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 Stittelaar 1 , Leon de Waal 1 , Edwin Veldhuis Kroeze 1 , Geert van Amerongen 1 , Judith MA van den Brand 2 , Erhard van der Vries 2 and Albert Osterhaus 1,2 1 Viroclinics Biosciences B.V., Rotterdam, the Netherlands; 2 Department of Viroscience, Erasmus MC, Rotterdam, the Netherlands. For questions please contact: Koert J. Stittelaar Abstract Influenza 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 dose The 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 route Ferrets 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 1x10 6 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 points In 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-outs A 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 ferrets Immunocompromised 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 0 0 1 HPAI H5N1 4 d.p.i. % Lung lesions 13 47 93 4.9 4.4 4.8 Log10 Lung TCID50 10 4 10 6 10 8 pH1N1 2009 H1N1 pre 2009 2 3 4 5 6 7 100 10 4 TCID50 Mortality (%) 75 50 25 0 0 1 2 3 Time (days after infection) 4 5 6 7 100 10 6 TCID50 75 50 25 0 0 1 2 3 4 5 6 7 100 10 8 TCID50 0 1 2 3 4 Peak ( o C) 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 2 Time (days after injection) Nasal swab Challenge ctrl. Inactivated Ag + Adj. Nasal swab Throat swab Throat swab Virus titer 3 4 LOD 6 5 4 3 2 0 1 2 Time (days after injection) Virus titer 3 4 LOD 6 5 4 3 2 Virus titer LOD 6 5 4 3 2 Virus titer LOD 0 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 Virus titer LOD 6 5 8 7 4 qRT-PCR TCID50 Nasal turbinate Virus titer LOD 6 5 8 7 4 qRT-PCR TCID50 Lung Virus titer LOD 6 5 8 7 4 qRT-PCR TCID50 Lung Virus titer 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 Route Time (days after infection) Lung gross path. HAI serum titers Lung Nose 2560 640 160 40 10 A / Netherlands / 602 / 2009 Endocine TM Antibody titer Saline Endocine TM Saline Endocine TM Saline A / Swine / Ned / 25 / 80 A / Swine / Italy / 14432 / 76 A / New Jersey / 08 / 76 10 8 6 4 2 Virus titer (Log10 TCID50/g) Chitosan Saline Chitosan Saline 6 5 4 3 2 1 Virus titer (Log10 TCID50/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 Necropsy Day 8 0 0.5 1 2 Time (Day after infection) 3 4 7 14 • Body weight loss • Body temperature • Activity Ferret activity score ΔX ΔY ΔZ ΔX ΔY ΔZ Cumulative Cumulative >6 date time Cumulative >6 with "-" without "-" Z Y X Time Date Measurement 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 vaccination 4 2 0 Immunocompetent Immunocompromised Immunocompromised + OS 0 3 6 9 Time (days after infection) 12 15 18 21 6 Viral load (Log10 vp/ml) 50%
Transcript of Ferret models to study clinical intervention strategies ... · severe CNS lesions (Bodewes, et al....
knows your target
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%
