ferret models to study clinical intervention strategies ... · severe cns lesions (bodewes, et al....

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The Fifth ESWI Influenza Conference, September 14-17, 2014 Riga, Latvia

ROTTERDAM SCIENCE TOWER IN THE NETHERLANDS

Ferret models to study clinical intervention strategies against influenza.Koert Stittelaar1, Leon de Waal1, Edwin Veldhuis Kroeze1, Geert van Amerongen1, Judith MA van den Brand2, Erhard van der Vries2 and Albert Osterhaus1,2

1 Viroclinics Biosciences B.V., Rotterdam, the Netherlands;2 Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands.

For questions please contact:

Koert J. Stittelaar

AbstractInfluenza is a moving target. Different influenza virus subtypes circulate across different avian and mammalian populations, drift, reassort, causing disease and spreading rapidly.

Ferrets have proven to be indispensable for influenza virus research. Ferrets can be infected with primary and cultured human and avian influenza virus isolates and develop a disease pattern which is very similar to that in humans. Ferrets have shown to be instrumental over a

broad spectrum of applications from the production of influenza-specific antiserum to the novel highly advanced immunocompromised model. The appropriateness of the different ferret models and their read out parameters for the assessment of clinical intervention strategies for influenza virus infection in humans, such as preventive vaccination and the use of antivirals are presented here in the context of high pathogenic avian influenza A/H5N1, pandemic influenza A/H1N1 and low pathogenic avian influenza A/H7N9 viruses.

• van den Brand JM, Stittelaar KJ, van Amerongen G, Rimmelzwaan GF, Simon J, de Wit E, Munster V, Bestebroer T, Fouchier RAM, Kuiken T, Osterhaus AD. Severity of pneumonia due to new H1N1 influenza virus in ferrets is intermediate between that due to seasonal H1N1 virus and highly pathogenic avian influenza H5N1 virus. J Infect Dis. 2010; 201:993-999.

• Veldhuis Kroeze EJ, van Amerongen G, Dijkshoorn ML, Simon JH, de Waal L, Hartmann IJ, Krestin GP, Kuiken T, Osterhaus AD, Stittelaar KJ. Pulmonary pathology of pandemic influenza A/H1N1 virus (2009)-infected ferrets upon longitudinal evaluation by computed tomography. J Gen Virol. 2011 Aug;92(Pt 8):1854-8.

• van den Brand JM, Stittelaar KJ, van Amerongen G, Reperant L, de Waal L, Osterhaus AD, Kuiken T. Comparison of temporal and spatial dynamics of seasonal H3N2, pandemic H1N1 and highly pathogenic avian influenza

H5N1 virus infections in ferrets. PLoS One. 2012;7(8):e42343.

• Veldhuis Kroeze EJ, Stittelaar KJ, Teeuwsen VJ, Dijkshoorn ML, van Amerongen G, de Waal L, Kuiken T, Krestin GP, Hinkula J, Osterhaus AD. Consecutive CT in vivo lung imaging as quantitative parameter of influenza vaccine efficacy in the ferret model. Vaccine. 2012 Dec 7;30(51):7391-4.

• van der Vries E, Stittelaar KJ, van Amerongen G, Veldhuis Kroeze EJ, de Waal L, Fraaij PL, Meesters RJ, Luider TM, van der Nagel B, Koch B, Vulto AG, Schutten M, Osterhaus AD. Prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets. PLoS Pathog. 2013 May;9(5):e1003343.

• Maltais AK, Stittelaar KJ, Veldhuis Kroeze EJ, van Amerongen G, Dijkshoorn ML, Krestin GP, Hinkula J, Arwidsson H, Lindberg A, Osterhaus AD. Intranasally administered Endocine™ formulated 2009 pandemic influenza H1N1 vaccine induces broad specific antibody responses and confers protection in ferrets. Vaccine. 2014 May 30;32(26):3307-15.

