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Background: The deadly virus COVID-19 has affected more than 1 crore people and claimed more than 5 lakh lives worldwide according to the World health organization. Though there are numerous treatment modalities available including anti-bacterials, antivirals, vaccines etc., none of them can be considered as effective cure for SARS CoV-2 virus as they are mostly non-specific in action. Aim(s): siRNA therapy can be considered as a significant treatment modality due to its specificity in action. The aim of this review is to explore siRNA as a potential treatment strategy for the treatment of COVID-19. Material(s) and Method(s): In this review we shall explore the targets of siRNA therapy which includes viral RNA-dependent RNA polymerase, helicase, protease and nucleoprotein N. siRNA related patents provide solutions for novel RNAi techniques, high expense of chemically synthetic siRNA, techniques for restraining SARS-CoV by disturbing RNA etc., siRNA-based drug delivery systems and limitations of nanocarrier delivery system were reviewed. siRNA is a gene silencer that targets highly conserved sequences which codes for protease 3CL (nsp5) and viral helicase (from 16-18 kbp). Conclusion(s): Thus, siRNA-based therapy is considered highly efficacious as it can hit the highly conserved regions of SARS-CoV-2 RNA.Copyright © 2023, Advanced Scientific Research. All rights reserved.
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BACKGROUND: At present, about half of the world's population is at risk of being infected with dengue virus (DENV). However, there are no specific drugs to prevent or treat DENV infection. Glycyrrhizae Radix et Rhizome, a well-known traditional Chinese medicine, performs multiple pharmacological activities, including exerting antiviral effects. The aim of this study was to investigate the anti-DENV effects of n-butanol extract from Glycyrrhizae Radix et Rhizome (GRE). METHODS: Compounds analysis of GRE was conducted via ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The antiviral activities of GRE were determined by the CCK-8 assay, plaque assay, qRT-PCR, Western blotting, and the immunofluorescence assay. The DENV-infected suckling mice model was constructed to explore the antiviral effects of GRE in vivo. RESULTS: Four components in GRE were analyzed by UHPLC-MS/MS, including glycyrrhizic acid, glycyrrhetnic acid, liquiritigenin, and isoliquiritigenin. GRE inhibited the attachment process of the virus replication cycle and reduced the expression of the E protein in cell models. In the in vivo study, GRE significantly relieved clinical symptoms and prolong survival duration. GRE also significantly decreased viremia, reduced the viral load in multiple organs, and inhibited the release of pro-inflammatory cytokines in DENV-infected suckling mice. CONCLUSIONS: GRE exhibited significant inhibitory activities in the adsorption stage of the DENV-2 replication cycle by targeting the envelope protein. Thus, GRE might be a promising candidate for the treatment of DENV infection.
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Since December 2019 the world has been dealing with a severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. The first SARS-CoV-2 vaccine was made available in Europe at the end of 2020. 202 volunteers from the vicinity of the University of Applied Sciences Wiener Neustadt took part in this study;their IgG levels recognizing the RBD of SARS-CoV-2 were determined. The aim was to evaluate the SARS-CoV-2 titer levels of vaccinated, recovered and vaccinated plus recovered persons. We could show that there is a significant difference in the antibody levels of vaccinated, vaccinated plus recovered and only recovered probands. Additionally, the highest antibody levels were found in triple vaccinated persons. Furthermore, the Moderna vaccine seems to have a higher immune response.Copyright © 2022
ABSTRACT
The present paper elucidates the conceivable application of two key molecules in SARS-CoV-2 detection of suspected infected persons. These molecules were selected on the basis of the strong interaction between ACE-2 and S protein that allows virus attachment to its host cells;on the other hand, specific immunocompetant effectors are generated by the human immune system during the infection. Several testing procedures are already being used to diagnose SARS-CoV-2 infection, particularly the RT-PCR technique. ELISA and LFIA are possible assays for the employment of shACE-2/ hAc-anti-S (the molecules of interest) as the main agents of the test that confer dual principal functions (capture and detection). The future diagnostic kits involving shACE-2 and hAc-anti-S will possibly be highly sensitive with rapid detection in addition to their advantage of relatively easy conception. They could be largely considered as technically advanced kits in regards to the current SARS-CoV-2 diagnostic immunoassays.Copyright © 2021 Bentham Science Publishers.
