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Establishment of replication-competent vesicular stomatitis virus-based recombinant viruses suitable for SARS-CoV-2 entry and neutralization assays.

ABSTRACT: Replication-competent vesicular stomatitis virus (VSV)-based recombinant viruses are useful tools for studying emerging and highly pathogenic enveloped viruses in level 2 biosafety facilities. Here, we used a replication-competent recombinant VSVs (rVSVs) encoding the spike (S) protein of SARS-CoV-2 in place of the original G glycoprotein (rVSV-eGFP-SARS-CoV-2) to develop a high-throughput entry assay for SARS-CoV-2. The S protein was incorporated into the recovered rVSV-eGFP-SARS-CoV-2 particles, which could be neutralized by sera from convalescent COVID-19 patients. The recombinant SARS-CoV-2 also displayed entry characteristics similar to the wild type virus, such as cell tropism and pH-dependence. The neutralizing titers of antibodies and sera measured by rVSV-eGFP-SARS-CoV-2 were highly correlated with those measured by wild-type viruses or pseudoviruses. Therefore, this is a safe and convenient screening tool for SARS-CoV-2, and it may promote the development of COVID-19 vaccines and therapeutics.

SUBMITTER: Li H

PROVIDER: S-EPMC7594855 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Establishment of replication-competent vesicular stomatitis virus-based recombinant viruses suitable for SARS-CoV-2 entry and neutralization assays.

Li Hongyue H Zhao Chaoyue C Zhang Yuhang Y Yuan Fei F Zhang Qi Q Shi Xuanling X Zhang Linqi L Qin Chengfeng C Zheng Aihua A

Emerging microbes & infections 20201201 1

Replication-competent vesicular stomatitis virus (VSV)-based recombinant viruses are useful tools for studying emerging and highly pathogenic enveloped viruses in level 2 biosafety facilities. Here, we used a replication-competent recombinant VSVs (rVSVs) encoding the spike (S) protein of SARS-CoV-2 in place of the original G glycoprotein (rVSV-eGFP-SARS-CoV-2) to develop a high-throughput entry assay for SARS-CoV-2. The S protein was incorporated into the recovered rVSV-eGFP-SARS-CoV-2 particle ...[more]

PMID: 32990161

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Project description:Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

Project description:BackgroundThe worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD.MethodsWe conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed.ResultsThe most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months.ConclusionsThis Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Project description:UnlabelledSeveral arenaviruses are known to cause viral hemorrhagic fever (VHF) in sub-Saharan Africa and South America, where VHF is a major public health and medical concern. The biosafety level 4 categorization of these arenaviruses restricts their use and has impeded biological studies, including therapeutic drug and/or vaccine development. Due to difficulties associated with handling live viruses, pseudotype viruses, which transiently bear arenavirus envelope proteins based on vesicular stomatitis virus (VSV) or retrovirus, have been developed as surrogate virus systems. Here, we report the development of a pseudotype VSV bearing each envelope protein of various species of arenaviruses (AREpv), including the newly identified Lujo virus (LUJV) and Chapare virus. Pseudotype arenaviruses generated in 293T cells exhibited high infectivity in various mammalian cell lines. The infections by New World and Old World AREpv were dependent on their receptors (human transferrin receptor 1 [hTfR1] and α-dystroglycan [αDG], respectively). However, infection by pseudotype VSV bearing the LUJV envelope protein (LUJpv) occurred independently of hTfR1 and αDG, indicating that LUJpv utilizes an unidentified receptor. The pH-dependent endocytosis of AREpv was confirmed by the use of lysosomotropic agents. The fusion of cells expressing these envelope proteins, except for those expressing the LUJV envelope protein, was induced by transient treatment at low pH values. LUJpv infectivity was inhibited by U18666A, a cholesterol transport inhibitor. Furthermore, the infectivity of LUJpv was significantly decreased in the Niemann-Pick C1 (NPC1)-deficient cell line, suggesting the necessity for NPC1 activity for efficient LUJpv infection.ImportanceLUJV is a newly identified arenavirus associated with a VHF outbreak in southern Africa. Although cell entry for many arenaviruses has been studied, cell entry for LUJV has not been characterized. In this study, we found that LUJpv utilizes neither αDG nor hTfR1 as a receptor and found unique characteristics of LUJV glycoprotein in membrane fusion and cell entry. Proper exclusion of cholesterol or some kinds of lipids may play important roles in LUJpv cell entry.

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Establishment of replication-competent vesicular stomatitis virus-based recombinant viruses suitable for SARS-CoV-2 entry and neutralization assays.

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Establishment of replication-competent vesicular stomatitis virus-based recombinant viruses suitable for SARS-CoV-2 entry and neutralization assays.

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