An ancestral vaccine induces anti-Omicron antibodies by hypermutation (preprint)

The immune escape of Omicron-sublineage 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 collected blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyzed their B-cell receptor (BCR) repertoires at six time points in total. Five Omicron variant-neutralizing BCR heavy chain (HC) clonotypes were tracked chronologically in identical vaccinees before and after the third dose. Before the third injection, all five BCR HC clonotypes showed reactivity to the ancestral receptor-binding domain (RBD), and two BCR HC clonotypes developed similar reactivity to the Omicron RBD. The other three BCR HC clonotypes showed minimal or reduced reactivity to the Omicron RBD before the third dose, which induced further somatic hypermutation (SHM) and dramatically increased their affinity for the Omicron RBD. In the public IGHV3-53/3-66 and IGHJ6 clonotypes found in 19 vaccinees (46%), concomitant reactivity to the ancestral and Omicron RBDs resulted from SHMs such as Y58F and F27V and diversification of HCDR3 by SHM. Our findings suggest that SHM occurrence in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against the immune escape of virus variants.

Lancemaside A from Codonopsis lanceolata is a broad-spectrum antiviral agent against SARS-CoV-2 and variants of concern (preprint)

Background: and Purpose 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. Here, we evaluated the potential inhibitory activity of CL extracts and their active compounds on SARS-CoV-2. Experimental Approach Pseudotyped SARS-CoV-2 entry assay and dose-response curve analysis with authentic SARS-CoV-2 and recombinant SARS-CoV-2 reporter virus expressing the nanoluciferase were carried out to investigate the effects of compounds against SARS-CoV-2 entry into host cells. Filipin cholesterol staining, SARS-CoV-2 Spike (S)-ACE2 binding assay, and S-mediated cell fusion assay using time-lapse imaging, flow cytometry, and split-GFP fusion were conducted to understand the inhibitory mechanisms. Key Results Lancemaside A (LA), a triterpenoid saponin isolated from CL, impeded the endosomal entry pathway of SARS-CoV-2 and its variants including Alpha, Beta, Delta, and Omicron with similar IC50 values of 2.23 ~ 3.37 μM as well as the TMPRSS2-mediated viral entry pathway with IC50 value of 3.92 μM. LA was also able to prevent the formation of S-induced multinucleated syncytia. Mechanically, LA altered the distribution of host cell membrane cholesterol and blocked the membrane fusion between SARS-CoV-2 and host cells. Conclusion and implications LA can be a broad-spectrum antiviral drug not only against SARS-CoV-2 but also against other novel enveloped viral pathogens that might arise in the future by targeting viral envelope fusion with the host cell membrane. Keywords SARS-CoV-2, Omicron, COVID-19, Lancemaside A, triterpenoid saponin, membrane fusion

Diagnostic Performance and Clinical Feasibility of a Novel One-Step RT-qPCR for Detecting Multiple Severe Acute Respiratory Syndrome Coronavirus (preprint)

The pandemic coronavirus disease 2019 (COVID-19) is characterized as an acute respiratory infection in the majority of cases and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other coronaviruses (CoVs) can infect humans, although the majority only cause mild respiratory symptoms. As early diagnosis of SARS-CoV-2 is critical to prevent further transmission events and to improve clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other CoVs in respiratory samples. Therefore, we developed and evaluated a novel quantitative reverse transcription PCR (RT-qPCR) assay, targeting the spike (S) and membrane (M) genes, to enable the rapid identification of SARS-CoV-2 including several new circulating variants, and other emerging pan-SARS-like CoVs. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays confirmed a limit of detection of 1 × 100 TCID50/ml and no cross-reaction with human coronaviruses or other respiratory viruses. We also validated our method using human clinical samples from COVID-19 patients and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic coronaviruses, including SARS-CoV-2, and may be useful for the identification of other pan-SARS-like CoVs of zoonotic origin.

TMPRSS2 and RNA-dependent RNA polymerase are effective targets of therapeutic intervention for treatment of COVID-19 caused by SARS-CoV-2 variants (B.1.1.7 and B.1.351) (preprint)

SARS-CoV-2 is a causative agent of 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 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. The viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development, thus the inhibitory effects of 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 UK 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.

