Accepted Manuscript

Title: Type I interferons in the pathogenesis and treatment of

canine diseases

Authors: Daniela Klotz, Wolfgang Baumgärtner, Ingo

Gerhauser

PII: S0165-2427(17)30326-4
DOI: http://dx.doi.org/10.1016/j.vetimm.2017.08.006
Reference: VETIMM 9664

To appear in: VETIMM

Received date: 4-7-2017

Revised date: 8-8-2017
Accepted date: 21-8-2017

Please cite this article as: Klotz, Daniela, Baumgärtner, Wolfgang, Gerhauser, Ingo,
Type I interferons in the pathogenesis and treatment of canine diseases.Veterinary
Immunology and Immunopathology http://dx.doi.org/10.1016/j.vetimm.2017.08.006

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Type I interferons in the pathogenesis and treatment of canine
diseases
Daniela Klotz1, Wolfgang Baumgärtner1,2, Ingo Gerhauser1,3

1 Department of Pathology, University of Veterinary Medicine Hannover, Hannover,

Germany
2 Center of Systems Neuroscience Hannover, Hannover, Germany

3 Corresponding author:
Dr. Ingo Gerhauser, PhD, Dipl. ECVP
Department of Pathology, University of Veterinary Medicine Hannover
Bünteweg 17, D-30559 Hannover, Germany
Tel.: +49 (0) 511 953 8660
Fax: +49 (0) 511 953 8675
E-mail: Ingo.Gerhauser@tiho-hannover.de

Running title: Interferons in dogs

Words: 6551
Figures: 4
Tables: 7
Supplementary Tables: 2

1
Abstract
Type I interferons (IFNs) such as IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω represent cytokines,
which are deeply involved in the regulation and activation of innate and adaptive immune
responses. They possess strong antiviral, antiproliferative, and immunomodulatory activities
allowing their use in the therapy of different viral diseases, neoplasms, and immune-
mediated disorders, respectively. Initially, treatment strategies were based on nonspecific
inducers of type I IFNs, which were soon replaced by different recombinant proteins. Drugs
with type I IFNs as active agents are currently used in the treatment of hepatitis B and C
virus infection, lymphoma, myeloid leukemia, renal carcinoma, malignant melanoma, and
multiple sclerosis in humans. In addition, recombinant feline IFN-ω has been approved for
the treatment of canine parvovirus, feline leukemia virus, and feline immunodeficiency virus
infections. However, the role of type I IFNs in the pathogenesis of canine diseases remains
largely undetermined so far, even though some share pathogenic mechanisms and clinical
features with their human counterparts. This review summarizes the present knowledge of
type I IFNs and down-stream targets such as Mx and 2`,5`-oligoadenylate synthetase
proteins in the pathogenesis of infectious and immune-mediated canine diseases. Moreover,
studies investigating the potential use of type I IFNs in the treatment of canine lymphomas,
melanomas, sarcomas, and carcinomas, canine distemper virus, parvovirus, and
papillomavirus infections as well as immune-mediated keratoconjunctivitis sicca and atopic
dermatitis are presented. A separate chapter is dedicated to the therapeutic potential of
IFN-λ, a type III IFN, in canine diseases. However, further future studies are still needed to
unravel the exact functions of the different subtypes of type I IFNs and their target genes in
healthy and diseased dogs and the full potential action of type I IFNs as treatment strategy.

Key words: dog; interferon stimulated genes; therapy; tumor; type I interferon; virus

2
Contents
1. Introduction........................................................................................................................3
2. Type I interferon pathway..................................................................................................4
3. Inducers of type I interferons............................................................................................6
4. Recombinant type I interferons..........................................................................................7
5. Functions of canine type I interferons and interferon stimulated genes………………………...8
6. Type I interferon treatment in dogs..................................................................................11
6.1 Type I interferons as anti-cancer drugs.......................................................................12
6.2 Type I interferons as antiviral drugs............................................................................14
6.3 Type I interferons in immune-mediated and idiopathic diseases...............................16
7. IFN-λ expression and function in canine diseases.............................................................17
8. Outlook.............................................................................................................................18
Acknowledgements...........................................................................................................18
References........................................................................................................................19

1. Introduction
Interferons (IFNs) were discovered in the 1950s and described as a substance, which
interferes with vaccinia and influenza virus replication (Isaacs and Lindenmann, 1957;
Nagano and Kojima, 1954). Soon after the confirmation of their wide antiviral activity,
several studies also demonstrated the strong anti-proliferative and immunomodulatory
actions of these proteins (Borden, 1979; Toy, 1983). IFNs represent a group of cytokines,
which are deeply involved in the regulation and activation of innate and adaptive immune
responses (Meyer, 2009). They can be subdivided into type I (IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-
ζ, IFN-κ, IFN-τ, and IFN-ω), type II (IFN-γ), and type III (IFN-λ) IFNs (Tables 1 and 2;
Supplementary Table 1). IFN-δ, IFN-ζ, and IFN-τ have been identified in pigs, mice, and
ruminants, respectively, but not in canines and humans (Woelk et al., 2007; Yang et al.,
2013). Furthermore, IFN-ω is present in humans, mice, felines, and cattle but genomic
analysis suggests the deletion of IFN-ω genes from the canine genome (Himmler et al., 1987;
Yang et al., 2013). Both gene conversion and gene duplication have shaped the evolution of
the IFN-α multigene family in eutherian mammals resulting in 13 subtypes in humans, 14 in
cattle, mice, and cats, 17 in pigs, and 18 in rats (Woelk et al., 2007). 10 functional IFN-α
genes and 2 truncated pseudogenes have been described in the dog genome, which are
3
clustered with canine IFN-ε, IFN-κ, and IFN-β on chromosome 11 (Yang et al., 2013).
However, the exact sequences, locations, and names of the different canine IFN-α subtypes
have not been reported and only 5 different IFN-α subtypes can be identified in the dog
genome based on published sequences (Fig. 1). In addition, the classification is solely based
on sequence homologies and information about functional properties of canine IFN-α
subtypes in comparison to other species is lacking.
The production of the first recombinant human (rh) type I IFN proteins in Escherichia (E.) coli
in 1980 raised hope to use them in the therapy of different neoplastic and viral diseases
(Nagata, 1980). In 1986, IFN-α2a (Roferon-A®) and IFN-α2b (IntronA®) were finally approved
by the Food and Drug Administration (FDA) for the treatment of hairy cell leukemia. The
treatment of the relapsing-remitting form of multiple sclerosis (MS) was revolutionized in
1995 by the approval of IFN-β1b (Betaferon®) in the European Union, which was followed by
the release of two formulations of IFN-β1a (Avonex® and Rebif®). Despite the development
of neutralizing anti-IFN antibodies in about 15% of patients, these drugs remain an
important element in the current guidelines of MS treatment strategies so far (Bertolotto,
2015). Moreover, common diseases of the canine central nervous system (CNS) including
demyelinating encephalitis (canine distemper) and vasculitis (steroid-responsive meningitis
arteritis) can share pathogenic mechanisms and clinical features with specific human
diseases (Beineke et al., 2009; Spitzbarth et al., 2012). Consequently, new therapeutic
approaches for these cytokine-driven, immune-mediated diseases, which target type I IFN
signaling, could be evaluated in clinical dog studies. However, in contrast to the intensive
research on the role of IFNs in human diseases, studies investigating the functions of these
highly active cytokines in dogs are rather limited. This review gives an update on the impact
of the type I IFN signaling pathway in the pathogenesis of various canine diseases and the
use of recombinant type I IFNs in their treatment. In addition, studies investigating the
functions and potential therapeutic use of canine IFN-λ, a type III IFN, are summarized.

