Improving immunological insights into the ferret model of human viral infectious disease

Abstract Ferrets are a well‐established model for studying both the pathogenesis and transmission of human respiratory viruses and evaluation of antiviral vaccines. Advanced immunological studies would add substantial value to the ferret models of disease but are hindered by the low number of ferret‐reactive reagents available for flow cytometry and immunohistochemistry. Nevertheless, progress has been made to understand immune responses in the ferret model with a limited set of ferret‐specific reagents and assays. This review examines current immunological insights gained from the ferret model across relevant human respiratory diseases, with a focus on influenza viruses. We highlight key knowledge gaps that need to be bridged to advance the utility of ferrets for immunological studies.


| FERRE TS A S AN INFLUENZ A PATHOG ENE S IS AND TR ANS MISS ION MODEL
Ferrets can be directly infected with influenza from human clinical isolates without prior adaptation. Influenza infection of ferrets recapitulates key hallmarks of human clinical disease, such as high fever accompanied by sweating, as well as respiratory symptoms such as rhinorrhea and sternutation. 35, 36 The shared susceptibility to influenza infection is based on similarity in respiratory tract physiology, where a predominance of α2,6-linked sialic acid (SA) receptors in the upper respiratory tract of ferrets mimics that of humans, 37-41 unlike α2,3-SA prevalent in other species such as mice. Ferrets are an extremely valuable model for studies on influenza pathogenesis 42,43 and both direct and aerosol transmission. 44,45 Critically, ferrets are a susceptible host for highly pathogenic avian strains of influenza with pandemic potential, such as H5N1 and H7N9, although disease severity in infected ferrets is somewhat variable. 42,[46][47][48] Similar variability in pathogenesis has been reported for some seasonal strains such as recent H3N2 isolates, 49,50 which display a range of disease severity in humans 51 but generally remain mild in ferrets. 52,53

| FERRE TS FOR INFLUENZ A SURVEILL AN CE AND VACCINE DE VELOPMENT
Ferrets play a critical role in annual seasonal influenza vaccine strain selection. Antigenic drift in circulating strains is monitored primarily using hemagglutination inhibition (HI) assays on serum from ferrets infected with recently circulating human viral isolates. 54,55 In both ferrets and humans, HI titres are a marker of protection from acquiring infection and currently the key immunological correlate for assessing potential vaccine effectiveness.

| FERRE TS A S AN IMMUNOLOG I C AL MODEL FOR S TUDYING INFLUENZ A
The utility of ferrets for incisive immunological studies is hampered by limited reagents to study ferret immunity and a paucity of background knowledge about the ferret immune system. Some insights into ferrets' immunological responses to influenza have been gained by indirect measurements of immune gene expression such as quantitative RT-PCR (qRT-PCR), transcriptome analysis or oligonucleotide microarrays. 22,[96][97][98][99][100] For example, assessing the differential expression levels of innate and adaptive immune genes in the lungs following primary or secondary 2009 H1N1pdm infection revealed upregulation of interferon-stimulated genes involved in antiviral responses such as C-X-C motif chemokine 10 (CXCL10), 2′-5′ oligoadenylate synthase 1 (OAS1), interferon regulatory factor 1 (IRF1) and radical S-adenosyl methionine domain containing 2 (RSAD2) as well as chemokines such as CXCL16 and C-C motif chemokines 3, 4 and 5 (CCL3, CCL4 and CCL5). 98 Similarly, the degree of disease severity, virus shedding and transmission in ferrets has been associated with tumour necrosis factor (TNF) and interleukin-6 (IL-6) mRNA expression in the upper respiratory tract. 97 However, mRNA levels can correlate poorly with protein levels, 101 and techniques such as flow cytometry, bead arrays and immunohistochemistry would facilitate direct measurement of immune marker expression. To date, flow cytometric or microscopy techniques have been limited in ferrets by the lack of suitable antibodies specific for ferret immune cell markers.
Screening for cross-species reactivity has identified antibody clones recognising ferret T-cell markers such as CD3 and CD8, and an intracellular B-cell marker CD79b 102-104 (summarised in Table 1).
The utility of such cross-reactive reagents has been shown experimentally. For example, prime-boost immunisation using DNA and adenoviral-based influenza vaccines provided effective protection in experimentally challenged ferrets, with protection correlating to the capacity of CD3+ T cells to express interferon gamma (IFN-γ) following in vitro stimulation on peripheral blood mononuclear cells (PBMCs) with HA peptide pools. 92 Caution should be taken when using antibodies developed for other species in ferret experiments, as there may be subsets of cells displaying variable reactivity to the antibodies. 105,106 In addition, currently available anti-ferret B-cell antibodies such as CD79α target intracellular epitopes, requiring fixation and permeabilisation. This limits downstream applications such as RT-PCR or the recovery of antigen-specific immunoglobulin sequences from sorted B cells. In the absence of cross-reactive clones, several groups have generated novel monoclonal antibodies specific for ferret cellular markers. For example, novel antiferret CD4-, CD8-and CD5-specific antibodies were derived by  109 and are reviewed in detail further below.
Using existing reagents, innate and adaptive immune responses to influenza infection 52,108,110 and immunisation 83,111 have been studied and provide important insights into ferret antiviral immunity.

