Type I Interferon Mimetics Bypass Vaccinia Virus Decoy Receptor Virulence Factor for Protection of Mice against Lethal Infection

Abstract

The canonical model of interferon (IFN) signaling focuses solely on the activation of STAT transcription factors which, according to the model, are initiated by the singular event of cross-linkage of the receptor extracellular domain by the IFN. The IFN has no further function beyond this. The model thus provides no approach to circumventing poxviruses decoy receptors that compete with the IFN receptors for IFNs. This simple event has allowed smallpox virus to decimate human populations throughout the ages. We have developed a noncanonical model of IFN signaling that has resulted in the development of small peptide mimetics to both types I and II IFNs. In this report, we focus on a type I IFN mimetic at positions 152 to 189, IFN-α1(152–189), which corresponds to the C terminus of human IFN-α1. This mimetic functions intracellularly and is thus not recognized by the B18R vaccinia virus decoy receptor. Mimetic synthesized with an attached palmitate (lipo-) for cell penetration protects mice from a lethal dose of vaccinia virus, while the parent IFN-α1 is ineffective. Unlike IFN-α1, the mimetic does not bind to the B18R decoy receptor. It further differs from the parent IFN in that it lacks the toxicity of weight loss and bone marrow suppression in mice while at the same time possessing a strong adjuvant effect on the immune system. The mimetic is thus an innate and adaptive immune regulator that is evidence of the dynamic nature of the noncanonical model of IFN signaling, in stark contrast to the canonical or classical model of signaling.

INTRODUCTION

Type I interferons (IFNs), the first definitively characterized immune system cytokines (1), are arguably also the most important cytokines in the host defense against viruses. Poxviruses are particularly effective in neutralizing or bypassing IFNs as a part of their evasion of host defense mechanisms (2, 3). These viruses have developed a plethora of tactics to evade the IFN system. In the case of vaccinia virus, soluble protein decoy receptors are produced to compete with cell membrane receptors for both type I and II IFNs (4, 5). Additionally, other immune evasion mechanisms include the production of complement binding protein, chemokine binding proteins, an interleukin 18 binding protein, a double-stranded RNA binding protein, a protein that binds to protein synthesis eukaryotic initiation factor 2 alpha (eIF-2α), and a tumor necrosis factor homolog (3). All of this suggests both versatility and possibly redundancy in poxvirus evasion of IFNs during infections.

We have discovered a noncanonical mechanism of IFN-γ signaling that has led to the development of a small peptide IFN-γ mimetic (6–9). The IFN-γ mimetic when internalized activates IFN-γ signal transduction by binding to the receptor subunit of the IFN-gamma receptor 1 (IFNGR1) cytoplasmic domain next to the JAK2 binding site (10). It does not recognize the receptor extracellular domain, and unlike the intact IFN-γ, it is not recognized by the poxvirus B8R protein decoy receptor (11). The IFN-γ mimetic peptide thus inhibited vaccinia virus replication in cell cultures and protected mice against overwhelmingly lethal doses of vaccinia virus (11, 12). This suggests that poxvirus IFN decoy receptors are of particular importance in blunting the antipoxvirus activity of IFNs.

Worldwide, it is estimated that smallpox has killed up to 500 million people in the 20th century (13). With the colonization of the Americas by Europeans, smallpox may have killed up to 90% of the South American population. Type I IFN is arguably the key host innate immune response to viral infections, but its ineffectiveness against the virus as a result of a protein, such as the B18R type I IFN decoy receptor of poxvirus (4), is illustrative of the simplicity that is the basis of how a virus virulence factor has had such a devastating effect on human life.