• Mann AJ, Noulin N, Catchpole A, Stittelaar KJ, de Waal L, Veldhuis Kroeze EJ, Hinchcliffe M, Smith A, Montomoli E, Piccirella S, Osterhaus AD, Knight A, Oxford JS, Lapini G, Cox R, Lambkin-Williams R. Intranasal H5N1 Vaccines, Adjuvanted with Chitosan Derivatives, Protect Ferrets against Highly Pathogenic Influenza Intranasal and Intratracheal Challenge. PLoS One. 2014 May 21;9(5):e93761.

• Kreijtz JH, Veldhuis Kroeze EJ, Stittelaar KJ, de Waal L, van Amerongen G, van Trierum S, van Run P, Bestebroer T, T Kuiken, Fouchier RA, Rimmelzwaan GF, Osterhaus AD. Low pathogenic avian influenza A(H7N9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies. Vaccine. 2013 Oct 9;31(43):4995-9.

References

Infection doseThe choice of infection dose of the influenza challenge virus in preclinical efficacy studies is important. For minimal variation in the test groups the challenge dose should be sufficiently high, but if the challenge dose is too high it will for instance be too difficult for a vaccine candidate to show protective efficacy and it might be falsely disqualified. The challenge dose influences a number of parameters like mortality, while there are also parameters like the lung viral load that are not influenced. We have carried out dose-finding experiments for pre-2009 influenza A virus H1N1, pandemic H1N1 and highly pathogenic avian influenza virus H5N1 (van den Brand, et al. 2010) and recently also for H7N9 (Kreijtz, et al. 2013).

Infection routeFerrets can be infected with influenza viruses via the respiratory route by intranasal, intratracheal inoculation, and by transmission. The most common route of infection used for ferrets is the intranasal route. Bodewes, et al, who reported that the method of virus inoculation is critical. Intratracheal inoculation with 1x106 TCID50 of influenza A H5N1 (A/Indonesia/5/05) resulted in severe bronchointerstitial pneumonia, while intranasal inoculation with same virus at the same dose induced moderate or severe CNS lesions (Bodewes, et al. 2011 AmJPath). We have shown that intratracheal challenge can be successfully used for efficacy testing also for intranasal vaccines (Maltais, et al. 2014 & Mann, et al. 2014).

Sampling & end pointsIn studies designed to evaluate the efficacy of antiviral agents against influenza, it is critical to collect respiratory tract samples for virological, pathological and molecular analyses at both the appropriate time point after infection or start of therapy as well as the appropriate location along the respiratory tract. This is because influenza virus infection is a highly dynamic process, both temporally and spatially (van den Brand, et al. 2012). Furthermore, experiments with repeated CT scans of the same animal instead of sacrificing multiple animals allows to study respiratory tract lesions of each individual animal compared with the situation before infection (veldhuis Kroeze, et al. 2011). We have shown that day-to-day CT monitoring is a valuable tool for the read-out of efficacy studies (veldhuis Kroeze, et al. 2012).

Clinical read-outsA number of clinical read-outs are relatively easy to obtain and to analyze from ferrets, including body weight and body temperature. However, signs of disease like loss of activity can be difficult to monitor. Therefore, we have developed a very sensitive activity sensor that overcomes the need for time-consuming observations and allows proper analysis of data that are obtained objectively. This activity sensor provides a valuable additional parameter to show the efficacy of countermeasures in the ferret model.

Immunocompromised ferretsImmunocompromised patients, such as transplant recipients on immune suppressive therapy, are a substantial and gradually expanding patient group. Unfortunately, existing antiviral strategies for treatment of influenza virus infections show limited effectiveness with frequent emergence of antiviral resistance. We developed an immunocompromised ferret model, with an immune suppressive regimen that mimics the regimen used in solid organ transplant recipients (van der Vries, et al. 2013). Like in immunocompromised patients, we have shown that these ferrets fail to control influenza virus replication. The immunocompromised ferrets provide a useful tool in the development of novel antiviral approaches for immunocompromised patients suffering from influenza. With this model we also can study mechanisms governing the development of mutations in therapy resistant viruses arising in immunocompromised patients and this model can be considered for “the animal rule”.

