scholarly journals Development of a Single-cycle Infectious SARS-CoV-2 Virus Replicon Particle System for use in BSL2 Laboratories

2021 ◽  
Author(s):  
Johnny Malicoat ◽  
Senthamizharasi Manivasagam ◽  
Sonia Zuñiga ◽  
Isabel Sola ◽  
Dianne McCabe ◽  
...  
Keyword(s):  
High Throughput ◽  
Envelope Glycoprotein ◽  
High Throughput Screening ◽  
Antiviral Drugs ◽  
Single Cycle ◽  
S Gene ◽  
Replicon Particle ◽  
Dual Reporter ◽  
Level 3 ◽  
Biosafety Level

Research activities with infectious severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The Spike (S) gene of SARS-CoV-2 encodes for the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G (ΔS-VRP(G)) as compared to cells expressing other viral glycoproteins including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G) infected cells with anti-CoV drugs remdesivir (nucleoside analog) or GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug-dose and cell-type dependent manner. Taken together, we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high throughput screening of antiviral drugs under BSL2 containment. Importance Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and antiviral drugs. By deleting the essential Spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high throughput screening of antiviral drugs under BSL2 containment.

scholarly journals High-Throughput Screening of a Diversity Collection Using Biodefense Category A and B Priority Pathogens

2012 ◽  
Vol 17 (7) ◽  
pp. 946-956 ◽  
Author(s):  
Esther W. Barrow ◽  
Patricia A. Clinkenbeard ◽  
Rebecca A. Duncan-Decocq ◽  
Rachel F. Perteet ◽  
Kimberly D. Hill ◽  
...  
Keyword(s):  
High Throughput ◽  
Broad Spectrum ◽  
High Throughput Screening ◽  
Inhibitory Concentration ◽  
Infectious Agents ◽  
Chemical Databases ◽  
Level 3 ◽  
Effective Use ◽  
Biosafety Level

One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute–based high-throughput screening (HTS) 96-well–based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 µg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.

scholarly journals Toward the Development of a Virus-Cell-Based Assay for the Discovery of Novel Compounds against Human Immunodeficiency Virus Type 1

2003 ◽  
Vol 47 (2) ◽  
pp. 501-508 ◽  
Author(s):  
Martin E. Adelson ◽  
Annmarie L. Pacchia ◽  
Malvika Kaul ◽  
Robert F. Rando ◽  
Yacov Ron ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Discovery ◽  
Human Immunodeficiency Virus Type ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cycle ◽  
Vector Systems ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.

scholarly journals Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

2020 ◽  
Author(s):  
Katharine H.D. Crawford ◽  
Rachel Eguia ◽  
Adam S. Dingens ◽  
Andrea N. Loes ◽  
Keara D. Malone ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Spike Protein ◽  
Single Cycle ◽  
Viral Particles ◽  
Main Target ◽  
Human Sera ◽  
Level 3 ◽  
Valuable Complement ◽  
Level 2 ◽  
Biosafety Level

AbstractSARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

scholarly journals High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Jonathan T. Sullivan ◽  
Chidananda Sulli ◽  
Alberto Nilo ◽  
Anila Yasmeen ◽  
Gabriel Ozorowski ◽  
...  
Keyword(s):  
Protein Engineering ◽  
High Throughput ◽  
Envelope Glycoprotein ◽  
High Throughput Screening ◽  
Neutralizing Antibody ◽  
Structural Basis ◽  
Vaccine Candidates ◽  
Clade B ◽  
Broadly Neutralizing Antibody ◽  
Hiv 1

ABSTRACT Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies. IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

scholarly journals Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013

2013 ◽  
Vol 18 (36) ◽  
Author(s):  
R A Perera ◽  
P Wang ◽  
M R Gomaa ◽  
R El-Shesheny ◽  
A Kandeil ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
The Novel ◽  
Excellent Correlation ◽  
Dromedary Camels ◽  
High Prevalence ◽  
Antibody Titres ◽  
Level 3 ◽  
High Throughput Assay ◽  
Biosafety Level

We describe a novel spike pseudoparticle neutralisation assay (ppNT) for seroepidemiological studies on Middle East respiratory syndrome coronavirus (MERS-CoV) and apply this assay together with conventional microneutralisation (MN) tests to investigate 1,343 human and 625 animal sera. The sera were collected in Egypt as a region adjacent to areas where MERS has been described, and in Hong Kong, China as a control region. Sera from dromedary camels had a high prevalence of antibody reactive to MERS-CoV by MERS NT (93.6%) and MERS ppNT (98.2%) assay. The antibody titres ranged up to 1,280 and higher in MN assays and 10,240 and higher in ppNT assays. No other investigated species had any antibody reactivity to MERS-CoV. While seropositivity does not exclude the possibility of infection with a closely related virus, our data highlight the need to attempt detection of MERS-CoV or related coronaviruses in dromedary camels. The data show excellent correlation between the conventional MN assay and the novel ppNT assay. The newly developed ppNT assay does not require Biosafety Level 3 containment and is thus a relatively high-throughput assay, well suited for large-scale seroepidemiology studies which are needed to better understand the ecology and epidemiology of MERS-CoV.

scholarly journals Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 513 ◽  
Author(s):  
Katharine H. D. Crawford ◽  
Rachel Eguia ◽  
Adam S. Dingens ◽  
Andrea N. Loes ◽  
Keara D. Malone ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Spike Protein ◽  
Single Cycle ◽  
Viral Particles ◽  
Human Sera ◽  
Level 3 ◽  
Vesicular Stomatitis ◽  
Valuable Complement ◽  
Level 2 ◽  
Biosafety Level

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

scholarly journals A trans-complementation system for SARS-CoV-2

2021 ◽  
Author(s):  
Xianwen Zhang ◽  
Yang Liu ◽  
Jianying Liu ◽  
Adam L. Bailey ◽  
Kenneth S. Plante ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Transgenic Mice ◽  
Viral Replication ◽  
Cell Line ◽  
High Throughput ◽  
Envelope Gene ◽  
Wild Type ◽  
Level 3 ◽  
Producer Cell ◽  
Biosafety Level

ABSTRACTThe biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

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