We have recently shown that type I IFN has a noncanonical signaling mechanism that is similar to that of IFN-γ (9, 14, 15). We preliminarily showed that long N-terminal-truncated type I IFNs failed to recognize the extracellular domain of their receptor, but if the truncated proteins were internalized, they induced an antiviral state similar to that of intact IFN (15). In this study, we have made a further N-terminal truncation of human IFN-α and determined its ability compared to that of the parent IFN to induce an antiviral state against vaccinia virus in culture and in the protection of mice against a lethal dose of vaccinia virus. We found that the small peptide mimetic of IFN-α was remarkably easy to produce in the context of the type I IFN noncanonical signal transduction mechanism. These findings stand in marked contrast to the complete absence of the development of any cytokine mimetic based on the classical model of signaling that places heavy emphasis on cross-linking of the receptor extracellular domain as a prerequisite to signaling by cytokines, such as the type I IFNs.

MATERIALS AND METHODS

Cell culture and virus.

BSC-40, L929, or WISH cells were obtained from ATCC (Manassas, VA) and propagated on Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. All cells were grown at 37°C in humidified atmosphere with 5% CO₂. The vaccinia virus Western Reserve strain was a kind gift from Richard Condit (University of Florida). Vaccinia virus was grown, purified on sucrose gradient, and titrated on BSC-40 cells, as described previously (16). EMC virus was grown and titrated on L929 cells, as described previously (11).

Peptides.

The peptides were synthesized on an Applied Biosystems 9050 automated peptide synthesizer using conventional fluorenylmethyloxycarbonyl chemistry, as described previously (6). The addition of a lipophilic group (palmitoyl-lysine) to the N terminus of the synthetic peptide was performed as a last step, using a semiautomated protocol. Peptides were characterized by mass spectrometry and were purified by high-performance liquid chromatography (HPLC). Lipo-IFN-α1(152–189) (a mimetic at residues 152–189) was synthesized by GenScript (Piscataway, NJ).

Mice.

All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. Female C57BL/6 mice (6 to 8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). Peptides dissolved in phosphate-buffered saline (PBS) in a volume of 100 μl were administered intraperitoneally. For intranasal administration, vaccinia virus was taken in a volume of 10 μl, and 5 μl was delivered in each of the nostrils of a lightly anesthetized mouse. Following infection, the mice were observed daily for signs of disease, such as lethargy, ruffled hair, weight loss, and eye secretions. Moribund mice were euthanized and counted as dead.

Measurement of intracellular and extracellular vaccinia virus formation.

BSC-40 cells were seeded and grown overnight to confluence. Peptides at the concentrations indicated were added to cells for 1 h, and then the cells were infected with vaccinia virus at a multiplicity of infection (MOI) of 5 for 1 h. This was followed by the addition of growth medium containing the same amount of peptides as before and incubation for the times indicated. The supernatants were harvested and the cells were scraped in 0.2 ml of cell lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% NP-40, 50 mM NaF, and 5 mM EDTA, followed by three cycles of freeze-thawing and sonication. The virus titer in the supernatant (extracellular) and cell extracts (intracellular) were measured by a plaque assay on BSC-40 cells.

Antiviral assay for EMC virus.

Antiviral assays for encephalomyocarditis virus (EMCV) were performed by using a cytopathic effect reduction assay. Murine L929 cells (3 × 10⁴ cells/well) were seeded in a 96-well plate and grown overnight to confluence for optimal growth. The supernatants from vaccinia virus-infected cells were mixed with lipo-IFN-α1(152–189) or IFN-α1 for 1 h and added to L929 cells for 4 h. The cells were then infected with encephalomyocarditis virus (MOI, 0.1) for 24 h, stained with crystal violet, and the absorbance was measured in a plate reader.

Binding assays.

Binding assays were performed as previously described (17), with minor modifications. Human IFN-α1 or IFN-α1(152–189) was coated at 1 μg per well in triplicate in a microtiter plate in binding buffer (in 0.1 M carbonate-bicarbonate [pH 9.6]). The wells were then washed in wash buffer (PBS containing 0.9% NaCl and 0.05% Tween 20), blocked with 1% bovine serum albumin (BSA) and 0.05% for 1 h at room temperature, washed three times with wash buffer, and incubated with various concentrations of B18R protein for 1 h at room temperature in blocking buffer. Following incubation, the wells were washed five times to remove unbound peptide. Following washing, a 1:500 dilution of antibody to His tag was added, incubated for 1 h, and the wells were washed. HRP-conjugated anti-rabbit IgG was added (1:1,000 dilution), incubated for 1 h, and washed. OPD was added, and color development was terminated by adding 2 N H₂SO₄. The absorbance at 490 nm was measured using a 450 microplate reader.

