New methods and reagents to improve the ferret model for human influenza infections

1. New methods and reagents to improve the ferret model for human influenza infections Martel C. a, Kirkeby S. a, Aasted B. a a Copenhagen University, Faculty of Life sciences, Institute of Veterinary Pathobiology, Denmark b Panum Institute, Institute of Odontology, Denmark Abstract:The ferret has been extensively used to study human influenza infections. However, its value as a model has suffered from the limited set of reagents and methods available for this animal. We have recently tested a large number of monoclonal antibodies cross-reacting with ferret CD markers (CD8, CD9, CD14, CD18, CD25, CD29, CD32, CD44, CD61, CD71, CD79b, CD88, CD104, CD172a and CD3) and cytokines (interferon-gamma, TNF-alpha, interleukine-4 and interleukine-8) for flow cytometry , as well as polyclonal antibodies cross-reacting with ferret immunoglobulins (IgA, IgG and IgM) for ELISA. Further improvements of the model will aim at establishing a reliable RT-PCR for ferret cytokines, as well as investigating the location of influenza receptors and viral particles in the respiratory tract via immunohistochemistry. • Material and methods: • Screening for monoclonal antibodies for flow cytometry : 53 mAbs against CD markers, known to cross-react with mink, were tested on ferret peripheral blood leucocytes. 4 antibodies against IL4, IL8, TNFa and IFNg were also tested on leucocytes after 4 hours of culture with PMA, ionomycin and brefeldin A. • Influenza receptor and goblet cell staining: lung samples were taken on normal ferrets and on animals infected with A/New Caledonia/20/99 (H1N1), then stained with Periodic Acid Schiff, Sambucus Nigra lectin directed against 2-6α-gal sialic acid, or monoclonal  IgG against NeuAcα2-6GalNAc-O The/Ser • Our own rabbit antibody preparation to mink IgG, a commercial rabbit anti-human IgM (Dako, Glostrup, Denmark A0425) and a goat antibody preparation to canine IgA (AbD Serotec, Oxford, UK AAI31), all cross-reacting with ferret immunoglobulins were absorbed and used for a sandwich ELISA in order to test their specificity for ferret Ig classes Results: Figure 1: Monoclonal antibodies directed against human CD markers cross-reacting with ferret Figure 2 : Bronchial wall from a normal ferret, stained for α gal 2-6 (A) and with periodic acid Schiff (B). C and D show a small bronchus from an infected ferret with similar stainings. Figure 3:ELISA of the 3 purified ferret Ig preparations (IgG, IgA and IgM) using mink immunoglobulin class specific reagents. Coating of the plates was made with a commercial anti-canine IgA, anti-human IgM or absorbed anti-mink IgG. • 21 mAbs directed at human CD markers cross-reacted with ferret leucocytes, along with a single anti-CD3 mAb produced against mink CD3 and 4 mAbs against major cytokines. (Fig.1) • Sambucus Nigra lectin, periodic acid Schiff staining and monoclonal  IgG against NeuAcα2-6GalNAc-O-The/Ser allow to study the distribution of influenza virus receptors and related structures in the ferret respiratory tract. (Fig. 2) • Anti-human IgM, anti-dog IGM, and anti-mink IgG were absorbed and found to cross-react with ferret Ig classes, allowing the creation of class-specific ELISA for ferret immunoglobulins. (Fig.3) Conclusion:This new set of reagents will prove invaluable in the study of influenza infections in the ferret. Monoclonal antibodies against cytokines and CD marker, along with class-specific Ig ELISAs will allow to take a look at finer immunological parameters, and histology/immunohistochemistry methods will help better understand the pathology of influenza virus in ferrets, and assess the effect of new vaccines in pre-clinical studies