Abstract
Human SARS-CoV-2 and avian infectious bronchitis virus (IBV) are highly
contagious and deadly coronaviruses, causing devastating respiratory
diseases in humans and chickens. The lack of effective therapeutics
exacerbates the impact of outbreaks associated with SARS-CoV-2 and IBV
infections. Thus, novel drugs or therapeutic agents are highly in
demand for controlling viral transmission and disease progression.
Mesenchymal stem cells (MSC) secreted factors (secretome) are safe and
efficient alternatives to stem cells in MSC-based therapies. This study
aimed to investigate the antiviral potentials of human Wharton’s jelly
MSC secretome (hWJ-MSC-S) against SARS-CoV-2 and IBV infections in
vitro and in ovo. The half-maximal inhibitory concentrations (IC[50]),
cytotoxic concentration (CC[50]), and selective index (SI) values of
hWJ-MSC-S were determined using Vero-E6 cells. The virucidal,
anti-adsorption, and anti-replication antiviral mechanisms of hWJ-MSC-S
were evaluated. The hWJ-MSC-S significantly inhibited infection of
SARS-CoV-2 and IBV, without affecting the viability of cells and
embryos. Interestingly, hWJ-MSC-S reduced viral infection by >90%, in
vitro. The IC[50] and SI of hWJ-MSC secretome against SARS-CoV-2 were
166.6 and 235.29 µg/mL, respectively, while for IBV, IC[50] and SI were
439.9 and 89.11 µg/mL, respectively. The virucidal and anti-replication
antiviral effects of hWJ-MSC-S were very prominent compared to the
anti-adsorption effect. In the in ovo model, hWJ-MSC-S reduced IBV
titer by >99%. Liquid chromatography-tandem mass spectrometry
(LC/MS-MS) analysis of hWJ-MSC-S revealed a significant enrichment of
immunomodulatory and antiviral proteins. Collectively, our results not
only uncovered the antiviral potency of hWJ-MSC-S against SARS-CoV-2
and IBV, but also described the mechanism by which hWJ-MSC-S inhibits
viral infection. These findings indicate that hWJ-MSC-S could be
utilized in future pre-clinical and clinical studies to develop
effective therapeutic approaches against human COVID-19 and avian IB
respiratory diseases.
Keywords: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),
infectious bronchitis virus (IBV), Wharton’s jelly stem cells,
secretome, coronaviruses
1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and
infectious bronchitis virus (IBV) are highly contagious coronaviruses
and leading causes of devastating respiratory tract diseases in humans
and chickens, respectively. Both SRAS-CoV-2 and IBV are enveloped
viruses, with a positive single-stranded RNA genome, and classified
into order Nidovirales, family Coronaviridae, and sub-family
Orthocoronavirinae. SARS-CoV-2 belongs to the Betacoronavirus genus,
while IBV belongs to the Gammacoronavirus genus [[58]1,[59]2,[60]3].
SARS-CoV-2 is the causative agent of the ongoing coronavirus disease 19
(COVID-19) pandemic and is currently the primary global health concern.
It initially emerged in December 2019 in Wuhan, a city in the center of
China, as devastating pneumonia with a previously unknown etiological
pathogen. Subsequently, SARS-CoV-2 has been identified as the causative
agent of COVID-19 and officially classified as a novel human
coronavirus [[61]1]. Like SARS-CoV-2, IBV infection is the cause of a
devastating upper-respiratory tract disease in chickens, known as
infectious bronchitis (IB) disease. The virus was initially isolated
from infected poultry in the USA in the 1930s [[62]4,[63]5]. IBV
infection in poultry flocks causes a substantial economic loss and
affects the meat quality and egg production [[64]6]. There are a
variety of vaccines developed against SARS-CoV-2 and IBV. However, the
high mutation rate and genetic recombination result in the frequent
emergence of new variants of these viruses, which significantly impact
the efficiency of the currently available vaccines [[65]3,[66]7]. The
emergence of new variants and the lack of effective therapeutic agents
for SARS-CoV-2 and IBV infections highlight the need for a proper
treatment to control the viral spread and mitigate disease progression.
Mesenchymal stem cells (MSCs) possess tremendous potential as
therapeutic agents for various human diseases
[[67]8,[68]9,[69]10,[70]11,[71]12]. This potential is not fully
harnessed because of the side effects associated with MCS-based therapy
(MSCT), which include, but are not limited to, tumorigenesis, immune
rejection, and infection [[72]13,[73]14,[74]15,[75]16]. MSCs secrete
many biologically active extracellular factors, including soluble
proteins, nucleic acids, and extracellular vesicles [[76]17]. These
secreted factors are collectively known as stem cells secretome. MSC
secretome (MSC-S) modulates the communications between stem cells and
surrounding cells [[77]18,[78]19]. Cell-free secretome was able to
mimic stem cells’ immunomodulatory and tissue-regenerative abilities
[[79]17,[80]18,[81]20]. Previous studies indicated that MSC-S could
successfully replace stem cells in various MSCT [[82]21,[83]22]. Using
cell-free secretome bypasses MSCT-associated side effects. In addition,
secretome has low immunogenicity compared to their cell of origin, so
it can aid in developing a disease-specific universal therapeutic
protocol [[84]19,[85]23,[86]24,[87]25,[88]26].
Human Wharton-jelly-derived MSCs (hWJ-MSCs) have recently gained more
attention for their superior proliferation rate, immune-privileged
characteristics, and lower carcinogenic profile after in vivo
transplantation [[89]27,[90]28,[91]29]. hWJ-MSCs have characteristics
of embryonic stem cells yet have no ethical concerns. Moreover, it can
be easily isolated from the readily available umbilical cord (UC).
These advantages grant hWJ-MSCs unique features compared to bone marrow
MSCs (BMSCs) and adipose tissue stem cells (ASCs) [[92]30,[93]31]. The
study aimed to evaluate the antiviral potential of hWJ-MSCs secretome
against SARS-CoV-2 and IBV infections, as well as to profile their
protein contents.
