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Epithelixin vitro Solutions for Respiratory
Diseases and Chemical Testing
Efficient replication of respiratory syncytial virus induces a decrease of mucociliary
clearance in human small airway 3D culture
Song Huang, Bernadett Boda, Rosy Bonfante, Jimmy Vernaz, Samuel Constant.
Epithelix, 18 Chemin des Aulx, Plan-Les-Ouates, CH-1228, Geneva, Switzerland
Introduction
Respiratory syncytial virus (RSV) infection causes upper
and lower respiratory tract infections and is the most
common cause of bronchiolitis and pneumonia in young
children. To understand RSV pathogenesis in humans and
to test new molecules for alleviating the diseases, an in
vitro RSV infection platform based on three dimensional
(3D) fully differentiated human airway epithelia cultured at
air-liquid interface was developed.
One such model, SmallAirTM, representing the small airway
epithelia with characteristic cell types (basal, ciliated and
Club cells) was successfully infected with clinical isolate of
RSV B.
Conclusion
These results demonstrate that SmallAirTM is a pertinent tool to perform anti-RSV drug screening via airborne or systemic delivery.
Effects of RSV B apical infection on the mucociliary clearance in SmallAirTM.
Mucociliary clearance was evaluated through microscopic monitoring of polystyrene microbeads (Sigma) applied
on the apical surface. Particle velocity was quantified from the recorded video using Image Pro Plus software.
Differences were tested by one-way ANOVA using Prism 6 GraphPad software and Dunnett’s multiple comparison
tests were used to compare every mean to control mean. A. RSV B was inoculated at 106/ml B. 107/ml
concentration. Reference antiviral, ribavirin was applied basolaterally (BL) or apically (AP) (n=2 and 2).
Determination of viral particle number in the apical
washes of SmallAirTM.
Genome copy (gc) number was determined using
quantitative PCR on the N gene of RSV (Essaidi-Laziosi et
al., 2016) at day 1, 2, 3 and 4. A. 105 and 106 gc of RSV B, in
100 ml, was inoculated alone or with apical vehicle treatment
on SmallAirTM cultures (n=2). B. Reference antiviral, ribavirin
was applied in the basolateral medium (BL) concomitantly
with the apical inoculation of 106/ml of RSV B and renewed
each day (n=2). C. Reference antiviral, ribavirin was applied
apically (Ap) with the inoculation of 106/ml of RSV B and
renewed each day (20 ml) (n=2). Differences were tested by
two-way ANOVA and Dunnett’s multiple comparison tests
using Prism 6 GraphPad software.
Effects of RSV B (106/ml) apical infection on
SmallAirTM functions.
A. Epithelial homeostatic barrier function was evaluated
through monitoring TEER (EVOMX) each day (n=2).
Reference antiviral, ribavirin was applied basolaterally
(BL) or apically (AP). B. Cytotoxicity was assessed using
measurement of LDH release (Sigma). Daily LDH
release was expressed in percentage of control positive,
10 % Triton X-100, treatment. Threshold limit value is 5
% cytotoxicity, which corresponds to a physiological LDH
release in SmallAirTM. C. General cilia motion was
recorded by a high speed camera and the beating
frequency was calculated using Cilia X software.
Summary
• With an initial inoculum of 105 or 106 RSV B viruses, the
genome copy number reached 1010 gc/ml 4 days post-
inoculation in the apical washes.
• The RSV B infection did not impair the tissue integrity
nor cause cytotoxicity.
• Solely the mucociliary clearance showed a dramatic
decrease at 4 days post-inoculation of RSV B (22 and 9
% of the non-infected control for 106 and 107/ml virus
inoculation, respectively).
• Ribavirin, as reference antiviral against RSV, applied
either apically (1-10-100 mM) or basally (10-100-1000
mM), inhibited viral replication in a dose-dependent
manner and partially prevented the decrease of the
mucociliary clearance.
Materials and Methods
SmallAir™, a fully differentiated 3D in vitro
cell model of the human small airway
epithelia. The cells were isolated from
bronchiolar region of human lungs.
After 45 days of culture at air-liquid
interface, the epithelia were fully
differentiated, both morphologically and
functionally.
Clinical isolate of Respiratory Syntitial Virus (RSV) B was inoculated at 105 and
106 genome copy in 100 ml on the apical side of SmallAirTM (SA069301) for 3
hours. After an apical wash, virus genome copy number was determined at 1, 2,
3 and 4 days post-inoculation on the apical side of the cultures. Reference
antiviral, ribavirin, was applied concomitantly with the virus either apically in 20 ml
(Ap; 10, 100, 1000 mM) or basally (BL; 1, 10, 100 mM). Different functional end
points were assessed at day 1, 2, 3 and 4 from both sides of cultures.
Results
Testing strategy
Transversal cross section of SmallAir™
with alcian blue and hematoxylin-eosin
staining (top) and Club cell specific CC-10
immunohistochemistry (below). Scale bar:
40 mm.
