Short peptide type I interferon mimetics: therapeutics for experimental allergic encephalomyelitis, melanoma, and viral infections

Abstract

The classical canonical model of interferon (IFN) signaling focuses solely on the activation of STAT transcription factors, which limits the model in terms of specific gene activation, associated epigenetic events, and IFN mimetic development. Accordingly, we have developed a noncanonical model of IFN signaling and report the development of short type I IFN peptide mimetic peptides based on the model. The mimetics, human IFNα1(152-189), human IFNβ(150-187), and ovine IFNτ(156-195) are derived from the C-terminus of the parent IFNs and function intracellularly based on the noncanonical model. Vaccinia virus produces a decoy IFN receptor (B18R) that inhibits type I IFN, but the IFN mimetics bypass B18R for effective antiviral activity. By contrast, both parent IFNs and mimetics inhibited vesicular stomatitis virus. The mimetics also possessed anti-tumor activity against murine melanoma B16 tumor cells in culture and in mice, including synergizing with suppressor of cytokine signaling 1 antagonist. Finally, the mimetics were potent therapeutics against experimental allergic encephalomyelitis, a mouse model of multiple sclerosis. The mimetics lack toxic side effects of the parent IFNs and, thus, are a potent therapeutic replacement of IFNs as therapeutics.

FIG. 1.

Inhibition of vaccinia virus (VV) and vesicular stomatitis virus (VSV) replication by type I IFN mimetics. (A) BSC40 cells were grown to confluence and treated with the C-terminal peptide mimetics of IFNs α, β, and τ for 2 h. Cells were then infected with 0.01 m.o.i. of VV for 1 h, followed by washing and addition of fresh medium. Forty eight hours later, cells were stained with crystal violet and plaques were counted. Percent inhibition was calculated with the plaques in media-only cells versus the various treatments. Intact IFNα1 added at 5,000 U/mL did not protect against VV infection. A scrambled IFNτ peptide was used as a negative control. Nonpalmitic refers to the peptide that was not conjugated with palmitic acid and, thus, does not gain entry across the plasma membrane. See Table 1 for mimetics of α, β, τ, and τ scrambled mimetic sequences of the corresponding IFNs. (B) L929 cells were seeded in a microtiter plate and incubated with the IFN mimetics of Table 1, including scrambled IFNτ peptide, or IFNα1 (5,000 U/mL) at the concentrations indicated for 2 h. Cells were then infected with 0.1 m.o.i. of VSV for 24 h. Cells were then stained with crystal violet, and the absorbance was read. IFN, interferon; m.o.i., multiplicity of infection.

FIG. 2.

Antimelanoma effects of the type I IFN mimetics. (A) Cell culture. B16F10 cells (20,000 in 0.1 mL) were incubated in microtiter plates with an equal volume of the IFNα1 and IFNβ mimetics as well as a scrambled (scr) mimetic (see Table 1) for 3 days, after which the cells were counted and compared with untreated cells. (B) IFN mimetic protection of C57BL/6 mice from B16F10 cells in the presence of suppressors of cytokine signaling 1 (SOCS1) antagonist. Female C57BL/6 mice (n=5) were injected i.p. with 10⁴ B16F10 cells. Starting from day 1, mice were injected i.p. with PBS, lipo-IFNα1(156–189) (100 μg), SOCS1 antagonist lipo-pJAK2(1001–1013) (100 μg), or a combination of the 2 on alternate days over a period of 14 days. As a control, scrambled peptide (scr) corresponding to the IFN mimetic was used at 100 μg per mouse. The survival of mice was followed over 40 days. A comparison of the combined treatment against the PBS injection on day 40 gave a P value of 0.001. PBS, phosphate-buffered saline.

FIG. 3.

Lipo-IFNα1(152–189) and lipo-JAK2(1001–1013) SOCS1 antagonist-protected mice show absence of peritoneal B16 tumors. Evidence of B16 tumors throughout the peritoneum of scrambled peptide/PBS-treated mice (top left) and absence of obvious tumors in IFN mimetic/SOCS1 antagonist-treated mice (top right). Also shown is a liver of scrambled peptide/PBS-treated mice (bottom left) with tumor nodules, which are absent from the combined mimetic/antagonist treatment of mice (bottom middle). Enlarged spleen with tumor nodules is seen in control mice (bottom right, left side of frame) but not in the combined treatment (bottom right, right side of frame).

FIG. 4.

Type I IFN mimetic lipo-IFNα1(152–189) lacked toxicity under conditions where the intact IFN was toxic. Mice (C57BL/6, n=3) were injected i.p. with murine IFNα1 (Δ, 5×10³ U/mouse), Lipo-IFNα1(152–189), 5×10³ U (100 μg, ■), or PBS (○), i.p. on alternate days. IFN activity refers to the antiviral activity assessed by the cytopathic effect of encephalomyocarditis virus on L cells. Body weight was measured daily. The average body weight is presented as a percentage of initial weight, and the standard deviation is shown. The weight loss seen with IFNα1-treated mice was not seen with the IFNα1 mimetic-treated mice. On day 11, the difference between the IFNα1 and IFNα1 mimetic showed a significance of P<0.05 by the Mann-Whitney rank test.

FIG. 5.

Type I IFN mimetics protect mice against the symptoms of EAE. SJL/J mice (n=5) were immunized with myelin basic protein. Twelve days later, they were injected i.p. with PBS (●) or 50 μg (2,500 U) each of lipo-IFNα1(152–189) (▴), lipo-IFNβ(150–187) (♦), lipo-IFNτ(156–195), (○) or scrambled lipo-IFNτ (□) on alternate days. Mice were followed daily. The mean daily severity of disease was graded as follows. 0, normal; 1, loss of tail tone; 2, hind leg weakness; 3, paraparesis; 4, paraplegia; 5, moribund; and 6, death. Statistics were performed using an unpaired Student's t-test. EAE, experimental allergic encephalomyelitis.

FIG. 6.

Intracellular signaling of IFN mimetic and SOCS1 antagonist. (A) IFN mimetics signal through TYK2 and STAT1α phosphorylation. WISH cells were treated with the peptides indicated, or the parent IFN for 10 min. Cell extracts were Western blotted with phospho-TYK2 or phospho-STAT1α. Membranes were stripped and re-probed with total TYK2 or STAT1α. The results are representative of at least 2 experiments. (B) B16F10 cells show a reduction in SOCS1 levels after treatment with SOCS1 antagonist. B16F10 cells were treated with the SOCS1 antagonist peptide, pJAK2, or its scrambled control peptide for 10 min. Cell extracts were Western blotted with SOCS1 antibody. Numbers under the lanes represent the relative intensities of bands as measured by the Image J program (NIH). Membrane was stripped and re-probed with Lamin B1, to ascertain equal loading of proteins.