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Background: Coronavirus-19 (COVID-19) pandemic is a worldwide public health problem causing 347,070 deaths from December 25, 2019, till May 25, 2020. Phospholipids are structural components of mammalian cytoskeleton and cell membranes. Phosphatidylglycerol is an anionic lipid found in mammalian membranes in low amounts (1-2%) of the total phospholipids. Also, phosphatidylglycerol suppresses viral attachment to the plasma membrane and subsequent replication in lung cells. Phosphatidylglycerol depletion caused by over expression of cytosolic phos-pholipase A2alpha induces lipid accumulation in lung alveoli and promotes acute respiratory distress syndrome (ARDS). An exogenous-surfactant replacement has been successfully achieved in ARDS and improved oxygenation and lung mechanics. Inhibition of cytosolic phospholipase A2alpha impairs an early step of COVID-19 replication. Aim(s): The present study was carried out to explain the correlation between the administration of exogenous artificial surfactant as well as cytosolic phospholipase A2alpha inhibitors to improve oxygenation and lung mechanics and inhibit COVID-19 replication. Method(s): Database research was carried out on Medline, Embase, Cochrane Library, country-spe-cific journals, and following-up WHO reports published between December 25, 2019-May 25, 2020. Result(s): Till 25 May 2020, coronavirus cases were 5,307,298, with 347,070 deathsand 2,314,849 recovered cases. According to the WHO reports, most COVID-19 deaths seen are in people who suffered from other chronic diseases characterized by phospholipidosis and phosphatidylglycerol deficiency, including hypertension, liver, heart, and lung diseases and diabetes. Phospholipases A2 (PLA2) catalyze the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids leading to enhanced inflammation and lung damage. Also, cytosolic phospholipase A2alpha inhibitors may reduce the accumulation of viral proteins and RNA. In addition, administration of exogenous phospholipid surfactant may help COVID-19 infected patients with ARDS to remove inflammatory mediators. Conclusion(s): The present study showed a relation between phosphatidylglycerol deficiency in COVID-19 infected patients with ARDS and/or chronic diseases and their mortality. These findings also showed an important approach for the prevention and treatment of COVID-19 infections by using cytosolic phospholipase A2alpha inhibitors and exogenous administration of a specific phos-pholipid surfactant.Copyright © 2021 Bentham Science Publishers.
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Several vaccine strategies are in use against the SARS-CoV2 virus which causes COVID-19. There are three major approaches to vaccine development: Using the whole virus (live virus, inactivated virus, or viral vector vaccines), using immunogenic parts of the virus, or using the genetic material of the virus. As of April 29, 2022, 10 vaccines with different mechanisms of action have received EUL by WHO.1 In the United States, 3 vaccines have received either emergency use authorization or FDA approval.2 The timing and number of injections in the primary series and timing and need for booster doses of these vaccines depend on the age of the person, underlying immunocompromised status.4 The vaccines have overall been demonstrated to be safe and effective in preventing COVID-19 infection and also in preventing serious COVID-19 infection. However they are rarely associated with some serious side effects. Thrombosis with thrombocytopenia syndrome (TTS) after Johnson & Johnson/ Janssen adenoviral vector vaccine is a rare complication that appears to affect women between the ages of 30 and 49 years. It has also been described with the Oxford/Astra Zeneca vaccine. Based on the available data there is no increased risk for TTS after mRNA vaccination, although cases of immune thrombocytopenia have bene reported after the Pfizer/ BioNTech and Moderna vaccines.3,4 An increased risk of Guillain-Barré syndrome has also been associated with this the Janssen vaccine.3 Myocarditis and pericarditis, particularly in male adolescents and young adults have been associated with the Pfizer and Moderna mRNA vaccines.5 Currently, antibody testing either post infection or post vaccination are not recommended for various reasons.6, 7 Preexposure prophylaxis with a combination of 2 long-acting antibodies, tixagevimab-cilgavimab target the receptor binding domain of the SARS-CoV-2 spike protein and inhibit virus attachment. In December 2021, the US FDA provided emergency use authorization for preexposure prophylaxis of COVID- 19 in patients 12 years or older weighing at least 40 kg, not currently infected with COVID-19 or have a known recent exposure and either have moderate to severe immunocompromise due to a medical condition or receipt of immunosuppressive treatments and may not mount an adequate immune response to COVID-19 vaccines, or in whom available vaccines are not recommended due to prior severe adverse reaction.8 This consists of 2 consecutive intramuscular injections at the same visit and the effi cacy appears to last for up to 6 months. It was granted marketing authorization in the EU in March 2022.