| FERRE TS A S AN IMMUNOLOG I C AL MODEL FOR OTHER EMERG ING VIR AL DISE A SE S
In addition to influenza, the ferret serves as a critical model for

SARS-CoV infection causes acute respiratory distress in humans
with mortality rates of up to 10%. 135  However, leucocyte counts and interferon-related gene expression were decreased upon re-infection, 5 suggesting that innate immune dysregulation is a possible mechanism of pathogenesis, though a protective antibody response was also evident during attempts to re-infect ferrets. 8

| Henipavirus
Emerging viruses belonging to the Paramyxoviridae family (Henipaviruses) can cause severe respiratory illness and/or encephalitis in humans. Ferrets infected with henipaviruses exhibit similar symptoms as humans including respiratory signs such as cough and nasal discharge, neural signs such as depression, 32 and high mortality rates with most experimentally infected ferrets succumbing within 1 week. 31 While the virus is detected in pharyngeal and rectal secretions, it is currently unclear if ferrets could serve as a transmission model for the disease. 31,32 Ferrets infected intranasally with henipaviruses similarly display clinical illness. 31,34 Assessment of immune gene expression by Leon et al 31 in both lungs and brain tissues of the infected ferrets revealed upregulation of macrophage markers such as CD40 and CD80 in both lung and brain tissues, whereas lymphocytic markers were unchanged in the lungs.

Ebola virus disease (EVD) is caused by a zoonotic virus from the
Filoviridae family of viruses. 28  Ferrets display hallmarks of pathological processes of human lethal infections such as petechial rashes, reticulated pallor of the liver and splenomegaly. 23,24 Transmission has also been reported in ferrets. 141 As for immunological studies, transcriptomic sequencing in ferrets

| K E Y K NOWLEDG E G APS TO ADDRE SS IN ORDER TO IMPROVE THE IMMUNOLOG I C AL UTILIT Y OF FERRE T MODEL S
While the ferret model has unique potential for informative studies into pathogenic viral infections as noted above, addressing several key knowledge gaps will substantially advance the ferret as an immunological model.

| Immunogenetics
There is a lack of well-annotated, ferret genomic sequence infor-

| Future T cell-specific reagents for ferrets
Future development of markers to delineate more T-cell subsets will increase the utility of the ferret as an immunological model; a recent report listed several important ferret T cell-specific antibodies to be in production at the CEIRS such as CD4, CCR7 , CD3e, CD40,   CD40L, CD44, CD62L, CD69, CD103, PD-1, CXCR3, CXCR5, IL-7R and IL-15R. 109

| Future B cell-specific reagents for ferrets
To increase our understanding of antibody responses in ferrets, flow cytometric reagents that are able to delineate B-cell subsets are required. Important ferret B cell-specific antibodies that are in production at the CEIRS include CD83, CD86, CD95, CD19, CD20, CD25, CD27, CD38, CD138 and FcR. 109

| Current and future markers for ferret myeloid lineage cells
Several markers defining innate cell populations in mice and humans such as CD11b 103,108 and CD14 102 have also been reported to cross-react with ferret leucocytes and have been utilised to characterise ferret innate immune responses. However, these markers have also been found to be expressed in non-myeloid lineages in humans, 150,151 and other markers such as CD16 152 and CD66 153 will be required to better define myeloid cell populations.

| Ferret immunoglobulin subclass and Fc receptors
Ferret immunoglobulin subclass and Fc receptors are not well studied. Currently, only one IgG subclass that has been identified in ferrets, while four different IgG subclasses have been identified in other carnivores such as dogs 154 and minks 155 163 While a ferret-specific T-cell IFN-γ expression assays 164 to measure CD8 T-lymphocyte activation have been developed and validated, the future elucidation of corresponding antibody/Fc receptor ferret orthologues will enable ferrets to be used to evaluate ADCC responses.

| Antigenic recognition of major influenza proteins
Epitope mapping studies using either human or murine monoclonal antibodies have greatly increased our understanding of influenza viral evolution and allowed the identification of major

| CON CLUS ION
Ferrets have tremendous utility for studying the pathogenesis and transmission of several human respiratory diseases and for pre-clinical evaluation of vaccines. Ferrets are a critically important model that directly impacts human seasonal influenza vaccine selection and the pre-clinical development of vaccines for other emerging diseases. However, knowledge gaps limit the in-depth assessment of any immune mechanisms that may underpin transmission, protection and/immunopathology of these viral diseases in the ferret model. There is an urgent need for novel reagents with well-validated and specific targets to resolve different immune cell populations. Improving the ferret model to enable the application of insightful modern immunological tools, such as single-cell B-cell receptor sequencing, next-generation sequencing platforms and other bioinformatics tools, will greatly enhance the informative value of the ferret model which will in turn lead to better immunological interventions for human respiratory diseases.

ACK N OWLED G EM ENTS
This work was supported by NHMRC programme grant #1052979 (SJK) and NHMRC project grant #1129099 (AKW). JW is supported by a Melbourne International Research Scholarship and Melbourne International Fee Remission Scholarship.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R CO NTR I B UTI O N S
JW performed literature review and prepared the manuscript. DL, SK and AW edited and provided comments. All authors contributed to the formulation of this scientific research topic.