2. Type I interferon pathway

The innate immune response can be activated by so-called pattern recognition receptors
(PRRs), which bind to molecules associated with infectious agents (pathogen-associated
molecular patterns, PAMPs) or cell components of damaged or dead cells (damage-
associated molecular patterns, DAMPs) (de Rivero Vaccari et al., 2014). These PRRs include
4
multiple toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like RNA helicases
such as RIG-I (DExD/H-box helicase 58, DDX58) and melanoma differentiation-associated
gene 5 (MDA5; interferon induced with helicase C domain 1, IFIH1) (Thompson and Locarnini,
2007). PRRs use specific adaptor proteins including myeloid-differentiation primary-response
gene 88 (MyD88) and toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-β
(TRIF) to activate transcription factors such as IFN regulatory factor (IRF)-3 and -7 and
nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB). These factors
translocate into the nucleus and initiate the transcription of type I IFNs and other pro-
inflammatory cytokines and chemokines (Carty et al., 2014; Kim and Maniatis, 1997; Schafer
et al., 1998; Yie et al., 1999). The high degree of similarity in the genomic structure of the 10
IRF family members in vertebrates including zebrafish, sticklebacks, frogs, anole lizards, and
dogs points to their important roles in the control of innate immune signaling pathways
(Huang et al., 2010) (Table 3; Supplementary Table 2).
Major producers of type I IFNs in the periphery are plasmacytoid dendritic cells (pDCs),
which show a high constitutive expression of IRF7 enabling them to rapidly synthesize large
amounts of IFN-α and IFN-β after viral infection (Delhaye et al., 2006). These type I IFNs bind
to ubiquitous membrane receptors, which are formed by a heterodimer of two
transmembrane proteins (IFNαR1, IFNαR2) (Kim et al., 1997). The signal transduction
cascade involves STAT1, STAT2, and the IFN regulatory factor 9 (IRF9), which form a complex
named IFN-stimulated gene factor 3 (ISGF3) and bind to the IFN-stimulated response
element (ISRE) in the promoter region of specific target genes (Honda et al., 2006). The wide
expression of STAT1 allows most cell types to mount type I IFN-dependent responses but
variations in the combinatorial association and abundance of IFNAR-JAK-STAT signaling
components control the magnitude and the selectivity of IFN responses (Bluyssen et al.,
1995; Ivashkiv and Donlin, 2014). Activation of the type I IFN pathway induces the
production of a wide array of antiviral proteins including the "classical” IFN stimulated genes
(ISGs) ISG15, protein kinase R (PKR; also known as EIF2AK2), Myxovirus resistance protein
(Mx), and 2`,5`-oligoadenylate synthetase (OAS) and the more recently detected effectors
apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), tripartite
motif containing 5 (TRIM5), zinc-finger antiviral protein (ZAP), RNA-specific adenosine
deaminase (ADAR), IFN-induced transmembrane protein (IFITM)1/2/3, tetherin (also known
as BST2), and viperin (also known as RSAD2) (Sadler and Williams, 2008; Schoggins and Rice,

5
2011). In addition, type I IFNs can stimulate IRF7 production, which triggers a positive
feedback loop (Sato et al., 1998). Recently, an in vitro study using cells infected with
vesicular stomatitis virus (VSV) identified a primate-specific microRNA (miR-576-3p), which is
induced by IRF3 and reduces IFN expression (Yarbrough et al., 2014). This negative feedback
mechanism sets an antiviral response threshold most likely to avoid excessive inflammation
(Yarbrough et al., 2014). However, viruses had to evolve mechanisms to impede the IFN
signaling cascade due to the high efficiency of ISGs in blocking viral replication
(Devasthanam, 2014). For instance, the morbillivirus V-protein inhibits the nuclear
translocation of STAT1 and STAT2 proteins but not STAT1 and STAT2 degradation or
phosphorylation (Palosaari et al., 2003; Röthlisberger et al., 2010; Svitek et al., 2014). NS1B
protein of influenza B virus binds to ubiquitin-like ISG15 proteins thereby inhibiting its
conjugation to an array of proteins (Sridharan et al., 2010). Besides, the species-specificity of
NS1B protein partly explains the restriction of influenza B viruses to humans, because it
blocks human and non-human primate but not murine and canine ISG15 proteins (Sridharan
et al., 2010). The major steps in the type I IFN signaling pathway are summarized in Figure 2.
The basal expression of STAT1 and selected ISGs (ISG15, Mx, OAS1, and PKR) in the
cerebellum of a healthy dog is shown in Figure 3.

3. Inducers of type I interferons

Soon after the discovery of IFNs, several in vitro studies have been performed to elucidate
the mechanisms of type I IFN induction and downstream signaling pathways. These studies
have been facilitated by simulating virus infections with polyriboinosinic-polyribocytidylic
acid (poly I:C), a synthetic analog of double-stranded (ds) RNA (Field et al., 1968). The
amount of poly I:C required for IFN induction varies considerably between primary kidney
cells isolated from dogs, humans, rabbits, calves and hamsters (Field et al., 1968). In general,
dog cells seem to be poor responders to poly I:C complexes (Smith et al., 1980). In addition,
a study using different domestic animal cell lines stimulated by eight rh IFNs demonstrated a
decreasing sensitivity of cells derived from ox, sheep, pig, cat, horse, and dog, the latter
being quite insensitive to most human IFNs (Bridgman et al., 1988). Nevertheless, the
activity of type I IFNs seems to depend more on the tissue of origin than the species of origin
of the stimulated cells (Hu et al., 1995).

6
The induction of IFNs has also been used as a treatment strategy in humans and animals. In
1970, tilorone hydrochloride was described as an orally active interferon inducer active
against several DNA and RNA viruses in mice (Krueger and Mayer, 1970). This agent
enhances the activity of Natural Killer (NK) cells and shows anti-pyretic, anti-fibrotic, anti-
inflammatory, and anti-tumor activity most likely by IFN induction, which was demonstrated
in several cell culture and animal studies (Zhang et al., 2015). Tilorone (trade names Amixin®,
Lavomax®, and others) is used in human medicine to treat virus infections including
influenza, viral hepatitis, and herpes, allergic encephalomyelitis, pulmonary tuberculosis as
well as urogenital and respiratory chlamydia. This drug has not been approved for veterinary
purposes but it has been demonstrated to prolong allografts in dogs and rats (Wildstein et
al., 1975). Interestingly, Yu Ping Feng San, an ancient chinese herbal decoction containing
Astragali Radix (Huangqi), Atractylodis Macrocephalae Rhizoma (Baizhu), and
Saposhnikoviae Radix (Fangfeng), triggers the transcription of several ISGs including RNaseL,
Mx2, PKR, and ISG15 in the murine macrophage cell line RAW 264.7 (Du et al., 2015). In
addition, this herbal mixture, traditionally used as remedy for infection and inflammation
symptoms, suppresses the neuraminidase activity of influenza virus A in Madin-Darby canine
kidney epithelial (MDCK) cells, thereby preventing viral release and spreading (Du et al.,
2015).