Measurement of anti-vaccinia antibody response by ELISA.

Microtiter plates were coated with 10⁶ PFU of purified UV-inactivated vaccinia virus (900,000 μJ/cm² for 5 min in a DNA cross-linker) in 100 μl of binding buffer (carbonate-bicarbonate [pH 9.6]) overnight at 4°C. The plates were blocked for 2 h at room temperature with PBS containing 5% fetal bovine serum. Mouse serum samples were serially diluted in PBS containing 0.1% Tween 20 (wash buffer). One-tenth milliliter of the diluted serum was added to each well. The plate was incubated for 2 h at room temperature and washed three times with wash buffer. Peroxidase-conjugated goat anti-mouse IgM (μ-chain specific) or IgG (γ-chain specific) (both from Santa Cruz Biotechnology, Santa Cruz, CA), diluted in a volume of 0.1 ml, was added to each well, incubated for 1 h, and washed five times with wash buffer. OPD in a volume of 0.1 ml was added and incubated for 15 min. The reaction was stopped by adding 50 μl of 2 N H₂SO₄. The optical density at 490 nm was determined using a microtiter plate reader.

Statistical analysis.

All experimental data were measured for statistical significance by Student's t test for peptide binding assays or Kaplan-Meier survival curve and log rank test for the mice studies, using the GraphPad Prism software from GraphPad Software, Inc., San Diego, CA.

RESULTS

IFN-α1 mimetic inhibits vaccinia virus replication.

We previously showed that an N-terminal truncation of human IFN-α1, IFN-α1(69–189), with a nine-arginine (R9) sequence for cell penetration, had broad antiviral activity (15). Lacking R9 for cell penetration, the N-terminal-truncated IFN-α1 lacked antiviral activity. IFN-α1(69–189) consists of 121 amino acid residues. By comparison, we showed that the murine IFN-γ mimetic, IFN-γ(95–132), consists of 38 residues. We thus synthesized a much more severe N-truncated IFN-α1 peptide, IFN-α1(152–189), of 38 residues with a palmitate fatty acid (lipo) attached for cell penetration (18) and determined if it could inhibit vaccinia virus replication in cell culture. This served two purposes: (i) it established the antiviral activity of the putative type I IFN mimetic and (ii) demonstrated that the mimetic can bypass the vaccinia virus B18R decoy receptor, which blocks the binding of type I IFNs to extracellular the IFN receptor domain (4). As shown in Fig. 1, lipo-IFN-α1(152–189) inhibited vaccinia virus replication in a dose-response manner, as determined by a one-step viral growth experiment in which virus replication was assessed both intracellularly and extracellularly. It is noteworthy that both a scrambled version of the IFN mimetic and intact IFN-α1 both failed to inhibit vaccinia virus replication. The scrambled mimetic showed the specificity for IFN mimetic inhibition, and the failure of IFN-α1 to inhibit confirmed the presence of the B18R decoy receptor, which was ineffective against the IFN mimetic.

FIG 1.

Inhibition of vaccinia virus replication by lipo-IFN-α1(152–189) as determined by one-step growth curve. BSC40 cells were grown to confluence and treated with the concentration of peptides indicated or the parent IFN for 2 h. The cells were then infected with 5 MOI of vaccinia virus for 1 h, followed by washing and addition of growth medium. Forty-eight hours later, the supernatant and cell extracts were obtained and titrated for the vaccinia virus. For details, see Materials and Methods. Error bars represent the standard error of the mean. The difference between untreated and treatment with different concentrations of IFN mimetic showed a P value of <0.01.

IFN mimetic is inhibitory over time.

