In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. ...Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. There are no approved ...vaccines or therapeutics for treating COVID-19. Here we report a humanized monoclonal antibody, H014, that efficiently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 at nanomolar concentrations by engaging the spike (S) receptor binding domain (RBD). H014 administration reduced SARS-CoV-2 titers in infected lungs and prevented pulmonary pathology in a human angiotensin-converting enzyme 2 mouse model. Cryo-electron microscopy characterization of the SARS-CoV-2 S trimer in complex with the H014 Fab fragment unveiled a previously uncharacterized conformational epitope, which was only accessible when the RBD was in an open conformation. Biochemical, cellular, virological, and structural studies demonstrated that H014 prevents attachment of SARS-CoV-2 to its host cell receptors. Epitope analysis of available neutralizing antibodies against SARS-CoV and SARS-CoV-2 uncovered broad cross-protective epitopes. Our results highlight a key role for antibody-based therapeutic interventions in the treatment of COVID-19.
The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical ...isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.
There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory ...symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.
High-efficiency rock-breaking is a problem that has long been studied in the oil- and gas-drilling industry. The successful use of ultrasonic technology in related fields has prompted us to study how ...to introduce ultrasonic technology into rock-breaking in oil and gas drilling. This paper introduces and discusses the successful cases of ultrasonic breaking technology in related fields, summarizes the three basic forms of ultrasonic action on rocks, namely, resonance, impact and cavitation, expounds the factors and laws that affect ultrasonic-assisted rock-breaking, and summarizes the research results reported in recent years. It is believed that, at present, the application of ultrasonic-assisted rock-breaking technology in the oil- and gas-drilling industry still faces some problems and challenges: first, the downhole high-temperature and high-pressure conditions will affect the effect of ultrasonic-assisted rock-breaking, and the related mechanisms and research are not clear; second, the impact of circulating media on ultrasonic-assisted rock-breaking is not clear; third, the problem of ultrasonic propagation and utilization in the downhole has not been well-solved; fourth, the stability of drilling tools and circulating media caused by high-frequency characteristics has not been well-solved. Therefore, it is suggested to increase research on the mechanism of ultrasonic-assisted rock-breaking with oil- and gas-drilling characteristics and the transmission and utilization of downhole ultrasonic energy in the future, and increase the development of supporting products to support the application of this technology in the oil and gas industry.
Viral subunit vaccines often contain immunodominant non-neutralizing epitopes that divert host immune responses. These epitopes should be eliminated in vaccine design, but there is no reliable method ...for evaluating an epitope's capacity to elicit neutralizing immune responses. Here we introduce a new concept 'neutralizing immunogenicity index' (NII) to evaluate an epitope's neutralizing immunogenicity. To determine the NII, we mask the epitope with a glycan probe and then assess the epitope's contribution to the vaccine's overall neutralizing immunogenicity. As proof-of-concept, we measure the NII for different epitopes on an immunogen comprised of the receptor-binding domain from MERS coronavirus (MERS-CoV). Further, we design a variant form of this vaccine by masking an epitope that has a negative NII score. This engineered vaccine demonstrates significantly enhanced efficacy in protecting transgenic mice from lethal MERS-CoV challenge. Our study may guide the rational design of highly effective subunit vaccines to combat MERS-CoV and other life-threatening viruses.
The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its ...spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life
; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.
Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the
half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.
Coiled tubing (CT) is widely used in drilling, workover, completion, fracturing and stimulation in the field of oil and gas exploration and development. During CT operation, the tubing will present a ...gas–liquid two-phase flow state. The prediction of frictional pressure drop for fluid in the tube is an important part of hydraulic design, and its accuracy directly affects the success of the CT technique. In this study, we analyzed the effects of the gas void fraction, curvature ratio and fluid inlet velocity on frictional pressure drop in CT, numerically. Experimental data verified simulated results. Flow friction sensitivity analysis shows the frictional pressure drop reaches its peak at a gas void fraction of 0.8. The frictional pressure gradient increases with the increase in curvature ratio. As the strength of secondary flow increases with the increase in inlet velocity, the increased trend of gas–liquid two-phase flow friction is aggravated. The correlation of friction factor for gas–liquid two-phase flow in coiled tubing is developed by regression analysis of simulation results. The research results can support high quality CT hydraulics design, through which the success of CT operations can be guaranteed.
Tuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)'s protective efficacy varies significantly ...among populations and regions. It is very urgent to develop more effective vaccines.
In this study, eleven candidate proteins of
were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in
cells. The vaccine's protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT.
Six immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with
, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ
T lymphocytes, CD3
IFN-γ
T lymphocytes, and the MP3RT-specific IgG antibody.
Taken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3
IFN-γ
T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.
The purpose of the current study was to define the role of MAX interactor 1 (Mxi1) in the pathogenesis of lung cancer and its underlying molecular mechanism. Bioinformatics analysis was performed to ...identify important regulatory pathway related to lung cancer. Dual luciferase reporter and ChIP assays were adopted to validate the interaction among Mxi1, miR-300 and KLF9. Loss- and gain-of-function studies were conducted to determine the roles of Mxi1, miR-300, and KLF9 in cell proliferation, migration, and invasion in vitro and their effects on myeloid-derived suppressor cell (MDSC) recruitment in vivo. Mxi1 was poorly expressed in lung cancer tissues and cells and its poor expression was associated with poor prognosis. Mxi1 inhibited miR-300 by suppressing its transcription. miR-300 suppressed the expression of KLF9, and KLF9 negatively regulated GADD34 expression in lung cancer cells. Mxi1 or KLF9 elevation or miR-300 repression inhibited lung cancer cell proliferation, as evidenced by reduced Ki67 and PCNA expression, and lowered invasion and migration. In vivo findings revealed that silencing KLF9 induced tumor growth by enhancing MDSC-mediated immunosuppression through upregulation of GADD34. Collectively, these findings suggest that Mxi1 can inhibit lung cancer progression by regulating the miR-300/KLF9 axis and GADD34-mediated immunosuppression.
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