Published Anti-SARS-CoV-2 In Vitro Hits Share Common Mechanisms of Action that Synergize with Antivirals (preprint)

The global efforts in the past few months have led to the discovery of around 200 drug repurposing candidates for COVID-19. Although most of them only exhibited moderate anti-SARS-CoV-2 activity, gaining more insights into their mechanisms of action could facilitate a better understanding of infection and the development of therapeutics. Leveraging large-scale drug-induced gene expression profiles, we found 36% of the active compounds regulate genes related to cholesterol homeostasis and microtubule cytoskeleton organization. The expression change upon drug treatment was further experimentally confirmed in human lung primary small airway. Following bioinformatics analysis on COVID-19 patient data revealed that these genes are associated with COVID-19 patient severity. The expression level of these genes also has predicted power on anti-SARS-CoV-2 efficacy in vitro, which led to the discovery of monensin as an inhibitor of SARS-CoV-2 replication in Vero-E6 cells. The final survey of recent drug-combination data indicated that drugs co-targeting cholesterol homeostasis and microtubule cytoskeleton organization processes more likely present a synergistic effect with antivirals. Therefore, potential therapeutics should be centered around combinations of targeting these processes and viral proteins.

Platycodin D prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection in vitro by hindering membrane fusion (preprint)

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to the global public health. Herbal medicines and their derived natural products have drawn much attention to treat COVID-19, but there has been no natural product showing inhibitory activity against SARS-CoV-2 infection with detailed mechanism. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection-routes via lysosome- and transmembrane protease, serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host-entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by a pharmacological inhibition or gene-silencing of NPC1, which is mutated in Niemann-Pick type C (NPC) patients displaying disrupted membrane cholesterol. Finally, readily available local foods or herbal medicines containing PG root show the similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that a brief disruption of membrane cholesterol can be a novel therapeutic approach against SARS-CoV-2 infection.

Sex differences in viral entry protein expression and host transcript responses to SARS-CoV-2 (preprint)

Epidemiological studies suggest that men exhibit a higher mortality rate to COVID-19 than women, yet the underlying biology is largely unknown. Here, we seek to delineate sex differences in the gene expression of viral entry proteins ACE2 and TMPRSS2, and host transcriptional responses to SARS-CoV-2 through large-scale analysis of genomic and clinical data.  We first compiled 220,000 human gene expression profiles from three databases and completed the meta-information through machine learning and manual annotation. Large scale analysis of these profiles indicated that male samples show higher expression levels of ACE2 and TMPRSS2 than female samples, especially in the older group (>60 years) and in the kidney. Subsequent analysis of 6,031 COVID-19 patients at Mount Sinai Health System revealed that men have significantly higher creatinine levels, an indicator of impaired kidney function. Further analysis of 782 COVID-19 patient gene expression profiles taken from upper airway and blood suggested men and women present distinct expression changes. Computational deconvolution analysis of these profiles revealed male COVID-19 patients have enriched kidney-specific mesangial cells in blood compared to healthy patients. Together, this study suggests biological differences in the kidney between sexes may contribute to sex disparity in COVID-19.

Robust and sensitive detection of SARS-CoV-2 using PCR based methods (preprint)

The World Health Organization (WHO) has declared the Coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of viral RNA by RT-PCR significantly varied according to the sequence of primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be significantly higher than RT-qPCR. These findings suggest that ddPCR could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.

Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells: Nafamostat is the most potent antiviral drug candidate (preprint)

Drug repositioning represents an effective way to control the current COVID-19 pandemic. Previously, we identified 24 FDA-approved drugs which exhibited substantial antiviral effect against SARS-CoV-2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. Comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC50 = 0.0022{micro}M).

Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs (preprint)

COVID-19 is an emerging infectious disease and was recently declared as a pandemic by WHO. Currently, there is no vaccine or therapeutic available for this disease. Drug repositioning represents the only feasible option to address this global challenge and a panel of 48 FDA-approved drugs that have been pre-selected by an assay of SARS-CoV was screened to identify potential antiviral drug candidates against SARS-CoV-2 infection. We found a total of 24 drugs which exhibited antiviral efficacy (0.1 M < IC50 < 10 M) against SARS-CoV-2. In particular, two FDA-approved drugs - niclosamide and ciclesonide - were notable in some respects. These drugs will be tested in an appropriate animal model for their antiviral activities. In near future, these already FDA-approved drugs could be further developed following clinical trials in order to provide additional therapeutic options for patients with COVID-19.