ABSTRACT Immunocompromised 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. METHODS Given 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. RESULTS Virus 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 DESIGN HRSV infections: Route, dose and volume of infection Cotton rats: intranasally-plus, 10 5 TCID 50 in 125 µl Ferrets: intratracheally, 10 5 TCID 50 in 3.0 ml Ferrets: intranasally, 10 5 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 Viral load qPCR (Log10) 0 3 6 9 12 15 18 21 1 2 3 4 5 6 Viral load qPCR (Log10) Viral load (Log10) 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 Virus culture RT-qPCR Virus culture 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. Viral Load (Log10) 8 6 4 2 0 Nose wash Turbinates Throat swab Trachea Bronchus Lung Viral Load (Log10) 8 6 4 2 0 Nose wash Turbinates Throat swab Trachea Bronchus Lung ND Viral Load (Log10) 8 6 4 2 0 Nose wash Turbinates Throat swab Trachea Bronchus Lung Viral Load (Log10) 8 6 4 2 0 Nose wash Turbinates Throat swab Trachea Bronchus Lung 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) Virus load qPCR (Log10) 0 3 6 9 12 15 18 21 1 2 3 4 5 6 Virus load qPCR (Log10) Time (days after infection) 0 3 6 9 12 15 18 1 2 3 4 5 6 Time (days after infection) 0 3 6 9 12 15 18 1 2 3 4 5 6 Viral load Culture (Log10) 0 3 6 9 12 15 18 21 1 2 3 4 5 6 Viral load Culture (Log10) 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 0 0 3 6 9 15 12 21 18 0 2 1 4 6 5 3 0 0 3 6 9 15 12 21 18 0 2 1 4 6 5 3 Viral load qPCR (Log10) Viral load qPCR (Log10) Time (days after infection) Time (days after infection) Day 0 Day 3 Titration in Cell Culture (Log10/ml) PCRLog10 VP/ml 0 2 1 3 4 5 6 6 7 8 3 2 1 0 4 5 1000 100 Plaque count / 0.1 mL (n; mean ± SD) 10 1 3 4 5 6 Log dilution (RSV stock titer 1E7.4 TCID 50 /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. Stittelaar 1 , L. de Waal 1 , E. Van der Vries 2 , G. van Amerongen 1 , W. Huisman 1 , E.J.B. Veldhuis Kroeze 1 , C.A. van Baalen 1 , P.L.A. Fraaij 2 , J.J. van Kampen 2 , M. Schutten 1 , R.L. de Swart 2 , A.D.M.E. Osterhaus 1 1 Viroclinics Biosciences B.V., Rotterdam, the Netherlands; 2 Erasmus MC, Rotterdam, the Netherlands. Correspondence: [email protected]