2. Materials and Method
2.1. Cells and Viruses
Vero-E6 (African green monkey kidney) cells were used to study the
antiviral effect of hWJ-MSC-S in vitro. Cells were propagated in
Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Waltham, MA, USA)
containing 10% fetal bovine serum (FBS) (GIBCO, Waltham, MA, USA), and
1% antibiotic-antimycotic (AA) mixture (GIBCO, Waltham, MA, USA) at 37
°C in a humidified atmosphere of 5% CO[2]. SARS-CoV-2,
hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)
[[94]32] and IBV, wild type IBV-EGY/CH/CV10-2019 [[95]33], viruses were
propagated in Vero-E6 cells and titrated using plaque titration assay
[[96]34].
2.2. Collection and Processing of Human Umbilical Cords (hUCs)
hUC samples (n = 20) were obtained from the department of obstetrics
and gynecology, Al-Azhar University Hospital in Assiut. This study was
approved by the ethical committees of faculty of medicine and the
faculty of science at Al-Azhar University in Assiut (APPROVAL
NUMBER/ID:202015). Informed consent was obtained from all individuals
who participated in the study. hUCs were collected under sterile
conditions after normal full-term healthy pregnancy from 20 healthy
mothers and transported to the laboratory in phosphate-buffered saline
(PBS) supplemented with antibiotics, 100 U/mL penicillin, and 100 μg/mL
streptomycin. At the laboratory, the outer surface of UC was sterilized
by 70% ethanol and the cord was washed twice with PBS and serum-free
DMEM (GIBCO, USA) to remove excess blood. The UC was cut longitudinally
with a sterile surgical scissor and Wharton’s Jelly (WJ) within the
cord was scraped using a scalpel. Then, the cord blood vessels (two
arteries and one vein) were removed, and the remaining cord tissue (CT)
was collected. WJ and CT were minced separately into 1–2 mm pieces
before digestion with the enzymatic blend for 30 min at 37 °C in a 5%
CO[2] incubator [[97]35].
2.3. Culturing of hUC-Derived Mesenchymal Stem Cells (hUC-MSCs)
Partially digested WJ and CT pieces were plated separately in a
six-well plate with DMEM/F12 (GIBCO, Waltham, MA, USA) supplemented
with 10% FBS and 1% AA solution. Plates were incubated at 37 °C in a 5%
CO[2] incubator [[98]35]. After 7 days, hUC pieces were removed, and
the culture medium was replaced with fresh ones. Cells were grown to
reach 80% confluency before passaging. The isolation was considered
successful if isolated cells were cultured and maintained up to the 5th
passage (P5) without any contamination. Isolated stem cells were
cryopreserved in FBS supplemented with 10% DMSO.
2.4. Flow Cytometry Characterization of hUC-MSCs
hUC-MSC cells were detached using 0.25% Trypsin-EDTA and collected by
centrifugation at 1000× g for 10 min. Then, 10^6 hWJ-MSCs in 100 μL
volume were stained with 10 μL of Peridinium-chlorophyll-protein
(Per-CP)-conjugated anti-CD105/Endoglin (Mouse IgG1; Clone 166707, R&D
Systems, McKinley Place, MN, USA), Carboxyfluorescein
(CFS)-conjugated-CD73 (Mouse IgG2B; Clone 606112, R&D Systems, McKinley
Place, MN, USA), allophycocyanin (APC) conjugated anti-CD90/Thy1 (Mouse
IgG2A; Clone Thy-1A1, R&D Systems, McKinley Place, MN, USA),
phycoerythrin (PE) conjugated anti-CD45 (Mouse IgG1; Clone 2D1, R&D
Systems, McKinley Place, MN, USA), and PE-CD34 (Mouse IgG1; Clone
QBEnd10, R&D Systems, McKinley Place, MN, USA) monoclonal antibodies
for 30 min. The viability of cells was monitored using
7-aminoactinomycin D (7-AAD) staining. A total of 50,000 events were
acquired and analyzed using the FACS Cantor flow cytometer (Becton
Dickinson Biosciences, Franklin Lakes, NJ, USA) and Kaluza analysis
software 1.5a (Beckman Coulter, Brea, CA, USA).
2.5. Collection of hWJ-MSC-S
hWJ-MSCs were grown in a complete growth medium to reach 80% confluency
before replacing the culture medium with a serum-free medium. After 48
h, Conditioned medium (CM) was harvested, centrifuged at 1000× g for 10
min to remove cells residues, and stored at −80 °C until used in
subsequent experiments.
2.6. Cytotoxicity Assay
The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) assay was performed to evaluate the cytotoxicity of hWJ-MSC-S in
Vero-E6 cells as previously described [[99]36]. Briefly, cells were
seeded in a 96-well plate in DMEM containing FBS (10%), and AA solution
(1%). After 24 h, the growth medium was aspirated, and cells were
washed twice with 1X PBS. Then, cells were treated with different
concentrations of hWJ-MSC-CM. At 24 h post-treatment, the medium was
removed, cells were washed with 1x PBS, MTT solution (20 μL/well of
stock solution) was added, and cells were incubated for 4 h. The
absorbance was measured at 450 nm. The percentage of cytotoxicity
compared to the untreated cells was determined with the following
equation:
[MATH:
% cytotoxicity=<
mrow>(absorbance of cells without treat
ment−absorbance of cells with treatment
)×100
absorbance of cells<
mo> without treatment <
/mrow> :MATH]
The plot of % cytotoxicity versus sample concentration was used to
calculate the concentration which exhibited 50% cytotoxicity (CC50).
2.7. Embryotoxicity
Embryotoxicity of hWJ-MSC-S was evaluated using toxicity assay in ovo
as previously described [[100]37,[101]38]. Briefly, different
concentrations of hWJ-MSC-S were inoculated in 0.2 mL volume into the
allantoic cavity of 10-day-old Specific pathogen-free embryonated
chicken eggs (SPF-ECEs) (10 eggs per concentration). The SPF-ECEs were
incubated at 37 °C for 5 days and inspected daily by candling to check
the embryo viability. Eggs inoculated by serum-free DMEM or left
without inoculation were used as negative and blank controls,
respectively.
