SARS-CoV-2 primed platelets-derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation.

BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive. METHODS: We explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting. RESULTS: COVID-19 patient-derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet-derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1ß/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement. CONCLUSIONS: We identified SARS-CoV-2 triggered platelets-derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

SARS-CoV-2 Spike Protein S1 Exposure Increases Susceptibility to Angiotensin II-Induced Hypertension in Rats by Promoting Central Neuroinflammation and Oxidative Stress.

The SARS-CoV-2 spike S1 subunit (S1) can cross the blood-brain barrier and elicit neuroinflammatory response independent of viral infection. Here we examined whether S1 influences blood pressure (BP) and sensitizes the hypertensive response to angiotensin (ANG) II by enhancing neuroinflammation and oxidative stress in hypothalamic paraventricular nucleus (PVN), a key brain cardiovascular regulatory center. Rats received central S1 or vehicle (VEH) injection for 5 days. One week after injection, ANG II or saline (control) was subcutaneously delivered for 2 weeks. S1 injection induced greater increases in BP, PVN neuronal excitation and sympathetic drive in ANG II rats but had no effects in control rats. One week after S1 injection, mRNA for proinflammatory cytokines and oxidative stress marker were higher but mRNA of Nrf2, the master regulator of inducible antioxidant and anti-inflammatory responses, was lower in the PVN in S1-injected rats than in VEH-injected rats. Three weeks after S1 injection, mRNA for proinflammatory cytokines and oxidative stress marker, microglia activation and reactive oxygen species in the PVN were comparable between S1 and VEH treated control rats but were elevated in two groups of ANG II rats. Notably, ANG II-induced elevations in these parameters were exaggerated by S1. Interestingly, ANG II increased PVN Nrf2 mRNA in VEH-treated rats but not in S1-treated rats. These data suggest that S1 exposure has no effect on BP, but post-S1 exposure increases susceptibility to ANG II-induced hypertension by downregulating PVN Nrf2 to promote neuroinflammation and oxidative stress and augment sympathetic excitation.

Low-Density Lipoprotein Receptor (LDLR) Is Involved in Internalization of Lentiviral Particles Pseudotyped with SARS-CoV-2 Spike Protein in Ocular Cells.

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 µM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.

A Chinese Herb Prescription "Fang-gan Decoction" Protects Against Damage to Lung and Colon Epithelial Cells Caused by the SARS-CoV-2 Spike Protein by Regulating the TGF-ß/Smad2/3 and NF-κB Pathways.

Objective To explore the effects and mechanisms of a traditional Chinese medicine (TCM) prescription, "Fang-gan Decoction" (FGD), in protecting against SARS-CoV-2 spike protein-induced lung and intestinal injuries in vitro and in vivo.Methods Female BALB/c mice and three cell lines pretreated with FGD were stimulated with recombinant SARS-CoV-2 spike protein (spike protein). Hematoxylin-eosin (HE) staining and pathologic scoring of tissues, cell permeability and viability, and angiotensin-converting enzyme 2 (ACE2) expression in the lung and colon were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of inflammatory factors in serum and cell supernatant. The expression of NF-κB p65, p-NF-κB p65, p-IκBα, p-Smad2/3, TGF-ß1, Caspase3, and Bcl-2 was evaluated by Western blotting.Results FGD protected against the damage to the lung and colon caused by the spike protein in vivo and in vitro according to the pathologic score and cell permeability and viability (P<0.05). FGD up-regulated ACE2 expression, which was reduced by the spike protein in the lung and colon, significantly improved the deregulation of inflammatory markers caused by the spike protein, and regulated the activity of TGF-ß/Smads and NF-κB signaling.Conclusion Traditional Chinese medicine has a protective effect on lung and intestinal tissue injury stimulated by the spike protein through possible regulatory functions of the NF-κB and TGF-ß1/Smad pathways with tissue type specificity.

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein-treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Age-dependent effects of the recombinant spike protein/SARS-CoV-2 on the M-CSF- and IL-34-differentiated macrophages in vitro.

The SARS-CoV-2 virus causes elevated production of senescence-associated secretory phenotype (SASP) markers by macrophages. SARS-CoV-2 enters macrophages through its Spike-protein aided by cathepsin (Cat) B and L, which also mediate SASP production. Since M-CSF and IL-34 control macrophage differentiation, we investigated the age-dependent effects of the Spike-protein on SASP-related pro-inflammatory-cytokines and nuclear-senescence-regulatory-factors, and CatB, L and K, in mouse M-CSF- and IL-34-differentiated macrophages. The Spike-protein upregulated SASP expression in young and aged male M-CSF-macrophages. In contrast, only young and aged male IL-34-macrophages demonstrated significantly reduced pro-inflammatory cytokine expression in response to the Spike-protein in vitro. Furthermore, the S-protein elevated CatB expression in young male M-CSF-macrophages and young female IL-34-macrophages, whereas CatL was overexpressed in young male IL-34- and old male M-CSF-macrophages. Surprisingly, the S-protein increased CatK activity in young and aged male M-CSF-macrophages, indicating that CatK may be also involved in the COVID-19 pathology. Altogether, we demonstrated the age- and sex-dependent effects of the Spike-protein on M-CSF and IL-34-macrophages using a novel in vitro mouse model of SARS-CoV-2/COVID-19.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Synthesis and immunogenicity assessment of a gold nanoparticle conjugate for the delivery of a peptide from SARS-CoV-2.

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S461-493) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH2 via glutaraldehyde-mediated coupling to obtain the AuNP-S461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S461-493; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo.

