ABSTRACTImmunocompromised individuals often experience severe disease caused by infections with influenza viruses or human respiratory syncytial virus (HRSV). In many cases infection results in prolonged virus shedding. The treatment of influenza A virus infected immuno-compromised patients is complicated by development of resistance to antiviral drugs. Antiviral approaches for the treatment of HRSV infection of the immunocompromised require further evaluation.

METHODSGiven the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompetent and immunocompromised ferrets with wild-type pandemic influenza virus A/H1N1 or a wild-type HRSV subgroup A. In influenza virus infected ferrets treatment with oseltamivir was evaluated and in HRSV infected ferrets Palivizumab was evaluated. The respiratory tract of the ferrets was sampled daily and respiratory tissues were collected during necropsies to determine viral loads by end-point titrations and qPCR.

RESULTSVirus could be isolated from throat swabs, nose swabs and respiratory tissues. For both influenza virus and HRSV virus loads waned from about 6 days post infection onward in the immunocompetent ferrets. However, the immunocompromised animals showed prolonged virus replication, similar to immunocompromised patients. Antiviral treatment resulted in reduced virus replication but also to resistance-related mutations

EXPERIMENTAL DESIGNHRSV infections: Route, dose and volume of infectionCotton rats: intranasally-plus, 105 TCID

50 in 125 µl

Ferrets: intratracheally, 105 TCID50

in 3.0 mlFerrets: intranasally, 105 TCID

50 in 125 µl

· Immune suppression in the immunocompromised ferrets was achieved by bi-daily oral administration of Tacrolimus, Cellcept and Prednisolone, a standard regimen for transplant patients (van der Vries, et al.).

· Viral loads were measured by RT-qPCR and by virus culture on HEp-2 cell monolayers using TCID

50 end-point

titration as well as RSV ViroSpot™ assay.

· Virus neutralizing antibody titers were measured by RSV ViroSpot™ assay and the classical VN assay.

· Immuno histochemistry (IHC) was performed on different tissue samples collected from the respiratory tract during necropsy at 4 d.p.i.; slides were stained with a Goat-anti-RSV polyclonal antibody.

Test virus:• RSV A Long

Inhibitor:• Antibody (Ab) dilutions (2-fold)• 60 minutes virus + Antibody• Virus/Ab on Hep-2 cell monolayer• Formalin fixation after 24 – 48 hours

knows your target

Cotton rats

4 d.p.i.

Nose

Trachea

Bronchus

Bronchiolus

Ferrets Immunocompromised Ferrets

0 3 6 9 12 15 18 21

1

2

3

4

5

6

Vira

l loa

d qP

CR

(Log

10)

0 3 6 9 12 15 18 21

1

2

3

4

5

6

Vira

l loa

d qP

CR

(Log

10)

Vira

l loa

d (L

og10

)

Ferrets:Immunocompetent vs.

Immunocompromised Throat swabs

Ferrets:Immunocompetent vs.

Immunocompromised Nose swabs

Time (days after infection) Time (days after infection)

Ferrets (IN):Broncheo Alveolar Lavage, 14 d.p.i.

5

4

3

2

1

0

Immunocompetent

RT-qPCR Virusculture

RT-qPCR Virusculture

Immunocompromised

Ferrets (IN) Immunocompromised Ferrets (IN)

HRSV replication in IN-inoculated (immunocompromised) ferrets

IN-inoculated Immuno-compromised Ferrets show HRSV in Broncho Alveolar Lavage at 14 d.p.i.

