An ancestral SARS-CoV-2 vaccine induces anti-Omicron variants antibodies by hypermutation.

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.

Rise in broadly cross-reactive adaptive immunity against human ß-coronaviruses in MERS-recovered patients during the COVID-19 pandemic.

To develop a universal coronavirus (CoV) vaccine, long-term immunity against multiple CoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, Middle East respiratory syndrome (MERS)-CoV, and future CoV strains, is crucial. Following the 2015 Korean MERS outbreak, we conducted a long-term follow-up study and found that although neutralizing antibodies and memory T cells against MERS-CoV declined over 5 years, some recovered patients exhibited increased antibody levels during the COVID-19 pandemic. This likely resulted from cross-reactive immunity induced by SARS-CoV-2 vaccines or infections. A significant correlation in antibody responses across various CoVs indicates shared immunogenic epitopes. Two epitopes-the spike protein's stem helix and intracellular domain-were highly immunogenic after MERS-CoV infection and after SARS-CoV-2 vaccination or infection. In addition, memory T cell responses, especially polyfunctional CD4+ T cells, were enhanced during the pandemic, correlating significantly with MERS-CoV spike-specific antibodies and neutralizing activity. Therefore, incorporating these cross-reactive and immunogenic epitopes into pan-CoV vaccine formulations may facilitate effective vaccine development.

Novel SARS-CoV-2 entry inhibitors, 2-anilinoquinazolin-4(3H)-one derivatives, show potency as SARS-CoV-2 antivirals in a human ACE2 transgenic mouse model.

The ongoing COVID-19 has not only caused millions of deaths worldwide, but it has also led to economic recession and the collapse of public health systems. The vaccines and antivirals developed in response to the pandemic have improved the situation markedly; however, the pandemic is still not under control with recurring surges. Thus, it is still necessary to develop therapeutic agents. In our previous studies, we designed and synthesized a series of novel 2-anilinoquinazolin-4(3H)-one derivatives, and demonstrated inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and MERS-CoV in vitro. We then conducted in vivo studies using modified compounds that are suitable for oral administration. These compounds demonstrated no toxicity in rats and inhibited viral entry. Here, we investigated the in vivo efficacy of these drug candidates against SARS-CoV-2. Three candidate drugs, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (1), N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-dichlorophenyl)acetamide (2), and N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-difluorophenyl)acetamide (3) were administered orally to hACE2 transgenic mice at a dose of 100 mg/kg. All three drugs improved survival rate and reduced the viral load in the lungs. These results show that the derivatives possess in vivo antiviral efficacy similar to that of molnupiravir, which is currently being used to treat COVID-19. Overall, our data suggest that 2-anilinoquinazolin-4(3H)-one derivatives are promising as potential oral antiviral drug candidates against SARS-CoV-2 infection.

Protocol for in vitro fluorescence assay of papain-like protease and cell-based immunofluorescence assay of coronavirus infection.

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.

Next-generation sequencing analysis of hepatitis C virus resistance-associated substitutions in direct-acting antiviral failure in South Korea.

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. CONCLUSION: 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.

Discovery of 2-aminoquinolone acid derivatives as potent inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to threaten human health and create socioeconomic problems worldwide. A library of 200,000 small molecules from the Korea Chemical Bank (KCB) were evaluated for their inhibitory activities against SARS-CoV-2 in a phenotypic-based screening assay to discover new therapeutics to combat COVID-19. A primary hit of this screen was the quinolone structure-containing compound 1. Based on the structure of compound 1 and enoxacin, which is a quinolone-based antibiotic previously reported to have weak activity against SARS-CoV-2, we designed and synthesized 2-aminoquinolone acid derivatives. Among them, compound 9b exhibited potent antiviral activity against SARS-CoV-2 (EC50 = 1.5 µM) without causing toxicity, while having satisfactory in vitro PK profiles. This study shows that 2-aminoquinolone acid 9b provides a promising new template for developing anti-SARS-CoV-2 entry inhibitors.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

A Randomized Trial of Nafamostat for Covid-19.