4. Recombinant type I interferons

The effects of the different type I IFN subtypes upon canine immune responses were initially
characterized in vitro by using rh proteins. Rh IFN-α suppresses B-cell differentiation in a
dose-dependent fashion and enhances cytotoxicity of NK cells and interleukin (IL)-2
production by activated T-lymphocytes in dogs (Krakowka et al., 1988). Rh IFN-α1, rh IFN-α2,
and rh IFN-ω exhibit similar anti-proliferative effects on MDCK cells, whereas only the two
IFN-α proteins significantly reduced VSV replication in this canine cell line (Kubes et al.,
1994). Species-specific anti-viral activities were also shown for chicken IFN-α, porcine IFN-
α1, human IFN-β, and murine IFN-β, which did not protect D-17 canine osteosarcoma cells
from VSV infection (Berger Rentsch and Zimmer, 2011).
The generation of recombinant canine (rc) IFNs necessary for species-specific studies in dogs
already started in the 1980s by the expression of mature canine IFN-α1 in E. coli (Himmler et
al., 1987). Rc IFN-α1 activates the JAK-STAT signaling pathway in murine fibroblasts (3T3)
7
and neuronal cells (NA) (Wang et al., 2014). In addition, a recombinant rabies virus
expressing rc IFN-α1 enhanced immune responses resulting in its attenuation and stronger
immunogenicity (Wang et al., 2014). A baculovirus system has been used to produce a high
amount of bioactive rc IFN-α4 bearing an histidine hexamer at the C-terminal region
(Ruttanapumma et al., 2006). Recently, canine IFN-α has been fused to different protein tags
to facilitate its expression and purification in E. coli and thereby its use in treating viral
diseases in dogs (Yang et al., 2015). Rc IFN-β has been produced by the infection of the
insect cell line Sf21 with a recombinant baculovirus (Iwata et al., 1996b). Interestingly, the
growth of a recombinant vaccinia virus expressing canine IFN-β was inhibited by the antiviral
activity of IFN-β indicating that this recombinant virus could be used as a suicide viral vector
(Nishikawa et al., 2000). Rc IFN-ε seems to have a relatively broad cross-species activity
because it strongly inhibits the replication of VSV in both homologous MDCK and
heterologous MDBK, human cervical carcinoma (HeLa), and Crandell Rees feline kidney
(CRFK) cell lines (Capes-Davis et al., 2010; Yang et al., 2013). Furthermore, rc IFN-ε exhibits a
significant anti-proliferative response in canine A72 and MDCK cells in a dose-dependent
manner (Yang et al., 2013). However, rc IFN-ε is approximately 16-fold less potent than rc
IFN-α7 in promoting NK cell cytotoxicity (Yang et al., 2013). Only one study investigated rc
IFN-κ so far and demonstrated antiviral activities in a VSV-MDCK virus-target cell system
(Yang et al., 2013). Table 4 summarizes the antiviral activities of rc IFN proteins on
homologous and heterologous cell lines (Ruttanapumma et al., 2006; Taira et al., 2005; Yang
et al., 2013).

5. Functions of canine type I interferons and interferon stimulated genes

In general, type I IFNs affect cell proliferation and differentiation, modulate the immune
response, inhibit angiogenesis, and promote apoptosis (Kotredes and Gamero, 2013; Maher
et al., 2007; Takaoka et al., 2003). Type I IFNs stimulate innate and adaptive immunity by
enhancing antigen presentation, inducing cytokines and chemokine production, augmenting
antibody production by B cells, and amplifying the effector function of T cells and NK cells
but also possess immunosuppressive properties, which are partly mediated by IL-10 and
programmed cell death 1 ligand 1 (PDL1) (Ahlenstiel et al., 2011; Ivashkiv and Donlin, 2014;
Kohlmeier et al., 2010; Rizza et al., 2010). IFNs also inhibit endocytosis in various cell lines,
which reduces the invasiveness of facultative intracellular bacteria and the internalization of
8
viruses such as VSV (Bukholm et al., 1990). These multifaceted properties of type I IFNs are
currently used in the treatment of human MS (Vosoughi and Freedman, 2010), chronic
hepatitis C virus infection (Tsubota et al., 2011), and certain types of cancer such as
melanoma in humans (Ferrantini et al., 2007; Gajewski and Corrales, 2015; Kohlmeier et al.,
2010; Rizza et al., 2010).
At the end of the 1970s, first studies were performed in dogs to evaluate type I IFN
production but did not differentiate between its subtypes. Serum type I IFN concentrations
reach a peak by 8 hours after inoculation of UV-inactivated Newcastle disease virus and
rapidly decline thereafter (Tsai and Appel, 1979). Lymphoid cells in the spleen and bone
marrow seem to contribute to this type I IFN production to a large extent, whereas
peripheral blood lymphocytes show minimal and macrophages no type I IFN production (Tsai
and Appel, 1983). Nevertheless, cells of the monocytic-phagocytic series and
immunocompetent cells derived from dog bone marrow, spleen, thymus, and lymph nodes
were all found to be capable of producing type I IFNs in vitro in response to inoculation of
viral and non-viral inducers (Georgadze et al., 1990). Type I IFNs disappear from the serum of
dogs experimentally infected with CDV 16 days after infection, but remain constantly
detectable in their cerebrospinal fluid (CSF) (Tsai et al., 1982). Consequently, it has been
suggested that type I IFNs in the CSF might be used as a marker for CDV persistence and
severity of CNS lesions (Kimoto, 1986; Tsai et al., 1982). The cellular origin of this type I IFN
production was not identified at this time but recent mouse studies demonstrated that
astrocytes, ependymal cells, and neurons seem to be the most important type I IFN
producers in the CNS (Delhaye et al., 2006; Detje et al., 2015). Nonetheless, IFN-ε and IFN-κ
were transcriptionally not up-regulated in the CNS of mice after infection with neurotropic
viruses such as Theiler's murine encephalomyelitis virus and La Crosse virus and only 3% of
neurons infected produced IFN-α and IFN-β (Delhaye et al., 2006). The role of type I IFNs in
canine diseases, which are typically not induced by viruses, has also been investigated. One
study documented the presence of IFN-α in the synovial fluid of osteoarthritic dogs but its
role remained undetermined (Salinardi et al., 2006). In addition, oligonucleotide microarrays
and real-time PCR was used to characterize the gene expression changes that occur in
advanced canine glaucoma (Jiang et al., 2010). However, genes with elevated expression in
the glaucomatous retina were largely associated with antigen presentation and protein
degradation, whereas IFNs and interleukins were not detected at abnormal levels.

9
Only few studies investigated the functions of classical ISGs in dogs and mainly concentrated
on canine Mx and OAS proteins (Fig. 4). Canine Mx1 and Mx2 proteins are localized in the
cytoplasm, whereas rodent Mx1 proteins have a nuclear localization signal (Nakamura et al.,
2005). Correspondingly, the canine Mx1 protein has high homology to human MxA (76.9%),
murine Mx2 (73.5%), and rat Mx2 (72.5%). The canine Mx2 protein is more homologous to
human MxB (74.5%). Nonetheless, canine Mx2 but not human MxB has an antiviral property
against VSV, which might be related to its cytoplasmic localization in a granular-like and not
diffuse pattern (Nakamura et al., 2005). Interestingly, Mx2 from multiple primates can inhibit
HIV-1, whereas Mx2 from dogs and sheep cannot (Busnadiego et al., 2014). Mx protein
expression has been demonstrated in various viral, protozoal, mycotic, and idiopathic canine
encephalitis including canine distemper, Chagas disease, aspergillosis, and granulomatous
meningoencephalomyelitis (Porter et al., 2006). Canine Mx proteins can be found in
astrocytes, macrophages/microglia, and neurons including Purkinje cells and granular layer
neurons in the cerebellum (Porter et al., 2006). Their functions have not been analyzed in
detail but human MxA has been shown to interfere with the production of new infectious
virus particles by binding to viral nucleocapsids and preventing replication of viral mRNA
(Haller et al., 2015). OAS proteins detect ds RNA and activate RNaseL, which degrades viral
RNA and thereby inhibits viral protein synthesis. There are three OAS genes (OAS1, OAS2,
OAS3) and similar to rodents two OAS-like (OASL) genes in the dog genome (Fig. 4), whereas
the human OASL2 gene represents a pseudogene (Perelygin et al., 2006). Eleven OAS1
subtypes (OAS1a-k) exist in rodents, which have not been described in other mammalian
species (Perelygin et al., 2006). One study detected unusually high OAS activity in the serum
of female beagle dogs, which was 10- to 100-fold higher than in cats, rabbits, and guinea pigs
but increased only 10-fold after infection with viruses or bacteria (Iwata et al., 2004). There
also seemed to be a negative correlation between OAS serum activity and age and/or
vaccination. Nevertheless, it could not be excluded that the dogs used in this study were
persistently infected with viruses without exhibiting any clinical signs (Iwata et al., 2004).
Recently, a microarray study demonstrated an upregulation of the ISGs IFN alpha-inducible
protein 6 (IFI6) and IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) in CDV
infected Schwann cells, olfactory ensheathing cells, CNS Schwann cell-like glia, and
fibroblasts . An upregulation of several ISGs including IFI6 and IFIT1 as well as Mx1, Mx2,

