Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo.

Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 â€‹ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 â€‹ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 â€‹mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 â€‹mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 â€‹mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

Novel Tetrahydroisoquinoline-Based Heterocyclic Compounds Efficiently Inhibit SARS-CoV-2 Infection In Vitro.

The ongoing COVID-19 pandemic has caused over six million deaths and huge economic burdens worldwide. Antivirals against its causative agent, SARS-CoV-2, are in urgent demand. Previously, we reported that heterocylic compounds, i.e., chloroquine (CQ) and hydroxychloroquine (HCQ), are potent in inhibiting SARS-CoV-2 replication in vitro. In this study, we discussed the syntheses of two novel heterocylic compounds: tert-butyl rel-4-(((3R,4S)-3-(1H-indol-3-yl)-1-oxo-2-propyl-1,2,3,4-tetrahydroisoquinolin-4-yl)methyl)piperazine-1-carboxylate (trans-1) and rel-(3R,4S)-3-(1H-indol-3-yl)-4-(piperazin-1-ylmethyl)-2-propyl-3,4-dihydroisoquinolin-1(2H)-one (trans-2), which effectively suppressed authentic SARS-CoV-2 replication in Vero E6 cells. Compound trans-1 showed higher anti-SARS-CoV-2 activity than trans-2, with a half maximal effective concentration (EC50) of 3.15 µM and a selective index (SI) exceeding 63.49, which demonstrated comparable potency to CQ or HCQ. Additional anti-SARS-CoV-2 tests on Calu-3 human lung cells showed that trans-1 efficiently inhibited viral replication (EC50 = 2.78 µM; SI: > 71.94) and performed better than CQ (EC50 = 44.90 µM; SI = 2.94). The time of an addition assay showed that the action mechanism of trans-1 differed from that of CQ, as it mainly inhibited the post-entry viral replication in both Vero E6 and Calu-3 cells. In addition, the differences between the antiviral mechanisms of these novel compounds and CQ were discussed.

Antiviral effects and tissue exposure of tetrandrine against SARS-CoV-2 infection and COVID-19.

Tetrandrine (TET) has been used to treat silicosis in China for decades. The aim of this study was to facilitate rational repurposing of TET against SARS-CoV-2 infection. In this study, we confirmed that TET exhibited antiviral potency against SARS-CoV-2 in the African green monkey kidney (Vero E6), human hepatocarcinoma (Huh7), and human lung adenocarcinoma epithelial (Calu-3) cell lines. TET functioned during the early-entry stage of SARS-CoV-2 and impeded intracellular trafficking of the virus from early endosomes to endolysosomes. An in vivo study that used adenovirus (AdV) 5-human angiotensin-converting enzyme 2 (hACE2)-transduced mice showed that although TET did not reduce pulmonary viral load, it significantly alleviated pathological damage in SARS-CoV-2-infected murine lungs. The systemic preclinical pharmacokinetics were investigated based on in vivo and in vitro models, and the route-dependent biodistribution of TET was explored. TET had a large volume of distribution, which contributed to its high tissue accumulation. Inhaled administration helped TET target the lung and reduced its exposure to other tissues, which mitigated its off-target toxicity. Based on the available human pharmacokinetic data, it appeared feasible to achieve an unbound TET 90% maximal effective concentration (EC90) in human lungs. This study provides insights into the route-dependent pulmonary biodistribution of TET associated with its efficacy.

Potent Anti-SARS-CoV-2 Efficacy of COVID-19 Hyperimmune Globulin from Vaccine-Immunized Plasma.

Coronavirus disease 2019 (COVID-19) remains a global public health threat. Hence, more effective and specific antivirals are urgently needed. Here, COVID-19 hyperimmune globulin (COVID-HIG), a passive immunotherapy, is prepared from the plasma of healthy donors vaccinated with BBIBP-CorV (Sinopharm COVID-19 vaccine). COVID-HIG shows high-affinity binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, the receptor-binding domain (RBD), the N-terminal domain of the S protein, and the nucleocapsid protein; and blocks RBD binding to human angiotensin-converting enzyme 2 (hACE2). Pseudotyped and authentic virus-based assays show that COVID-HIG displays broad-spectrum neutralization effects on a wide variety of SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), and Omicron (B.1.1.529) in vitro. However, a significant reduction in the neutralization titer is detected against Beta, Delta, and Omicron variants. Additionally, assessments of the prophylactic and treatment efficacy of COVID-HIG in an Adv5-hACE2-transduced IFNAR-/- mouse model of SARS-CoV-2 infection show significantly reduced weight loss, lung viral loads, and lung pathological injury. Moreover, COVID-HIG exhibits neutralization potency similar to that of anti-SARS-CoV-2 hyperimmune globulin from pooled convalescent plasma. Overall, the results demonstrate the potential of COVID-HIG against SARS-CoV-2 infection and provide reference for subsequent clinical trials.