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Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteropathogenic coronavirus that causes severe diarrhea in neonatal piglets, leading to serious economic losses to the pig industries. At present, there are no effective control measures for SADS, making an urgent need to exploit effective antiviral therapies. Here, we confirmed that Aloe extract (Ae) can strongly inhibit SADS-CoV in Vero and IPI-FX cells in vitro. Furthermore, we detected that Emodin from Ae had anti-SADS-CoV activity in cells but did not impair SADS-CoV infectivity directly. The time-of-addition assay showed that Emodin inhibits SADS-CoV infection at the whole stages of the viral replication cycle. Notably, we found that Emodin can significantly reduce virus particles attaching to the cell surface and induce TLR3 (p < 0.001), IFN-λ3 (p < 0.01), and ISG15 (p < 0.01) expressions in IPI-FX cells, indicating that the anti-SADS-CoV activity of Emodin might be due to blocking viral attachment and the activation of TLR3-IFN-λ3-ISG15 signaling axis. These results suggest that Emodin has the potential value for the development of anti-SADS-CoV drugs.
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COVID19 is characterized by alteration of smell and taste perception. It was investigated the presence of alteration for cold, heat, and food consistency perception in COVID-19 patients and the possible correlation with the most commonly used biomarkers in a routine way (CRP, IL-6, IL-1, PSP, PCT) to show if the local inflammatory process may induce alteration of cold, heat, and food consistency perception in COVID-19. From March 2020 to April 2021, a survey about taste and oral tactile/thermal sensitivity dysfunction was carried out on 1155 patients with mild or moderate COVID-19, while patients admitted to intensive care were excluded because they could not answer questions from health professionals. Positive RT-PCR confirmed all diagnoses. CRP, IL-6, IL-1, PSP, and PCT were detected for each patient. Taste and Smell Questionnaire Section of the US NHANES 2011−2014 protocol (CDC 2013b) was used for gustatory function assessment. The study was approved by the Ethics committee of AOU Policlinico Consorziale di Bari (No. 6388 COVID19 DOM-protocol number 0034687/12-05-2020), and written informed consent was obtained. A total of 208 patients referred only to alteration to taste (208/821). 75/208 ageusia, 71/208 ipoageusia, 21/208 fantageusia, 19/208 dysgeusia, 22/208 parageusia. Significant correlations between CRP, IL-6, IL-1, PSP, PCT levels and type of dysfunctions about the perception of heat, cold and food consistency have not been found (P>0.05). Loss of taste can be due to the action of inflammatory mediators on gustatory buds, while alteration of cold, heat and food consistency perception may be due to direct damage of filiform papillae, allowing virus binding through the spike protein to ACE2 receptors of filiform papillae cells.