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1ß Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1ß. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1ß by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1ß production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Oral immunization with a novel attenuated Salmonella Gallinarum encoding infectious bronchitis virus spike protein induces protective immune responses against fowl typhoid and infectious bronchitis in chickens.

Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and infectious bronchitis (IB) are two economically important avian diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant, JOL967, to deliver spike (S) protein 1 of IB virus (V) to elicit protective immunity against both FT and IB in chickens. The codon optimized S1 nucleotide sequence was cloned in-frame into a prokaryotic constitutive expression vector, pJHL65. Subsequently, empty pJHL65 or recombinant pJHL65-S1 plasmid was electroporated into JOL967 and the resultant clones were designated as JOL2068 and JOL2077, respectively. Our results demonstrated that the chickens vaccinated once orally with JOL2077 elicited significantly (p < 0.05) higher IBV-specific humoral and cell-mediated immunity compared to JOL2068 and PBS control groups. Consequently, on challenge with the virulent IBV strain at 28th day post-vaccination, JOL2077 vaccinated birds displayed significantly (p < 0.05) lower inflammatory lesions in virus-targeted tissues compared to control groups. Furthermore, 33.3% (2 of 6) of birds vaccinated with JOL2077 vaccine had shown virus recovery from tracheal tissues compared to 100% (6 of 6) recovery obtained in both the control groups. Against wild-type SG lethal challenge, both JOL2077 and JOL2068 vaccinated groups exhibited only 10% mortality compared to 80% mortality observed in PBS control group. In conclusion, we show that JOL2077 can induce efficient IBV- and carrier-specific protective immunity and can act as a bivalent vaccine against FT and IB. Further studies are warranted to investigate the potential of JOL2077 vaccine in broiler and young layer birds.

Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.

Purpose: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway. Methods: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration. Results: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure. Conclusion: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.

Protein nanopore reveals the renin-angiotensin system crosstalk with single-amino-acid resolution.

The discovery of crosstalk effects on the renin-angiotensin system (RAS) is limited by the lack of approaches to quantitatively monitor, in real time, multiple components with subtle differences and short half-lives. Here we report a nanopore framework to quantitatively determine the effect of the hidden crosstalk between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) on RAS. By developing an engineered aerolysin nanopore capable of single-amino-acid resolution, we show that the ACE can be selectively inhibited by ACE2 to prevent cleavage of angiotensin I, even when the concentration of ACE is more than 30-fold higher than that of ACE2. We also show that the activity of ACE2 for cleaving angiotensin peptides is clearly suppressed by the spike protein of SARS-CoV-2. This leads to the relaxation of ACE and the increased probability of accumulation of the principal effector angiotensin II. The spike protein of the SARS-CoV-2 Delta variant is demonstrated to have a much greater impact on the crosstalk than the wild type.

SARS-CoV-2 Entry Inhibitors Targeting Virus-ACE2 or Virus-TMPRSS2 Interactions.

COVID-19 is an infectious disease caused by SARS-CoV-2. The life cycle of SARS-CoV-2 includes the entry into the target cells, replicase translation, replicating and transcribing genomes, translating structural proteins, assembling and releasing new virions. Entering host cells is a crucial stage in the early life cycle of the virus, and blocking this stage can effectively prevent virus infection. SARS enters the target cells mediated by the interaction between the viral S protein and the target cell surface receptor angiotensin- converting enzyme 2 (ACE2), as well as the cleavage effect of a type-II transmembrane serine protease (TMPRSS2) on the S protein. Therefore, the ACE2 receptor and TMPRSS2 are important targets for SARS-CoV-2 entry inhibitors. Herein, we provide a concise report/information on drugs with potential therapeutic value targeting virus-ACE2 or virus-TMPRSS2 interactions to provide a reference for the design and discovery of potential entry inhibitors against SARS-CoV-2.

Marine algal antagonists targeting 3CL protease and spike glycoprotein of SARS-CoV-2: a computational approach for anti-COVID-19 drug discovery.

The COVID-19 pandemic has severely destructed human life worldwide, with no suitable treatment until now. SARS-CoV-2 virus is unprecedented, resistance against number of therapeutics and spreading rapidly with high mortality, which warrants the need to discover new effective drugs to combat this situation. This current study is undertaken to explore the antiviral potential of marine algal compounds to inhibit the viral entry and its multiplication using computational analysis. Among the proven drug discovery targets of SARS-CoV-2, spike glycoprotein and 3-chymotrypsin-like protease are responsible for the virus attachment and viral genome replication in the host cell. In this study, the above-mentioned drug targets were docked with marine algal compounds (sulfated polysaccharides, polysaccharide derivatives and polyphenols) using molecular docking tools (AutoDockTools). The obtained results indicate that κ-carrageenan, laminarin, eckol, trifucol and ß-D-galactose are the top-ranking compounds showing better docking scores with SARS-CoV-2 targets, than the current experimental COVID-19 antiviral drugs like dexamethasone, remdesivir, favipiravir and MIV-150. Further, molecular dynamic simulation, ADMET and density functional theory calculations were evaluated to substantiate the findings. To the best of our knowledge, this is the first report on in silico analysis of aforesaid algal metabolites against SARS-CoV-2 targets. This study concludes that these metabolites can be curative for COVID-19 in the hour of need after further validations in in vitro and in vivo testings.Communicated by Ramaswamy H. Sarma.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo.

"Pan-coronavirus" antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound's broad-spectrum antiviral activity and the lack of resistant mutants.