2.8. Determination of Viral Inhibitory Concentration 50 (IC[50])
The IC50 value of hWJ-MSC-S was determined as previously described
[[102]39,[103]40,[104]41]. Briefly, Vero-E6 cells were seeded at a
concentration of 2.4 × 10^4 in 96-well plates and incubated overnight
at 37 °C and 5% CO[2] condition. Serial dilutions of hWJ-MSC-S were
mixed with 10^3 PFU of virus (SRAS-CoV-2 or IBV) and incubated at room
temperature (RT) for 1 h. Cell monolayers were washed with PBS and
inoculated with hWJ-MSC-CM/virus mixtures, and incubated at 37 °C and
5% CO[2] for 72 h. Next, cells were fixed with 100 μL of 4%
paraformaldehyde for 20 min and stained with 0.1% crystal violet in
distilled water for 15 min at RT. The crystal violet dye was then
dissolved using 100 μL absolute methanol per well, and the optical
density was measured at 570 nm using Anthos Zenyth 200 rt plate reader
(Anthos Labtec Instruments, Heerhugowaard, The Netherlands) [[105]36].
The IC[50] of the hWJ-MSC-S is that required to reduce the
virus-induced cytopathic effect (CPE) by 50%, compared to the untreated
virus control. The infection assay was performed in BSL-3 facility.
2.9. Plaque-Reduction Assay
Plaque-reduction assay was used to determine the antiviral activity of
hWJ-MSC-S as previously described [[106]42]. Briefly, Vero-E6 cells
were seeded at a concentration of 1 × 10^5 in a 6-well plate and
incubated overnight at 37 °C. Following this, 200 µL (10^3 PFU) of the
virus was mixed with different non-toxic concentrations of hWJ-MSC-S
and incubated at RT for 1 h before inoculation onto the monolayer of
Vero-E6 cells. One hour later, the supernatant was aspirated, cells
were washed, overlaid with DMEM medium supplemented with 2% agarose, 1%
AA mixture, and 4% bovine serum albumin (BSA), and incubated at 37 °C.
Three days later, the overlay medium was discarded, and cells were
fixed for 1 h in 10% formalin solution and stained with 0.1% crystal
violet working solution. The infection experiment was performed in
BSL-3 facility. The plaque-forming units (PFUs) were counted, and
percentages of reduction were calculated according to the following
equation:
[MATH: % Viral inhibition=(<
mrow>PFU number of virus control − PFU number after<
/mi> the treatment wit
h hWJ−MSC−
CM) × 100PFU number of virus control :MATH]
2.10. Determination of the Mode of Action
The mode of antiviral activity of hWJ-MSC-S was determined by
evaluation of its virucidal, anti-adsorption, and anti-replication
effects in Vero-E6 cells as previously described [[107]36,[108]43]. To
evaluate the virucidal activity, 200 µL (10^3 PFU) of the virus was
mixed with different non-toxic concentrations of hWJ-MSC-S and
incubated at RT for 1 h before infecting the cell monolayer for 1 h at
37 °C. To determine the anti-adsorption effect, the cell monolayer was
pre-treated with different concentrations of hWJ-MSC-S for 2 h at 4 °C
before infection with 200 µL (10^3 PFU) of the virus for 1 h at 37 °C.
In another set of experiments, the cell monolayer was infected with 200
µL (10^3 PFU) of the virus at first for 1 h at 37 °C and then treated
with hWJ-MSC-S for 1 h at 37 °C to determine the anti-replication
effect. In all protocols, the supernatant was aspirated, cells were
washed, and incubated with DMEM medium supplemented with 2% agarose, 1%
AA mixture, and 4% BSA at 37 °C. Three days later, the overlay medium
was discarded, and cells were fixed for 1 h in 10% formalin solution
and stained with 0.1% crystal violet working solution. The PFUs were
counted, and the percentages of reduction were calculated as described
above.
2.11. In Ovo Anti-IBV Activity of hWJ-MSC-S
IBV (10^5 EID[50]/0.1 mL) was mixed with 0.1 mL of different non-toxic
concentrations of hWJ-MSC-S and incubated at RT for 1 h before
inoculation into the allantoic cavity of 10-day-old SPF-ECEs (five eggs
per concentration). Inoculated SPF-ECEs were incubated at 37 °C for 5
days and inspected daily by candling to check the embryo viability
[[109]44]. SPF-ECEs inoculated by DMEM or left without inoculation were
used as a negative and blank control, respectively. Three days
post-inoculation, allantoic fluid was collected and subjected to RNA
extraction and reverse transcription-quantitative real-time polymerase
chain reaction (RT-qPCR) to detect IBV copies.
2.12. RNA Extraction and RT-qPCR to Detect IBV
Viral RNA was extracted from allantoic fluids using the QIAamp Viral
RNA mini kit (Qiagen, Hilden, Germany) as per the manufacturer’s
recommendations. The RNA concentration was measured with a NanoDrop
ND-2000 spectrophotometer (Thermo Fisher Scientific< Waltham, MA, USA).
The forward primer IBV (IBV-F: 5′-ATGCTCAACCTTGTCCCTAGCA-3′), reverse
primer (IBV-R: 5′-TCAA-ACTGCGGATCA-TCACGT-3′), and TaqMan^® probe
(IBV-TM: FAM-TTGGAAGTAGAGTGACGCC-CAAACTTCA-BHQ1) specific to IBV N gene
were used to determine IBV copy numbers in one step RT-qPCR [[110]7].
The master mix was utilized in a total volume of 25 µL containing 12.5
µL of QuantiTect RT-PCR kit (Qiagen, Hilden, Germany), 0.5 µL of each
primer (50 pmol), 0.125 µL (30 pmol) of the probe, 0.25 µL of
RT-enzyme, 8.125 µL of RNase-free water and 3 µL of RNA template
[[111]45]. The reaction was performed and analyzed using a Stratagene
MX3005P real-time PCR machine (Agilent technologies, Santa Clara, CA,
USA).
