SARS-CoV-2 Research, Vaccine and Therapeutic Development
In addition to the need for fast development of assays to detect SARS-CoV-2 and identify exposed individuals, the COVID-19 crises has led to massive worldwide efforts to develop drug treatments and vaccines effective against SARS-CoV-2. A more comprehensive understanding of the biology of the SARS-CoV-2 virus is also needed. We support scientists working to develop vaccines and to answer questions about viral pathology and treatment including:
- How does the virus enter human cells?
- How does the virus make people sick?
- What treatments can be used to alleviate symptoms?
- How can immunity to the virus be gained?
We offer collaborative support and a broad portfolio of reagents that are used in research labs studying coronaviruses and other emerging viral diseases.
Tracking and Monitoring Viral Activity in Cells
Understanding the interactions between viral pathogens and host cells, and monitoring the effect of viral activity on cells, are essential to the development of effective anti-viral treatments or vaccines. Promega technologies are used in studies monitoring key steps in viral pathogenesis, including detecting virus interactions with host cell surface receptors, tracking and monitoring production of viral nucleic acids and proteins within the cell, and monitoring host cell viability and metabolism.
Detect Virus:Host Protein:Protein Interactions
Understanding how the SARS-CoV-2 virus enters host cells is a potential first step to developing a treatment or preventing infection. Viral entry is dependent on protein:protein interactions between a host cell surface receptor and the viral proteins. Protein:protein interaction assays are a valuable tool for studying these interactions.
NanoBiT® and NanoBRET™ technologies provide the sensitivity necessary to detect protein:protein interactions at the concentrations expressed in vivo. Both are bioluminescence-based methods that are particularly useful in viral studies due to the small size and bright signal of the luciferase used. For more details, see the references and resources below.
NanoLuc technologies applied to virology
Reporter technologies are creating ways to study virus:host interactions in more detail than ever before.
Protein Interaction Detection Systems
Lumit™ SARS CoV-2 Spike RBD: hACE2 Immunoassay
A homogeneous bioluminescent assay that measures interaction between SARS-CoV-2 spike protein receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2).
NanoBiT® PPI Starter Systems
Complementation reporter for detection of protein interactions in live cells.
N2014, N2015, N2016
NanoBRET™ PPI Starter Systems
Live-cell protein:protein interaction assay. Includes vectors, controls and detection reagent for BRET-based PPI.
N1811, N1821
References
Protein Interactions in Viral Research
Rawle, D.J., et al. (2018) HIV-1 uncoating and reverse transcription require eEF1A binding to surface-exposed acidic residues of the reverse transcriptase thumb domain. mBio 9, e00316-18.
Miyakawa, K., et al. (2017) The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein. Nature Comm. 8, 14259.
SARS-CoV-2 Protein Interactions
Azad, T., et al. (2020) Nanoluciferase complementation-based biosensor reveals the importance of N- linked glycosylation of SARS-CoV-2 Spike for viral entry . Research Square . DOI: 10.21203/rs.3.rs-58455/v1
Lima, M., et al. (2021) Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2. Biochem. Biophys. Res. Comm. 534, 485-490.
Zhang, H., et al. (2020) Proteasome activator PA28γ-dependent degradation of coronavirus disease (COVID-19) nucleocapsid protein. Biochem. Biophys. Res. Comm. 529, 251-256.
Track Recombinant Viruses and Detect Reporter Activity
Luciferase reporters provide a simple and convenient method for monitoring the effect of potential treatments, creating in vitro assays for detecting viral antibodies, and for imaging viruses in animal models. The small size of NanoLuc® Luciferase is especially useful for the creation of reporter viruses.
Other reporters, including firefly and Renilla luciferase, are often used in the creation of pseudotyped viruses. Pseudotyped virus particles are made by replacing the host-binding protein with a host-binding protein of another virus and adding a genetic reporter, allowing researchers to study the viral entry process. Pseudotyped viruses are also used to study inhibitors of the virus, such as antibodies and small molecule compounds.
Remdesivir as a potential treatment for SARS-CoV-2
Luciferase Reporter Assays
Nano-Glo® Luciferase Assay System
Add-mix-measure assay for NanoLuc® Luciferase with >2 hour half-life in most applications.
N1110, N1120, N1130, N1150
Nano-Glo® Live Cell Assay
Measures NanoBiT® or NanoLuc® luminescence in living cells for up to 2 hours.
N2011, N2012, N2013
Bright-Glo™ Luciferase Assay System
A sensitive reporter assay for continuous-processing on robotic systems.
E2610, E2620, E2650
References
Pseudovirus studies using reporter assays
Millet, J.K., et al. (2019) Production of pseudotyped particles to study highly pathogenic coronaviruses in a biosafety level 2 setting. J. Vis. Exp. (145), e59010.
Use of pseudovirus to study receptor binding
Walls, A.C., et al. (2020) Structure, function and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell online 9 March 2020.
