Academic Editor: Sang Heui Seo
Background: SARS-CoV-2 is the coronavirus responsible for the COVID-19
pandemic. Although it poses a substantial public health threat, antiviral
regimens against SARS-CoV-2 remain scarce. Here, we evaluated the antiviral
potential of UV-4B, a host targeting antiviral, against SARS-CoV-2 in clinically
relevant human cell lines. Methods: Cells derived from human lung (A549
cells transfected with human angiotensin converting enzyme 2 receptor (ACE2;
ACE2-A549)) and colon (Caco-2) were infected with either a wild type or beta
variant strain of SARS-CoV-2 and exposed to various concentrations of UV-4B.
Supernatant was sampled daily and viral burden was quantified by plaque assay on
Vero E6 cells. Results: Therapeutically feasible concentrations of UV-4B
inhibited the replication of the wild type strain in ACE2-A549 and Caco-2 cells
yielding EC
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the novel coronavirus responsible for the COVID-19 pandemic [1]. Since its emergence in late 2019, SARS-CoV-2 has spread to every continent, causing more than 150 million cases and over 3 million deaths as of April 2021 [2]. Most infected patients will experience mild to moderate disease however, risk for severe infection increases in older patients and those with comorbid conditions [3]. Infected patients commonly exhibit symptoms including fever, muscle pain, headache, cough, shortness of breath, and gastrointestinal (GI) distress. Severe cases of infection may lead to pneumonia, acute respiratory distress syndrome (ARDS), and respiratory failure [1, 3].
Throughout the course of the pandemic, several variant strains of SARS-CoV-2 have emerged. The circulation of variants of concern include those from the United Kingdom, South Africa, Brazil, and India. The variants contain mutations in the spike protein that not only increase viral transmissibility, but also potentially render these strains less susceptible to the protective effect elicited by approved vaccines and monoclonal antibody therapies [4, 5], highlighting the urgent need for antiviral regimens with activity against multiple strains of SARS-CoV-2.
In spite of SARS-CoV-2’s emergence as a major threat to global public health, antiviral therapies approved to treat infection remain scarce. Currently, mild to moderate cases of infection are managed through supportive care measures and, only one antiviral, the RNA polymerase inhibitor remdesivir, has received FDA approval for the treatment of severe cases of COVID-19 infection requiring hospitalization [6, 7].
UV-4B, the hydrochloride salt form of the iminosugar UV-4
(N-(9-methoxynonyl)-1-deoxynojirimycin; MON-DNJ), is a broad-spectrum antiviral
that has demonstrated virustatic or virucidal activity against several RNA
viruses including dengue virus and influenza virus [8, 9, 10]. UV-4B is a host
targeting antiviral that disrupts the viral replication cycle through competitive
inhibition of host cell endoplasmic reticulum
Here, the antiviral activity of UV-4B was evaluated against a wild type strain (USA-WA1/2020) and the beta variant strain of SARS-CoV-2 in two human cell lines. Angiotensin converting enzyme 2 (ACE2) receptor expressing A549 cells (ACE2-A549) are a human lung cell line that were selected since SARS-CoV-2 predominantly targets the lung and other tissues of the respiratory tract [11]. Caco-2 (human colorectal adenocarcinoma) cells, which naturally express the ACE2 receptor, were employed because SARS-CoV-2 infection is known to cause gastrointestinal distress and because of evidence supporting viral replication in the GI tract [11, 12, 13].
The ultimate goal of this study is to demonstrate the antiviral activity of UV-4B against wild type and beta variant strains of SARS-CoV-2 in two human cell lines that serve as in vitro models of tissues targeted by SARS-CoV-2 in man [11, 12, 13], providing a potential therapeutic option for the treatment of COVID-19.
A549 cells stably expressing the angiotensin converting enzyme 2 (ACE2) receptor
(ACE2-A549 cells) [14] were a kind gift from Dr. Shinji Makino. ACE2-A549 cells
were maintained in Dulbecco’s modified Eagle medium (DMEM) (Hyclone; Logan, UT,
USA) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich; St. Louis,
MO, USA) and 1% penicillin-streptomycin solution (Hyclone; Logan, UT, USA).