2.13. Mass Spectrometry Analysis of hWJ-MSC-CM
Proteins were precipitated from hWJ-MSC-S samples collected with
four-times chilled Acetone. After incubation at −80 °C for 30 min and
at −20 °C overnight, samples were centrifuged at 10,000 rpm for 30 min.
The protein extract of hWJ-MSC-S was denaturated by placing 50 μL lysis
solution (8 M urea, 500 mM Tris HCl, pH 8.5) with complete
ultra-proteases (Roche, Mannheim, Germany). Samples were incubated at
37 °C for 1 h with occasional vortex, and then centrifuged at 12,000
rpm for 20 min. The protein concentration of hWJ-MSC-S was assayed
using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA)
at Å562 nm before digestion. Next, 30 µg of hWJ-MSC-S protein was
subjected to in-solution digestion. In brief, protein pellets were
re-suspended in an 8 M urea lysis solution and reduced with 5 mM tris
2-carboxyethyl phosphine (TCEP) for 30 min. The alkylation of cysteine
residues was performed using 10 mM iodoacetamide for 30 min in a dark
area. Samples were diluted to a final concentration of 2 M urea with
100 mM Tris-HCl, pH 8.5, before digestion with trypsin [[112]46]. For
endopeptidase digestion, modified procaine trypsin (Sigma, Darmstadt,
Germany) was added at 30: 1 (protein: protease mass ratio) and
incubated overnight in a thermo-shaker at 600 rpm at 37 °C. The
digested peptide solution was acidified using 90% formic acid to a
final pH of 2.0. The resultant peptide mixture herein was cleaned up
using the stage tip as discussed earlier [[113]46]. Each sample was run
in triplicates. Nano-LC MS/MS analysis was performed using Triple TOF
5600 + (AB Sciex, Darmstadt, Canada) interfaced at the front end with
Eksigent nanoLC 400 autosamplers with Ekspert nanoLC 425 pump. On trap
and elute mode, peptides were trapped on CHROMXP C18CL 5 μm (10 × 0.5
mm) (AB Sciex, Darmstadt, Germany). MS and MS/MS ranges were 400–1250
m/z and 170–1500 m/z, respectively. A design of 120-min linear gradient
3–80% solution (80% ACN, 0.2% formic acid) was used. The 40 most
intense ions were sequentially selected under data-dependent
acquisition (DDA) mode with a charge state 2–5. For each cycle, survey
full scan MS and MS/MS spectra were acquired at a resolution of 35.000
and 15.000, respectively. To ensure accuracy, external calibration was
scheduled and run during sample batches to correct possible TOF
deviation.
2.14. Bioinformatics Analysis
Mascot generic format (mgf) files were generated from the raw file
using the script supplied by AB Sciex. MS/MS spectra were searched
using X Tandem in Peptide shaker (version 1.16.26) against Uniprot
Homosapiens (Swiss-prot and TrEMBL database containing 224,139
proteins) with target and decoy sequences. The search space included
all fully and semi-tryptic peptide candidates with a maximum of 2
missed cleavages. Precursor mass and fragment mass were identified with
an initial mass tolerance of 20 ppm and 10 ppm, respectively. The
carbamidomethylation of cysteine (+57.02146 amu) was considered as a
static modification and oxidation at methionine (+15.995), acetylation
of protein N- terminal and K (+42.01 amu), and pyrrolidone from
carbamidomethylated C (−17.03 amu) as variable modification. To assure
a high-quality result, the false discovery rate (FDR) was kept at 1% at
the protein level. Final assembly and emerging of sample replicates
were applied to generate the final outputs of each sample using
in-house software ‘ProteoSelector’
([114]https://www.57357.org/en/department/proteomics-unit-dept/in-house
-bioinformatics-tools/ (accessed on 3 December 2021)) ([115]Tables S1
and S2). Normalized spectral abundance factor (NSAF) was averaged for
each protein [[116]47,[117]48].
2.15. Gene Ontology and Pathway Enrichment Analysis
Gene ontology was performed on uniquely retrieved genes from the
consolidated proteome profile of hWJ-MSC-S and searched against gene
ontology database biological process, molecular function, cellular
component, and protein class analysis was utilized with Fisher’s Exact
test with significance level 0.05 and 5% FDR. Or pathway enrichment
analysis, Enrichr’s web-based tool was used for pathway enrichment
analysis with a COVID-19 database search [[118]49,[119]50]. The
retrieved result was filtered at an adjusted p-value of 0.05 and an
odds ratio of more than 1.
2.16. Statistical Analysis
Data were shown as means ± standard deviation (SD) of three independent
experiments. One-way ANOVA and Student’s t-test were used to compare
different treatments and quantify significance using Prism (5.0;
GraphPad Software, La Jolla, CA, USA). p values < 0.05 were considered
statistically significant. Graphics were drawn by R [[120]51] and
Prism.
3. Results
3.1. Isolation and Characterization of hUCMSC
The overall isolation of UC-MSCs was successful in 15 out of 20 (75%)
of the collected umbilical cords. Cells were successfully isolated from
12 out of 15 (80%) and 6 out of 15 (40%) of the collected WJ and CT
samples, respectively. The isolation rate was significantly higher in
WJ than in CT, as shown in [121]Figure 1A. Isolated cells showed normal
spindle-shaped stem cells with adherent properties ([122]Figure 1B).
There were no cell morphology differences between WJ- and CT-derived
stem cells. Flow cytometry characterization of isolated UC-MSCs showed
that they were positive for the expression of MSC markers CD73, CD105,
and CD90, whereas they were negative for hematopoietic stem cell
markers CD45 and CD34 ([123]Figure 1C). hWJ-MSCs were further
maintained for 48 h to collect their secretome, as illustrated in
[124]Figure 1D.
Figure 1.
