Correction: Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2.

[This corrects the article DOI: 10.1371/journal.pntd.0001636.].

Human ACE2 protein is a molecular switch controlling the mode of SARS-CoV-2 transmission.

BACKGROUND: Human angiotensin-converting enzyme 2 (hACE2) is the receptor mediating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. hACE2 expression is low in the lungs and is upregulated after SARS-CoV-2 infection. How such a hACE2-limited pulmonary environment supports efficient virus transmission and how dynamic hACE2 expression affects SARS-CoV-2 infection are unclear. METHODS: We generated stable cell lines with different expression levels of hACE2 to evaluate how the hACE2 expression level can affect SARS-CoV-2 transmission. RESULTS: We demonstrated that the hACE2 expression level controls the mode of SARS-CoV-2 transmission. The hACE2-limited cells have an advantage for SARS-CoV-2 shedding, which leads to cell-free transmission. By contrast, enhanced hACE2 expression facilitates the SARS-CoV-2 cell-to-cell transmission. Furthermore, this cell-to-cell transmission is likely facilitated by hACE2-containing vesicles, which accommodate numerous SARS-CoV-2 virions and transport them to neighboring cells through intercellular extensions. CONCLUSIONS: This hACE2-mediated switch between cell-free and cell-to-cell transmission routes provides SARS-CoV-2 with advantages for either viral spread or evasion of humoral immunity, thereby contributing to the COVID-19 pandemic and pathogenesis.

Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Natural fucoidans inhibit coronaviruses by targeting viral spike protein and host cell furin.

Fucoidans are a class of long chain sulfated polysaccharides and have multiple biological functions. Herein, four natural fucoidans extracted from Fucus vesiculosus, F. serratus, Laminaria japonica and Undaria pinnatifida, were tested for their HCoV-OC43 inhibition and found to demonstrate EC50 values ranging from 0.15 to 0.61 µg/mL. That from U. pinnatifida exhibited the most potent anti-HCoV-OC43 activity with an EC50 value of 0.15 ± 0.02 µg/mL, a potency largely independent of its sulfate content. Comparison of the gene expression profiles of fucoidan-treated and untreated cells infected with HCoV-OC43 revealed that fucoidan treatment effectively diminished HCoV-OC43 gene expressions associated with induced chemokines, cytokines and viral activities. Further studies using a highly fucoidan-resistant HCoV-OC43 determined that fucoidan inhibited HCoV-OC43 infection via interfering with viral entry and led to the identification of the specific site on the N-terminal region of spike protein, that located adjacent to the host cell receptor binding domain, targeted by the virus. Furthermore, in a SARS-CoV-2 pseudovirus neutralization assay, fucoidan also blocked SARS-CoV-2 entry. In vitro and in vivo, fucoidan decreased SARS-CoV-2 viral loads and inhibited viral infection in Calu-3 or Vero E6 cells and SARS-CoV-2 infected hamsters, respectively. Fucoidan was also found to inhibit furin activity, and reported furin inhibitors were found to inhibit viral infection by wild type HCoV-OC43 or SARS-CoV-2. Accordingly, we conclude that fucoidans inhibit coronaviral infection by targeting viral spike protein and host cell furin to interfere with viral entry.

Longitudinal neutralizing antibody responses after SARS-CoV-2 infection: A convalescent cohort study in Taiwan.

BACKGROUND: Understanding the neutralizing antibody (NAb) titer against COVID-19 over time is important to provide information for vaccine implementation. The longitudinal NAb titer over one year after SARS-CoV-2 infection is still unclear. The purposes of this study are to evaluate the duration of the neutralizing NAb titers in COVID-19 convalescents and factors associated with the titer positive duration. METHODS: A cohort study followed COVID-19 individuals diagnosed between 2020 and 2021 May 15th from the COVID-19 database from the Taiwan Centers for Disease Control. We analyzed NAb titers from convalescent SARS-CoV-2 individuals. We used generalized estimating equations (GEE) and a Cox regression model to summarize the factors associated with NAb titers against COVID-19 decaying in the vaccine-free population. RESULTS: A total of 203 convalescent subjects with 297 analytic samples were followed for a period of up to 588 days. Our study suggests that convalescent COVID-19 in individuals after more than a year and four months pertains to only 25% of positive titers. The GEE model indicates that longer follow-up duration was associated with a significantly lower NAb titer. The Cox regression model indicated the disease severity with advanced condition was associated with maintaining NAb titers (adjusted hazard ratio: 2.01, 95% CI: 1.11-3.63) and that smoking was also associated with higher risk of negative NAb titers (adjusted hazard ratio: 0.55, 95% CI: 0.33-0.92). CONCLUSIONS: Neutralizing antibody titers diminished after more than a year. The antibody titer response against SARS-CoV-2 in naturally convalescent individuals provides a reference for vaccinations.