Caco-2 cells (ATCC HTB-37) were purchased from the American Type Culture
Collection (ATCC; Manassas, VA, USA) and cultured in minimum essential medium
(MEM) (Corning Cellgro; Manassas, VA, USA) supplemented with 20% FBS (Sigma
Aldrich; St. Louis, MO, USA) and 1% penicillin-streptomycin solution (Hyclone;
Logan, UT, USA). Finally, Vero E6 cells (ATCC CRL-1586) were obtained from the
American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in MEM
(Corning Cellgro; Manassas, VA, USA) supplemented with 10% FBS (Sigma Aldrich;
St. Louis, MO, USA) and 1% penicillin-streptomycin solution (Hyclone; Logan, UT,
USA). Cells were incubated at 37
The wild type SARS-CoV-2 strain, isolate USA-WA1/2020, and beta variant, isolate hCoV-19/South Africa/KRISP-EC-K005321/2020 were obtained from Biodefense and Emerging Infectious Research Resources Repository (BEI Resources; Manassas, VA, USA). Viral stocks were propagated on Vero E6 cells as previously described [15].
UV-4B was kindly provided by Emergent BioSolutions (Gaithersburg, MD, USA).
UV-4B stocks of 2809.8
ACE2-A549 or Caco-2 cells were plated onto six-well plates at a cell density of
1
UV-4B cytotoxicity was measured with the commercially available WST-1 cell
proliferation assay (Roche Diagnostics GmbH; Mannheim, Germany). ACE2-A549 or
Caco-2 cells were seeded onto 96-well plates at concentrations of 5000, and
15,000 cells/well, respectively and incubated overnight at 37
EC
Cell proliferation assays were conducted in both cell lines to evaluate UV-4B
for potential cytotoxicity. Our results demonstrated UV-4B was not toxic to
ACE2-A549 cells (CC
Inhibitory Sigmoid E |
|||||||
R |
E |
E |
Hill | EC |
CC | ||
ACE2-A549 Cells | |||||||
Wild-Type | 0.999 | 14.860 (0.14) |
6.865 (0.09) | 1.709 (0.12) | 2.694 (0.14) | ||
Beta Variant | 0.999 | 19.020 (0.23) | 6.643 (0.17) | 1.464 (0.11) | 4.369 (0.27) | ||
Caco-2 Cells | |||||||
Wild-Type | 0.971 | 13.08 (0.66) | 6.819 (0.66) | 0.866 (0.45) | 2.489 (1.32) | ||
Beta Variant | 0.997 | 12.81 (0.19) | 7.686 (0.16) | 1.907 (0.44) | 6.816 (0.70) | ||
Effect of UV-4B on cell viability of ACE2-A549 and Caco-2 cells. ACE2-A549 (A) and Caco-2 (B) cells were treated with different concentrations of UV-4B. Cell viability was measured after three days using the commercially available WST-1 assay according to manufacturer recommendations. Cell viability is reported as percent cell viability relative to an untreated control. Columns represent the mean of 6 independent samples, error bars represent one standard deviation.
