRSS2) and RdRp inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the United Kingdom and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 and RdRp are equally effective against the two variants of concern. IMPORTANCE The COVID-19 pandemic is causing unprecedented global problems in both public health and human society. While some vaccines and monoclonal antibodies were successfully developed very quickly and are currently being used, numerous variants of the causative SARS-CoV-2 are emerging and threatening the efficacy of vaccines and monoclonal antibodies. In order to respond to this challenge, we assessed antiviral efficacy of small-molecule inhibitors that are being developed for treatment of COVID-19 and found that they are still very effective against the SARS-CoV-2 variants. Since most small-molecule inhibitors target viral or host factors other than the mutated sequence of the viral spike protein, they are expected to be potent control measures against the COVID-19 pandemic.

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform.

We previously reported the potent antiviral effect of the 2-aminoquinazolin-4-(3H)-one 1, which shows significant activity (IC50 = 0.23 μM) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with no cytotoxicity. However, it is necessary to improve the in vivo pharmacokinetics of compound 1 because its area under the curve (AUC) and maximum plasma concentration are low. Here, we designed and synthesized N-substituted quinazolinone derivatives that had good pharmacokinetics and that retained their inhibitory activity against SARS-CoV-2. These compounds were conveniently prepared on a large scale through a one-pot reaction using Dimroth rearrangement as a key step. The synthesized compounds showed potent inhibitory activity, low binding to hERG channels, and good microsomal stability. In vivo pharmacokinetic studies showed that compound 2b had the highest exposure (AUC24h = 41.57 μg∙h/mL) of the synthesized compounds. An in vivo single-dose toxicity evaluation of compound 2b at 250 and 500 mg/kg in rats resulted in no deaths and an approximate lethal dose greater than 500 mg/kg. This study shows that N-acetyl 2-aminoquinazolin-4-(3H)-one 2b is a promising lead compound for developing anti-SARS-CoV-2 agents.

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ± 0.75 μM) than PAC (25.90 ± 0.81 μM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25–26 μM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ± 1.089 μM) than PAC (21.022 ± 1.202 μM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

The rhizome of Dryopteris crassirhizoma Nakai. (Dryopteridaceae) has been used in traditional medicine in East Asia and has recently been reported to have anticancer, anti-inflammation, and antibacterial activity as well as antiviral activity. Natural phloroglucinols from D. crassirhizoma, dryocrassin ABBA and filixic acid ABA were reported to inhibit influenza virus infection with an inhibitory activity on neuraminidase. In this study, we found that dryocrassin ABBA and filixic acid ABA have an inhibitory activity against the main protease of SARS-CoV-2. Therefore, dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against SARS-CoV-2 infection in Vero cells dose-dependently using the immunofluorescence-based antiviral assays. Moreover, these compounds inhibited SARS-CoV and MERS-CoV infection, suggesting their broad-spectrum anticoronaviral activity. In addition, a 5-day repeated-dose toxicity study of dryocrassin ABBA and filixic acid ABA suggested that an approximately lethal dose of these compounds in mice was >10 mg/kg. Pharmacokinetic studies of dryocrassin ABBA showed good microsomal stability, low hERG inhibition, and low CYP450 inhibition. In vivo pharmacokinetic properties of dryocrassin ABBA showed a long half-life (5.5–12.6 h) and high plasma exposure (AUC 19.3–65 μg·h/mL). Therefore, dryocrassin ABBA has therapeutic potential against emerging coronavirus infections, including COVID-19.

Background/AimsThe treatment with daclatasvir plus asunaprevir (DCV+ASV) is associated with potent antiviral effects in patients with genotype 1b hepatitis C virus (HCV) infection. We investigated the real-world efficacy, changes in liver stiffness and noninvasive fibrosis markers, and the safety of DCV+ASV treatment in Korean patients.Methods : In total, 363 patients with chronic hepatitis C were treated with DCV+ASV between August 2015 and January 2017. Finally, we analyzed the data of 270 patients who were monitored for at least 12 weeks after the end of treatment.Results : The mean age was 60.7 years, and females predominated (60.4%). Most patients (64.8%) were treatment-naïve, and 56 patients (20.7%) had cirrhosis. Two hundred fifty-seven (95.2%) and 251 (93.0%) patients achieved end-of-treatment responses and sustained virological responses at 12 weeks posttreatment (SVR12), respectively. The SVR12 rates were higher in patients who were