75

50

25

00 1

HPAI H5N14 d.p.i.

% Lung lesions 13 47 93

4.9 4.4 4.8 Log10Lung TCID50

104 106 108

pH1N1 2009

H1N1 pre 2009

2 3 4 5 6 7

100104 TCID50

Mor

talit

y (%

)

75

50

25

00 1 2 3

Time (days after infection)

4 5 6 7

100106 TCID50

75

50

25

00 1 2 3 4 5 6 7

100108 TCID50

0 1 2 3 4 Peak (oC) Tine (h) RLW Area (%) Lung Nose Lung Nose

1 40,8 33 2,08 50 + + - 6,8 2,9

2 41,3 31 1,21 30 + + - 3,2 <1,7

3 FD 41,7 24 2,52 50 + + - 5,7 3,8

4 FD 41,0 24 1,74 90 + + - 5,7 4,7

5 41,3 24 1,7 50 + + - 6 <1,7

6 FD 40,1 21 3,94 90 + + + 6,7 5,8

7 Euth 41,8 23 3,87 90 + + + 4,9 6,8

8 FD 41,2 24 2,99 90 + + + + 5,6 5,3

9 FD 41,8 16 2,28 90 + + + + 5,3 5,4

10^5

10^6

10^7

10^8

Time (days after infection)IT Dose (TCID50)Ferret

Body temperature Lung gross path. Immuno staining TCID50 (Log10/g)

6

5

4

3

2

0 1 2Time (days after injection)

Nasal swab

Challenge ctrl. Inactivated Ag + Adj.

Nasal swab

Throat swabThroat swab

Viru

s ti

ter

3 4

LOD

6

5

4

3

2

0 1 2Time (days after injection)

Viru

s ti

ter

3 4

LOD

6

5

4

3

2

Viru

s ti

ter

LOD

6

5

4

3

2

Viru

s ti

ter

LOD0 1 2

Time (days after injection)3 4 0 1 2

Time (days after injection)3 4

6

5

8

7

4

qRT-PCR TCID50

Nasal turbinate

Challenge ctrl. Inactivated Ag + Adj.

Viru

s ti

ter

LOD

6

5

8

7

4

qRT-PCR TCID50

Nasal turbinate

Viru

s ti

ter

LOD

6

5

8

7

4

qRT-PCR TCID50

Lung

Viru

s ti

ter

LOD

6

5

8

7

4

qRT-PCR TCID50

Lung

Viru

s ti

ter

LOD

H7N9 – A/Anhui/1/2013

H7N9 – A/Anhui/1/2013 5.5 Log10 TCID50 I.T.

0 1 2 3 4 5 RLW Area (%) pre day 1 day 2 day 3 day 4 day 5 Lung Nose

1 Euth. 0,6 10 <1.1 2,9 4,6 4,9 5,1 4,5 <1,3 5,9

2 Euth. 0,5 10 <1.1 2,0 4,3 4,5 4,0 3,5 <1,4 5,2

3 Euth. 0,5 10 <1.1 3,1 5,0 5,3 5,2 4,9 <1,3 6,3

4 Euth. 0,5 10 <1.1 2,4 5,1 5,2 4,8 4,6 1,2 6,1

5 Euth. 0,6 10 <1.1 2,7 4,7 4,5 4,9 4,4 <1,3 5,9

6 Euth. 0,4 10 <1.1 2,7 5,0 5,4 4,9 4,0 <1,3 5,2

7 Euth. 2,4 90 <1.1 <1.1 <1.1 3,1 2,8 1,8 5,2 3,5

8 FD 1,4 90 <1.1 <1.1 1,5 1,8 N/A N/A 5,0 4,5

9 FD 2,0 100 <1.1 <1.1 <1.1 4,6 N/A N/A 5,3 <1,5

10 FD 2,5 100 <1.1 <1.1 <1.1 1,3 <1.1 <1.1 4,9 4,7

11 FD 1,1 100 <1.1 <1.1 4,9 N/A N/A N/A 5,0 4,5

12 FD 1,9 90 <1.1 <1.1 <1.1 1,2 <1.1 N/A 5,6 <1,2

I.T.