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Background: The dominance of SARS-CoV-2 Variants of Concern (VOC) and Interest (VOI) has challenged the efficacy of public health strategies to control the current pandemic. Astodrimer sodium is a broad-spectrum antiviral dendrimer that has been formulated as a topical nasal spray to help reduce exposure to infectious viral load in the nasal cavity. Astodrimer sodium showed antiviral and virucidal activity against early pandemic isolates of SARS-CoV-2 in vitro and after nasal administration in vivo. The current studies assessed the spectrum of activity of astodrimer sodium against emerging variants of SARS-CoV-2 and other pandemic viruses. Methods: Assays utilized hACE2+ and hTMPRSS2+ HEK-293T cells, Calu-3 and Vero E6 cells. Time of addition studies involved adding astodrimer sodium 1 hour prior to, at the time of, or 1-hour post-infection. Coronavirus spike receptor binding domain (RBD) or S1 binding studies were analysed by ELISA or confocal microscopy. Virucidal studies involved exposing 105 SARS-CoV-2 PFU to 10mg/mL astodrimer sodium for 0.5, 1, 5, 15 and 30 mins. Results: Astodrimer sodium demonstrated potent antiviral and virucidal activity against SARS-CoV-2 VOC α, β, δ and γ, and VOI κ in Vero E6 and Calu-3 cells. Astodrimer sodium reduced infectious viral load of all variants by >99.9% vs virus control. The pan-SARS-CoV-2 activity of astodrimer sodium occurred despite multiple mutations and deletions in the viral spike protein of each variant. The attachment of SARS-CoV-2 early pandemic virus isolates, Wuhan-Hu-1 and USA-WA-1/2020, and SARS-CoV-1 Spike binding to ACE2, as well as attachment of Middle Eastern respiratory syndrome (MERS) coronavirus spike protein to its cellular receptor, was inhibited by astodrimer sodium. Astodrimer sodium did not prevent attachment of the SARS-CoV-2 VOC α and β spike S1, or γ RBD spike protein, to the ACE2 receptor in vitro. Conclusion: Astodrimer sodium mimics negatively charged glycosaminoglycans and provides a potent antiviral and virucidal barrier to viral attachment and entry. The potent broad-spectrum anti-pandemic coronavirus and virucidal efficacy of astodrimer sodium against whole virus is likely due to blocking multiple electrostatic interactions of the spike protein that are not negated by minor or major changes to the isolated RBD of SARS-CoV-2 VOC α, β and γ alone. Astodrimer sodium has the potential to block the binding of pan-SARS-CoV-2, thus reducing the potential for the development of COVID-19.
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Respiratory viral infections are a major public health concern because of their global occurrence, ease of spread and considerable morbidity and mortality. Medical treatments for viral respiratory diseases primarily involve providing relief from symptoms like pain and discomfort rather than treating the infection. Very few antiviral medications have been approved with restrictive usage, high cost, unwanted side effects and limited availability. Plants with their unique metabolite composition and high remedial values offer unique preventive and therapeutic efficacy in treatment of viral infections. The present review is focused on the types and mode of action of plant secondary metabolites that have been used successfully ί in the treatment of infections caused by respiratory viruses like Influenza, SARS, MERS, RSV etc. Plant metabolites such as phenolics, alkaloids, terpenoids and oligosaccharides inhibit attachment and entry of the virus. Others such as flavonoids, viz quercetin and baicalein, alkaloids viz sanguinarine, berberine and emetine, specific lipids and fatty acids prevent viral replication and protein synthesis. These metabolites have the potential to be used as lead molecules that can be optimized to develop potent drugs for effectively combating pandemics caused by respiratory viruses.
