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

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.

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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

10.1101/2021.01.29.428442 ◽

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.

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Use of Vesicular Stomatitis Virus Pseudotypes Bearing Hantaan or Seoul Virus Envelope Proteins in a Rapid and Safe Neutralization Test

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.1.154-160.2003 ◽

2003 ◽

Vol 10 (1) ◽

pp. 154-160 ◽

Cited By ~ 50

Author(s):

Michiko Ogino ◽

Hideki Ebihara ◽

Byoung-Hee Lee ◽

Koichi Araki ◽

Åke Lundkvist ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Protein Gene ◽

Fluorescent Protein ◽

Neutralization Test ◽

Hemorrhagic Fever ◽

Envelope Glycoproteins ◽

Green Fluorescent Protein Gene ◽

Virus Envelope ◽

Vesicular Stomatitis

ABSTRACT A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 105 to 106/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVΔG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4°C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

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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

npj Vaccines ◽

10.1038/s41541-021-00352-1 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Delphine C. Malherbe ◽

Drishya Kurup ◽

Christoph Wirblich ◽

Adam J. Ronk ◽

Chad Mire ◽

...

Keyword(s):

Weight Loss ◽

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Specific Binding ◽

Vaccine Dose ◽

Spike Protein ◽

Hamster Model ◽

Rapid Control ◽

Vesicular Stomatitis ◽

Vectored Vaccine

AbstractThe 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 models 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.

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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

10.20944/preprints202005.0379.v1 ◽

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.

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

Viruses ◽

10.3390/v12050513 ◽

2020 ◽

Vol 12 (5) ◽

pp. 513 ◽

Cited By ~ 77

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.

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Novel surrogate virus neutralization test reveals low serum neutralizing anti-SARS-CoV-2-S antibodies levels in mildly affected COVID-19 convalescents

10.1101/2020.07.12.20151407 ◽

2020 ◽

Author(s):

Berislav Bošnjak ◽

Saskia Catherina Stein ◽

Stefanie Willenzon ◽

Anne Katrin Cordes ◽

Wolfram Puppe ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Clinical Symptoms ◽

Neutralization Test ◽

Antibody Therapy ◽

Neutralization Assay ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Iga Antibodies ◽

Vesicular Stomatitis

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells using surface-expressed angiotensin-converting enzyme 2 (ACE2). We developed a surrogate neutralization test (sVNT) to assess at what degree serum antibodies interfere with the binding of SARS-CoV-2-S-RBD to ACE2. The sVNT revealed neutralizing anti-SARS-CoV-2-S antibodies in the sera of 90% of mildly and 100% of severely affected coronavirus-disease-2019 (COVID-19) convalescent patients. Importantly, sVNT results correlated strongly to the results from pseudotyped-vesicular stomatitis virus-vector-based neutralization assay and to levels of anti-SARS-CoV-2-S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies also correlated to duration and severity of clinical symptoms, but not patient age or gender. These findings together with the sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

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Five Commercial Immunoassays for SARS-CoV-2 Antibody Determination and Their Comparison and Correlation with the Virus Neutralization Test

Diagnostics ◽

10.3390/diagnostics11040593 ◽

2021 ◽

Vol 11 (4) ◽

pp. 593

Author(s):

Václav Šimánek ◽

Ladislav Pecen ◽

Zuzana Krátká ◽

Tomáš Fürst ◽

Hana Řezáčková ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Test ◽

Young Patients ◽

Virus Neutralization Test ◽

Virus Neutralization ◽

Age Dependent ◽

Previous Contact ◽

Dependent Elderly ◽

Protein Assays ◽

Antibody Determination

There is an ongoing debate as to whether SARS-CoV-2 antibodies can be found in patients who have recovered from COVID-19 disease. Currently, there is no consensus on whether the antibodies, if present, are protective. Our regular measurements of SARS-CoV-2 antibodies, starting in July 2020, have provided us with the opportunity of becoming acquainted with the five different immunoassays. A total of 149 patients were enrolled in our study. We measured the samples using each immunoassay, then performing a virus neutralization test and comparing the results of SARS-CoV-2 antibodies with this test. We observed that the production of neutralizing antibodies is age-dependent. Elderly patients have a higher proportion of high neutralizing titers than young patients. Based on our results, and in combination with the literature findings, we can conclude that the serological SARS-CoV-2 antibody measurement is a helpful tool in the fight against COVID-19. The assays can provide information about the patient’s previous contact with the virus. Anti-spike protein assays correlate well with the virus neutralization test and can be used in the screening of potential convalescent plasma donors.

