Abstract

1. Introduction

Interferons (IFNs) are critical effectors of both innate and adaptive immune responses, associated with the development of immune cell populations and their activation to respond to pathogens, cancers and other insults. IFNs are classified according to the receptors through which they signal (Fig. 1 ). Type I IFNs (-α, -β, -δ, -ε, -ζ, -κ, -τ, and -ω), signal through the IFN-α/β receptor (IFNAR), and are one of three major classes of IFNs, the other two being type II IFN (-γ) and type III IFNs (-λ1, -λ2, -λ3). Type I IFNs were discovered for their effectiveness to inhibit virus replication [1], and have since been shown to exert critical effects on the development and activation of immune cell subsets. Given both their antiviral and immunomodulatory effects, type I IFNs, alone, or in combination with other therapies, have been examined clinically in a variety of chronic and acute viral infections. This review will highlight the antiviral and immunomodulatory effects of type I IFNs, the mechanisms by which viruses inhibit and evade a host type I IFN response, and describe recent therapeutic applications for recombinant type I IFNs in the treatment of viral infections.

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Fig. 1

IFNs and their cognate receptors. IFNs are classified based on the receptors through which they signal. Type I IFNs (-α, -β, -δ, -ε, -ζ, -κ, -τ, and -ω) bind to and activate the IFN-α/β receptor (IFNAR) complex. Type II IFN (-γ) activates the IFN-γ receptor 1 and type III IFNs (-λ1, -λ2, -λ3) signal through a receptor complex made up of IL28RA and IL10R2.

1.1. Induction of type I IFNs, receptor activation and signaling

Type I IFNs are induced following detection of pathogen-associated molecular patterns (PAMPs) and damage/danger-associated molecular patterns (DAMPs) by innate pattern recognition receptors (PRRs). Expressed by both immune and non-immune cells, PRRs comprise Toll-like receptors (TLRs), C-type lectin receptors (CLRs), retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), Aim2-like receptors (ALRs) and cyclic GMP-AMP synthase (cGAS), which together bind a diverse range of extracellular and endosomal PAMPs and DAMPs (Fig. 2 ). PRR signaling results in the expression of type I IFNs and pro-inflammatory cytokines mediated by three essential transcription factors: IFN regulatory factor (IRF)3, IRF7, and nuclear factor-κB (NF-κB) [[2], [3], [4], [5], [6]]. Virus-inducible IFN-β expression is upregulated by the formation of an IFN-β enhanceosome, which is comprised of NF-κB, IRF3, IRF7 and c-Jun at the IFN-β promoter [7,8]. The induction of type I IFNs provides the first line of defense against many diverse pathogens. Indeed, IFN dysregulation can lead to increased virus susceptibility: RIG-I^-/- and MDA5^-/- mice produce lower levels of type I IFNs and are more susceptible to infection by RNA viruses, including Japanese encephalitis virus (JEV), encephalomyocarditis virus (EMCV), and West Nile virus (WNV) [9,10].

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Fig. 2

Pattern Recognition Receptor activation leads to type I IFN production. Binding of PAMPs and DAMPs to host PRRs (TLRs, CLRs, RLRs, NLRs, ALRs, and cGAS) induces signaling cascades that activate IRF3, IRF7 and NF-κB, resulting in the production of type I IFNs-α/β and pro-inflammatory cytokines.

Type I IFNs bind to their cognate transmembrane receptor, IFNAR, comprised of an IFN-α/β receptor alpha chain (IFNAR1) and an IFN-α/β receptor beta chain (IFNAR2). Receptor binding leads to activation of multiple intracellular signaling cascades (Fig. 3 ). Best known is activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, whereby IFNAR-associated JAK1 and TYK2 participate in the recruitment of STATs (1–6) to IFNAR and their subsequent phosphorylation-activation to form homo- or heterodimers [[11], [12], [13], [14]]. Unlike other STAT dimers, STAT1-STAT2 heterodimers also bind IRF9 to form the IFN-stimulated gene (ISG) factor 3 (ISGF3) complex [15,16]. In the nucleus, ISGF3 binds to IFN-sensitive response elements (ISREs), 5′-AGTTTN₃TTTC-3′ [15,16], while other STAT dimers bind to IFN-γ activated sequence (GAS) elements, 5′-TTCN₃GAA-3′, to initiate transcription of ISGs [17]. Several non-canonical pathways are also activated by type I IFNs, including the p38-associated mitogen-activated protein kinase (MAPK) signaling pathway [18] to modulate histone modification and early gene expression [19], and the phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) pathway, to regulate mTORC1 activation, protein synthesis and cap-dependent mRNA translation [20][D. Saleiro et al, this issue].