Correction: Lee et al. The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b. Pharmaceutics 2022, 14, 1511.

In the original publication [...].

Upregulation of PD-L1 by SARS-CoV-2 promotes immune evasion.

Patients with severe COVID-19 often suffer from lymphopenia, which is linked to T-cell sequestration, cytokine storm, and mortality. However, it remains largely unknown how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS-CoV-2-infected cells. We adopted a reverse time-order gene coexpression network approach to analyze time-series RNA-sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS-CoV-2-activated nuclear factor-κB (NF-κB) and interferon regulatory factor 1 (IRF1) pathways contributing to viral infection and COVID-19 severity through epigenetic analysis of H3K4me3 chromatin immunoprecipitation sequencing. Cross-referencing our transcriptomic and epigenomic data sets revealed that coupling NF-κB and IRF1 pathways mediate programmed death ligand-1 (PD-L1) immunosuppressive programs. Interestingly, we observed higher PD-L1 expression in Omicron-infected cells than SARS-CoV-2 infected cells. Blocking PD-L1 at an early stage of virally-infected AAV-hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS-CoV-2-mediated NF-κB and IRF1-PD-L1 axis may represent an alternative strategy to reduce COVID-19 severity.

A receptor-binding domain-based nanoparticle vaccine elicits durable neutralizing antibody responses against SARS-CoV-2 and variants of concern.

Numerous vaccines have been developed to address the current COVID-19 pandemic, but safety, cross-neutralizing efficacy, and long-term protectivity of currently approved vaccines are still important issues. In this study, we developed a subunit vaccine, ASD254, by using a nanoparticle vaccine platform to encapsulate the SARS-CoV-2 spike receptor-binding domain (RBD) protein. As compared with the aluminum-adjuvant RBD vaccine, ASD254 induced higher titers of RBD-specific antibodies and generated 10- to 30-fold more neutralizing antibodies. Mice vaccinated with ASD254 showed protective immune responses against SARS-CoV-2 challenge, with undetectable infectious viral loads and reduced typical lesions in lung. Besides, neutralizing antibodies in vaccinated mice lasted for at least one year and were effective against various SARS-CoV-2 variants of concern, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, particle size, polydispersity index, and zeta-potential of ASD254 remained stable after 8-month storage at 4°C. Thus, ASD254 is a promising nanoparticle vaccine with good immunogenicity and stability to be developed as an effective vaccine option in controlling upcoming waves of COVID-19.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b.

Ciclesonide is an inhaled corticosteroid used to treat asthma and has been repurposed as a treatment for mildly ill COVID-19 patients, but its precise mechanism of action is unclear. Herein, we report that ciclesonide blocks the coronavirus-induced production of the cytokines IL-6, IL-8, and MCP-1 by increasing IκBα protein levels and significantly decreasing p65 nuclear translocation. Furthermore, we found that the combination of ciclesonide and dbq33b, a potent tylophorine-based coronavirus inhibitor that affects coronavirus-induced NF-κB activation a little, additively and synergistically decreased coronavirus-induced IL-6, IL-8, and MCP-1 cytokine levels, and synergistically inhibited the replication of both HCoV-OC43 and SARS-CoV-2. Collectively, the combination of ciclesonide and dbq33b merits consideration as a treatment for COVID-19 patients who may otherwise be overwhelmed by high viral loads and an NF-κB-mediated cytokine storm.