Vira

l Loa

d (L

og10

) 8

6

4

2

0

Nose w

ash

Turbinates

Throat swab

Trachea

BronchusLung

Vira

l Loa

d (L

og10

) 8

6

4

2

0

Nose w

ash

Turbinates

Throat swab

Trachea

BronchusLung

ND Vira

l Loa

d (L

og10

) 8

6

4

2

0

Nose w

ash

Turbinates

Throat swab

Trachea

BronchusLung

Vira

l Loa

d (L

og10

) 8

6

4

2

0

Nose w

ash

Turbinates

Throat swab

Trachea

BronchusLung

4 d.p.i Cotton rats

Ferrets are, like cotton rats, susceptible to primary HRSV

HRSV replication in IT-inoculated (immunocompromised) ferrets

Immunocompromised ferrets are highly susceptible to HRSV and show persistent virus replication

Immunestaining of HRSV-infected cells in different parts of the respiratory tract of cotton rats and ferrets

4 d.p.i. Ferrets (IT)

4 d.p.i. Immunocompromised Ferrets (IT)

21 d.p.i. Immunocompromised Ferrets (IT)

RT-qPCR Virus culture

Immunocompromised Ferrets (IT)Ferrets (IT)

Throat swabs (RT-qPCR ) Nose swabs (RT-qPCR)

Throat swabs (Virus culture) Nose swabs (Virus culture)

Viru

s lo

ad q

PCR

(Log

10)

0 3 6 9 12 15 18 21

1

2

3

4

5

6

Viru

s lo

ad q

PCR

(Log

10)

Time (days after infection)0 3 6 9 12 15 18 21

1

2

3

4

5

6

Time (days after infection)

0 3 6 9 12 15 18 21

1

2

3

4

5

6

Vira

l loa

d C

ultu

re (L

og10

)

0 3 6 9 12 15 18 21

1

2

3

4

5

6

Vira

l loa

d C

ultu

re (L

og10

)

Time (days after infection) Time (days after infection)

Transmission experiments ongoing

Throat swabs (RT-qPCR) Nose swabs (RT-qPCR)

CONCLUSION• Immunocompetent and immunocompromised ferrets represent an attractive animal model

to evaluate specific and broadly reactive antivirals against influenza viruses as well as HRSV.• Stability testing of RSV by virus titration and qPCR in (pre) clinical samples enables

optimization of sample logistics for clinical trials at remote clinical sites.

VIROSPOT RSV ViroSpot neutralization and virus titration

Stability of RSV in (pre) clinical samples collected at

remote clinical site

21 direct 21 frozen 26 direct 26 frozen

00 3 6 9 1512 2118

0

2

1

4

6

5

3

00 3 6 9 1512 2118

0

2

1

4

6

5

3

Vira

l loa

d qP

CR (L

og10

)Vi

ral l

oad

qPCR

(Log

10)

Time (days after infection)

Time (days after infection)

Day 0

Day 3

Titration in Cell Culture (Log10/ml)

PC

RLo

g10

VP

/ml

0 21 3 4 5 6

6

7

8

3

2

1

0

4

5

1000

100

Plaq

ue c

ount

/ 0.

1 m

L (n

; mea

n ±

SD)

10

13 4 5 6

Log dilution(RSV stock titer 1E7.4 TCID50/mL)

103*1E4/0.1 mL =

1E7.01 PFU/mL

At day 1, RSV-infected cell COUNTS in range of 30 - 300 per well to calculate virus concentrations (PFU/mL).

Nasopharyngeal swabs Throat PCR

Throat titration

Optimization of sample logistics

for clinical trials

Human Respiratory Syncytial Virus Infections in Immunocompetent and Immunocompromised Ferrets: a novel model for antiviral testing

No. 102

K.J. Stittelaar1, L. de Waal1, E. Van der Vries2, G. van Amerongen1, W. Huisman1, E.J.B. Veldhuis Kroeze1, C.A. van Baalen1, P.L.A. Fraaij2 , J.J. van Kampen2 , M. Schutten1 , R.L. de Swart2 , A.D.M.E. Osterhaus1

1Viroclinics Biosciences B.V., Rotterdam, the Netherlands; 2Erasmus MC, Rotterdam, the Netherlands. Correspondence: stittelaar@viroclinics.com