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Fig. 3

Type I IFN signaling. IFNs-α/β bind to IFNAR, inducing the phosphorylation-activation of tyrosine kinases JAK1 and TYK2. JAK1 and TYK2 activation initiates multiple canonical and non-canonical signaling cascades that are critical for the regulation of cellular processes and the expression of ISGs for the innate immune response.

In humans, IFN-inducible transcriptional and translational regulation of ISGs results in the expression of over 7000 genes, that contribute to cellular processes including metabolism, survival, migration, activation and, importantly, innate host defense against viral infections [21]. Notably, many ISGs have been identified with functions that interfere with different stages of viral replication and transmission (Table 1 ). Interestingly, in vitro studies that examined the effects of IFN-ß against Coxsackievirus B3 infection, identified a novel function of IFN-ß in regulating glucose metabolism, mediated by activation of the PI3K/AKT signaling pathway, important for the induction of a rapid antiviral response [59].

Table 1

Antiviral ISGs with known functions.

ISG(s)	Function(s)	Reference(s)
APOBEC3	Cytidine deamination of single-stranded viral DNA (deoxycytidine to deoxyuridine) to inhibit retrovirus replication.	[22,23]
BST2	Binds and inhibits the release of budding progeny virions.	[24,25]
DDX58	RIG-I detects ssRNA to induce MAVS and IRF-dependent type I IFN production.	[9,[26], [27], [28]]
DDX60	Enhances RIG-I and MDA5-dependent type I IFN production.	[29,30]
EIF2AK2	Detects dsRNA and phosphorylates EIF2α to inhibit both cellular and viral mRNA translation.	[31,32]
IFIH1	Detects dsRNA to induce MAVS and IRF-dependent type I IFN production.	[26,28]
IFITM1, IFITM2, and IFITM3	Inhibit viral entry. IFITM3 inhibits the formation of fusion pores in the late endosome.	[[33], [34], [35]]
IRF1 and IRF7	Induce ISG expression in the absence of type I IFN signaling.	[36,37]
ISG15	Regulates host and viral protein function by ISGylation.	[[38], [39], [40]]
ISG20	Cleaves ssRNA to inhibit viral RNA synthesis and replication.	[41,42]
MX1	Forms oligomeric ring structures that bind viral nucleoproteins to inhibit replication.	[[43], [44], [45]]
OAS1, OAS2, and OAS3	Detect dsRNA and synthesize 2′–5′ olygoadenylates, which are the substrate for RNaseL activation.	[[46], [47], [48]]
OASL	Enhances RIG-I activation.	[49]
RSAD2	Restricts viral budding by modulating lipid synthesis.	[50]
SAMHD1	Depletes intracellular dNTPs to inhibit viral replication.	[[51], [52], [53], [54]]
TRIM5	Binds virus capsid proteins to inhibit viral infection.	[55]
TRIM25	Ubiquitinates RIG-I to enhance type I IFN induction.	[56]
ZC3HAV1	Inhibits viral mRNA expression and enhances RIG-I-dependent type I IFN induction.	[57,58]

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2. Viral antagonism of type I IFN responses

Given the effectiveness of IFN-inducible antiviral responses in limiting infection and viral replication in a largely cell-independent manner, irrespective of the virus, viruses have developed different mechanisms to inhibit type I IFN induction and signaling, and the activity of antiviral ISGs (Table 2 ). Viruses may encode in their genomes multiple different proteins that target different facets of type I IFN-inducible antiviral responses, or a single multifunctional protein: herpes simplex virus (HSV) encodes several proteins - ICP0, ICP27, ICP34.5, US11 - directed against different IFN-inducible targets, whereas influenza A virus (IAV), encodes a multifunctional viral protein - NS1 - with the capacity to interfere with multiple host pathways and proteins involved in an antiviral response.

