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

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:

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

Figure 1: Monoclonal antibodies directed against human CD markers cross-reacting with ferret

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.

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.

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.

CD14

CD172a

CD44

CD8 CD29

CD88

CD104

CD25

CD32

CD9

CD61

IgMIgG2a IgG2bIgG1 CD3

CD18

CD79bCD71

TNF-alpha IL-4 IFN-gammaIL-8

Anti IgG

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0,5

1000 2000 4000 8000 16000 32000 64000

Dilution

OD

IgG

IgA

IgM

negative

Anti IgA

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1000 2000 4000 8000 16000 32000 64000

Dilution

OD

IgG

IgA

IgM

negative

Anti IgM

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1000 2000 4000 8000 16000 32000 64000

Dilution

OD

IgG

IgA

IgM

negative

•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)