10
OAS1, ISG15, PKR, and TRIM22 was also found in the cerebellum of CDV-infected dogs
(Ulrich et al., 2014).

6. Type I interferon treatment in dogs

Type I and II IFNs are cytokines of major interest in human and veterinary medicine due to
their antiviral, anti-tumor and immunomodulatory effects in various diseases including
canine papillomatosis and canine pyoderma (Foster, 2004). Canine type I and II IFNs also
inhibit the growth of Neospora caninum tachyzoites in MDCK cells in a dose-dependent
manner but IFN-γ inhibits the parasite growth with greater efficacy than IFN-α or IFN-β
(Nishikawa et al., 2001). Moreover, low dose oral administration of canine IFN-α4 reduced
periodontopathic bacterial counts and induced improvement of naturally occurring gingival
inflammation in dogs (Ito et al., 2010).
Several studies have investigated the pharmacokinetics of type I IFNs in dogs. One study
compared the pharmacokinetic profiles of recombinant IFN-α administered via the
intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.), and oral (p.o.) route to 15 male
beagle dogs (Gibson et al., 1985). The IFN-α serum concentration was below the detection
limit after oral administration and the systemic bioavailability for the i.m. and s.c. routes was
42% when compared with i.v. infusion. The elimination half-lives of IFN-α ranged from 4.5 to
9.5 h (Gibson et al., 1985). Preliminary studies in healthy dogs on the intravesical instillation
of rh IFN-β for the treatment of canine bladder cancer demonstrated absence of systemic
bioavailability (Hara, 1989). Similarly, IFNs can be given via the inhalation route to achieve a
pulmonary topical effect (Mallet et al., 1997). Oromucosally administered IFN-α can
stimulate cells of the local mucosa-associated lymphoid tissue to produce endogenous IFN-α
and IFN-γ thereby inducing the expansion of antigen-specific CD4+ and CD8+ T lymphocytes
(Dec and Puchalski, 2008). Furthermore, the production of cytokines such as TNF, IL-6, and
IL-12 by expanded T lymphocytes can lead to systemic effects of oromucosally administered
IFN-α (Tompkins, 1999). Interestingly, human IFN-β is resorbed after intranasal
administration and shows a slow decline of plasma concentrations in dogs and rats but not
rabbits, which is most likely attributed to species-specific differences of absorption rate
constant (Igawa et al., 1990). Consequently, the oromucosal administration of human and
animal type I IFN might be used as a prophylactic or therapeutic agent in veterinary medicine
for viral diseases of the respiratory tract such as influenza (Dec and Puchalski, 2008). The
11
major potential disadvantages of type I IFN use in dogs are the expense and poor owner
compliance with daily injections. Neutralizing antibodies may eventually also be produced
against type I IFN, which might limit efficacy necessitating the use of higher doses (Clifford et
al., 2000). However, the co-administration of human IFN-α with a higher tacrolimus dose
(0.1 mg/kg) in Beagle dogs suppresses the production of these neutralizing IgG antibodies,
which minimizes this potential disadvantage of type I IFN therapy (Yamazaki et al., 2010).
Tacrolimus (also known as FK506 or fujimycin) represents an immunosuppressive 23-
membered macrocyclic polyketide, which was first isolated in 1984 from a culture broth of
Streptomyces tsukubaensis and inhibits IL-2 transcription and thereby the development and
proliferation of T cells (Ban et al., 2016). Drugs approved in the European Union containing
interferons for therapy of human and animal including canine diseases are summarized in
Table 5.

6.1 Type I interferons as anti-cancer drugs

The anti-proliferative properties of type I IFNs have been known for decades and initiated
first clinical trials in human lymphoma, osteosarcoma, and melanoma patients (Borden,
1979). The promising results of these studies resulted in the evaluation of treatment options
of type I IFNs in the therapy of canine neoplastic diseases. One in vitro study investigated the
single and combined effects of human IFNs and cobalt-60 radiation on canine and feline
tumor cells derived from osteosarcoma, melanoma, thyroid carcinoma, nasal
adenocarcinoma, squamous cell carcinoma, and lymphosarcoma (Kessler et al., 1996). In
general, the sensitivity to IFNs was higher in cell lines derived from round cell tumors
compared to those derived from solid tumors. Incubation of lymphosarcoma cells with 100-
1000 U/ml IFN-α2 resulted in survival rates between 78 and 82% but higher cell kills were
found in combination with cobalt-60 radiation. Interestingly, even small single doses of
human IFNs showed significant effects on the investigated tumor cell lines in vitro (Kessler et
al., 1996). Interestingly, therapy of a dog with epitheliotropic lymphoma using rh IFN-α2a
resulted in rapid resolution of clinical signs and a 10-week disease-free interval (Tzannes et
al., 2008). Unfortunately, the lymphoma recurred at 17 weeks and did not respond to rescue
chemotherapy. Nevertheless, treatment of additional oral lesions with localized
radiotherapy and increased IFN dosages allowed another 12 weeks of stable disease
(Tzannes et al., 2008). IFN-α2a might also be used in the treatment of canine
12
hemangiosarcoma due to its inhibitory effect on angiogenesis via suppression of basic
fibroblastic growth factor (bFGF) and vascular endothelial growth factor (VEGF) production
(Clifford et al., 2000). Interestingly, an inhibition of angiogenesis was also postulated as an
important oncolytic mechanism of CDV in a permanently infected histiocytic sarcoma (DH82)
cell line (Pfankuche et al., 2017). Moreover, this effect seems to be mediated by a
downregulation of VEGF, which was shown in a microarray analysis of CDV-infected
compared to uninfected DH82 cells in addition to a more than 100-fold upregulation of the
ISG TRIM22 (Pfankuche et al., 2017).
Encouraging result were also found in a study investigating the treatment of fibrosarcomas
(8 dogs), osteosarcomas (5 dogs), myxosarcomas (2 dogs), and liposarcomas (1 dog) using a
combined therapy with canine IFN-β, which prevented or delayed local relapses and the
formation of metastases resulting in relatively long survival times (Finocchiaro et al., 2011).
IFN-β gene transfer into human and canine melanoma cells using lipofection induces a
strong cytotoxic bystander effect indicating a high clinical potential (Rossi et al., 2015).
Similarly, IFN-β-overexpressing canine adipose tissue-derived mesenchymal stem cells (cAT-
MSCs) showed significant pro-apoptotic and growth-inhibitory effects on canine melanoma
cells in vitro (Han et al., 2015). Moreover, cAT-MSCs producing IFN-β and cisplatin
synergistically reduced the tumor volume in BALB/c nude mice xenografted with the canine
melanoma cell line LMeC (Ahn et al., 2013).
Several studies investigated the growth inhibition activities of recombinant feline (rFe) IFN-ω
on tumor cells of different species. The rFe IFN-ω had a dose-dependent inhibitory effect on
the cell growth and colony formation of various cell lines derived from canine tumors
including oral acanthomatous epulis, mammary benign mixed tumor, squamous cell
carcinoma, and malignant melanoma (Tateyama et al., 1995). The anti-proliferative and
colony inhibiting activities of rFe IFN-ω on canine and feline cells were not found in human
liver (Chang), monkey kidney (Vero), and hamster lung (HmLu-1) and kidney (BHK-21) cells
(Priosoeryanto et al., 1995). The in vitro antitumor activity of rFe IFN-ω on feline and canine
mammary carcinoma cells further increased in combination with conventional anticancer
drugs such as anthracyclines (Penzo et al., 2009). Table 6 gives an overview about studies
performed to determine the potential of type I IFNs as anti-cancer drugs. In general, clinical
studies lack control animals and are based on a too small number of investigated cases to
draw final conclusions about the effectiveness of IFN tumor therapy.