Covalently Engineered Protein Minibinders with Enhanced Neutralization Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of escaping mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly challenged our efforts in fighting against the COVID-19 pandemic. A broadly neutralizing reagent against these concerning variants is thus highly desirable for the prophylactic and therapeutic treatments of SARS-CoV-2 infection. We herein report a covalent engineering strategy on protein minibinders for potent neutralization of the escaping variants such as B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) through in situ cross-linking with the spike receptor binding domain (RBD). The resulting covalent minibinder (GlueBinder) exhibited enhanced blockage of RBD-human angiotensin-converting enzyme 2 (huACE2) interaction and more potent neutralization effect against the Delta variant than its noncovalent counterpart as demonstrated on authentic virus. By leveraging the covalent chemistry against escaping mutations, our strategy may be generally applicable for restoring and enhancing the potency of neutralizing antibodies to SARS-CoV-2 and other rapidly evolving viral targets.

Establishment of Human Pluripotent Stem Cell-Derived Skin Organoids Enabled Pathophysiological Model of SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) patients with impact on skin and hair loss are reported. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected in the skin of some patients; however, the detailed pathological features of skin tissues from patients infected with SARS-CoV-2 at a molecular level are limited. Especially, the ability of SARS-CoV-2 to infect skin cells and impact their function is not well understood. A proteome map of COVID-19 skin is established here and the susceptibility of human-induced pluripotent stem cell (hiPSC)-derived skin organoids with hair follicles and nervous system is investigated, to SARS-CoV-2 infection. It is shown that KRT17+ hair follicles can be infected by SARS-CoV-2 and are associated with the impaired development of hair follicles and epidermis. Different types of nervous system cells are also found to be infected, which can lead to neuron death. Findings from the present work provide evidence for the association between COVID-19 and hair loss. hiPSC-derived skin organoids are also presented as an experimental model which can be used to investigate the susceptibility of skin cells to SARS-CoV-2 infection and can help identify various pathological mechanisms and drug screening strategies.

Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11-RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq.

B cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11chigh CD95high) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

Comparative Antiviral Efficacy of Viral Protease Inhibitors against the Novel SARS-CoV-2 In Vitro.

The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is of urgent demand to cure the huge number of patients. Virus-encoded proteases are considered potential drug targets. The human immunodeficiency virus protease inhibitors (lopinavir/ritonavir) has been recommended in the global Solidarity Trial in March launched by World Health Organization. However, there is currently no experimental evidence to support or against its clinical use. We evaluated the antiviral efficacy of lopinavir/ritonavir along with other two viral protease inhibitors in vitro, and discussed the possible inhibitory mechanism in silico. The in vitro to in vivo extrapolation was carried out to assess whether lopinavir/ritonavir could be effective in clinical. Among the four tested compounds, lopinavir showed the best inhibitory effect against the novel coronavirus infection. However, further in vitro to in vivo extrapolation of pharmacokinetics suggested that lopinavir/ritonavir could not reach effective concentration under standard dosing regimen [marketed as Kaletra®, contained lopinavir/ritonavir (200 mg/50 mg) tablets, recommended dosage is 400 mg/10 mg (2 tablets) twice daily]. This research concluded that lopinavir/ritonavir should be stopped for clinical use due to the huge gap between in vitro IC50 and free plasma concentration. Nevertheless, the structure-activity relationship analysis of the four inhibitors provided further information for de novel design of future viral protease inhibitors of SARS-CoV-2.

Anti-SARS-CoV-2 Potential of Artemisinins In Vitro.

The discovery of novel drug candidates with anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) potential is critical for the control of the global COVID-19 pandemic. Artemisinin, an old antimalarial drug derived from Chinese herbs, has saved millions of lives. Artemisinins are a cluster of artemisinin-related drugs developed for the treatment of malaria and have been reported to have multiple pharmacological activities, including anticancer, antiviral, and immune modulation. Considering the reported broad-spectrum antiviral potential of artemisinins, researchers are interested in whether they could be used to combat COVID-19. We systematically evaluated the anti-SARS-CoV-2 activities of nine artemisinin-related compounds in vitro and carried out a time-of-drug-addition assay to explore their antiviral mode of action. Finally, a pharmacokinetic prediction model was established to predict the therapeutic potential of selected compounds against COVID-19. Arteannuin B showed the highest anti-SARS-CoV-2 potential with an EC50 of 10.28 ± 1.12 µM. Artesunate and dihydroartemisinin showed similar EC50 values of 12.98 ± 5.30 µM and 13.31 ± 1.24 µM, respectively, which could be clinically achieved in plasma after intravenous administration. Interestingly, although an EC50 of 23.17 ± 3.22 µM was not prominent among the tested compounds, lumefantrine showed therapeutic promise due to high plasma and lung drug concentrations after multiple dosing. Further mode of action analysis revealed that arteannuin B and lumefantrine acted at the post-entry step of SARS-CoV-2 infection. This research highlights the anti-SARS-CoV-2 potential of artemisinins and provides leading candidates for anti-SARS-CoV-2 drug research and development.