SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus

Havranek, Katherine E.; Jimenez, Ariana R; Acciani, Marissa Danielle; Mendoza, Maria Fernanda Lay; Ballista, Judith Mary Reyes; Diaz, Darren; Brindley, Melinda A.

doi:10.3390/v12121465

Cited by 36 publications

(38 citation statements)

References 63 publications

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“…However, when low levels of ACE2 (50 to 250 ng of plasmid) were co-expressed with either AXL or TIM-1, the combinations enhanced infection over that observed with ACE2 alone. TIM-1-enhanced ACE2-dependent recombinant VSV/Spike (rVSV/Spike) (21) infection, and enhancement occurred over a wider range of ACE2 concentrations than AXL-enhanced ACE2-dependent infection, with AXL consistently enhancing infection when 250 ng of ACE2 plasmid was transfected ( Fig. 1B-C, S1C ).…”

Section: Resultsmentioning

confidence: 99%

Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

Bohan

Ert

Ruggio

et al. 2021

Preprint

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Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.

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Section: Resultsmentioning

confidence: 99%

Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

Bohan

Ert

Ruggio

et al. 2021

Preprint

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“…10 Briefly, pseudotyped virus particles containing a luciferase reporter for detection were made from a modified vesicular stomatitis virus (VSVΔG) backbone expressing the full-length spike glycoprotein of SARS-CoV-2 (MN908947, Wuhan-Hu-1) from which the last 19 amino acids of the cytoplasmic tail were removed. 11 Seven two-fold serial dilutions of heat-inactivated serum samples were prepared in 96-well roundbottom transfer plates (Corning). Pseudotyped virus was added to the serum dilutions at a target working dilution and incubated at 37°C with 5% CO2 for 60 ± 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

Safety and Immunogenicity of an Inactivated Recombinant Newcastle Disease Virus Vaccine Expressing SARS-CoV-2 Spike: Interim Results of a Randomised, Placebo-Controlled, Phase 1/2 Trial

Pitisuttithum

Mercer

White

et al. 2021

Preprint

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Production of affordable coronavirus disease 2019 (COVID-19) vaccines in low- and middle-income countries is needed. NDV-HXP-S is an inactivated egg-based Newcastle disease virus vaccine expressing the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is being developed in Thailand, Vietnam, and Brazil; herein are initial results from Thailand. This phase 1 stage of a randomised, dose-escalation, observer-blind, placebo-controlled, phase 1/2 trial was conducted at the Vaccine Trial Centre, Mahidol University (Bangkok). Healthy adults aged 18-59 years, non-pregnant and negative for SARS-CoV-2 antibodies were eligible. Participants were block randomised to receive one of six treatments by intramuscular injection twice, 28 days apart: 1 microgram (mcg) +/- CpG1018 (a toll-like receptor 9 agonist), 3 mcg +/- CpG1018, 10 mcg, or placebo. Participants and personnel assessing outcomes were masked to treatment. The primary outcomes were solicited and spontaneously reported adverse events (AEs) during 7 and 28 days after each vaccination, respectively. Secondary outcomes were immunogenicity measures (anti-S IgG and pseudotyped virus neutralisation). An interim analysis assessed safety at day 57 in treatment-exposed individuals and immunogenicity through day 43 per protocol. ClinicalTrials.gov (NCT04764422). Between March 20 and April 23, 2021, 377 individuals were screened and 210 were enrolled (35 per group); all received dose one; five missed dose two. The most common solicited AEs among vaccinees, all predominantly mild, were injection site pain (<63%), fatigue (<35%), headache (<32%), and myalgia (<32%). The proportion reporting a vaccine-related AE ranged from 5.7% to 17.1% among vaccine groups and was 2.9% in controls; there was no vaccine-related serious adverse event. The immunogenicity of the 10 mcg formulation ranked best, followed by 3 mcg+CpG1018, 3 mcg, 1 mcg+CpG1018, and 1 mcg formulations. On day 43, the geometric mean concentrations of 50% neutralising antibody ranged from 122.23 IU/mL (1 mcg, 95% CI 86.40-172.91) to 474.35 IU/mL (10 mcg, 95% CI 320.90-701.19), with 93.9% to 100% of vaccine groups attaining a => 4-fold increase over baseline. NDV-HXP-S had an acceptable safety profile and potent immunogenicity. The 3 mcg and 3 mcg+CpG1018 formulations advanced to phase 2.

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“…To further assess the ability of the PS receptors to facilitate ACE2-dependent infection, the amount of transfected PS receptor plasmid was held constant with increasing concentrations of ACE2. While both PS receptors enhanced ACE2-dependent virus infection, TIM-1 enhanced recombinant replication-competent VSV that encoded SARS-CoV-2 spike (rVSV/ Spike) [20] infection over a wider range of ACE2 concentrations than AXL did, with AXL consistently enhancing infection only at 250 ng of transfected ACE2 plasmid ( Figs 1B, 1C and S1C). The more limited ability of AXL to potentiate was not due to limiting Gas6 in the media as the addition of Gas6 to media did not enhance the effect.…”

Section: Ps Receptors Enhance Ace2-dependent Sars-cov-2 Infectionmentioning

confidence: 98%

Phosphatidylserine receptors enhance SARS-CoV-2 infection

et al. 2021

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Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

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SARS-CoV-2 Spike Alterations Enhance Pseudoparticle Titers and Replication-Competent VSV-SARS-CoV-2 Virus

Cited by 36 publications

References 63 publications

Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19

Safety and Immunogenicity of an Inactivated Recombinant Newcastle Disease Virus Vaccine Expressing SARS-CoV-2 Spike: Interim Results of a Randomised, Placebo-Controlled, Phase 1/2 Trial

Phosphatidylserine receptors enhance SARS-CoV-2 infection

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