13
6.2 Type I interferons as antiviral drugs
The ability of type I IFNs to interfere with the growth of influenza virus enormously
contributed to their discovery (Isaacs and Lindenmann, 1957; Isaacs et al., 1957). Several
ISGs mediate the antiviral activity of type I IFNs, for instance TRIM22 targets the influenza A
virus nucleoprotein for degradation (Di Pietro et al., 2013). Human MxA blocks nuclear
translocation of nucleocapsids and replication of genomes of influenza A viruses (Haller et
al., 2015). Interestingly, the high yields of influenza A virus in MDCK cells might partially be
caused by a lack of anti-influenza activity of canine Mx proteins (Seitz et al., 2010). Recent
studies demonstrated that autophagy and p53 are implicated in IFN-α induction and
subsequent type I IFN-mediated immune responses against influenza A virus infections,
respectively (Law et al., 2010; Zhu et al., 2014). Furthermore, a stable knockdown of IRF7 can
increase influenza virus production in MDCK cells through mechanisms other than inhibition
of type I induction (Hamamoto et al., 2013). Few studies investigated the interactions of
canine influenza viruses with type I IFN signaling. Avian-like H3N2 canine influenza virus, a
newly emerged etiological agent for respiratory infections in dogs, induces the expression of
IFN-β and several ISGs including Mx1, Mx2, TRIM22, ISG15, DDX58, and OAS1-3 (Park et al.,
2015; Su et al., 2015). Expression levels of these ISGs were increased at 12 hours and 4 days
post infection (dpi) and lowered again at 7 dpi maybe due to inhibited virus replication by
the host response (Su et al., 2015). Nevertheless, this fast IFN-β response is blocked by the
NS1 protein of canine influenza virus by inhibiting NF-κB and IRF3 signaling pathways (Su et
al., 2016). However, the well-described inhibitory effects of type I IFNs on influenza A virus
replication prompted several clinical trials in humans, which demonstrated that the
application of rh IFN-α2b by a nasal spray can help to prevent an infection (Gao et al., 2010;
Yu et al., 2005). Consequently, future studies should also analyze the applicability of type I
IFN treatment in viral respiratory diseases of dogs.
In contrast to influenza viruses, canine papillomaviruses do not induce an up-regulation of
type I IFNs and pro-inflammatory cytokines in their target cells (Luff et al., 2013). In addition,
an infection of canine keratinocytes does not result in an up-regulation of ISGs including IFN
inducible gene 16 (IFI16) and interferon-induced protein with tetratricopeptide repeats 1
(IFIT1) at 2, 4, or 6 dpi (Luff et al., 2014). Nevertheless, therapy of a Miniature Schnauzer and
an American Staffordshire Terrier suffering from papillomavirus-induced pigmented
14
epidermal plaques with low doses of rh IFN-α2a for 1-2 years resulted in a regression of
lesions and prevention of recurrence (Stokking et al., 2004). Unfortunately, the same
treatment failed in a similar case in a Pomeranian, in which squamous cell carcinoma arose
within several plaques (Stokking et al., 2004). Moreover, the treatment of multiple
papillomavirus-associated epidermal hamartomas and squamous cell carcinomas in situ in a
4-year-old spayed female toy fox terrier with IFN-α had to be discontinued after 2 weeks,
when the dog developed diarrhea and an increase in liver enzymes (Callan et al., 2005).
However, it remained uncertain whether IFN-α therapy was the cause of these problems.
The combination of ribavirin and IFN-α exhibited high antiviral activity for the intra- and
extracellular stages of the replicative cycle of CDV in Vero cells (Carvalho et al., 2014). A
combination of IFN with clarithromycin, hydrocodone, and vitamin C was described as
treatment in a 9-week-old male Australian Shepherd mix with a chronic cough due to CDV
infection but the outcome for this dog remains unknown (Townsell et al., 2015). Generally,
the therapeutic value of IFN therapy can be hardly assessed by the above described case
reports, which lack proper controls.
In contrast to few preliminary studies in other viruses, the efficacy of type I IFN treatment is
well documented in CPV infection. Clinical effects of rFe IFN-ω on experimental parvovirus
infection has already been found in the 1990s (Ishiwata et al., 1998). This treatment shifted
the enteritis from a severe to mild form and improved vomiting and anorexia in Beagle dogs
infected with CPV-2. Another study demonstrated that rFe IFN-ω enhances the cellular
immunity of macrophages and natural killer cells in healthy dogs (Kuwabara et al., 2006). A
rFe IFN-ω preparation (INTERCAT™) also represents the first veterinary antiviral agent, which
has been approved as a treatment agent for feline calicivirus infection in 1994 and for CPV
infection in 1997 in Japan (Minagawa et al., 1999). Subsequently, a similar product
(Virbagen® Omega) was licensed by the European Medicines Agency (EMEA) as a remedy to
treat CPV, feline leukemia virus (FeLV), and feline immunodeficiency virus (FIV) infections.
The applicability of feline IFNs in dogs was confirmed in further studies despite earlier
descriptions of a high degree of species specificity in these two companion animals (Iwata et
al., 1996a). An experimental trial with 36 4-month-old Beagle dogs and a field trial
conducted on 93 dogs showed significant efficacy on CPV-2 infection, which was
characterized by clinical improvement and strong reduction of mortality from 61.9% (13/21)
in controls to 19.4% (14/72) in IFN-ω treated animals (Minagawa et al., 1999). Similarly, a

15
challenge trial of 8-9-week-old Beagle pups inoculated oro-nasally with a virulent strain of
CPV-2b and a placebo-controlled field trial with 94 dogs suffering from parvoviral enteritis
confirmed that rFe IFN-ω was effective in improving clinical signs and reducing mortality
without inducing drug-associated side effects (de Mari et al., 2003; Martin et al., 2002).
Table 7 gives an overview about studies performed to determine the potential of type I IFNs
as antiviral drugs.