Cardiotonic steroids are steroid-like natural compounds known to inhibit Na+/K+-ATPase pumps. To develop a broad-spectrum antiviral drug against the emerging coronavirus infection, this study assessed the antiviral properties of these compounds. The activity of seven types of cardiotonic steroids against the MERS-CoV, SARS-CoV, and SARS-CoV-2 coronavirus varieties was analyzed using immunofluorescence antiviral assay in virus-infected cells. Bufalin, cinobufagin, and telocinobufagin showed high anti-MERS-CoV activities (IC50, 0.017~0.027 μM); bufalin showed the most potent anti-SARS-CoV and SARS-CoV-2 activity (IC50, 0.016~0.019 μM); cinobufotalin and resibufogenin showed comparatively low anti-coronavirus activity (IC50, 0.231~1.612 μM). Differentially expressed genes in Calu3 cells treated with cinobufagin, telocinobufagin, or bufalin, which had high antiviral activity during MERS-CoV infection were analyzed using QuantSeq 3′ mRNA-Seq analysis and data showed similar gene expression patterns. Furthermore, the intraperitoneal administration of 10 mg/kg/day bufalin, cinobufagin, or digitoxin induced 100% death after 1, 2, and 4 days in 5-day repeated dose toxicity studies and it indicated that bufalin had the strongest toxicity. Pharmacokinetic studies suggested that telocinobufagin, which had high anti-coronavirus activity and low toxicity, had better microsomal stability, lower CYP inhibition, and better oral bioavailability than cinobufagin. Therefore, telocinobufagin might be the most promising cardiotonic steroid as a therapeutic for emerging coronavirus infections, including COVID-19.

Since its discovery in 1989, hepatitis C virus (HCV) has been intensively investigated to understand its biology and develop effective antiviral therapies. The efforts of the previous 25 years have resulted in a better understanding of the virus, and this was facilitated by the development of in vitro cell culture systems for HCV replication. Antiviral treatments and sustained virological responses have also improved from the early interferon monotherapy to the current all-oral regimens using direct-acting antivirals. However, antiviral resistance has become a critical issue in the treatment of chronic hepatitis C, similar to other chronic viral infections, and retreatment options following treatment failure have become important questions. Despite the clinical challenges in the management of chronic hepatitis C, substantial progress has been made in understanding HCV, which may facilitate the investigation of other closely related flaviviruses and lead to the development of antiviral agents against these human pathogens.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea that is characterized by severe hemorrhage and a high fatality rate. Currently, no specific vaccine or treatment has been approved for this disease. To develop a therapeutic agent for SFTS, we isolated antibodies from a phage-displayed antibody library that was constructed from a patient who recovered from SFTS virus (SFTSV) infection. One antibody, designated as Ab10, was reactive to the Gn envelope glycoprotein of SFTSV and protected host cells and A129 mice from infection in both in vitro and in vivo experiments. Notably, Ab10 protected 80% of mice, even when injected 5 days after inoculation with a lethal dose of SFTSV. Using cross-linker assisted mass spectrometry and alanine scanning, we located the non-linear epitope of Ab10 on the Gn glycoprotein domain II and an unstructured stem region, suggesting that Ab10 may inhibit a conformational alteration that is critical for cell membrane fusion between the virus and host cell. Ab10 reacted to recombinant Gn glycoprotein in Gangwon/Korea/2012, HB28, and SD4 strains. Additionally, based on its epitope, we predict that Ab10 binds the Gn glycoprotein in 247 of 272 SFTSV isolates previously reported. Together, these data suggest that Ab10 has potential to be developed into a therapeutic agent that could protect against more than 90% of reported SFTSV isolates.

Hepatitis C virus (HCV) infects ~71 million people worldwide, and 399,000 people die annually due to HCV-related liver cirrhosis and hepatocellular carcinoma. The use of direct-acting antivirals results in a sustained virologic response in >95% of patients with chronic HCV infection. However, several issues remain to be solved to eradicate HCV. At the 26th International Symposium on Hepatitis C Virus and Related Viruses (HCV2019) held in Seoul, South Korea, October 5−8, 2019, virologists, immunologists, and clinical scientists discussed these remaining issues and how we can achieve the elimination of HCV.

Stephania tetrandra and other related species of Menispermaceae are the major sources of the bis-benzylisoquinoline alkaloids tetrandrine (TET), fangchinoline (FAN), and cepharanthine (CEP). Although the pharmacological properties of these compounds include anticancer and anti-inflammatory activities, the antiviral effects of these compounds against human coronavirus (HCoV) remain unclear. Hence, the aims of the current study were to assess the antiviral activities of TET, FAN, and CEP and to elucidate the underlying mechanisms in HCoV-OC43-infected MRC-5 human lung cells. These compounds significantly inhibited virus-induced cell death at the early stage of virus infection. TET, FAN, and CEP treatment dramatically suppressed the replication of HCoV-OC43 as well as inhibited viral S and N protein expression. The virus-induced host response was reduced by compound treatment as compared with the vehicle control. Taken together, these findings demonstrate that TET, FAN, and CEP are potential natural antiviral agents for the prevention and treatment of HCoV-OC43 infection.

