Moving Forward: Recent Developments for the Ferret Biomedical Research Model

Since the initial report in 1911, the domestic ferret has become an invaluable biomedical research model. While widely recognized for its utility in influenza virus research, ferrets are used for a variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens.

populations. It is likely that new models and transgenic animals will be developed in the near future.
The sequencing of the ferret genome (15) was instrumental in advancing functional genomic analysis. Numerous groups developed reagents to monitor gene-specific mRNA expression levels via TaqMan-based or Sybr green-based real-time reverse transcription-PCR assays for a plethora of targets. Many of these primers are available free of charge through the National Institute of Allergy and Infectious Diseases (NIAID) established BEI Resources (https://www.beiresources.org/Home.aspx). Bruder et al. described the development of an expression microarray platform that included the identification of 41 genes with consistent baseline transcription profiles across tissues that could be used as housekeeping genes (16). Our group developed and is validating a FLUIDIGM panel with 144 distinct immune response and lung injury and repair genes. Beyond transcription, Tisoncik-Go et al. described an integrated omics analysis that profiles lipids, metabolites, and proteins in the respiratory compartments of influenza virus-infected ferrets (17). Combined, these tools provide powerful resources to the research community.

THE NEXT FRONTIER: THE IMMUNE RESPONSE
Despite its relevance for biomedical research, there are limitations of the ferret model for immunologic studies due to the dearth of reagents. Screening of commercially available antibodies for cross-reactivity with markers on innate and adaptive cell subsets and cytokines in ferrets has yielded limited success ( Table 2). To resolve this, a group of researchers from around the world are working together to develop validated reagents and assays to improve our understanding of the innate and adaptive immune responses in the ferret.
To date, recombinant proteins representing a range of intrinsic, innate, and adaptive immune markers are under development, and some are already available from commercial sources (18,19). These include type I and III interferons (IFNs), RIG-I and Toll-like receptors, cytokines, and chemokines, as well as cell surface markers for immune and nonimmune cells. In terms of adaptive immune responses, Kirchenbaum and Ross recently developed a monoclonal antibody against the ferret B cell receptor light chain that is useful in distinguishing kappa versus lambda B cell responses (20,21). Enzymelinked immunosorbent spot (ELISpot) and flow cytometric assays have been developed to quantify the isotypes of antibody-secreting cells (IgG or IgA) (22), pan-B cells (CD20 ϩ , CD79␣ ϩ ), and Ig ϩ B cells (18,19). T cell phenotyping has been limited to quantification of overall CD3 ϩ T cells, including CD4 ϩ and CD8 ϩ subsets, by flow cytometric assays and identification of antigen-specific effector responses by detecting IFN-␥ secretion in flow-based intracellular cytokine secretion assays or ELISpot assays (18). An in vivo depletion of CD8 T cells using a cross-reactive human monoclonal antibody has been shown to delay influenza virus clearance (23). To increase our toolbox, the Centers for Excellence in Influenza Research and Surveillance (CEIRS) network has undertaken a large project to rapidly produce monoclonal antibodies and develop assays to support the universal influenza vaccine initiative (24). Antibodies in production include B cell markers (CD83, CD86, CD95, CD19, CD20, CD25, CD27, CD38, CD138, CXCR5, and FcR), T cell markers (CD4, CCR7, CD3e, CD40, CD40L, CD44, CD62L, CD69, CD103, PD-1, CXCR3, interleukin-7 receptor [IL-7R], and IL-15Ra) and others (CXCR4, CD140, IL-2, IL-21, and IL-4). These much-needed reagents will facilitate efforts to establish immunologic assays to interrogate the innate and adaptive immune responses to infection and vaccination at the level of detail that is routinely applied to studies of mouse or human immunology. Importantly, the ferret model will allow correlates of protection to be established after vaccination and infection in conjunction with transmission studies, which are not available in the mouse models. Additionally, the longer life span of the ferret relative to the mouse will allow analysis of the evolution of the immune response to sequential infection and/or vaccination (25), permitting more accurate modeling of the immune response in humans.

WAYS FORWARD
While there has been exciting progress, much work remains to move the ferret model forward. Toward this goal, the CEIRS group has produced fibroblasts and primary nasal and tracheal epithelial cells and cell lines, established a repository of defined tissues and cell types (Table 3), and are working with the J. Craig Venter Institute to define the ferret major histocompatibility complex (MHC). An exciting achievement is the completion of the PacBio sequencing of the ferret MHC (Granger Sutton, personal communication). While these are important steps, the ultimate goal is to provide the biomedical research community with validated reagents and protocols they can trust to ensure the rigor and reproducibility in experiments utilizing the ferret model. In support of this goal, many of the reagents created through the CEIRS network will be made publicly available through the CEIRS Data Processing and Coordinating Center (DPCC) website (http://www.niaidceirs.org/resources/ceirs-reagents/).

ACKNOWLEDGMENTS
We thank everyone involved in Team Ferret, whose names we will not list for fear we might miss someone, as well as others producing reagents for the ferret model. We also thank Diane Post (NIAID) and the members of the CEIRS network for feedback, advice, and constructive criticism.