A multitope SARS-CoV-2 vaccine provides long-lasting B cell and T cell immunity against Delta and Omicron variants.

BackgroundThe Delta and Omicron variants of SARS-CoV-2 are currently responsible for breakthrough infections due to waning immunity. We report phase I/II trial results of UB-612, a multitope subunit vaccine containing S1-RBD-sFc protein and rationally designed promiscuous peptides representing sarbecovirus conserved helper T cell and cytotoxic T lymphocyte epitopes on the nucleocapsid (N), membrane (M), and spike (S2) proteins.MethodWe conducted a phase I primary 2-dose (28 days apart) trial of 10, 30, or 100 µg UB-612 in 60 healthy young adults 20 to 55 years old, and 50 of them were boosted with 100 µg of UB-612 approximately 7 to 9 months after the second dose. A separate placebo-controlled and randomized phase II study was conducted with 2 doses of 100 µg of UB-612 (n = 3,875, 18-85 years old). We evaluated interim safety and immunogenicity of phase I until 14 days after the third (booster) dose and of phase II until 28 days after the second dose.ResultsNo vaccine-related serious adverse events were recorded. The most common solicited adverse events were injection site pain and fatigue, mostly mild and transient. In both trials, UB-612 elicited respective neutralizing antibody titers similar to a panel of human convalescent sera. The most striking findings were long-lasting virus-neutralizing antibodies and broad T cell immunity against SARS-CoV-2 variants of concern (VoCs), including Delta and Omicron, and a strong booster-recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron VoCs.ConclusionUB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines.Trial RegistrationClinicalTrials.gov: NCT04545749, NCT04773067, and NCT04967742.FundingUBI Asia, Vaxxinity Inc., and Taiwan Centers for Disease Control, Ministry of Health and Welfare.

Humoral Immunogenicity and Reactogenicity of the Standard ChAdOx1 nCoV-19 Vaccination in Taiwan.

BACKGROUND: The ChAdOx1 nCoV-19 vaccine has been widely administered against SARS-CoV-2 infection; however, data regarding its immunogenicity, reactogenicity, and potential differences in responses among Asian populations remain scarce. METHODS: 270 participants without prior COVID-19 were enrolled to receive ChAdOx1 nCoV-19 vaccination with a prime-boost interval of 8-9 weeks. Their specific SARS-CoV-2 antibodies, neutralizing antibody titers (NT50), platelet counts, and D-dimer levels were analyzed before and after vaccination. RESULTS: The seroconversion rates of anti-RBD and anti-spike IgG at day 28 after a boost vaccination (BD28) were 100% and 95.19%, respectively. Anti-RBD and anti-spike IgG levels were highly correlated (r = 0.7891), which were 172.9 ± 170.4 and 179.3 ± 76.88 BAU/mL at BD28, respectively. The geometric mean concentrations (GMCs) of NT50 for all participants increased to 132.9 IU/mL (95% CI 120.0-147.1) at BD28 and were highly correlated with anti-RBD and anti-spike IgG levels (r = 0.8248 and 0.7474, respectively). Body weight index was statistically significantly associated with anti-RBD IgG levels (p = 0.035), while female recipients had higher anti-spike IgG levels (p = 0.038). The GMCs of NT50 declined with age (p = 0.0163) and were significantly different across age groups (159.7 IU/mL for 20-29 years, 99.4 IU/mL for ≥50 years, p = 0.0026). Injection-site pain, fever, and fatigue were the major reactogenicity, which were more pronounced after prime vaccination and in younger participants (<50 years). Platelet counts decreased and D-dimer levels increased after vaccination but were not clinically relevant. No serious adverse events or deaths were observed. CONCLUSION: The vaccine is well-tolerated and elicited robust humoral immunity against SARS-CoV-2 after standard prime-boost vaccination in Taiwanese recipients.

NK cell receptor and ligand composition influences the clearance of SARS-CoV-2.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling.

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.

Safety and immunogenicity of CpG 1018 and aluminium hydroxide-adjuvanted SARS-CoV-2 S-2P protein vaccine MVC-COV1901: interim results of a large-scale, double-blind, randomised, placebo-controlled phase 2 trial in Taiwan.