An IFN effect on virus replication is stable over time, so we were interested in assessing the effects of lipo-IFN-α1(152–189) over time. As shown in Fig. 2, the mimetic stably inhibited vaccinia virus replication in a one-step growth curve, as assessed by intracellular and released virus measurement over time. A scrambled version of the mimetic was ineffective in comparison to PBS-treated infected cells. The mimetic thus induced a stable antiviral effect over time, which is particularly significant given that poxviruses like vaccinia virus are resistant to IFNs.

FIG 2.

Time course of inhibition of vaccinia virus replication by lipo-IFN-α1(152–189). BSC-40 cells were grown to confluence and then treated with 30 μM lipo-IFN-α1(152–189), or IFN scrambled peptide for 1 h. The treated cells were compared with untreated cells for virus replication. The cells were then infected with vaccinia virus at an MOI of 5 for 1 h. Virus was washed, fresh growth medium was added, and cells were allowed to grow for the times indicated. Cell extracts (A) and supernatants (B) were titrated for the amount of extracellular and intracellular virus, respectively. For details, see Materials and Methods. Error bars represent the standard error of the mean. The difference between untreated and IFN mimetic-treated samples showed a P value of <0.01.

Culture supernatants from vaccinia virus-infected cells inhibit IFN-α1 but not lipo-IFN-α1(152–189) antiviral activity.

Poxviruses, such as vaccinia virus, produce soluble decoy receptors to type I and type II IFNs, which effectively block IFN function in innate and adaptive host defenses. We thus determined if the supernatant from cultures infected with vaccinia virus would differentially affect the antiviral effects of IFN-α1 and its mimetic lipo-IFN-α1(152–189) against EMC virus, a virus that does not produce IFN decoy receptors. Such supernatants would be expected to contain B18R, the type I IFN decoy receptor (4). Lipo-IFN-α1(152–189) used at 500 U in a one-step EMC virus infectivity experiment completely inhibited EMC virus replication in the presence of supernatant that contained the B18R protein (Fig. 3A). IFN-α1, the parent IFN, at 500 U had no effect on EMC virus replication, probably due to binding to B18R in the supernatant. The mimetic, in contrast, does not recognize the receptor extracellular domain and was thus unaffected by B18R. A scrambled mimetic control failed to inhibit EMC virus, not due to B18R, but because it lacked antiviral activity. The lack of functional recognition of B18R is evidence supporting the idea the type I IFN mimetics do not exert their antiviral effects via interaction with the receptor extracellular domain even though they are part of the C terminus of the IFN.

FIG 3.

Effect of B18R containing supernatants from vaccinia virus infected cells on IFN-α1 and IFN-α1 mimetic antiviral activity in the context of binding to B18R. (A) BSC40 cells (2 × 10⁵) in 0.5 ml were infected with vaccinia virus (MOI, 5) for 24 h and the supernatants were harvested. Fifty microliters of supernatant was mixed with lipo-IFN-α1(152–189) (500 U, 10 μg) or IFN-α1 (500 U) for 1 h and added to L929 cells (3 × 10⁴) in a microtiter dish for 4 h. The cells were then infected with encephalomyocarditis (EMC) virus (MOI, 0.1) for 24 h, stained with crystal violet, and the absorbance was measured. (B) The vaccinia virus decoy receptor for type I IFN binds to intact IFN but not to the IFN mimetic. Human IFN-α1 (□) or IFN-α1(152–189) (●) was coated at 1 μg per well in triplicate in a microtiter plate. Following blocking with 1% BSA, various concentrations of this tagged B18R protein were added and incubated for 1 h. Following washing, a 1:500 dilution of rabbit antibody to His tag was added, incubated for 1 h, and the plates were washed. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was added (1:1,000 dilution), incubated for 1 h, and washed. O-Phenylenediamine (OPD) was added, and color development was terminated by addition of 2 N H₂SO₄, and the absorbance was measured in a plate reader. For details, see Materials and Methods. Error bars represent the standard error of the mean. The difference between untreated and IFN mimetic-treated samples showed a P value of <0.01.

