IFN-α Subtypes Research Articles

Treatment with interferon-alpha delays disease in swine infected with a highly virulent CSFV strain

Interferon-alpha (IFNα) can effectively inhibit or abort a viral infection within the host. It has been reported that IFN induction and production is hindered during classical swine fever virus (CSFV) infection. Most of those studies have been performed in vitro, making it difficult to elucidate the actual role of IFNs during CSFV infection in swine. Here, we report the effect of IFNα treatment (delivered by a replication defective recombinant human adenovirus type 5, Ad5) in swine experimentally infected with highly virulent CSFV strain Brescia. Treatment with two different subtypes of IFNα delayed the appearance of CSF-related clinical signs and virus replication although it did not prevent lethal disease. This is the first report describing the effect of IFNα treatment during CSFV infection in swine.

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

High-throughput quantitative real-time RT-PCR assay for determining expression profiles of types I and III interferon subtypes.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

Cloning, expression and antiviral activity of mink alpha-interferons.

BackgroundAs a key link between innate and adaptive immune responses, the interferon (IFN) system is the first line of defense against viral infection. IFN, and in particular, IFN-α, has been used clinically as an effective therapeutic agent for viral infections. However, different subtypes of IFN-α demonstrate distinct antiviral activity. Therefore, it is important to identify IFN-α subtypes with high antiviral activity for the development of genetically engineered antiviral drugs.ResultsIn this study, we cloned the genes for 13 IFN-α subtypes from peripheral blood lymphocytes of the mink. The homologies of the 13 mink IFN-α genes were 93.6–99.3% and 88.8–98.4% at the nucleotide and amino acid sequence levels, respectively. In contrast to human and canine IFN-α subtypes, most mink IFN-α subtypes contained two N-glycosylation sites. We expressed and purified 13 mink IFN-α subtypes in Escherichia coli. The cytopathic effect inhibition assay showed that all the 13 recombinant mink IFN-α subtypes inhibited the propagation of vesicular stomatitis virus in WISH cells, with IFN-α2 and IFN-α12 demonstrating the highest activities. Furthermore, recombinant mink IFN-α2 and IFN-α12 significantly suppressed the propagation of canine distemper virus in Vero cells, with IFN-α2 demonstrating the highest activity.ConclusionsWe identified the mink IFN-α2 subtype as a promising candidate for the development of effective antiviral drugs.

Induction of IFN-αSubtypes and Their Antiviral Activity in Mumps Virus Infection

Human type I interferons (IFNs) comprise one IFN-β, -ω, -κ, and -ɛ and 12 different IFN-α subtypes, which play an important role in early host antiviral response. Despite their high structural homology and signaling through the same receptor, IFN-α subtypes exhibit different antiviral, antiproliferative, and immunomodulatory activities. Differences in the production of IFN-α subtypes therefore determine the quality of an antiviral response. In this study, we investigated the pattern of IFN-α subtypes induced in infection with different mumps virus (MuV) strains and examined the MuV sensitivity to the action of IFN-α subtypes. We found that all IFN-α subtypes are being expressed in response to MuV infection with a highly similar IFN-α subtype pattern between the virus strains. We assessed an antiviral activity of several IFN-α subtypes: IFN-α1, IFN-α2, IFN-α4, IFN-α6, IFN-α8, IFN-α14, IFN-α17, and IFN-α21. Although they were all effective in suppressing MuV replication, the intensity and pattern of their action varied between MuV strains. Our results indicate that the overall IFN antiviral activity as well as the activity of specific IFN-α subtypes against MuV depend on a virus strain.

Production and characterization of thirteen human type-I interferon-α subtypes

Thirteen human interferon-α (IFNα) subtypes were expressed in Escherichiacoli and purified using an N-terminal affinity tag from the prodomain of subtilisin. IFNα subtypes were expressed in soluble form and purified from cell lysates or refolded and purified from inclusion bodies. Proteins produced by either protocol exhibited biological activities equal to or greater than commercially prepared IFNα preparations. The IFNαs were used to produce an anti-IFNα16 antibody (MAb-1B12) that specifically neutralized the biological activity of IFNα16, but not the 12 other IFNαs. Using MAb-1B12, and a previously generated IFNAR1/IFNAR2-FChk heterodimer, an assay was developed to determine total type I IFN biological activity and IFNα16-derived biological activity in an unknown sample.