BACKGROUND: MVC-COV1901, a recombinant protein vaccine containing pre-fusion-stabilised spike protein S-2P adjuvanted with CpG 1018 and aluminium hydroxide, has been shown to be well tolerated with a good safety profile in healthy adults aged 20-49 years in a phase 1 trial, and provided a good cellular and humoral immune responses. We present the interim safety, tolerability, and immunogenicity results of a phase 2 clinical trial of the MVC-COV1901 vaccine in Taiwan. METHODS: This is a large-scale, double-blind, randomised, placebo-controlled phase 2 trial done at ten medical centres and one regional hospital in Taiwan. Individuals aged 20 years or older who were generally healthy or had stable pre-existing medical conditions were eligible for enrolment. Exclusion criteria included (but were not limited to) travel overseas within 14 days of screening, intention to travel overseas within 6 months of the screening visit, and the absence of prespecified medical conditions, including immunosuppressive illness, a history of autoimmune disease, malignancy with risk to recur, a bleeding disorder, uncontrolled HIV infection, uncontrolled hepatitis B and C virus infections, SARS-CoV-1 or SARS-CoV-2 infections, an allergy to any vaccine, or a serious medical condition that could interfere with the study. Study participants were randomly assigned (6:1) to receive two doses of either MVC-COV1901 or placebo, administered via intramuscular injection on day 1 and day 29. MVC-COV1901 contained 15 µg of S-2P protein adjuvanted with 750 µg CpG 1018 and 375 µg aluminium hydroxide in a 0·5 mL aqueous solution, and the placebo contained the same volume of saline. Randomisation was done centrally by use of an interactive web response system, stratified by age (≥20 to <65 years and ≥65 years). Participants and investigators were masked to group assignment. The primary outcomes were to evaluate the safety, tolerability, and immunogenicity of MVC-COV1901 from day 1 (the day of the first dose) to day 57 (28 days after the second dose). Safety was assessed in all participants who received at least one dose. Immunogenicity was assessed by measuring geometric mean titres (GMTs) and seroconversion rates of neutralising antibody and antigen-specific IgG in the per-protocol population. This study is registered with ClinicalTrials.gov, NCT04695652. FINDINGS: Of 4173 individuals screened between Dec 30, 2020, and April 2, 2021, 3854 were enrolled and randomly assigned: 3304 to the MVC-COV1901 group and 550 to the placebo group. A total of 3844 participants (3295 in the MVC-COV1901 group and 549 in the placebo group) were included in the safety analysis set, and 1053 participants (903 and 150) had received both doses and were included in the per-protocol immunogenicity analysis set. From the start of this phase 2 trial to the time of interim analysis, no vaccine-related serious adverse events were recorded. The most common solicited adverse events in all study participants were pain at the injection site (2346 [71·2%] of 3295 in the MVC-COV1901 group and 128 [23·3%] of 549 in the placebo group), and malaise or fatigue (1186 [36·0%] and 163 [29·7%]). Fever was rarely reported (23 [0·7%] and two [0·4%]). At 28 days after the second dose of MVC-COV1901, the wild-type SARS-CoV-2 neutralising antibody GMT was 662·3 (95% CI 628·7-697·8; 408·5 IU/mL), the GMT ratio (geometric mean fold increase in titres at day 57 vs baseline) was 163·2 (155·0-171·9), and the seroconversion rate was 99·8% (95% CI 99·2-100·0). INTERPRETATION: MVC-COV1901 has a good safety profile and elicits promising immunogenicity responses. These data support MVC-COV1901 to enter phase 3 efficacy trials. FUNDING: Medigen Vaccine Biologics and Taiwan Centres for Disease Control, Ministry of Health and Welfare.

Structure-guided antibody cocktail for prevention and treatment of COVID-19.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.

Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters.

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

Remdesivir and Cyclosporine Synergistically Inhibit the Human Coronaviruses OC43 and SARS-CoV-2.

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC50 values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC50 values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC50 values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

Small Structural Proteins E and M Render the SARS-CoV-2 Pseudovirus More Infectious and Reveal the Phenotype of Natural Viral Variants.

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.