scholarly journals Effective screening of SARS-CoV-2 neutralizing antibodies in patient serum using lentivirus particles pseudotyped with SARS-CoV-2 spike glycoprotein

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ritesh Tandon ◽  
Dipanwita Mitra ◽  
Poonam Sharma ◽  
Martin G. McCandless ◽  
Stephen J. Stray ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Transduction Efficiency ◽  
Pseudotyped Virus ◽  
Spike Glycoprotein ◽  
Highly Pathogenic ◽  
Mammalian Cell Lines ◽  
Serological Studies ◽  
Pathogenic Viruses ◽  
Vesicular Stomatitis

Abstract Pseuodotyped particles have significant importance and use in virology as tools for studying the biology of highly pathogenic viruses in a lower biosafety environment. The biological, chemical, and serological studies of the recently emerged SARS-CoV-2 will be greatly aided by the development and optimization of a suitable pseudotyping system. Here, we pseudotyped the SARS-CoV-2 Spike glycoprotein (SPG) on a traditional retroviral (MMLV) as well as a third generation lentiviral (pLV) vector and tested the transduction efficiency in several mammalian cell lines expressing SARS-CoV-2 receptor hACE2. While MMLV pseudotyped the vesicular stomatitis virus G glycoprotein (VSV-G) efficiently, it could not pseudotype the full-length SPG. In contrast, pLV pseudotyped both glycoproteins efficiently; however, much higher titers of pLV-G particles were produced. Among all the tested mammalian cells, 293Ts expressing hACE2 were most efficiently transduced using the pLV-S system. The pLV-S particles were efficiently neutralized by diluted serum (>:640) from recently recovered COVID-19 patients who showed high SARS-CoV-2 specific IgM and IgG levels. In summary, pLV-S pseudotyped virus provides a valid screening tool for the presence of anti SARS-CoV-2 specific neutralizing antibodies in convalescent patient serum.

scholarly journals Effective Screening of SARS-CoV-2 Neutralizing Antibodies in Patient Serum using Lentivirus Particles Pseudotyped with SARS-CoV-2 Spike Glycoprotein

2020 ◽  
Author(s):  
Ritesh Tandon ◽  
Dipanwita Mitra ◽  
Poonam Sharma ◽  
Stephan J Stray ◽  
John T Bates ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Transduction Efficiency ◽  
Pseudotyped Virus ◽  
Spike Glycoprotein ◽  
Highly Pathogenic ◽  
Mammalian Cell Lines ◽  
Serological Studies ◽  
Pathogenic Viruses ◽  
Vesicular Stomatitis

Pseuodotyped particles have significant importance and use in virology as tools for studying the biology of highly pathogenic viruses in a lower biosafety environment. The biological, chemical, and serological studies of the recently emerged SARS-CoV-2 will be greatly aided by the development and optimization of a suitable pseudotyping system. Here, we pseudotyped the SARS-CoV-2 Spike glycoprotein (SPG) on a retroviral (MMLV) as well as a third generation lentiviral (pLV) vector and tested the transduction efficiency in several mammalian cell lines expressing SARS-CoV-2 receptor hACE2. While MMLV pseudotyped the vesicular stomatitis virus G glycoprotein (VSV-G) efficiently, it could not pseudotype SPG. In contrast, pLV pseudotyped both glycoproteins efficiently; however, much higher titers of pLV-G particles were produced. Among all the tested mammalian cells, 293Ts expressing hACE2 were most efficiently transduced using the pLV-S system. The pLV-S particles were efficiently neutralized by diluted serum (>:640) from a recently recovered COVID-19 patient who showed high SARS-CoV-2 specific IgM and IgG levels. In summary, pLV-S pseudotyped virus provides a valid screening tool for the presence of anti SARS-CoV-2 specific neutralizing antibodies in convalescent patient serum.

scholarly journals Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

2020 ◽  
Author(s):  
Ritesh Tandon ◽  
Joshua S. Sharp ◽  
Fuming Zhang ◽  
Vitor H. Pomin ◽  
Nicole M. Ashpole ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Heparan Sulfate ◽  
Mammalian Cells ◽  
Sulfated Polysaccharides ◽  
Transduction Efficiency ◽  
Pseudotyped Virus ◽  
Spike Glycoprotein ◽  
Viral Activity ◽  
Recent Emergence ◽  
The Impact

