A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storm is closely associated with coronavirus disease 2019 (COVID-19) severity and lethality. However, drugs that are effective against inflammation to treat lethal COVID-19 are still urgently needed. Here, we constructed a SARS-CoV-2 spike protein-specific CAR, and human T cells infected with this CAR (SARS-CoV-2-S CAR-T) and stimulated with spike protein mimicked the T-cell responses seen in COVID-19 patients, causing cytokine storm and displaying a distinct memory, exhausted, and regulatory T-cell phenotype. THP1 remarkably augmented cytokine release in SARS-CoV-2-S CAR-T cells when they were in coculture. Based on this "two-cell" (CAR-T and THP1 cells) model, we screened an FDA-approved drug library and found that felodipine, fasudil, imatinib, and caspofungin were effective in suppressing the release of cytokines, which was likely due to their ability to suppress the NF-κB pathway in vitro. Felodipine, fasudil, imatinib, and caspofungin were further demonstrated, although to different extents, to attenuate lethal inflammation, ameliorate severe pneumonia, and prevent mortality in a SARS-CoV-2-infected Syrian hamster model, which were also linked to their suppressive role in inflammation. In summary, we established a SARS-CoV-2-specific CAR-T-cell model that can be utilized as a tool for anti-inflammatory drug screening in a fast and high-throughput manner. The drugs identified herein have great potential for early treatment to prevent COVID-19 patients from cytokine storm-induced lethality in the clinic because they are safe, inexpensive, and easily accessible for immediate use in most countries.

Erratum: A cell-penetrating whole molecule antibody targeting intracellular HBx suppresses hepatitis B virus via TRIM21-dependent pathway: Erratum.

[This corrects the article DOI: 10.7150/thno.20047.].

Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells.

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Several FDA-Approved Drugs Effectively Inhibit SARS-CoV-2 Infection in vitro.

To identify drugs that are potentially used for the treatment of COVID-19, the potency of 1403 FDA-approved drugs were evaluated using a robust pseudovirus assay and the candidates were further confirmed by authentic SARS-CoV-2 assay. Four compounds, Clomiphene (citrate), Vortioxetine, Vortioxetine (hydrobromide) and Asenapine (hydrochloride), showed potent inhibitory effects in both pseudovirus and authentic virus assay. The combination of Clomiphene (citrate), Vortioxetine and Asenapine (hydrochloride) is much more potent than used alone, with IC50 of 0.34 µM.

A unique B cell epitope-based particulate vaccine shows effective suppression of hepatitis B surface antigen in mice.

OBJECTIVE: This study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models. METHODS: A series of peptides and carrier proteins were evaluated in HBV-tolerant mice to obtain an optimised therapeutic molecule. The immunogenicity, therapeutic efficacy and mechanism of the candidate were investigated systematically. RESULTS: Among the HBsAg-aa119-125-containing peptides evaluated in this study, HBsAg-aa113-135 (SEQ13) exhibited the most striking therapeutic effects. A novel immunoenhanced virus-like particle carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg) was created and used to display SEQ13, forming candidate molecule CR-T3-SEQ13. Multiple copies of SEQ13 displayed on the surface of this particulate antigen promote the induction of a potent anti-HBs antibody response in mice, rabbits and cynomolgus monkeys. Sera and purified polyclonal IgG from the immunised animals neutralised HBV infection in vitro and mediated efficient HBV/hepatitis B virus surface antigen (HBsAg) clearance in the mice. CR-T3-SEQ13-based vaccination induced long-term suppression of HBsAg and HBV DNA in HBV transgenic mice and eradicated the virus completely in hydrodynamic-based HBV carrier mice. The suppressive effects on HBsAg were strongly correlated with the anti-HBs level after vaccination, suggesting that the main mechanism of CR-T3-SEQ13 vaccination therapy was the induction of a SEQ13-specific antibody response that mediated HBV/HBsAg clearance. CONCLUSIONS: The novel particulate protein CR-T3-SEQ13 suppressed HBsAg effectively through induction of a humoural immune response in HBV-tolerant mice. This B cell epitope-based therapeutic vaccine may provide a novel immunotherapeutic agent against chronic HBV infection in humans.

Structural and functional analyses of hepatitis B virus X protein BH3-like domain and Bcl-xL interaction.

Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.

Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus.

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF50) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R2 = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF50 ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R2 = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations: HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; Anti-HBc: Hepatitis B core antibody; qAnti-HBs: quantitative hepatitis B surface antibody; qAnti-HBc: quantitative hepatitis B core antibody; qHBeAg: quantitative hepatitis B e antigen; NAT: neutralization activity; HBIG: hepatitis B immune globulin; NTCP: Na+-taurocholate cotransporting polypeptide; IRES: internal ribosome entry site; ccHBV: cell culture derived hepatitis B virus; GE/cell: genome equivalent per cell; MOI: multiplicity of infection; Dpi: day post infection; HepG2-TetOn: a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC: receiver operating characteristic curve; AUROC: area under receiver operating characteristic curve; LLOQ: the lower limits of quantification; MDF50: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration.

A cell-penetrating whole molecule antibody targeting intracellular HBx suppresses hepatitis B virus via TRIM21-dependent pathway.

Rationale: Monoclonal antibodies (mAbs) mostly targeting extracellular or cell surface molecules have been widely used in the treatment of various diseases. However, mAbs cannot pass through the cell membrane as efficiently as small compounds, thus limiting their use against intracellular targets. Methods to shuttle antibodies into living cells may largely expand research and application in areas based on mAbs. Hepatitis B virus X protein (HBx) is an important intracellular multi-functional viral protein in the life cycle of hepatitis B virus (HBV). HBx plays essential roles in virus infection and replication and is strongly associated with HBV-related carcinogenesis. Methods: In this study, we developed a cell-penetrating whole molecule antibody targeting HBx (9D11-Tat) by the fusion of a cell penetrating peptide (CPP) on the C-terminus of the heavy chain of a potent mAb specific to HBx (9D11). The anti-HBV effect and mechanism of 9D11-Tat were investigated in cell and mouse models mimicking chronic HBV infection. Results: Our results demonstrated that the recombinant 9D11-Tat antibody could efficiently internalize into living cells and significantly suppress viral transcription, replication, and protein production both in vitro and in vivo. Further analyses suggested the internalized 9D11-Tat antibody could greatly reduce intracellular HBx via Fc binding receptor TRIM21-mediated protein degradation. This process simultaneously stimulated the activations of NF-κB, AP-1, and IFN-ß, which promoted an antiviral state of the host cell. Conclusion: In summary, our study offers a new approach to target intracellular pathogenesis-related protein by engineered cell-penetrating mAb expanding their potential for therapeutic applications. Moreover, the 9D11-Tat antibody may provide a novel therapeutic agent against human chronic HBV infection.