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 )

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* * *

ns

****

**

*

A

CC

B

BB

A

A

SmallAirTM

T E E R o f S m a l lA i rT M

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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

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4

5

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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

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R S V B

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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

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