[125]Figure 1
[126]Open in a new tab
Isolation and characterization of hUCMSCs. (A) Percentage of the
isolation success rate of hUCMSCs. (B) Morphology of hWJ-MSCs was
observed under light microscopy at 200× magnification. (C) Flow
cytometry characterization of hWJ-MSCs demonstrates positive expression
of mesenchymal stem cells markers: CD105, CD73, CD90, and negative
expression of hematopoietic stem cells markers: CD45, and CD34. (D)
Schematic depicting the collection of hWJ-MSCs secretome.
3.2. Cytotoxicity and Antiviral Activity of hWJ-MSC-S
The cytotoxicity and antiviral activity of hWJ-MSC-S against SARS-CoV-2
and IBV were evaluated using the Vero-E6 cell line. The half-maximal
inhibitory concentration (IC[50]), half-maximal cytotoxicity
concentration (CC[50]), and selective index (SI) of hWJ-MSC-S were
calculated. To determine CC[50], Vero-E6 cells were treated with
different concentrations of hWJ-MSC-S, and the cytotoxicity levels were
measured using an MTT assay. The CC50 value of hWJ-MSC-S was 39,200
µg/mL ([127]Figure 2A). To evaluate the antiviral effect of hWJ-MSC-CM,
SARS-CoV-2 and IBV were individually treated with multiple non-toxic
concentrations of hWJ-MSC-S before infecting Vero-E6 cells. The
antiviral activity of hWJ-MSC-S was determined using a cytopathic
effect assay, and plaque-reduction assay. hWJ-MSC-S significantly
reduced the cytopathic effect induced by SARS-CoV-2 and IBV infection
in Vero-E6 ([128]Figure 2B). IC50 values of hWJ-MSC-S against
SARS-CoV-2 and IBV were 166.6 µg/mL and 439.9 µg/mL, respectively
([129]Figure 2B). In addition, the SI (CC50/IC50) value of hWJ-MSC-S
against SARS-CoV-2 and IBV was 235.29 and 89.11, respectively
([130]Table 1). Similarly, the plaque reduction assay showed that
plaque formed by SARS-CoV-2 and IBV was reduced by hWJ-MSC-S in a
dose-dependent manner ([131]Figure 2C). hWJ-MSC-S reduced PFU/mL of
SARS-CoV-2 and IBV by >90%, at a concentration of 1000 µg/mL
([132]Figure 2C), while at a concentration of 250 µg/mL of hWJ-MSC-S,
there was a variation in the viral inhibition between SARS-CoV-2 and
IBV ([133]Figure 2C). At a 125 µg/mL concentration, the antiviral
activities of hWJ-MSC-S against both SARS-CoV-2 and IBV were
significantly decreased.
Figure 2.
[134]Figure 2
[135]Open in a new tab
Cytotoxicity and antiviral activity of hWJ-MSC-S. (A) Cytotoxicity
concentration (CC[50]) of hWJ-MSC-S on Vero-E6 cells. Cells were
treated with different concentrations of hWJ-MSC-S for 24 h. The
cytotoxicity levels were measured using an MTT assay. (B) Half-maximal
inhibitory concentration (IC[50]) of hWJ-MSC-S against SARS-CoV-2
infection (red line) and IBV infection (green line) in Vero-E6 cells.
Virus incubated with different concentrations of hWJ-MSC-S for 1 h
before infecting Vero-E6 cells. The IC[50] was calculated as the
concentration of hWJ-MSC-S that was required to reduce the
virus-induced cytopathic effect (CPE) by 50%, compared to the virus
control. (C) Reduction in plaque formation after treatment of
SARS-CoV-2 and IBV with different concentrations of hWJ-MSC-S (125,
2,505,001,000 µg/mL). IC[50] and CC[50] values were calculated using
nonlinear regression analysis by plotting log inhibitor versus
normalized response (Variable slope). Results are shown as means ± SD
of three independent experiments, each run in triplicate. The p value
** p < 0.01 indicates the significant correlation between the antiviral
activities of hWJ-MSC-S against SARS-CoV-2 vs IBV.
Table 1.
CC[50], IC[50], and SI of hWJ-MSC-S.
Virus Cell CC[50]
(µg/mL) IC[50]
(µg/mL) SI
(CC[50]/IC[50])
SARS-CoV-2 Vero-E6 39,200 166.6 235.29
IBV Vero-E6 3900 439.9 8.87
[136]Open in a new tab
CC[50], 50% cytotoxic concentration; IC[50,] 50% inhibitory
concentration; SI, Selective index (CC[50]/IC[50]).
3.3. Determination of the Antiviral Mechanism of hWJ-MSC-S
The hWJ-MSC-S mode of action was determined by investigating the
antiviral activity of hWJ-MSC-S against SARS-CoV-2 and IBV infection in
Vero-E6 cells, using three different antiviral protocols: (i)
virucidal, (ii) inhibition of viral adsorption, and (iii) inhibition of
viral replication, as illustrated in [137]Figure 3A. Although hWJ-MSC-S
inhibited SARS-CoV-2 infection in all antiviral protocols, there was a
significant variation in the percentage of inhibition among different
antiviral assays and hWJ-MSC-S concentrations ([138]Figure 3B,C,
[139]Figures S1 and S2). At a 1000 µg/mL concentration, hWJ-MSC-S
inhibited SARS-CoV-2 infection by >95%, 85%, and 42% in virucidal,
anti-replication, and anti-adsorption protocols, respectively
([140]Figure 3B and [141]Figure S1), whereas 500 µg/mL and 250 µg/mL
concentrations of hWJ-MSC-S inhibited viral infection by >70%, >60%,
and <50% in virucidal, anti-replication, and anti-adsorption protocols,
respectively. These results indicated that the hWJ-MSC-S inhibited
SARS-CoV-2 infection directly by inactivating the virion and indirectly
by inhibiting viral replication. Interestingly, hWJ-MSC-S inhibited IBV
infection similarly to SARS-CoV-2. At 1000 µg/mL concentration,
hWJ-MSC-S inhibited IBV infection by >90% using virucidal and
anti-replication protocols, and >50% inhibition was observed in the
anti-adsorption protocol ([142]Figure 3C and [143]Figure S2), while at
500 µg/mL concentration of hWJ-MSC-S, viral inhibition was >60% in
virucidal and anti-replication protocols, and <40% in the
anti-adsorption protocol. At 250 and 125 µg/mL concentrations, the
antiviral effect of hWJ-MSC-S against SARS-CoV-2 and IBV was
significantly reduced ([144]Figure 3B,C, [145]Figures S1 and S2).