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Background. ADG20 is a fully human IgG1 monoclonal antibody engineered to have potent and broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like CoVs with pandemic potential and an extended half-life. ADG20 is administered intramuscularly (IM). A QSP/PBPK model was constructed to support dose selection for a Phase 2/3 trial of ambulatory patients with mild to moderate COVID-19 (STAMP: NCT04805671). Methods. A QSP/PBPK model was used to simulate receptor occupancy (RO) and drug exposure in the upper airway (nasopharyngeal/oropharyngeal epithelial lining fluid [ELF] compartment). RO was linked to an existing viral dynamic model to enable the prediction of the natural time course of viral load and the effect of ADG20 on viral clearance and infectivity rate. RO was calculated using: 1) in vitro ADG20-SARS-CoV-2 binding kinetics (association rate constant (kon) of 1.52E+06 M-1•s1 and dissociation rate constant (koff) of 2.81E-04 s-1 from a Biacore assay;2) time course of ADG20 concentrations in ELF;and 3) time course of viral load following ADG20 administration. Molar SARS-CoV-2 viral binding site capacity was calculated assuming 40 spike proteins per virion, 3 binding sites per spike, and an initial viral load of log 107 copies/mL for all patients. The QSP/PBPK model and a 2018 CDC reference body weight distribution (45-150 kg) were used to simulate 1000 concentration-time profiles for a range of candidate ADG20 regimens. ADG20 regimens were evaluated against 2 criteria: 1) ability to attain near complete ( >90%), and durable (28-day) SARS-CoV-2 RO in the ELF;and 2) ability to maintain ELF ADG20 concentrations relative to a concentration (0.5 mg/L) associated with 100% viral growth suppression in an in vitro post-infection assay. Results. A single 300 mg IM ADG20 dose met the dose selection criteria in terms of RO (Figure A) and viral growth suppression (Figure B). Conclusion. These data support the evaluation of an ADG20 300 mg IM dose for the treatment of mild to moderate COVID-19. ADG20 is forecasted to attain near complete ( >90%) SARS-CoV-2 RO in the ELF and maintain ELF ADG20 concentrations above that associated with 100% viral growth suppression in vitro.
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Background: SARs-CoV- 2 infection recruits high numbers of neutrophils that extrude neutrophil extracellular traps (NETs), webs of extracellular DNA coated with citrullinated histones (cit-His) and antimicrobial proteins. NETs have also been shown to entrap virions, concentrate antiviral proteins, and inactivate viruses. However, when NETs are degraded, they release NET degradation products (NDPs) such as cit-His, cell-free (cf) DNA, myeloperoxidase (MPO) and neutrophil elastase (NE) that can be toxic to the host. Our group and others have found that NETs and NDPs are highly prominent in patients with severe COVID-19 and are associated with the development of respiratory failure (Figure 1). Platelet factor 4 (PF4) is a highly-positively charged, platelet-specific chemokine that aggregates polyanionic molecules like heparin and DNA. We have shown that PF4 binds to NETs, reducing the release of NDPs by preventing NET digestion by circulating nucleases. Importantly, PF4-NET complexes markedly enhance gram-positive and -negative bacterial entrapment, likely by bridging the negatively charged polyanionic phosphoribose backbone of the NET DNA scaffold to polyanionic surface molecules in the bacterial cell wall. Treatment with PF4 improved outcomes in lipopolysaccharide endotoxemia and cecal ligation and puncture models of murine sepsis. Objectives: The objective of this study was to investigate whether PF4 binding to NETs is similarly protective in SARs-CoV- 2 infection by preventing the degradation of NETs and by enhancing NET-mediated viral capture. Methods: We generated NET-lined microfluidic channels. Neutrophils were isolated from healthy human donors, adhered to fibronectin-coated channels, and incubated with phorbol myristate acetate (PMA) to induce the release of NETs. Channels were then treated with buffer alone or PF4 (100 μg/ml) to compact NETs, after which gamma-irradiated SARS-CoV- 2 (1 x 107 PFU) were infused at 2 dynes/cm2 for 1 hour. Viral particles were then labeled with SARS-CoV- 2 guinea pig antiserum and visualized with a fluorescently-labeled secondary antibody. Viral binding to NETs was quantified using confocal microscopy. Results: Similar to that seen with bacterial attachment to NETs, we observed scant viral binding to non-compact NETs. In contrast, there was abundant binding of SARs-CoV- 2 aggregates to PF4 compacted NETs (Figure 2). Conclusions: These findings demonstrate that PF4 plays a crucial role in NET-mediated viral capture and suggest that PF4-NET complexes may be part of the physiologic mechanism by which viral spread is contained in the host. Moreover, we have previously shown that an Fc-modified version of KKO, a monoclonal antibody directed against complexes of PF4 and polyanions, markedly enhanced the protective effects of PF4 in vitro and in murine models of sepsis. Therefore, we will examine whether PF4 plus modified KKO infusions are able to limit SARS-CoV- 2 viremia, preventing the pneumonitis and multi-system organ dysfunction of severe COVID-19. (Figure Presented).
