Expression of porcine fusion protein IRF7/3(5D) efficiently controls foot-and-mouth disease virus replication.

UNLABELLED: Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-ß, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log10 units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV. IMPORTANCE: FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

TBK1-targeted suppression of TRIF-dependent signaling pathway of toll-like receptor 3 by auranofin.

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The stimulation of TLRs by microbial components triggers two branches of downstream signaling pathways: myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-beta (TRIF)-dependent signaling pathways. Auranofin, a sulfur-containing gold compound (Au[I]), has been widely used for the treatment of rheumatoid arthritis. Since dysregulation of TLRs can lead to severe systemic inflammatory and joint destructive process in rheumatoid arthritis, auranofin-mediated modulation of TLR activation may have therapeutic potential against such diseases. Previously, we demonstrated that auranofin suppressed TLR4 signaling pathway by inhibiting TLR4 dimerization induced by LPS. Here, we examined the effect of auranofin on signal transduction via the TRIF-dependent pathway induced by a TLR3 agonist. Auranofin inhibited nuclear factor-kappaB and interferon (IFN) regulatory factor 3 (IRF3) activation induced by polyinosinic-polycytidylic acid (poly[I:C]). Auranofin inhibited poly[I:C]-induced phosphorylation of IRF3 as well as IFN-inducible genes such as IFN inducible protein-10. Furthermore, auranofin inhibited TBK1 kinase activity in vitro. All the results suggest that auranofin suppress TLR signaling at multiple steps.

Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma-inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction.

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.

Inhibition of staphylococcal enterotoxin A-induced superantigenic and lethal activities by a monoclonal antibody to toxic shock syndrome toxin-1.

Staphylococcal toxic shock syndrome is caused by a family of related superantigens that includes toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (SEs) A, B, and C. The cross-inhibitory activity against SEA by a novel anti-TSST-1 monoclonal antibody (MAb), MAb5, which also cross-inhibits SEB-induced superantigenic activities, was investigated. MAb5 blocked SEA binding to human monocytes, cross-neutralized SEA-induced T cell mitogenesis and TNF-alpha secretion in human peripheral blood mononuclear cells, and prevented lethality in mice. Epitope mapping revealed that MAb5 binds to residues SEA(154-161) within the central alpha helix that is structurally highly conserved among TSST-1, SEA, and SEB. The cross-inhibitory activity of MAb5 is likely due to steric hindrance of this conserved motif, although the precise function of this motif shared among related staphylococcal superantigens remains to be further elucidated.

Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response.

An interleukin-18 binding protein (IL-18BP) was purified from urine by chromatography on IL-18 beads, sequenced, cloned, and expressed in COS7 cells. IL-18BP abolished IL-18 induction of interferon-gamma (IFNgamma), IL-8, and activation of NF-kappaB in vitro. Administration of IL-18BP to mice abrogated circulating IFNgamma following LPS. Thus, IL-18BP functions as an inhibitor of the early Th1 cytokine response. IL-18BP is constitutively expressed in the spleen, belongs to the immunoglobulin superfamily, and has limited homology to the IL-1 type II receptor. Its gene was localized on human chromosome 11q13, and no exon coding for a transmembrane domain was found in an 8.3 kb genomic sequence. Several Poxviruses encode putative proteins highly homologous to IL-18BP, suggesting that viral products may attenuate IL-18 and interfere with the cytotoxic T cell response.

Corticosteroids enhance the capacity of macrophages to induce Th2 cytokine synthesis in CD4+ lymphocytes by inhibiting IL-12 production.

We investigated the effects of corticosteroids on IL-12 production by mouse splenic adherent cells and the subsequent capacity of these cells to induce cytokine production by CD4+ T cells. To distinguish the effects of corticosteroids on APCs from those on T cells, only the APCs and not the T cells were exposed to corticosteroids. Treatment of splenic adherent cells with dexamethasone greatly inhibited production of IL-12, a cytokine known to enhance IFN-gamma synthesis and decrease IL-4 synthesis by CD4+ T cells. The reduction in IL-12 production by corticosteroid-treated macrophages decreased their ability to induce IFN-gamma and increased their ability to induce IL-4 synthesis in Ag-primed CD4+ T cells. Splenic adherent cells from mice treated in vivo with dexamethasone also displayed a reduced capacity to produce IL-12. These results help to resolve previous conflicting observations regarding the effects of corticosteroids on cytokine production by T cells, and indicate that while corticosteroids may directly inhibit Th1 and Th2 cytokine production in T cells, corticosteroids, by reducing IL-12 production in APCs, have the potential to indirectly enhance Th2 cytokine synthesis. Therefore, treatment of diseases such as allergy with chronic corticosteroids may indirectly exacerbate the course of the disease, which is caused primarily by the overproduction of Th2 cytokines in allergen-specific CD4+ T cells.

IFN-gamma-inducing factor/IL-18 administration mediates IFN-gamma- and IL-12-independent antitumor effects.