Related to the above, we carried out enzyme-linked immunosorbent assay (ELISA) binding with IFN-α1 and IFN-α1(152–189) with recombinant B18R. As shown in Fig. 3B, IFN-α1 but not IFN-α1(152–189) bound B18R, which is consistent with the vaccinia culture supernatant functional results, suggesting that the mimetic does not recognize the type I IFN extracellular domain.

IFN mimetic protects mice against lethal vaccinia virus infection.

Based on the above cell culture results, we determined if lipo-IFN-α1(152–189) could protect mice against vaccinia virus infection. Accordingly, mice infected intranasally with 2 × 10⁶ PFU of vaccinia virus were treated by intraperitoneal (i.p.) injection with IFN-α1 (5,000 U), IFN-α1 mimetic (5,000 U), and scrambled mimetic for 6 consecutive days, starting the day of virus exposure, as described in Fig. 4A. Consistent with the cell culture results, all of the mice treated with the IFN mimetic survived, while the IFN-α1-, scrambled mimetic-, and PBS-treated mice all died by day 10. The IFN mimetic is thus a potent therapeutic against a poxvirus under conditions in which the parent IFN is without effect.

FIG 4.

Lipo-IFN-α1(152–189) protects mice against vaccinia virus, while intact IFN does not. (A) Mice (C57BL/6, n = 5) were infected intranasally (i.n.) with 2 × 10⁶ PFU of vaccinia virus. Starting on day 0, PBS, lipo-IFN-α1(152–189) (5000 U, 100 μg), scrambled mimetic (100 μg), or IFN-α1 (5,000 U) was administered i.p. in a volume of 100 μl for six consecutive days. The survival of mice was followed. (B). Clinical scores of mice infected with vaccinia virus and treated with IFN-α1(152–189), IFN-α1, or scrambled peptide. C57BL/6 mice (n = 5) were infected intranasally with 2 × 10⁶ PFU of vaccinia virus. Starting on day 0, PBS, lipo-IFN-α1(152–189) (5,000 U, 100 μg), scrambled mimetic (100 μg), or IFN-α1 (5,000 U) was administered i.p. in a volume of 100 μl for six consecutive days. The following criteria were used to measure the clinical score of mice: 1, ruffled hair, hunched posture; 2, weight loss of ≥15%; 3, moribund; 4, death. For details, see Materials and Methods. The difference between PBS-injected mice and IFN mimetic treatment showed a P value of <0.001.

In addition to assessing mice for mortality, we also monitored them in terms of morbidity on a scale of 1 to 4, with 1 indicating ruffled hair and 4 representing death. Mice treated with lipo-IFN-α1(152–189) were essentially asymptomatic over the 12-day period of observation. In contrast, the scrambled mimetic-treated mice showed a graded morbidity response starting at day 4 that increased in discomfort, with death by day 9 (Fig. 4B). These results reflect how the IFN mimetic kept vaccinia virus effects on the mice in check throughout the period of observation, reflecting its total control of virus. We feel that this is significant in terms of possible prophylactic treatment of individuals exposed to monkeypox virus or smallpox virus.

IFN-α1(152–189) mimetic possesses adjuvant activity.

In addition to its protective effects against vaccinia virus infections, we were also interested in determining if lipo-IFN-α1(152–189) possessed adjuvant activity against infectious virus and bovine serum albumin (BSA), a weak immunogen in mice (19). As shown in Fig. 5, mice injected with infectious vaccinia virus and lipo-IFN-α1(152–189) showed a 5- to 8-fold-greater production of IgM (Fig. 5A) and IgG (Fig. 5B) antibodies to virus at 2 to 3 weeks following vaccination at the highest serum concentration. As expected, a scrambled type I IFN mimetic lacking antiviral activity did not demonstrate any significant production of vaccinia virus-specific IgM and IgG antibodies. Thus, the type I IFN mimetic probably possessed adjuvant activity and functioned directly as an antiviral.