6.3 Type I interferons in immune-mediated and idiopathic diseases

Several studies investigated the therapeutic potential of the immunomodulatory properties
of type I IFNs in immune-mediated diseases and idiopathic disorders of dogs. The oral
administration of low-doses of human IFN-α to dogs suffering from immune-mediated
keratoconjunctivitis sicca resulted in a favorable response in 55% (11/20) of all dogs treated
without side effects noted (Gilger et al., 1999). Dogs responding to IFN-α treatment showed
increased wetting of the eyes, decreased mucus discharge, fewer signs of discomfort, and in
7 of 11 dogs increased Schirmer's tear test (Gilger et al., 1999). Another pilot study tested
human IFN-α for the management of idiopathic recurrent superficial pyoderma in dogs
(Thompson et al., 2004). The oral use of rh IFN-α2b at 1,000 IU/ml/day provided only a
transient benefit in this disease (Thompson et al., 2004). Atopic dermatitis (AD) is a common
allergic skin disease of dogs, which is associated with IgE antibodies most commonly directed
against environmental allergens (Halliwell, 2006). Interestingly, this canine disease
represents the best model to study both pathogenesis and microbiome modifications in
human atopic dermatitis (Plager et al., 2012; Santoro and Rodrigues Hoffmann, 2016).
Canine AD and related human eosinophilic allergic diseases share gene transcription
abnormalities also affecting IFN-inducible genes (Plager et al., 2012). A double-blind
controlled study demonstrated that rFe IFN-ω may help in the long-term management of
canine AD (Carlotti et al., 2009). The oral and subcutaneous therapy of canine AD with rFe
IFN-ω can improve the pruritus scores as well as the extent and severity of lesions (Litzlbauer
et al., 2014). However, allergen avoidance, allergen-specific immunotherapy, and oral
treatment with glucocorticoids, ciclosporin, and oclacitinib have the highest evidence for
efficacy, whereas additional studies are necessary to determine for which dogs and at which
stage of disease recombinant IFNs should be included in the treatment of canine AD (Olivry
and Bizikova, 2013; Olivry et al., 2015; Saridomichelakis and Olivry, 2016).
16
7. IFN-λ expression and function in canine diseases
Type III IFNs were described in 2003 and members of this IFN group have been identified in
humans, monkeys, mice, chicken, frogs, cattle, bats, and canines (Diaz-San Segundo et al.,
2011; Fan et al., 2014; Kotenko et al., 2003; Sheppard et al., 2003). IFN-λs are mainly
produced by epithelial cells of the lung, intestine, and kidney after activation of IRF-3 and
IRF-7 transcription factors (Donnelly and Kotenko, 2010). The IFN- receptor represents a
dimer of IFN-R1 and IL-10R2 proteins, which signal via STAT proteins and induce ISG
transcription similar to type I IFNs (Donnelly and Kotenko, 2010; Yang et al., 2010a). Humans
possess three subtypes of type III IFNs, which are referred to as IFN-1, IFN-2, and IFN-3
or IL-29, IL-28a, and IL-28b, respectively, whereas only IFN-1 has been identified in dogs
(Fan et al., 2014). Rc IFN-λ1 has antiviral and anti-proliferative effects on MDCK cells
underlining its potential as antiviral and anticancer agent (Ichihashi et al., 2013).
Furthermore, IFN- might be involved in the differentiation of early B and T cells (Yang et al.,
2010a).
Fibroblasts, endothelial cells, monocytes, and T cells express IFN-receptors but do not
respond to IFN-s (Ichihashi et al., 2013; Witte et al., 2009). Consequently, treatment of
infectious diseases and tumors with this type III IFN might result in less toxicity than type I
IFNs. However, IFN-stimulates STAT3 in canine mammary tumor cells, which results in
increased expression of angiogenic factors, epithelial-mesenchymal transition (EMT), and
increased cell migration in vitro (Mucha et al., 2014). Similarly, a high-throughput mRNA and
miRNA profiling of EMT demonstrated an up-regulation of ISGs in Ras-transformed MDCK
cells (Shukla et al., 2015). The expression of human IFN-λ is also related to poor prognosis in
bladder, blood, and breast cancer (Yang et al., 2010b). Moreover, human IFN-λs seem to be
under the transcriptional control of the tumor-related transcriptional factors steroidogenic
factor-1 (SF-1), Wilms tumor 1 (WT-1), and P53 and might even take part in cancer
development (Yang et al., 2010b). Therefore, the therapeutic potential of IFN-λs in canine
and human neoplastic diseases might be limited. However, a recent study showed that IFN-λ
and IL-22 act synergistically for the induction of ISGs such as Mx1, PKR, and ISG15 in murine
intestinal epithelial cells (IEC6) and curtail replication of VSV, poliovirus, and rotavirus
indicating therapeutic potential as antiviral drug (Hernandez et al., 2015). Rc IFN-λ1 also
induces expression of Mx1 and OAS1 in a dose-dependent manner and shows antiviral
17
activity on both homologous and heterologous cells (Table 4) (Fan et al., 2014; Ichihashi et
al., 2013).

8. Outlook
Since the detection of type I IFNs 60 years ago abundant studies have been performed to
elucidate their functions and therapeutic potential. The antiviral, antiproliferative, and
immunomodulatory capacities of type I IFNs are already used in the therapy of different viral
diseases, neoplasms, and immune-mediated disorders of humans and animals. However,
systematic experimental and field studies with an adequate number of control animals,
similar to the studies on canine parvoviral enteritis, are needed to determine the efficacy of
type I IFN treatment for these diseases. The medical costs, which have to be spent for a long-
term treatment with type I IFNs, are also limiting its use in veterinary medicine and have to
be reduced. Furthermore, there are outstanding questions in basic and clinical research left
to be answered in detail:
1. Which cells produce the different subtypes of type I IFN?
2. Which cells are targeted by the different subtypes of type I IFN?
3. Which genes are transcriptionally activated by the different subtypes of type I IFN?
4. What are the precise interactions between type I IFNs and other signaling cascades?
5. Are there species-specific differences in the properties of type I IFNs?
6. Which cross-species activities have the different subtypes of type I IFN?
7. Which pharmacokinetics exhibit the different type I IFN subtypes in dogs and other
species?
8. Which side effects are induced by a type I IFN treatment in animals?
9. Can the different type I IFN subtypes be used to target specific cells or organs and/or
limit side effects?
Detailed information of the properties of the different type I IFN subtypes in dogs and other
species are fundamental to understand their role in the pathogenesis of infectious and non-
infectious diseases and unravel the full potential of type I IFNs as treatment strategy.

Acknowledgments
The authors are thankful to Bettina Buck, Petra Grünig, Caroline Schütz, Kerstin Schöne, and
Claudia Herrmann for excellent technical assistance.
18
Competing interests
The authors declare that they have no competing interests.

Authors’ contributions
DK performed the literature search, designed the figures and tables, and helped to draft the
manuscript. WB helped to design and draft the manuscript. IG drafted the manuscript and
helped to design the figures and tables. All authors read and approved the final manuscript.

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Figure legends

Figure 1: Schematic drawing of canine interferon gene loci.

Canine type I (IFNA, IFNB, IFNE, IFNK), type II (IFNG), and type III (IFNL) interferon genes are

located on chromosome 11, 10, and 24 in the canine genome, respectively. The five

functional IFNAs (black arrows) form a gene cluster on chromosome 11 with outer limits

defined by IFNB and IFNE. The IFNK gene is located 4.7 Mbp from this gene cluster on

chromosome 11. The direction of transcription is visualized by the direction of the arrows.

MX, Myxovirus resistance protein; OAS, 2`,5`-oligoadenylate synthetase; OASL, OAS-like.

Figure 2: Type I interferon signaling pathway

Left: The infection of cells by viruses can be recognized by specific receptor molecules, which

are located in the cytoplasm or endosomes. These receptors including TLR3, TLR7, MDA5,

and PKR detect virus-specific ssRNA or dsRNA and activate transcription factors such as IRF1,

IRF3, IRF5, IRF7, and NF-κB, which translocate into the nucleus to induce the transcription of

IFN-α and IFN-β. Right: Type I IFNs bind to a membranous receptor and activate the JAK-

STAT signaling pathway causing the expression of target genes including ISG15, PKR, OAS,

and Mx. The latter acts in a positive feedback loop to stimulate type I IFN transcription.

IFN, interferon; IFNAR, type I IFN receptor; IRF, IFN regulated factor; ISG, IFN stimulated

gene; JAK, Janus family kinase; NF-κB, nuclear factor of kappa light polypeptide gene

enhancer in B cells; MDA5, melanoma differentiation-associated gene 5; MyD88, myeloid

differentiation primary response gene 88; Mx, Myxovirus (Influenza Virus) resistance

protein; OAS, 2`,5`-oligoadenylate synthetase; PKR, protein kinase R; STAT, signal transducer

and activator of transcription; TLR, toll-like receptor; TRIF, Toll/interleukin-1 receptor

domain-containing adapter-inducing IFN-β; TYK, non-receptor tyrosine-protein kinase.