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Comparison of Four SARS-CoV-2 Neutralization Assays

Vaccines ◽

10.3390/vaccines9010013 ◽

2020 ◽

Vol 9 (1) ◽

pp. 13

Author(s):

Lydia Riepler ◽

Annika Rössler ◽

Albert Falch ◽

André Volland ◽

Wegene Borena ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

The Other ◽

In Vitro Assays ◽

The Novel ◽

Effective Protection ◽

Vaccine Candidates ◽

Vesicular Stomatitis ◽

Novel Coronavirus

Neutralizing antibodies are a major correlate of protection for many viruses including the novel coronavirus SARS-CoV-2. Thus, vaccine candidates should potently induce neutralizing antibodies to render effective protection from infection. A variety of in vitro assays for the detection of SARS-CoV-2 neutralizing antibodies has been described. However, validation of the different assays against each other is important to allow comparison of different studies. Here, we compared four different SARS-CoV-2 neutralization assays using the same set of patient samples. Two assays used replication competent SARS-CoV-2, a focus forming assay and a TCID50-based assay, while the other two assays used replication defective lentiviral or vesicular stomatitis virus (VSV)-based particles pseudotyped with SARS-CoV-2 spike. All assays were robust and produced highly reproducible neutralization titers. Titers of neutralizing antibodies correlated well between the different assays and with the titers of SARS-CoV-2 S-protein binding antibodies detected in an ELISA. Our study showed that commonly used SARS-CoV-2 neutralization assays are robust and that results obtained with different assays are comparable.

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Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants

10.21203/rs.3.rs-400230/v1 ◽

2021 ◽

Author(s):

Satoshi Ikegame ◽

Mohammed Siddiquey ◽

Chuan-Tien Hung ◽

Griffin Haas ◽

Luca Brambilla ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Cell Line ◽

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

The Novel ◽

Serum Samples ◽

Egfp Reporter ◽

Vesicular Stomatitis ◽

Reduced Activity

Abstract The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4–8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

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Safety, Immunogenicity and Efficacy of a Recombinant Vesicular Stomatitis Virus Vectored Vaccine Against Severe Fever with Thrombocytopenia Syndrome and Heartland Bandaviruses

10.1101/2021.11.29.470508 ◽

2021 ◽

Author(s):

Phillip Hicks ◽

Jonna B. Westover ◽

Tomaz B Manzoni ◽

Brianne Roper ◽

Gabrielle L Rock ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Neutralizing Antibodies ◽

Genetic Manipulation ◽

Case Fatality ◽

Vaccine Platform ◽

Vesicular Stomatitis ◽

Immunocompetent Mice ◽

Vectored Vaccine ◽

Severe Fever ◽

Promising Vaccine Candidate

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a recently emerged tickborne virus in east Asia with over 8,000 confirmed cases. With a high case fatality ratio, SFTSV has been designated a high priority pathogen by the WHO and the NIAID. Despite this, there are currently no approved therapies or vaccines to treat or prevent SFTS. Vesicular stomatitis virus (VSV) represents an FDA-approved vaccine platform that has been considered for numerous viruses due to its low sero-prevalence in humans, ease in genetic manipulation and promiscuity in incorporating foreign glycoproteins into its virions. In this study, we developed a recombinant VSV (rVSV) expressing the SFTSV glycoproteins Gn/Gc (rVSV-SFTSV) and assessed its safety, immunogenicity and efficacy in mice. We demonstrate that rVSV-SFTSV is safe when given to immunocompromised animals and is not neuropathogenic when injected intracranially into young immunocompetent mice. Immunization of Ifnar-/- mice with rVSV-SFTSV resulted in high levels of neutralizing antibodies and protection against lethal SFTSV challenge. Additionally, passive transfer of sera from immunized IFNAR-/- mice into naïve animals was protective when given pre- or post-exposure. Finally, we demonstrate that immunization with rVSV-SFTSV cross protects mice against challenge with the closely related Heartland virus despite low neutralizing titers to the virus. Taken together, these data suggest that rVSV-SFTSV is a promising vaccine candidate.

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