FIG 5.

Adaptive immune response in mice infected with vaccinia virus and treated with lipo-IFN-α1(152–189) or noninfected mice treated with scrambled peptide. Microtiter plates were coated with UV-inactivated vaccinia virus. Serum samples (50 μl) collected from mice treated as indicated were diluted and added to wells. After washing to remove nonspecific binding, secondary anti-mouse IgM (A) or IgG (B) conjugated to HRP was added, followed by the addition of OPD and absorbance measurement. The dilutions of serum for IgM were 1:50, 1:200, and 1:1,000 for 1, 2, and 3, respectively. The dilutions of serum for IgG were 1:100, 1:500, and 1:5,000 for 1, 2, and 3, respectively. For details, see Materials and Methods. Error bars represent the standard error of the mean. The difference between scrambled peptide and treatment with IFN mimetics showed a P value of <0.01.

Vaccinia virus is a strong immunogen (3), so a similar experiment was also performed using BSA, which is a relatively poor immunogen (19). The mimetic induced significantly greater lymphocyte proliferation at 4 weeks compared to that in the scrambled peptide-treated mice (Fig. 6A). Similarly, the antibody response was significantly increased at 2 to 3 weeks following the injection of BSA and mimetic (Fig. 6B). Scrambled mimetic control lacked adjuvant activity. These results show that the type I IFN mimetic possessed adjuvant activity in both the cellular and humoral responses.

FIG 6.

IFN-α1(152–189) exhibits adjuvant properties at both cellular and humoral levels against a weak antigen. (A). Spleens were harvested from C57BL/6 mice (n = 3) 4 weeks after immunization with BSA as a weak antigen and treatment with IFN-α1(152–189) peptide or the control peptide. Untreated mice were included as a control. Splenocytes (5 × 10⁵ cells per well) in microtiter plates were incubated with (+) or without (−) BSA (50 μg/ml) for 72 h. CellTiter aqueous one-cell proliferation assay (Promega, Madison, WI) was added, and absorbance was read to measure the proliferation. Scram, scrambled. (B). Mice were immunized using BSA as an antigen in the presence of IFN-α1(152–189) peptide or the control peptide. After 2 and 3 weeks, blood was drawn from the mice and measured for the presence of BSA-specific antibodies in an ELISA format. The proliferation and ELISAs were carried out as previously described (12, 33). Error bars represent the standard error of the mean. The difference between untreated and IFN mimetic-treated samples showed a P value of <0.01.

DISCUSSION

IFNs are arguably the most effective host defense against viral infections at both the innate and adaptive phases of the immune response, but the poxviruses represent a particular challenge to both type I and type II interferons. Poxviruses, such as the vaccinia virus, produce decoy receptors that block the interaction of both type I and type II IFN with their cognate receptors on the cell membrane. There are other host evasion mechanisms of poxviruses, but IFN decoy receptors may be of particular significance in bypassing the IFN system (4, 5). Thus, poxviruses have used a remarkably simple mechanism to wreak havoc upon civilization over the ages, causing an estimated 300 to 500 million deaths as recently as the 20th century (13).

We have recently developed type I IFN mimetics corresponding to human IFN-α1, IFN-β, and ovine IFN-τ that inhibit vaccinia virus replication in cell cultures (15, 18). The parent IFNs were without effect against vaccinia virus. In this study, we focused on one of the type I IFN mimetics, lipo-IFN-α1(152–189), for its ability to protect mice from a lethal dose of vaccinia virus. The mimetic consists of the C-terminal sequence 152 to 189 of IFN-α1 and must be internalized (via the lipo group) by the cell in order to be functional. Built into the mechanism of action of lipo-IFN-α1(152–189) is the mechanism for bypassing the B18R decoy receptor of vaccinia virus. The IFN-α1 mimetic does not recognize the type I IFN extracellular domain or the B18R decoy receptor, while its poxvirus nonfunctional parent IFN, IFN-α1, does recognize B18R.