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 μg/L, 1.08 mg/L, 1.77 μg/L, and 5.86 mg/L respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes. Importance The recent emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) in Wuhan, China in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in anti-viral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

scholarly journals Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

2020 ◽  
Author(s):  
Sabari Nath Neerukonda ◽  
Russell Vassell ◽  
Rachel Herrup ◽  
Shufeng Liu ◽  
Tony Wang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
293T Cell ◽  
Neutralization Assay ◽  
Target Cells ◽  
Highly Pathogenic ◽  
Angiotensin Converting Enzyme 2 ◽  
Pathogenic Viruses ◽  
Screening Assays ◽  
Assay Precision ◽  
Transmembrane Serine Protease

AbstractPseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

scholarly journals Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

2020 ◽  
Author(s):  
Ritesh Tandon ◽  
Joshua S. Sharp ◽  
Fuming Zhang ◽  
Vitor H. Pomin ◽  
Nicole M. Ashpole ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Intranasal Administration ◽  
Prophylactic Treatment ◽  
Sulfated Polysaccharides ◽  
Transduction Efficiency ◽  
Pseudotyped Virus ◽  
Response Curves ◽  
Angiotensin Converting Enzyme 2 ◽  
Effective Inhibition ◽  
The Impact

AbstractSevere acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 µg/L, 1.08 mg/L, 1.77 µg/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

scholarly journals Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248348
Author(s):  
Sabari Nath Neerukonda ◽  
Russell Vassell ◽  
Rachel Herrup ◽  
Shufeng Liu ◽  
Tony Wang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
293T Cell ◽  
Neutralization Assay ◽  
Target Cells ◽  
Highly Pathogenic ◽  
Angiotensin Converting Enzyme 2 ◽  
Pathogenic Viruses ◽  
Screening Assays ◽  
Assay Precision ◽  
Transmembrane Serine Protease

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

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.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Immune Serum Produced by DNA Vaccination Protects Hamsters against Lethal Respiratory Challenge with Andes Virus

2007 ◽  
Vol 82 (3) ◽  
pp. 1332-1338 ◽  
Author(s):  
Jay W. Hooper ◽  
Anthony M. Ferro ◽  
Victoria Wahl-Jensen
Keyword(s):  
Dna Vaccine ◽  
Neutralizing Antibodies ◽  
Lethal Dose ◽  
Case Fatality ◽  
Immune Serum ◽  
Human Infection ◽  
M Gene ◽  
Hamster Model ◽  
Highly Pathogenic ◽  
Andes Virus

ABSTRACT Hantavirus pulmonary syndrome (HPS) is a highly pathogenic disease (40% case fatality rate) carried by rodents chronically infected with certain viruses within the genus Hantavirus of the family Bunyaviridae. The primary mode of transmission to humans is thought to be inhalation of excreta from infected rodents; however, ingestion of contaminated material and rodent bites are also possible modes of transmission. Person-to-person transmission of HPS caused by one species of hantavirus, Andes virus (ANDV), has been reported. Previously, we reported that ANDV injected intramuscularly causes a disease in Syrian hamsters that closely resembles HPS in humans. Here we tested whether ANDV was lethal in hamsters when it was administered by routes that more accurately model the most common routes of human infection, i.e., the subcutaneous, intranasal, and intragastric routes. We discovered that ANDV was lethal by all three routes. Remarkably, even at very low doses, ANDV was highly pathogenic when it was introduced by the mucosal routes (50% lethal dose [LD50], ∼100 PFU). We performed passive transfer experiments to test the capacity of neutralizing antibodies to protect against lethal intranasal challenge. The neutralizing antibodies used in these experiments were produced in rabbits vaccinated by electroporation with a previously described ANDV M gene-based DNA vaccine, pWRG/AND-M. Hamsters that were administered immune serum on days −1 and +5 relative to challenge were protected against intranasal challenge (21 LD50). These findings demonstrate the utility of using the ANDV hamster model to study transmission across mucosal barriers and provide evidence that neutralizing antibodies produced by DNA vaccine technology can be used to protect against challenge by the respiratory route.

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