BACKGROUND: Nafamostat mesylate is a potent in vitro antiviral agent that inhibits the host transmembrane protease serine 2 enzyme used by severe acute respiratory syndrome coronavirus 2 for cell entry. METHODS: This open-label, pragmatic, randomized clinical trial in Australia, New Zealand, and Nepal included noncritically ill hospitalized patients with coronavirus disease 2019 (Covid-19). Participants were randomly assigned to usual care or usual care plus nafamostat. The primary end point was death (any cause) or receipt of new invasive or noninvasive ventilation or vasopressor support within 28 days after randomization. Analysis was with a Bayesian logistic model in which an adjusted odds ratio <1.0 indicates improved outcomes with nafamostat. Enrollment was closed due to falling numbers of eligible patients. RESULTS: We screened 647 patients in 21 hospitals (15 in Australia, 4 in New Zealand, and 2 in Nepal) and enrolled 160 participants from May 2021 to August 2022. In the intention-to-treat population, the primary end point occurred in 8 (11%) of 73 patients with usual care and 4 (5%) of 82 with nafamostat. The median adjusted odds ratio for the primary end point for nafamostat was 0.40 (95% credible interval, 0.12 to 1.34) with a posterior probability of effectiveness (adjusted odds ratio <1.0) of 93%. For usual care compared with nafamostat, hyperkalemia occurred in 1 (1%) of 67 and 7 (9%) of 78 participants, respectively, and clinically relevant bleeding occurred in 1 (1%) of 73 and 7 (8%) of 82 participants. CONCLUSIONS: Among hospitalized patients with Covid-19, there was a 93% posterior probability that nafamostat reduced the odds of death or organ support. Prespecified stopping criteria were not met, precluding definitive conclusions. Hyperkalemia and bleeding were more common with nafamostat. (Funded by ASCOT and others; ClinicalTrials.gov number, NCT04483960.)

Lancemaside A from Codonopsis lanceolata: Studies on Antiviral Activity and Mechanism of Action against SARS-CoV-2 and Its Variants of Concern.

Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system. We demonstrated that this inhibitory effect of LA was due to the alteration of membrane cholesterol and blockade of the membrane fusion between SARS-CoV-2 and host cells by filipin staining and cell-based membrane fusion assays. Our findings also showed that LA, as a membrane fusion blocker, could impede the endosomal entry pathway of SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in Vero cells with similar of IC50 values ranging from 2.23 to 3.37 µM as well as the TMPRSS2-mediated viral entry pathway in A549 cells overexpressing ACE2 and TMPRSS2 with IC50 value of 3.92 µM. We further demonstrated that LA could prevent the formation of multinucleated syncytia arising from SARS-CoV-2 spike protein-mediated membrane fusion. Altogether, the findings reported here suggested that LA could be a broad-spectrum anti-SARS-CoV-2 therapeutic agent by targeting the fusion of viral envelope with the host cell membrane.

Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses.

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.

Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibits the infection of SARS-CoV-2 variants and syncytium formation.

The continuous emergence of SARS-CoV-2 variants prolongs COVID-19 pandemic. Although SARS-CoV-2 vaccines and therapeutics are currently available, there is still a need for development of safe and effective drugs against SARS-CoV-2 and also for preparedness for the next pandemic. Here, we discover that astersaponin I (AI), a triterpenoid saponin in Aster koraiensis inhibits SARS-CoV-2 entry pathways at the plasma membrane and within the endosomal compartments mainly by increasing cholesterol content in the plasma membrane and interfering with the fusion of SARS-CoV-2 envelope with the host cell membrane. Moreover, we find that this functional property of AI as a fusion blocker enables it to inhibit the infection with SARS-CoV-2 variants including the Alpha, Beta, Delta, and Omicron with a similar efficacy, and the formation of syncytium, a multinucleated cells driven by SARS-CoV-2 spike protein-mediated cell-to-cell fusion. Finally, we claim that the triterpene backbone as well as the attached hydrophilic sugar moieties of AI are structurally important for its inhibitory activity against the membrane fusion event. Overall, this study demonstrates that AI is a natural viral fusion inhibitor and proposes that it can be a broad-spectrum antiviral agent against current COVID-19 pandemic and future outbreaks of novel viral pathogens.