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Background: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has been responsible for the global pandemic and disease known as COVID-19. Clinical studies have found >50% of COVID-19 patients report gastrointestinal (GI) symptoms, with some studies suggesting longer viral clearance in patients with GI symptoms. SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for viral attachment and intracellular entry, which is expressed in the intestine, making the GI tract a potential route of infection. Aim: To determine the effect of microbial colonization on colonic ACE2 expression using a humanized mouse model. Methods: Human stool was collected from healthy volunteers and individuals with irritable bowel syndrome, a common gastrointestinal disorder, and fecal slurries were prepared. 4-6 week old female Swiss-Webster mice (N=6/group) were gavaged with fecal slurries and maintained in sterile ex-isolator cages for 6 weeks. Posthumanization stool was collected along with proximal colonic tissue. Shotgun metagenomics was done on mouse pellets. RNAseq on tissue performed at a depth of 20 million reads/ sample using NovaSeq 6000. MAP-Rseq workflow using the mm10 genome was done to identify differentially expressed genes. Associations of ACE2 expression with α –diversity between microbiome from healthy and irritable bowel syndrome humanized mice was done using linear modeling with compositional associations assessed using PERMANOVA (BrayCurtis distance). To identify taxa associated with ACE2 expression, a permutation-based approach using the F-statistic of a linear model was used with false discovery rate (FDR) to correct for multiple testing. Results: Humanized mice demonstrated significantly lower colonic ACE2 expression compared to the germ-free mice (333.4 ± 191.1 vs. 1914 4 ± 309.9, FDR<0.001). However, ACE2 expression was similar post-humanization across all mouse groups regardless of stool used for humanization, despite decreased diversity in stool from irritable bowel syndrome patients and compositional differences from healthy volunteer stool. No associations between microbiome α-diversity (Shannon p=0.825, observed p= 0.400, InvSimpson p=0.512), β-diversity (p=0.568) or individual taxa were seen with ACE2 expression. Conclusions: Commensal microbial colonization significantly suppresses colonic ACE2 expression. However, in this pilot study, mice colonized with dysbiotic and healthy microbial communities had similar ACE2 expression. Future studies will have to explore the role of commensal microbes on gastrointestinal expression of ACE2 which may in turn reflect predisposition for infection or intestinal involvement with SARS-CoV-2. Supported by DK103911, DK120745.