27
Figure 3: Interferon stimulated gene expression in the cerebellum of a healthy dog.

A-F: Serial sections from the same area of the cerebellum of a healthy beagle dog. Arrows

indicate Purkinje cells. A: Hematoxylin and eosin staining of normal cerebellar tissue. B:

Strong STAT1 protein expression in the nuclei of ependymal cells. STAT1-positive nuclei are

also found in the cerebellar white matter. The Purkinje cells are negative for STAT1. C:

Strong ISG15 expression in ependymal cells. Weak ISG15 expression in Purkinje cells. D:

Weak expression of Mx protein in ependymal cells. All other cells are negative for Mx

protein. E: Strong OAS protein expression in Purkinje and ependymal cells. Weak OAS

protein expression in other gray and white matter cells. F: Strong PKR protein expression

within Purkinje, endothelial, and ependymal cells. B-F: Immunohistochemistry visualized by

the avidin-biotin-peroxidase complex method with 3,3-diaminobenzidine as substrate and

counterstaining with Mayer’s hematoxylin. GM, gray matter; ISG, interferon stimulated

gene; Mx, Myxovirus resistance protein; OAS, 2`,5`-oligoadenylate synthetase; PC, Plexus

choroideus; PKR, protein kinase R; STAT, signal transducer and activator of transcription;

WM, white matter. Bars, 100 μm.

Figure 4: Schematic drawing of canine MX and OAS gene loci.

Canine MX and OAS genes are located on chromosome 26 and 31, respectively. Each gene is

represented by its abbreviated name and the arrows denote the direction of transcription.

MX, Myxovirus resistance protein; OAS, 2`,5`-oligoadenylate synthetase; OASL, OAS-like.

28
29
30
 Tables

Table 1: Main characteristics of canine interferons

Characteristics IFN- IFN- IFN- IFN- IFN- IFN-

IFN type Type I Type I Type I Type I Type II Type III
1 Exon 1 Exon
Gene structure 1 Exon (Intronless) 2 Exons 4 Exons
(Intronless) (Intronless)
Number of
5 1 1 1 1 1
subtypes
Pseudogenes* 2 0 0 0 0 2
Length of
mature 172-191 186 147 209 166 190
peptide
IFNAR IFNGR IFNLR
Receptor
(IFNAR1/IFNAR2) (IFNGR1/IFNGR2) (IL28RA/IL10RB)
IFN, Interferon; IFNAR, IFN- receptor; IFNGR, IFN- receptor; IFNLR, IFN- receptor;

IL28RA, Interleukin 28 receptor subunit ; IL10RB, Interleukin 10 receptor subunit ;

* IFN-α: described in Yang et al. 2013; IFN-λ: described in Fan et al. 2014

Table 2: Comparison of canine interferon genes and proteins with their human and murine

counterparts

% identity % identity % identity / % identity /

Length
Gene with human with mouse % positives with % positives with
(dog)
gene gene human protein mouse protein
IFNA1 564 72 68 52 / 70 48 / 66
IFNA2 519 73 68 53 / 65 46 / 64
IFNA4* 564 72 65 55 / 71 47 / 64
IFNA7 564 71 68 52 / 63 48 / 66
IFNA10§ 576 70 - 51 / 68 -
IFNB 561 75 66 60 / 75 44 / 59
IFNE 444 83 72 74 / 82 57 / 70
IFNK 630 80 76 71 / 83 38 / 53
IFNG 501 78 68 66 / 78 45 / 67
IFNL# 573 80 77 73 / 82 61 / 74
IFN, Interferon; IFNA, IFN-; IFNB, IFN-; IFNE, IFN-; IFNK, IFN-, IFNG, IFN-; IFNL, IFN-;

*Canine IFNA4 is identical to canine IFNA3, IFNA5, IFNA6, and IFNA8; §Predicted sequence;

#Canis lupus familiaris and Homo sapiens: IFNL1; Mus musculus: IFNL2
31
 Table 3: Comparison of canine IRF genes and proteins with their human and murine

counterparts

Gene % identity % identity % identity / % identity /

Length Number Chromosome, with with % positives % positives
Gene
(Exon of Exons (Region) human mouse with human with mouse
Length) gene gene protein protein
7329 Chromosome 11, 88 82
IRF1* 10 89 82
(966) (20773801..20781129) 93 90
78547 Chromosome 16, 95 94
IRF2* 13 92 90
(1119) (46053138..46131684) 96 97
4050 Chromosome 1, 74 66
IRF3* 6 82 76
(846) (106875016..106879065) 82 76
21274 Chromosome 35, 94 92
IRF4* 11 90 86
(2421) (760367..781640) 97 96
11216 Chromosome 14, 83 84
IRF5* 11 85 83
(1818) (7723693..7734908) 85 86
19433 Chromosome 7, 98 96
IRF6* 9 93 90
(1389) (8447826..8467258) 98 97
4101 Chromosome 18, 81 81
IRF7* 7 83 81
(1077) (25709006..25713106) 87 92
23233 Chromosome 5, 92 88
IRF8* 9 90 85
(1281) (66789265..66812497) 94 92
5094 Chromosome 8, 80 70
IRF9* 9 84 77
(1233) (4152109..4157202) 87 80
IRF, Interferon regulatory factor; *Predicted in dog

32
 Table 4: Antiviral activities of recombinant canine interferon proteins on homologous and

heterologous cell lines.

Canine Bovine Rabbit Feline Human

Canine Chicken Feline
kidney kidney kidney kidney cervical
Interferon fibroblasts embryo macrophages
cells cells cells cells carcinoma
(A72) fibroblasts (Fcwf-4)
(MDCK) (MDBK) (RK-13) (CRFK) (HeLa)
Rc IFN-α4 n.d. VSV VSV n.d. n.d. n.d. n.d. n.d.
CAV-1
CDV
Rc IFN-α7 CHV-1 n.d. n.d. VSV* VSV* n.d. VSV* n.d.
CPV IAV
VSV
CAV-1
Rc IFN-α8 CHV-1* n.d. n.d. VSV* VSV* n.d. VSV* n.d.
VSV
CDV
Rc IFN-ε CPV IAV n.d. n.d. VSV n.d. VSV n.d. VSV
VSV
Rc IFN-κ VSV n.d. n.d. n.d. n.d. n.d. n.d. n.d.
CPV IAV
Rc IFN-λ1 n.d. n.d. VSV n.d. n.d. n.d. VSV
VSV
CAV, canine adenovirus; CDV, canine distemper virus; CHV, canine herpes virus; CPV, canine

parvovirus; IAV, influenza A virus; RC, recombinant canine; VSV, vesicular stomatitis virus;

n.d., not determined; *Lack of antiviral activity of a recombinant interferon protein against a

specific virus is indicated by crossed out letters.