We evaluated the mechanism of the antitumor effects of mouse rIFN-gamma-inducing factor/IL-18 protein on the growth of mouse tumor cell lines in vivo. Mice received IL-18 before or after challenge with CL8-1, a mouse melanoma cell line. Both regimens significantly suppressed tumor growth and reduced the number of mice with growth of tumor from 60% (3/5) to 20% (1/5). Furthermore, IL-18 administered before and after tumor inoculation completely abrogated the establishment of CL8-1 in all animals. IL-18 administration also significantly suppressed the growth of MCA205, a sarcoma cell line, even when treatment was delayed to 7 days following tumor inoculation. Although IL-18/IL-12 combination therapy had the most significant and immediate antitumor effects, many mice so treated succumbed with markedly elevated serum IFN-gamma levels. The antitumor effects of IL-18 were abrogated almost completely when NK cells were eliminated using anti-asialo GM1 Ab administration, but only marginally impaired in IFN-gamma or IL-12 gene-disrupted mice. Immunohistochemical staining revealed that the number of the CD8+ T cells, but not CD4+ T cells, found at the tumor site was reduced in animals treated with IL-18. These results indicate that IL-18 has potent antitumor effects mediated by CD4+ T cells and NK cells, but in IFN-gamma- and IL-12-independent pathways.

Killing of Leishmania donovani amastigotes by poly ICLC in hamsters.

In vitro as well as in vivo studies suggest that cytokine-induced synthesis of nitric oxide (NO) from L-arginine is a major effector mechanism against intracellular pathogens. In this study, we demonstrate that golden hamsters infected with Leishmania donovani amastigotes upon treatment with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly ICLC), a potent interferon inducer and immune enhancer, in combination with L-arginine, develop the capacity to eliminate intracellular pathogens. This antileishmanial activity of poly ICLC was suppressed by N w nitro-L-arginine (N w NLA), an inhibitor of inducible NO synthase. Furthermore, prolonged treatment of infected animals with L-arginine alone for 5 days more after 5 day treatment with poly ICLC plus L-arginine increased the antileishmanial activity compared with 5 day treatment with poly ICLC plus L-arginine, suggesting that inducible NO synthase, once activated, produces NO for 5 days more. Our results suggest that an L-arginine-dependent, NO-mediated mechanism is probably responsible for the antileishmanial action of poly ICLC.

Dissociation of xanthine oxidase induction and cytochrome P450 depression during interferon induction in the rat.

Interferon (IFN) and IFN inducers down-regulate hepatic cytochrome P450 (P450) through a pretranslational mechanism involving depression of P450 mRNA levels and a subsequent decrease in P450 synthesis. Current evidence suggests that interferon induces the synthesis of a protein which subsequently mediates the down-regulation of P450. Xanthine oxidase (XO) activity is induced by interferons in rodents, and the XO inhibitor allopurinol (AP) inhibits the down-regulation of P450 by interferons in the mouse and hamster so it has been proposed as the putative intermediate protein. In studies undertaken in rats to further characterize the molecular basis of the protective effect of AP, we observed that AP (20 and 50 mg/kg) did not protect against down-regulation of P450 by the interferon inducer polyinosinic-polycytidylic acid (10 mg/kg). In fact, at 50 mg/kg AP had an additive effect on the depression of CYP2E1. Total XO induction in the rat was only 30-50% compared with 100-500% in mice and hamsters, and this induction was inhibited completely by AP. Therefore, XO does not mediate the down-regulation of hepatic cytochrome P450 by interferons in the rat.

Inhibition of interferon-alpha/beta induction by crystalline cadmium sulfide.

The induction of interferon-alpha/beta by Newcastle disease virus in L-929 cells was inhibited by pretreatment of the cell cultures with the crystalline form of cadmium sulfide. As little as 10 micrograms/ml of crystalline cadmium sulfide was sufficient to inhibit the induction of interferon. Cell viability was not affected by this treatment as measured by trypan blue dye-exclusion viability staining. Between 6 and 24 hr of preincubation with crystalline cadmium sulfide was required for significant inhibition of interferon induction.

Hyporeactivity to interferon induction: characterization of a hyporeactive factor in the serum of encephalomyocarditis virus-infected mice.

Mice infected with encephalomyocarditis virus develop a severe state of hyporeactivity to interferon induction. One mechanisms possibly responsibile for development of hyporesponsiveness in these animals is a circulating factor which can be detected in their serum 96 h after encephalomyocarditis virus infection (at the time of peak hyporeactivity in vivo). This report describes some of the physiocochemical characteristics of this serum hyporeactive factor (SHF). SHF is a protein with a molecular weight between 20,000 and 40,000 that was extremely labile at 56 C, losing greater than 90% of its biological activity in 8 min, but stable at 37 c for at least 4 h. Hyporeactive factor was also stable over a pH range of 2 to 11 for 48 h at 4 C. These results suggest that SHF is physicochemically similar to interferon. However, no interferon could be detected in the SHF preparation, and no loss in biological activity was observed when the serum factor was incubated with anti-interferon antibody, suggesting that they are separate substances.