Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable ...therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.
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Described here is the first reported therapeutic based on lipid-nanoparticle delivery of small interfering RNAs targeted to inhibit the genome of SARS-COV-2 as a treatment for COVID-19.
Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, ...including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.
The highly oxygenated pimarane diterpenoids basimarols A, B, and C (3–5) were isolated from the plant species Basilicum polystachyon, which was collected within the Australian arid zone. Structure ...elucidation was performed using a suite of spectroscopic techniques, including X-ray crystallography. Anticancer and anti-DENV activity of 3–5 was explored, but only limited activity was observed. More extensive antiviral evaluation of stachyonic acid A (1), which was also isolated from B. polystachyon, revealed broad spectrum antiviral activity against West Nile virus (Kunjin strain, WNVKun) and human influenza viruses H1N1 and H3N2.
Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein ...receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect divergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.
The ongoing SARS-CoV-2 pandemic continues to pose an enormous health challenge globally. The ongoing emergence of variants of concern has resulted in decreased vaccine efficacy necessitating booster ...immunizations. This was particularly highlighted by the recent emergence of the Omicron variant, which contains over 30 mutations in the spike protein and quickly became the dominant viral strain in global circulation. We previously demonstrated that delivery of a SARS-CoV-2 subunit vaccine via a high-density microarray patch (HD-MAP) induced potent immunity resulting in robust protection from SARS-CoV-2 challenge in mice. Here we show that serum from HD-MAP immunized animals maintained potent neutralisation against all variants tested, including Delta and Omicron. These findings highlight the advantages of HD-MAP vaccine delivery in inducing high levels of neutralising antibodies and demonstrates its potential at providing protection from emerging viral variants.
INTRODUCTIONMicroneedles are emerging as a promising technology for vaccine delivery, with numerous advantages over traditional needle and syringe methods. Preclinical studies have demonstrated the ...effectiveness of MAPs in inducing robust immune responses over traditional needle and syringe methods, with extensive studies using vaccines targeted against different pathogens in various animal models. Critically, the clinical trials have demonstrated safety, immunogenicity, and patient acceptance for MAP-based vaccines against influenza, measles, rubella, and SARS-CoV-2.AREAS COVEREDThis review provides a comprehensive overview of the different types of microarray patches (MAPs) and analyses of their applications in preclinical and clinical vaccine delivery settings. This review also covers additional considerations for microneedle-based vaccination, including adjuvants that are compatible with MAPs, patient safety and factors for global vaccination campaigns.EXPERT OPINIONMAP vaccine delivery can potentially be a game-changer for vaccine distribution and coverage in both high-income and low- and middle-income countries. For MAPs to reach this full potential, many critical hurdles must be overcome, such as large-scale production, regulatory compliance, and adoption by global health authorities. However, given the considerable strides made in recent years by MAP developers, it may be possible to see the first MAP-based vaccines in use within the next 5 years.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global ...spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.
Abstract
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), ...infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.
Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type ...2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.
The COVID-19 pandemic has highlighted the need for vaccines capable of providing rapid and robust protection. One way to improve vaccine efficacy is delivery via microarray patches, such as the ...Vaxxas high-density microarray patch (HD-MAP). We have previously demonstrated that delivery of a SARS-CoV-2 protein vaccine candidate, HexaPro, via the HD-MAP induces potent humoral immune responses. Here, we investigate the cellular responses induced by HexaPro HD-MAP vaccination. We found that delivery via the HD-MAP induces a type one biassed cellular response of much greater magnitude as compared to standard intramuscular immunization.
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