Abl is a central regulator of multiple cellular processes controlling actin dynamics, proliferation, and differentiation. Here, we showed that knockdown of Abl impaired hepatitis C virus (HCV) propagation. Treatment of Abl tyrosine kinase-specific inhibitor, imatinib and dasatinib, also significantly decreased HCV RNA and protein levels in HCV-infected cells. We showed that both imatinib and dasatinib selectively inhibited HCV infection at the entry step of HCV life cycle, suggesting that Abl kinase activity may be necessary for HCV entry. Using HCV pseudoparticle infection assays, we verified that Abl is required for viral entry. By employing transferrin uptake and immunofluorescence assays, we further demonstrated that Abl was involved in HCV entry at a clathrin-mediated endocytosis step. These data suggest that Abl may represent a novel host factor for HCV entry.

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe aggravating respiratory failure in infected patients, frequently resulting in mechanical ventilation. As limited therapeutic antibody is accumulated in lung tissue following systemic administration, inhalation is newly recognized as an alternative, possibly better, route of therapeutic antibody for pulmonary diseases. The nebulization process, however, generates diverse physiological stresses, and thus, the therapeutic antibody must be resistant to these stresses, remain stable, and form minimal aggregates. We first isolated a MERS-CoV neutralizing antibody that is reactive to the receptor-binding domain (RBD) of spike (S) glycoprotein. To increase stability, we introduced mutations into the complementarity-determining regions (CDRs) of the antibody. In the HCDRs (excluding HCDR3) in this clone, two hydrophobic residues were replaced with Glu, two residues were replaced with Asp, and four residues were replaced with positively charged amino acids. In LCDRs, only two Leu residues were replaced with Val. These modifications successfully generated a clone with significantly greater stability and equivalent reactivity and neutralizing activity following nebulization compared to the original clone. In summary, we generated a MERS-CoV neutralizing human antibody that is reactive to recombinant MERS-CoV S RBD protein for delivery via a pulmonary route by introducing stabilizing mutations into five CDRs.

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

A tuning method was proposed for automatic lighting (auto-lighting) algorithms derived from the steepest descent and conjugate gradient methods. The auto-lighting algorithms maximize the image quality of industrial machine vision by adjusting multiple-color light emitting diodes (LEDs)—usually called color mixers. Searching for the driving condition for achieving maximum sharpness influences image quality. In most inspection systems, a single-color light source is used, and an equal step search (ESS) is employed to determine the maximum image quality. However, in the case of multiple color LEDs, the number of iterations becomes large, which is time-consuming. Hence, the steepest descent (STD) and conjugate gradient methods (CJG) were applied to reduce the searching time for achieving maximum image quality. The relationship between lighting and image quality is multi-dimensional, non-linear, and difficult to describe using mathematical equations. Hence, the Taguchi method is actually the only method that can determine the parameters of auto-lighting algorithms. The algorithm parameters were determined using orthogonal arrays, and the candidate parameters were selected by increasing the sharpness and decreasing the iterations of the algorithm, which were dependent on the searching time. The contribution of parameters was investigated using ANOVA. After conducting retests using the selected parameters, the image quality was almost the same as that in the best-case parameters with a smaller number of iterations.

Genotype 2a JFH1 virus has substantially contributed to the progress of HCV biology by allowing entire viral life cycle of HCV in cell culture. Using this genotype 2a virus, casein kinase II (CKII) was previously identified as a crucial host factor in virus assembly by phosphorylating NS5A. Since most of the prior studies employed genotype 2a JFH1 or JFH1-based intragenotypic chimera, we used genotype 1a H77S to study virus assembly. CKII inhibition by chemical inhibitors enhanced H77S virus production in contrast to that of JFH1 virus, but genetic inhibition of CKII by siRNA did not change H77S virus titer significantly. The different outcomes from these two approaches of CKII inhibition suggested that nonspecific target kinase of CKII inhibitors plays a role in increasing H77S virus production and both viral and host factors were investigated in this study. Our results emphasize substantial differences among the HCV genotypes that should be considered in both basic research and clinical practices.

Dimerization of hypoxia-inducible factor-1 beta (HIF-1β) [aryl hydrocarbon receptor nuclear translocator (ARNT)] with HIF-1α is involved in various aspects of cancer biology, including proliferation and survival under hypoxic conditions. We investigated the in vitro mechanism by which silencing of HIF-1β leads to the suppression of tumor cell growth and cellular functions. Various hepatocellular carcinoma (HCC) cell lines (Huh-7, Hep3B, and HepG2) were transfected with small interfering RNA (siRNA) against HIF-1β (siHIF-1β) and cultured under hypoxic conditions (1% O2 for 24 h). The expression levels of HIF-1β, HIF-1α, and growth factors were examined by immunoblotting. Tumor growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and tumor activity was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling, tumor cell invasion, and migration assays. Under hypoxic conditions, silencing of HIF-1β expression suppressed tumor cell growth and regulated the expression of tumor growth-related factors, such as vascular endothelial growth factor, epidermal growth factor, and hepatocyte growth factor. Suppression of tumor cell invasion and migration was also demonstrated in HIF-1β-silenced HCC cell lines. Silencing of HIF-1β expression may induce anti-tumor effects under hypoxic conditions in HCC cell lines.