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 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 Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 386 ◽  
Author(s):  
Ferdinand Zettl ◽  
Toni Luise Meister ◽  
Tanja Vollmer ◽  
Bastian Fischer ◽  
Jörg Steinmann ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Pearson Correlation ◽  
Spike Protein ◽  
Virus Neutralization ◽  
Pseudotype Virus ◽  
Vesicular Stomatitis ◽  
Level 1 ◽  
Biosafety Level

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, a new member of the genus Betacoronavirus, is a pandemic virus, which has caused numerous fatalities, particularly in the elderly and persons with underlying morbidities. At present, there are no approved vaccines nor antiviral therapies available. The detection and quantification of SARS-CoV-2-neutralizing antibodies plays a crucial role in the assessment of the immune status of convalescent COVID-19 patients, evaluation of recombinant therapeutic antibodies, and the evaluation of novel vaccines. To detect SARS-CoV-2-neutralizing antibodies, classically, a virus-neutralization test has to be performed at biosafety level 3, considerably limiting the general use of this test. In the present work, a biosafety level 1 pseudotype virus assay based on a propagation-incompetent vesicular stomatitis virus (VSV) has been used to determine the neutralizing antibody titers in convalescent COVID-19 patients. The neutralization titers in serum of two independently analyzed patient cohorts were available within 18 h and correlated well with those obtained with a classical SARS-CoV-2 neutralization test (Pearson correlation coefficients of r = 0.929 and r = 0.939, respectively). Most convalescent COVID-19 patients had only low titers of neutralizing antibodies (ND50 < 320). The sera of convalescent COVID-19 patients also neutralized pseudotype virus displaying the SARS-CoV-1 spike protein on their surface, which is homologous to the SARS-CoV-2 spike protein. In summary, we report a robust virus-neutralization assay, which can be used at low biosafety level 1 to rapidly quantify SARS-CoV-2-neutralizing antibodies in convalescent COVID-19 patients and vaccinated individuals.

scholarly journals Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2

2020 ◽  
Author(s):  
James Brett Case ◽  
Paul W. Rothlauf ◽  
Rita E. Chen ◽  
Zhuoming Liu ◽  
Haiyan Zhao ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Spike Protein ◽  
Glycoprotein Gene ◽  
Level 3 ◽  
Focus Reduction ◽  
High Throughput Imaging ◽  
Biosafety Level

ABSTRACTAntibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune γ-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.

Evaluation of Neutralizing Antibodies against Highly Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of Neutralizing Antibodies Using Vesicular Stomatitis Virus (Vsv) Pseudovirus-Based Assay

2020 ◽  
Author(s):  
Sarah A. Almahboub ◽  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
M-Zaki ElAssouli ◽  
Anwar M. Hashem
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Highly Pathogenic ◽  
S Protein ◽  
Level 3 ◽  
Vesicular Stomatitis ◽  
Biosafety Level ◽  
Detailed Protocol ◽  
S Proteins

Emerging highly pathogenic human coronaviruses (CoVs) represent a serious ongoing threat to the public health worldwide. The spike (S) proteins of CoVs are surface glycoproteins that facilitate viral entry into host cells via attachment to their respective cellular receptors. The S protein is believed to be a major immunogenic component of CoVs and a target for neutralizing antibodies (nAbs) and most candidate vaccines. Development of a safe and convenient assay is thus urgently needed to determine the prevalence of CoVs nAbs in the population, to study immune response in infected individuals, and to aid in vaccines and viral entry inhibitors evaluation. While live virus-based neutralization assays are used as gold standard serological methods to detect and measure nAbs, handling of highly pathogenic live CoVs requires strict bio-containment conditions in biosafety level-3 laboratories. On the other hand, use of replication-incompetent pseudoviruses bearing CoVs S proteins could represent a safe and useful method to detect nAbs in serum samples under biosafety level-2 conditions. Here, we describe a detailed protocol of a safe and convenient assay to generate vesicular stomatitis virus (VSV)-based pseudoviruses to evaluate and measure nAbs against highly pathogenic CoVs. The protocol covers methods to produce VSV pseudovirus bearing the S protein of the Middle East respiratory syndrome-CoV (MERS-CoV) and the severe acute respiratory syndrome-CoV-2 (SARS-CoV-2), pseudovirus titration, and pseudovirus neutralizing assay. Such assay could be adapted by different laboratories and researchers working on highly pathogenic CoVs without the need to handle live viruses in biosafety level-3 environment.