33
 Table 5: Interferons approved in the European Union for therapy of human and animal

including canine diseases

European
Approved Administration
Agent Trade Manufacturer Indications
for route*
Name
IFN-α2a Roferon Roche Pharma Human Hepatitis B and C virus s.c.
A® AG infection, cutaneous T-cell-
lymphoma, hairy cell
leukemia, myeloid
leukemia, renal carcinoma,
follicular non-Hodgkin
lymphoma, malignant
melanoma
IFN-α2b Intron A® SP Labo N.V. Human Hepatitis B and C virus s.c. / i.v.
infection, hairy cell
leukemia, myeloid
leukemia,
follicular lymphomas,
carcinoid tumor, malignant
melanoma
IFN alfacon-1 Infergen® Yamanouchi Human Hepatitis C virus infection s.c.
Pharma Co
Peginterferon- Pegasys® Roche Pharma Human Hepatitis B and C virus s.c.
α2a AG infection
Peginterferon- PegIntron® SP Labo N.V. Human Hepatitis C virus infection s.c.
α2b
IFN-β1a Avonex® Biogen Human Multiple Sclerosis i.m.
IFN-β1a Rebif® Merck Serono Human Multiple Sclerosis s.c.
S.p.A.
IFN-β1b Betaferon® Bayer Pharma Human Multiple Sclerosis s.c.
AG
Peginterferon- Plegridy® Biogen Human Multiple Sclerosis s.c.
β1a
IFN-γ1b Imukin® Böhringer Human Septic Granulomatosis, s.c.
Ingelheim Osteopetrosis
Type I IFN Zylexis® Pfizer Animal Dogs, Cats, Herpes virus and calicivirus s.c. (dogs, cats)
(Inactivated Health Cattle, Pigs, infections, stress-induced
Parapoxvirus Horses respiratory and intestinal i.m. (cattle, pigs,
ovis, diseases horses)
Strain D 1701)
IFN-ω Virbac® Virbac S.A. Dogs Canine Parvovirus s.c. / i.v.
Omega
Cats Feline Leukemia Virus,
Feline Immunodeficiency
Virus

34
* i.m., intramuscular; i.v., intravenous; s.c., subcutaneous. Virbac® Omega represents a

feline recombinant protein and is the only solely on interferon based drug, that is approved

for veterinary purposes. Gray shading, veterinary licensed drug.

35
 Table 6: Studies investigating the potential of type I IFNs as anti-cancer drugs

Interferon Disease Method Summary Reference

 Rh IFN-α2a  Epitheliotropic  In vivo  Rapid and complete Tzannes et al.,
Lymphoma  Daily treatment of remission of lesions primarily 2008
one dog by due to IFN therapy
subcutaneous  Remission and survival similar
injection (4.8 – 6.4 to or greater than that
x 104 IU/kg) obtained with conventional
chemotherapy
 Rh IFN-α2a  Lymphosarcoma  In vitro  Higher sensitivity of Kessler et al.,
 Rh IFN-α2b  Melanoma  Survival rate after hematopoietic tumors and 1996
 Rh IFN-γ  Nasal IFN treatment melanomas to IFN compared
adenocarcinoma to other tumor types
 Osteosarcoma
 Squamous cell
carcinoma,
 Thyroid
carcinoma
 Canine IFN-β  Osteosarcoma  In vivo  Treatment without significant Finocchiaro et
 Fibrosarcoma  Intra- and/or adverse side effects al., 2011
 Liposarcoma peritumoral  Treatment delays or prevents
 Myxosarcoma injection (2 mg/ml post-surgical recurrence and
Plasmid DNA, 1-4 metastases
ml depending on  Treatment increases the
tumor size) overall survival time
 Human IFN-β  Melanoma  In vitro  Significant cytotoxic effect of Rossi et al.,
 Canine IFN-β  Lipofection of cells IFN-β on melanoma cells 2015
 Cell growth assay  Lipofection with IFN-gene
 Bystander effect more effective than addition
assay of rh IFN-protein
 Fluorescence flow  Remarkable bystander effect
cytometry of ROS of rh IFN-with increased cell
death rates and intracellular
ROS concentration
 Canine IFN-β  Melanoma  In vitro  Pro-apoptotic and growth- Han et al.,
 IFN- inhibitory effects of IFN-β on 2015
overexpressing canine melanoma cells
adipose tissue-
derived
mesenchymal stem
cells
 Cell-cycle analysis
 Quantification of
apoptotic cells

36
 Table 6 continued

Interferon Disease Method Summary Reference

 Canine IFN-β  Melanoma  In vitro  IFN-β inhibits growth of Ahn et al.,
 Co-culture of IFN- melanoma cells in vitro 2013
overexpressing
adipose tissue-
derived
mesenchymal stem
cells and canine
melanoma cell line
(LMeC)


In vivo  Combination treatment of

BALB/c nude mice IFN-β and low-dose cisplatin

Xenograft model induces high levels of

TUNEL assay for apoptosis of transplanted
apoptotic cells melanoma cells in vivo
 RFe IFN-ω  Oral  In vitro  RFe IFN-ω reduces cell Tateyama et
acanthomatous  Viable cell counting proliferation and colony- al., 1995
epulis  Colony-forming forming ability of canine cells
 Mammary benign inhibition assay especially mammary benign
mixed tumor mixed tumor cells
 Squamous cell  Activity of rFe IFN-ω
carcinoma dependent on target cell
 Malignant type, rather than on true
Melanoma species differences
 RFe IFN-ω  Oral  In vitro  Antiproliferative and colony Priosoeryanto
acanthomatous  Viable cell counting inhibiting activities of rFe IFN- et al., 1995
epulis  Colony-forming ω on canine cells are cell-
 Mammary benign inhibition assay specific and dose-dependent
mixed tumor  Human, monkey and hamster
cells resistant to rFe IFN-ω
 RFe IFN-ω  Mammary tumor  In vitro  Antitumor activity of rFe IFN- Penzo et al.,
 Viable cell counting ω on mammary carcinoma 2009
 Sphere assay cells with dose-dependent
and target cell-specific action
 Canine mammary tumor cells
more sensitive to rFe IFN-ω
than feline counterparts

37
 Table 7: Studies investigating the potential of type I IFNs as antiviral drugs
Interferon Disease Method Summary Reference
 RFe IFN-ω  Canine  In vivo  RFe IFN-ω rapidly shifts enteritis Ishiwata et
Parvovirus  Intravenous treatment of from a severe in a mild form al., 1998
experimentally infected dogs  RFe IFN-ω improves vomiting and
once daily for three days (1 x anorexia
106 IU/kg/day)
 RFe IFN-ω  Canine  In vivo  RFe IFN-ω improves clinical signs Minagawa et
Parvovirus  Intravenous treatment of and reduces mortality al, 1999
experimentally and naturally  RFe IFN-ω inhibits decrease of
infected dogs once daily for leukocytes and promotes early
three days (1-5 x 106 recovery
IU/kg/day)
 RFe IFN-ω  Canine  In vivo  RFe IFN-ω improves clinical signs Martin et al.,
Parvovirus  Intravenous treatment of and reduces mortality 2002
experimentally infected dogs
once daily for three days (2.5
x 106 IU/kg/day)
 RFe IFN-ω  Canine  In vivo  RFe IFN-ω significantly reduces De Mari et al.,
Parvovirus  Intravenous treatment of mortality 2003
naturally infected dogs once
daily for three days (2.5 x 106
IU/kg/day)
 IFN-α2a  Canine  In vivo  Papilomatous skin lesions Stokking et
Papilloma  Oral treatment of three dogs regressed in two dogs al., 2004
Virus with low dose IFN-α2a (1 x  New lesions developed in one dog
103 IU/dog/day) on a 21-day 12 months after discontinuation
on, 7-day off schedule of therapy
 Squamous cell carcinoma arose
within third dog
 IFN-α  Canine  In vivo  No remarkable effect on Callan et al.,
Papilloma  Subcutaneous treatment of papillomatous skin lesions or 2005
Virus one dog with IFN-α three arising squamous cell carcinomas
times weekly for two weeks in situ
6 2
(1 x 10 IU/m /day)  Discontinuation of therapy after
two weeks due to development of
diarrhea and increased liver
enzymes
 Unclear if IFN-α therapy caused
clinical symptoms
 Rh IFN-α  Canine  In vitro  Strong efficacy of rh IFN-α against Carvalho et
Distemper  Microscopic evaluation of canine distemper virus al., 2014
Virus Vero cell viability and  Combination of ribavirin and rh
morphology IFNα exhibits antiviral activity for
 Measurement of the intra- and extracellular stages
mitochondrial enzymes of the replicative cycle
reduction
 IFN  Canine  In vivo  Outcome and treatment protocol Townsell et
(Type of IFN Distemper  Treatment of one dog with for this dog not reported al., 2015
not Virus distemper suffering from
reported) respiratory symptoms

38