Review
Andersen, P.I. (2020) Discovery and development of safe-in-man broad-spectrum antiviral agents. Int. J. Inf. Dis. 93, 268-75.
Advantages of a small, bright luciferase reporter
Recombinant reporter viruses are important tools for furthering our understanding of viral life cycles and lethality in cell and animal models. Reporter viruses make it easier to follow infection in the same animal over time and quantify events such as cellular entry and replication.
Insertion of large reporter genes (e.g., firefly luciferase) into the genome often causes defects in viral processes. Because of their small size, NanoLuc® and HiBiT tags can be stably inserted into the viral genome without disrupting the natural biology of the virus.
Detect and Monitor Viral Copy Number
PCR-based methods, such as endpoint and real-time PCR and RT-PCR, are fundamental tools used in the development of viral detection tests, and for analysis of viral genomes.
GoTaq® PCR and qPCR systems and GoScript™ Reverse Transcriptase offer robust and reproducible amplification and reverse transcription for detecting and amplifying sequences for numerous viral targets.
The GoTaq® 1-Step RT-qPCR System is approved for use with CDC protocol for SARS-CoV-2 Detection. PCR Master Mix has also been used in the development of a SARS-CoV-2 PCR diagnostic test.
CDC SARS-CoV-2 Test Protocol
PCR, qPCR and RT-PCR Reagents
GoTaq® Probe qPCR and RT-qPCR Systems
Ready-to-use master mixes for probe-based qPCR or RT-qPCR.
A6101, A6102, A6120, A6121, A6110
PCR Optimization Kit
Preformulated PCR master mix components to determine optimal PCR amplification conditions.
D2381
GoScript™ Reverse Transcriptase
Optimized, dependable reverse transcription. Available as kit, master mix or standalone enzyme.
A5003, A5004, A5000, A5001, A2790, A2791, A2800, A2801
References
GoTaq® 1-Step RT-qPCR System
Nelson, A.C. et al. (2020) Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods. doi: https://doi.org/10.1101/2020.04.02.022186
Driouich, J.-S., et al. (2018) SuPReMe: A rapid reverse genetics method to generate clonal populations of recombinant RNA viruses. Emerg. Microb. Infect. 7, 40
qPCR for Detection of Viral RNA
Cai, M. et al. (2020) The R251K Substitution in Viral Protein PB2 Increases Viral Replication and Pathogenicity of Eurasian Avian-like H1N1 Swine Influenza Viruses. Viruses 12(1), 52.
Si,L., et al. (2020) Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics. BioRxiv, April 2020. DOI: https://doi.org/10.1101/2020.04.13.039917
Viral Structure and Characterization
Wicht, O. et al. (2014) Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture. J. Virol. 88(14), 7952–7961.
Monitor Host Cell Function
Monitoring compounds for antiviral activity includes testing for viral-induced cytopathic effects (CPE) in host cells. A viability assay amenable to high-throughput analysis such as CellTiter-Glo® is useful for testing hundreds or thousands of compounds at once, reducing the time needed to analyze the effect of potential treatments. Viability assays are also used to support studies investigating the mechanism of action of viruses.
In addition to cytopathic effects, viral infection induces changes in cellular metabolism. In many infectious diseases, viruses reprogram host cell metabolism to support viral replication. These virus-induced metabolic changes can be understood using assays that monitor nutrient uptake and changes in co-factors such as NADPH, among others.
Characterization of proteins by mass spectrometry is another approach used to monitor virus-induced changes in host cells. Analysis of changes in cellular proteins helps researchers understand how viruses interact with cellular pathways.
Using Cell Viability Assays in Screens for SARS-CoV-2 Drug Candidates
Dr. Colleen Jonsson of the University of Tennessee Health Science Center has been studying highly pathogenic human viruses for more than three decades. She has led several cross-institutional projects using high-throughput screens to discover small molecules that could be used as antiviral drugs. And now, she’s using that experience to find an antiviral therapeutic against SARS-CoV-2. For her high-throughput screens, Dr. Jonsson used the CellTiter-Glo® Cell Viability Assay to determine whether the small molecules inhibit virus-induced cell death. Read more here.
"The CellTiter-Glo Assay provides a very robust, easy-to-use reagent for high-throughput screening of small molecules for antiviral activity against viruses such as SARS CoV and SARS CoV-2 that cause a cytopathic effect in cell lines."
Dr. Colleen Jonsson, Director of the Regional Biocontainment Laboratory and Director of the Institute for the Study of Host-Pathogen Systems, University of Tennessee Health Science Center.
How the coronavirus enters cells and how to block it
Read about viral host interactions, and how cell viability assays support these studies.
Solutions for Cell Viability, Metabolism and Proteomics Studies
CellTiter-Glo® Luminescent Cell Viability Assay
Determine the number of viable cells in culture based on quantitation of ATP present.