S m a l lA i rT M
R S V B d i l u t i o n
02
04
06
08
0
10
0
0
1
2
3
4
5
6
7
8
9
1 0
1 1
R S V B + v e h i c l e E 6 / m l
R S V B + v e h i c l e E 7 / m l
T i m e p o s t - i n o c u l a t i o n ( h o u r s )
RS
VB
ge
no
me
co
py
nu
mb
er
(lo
g 1
0)
R S V B E 6 / m l
R S V B E 7 / m l
L D H r e le a s e o f S m a l lA i rT M
E 6 / m l R S V B
Cy
toto
xic
ity
%
[10
% T
rit
on
X-1
00
]
Mo
ck
(- )
RS
VB
RS
VB
+ 1
mM
BL
Ri b
av
i ri n
RS
VB
+ 1
0 m
M B
L R
i ba
vi r
i n
RS
VB
+ 1
00
mM
BL
Ri b
av
i ri n
Ve
hi c
l e (
- )
RS
VB
+ V
eh
i cl e
RS
VB
+ 1
0 m
M A
P R
i ba
vi r
i n
RS
VB
+ 1
00
mM
AP
Ri b
av
i ri n
RS
VB
+ 1
00
0 m
M A
P R
i ba
vi r
i n
0
2
4
6
8
1 0
D a y 1
D a y 2
D a y 3
D a y 4
C B F o f S m a l l lA i rT M
E 6 / m l R S V B
Cil
ia B
ea
tin
g F
re
qu
en
cy
[H
z]
Mo
ck
(- )
RS
VB
RS
VB
+ 1
mM
BL
Ri b
av
i ri n
RS
VB
+ 1
0 m
M B
L R
i ba
vi r
i n
RS
VB
+ 1
00
mM
BL
Ri b
av
i ri n
Ve
hi c
l e (
- )
RS
VB
+ V
eh
i cl e
RS
VB
+ 1
0 m
M A
P R
i ba
vi r
i n
RS
VB
+ 1
00
mM
AP
Ri b
av
i ri n
RS
VB
+ 1
00
0 m
M A
P R
i ba
vi r
i n
0
2
4
6
8
1 0
D a y 1
D a y 2
D a y 3
D a y 4
M C C o f S m a l lA i rT M
E 6 / m l R S V B a t D a y 4
Pa
rti
cle
s v
elo
cit
y [
µm
/s]
Mo
ck
(- )
RS
VB
RS
VB
+ 1
mM
BL
Ri b
av
i ri n
RS
VB
+ 1
0 m
M B
L R
i ba
vi r
i n
RS
VB
+ 1
00
mM
BL
Ri b
av
i ri n
Ve
hi c
l e (
- )
RS
VB
+ V
eh
i cl e
RS
VB
+ 1
0 m
M A
P R
i ba
vi r
i n
RS
VB
+ 1
00
mM
AP
Ri b
av
i ri n
RS
VB
+ 1
00
0 m
M A
P R
i ba
vi r
i n
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
M C C o f S m a l lA i rT M
E 7 / m l R S V B a t D a y 4
Pa
rti
cle
s v
elo
cit
y [
µm
/s]
Mo
ck
(- )
E7
/ml
E7
/ml
+ 1
mM
BL
Ri b
av
i ri n
E7
/ml
+ 1
0 m
M B
L R
i ba
vi r
i n
E7
/ml
+ 1
00
mM
BL
Ri b
av
i ri n
Ve
hi c
l e (
- )
E7
/ml
+ V
eh
i cl e
(+
)
E7
/ml
+ 1
0 m
M A
P R
i ba
vi r
i n
E7
/ml
+ 1
00
mM
AP
Ri b
av
i ri n
E7
/ml
+ 1
00
0 m
M A
P R
i ba
vi r
i n
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
* * *
ns
****
**
*
A
CC
B
BB
A
A
SmallAirTM
T E E R o f S m a l lA i rT M
E 6 / m l R S V B
TE
ER
[%
of
no
n-in
fec
ted
]
Mo
ck
(- )
RS
VB
RS
VB
+ 1
mM
BL
Ri b
av
i ri n
RS
VB
+ 1
0 m
M B
L R
i ba
vi r
i n
RS
VB
+ 1
00
mM
BL
Ri b
av
i ri n
Ve
hi c
l e (
- )
RS
VB
+ V
eh
i cl e
RS
VB
+ 1
0 m
M A
P R
i ba
vi r
i n
RS
VB
+ 1
00
mM
AP
Ri b
av
i ri n
RS
VB
+ 1
00
0 m
M A
P R
i ba
vi r
i n
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
D a y 1
D a y 2
D a y 3
D a y 4
Apical inoculation
Virus genome copy number
Transepithelial electrical resistance (TEER)
Cilia beating frequency (CBF)
Mucociliary clearance (MCC)
Cytotoxicity(LDH release)
AP antiviral
BL antiviral
or
S m a l lA i rT M
E 6 / m l R S V B w i t h A p R ib a v i r in
02
04
06
08
0
10
0
0
1
2
3
4
5
6
7
8
9
1 0
1 1
R S V B + v e h i c le
R S V B + 1 0 m M A p R ib a v i r i n
R S V B + 1 0 0 m M A p R ib a v i r i n
R S V B + 1 0 0 0 m M A p R ib a v i r i n
I n p u t
T i m e p o s t - i n o c u l a t i o n ( h o u r s )
RS
VB
ge
no
me
co
py
nu
mb
er
(lo
g 1
0) * * * *
S m a l lA i rT M
E 6 / m l R S V B w i t h B L R ib a v i r in
02
04
06
08
0
10
0
0
1
2
3
4
5
6
7
8
9
1 0
1 1
R S V B
R S V B + 1 m M B L R ib a v i r i n
R S V B + 1 0 m M B L R ib a v i r i n
R S V B + 1 0 0 m M B L R ib a v i r i n
I n p u t
T i m e p o s t - i n o c u l a t i o n ( h o u r s )
RS
VB
ge
no
me
co
py
nu
mb
er
(lo
g 1
0)
****
****
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