scholarly journals A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adrian Creanga ◽  
Rebecca A. Gillespie ◽  
Brian E. Fisher ◽  
Sarah F. Andrews ◽  
Julia Lederhofer ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Depth Profiling ◽  
Influenza Viruses ◽  
Effective Vaccine ◽  
Viral Gene ◽  
Broadly Neutralizing Antibodies ◽  
Level 2 ◽  
Biosafety Level ◽  
Major Target

AbstractBroadly neutralizing antibodies (bnAbs) have been developed as potential countermeasures for seasonal and pandemic influenza. Deep characterization of these bnAbs and polyclonal sera provides pivotal understanding for influenza immunity and informs effective vaccine design. However, conventional virus neutralization assays require high-containment laboratories and are difficult to standardize and roboticize. Here, we build a panel of engineered influenza viruses carrying a reporter gene to replace an essential viral gene, and develop an assay using the panel for in-depth profiling of neutralizing antibodies. Replication of these viruses is restricted to cells expressing the missing viral gene, allowing it to be manipulated in a biosafety level 2 environment. We generate the neutralization profile of 24 bnAbs using a 55-virus panel encompassing the near-complete diversity of human H1N1 and H3N2, as well as pandemic subtype viruses. Our system offers in-depth profiling of influenza immunity, including the antibodies against the hemagglutinin stem, a major target of universal influenza vaccines.

Achieving Biosafety Level 3 through the Use of Biosafety Level 2 Facilities and Biosafety Level 3 Practices: A Prevalence Survey of Medical Research and Academic Institutions

1997 ◽  
Vol 2 (4) ◽  
pp. 43-46 ◽  
Author(s):  
Robert J. Emery ◽  
Pek Lee ◽  
James Garman
Keyword(s):  
Research Work ◽  
Health And Safety ◽  
Mail Survey ◽  
Prevalence Survey ◽  
Strict Adherence ◽  
Level 3 ◽  
Level 2 ◽  
Safety Guidelines ◽  
Biosafety Level ◽  
Biosafety Level 2

Heightened interest in pathogens with the potential for aerosol transmission and for which prevention and medical treatment is not readily available has resulted in a need for more work environments that meet Biosafety Level 3 (BSL 3) criteria. Recognizing that the facility-based criteria for BSL 3 cannot be achieved by some existing laboratories, the Centers for Disease Control and Prevention (CDC) and National Institutes of Health (NIH) biological safety guidelines provide an option for attaining BSL 3 status through the use of Biosafety Level 2 (BSL 2) facilities and strict adherence to BSL 3 practices (BSL 2/3). Inherent to this provision is a greater emphasis on safe work practices. Since the extent to which this approach is actually used in practice is not known, a nationwide mail survey of medical academic and research institutions was conducted to provide an objective indication of the proportion of BSL 3 operations actually being carried out in the BSL 2/3 mode. The results obtained indicate that 2% of activities designated as BSL 3 in the study population actually achieve this level of protection using the BSL 2/3 approach. The findings quantitatively estimate for the first time the proportion of BSL 3 activities being carried out in this fashion, and can serve as a reference point for future studies to evaluate usage trends. The results also demonstrate the utility of flexible, performance-based health and safety guidelines, as a significant amount of clinical and research work is being accommodated with the BSL 2/3 provision.

scholarly journals A single dose of replication-competent VSV-vectored vaccine expressing SARS-CoV-2 S1 protects against virus replication in a hamster model of severe COVID-19

2021 ◽  
Author(s):  
Delphine C. Malherbe ◽  
Drishya Kurup ◽  
Christoph Wirblich ◽  
Adam J. Ronk ◽  
Chad Mire ◽  
...  
Keyword(s):  
Animal Model ◽  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Specific Binding ◽  
Vaccine Dose ◽  
Spike Protein ◽  
Hamster Model ◽  
Rapid Control ◽  
Vesicular Stomatitis ◽  
Vectored Vaccine