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Background: SARS-Cov-2 infection (COVID-19) and associated gastrointestinal manifestations have been well documented during the pandemic. To date, several centers have reported isolated cases of COVID-19 and its effect on the pancreas. Here, we present a case series of 13 patients with acute pancreatitis (AP) due to COVID-19, which represents one of the larger case series to date. Methods: A retrospective review was performed from 3/1/2020 through 4/1/2020 at 4 NYC academic medical centers. Patients with a diagnosis of AP and COVID-19 were included. AP was diagnosed based on AGA criteria. COVID-19 infection was confirmed via nasopharyngeal viral PCR testing. All patients with a prior history of AP were excluded. Patients with apparent/suspected etiologies of AP (including gallstones, alcohol, hypertriglyceridemia, post ERCP, medication, and other viral etiologies) were excluded. 13 patients met our inclusion and exclusion criteria. Outcomes studied included mortality, ICU admission, length of stay, BISAP scores on admission and at 48 hours. Results: 7 of the 13 patients in this cohort were African American, 8 of 13 were men, and the median age was 51 years of age. The youngest patient was 18 years old and the oldest patient was 71 years old. Of the 13 patients, 5 patients died during their hospital course. Of those 5 who passed, 4 were African American, and all 5 were > 50 years of age. 6 of the 13 required ICU level of care. The mean length of stay for all patients was 23 days. On admission, 4 patients had BISAP scores > 3, at 48 hours 3 patients had BISAP scores > 3. Discussion: We report the characteristics of 13 patients with confirmed SARS-Cov-2 infection and AP without other common etiologies. We suspect that SARS-Cov-2 was a direct cause of AP in these patients. 5 patients died (38.5%) due to multiorgan failure from Acute Respiratory Distress Syndrome. Patients with COVID-19 and AP had a higher mortality rate than the overall mortality reported with COVID-19 during the same period. The mortality of patients in our series far exceeds the reported mortality in mild or moderate AP (less than 1%)1,2. Currently molecular theories suggest that viral attachment to ACE-2 receptors on pancreatic acinar cells leads to apoptosis, inhibition of nitric oxide production, and programmed cell death that ultimately leads to AP. Conclusion: This case series indicates a possible association between COVID-19 and AP and the increased mortality in this subset of patients. Further research is needed concerning the molecular mechanisms and clinical management of this entity. Larger studies are needed to confirm the worse outcomes with AP associated with COVID-19. Ref: 1. Russo MW et al. Digestive and liver diseases statistics, 2004. Gastroenterology. 2004;126:1448–53. 2. Triester SL et al. Prognostic factors in acute pancreatitis. J Clin Gastroenterol. 2002;34:167–76.
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Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2. Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA. Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6;Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6;Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3;Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3;Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK;65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05;78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03;n=6;Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation. Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. [Formula presented] Disclosures: Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding;BMS: Research Funding;Kiadis Pharma: Rese rch Funding.
ABSTRACT
Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2-5 µM range, were identified. Further studies demonstrated that these "kite-shaped" molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , COVID-19/virology , Leukemia Virus, Murine/drug effects , SARS-CoV-2 , Virus Internalization/drug effects , Animals , Cell Line , Chlorocebus aethiops , Drug Discovery/methods , Drug Repositioning , Drug Synergism , Humans , Leukemia Virus, Murine/metabolism , Mice , Molecular Docking Simulation , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Attachment/drug effectsABSTRACT
Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4+ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4+ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4+ T cells.
Subject(s)
HIV Infections/metabolism , Hyaluronan Receptors/metabolism , Leukosialin/metabolism , Membrane Glycoproteins/metabolism , Cell Membrane/metabolism , HIV Infections/genetics , HIV-1/genetics , HIV-1/metabolism , HIV-1/pathogenicity , Host-Pathogen Interactions , Humans , Hyaluronan Receptors/genetics , Leukosialin/genetics , Membrane Glycoproteins/genetics , Membrane Proteins/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Virion/metabolism , Virus Assembly , Virus Attachment , gag Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Viruses are obligate intracellular parasites and have evolved to enter the host cell. To gain access they come into contact with the host cell through an initial adhesion, and some viruses from different genus may use heparan sulfate proteoglycans for it. The successful inhibition of this early event of the infection by synthetic molecules has always been an attractive target for medicinal chemists. Numerous reports have yielded insights into the function of compounds based on the dispirotripiperazine scaffold. Analysis suggests that this is a structural requirement for inhibiting the interactions between viruses and cell-surface heparan sulfate proteoglycans, thus preventing virus entry and replication. This review summarizes our current knowledge about the early history of development, synthesis, structure-activity relationships and antiviral evaluation of dispirotripiperazine-based compounds and where they are going in the future.