G7570, G7571, G7572, G7573
Glucose-Glo™ Assay
Easily detect glucose from a variety of biological samples.
J6021, J6022
Trypsin/Lys-C Mix, Mass Spec Grade
Cleavage sites: C-terminal of Arg and Lys. Optimal pH: 8.
V5071, V5072, V5073
References
Evaluation of SARS-CoV-2 inhibitors using cell viability assay
Hoffman, M., et al. (2020) SARS-CoV-2 Cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell online 5 Mar 2020.
Sheahan, T. et al. (2020) An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and mutlipe endemic, epidemic and bat coronavirus BioRxIV March 2020.
Overview of SARS-CoV-2 metabolic impact
Bornstein, S. et al. (2020) Endocrine and metabolic link to coronavirus infection. Nat. Rev. Endocrinol. https://www.nature.com/articles/s41574-020-0353-9
Proteomics in SARS-CoV-2 infection
Bojkova, D. et al. (2020)SARS-CoV-2 infected host cell proteomics reveal potential therapy targets. DOI: 10.21203/rs.3.rs-17218/v1 Accessed April 17, 2020.
Monitor Immune Response
A key question asked by viral researchers is how a host’s immune system responds to the viral infection, including both adaptive and innate immunity. The innate immune response involves activation of inflammasomes and cytokine release. While this cytokine release is therapeutic, a “cytokine storm” can be an adverse side effect in patients that must be managed with additional therapeutics designed to block the efficacy of the released cytokines such as IL-6 and IL-15.
In addition to the innate immune response, humoral and cellular responses play key roles in host response. These questions can be answered with reporter bioassays as well as assays to detect the activity of caspase-1 and levels of cytokines in cell-based research applications.
Bioassays and Inflammation Assays
Caspase-Glo® 1 Inflammasome Assay
A homogeneous, bioluminescent method to selectively measure the activity of caspase-1, an essential component of the inflammasome.
G9951, G9952, G9953
ADCC Reporter Bioassays, V Variant
Measure potency and stability of antibodies mediating ADCC through the high-affinity human FcγRIIIa-V receptor.
G7015, G7014, G7010, G7016, G7013, G7018
IL-6 Bioassay
Measure the potency and stability of biologics designed to activate or inhibit the interaction of IL-6 and IL-6R.
J2992, JA2501, JA2505
Lumit™ Cytokine Detection Immunoassays
Assays to measure target analytes in cell culture samples with a simple, no-wash assay protocol.
References
Inflammasome activation in viral infection
Soderholm, S. et al. (2016) Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages (2016) Molecular & Cellular Proteomics 15(10), 3203–3219.
Inflammasome activation in SARS-CoV-2 infection
Conti P. et al. (2020) Induction of pro-inflammatory cytokines (IL-1 and IL-6) and lung inflammation by Coronavirus-19 (CoV-19 or SARS-CoV-2): anti-inflammatory strategies.J. Biol. Regul. Homeost. Agents Mar 14; 34(2) doi: 10.23812/CONTI-E [Epub ahead of print].
Use of a reporter bioassays to study neutralizing antibodies
Pinto, D., et al. (2020) Structural and functional analysis of a potent sarbecorvirus neutralizing antibody. bioRxiv posted 9 Apr 2020.
RNA Production for Vaccine Research and Development
Transcribed RNA is required for vaccine production, viral standards, and basic viral research. For in vivo and in vitro studies, RiboMAX® RNA Production System generates a large quantity of high-quality RNA or mRNA from a DNA template without the need for mammalian cells or cell components. These in-vitro transcribed viral RNA or mRNA transcripts, typically encoding a disease-specific antigen such as the spike protein of a coronavirus, may be used as inoculation material for viral infection studies. If the transcribed mRNA is to be used as a therapeutic, the mRNA encoding a desirable protein can be packaged as necessary for delivery to the tissue of interest.
References
Generation of Coronavirus RNA
Eriksson K.K., et al. (2008) Generation of Recombinant Coronaviruses Using Vaccinia Virus as the Cloning Vector and Stable Cell Lines Containing Coronaviral Replicon RNAs. In: Cavanagh D. (eds) SARS- and Other Coronaviruses. Methods in Molecular Biology (Methods and Protocols), vol 454. Humana Press, Totowa, NJ.
RNA preparation
Ding X., et al. (2020) All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-Cov-2 and HIV virus. BioRxiv accessed April 13 2020. doi.org/10.1101/2020.03.19.998724
Standard RNA production with RiboMAX
Xu, L. et al. (2009) Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-Time Quantitative Reverse Transcription-PCR Assays. Virologica Sinica, 24 (3), 187-193. DOI 10.1007/s12250-009-3021-8
Bioluminescence Detection
The bioluminescence technology used in Promega reagents for viral research make the assays highly sensitive and simple to use. GloMax® plate-readers come with pre-loaded protocols that make reading and interpreting results from Promega luminescence assays even simpler.