SUMMARYThe development of effective countermeasures against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic, is a priority. We designed and produced ConVac, a replication-competent vesicular stomatitis virus (VSV) vaccine vector that expresses the S1 subunit of SARS-CoV-2 spike protein. We used golden Syrian hamsters as animal model of severe COVID-19 to test the efficacy of the ConVac vaccine. A single vaccine dose elicited high levels of SARS-CoV-2 specific binding and neutralizing antibodies; following intranasal challenge with SARS-CoV-2, animals were protected from weight loss and viral replication in the lungs. No enhanced pathology was observed in vaccinated animals upon challenge, but some inflammation was still detected. The data indicate rapid control of SARS-CoV-2 replication by the S1-based VSV-vectored SARS-CoV-2 ConVac vaccine.

scholarly journals Pseudoparticle Neutralization Assay for Detecting Ebola- Neutralizing Antibodies in Biosafety Level 2 Settings

2015 ◽  
Vol 61 (6) ◽  
pp. 885-886 ◽  
Author(s):  
Alex W H Chin ◽  
Ranawaka A P M Perera ◽  
Yi Guan ◽  
Peter Halfmann ◽  
Yoshihiro Kawaoka ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Level 2 ◽  
Biosafety Level ◽  
Biosafety Level 2

scholarly journals Penicillin-Susceptible, Oxidase-Negative, Nonhemolytic, NonmotileBacillus megateriumin Disguise ofBacillus anthracis

2017 ◽  
Vol 2017 ◽  
pp. 1-4
Author(s):  
Shih Keng Loong ◽  
Boon Teong Teoh ◽  
Jefree Johari ◽  
Chee Sieng Khor ◽  
Juraina Abd-Jamil ◽  
...  
Keyword(s):  
Public Health ◽  
Environmental Conditions ◽  
Health Management ◽  
Bacterial Pathogen ◽  
16S Rdna Sequencing ◽  
Diagnostic Laboratory ◽  
Rdna Sequencing ◽  
Level 3 ◽  
Level 2 ◽  
Biosafety Level

Bacillus anthracisis a bacterial pathogen of major concern. The spores of this bacteria can survive harsh environmental conditions for extended periods and are well recognized as a potential bioterror weapon with significant implications. Accurate and timely identification of thisBacillusspecies in the diagnostic laboratory is essential for disease and public health management. Biosafety Level 3 measures and ciprofloxacin treatment were instituted whenB. anthraciswas suspected from a patient with gangrenous foot. 16S rDNA sequencing was performed to accurately identify the suspected bacterium, due to the superiority of this method to accurately identify clinically isolated bacteria.B. megateriumwas identified as the causative agent and the organism was subsequently treated as a Biosafety Level 2 pathogen.

scholarly journals Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories

2019 ◽  
Vol 57 (2) ◽  
pp. 601-607
Author(s):  
Slavica Vaselek ◽  
Jorian Prudhomme ◽  
Jitka Myskova ◽  
Tereza Lestinova ◽  
Tatiana Spitzova ◽  
...  
Keyword(s):  
Leishmania Infantum ◽  
Blood Feeding ◽  
Sand Fly ◽  
Level 3 ◽  
Infection Parameters ◽  
Leishmania Amastigotes ◽  
The Individual ◽  
Level 2 ◽  
Biosafety Level ◽  
Biosafety Level 2

Abstract Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different levels of biosafety protection. Protocol originally designed for a biosafety level 2 facility was modified for biosafety level 3 facility and infection parameters were compared. Particularly, specially designed plastic containers were used for blood feeding; feeders were placed outside the sand fly cage, on the top of the mesh; feeding was performed inside the climatic chamber; separation of engorged females was done in Petri dishes kept on ice; engorged females were kept in the cardboard containers until dissection. All experiments, conducted in both laboratories, resulted in fully developed late stage infections with high number of parasites and colonization of the stomodeal valve. We demonstrated that protocol originally designed for biosafety level 2 facilities can be successfully modified for other biosafety facilities, depending on the special requirements of the individual institution/laboratory.

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