3. Type I IFNs as broad-spectrum antivirals

Newly emerging and re-emerging virus infections pose a serious threat to global health (Fig. 4 ). In the absence of targeted vaccines for newly emerging virus infections, or sufficient vaccine availability for re-emerging virus infections, there is an obvious need for direct antivirals to deploy during a pandemic. Viruses mutate to become resistant to pathogen-specifc antivirals, necessitating the development of broad-spectrum pleiotropic antivirals. Type I IFNs are prototypical candidate broad-spectrum antivirals, specifically because of their rapid induction in response to any and all virus infections, the pleiotropic nature of their effects on inhibition of different stages of viral replicative cycles and their effects on activating immune cells to clear virus infections. Not surprisingly, therefore, as mentioned above, viruses have evolved to evade an IFN response, specifically because it is so critical for the host immune response to infection. However, given the pleiotropic nature of the antiviral effects of IFNs, virus-targeted inhibition of some of these elements may still permit a partial - and effective - IFN response. To date, however, type I IFNs have seen limited clinical use for the treatment of acute and chronic infections.

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Fig. 4

Extent of global viral infections. Graphical depiction of global viral outbreaks and summary of the impact of global viral infections based on information gathered from the World Health Organization (WHO) [196]. CHIKV (grey), chikungunya virus; EBOV (red), Ebola virus; IAV (purple), influenza A virus; MERS-CoV (blue), Middle-East respiratory syndrome coronavirus; LASV (pink), Lassa virus; RVFV (blue-purple), Rift Valley fever virus; SARS-CoV (yellow-green), severe acute respiratory syndrome coronavirus; WNV (green), West Nile virus; YFV (brown), yellow fever virus; ZIKV (gold), Zika virus; DENV, dengue virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HSV-1/2, herpes simplex virus type 1/2; PV, poliovirus.

Pegylated recombinant IFN-α-2a/2b (PEG-INF) in combination with ribavirin, a nucleoside inhibitor of viral RNA synthesis, remains the standard of care for treatment for chronic hepatitis C virus (HCV) infection in those jurisdictions where direct antiviral agents (DAA) are unavailable. Type I IFNs were first approved for single-agent therapeutic use in the context of HCV infection in the 1990s. A major limitation for the clinical use of type I IFNs for HCV has been the prevalence of adverse events, with the most common adverse events including fatigue, headache, pyrexia, rigors, myalgia, nausea, abdominal pain, anxiety, depression, psychosis and insomnia [197]. Serious adverse events include neutropenia, thrombocytopenia, hyper- and hypothyroidism, pancreatitis, type I diabetes mellitus, and irreversible pulmonary hypertension [[198], [199], [200]]. Notably, these adverse advents are associated with sustained IFN treatment. Pegylation of recombinant IFN improved pharmacokinetics to allow for longer dosing intervals [201]. The clinical efficacy of IFN therapy is determined by measuring serum HCV RNA levels, where a sustained virological response (SVR) is defined by undetectable HCV RNA levels at 24 weeks following the completion of the IFN therapy. Patients infected with HCV genotype (G)2, G2, or G3, exhibit SVR rates of between 76–82% [[202], [203], [204], [205]], in comparison to SVR rates of approximately 67% and 40–50% for patients infected with HCV G4 and G1, respectively [[206], [207], [208], [209], [210], [211]]. Furthermore, patients with HCV G1/G2/G3 infections who exhibit a rapid virological response (RVR), defined by undetectable serum HCV RNA after 4 weeks of treatment, are most likely to achieve a SVR [212,213]. In addition to RVR, other predicators of SVR include low expression of the suppressor of cytokine signaling, SOCS3, mRNA and protein in the liver [214], whereas elevated SOCS3 expression [215], and polymorphisms in MX1 and IFNAR1 gene promoter regions are associated with non-response [216,217]. A lack of variation in the gene sequence encoding the PKR-binding domain within E2 and NS5A of HCV G1/G4 may contribute to the increased resistance to PEG-INF and ribavirin combination therapy [218].