The classical model of cytokine signaling dominates our view of specific gene activation by cytokines, such as the IFNs (20, 21). According to the model, IFN activates the cell solely via interaction with the extracellular domain of the receptor. This results in the activation of receptor or receptor-associated tyrosine kinases (JAKs) and the phosphorylation and dimerization of STAT transcription factors that dissociate from the receptor cytoplasmic domain and translocate to the nucleus. Specific gene activation is ascribed solely to the activated STATs despite the fact that it is not uncommon that functionally different ligands may activate the same STAT(s) (7–9). Also, the important recent observation that activated JAKs from cytokine signaling, like activated STATs, similarly undergo nuclear translocation and perform epigenetic functions (22, 23) is not addressed by the classical JAK/STAT signaling model. Elegant structure studies of IFN-γ and IFN-α complexed to a receptor have not resulted in the advancement in signal transduction and understanding of specific gene activation (24, 25). For example, there are no IFN mimetics based on these structure studies, which raises questions about claims that receptor cross-linking by IFN is the sole requirement for subsequent signaling events.

Our studies on IFN signaling have resulted in the development of a noncanonical more complex signaling model that is akin to that of steroid hormone/steroid receptor signaling (26). We have shown that ligand receptors, activated JAKs, and STATs are all associated with specific gene activation, including activated JAK epigenetic events (7–9). We showed that activated JAK2 for IFN-γ and activated tyrosine kinase 2 (TYK2) for type I IFNs phosphorylated histone H3 at tyrosine 41 in genes that are specifically activated by the IFNs (14, 15). This caused the release of the histone inhibitory protein 1α (HPIα). Thus, we found that activated STATs and JAKs functioned coordinately in the nucleus in terms of gene activation in the context of associated epigenetics.

Important for the study here, the noncanonical model first resulted in the development of the IFN-γ mimetic, lipo-IFN-γ(95–132), and subsequently in the development of type I IFN mimetics, like lipo-IFN-α1(152–189) (15). This is based primarily on the IFN-γ studies in which we showed that the C terminus of the IFN recognized the cytoplasmic domain of the IFNGR1 receptor subunit. Internalized, the C-terminal sequence was sufficient for inducing antiviral activity, and the fact that the extracellular receptor domain was not recognized made the IFN-γ mimetic functional in the presence of the vaccinia virus B8R protein (11, 12). This led to similar findings with the type I IFN mimetics, which did not recognize B18R protein of vaccinia virus.

The IFN-γ mimetic antiviral activity was confirmed shortly after its discovery (27). More recently, the IFN-γ mimetic has been developed for the treatment of hepatic fibrosis (28, 29). IFN-γ has been shown to be a potent antifibrotic, but its use in the treatment of hepatic fibrosis is limited by matters, such as undesirable side effects (28, 29). The undesirable side effects of IFNs have been shown to be due to extracellular receptor interaction (30), so in the absence of such binding by the IFN-γ mimetic, it functioned when targeted to hepatic tissue as a potent antifibrotic without the undesirable side effects. It was shown previously that the type I IFN expressed intracellularly exhibits biologically activity (31, 32), which is consistent with our noncanonical model of type I IFN signaling.

We have shown that type I IFN mimetics, like lipo-IFN-α1(152–189), similarly lack undesirable side effects in mice, such as the loss of weight and bone marrow suppression (15). We took advantage of this in the treatment of experimental allergic encephalomyelitis (EAE), a mouse model of multiple sclerosis, and in protecting mice against melanoma (18). IFN-α mimetic similarly possessed potent adjuvant effects against BSA, a weak antigen. Vaccinia virus is a potent antigen and might thus mask mimetic adjuvancy, so the rationale behind the use of BSA as a test of adjuvancy was its weak immunogenicity. IFN-α1 mimetic showed clear adjuvancy for BSA at both the cellular and humoral levels. Type I IFN mimetics, like that of the IFN-α1 mimetic, are thus potent antivirals against an IFN-resistant poxvirus, lack toxicity, and function as adjuvants when used as antivirals.