UV-4B effectively inhibited replication of the wild type SARS-CoV-2 strain in
ACE2-A549 cells, yielding an EC
Antiviral activity of UV-4B against SARS-CoV-2 in ACE2-A549, and
Caco2 cells. ACE2-A549 (A) and Caco-2 (B) cells were infected with the wild type
strain of SARS-CoV-2 at multiplicities of infection (MOIs) of 0.03, and 0.006,
respectively. ACE2-A549 (C) and Caco-2 (D) cells were infected with the beta
variant strain of SARS-CoV-2 at MOIs of 0.0075 and 0.006, respectively. Infected
cells were treated with different concentrations of UV-4B at concentrations
ranging from 0–400
ACE2-A549 and Caco-2 cells infected with the beta variant strain of SARS-CoV-2
were also sensitive to UV-4B’s antiviral effect but the degree of inhibition
elicited by drug treatment varied slightly between both strains. In ACE2-A549
cells, UV-4B exhibited an EC
The emergence of SARS-CoV-2 in late 2019 initiated a global health crisis that
has affected millions [1]. The appearance of viral variants with the potential to
escape the protective effect afforded by currently available vaccines and
monoclonal antibody therapies highlights the urgent need for antiviral regimens
that exhibit activity against multiple strains of SARS-CoV-2. In this work, the
UV-4B inhibited replication of a wild type and the beta variant strain of
SARS-CoV-2 in ACE2-A549 and Caco-2 cells at low micromolar concentrations in the
absence of cytotoxicity. Further investigation of UV-4B’s anti-SARS-CoV-2
activity is warranted for several reasons. First, because potent activity against
both viral strains was achieved at therapeutically feasible concentrations. A
phase I trial evaluation of oral UV-4B safety, tolerability, and pharmacokinetics
(NCT02061358) showed a one-time dose of 1000 mg was associated with a C
Although therapeutically feasible concentrations of UV-4B inhibited replication of a wild type and variant strain of SARS-CoV-2, the degree of viral inhibition varied between strains in both ACE2-A549 and Caco-2 cell lines since the beta variant was less sensitive to UV-4B effect at the lowest concentrations evaluated. Spike protein mutations present in the beta strain associated with greater viral transmissibility may contribute to between strain variability in UV-4B susceptibility. For example, the D614G mutation may enhance viral infectivity and increase viral entry by increasing spike protein density of released viral particles [22]. The D614G mutation is also predicted to enhance viral virulence by increasing glycosylation at the nearby 616 residue [23, 24]. This variant also expresses several mutations (N501Y, E484K, and K417N substitutions) in the receptor binding domain that are associated with enhanced binding affinity to the ACE2 receptor [4, 5]. It is plausible that alpha-glucosidase enzymes are only partially inhibited at low UV-4B concentrations and the mutations in the variant strain that enhance viral infectivity or increase binding affinity to the ACE2 receptor nullify drug effect at these concentrations.
A potential limitation to this study was all UV-4B regimens were evaluated at static drug concentrations, this does not accurately represent the pharmacokinetic profiles achieved following drug administration in man. Future studies using the hollow fiber infection model will allow for the simulation of pharmacokinetic (PK) profiles associated with oral UV-4B administration in man and provide insight into this drug’s anti-SARS-CoV-2 activity under dynamic concentrations.
The results of this study demonstrate that UV-4B substantially inhibited the replication of both a wild type and a variant strain of SARS-CoV-2 at therapeutically feasible concentrations. Future studies will focus on evaluating UV-4B for its effectiveness against other mutant strains of SARS-CoV-2 as monotherapy as well as in combination with other anti-SARS-CoV-2 agents.
SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; COVID-19,
coronavirus disease 2019; ARDS, acute respiratory distress syndrome; ACE2,
angiotensin converting enzyme 2; ACE2-A549, ACE2 receptor expressing A549 cells;
DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; GI,
Gastrointestinal; MEM, minimum essential medium; MOI, multiplicity of infection;
PBS, phosphate buffered saline; PK, pharmacokinetic; PFU/mL, plaque forming units
per milliliter; EC
Conceptualization, ANB; methodology, EJF and ANB; formal analysis, EJF and ANB; investigation, EJF; resources, ANB and KLW; writing-original draft, EJF and ANB; writing-review and editing, EJF, KLW and ANB; supervision, ANB.
Not applicable.
The authors would like to thank Anthony Treston from Emergent BioSolutions for reviewing the manuscript. The following reagent was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, isolate hCoV-19/South Africa/KRISP-EC-K005321/2020, NR-54008, contributed by Alex Sigal and Tulio de Oliveira. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, isolate USA-WA1/2020, NR-52281.
This research was funded by the Institute for Therapeutic Innovation, University of Florida.
The authors declare no conflict of interest. KLW is employed by Emergent BioSolutions.