PEG-INF has also shown therapeutic efficacy in patients with chronic hepatitis B virus (HBV) infection, as evidenced by seroconversion from hepatitis B e-antigen (HBeAg) positivity (active HBV replication) to HBeAg negativity and detectable levels of anti-HBe antibodies [[219], [220], [221]]. In a study conducted by Keating et al. [221], 42% of HBV infected patients treated with PEG-INF achieved seroconversion, while 12% cleared the virus one year following onset of treatment. In the same study, inactive HBV was detected in another 17% of treated patients.

In a single patient case study of chronic hepatitis E virus (HEV) infection, weekly PEG-INF treatment resulted in a decrease in serum HEV RNA by week 2 and a complete virological response by week 4 [222]. In the same patient, serum HEV RNA remained undetectable after 5 months. An SVR was seen in another patient with chronic HEV infection following PEG-INF treatment, with undetectable serum HEV RNA by week 3 that persisted for 6 months after cessation of PEG-INF treatment [223]. PEG-INF treatment induces ISG transcription and clearance of HEV infection in humanized mice [224].

Apart from their clinical use for chronic virus infections, little consideration has been given to the application of type I IFNs for severe acute virus infections. Between November 2002 and August 2003 over 8000 cases of severe acute respiratory syndrome coronoavirus (SARS-CoV) infection were reported worldwide, resulting in 916 deaths [196]. In the absence of any vaccine or approved DAAs, treatment was limited to corticosteroids, ribavirin and supportive care [225]. IFN alfacon-1 is a synthetic IFN-α, engineered such that at each position along the 165 amino acids of the protein the most frequently represented amino acid among all the IFN-α subtypes is present [226]. Results from in vitro and in vivo studies identify that IFN alfacon-1 consistently exhibits superior antiviral potency compared with other IFN-α subtypes, attributed to its higher binding affinity for IFNAR [[226], [227], [228]]. During the SARS outbreak of 2003, in a pilot clinical study in Toronto, Canada, evidence was provided that treatment with IFN alfacon-1 was associated with more rapid resolution of radiographic lung abnormalities and better oxygen saturation compared with those infected patients who received only corticosteroids [229]. IFN-treated patients exhibited lower levels of creatine kinase and more rapid return of lactate dehyrogenase levels to normal, indicative of improvement to SARS-associated lung parenchymal disease. Notably, given the short treatment time of 10 days, patients tolerated IFN treatment well with minimal adverse events and no exacerbation of any virus-associated symptoms. The single clinical adverse event reported was fever, likely associated with the underlying disease. Subsequent in vitro studies using human bronchial epithelial cells revealed that type I IFN treatment directly inhibits SARS-CoV replication [230,231], overriding the inhibitory effects of the virally encoded factors Nsp1, Nsp3 and ORF6. In non-human primate studies, administration of IFN prior to SARS-CoV infection in cynomolgus macaques limited viral replication and pulmonary damage [226].