Deciphering COVID-19 host transcriptomic complexity and variations for therapeutic discovery against new variants.

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.

Need for a Standardized Translational Drug Development Platform: Lessons Learned from the Repurposing of Drugs for COVID-19.

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.

Synthesis and structure-activity relationship study of saponin-based membrane fusion inhibitors against SARS-CoV-2.

We previously discovered that triterpenoid saponin platycodin D inhibits the SARS-CoV-2 entry to the host cell. Herein, we synthesized various saponin derivatives and established a structure-activity relationship of saponin-based antiviral agents against SARS-CoV-2. We discovered that the C3-glucose, the C28-oligosaccharide moiety that consist of (→3)-ß-d-Xyl-(1 â†’ 4)-α-l-Rham-(1 â†’ 2)-ß-d-Ara-(1 â†’ ) as the last three sugar units, and the C16-hydroxyl group were critical components of saponin-based coronavirus cell entry inhibitors. These findings enabled us to develop minimal saponin-based antiviral agents that are equipotent to the originally discovered platycodin D. We found that our saponin-based antiviral agents inhibited both the endosomal and transmembrane protease serine 2-mediated cell surface viral entries. Cell fusion assay experiment revealed that our newly developed compounds inhibit the SARS-CoV-2 entry by blocking the fusion between the viral and host cell membranes. The effectiveness of the newly developed antiviral agents over various SARS-CoV-2 variants hints at the broad-spectrum antiviral efficacy of saponin-based therapeutics against future coronavirus variants.

Optimization of 2-Aminoquinazolin-4-(3H)-one Derivatives as Potent Inhibitors of SARS-CoV-2: Improved Synthesis and Pharmacokinetic Properties.

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.

Targeting the Receptor-Binding Motif of SARS-CoV-2 with D-Peptides Mimicking the ACE2 Binding Helix: Lessons for Inhibiting Omicron and Future Variants of Concern.

The COVID-19 pandemic continues to spread around the world, with several new variants emerging, particularly those of concern (VOCs). Omicron (B.1.1.529), a recent VOC with many mutations in the spike protein's receptor-binding domain (RBD), has attracted a great deal of scientific and public interest. We previously developed two D-peptide inhibitors for the infection of the original SARS-CoV-2 and its VOCs, alpha and beta, in vitro. Here, we demonstrated that Covid3 and Covid_extended_1 maintained their high-affinity binding (29.4-31.3 nM) to the omicron RBD. Both D-peptides blocked the omicron variant in vitro infection with IC50s of 3.13 and 5.56 µM, respectively. We predicted that Covid3 shares a larger overlapping binding region with the ACE2 binding motif than different classes of neutralizing monoclonal antibodies. We envisioned the design of D-peptide inhibitors targeting the receptor-binding motif as the most promising approach for inhibiting current and future VOCs of SARS-CoV-2, given that the ACE2 binding interface is more limited to tolerate mutations than most of the RBD's surface.

Comparison of Waning Neutralizing Antibody Responses Against the Omicron Variant 6 Months After Natural Severe Acute Respiratory Syndrome Coronavirus 2 Infection (With or Without Subsequent Coronavirus Disease 2019 [COVID-19] Vaccination) Versus 2-Dose COVID-19 Vaccination.

Following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, subsequent ChAdOx1 nCoV-19 vaccination induced similar neutralizing antibody levels against the original strain but significantly higher levels against the Omicron variant compared to those who were not vaccinated. Prior SARS-CoV-2 infection exhibited higher neutralization antibody titers than vaccination alone for both original strains and the Omicron variant.

Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2.

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.