Figure 3.
[146]Figure 3
[147]Open in a new tab
Antiviral mechanism of hWJ-MSC-S. (A) Schematic depicting the
experimental protocols used for the assessment of antiviral mechanisms
of hWJ-MSC-S. Virucidal, anti-adsorption, and anti-replication
activities of hWJ-MSC-S against SARS-CoV-2 (B) and IBV (C) were
evaluated in Vero E6 cells and measured by plaque reduction assay.
Results are shown as means ± SD of three independent experiments, each
run in triplicate. The p value; * p < 0.05, ** p < 0.01, *** p < 0.001
indicated the significant correlation among antiviral assays.
3.4. Determination of in Ovo Anti-IBV Activity of hWJ-MSC-S
The embryotoxicity and antiviral activity of hWJ-MSC-S against IBV were
evaluated using 10-day-old SPF-ECEs. The lethal dose 50 (LD[50]) was
assessed by inoculating SPF-ECEs with various hWJ-MSC-S concentrations
and embryo viability was checked daily. The LD[50] was calculated as
the concentration that causes the death of 50% of inoculated embryos.
The LD[50] value of hWJ-MSC-S was 4121 µg/mL ([148]Figure 4A). To
evaluate the inhibitory effect of hWJ-MSC-S against IBV in the in ovo
model, IBV was treated with different non-toxic concentrations of
hWJ-MSC-S for 1 h before inoculation into the allantoic cavity of
SPF-ECEs, as illustrated in [149]Figure 4B. After 72 h, the allantoic
fluid was collected from all groups and subjected to RT-qPCR to
evaluate the reduction in IBV titer. hWJ-MSC-S significantly reduced
the virus titer ([150]Figure 4C). Treating IBV with hWJ-MSC-S at a 1000
µg/mL concentration significantly reduced the viral titer in the
allantoic fluid of SPF-ECEs. Viral titer was reduced from 763,000 ±
32,638 EID[50]/[mL] to 38.333 ± 6.0093 EID[50]/[mL] ([151]Figure 4C).
At a concentration of 500 µg/mL, hWJ-MSC-S reduced IBV titer from
763,000 ± 32,638 EID[50]/[mL] to 1896 ± 926.1 EID[50]/[mL]. In
addition, 250 µg/mL of hWJ-MSC-S reduced IBV titer from 763,000 ±
32,638 EID[50]/[mL] to 20,380 ± 2885 EID[50]/[mL], while 125 µg/mL of
hWJ-MSC-S reduced IBV titer from 763,000 ± 32,638 EID[50]/[mL] to
437,700 ± 18,480 EID[50]/[mL] ([152]Figure 4C). Percentages of IBV
inhibition were >99%, >97%, >96% at hWJ-MSC-S concentrations of 1000
µg/mL, 500 µg/mL, and 250 µg/mL, respectively ([153]Figure 4D),
whereas, at a 125 µg/mL concentration, the viral inhibition by
hWJ-MSC-S significantly dropped to 40% ([154]Figure 4D).
Figure 4.
[155]Figure 4
[156]Open in a new tab
In ovo toxicity and anti-IBV activity of hWJ-MSC-S. (A) Lethal Dose 50
(LD[50]) of hWJ-MSC-S in SPF-ECEs. The LD[50] was assessed by
inoculating SPF-ECEs with different hWJ-MSC-S concentrations and embryo
viability was checked daily. The LD[50] was calculated as the
concentration that causes the death of 50% of inoculated embryos. (B)
Schematic depicting the experimental protocols used for the assessment
of in ovo toxicity and anti-IBV activity of hWJ-MSC-S. (C) Reduction in
viral titer after treatment of IBV with different concentrations of
hWJ-MSC-S (125, 250, 500, 1000 µg/mL). (D) Percentages of IBV
inhibition after treatment with different concentrations of hWJ-MSC-S
(125, 250, 500, 1000 µg/mL). Results are shown as means ± SD of three
independent experiments, each run in triplicate.
3.5. Proteomics Profiling and Gene Ontology of hWJ-MSC-S
Proteomics profiling of hWJ-MSC-S was run in triplicate. NSAF metrics
represent protein abundance. [157]Figure 5A and [158]Table S2 show NSAF
abundancies of the top 50 proteins, clustered using the Ward algorithm
[[159]52]. A complete proteomics profile was obtained by merging the
three replicates. As a result, 259 proteins mapped to 252 genes were
listed. Additional data could be found in [160]supplementary data.
Next, we parsed the gene ontology of the proteome profile.
Interestingly, results showed significant enriched biological processes
involved in immune response activation, namely: Annexin A1, Heat shock
protein 90, and collagen alpha 1. We also reported a significant
opsonization component, including complement C3, Pentraxin-related
protein, and Spondin. Regulation of leukocyte migration was
overexpressed with Thrombospondin-1, Plasminogen activator inhibitor 1,
and extracellular matrix protein 1 ([161]Figure 5B and [162]Table S3).
Protein class analysis showed significant fractions involved in defense
immune proteins ([163]Figure 6A). Finally, pathway enrichment analysis
showed significantly activated pathways, characteristic of COVID-19
([164]Figure 6B and [165]Table S4).
Figure 5.
[166]Figure 5
[167]Open in a new tab
(A) Heatmap for the top 50 NSAF proteins for the three replicates.