Most recently, during the unprecedented 2013–2016 Ebola virus (EBOV) disease outbreak in West Africa, there were no approved therapies or vaccine available. Earlier studies in non-human primates provided evidence that IFN-α/β treatment reduced blood viremia and prolonged the survival of infected animals, [232,233], this despite the viral encoded IFN inhibitory factors, VP24 and VP35. Moreover, when combined with a monoclonal antibody cocktail directed against EBOV glycoproteins (ZMab), adenovirus-vectored IFN-α therapy extended the treatment window post-infection of non-human primates and suppressed viremia, leading to survival and robust immune responses [234]. Using transcription-competent virus-like particles (trVLP) to model EBOV Zaire infection in vitro, requiring only level 2 containment, McCarthy et al. screened a panel of candidate antiviral drugs and assessed their ability to suppress viral replication in human 293T cells [235]. IFN-α and IFN-β, along with the estrogen receptor modulator, toremifene, and number of viral polymerase inhibitors - lamivudine, zidovudine, tenofovir, and favipiravir - inhibited trVLP replication when administered 24h post-infection. Notably, IFN-ß exhibited the most potent antiviral activity. Two- and three-drug combinations were tested using the same in vitro model. The two- and three-drug combinations that most potently inhibited EBOV trVLP replication were IFN-β + lamivudine, and IFN-β + lamivudine+zidovudine, respectively. Moreover, these same drug combinations inhibited infectious recombinant EBOV Zaire replication in 293T cells [235]. Given the superior antiviral potency of IFN-β in both the trVLP studies and when used against EBOV Zaire, IFN-β was administered to 9 patients infected with EBOV in a proof-of-concept, single arm clinical study in Guinea, during the EBOV disease outbreak [236]. When compared to EBOV infected patients who only received standard supportive care at the same treatment facility, IFN-β treatment was associated with faster viral clearance from the blood, earlier resolution of disease symptoms and better survival [235]. Patients tolerated IFN treatment well with no exacerbated disease symptoms. These preliminary clinical findings provide a rationale for further clinical evaluation of IFN-ß for the treatment of EBOV infection.

Type I IFNs have also been shown to inhibit acute influenza A virus (IAV) infections, in vitro [155,237]. In primary human lung explants, IFN alfacon-1 inhibited avian H5N1 and pandemic H1N1 IAV replication, while upregulating the expression of several antiviral ISGs - PKR, OAS, and ISG15 [155]. Treatment of IAV-infected human lung A549 cells with IFN-β inhibits viral replication in a dose-dependent manner [237]. In the context of another acute virus infection, West Nile Virus, Type I IFNs have been shown to increase incorporation of microRNAs (miRNAs) into extracellular vesicles in A549 cells [238]. These enriched miRNAs regulate genes involved in antiviral and pro-inflammatory responses.

The orally administered, low molecular weight IFNAR2 agonist CDM-3008, has been shown to mimic the action of IFN-α by inhibiting both HCV [239] and HBV [240] infection in vitro. CDM-3008 is able to induce JAK-STAT signaling and upregulate ISG expression. Adenovirus-vectored IFN-alfacon-1, which has long-lasting antiviral activity, protects mice, hamsters and non-human primates in animal models of enterovirus (EV) 71 [241], EBOV [234,242], Chikungunya virus (CHIKV) [243], and Rift Valley fever virus (RVFV) infection [244]. A single dose of adenovirus-vectored IFN-α (DEF201), given intranasally within 6h post-RVFV challenge, significantly reduced viral loads in the serum, liver and spleen of hamsters [244]. DEF201, when administered prophylactically 21 days to 24h prior to CHIKV challenge in mice, reduced CHIKV viral titers [243]. Furthermore, administration of DEF201 at 6h and 12h post-lethal EV71 infection of mice resulted in full and partial protection, respectively [241]. Viewed together, these in vivo studies provide a rationale for the evaluation of the prophylactic and therapeutic effects of adenovirus-vectored IFN-α for the treatment of severe acute virus infections when vaccines and/or approved antiviral agents are unavailable.

4. Concluding remarks

The preceding serves to illustrate the pleiotropic nature of type I IFNs in inhibiting virus replication, irrespective of the virus. Virus outbreaks pose a serious threat to global health, as exemplified by the recent outbreaks of SARS CoV, avian H5N1 influenza, Zika virus, WNV and EBOV. In the absence of a vaccine targeted against a newly emerging or re-emerging virus, antiviral drugs serve to limit viral spread. Viruses mutate to specifically evade pathogen-specific antivirals, a case in point being the emergence of Tamiflu-resistant influenza N1 strains [245]. A preferred strategy to limit virus outbreaks would be to deploy broad-spectrum antiviral agents that would exhibit pleiotropic effects [21], including invoking metabolic events important for the induction of a rapid antiviral response [59], targeting different stages of a virus replicative cycle and also invoking a robust immune response against the virus, regardless of the virus. Type I IFNs present as ideal broad-spectrum antiviral candidates and in limited clinical studies have demonstrated therapeutic effectiveness against severe acute virus infections. Their further evaluation is warranted.

References