Nose swabs (qRT-PCR Log10/ml) TCID50 (Log10/g)

I.N.

Ferret RouteTime (days after infection) Lung gross path.

HAI serum titers Lung Nose2560

640

160

40

10

A / Netherlands / 602 / 2009

EndocineTM

Ant

ibod

y ti

ter

Saline EndocineTM Saline EndocineTM Saline

A / Swine / Ned / 25 / 80A / Swine / Italy / 14432 / 76A / New Jersey / 08 / 76

10

8

6

4

2

Viru

s ti

ter (

Log1

0 TC

ID50

/g)

Chitosan Saline Chitosan Saline

6

5

4

3

2

1

Viru

s ti

ter (

Log1

0 TC

ID50

/g)

Lung Nose

(IN) Intranasal vs. (IT) Intratracheal

H5N1 A/Vietnam/1194/2004 5 Log10 TCID50

Intranasally Administered Endocine™ Formulated 2009 Pandemic Influenza H1N1 Vaccine Induces Broad Specific Antibody Responses and Confers Protection in Ferrets.

Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against Highly Pathogenic Influenza intranasal and intratracheal challenge.

Infection Day 0

Start Delayed Treatment Day 2

NecropsyDay 8

0 0.5 1 2Time (Day after infection)

3 4 7 14

• Body weight loss

• Body temperature

• Activity

Ferret activity score

ΔX ΔY ΔZ ΔX ΔY ΔZ CumulativeCumulative

>6 date timeCumulative

>6

with "-" without "-"

ZYXTimeDateMeasurement

253 22.12.13 04:29:15 -0,52 -10,72 78,75

254 22.12.13 04:29:30 2,87 -8,09 79,04 3 3 0 3 3 0 6 1

255 22.12.13 04:29:45 1,09 -7,24 81,68 -2 1 3 2 1 3 5 0

256 22.12.13 04:30:00 -0,97 -9,25 79,77 -2 -2 -2 2 2 2 6 0 22.12.13 04:30 1

257 22.12.13 04:39:00 ____ ____ ____

258 22.12.13 04:39:15 -14,57 -37,91 37,53

259 22.12.13 04:39:30 -2,4 -32,6 55,01 12 5 17 12 5 17 35 1

260 22.12.13 04:39:45 -12,24 -59,29 18,47 -10 -27 -37 10 27 37 73 1

261 22.12.13 04:40:00 -34,18 -39,18 -16,64 -22 20 -35 22 20 35 77 1 22.12.13 04:40 3

262 22.12.13 04:49:00 ____ ____ ____

263 22.12.13 04:49:15 -18,63 -17,09 -54,28

264 22.12.13 04:49:30 -18,33 -17,19 -54,49 0 0 0 0 0 0 1 0

265 22.12.13 04:49:45 -18,58 -17,93 -53,49 0 -1 1 0 1 1 2 0

266 22.12.13 04:50:00 -17,79 -17,87 -54,34 1 0 -1 1 0 1 2 0 22.12.13 04:50 0

267 22.12.13 04:59:00 ____ ____ ____

40

30

20

10

0

D-6 D-4 D-2 D0 D2 D4

50

Unprotected challenge ctrl.

40

30

20

10

0

D-6 D-4 D-2 D0 D2 D4

50

Protected by vaccination4

2

0

Immunocompetent

Immunocompromised

Immunocompromised + OS

0 3 6 9Time (days after infection)

12 15 18 21

6

Vira

l loa

d (L

og10

vp/

ml)

50%