Clustered by Ward algorithm and scaled by each replicate. Associated
scale represents scales values of NSAF metric. (B) Gene ontology
analysis. The upper side (pie chart) of the figure shows the biological
process, molecular function, and cellular component total protein
percentage hits in each analysis. The bar plot shows the top 20 hits of
enrichment biological process (red), molecular function (blue), and
cellular component (yellow). Y axis represents enriched fold change.
Figure 6.
[168]Figure 6
[169]Open in a new tab
(A) Pathway enrichment analysis illustrates the top 10 odds ratio
pathways against the COVID-19 database. The pathway was sorted by odds
ratio from larger to smaller ones. The size of each point is
proportional to the adjusted p-value (adjusted p-value < 0.05). (B) The
pie chart represents the percentage of proteins involved in specific
protein classes.
4. Discussion
SARS-CoV-2 and IBV are highly infectious coronaviruses and leading
causes of morbidity and mortality rates in infected hosts
[[170]53,[171]54,[172]55]. Currently, there are no effective methods or
proven therapies to control the viral spread and mitigate disease
progression for SARS-CoV-2 and IBV infections
[[173]56,[174]57,[175]58]. Furthermore, inherited mutagenic
characteristics of SARS-CoV-2 and IBV limit the efficacy of available
vaccines and exacerbate the disease situations [[176]59,[177]60]. Thus,
the development of effective antiviral therapeutic approaches for
SARS-CoV-2 and IBV infections is a critical step toward controlling
COVID-19 and IB diseases. Secreted factors of stem cells are gaining
more attention as alternatives to stem cells to avoid the associated
side effects. Exploiting stem cell secretome offers a variety of
advantages, including, but not limited to, improved safety and
efficacy, flexibility in the storage and handling, and significantly
enhanced immune tolerance [[178]61,[179]62,[180]63,[181]64]. The
antiviral potentials of hWJ-MSC-S against SARS-CoV-2 and IBV are mainly
unexplored.
In this study, we investigated the antiviral activity of hWJ-MSC-S
against SARS-CoV-2 and IBV infections. Firstly, we isolated and
characterized Wharton’s Jelly mesenchymal stem cells and collected
their secretome. Secondly, we demonstrated the antiviral inhibitory
effects of hWJ-MSC-S on SARS-CoV-2 and IBV infections. Thirdly, we
showed that hWJ-MSC-S inhibits viral infection, mainly through the
inactivation of virus particles and the inhibition of viral
replication. Fourthly, in ovo antiviral activity of hWJ-MSC-S against
IBV was demonstrated by its ability to decrease viral titers and to
protect chicken embryos. Fifthly, we identified the protein composition
of hWJ-MSC-s and pinpointed the major immunomodulatory key players and
significant pathways involved.
In this study, the isolation rate of mesenchymal stem cells from
Wharton’s Jelly was more efficient than cord lining
[[182]35,[183]65,[184]66]. This could be attributed to hWJ-MSC’s high
proliferation and growth rates and highlights their therapeutic
potential [[185]67,[186]68]. hWJ-MSC-S exhibited very low cytotoxicity
and embryotoxicity levels and its CC[50] and LD[50] values were very
high. Accordingly, this result confirms the previous findings that have
proven the safety and efficacy of stem-cell-secreted factors [[187]62].
hWJ-MSC-S significantly inhibited SARS-CoV-2 and IBV infections in a
dose-dependent manner.
Interestingly, hWJ-MSC-S reduced plaque formed by SARS-CoV-2 and IBV in
Vero-E6 cells by more than 90%. Similarly, in the in ovo model,
hWJ-MSC-S significantly reduced viral titer and enhanced embryo
survival compared to the control (p < 0.0001). Although hWJ-MSC-S
similarly inhibited SARS-CoV-2 and IBV infections, there were
variations in the inhibitory concentrations between the two viruses. It
showed markedly higher IC[50] and SI against SARS-CoV-2 in comparison
to IBV. In addition to that, IBV was less sensitive to inactivation by
hWJ-MSC-S. This suggests that hWJ-MSC-S may exploit different
strategies to inhibit SARS-CoV-2 and IBV infections. Further, the
differences in sensitivity to hWJ-MSC-S between the two viruses could
be attributed to the nature of each virion. Moreover, IBV is relatively
resistant to inactivation with soluble antiviral factors compared to
other coronaviruses, such as SARS-CoV [[188]69,[189]70].
The hWJ-MSC-S inhibited SARS-CoV-2 and IBV infections using different
strategies—directly by virucidal effect and indirectly via inhibiting
viral replication. This variation in the mode of action could be
ascribed to the wide range of molecules that have been identified in
hWJ-MSC-S proteomic analysis ([190]Figure 5 and [191]Figure 6). In
general, stem cell secretome contains various bioactive factors,
including paracrine molecules (such as growth factors, cytokines, and
microRNAs(miRNAs)) and extracellular vesicles (microvesicles and
exosomes). These bioactive molecules modulate the activities of stem
cell secretome [[192]71,[193]72]. Our proteomic data revealed a
significant enrichment of immunomodulatory and antiviral proteins,
including Thymosin beta-4 (Tβ4), Peptidyl-prolyl cis-trans isomerase A
(PPIase A), and tissue metalloproteinase inhibitors 1 (TIMP1) in
hWJ-MSC-S. Tβ4 is an anti-inflammatory and antioxidant protein that has
been shown to inhibit coronavirus mouse hepatitis virus (CoV MHV)
Infection in vivo [[194]73]. PPIase A, also known as cyclophilin A
(CypA), is an enzyme that catalyzes the cis-trans isomerization of
proline imidic peptide bonds in oligopeptides [[195]74,[196]75]. The
TIMP1 is a zinc-dependent enzyme known to inhibit MMP activity
[[197]76]. Mesenchymal stem cells (MSCs) are known to secrete high
levels of TIMPs [[198]77].
Tβ4 has been reported to significantly increase the survival of
CoV-MHV-A59-infected mice by inhibiting viral replication and
alleviating infection-associated immune activation and liver damage
[[199]73]. In addition, levels of serum Tβ4 have been associated with
hepatitis B virus-related liver failure, and higher Tβ4 values
indicated improvement and recovery from the disease [[200]78].
Interestingly, thymosin treatment has been used recently to alleviate
the severity of COVID-19 symptoms [[201]79]. Like Tβ4, CypA is involved
in the life cycles of several viruses, including SARS-CoV, CoV-229E,
CoV-NL63, FCoV, HIV-1, HCV, and influenza virus
[[202]74,[203]75,[204]80,[205]81]. The CypA has been shown to impair
influenza virus replication through degradation of the viral M1 protein
[[206]75,[207]82]. Recently, CypA and TIMP1 have been suggested as
potential treatments for SARS-CoV-2 infection and COVID-19
[[208]83,[209]84,[210]85,[211]86,[212]87].
Like stem cell-derived soluble factors, extracellular vesicles (EVs) in
stem cell secretome have been shown to carry out immunomodulatory and
antiviral activity [[213]88,[214]89,[215]90,[216]91]. UC-MSC-derived
exosomes inhibited SRAS-CoV-2 infection and associated lung
inflammation [[217]92,[218]93,[219]94]. Furthermore, exosomes
competitively blocked SARS-CoV-2 entry via its surface-associated human
angiotensin-converting enzyme 2 (hACE2) [[220]95]. Given the
well-documented immunomodulatory and antiviral activities of stem cell
soluble factors and EV, the antiviral effects of hWJ-MSC-S on
SARS-CoV-2 and IBV may be mediated by its soluble factors (including
Tβ4 and CypA and TIMP1) and EVs (MVs and EXos). These findings imply
that hWJ-MSC-S-based therapeutic agents can potentially be used in
future studies to target SARS-CoV-2 and IBV infections and their
associated diseases. The low cytotoxicity and embryotoxicity profiles
of hWJ-MSC-S indicated that it could be used safely in vivo studies,
particularly to treat IBV-infected poultry flocks. Although drinking
water and feed medication are common practices for the treatment of
poultry diseases, for hWJ-MSC-S-based anti-IBV therapies, the
parenteral administration would be the main route. This is because the
biologically active extracellular paracrine factors of hWJ-MSC-S could
be deactivated by impurities in drinking water and feed additives.
Moreover, a lyophilized form of hWJ-MSC-S could be more suitable for
drug administration to poultry flocks. Further, hWJ-MSC-S could be used
therapeutically to treat IBV-infected chickens, as well as prophylaxis
to control IBV infection in chicken flocks.
5. Conclusions
In this work, we have demonstrated the antiviral activity of hWJ-MSC-S
against two medically important coronaviruses: human SARS-CoV-2 and
avian IBV. The hWJ-MSC-S significantly inhibited SARS-CoV-2 and IBV
infection in vitro and in ovo, respectively. Our antiviral mechanistic
studies suggest that the antiviral effects of hWJ-MSC-S are mainly
mediated via virucidal and anti-replication mechanisms. Using proteome
profiling and gene ontology, we have identified various
immunomodulatory and antiviral bioactive factors in hWJ-MSC-S. This
study provides a perspective on new antiviral therapeutics against
SARS-CoV-2 and IBV infections, using hWJ-MSC-S-based approaches.
Findings from this study may, thus, have implications for developing
effective strategies to combat COVID-19, IB, and future coronavirus
outbreaks. While this study aimed to evaluate the antiviral effect of
hWJ-MSC-secreted factors collectively, it will be particularly
meaningful to determine the role of an individual secreted factor in
the hWJ-MSC-S-mediated antiviral effect.
Supplementary Materials
The following supporting information can be downloaded at:
[221]https://www.mdpi.com/article/10.3390/cells11091408/s1. Table S1:
Detailed proteome profile of hWJ-MSC-S; Table S2: Calculated NSAF
metrics for hWJ-MSC-S proteome; Table S3: Gene ontology of hWJ-MSC-S;
Table S4: Pathway enrichment analysis of hWJ-MSC-S; Figure S1:
Comparison between different concentrations of WJ-MSC-S against
SARS-CoV-2 using different antiviral assays; Figure S2: Comparison
between different concentrations of WJ-MSC-S against IBV using
different antiviral assays.
[222]Click here for additional data file.^ (454.8KB, zip)
Author Contributions
Conceptualization, H.A.M.H.; methodology, H.A.M.H., A.A.T. and S.M.;
validation, H.A.M.H., A.A.T., M.A.D., A.M.E.-A., A.A.W., A.S.,
M.A.A.H., A.A.E., S.M., H.A., U.A.R., M.M.A., M.A.-W. and K.M.S.;
investigation, M.A.A.H., A.O., A.M.A., H.A.M.H., A.A.T., S.M., H.A.,
M.A.D. and E.K.B.; resources, H.A.M.H. and A.A.T.; data curation,
M.A.A.H., A.O., A.M.A., K.M.S., A.S. and M.A.D.; writing—original draft
preparation, H.A.M.H.; writing—review and editing, H.A.M.H., M.A.A.H.,
A.A.T., S.M. and S.S.; visualization, M.A.A.H., M.A.D., A.O., A.M.A.
and K.M.S.; supervision, H.A.M.H., A.A.T., S.S. and S.M.; project
administration, H.A.M.H.; funding acquisition, H.A.M.H. and A.A.T. All
authors have read and agreed to the published version of the
manuscript.
Funding
This paper is based upon work supported by the Academy of Scientific
Research & Technology (ASRT), under Ideation Fund, grant ID (7213).
Institutional Review Board Statement
This study was approved by the ethical committees of the faculty of
medicine and faculty of science at Al-Azhar University in Assiut
(APPROVAL NUMBER/ID:202015).
Informed Consent Statement
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement
The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE [[223]96] partner repository
with the dataset identifier PXD030966 and 10.6019/PXD030966.
Conflicts of Interest
The authors declare no conflict of interest.
Footnotes
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References