How type I interferons work in multiple sclerosis and other diseases: some unexpected mechanisms.

Type I interferons (IFNs) are important in innate and adaptive immunity. They are used to treat virus infections, cancer, and multiple sclerosis (MS). There are 5 type I IFN families in humans-IFN-α with 13 subtypes, plus IFN-β, ɛ, κ, and ω. Because their receptor binding affinities vary, these IFNs have different gene induction profiles and quite variable therapeutic effects. IFN-α subtypes may each be specific for certain viruses, but can be neurotoxic. IFN-β induces IFN-α, plus has additional direct effects on target cells. IFN-β was the first therapy approved that could change the course of MS. It has broader specificity than IFN-α, enhances cognition in MS, and may be neuroprotective and can potentially enhance fertility in women. Priming the IFN signaling system with an injection of IFN-β can enhance subnormal type I IFN signals in MS. Many other commonly used drugs and vitamins may potentiate clinical benefits of IFN-β.

FRI0378 Potent, Broad, and Specific Neutralizing Capacities of Polyclonal Anti-Interferon Alpha Antibodies Induced by IFN Kinoid in SLE Patients

BackgroundNeovacs developed the Interferon alpha Kinoid (IFN-K), a therapeutic vaccine composed of IFNα2b coupled to a carrier protein, the keyhole limpet hemocyanin. A placebo-controlled phase I/II study was conducted comparing...

Modified interferon-α subtypes production and chemokine networks in the thymus during acute simian immunodeficiency virus infection, impact on thymopoiesis

Thymus dysfunction characterizes human/simian immunodeficiency virus (SIV) infections and contributes to physiopathology. However, both the mechanisms involved in thymic dysfunction and its precise timing remain unknown. We here analyzed thymic function during acute SIV infection in rhesus macaques. Rhesus macaques were intravenously infected with SIVmac251 and bled every 2/3 days or necropsied at different early time points postinfection. Naive T-cell counts were followed by flow cytometry and their T-cell receptor excision circle content evaluated by qPCR. Thymic chemokines were quantified by reverse transcription-qPCR and localized by in-situ hybridization in thymuses collected at necropsy. Thymic interferon alpha (IFN-α) subtype production was quantified by reverse transcription-qPCR combined to heteroduplex tracking assay. The effect of thymic IFN-α subtypes was tested on sorted triple negative thymocytes cultured on OP9-hDL1 cells. A reduced intrathymic proliferation history characterizes T cells produced during the first weeks of infection. Moreover, we evidenced a profound alteration of both chemokines and IFN-α subtypes transcriptional patterns in SIV-infected thymuses. Finally, we showed that IFN-α subtypes produced in the infected thymuses inhibit thymocyte proliferation, still preserving their differentiation capacity. Thymopoiesis is deeply impacted from the first days of SIV infection. Reduced thymocyte proliferation - a time-consuming process - together with modified chemokine networks is consistent with thymocyte differentiation speed-up. This may transiently enhance thymic output, thus increasing naive T-cell counts and diversity and the immune competence of the host. Nonetheless, long-lasting modification of thymic physiology may lead to thymic exhaustion, as observed in late primary HIV infection.

The Recombinant Vaccinia Virus Gene Product, B18R, Neutralizes Interferon Alpha and Alleviates Histopathological Complications in an HIV Encephalitis Mouse Model

Interferon-alpha (IFN-α) has been identified as a neurotoxin that plays a prominent role in human immunodeficiency virus (HIV)-associated neurocognitive disorders and HIV encephalitis (HIVE) pathology. IFN-α is associated with cognitive dysfunction in other inflammatory diseases where IFN-α is upregulated. Trials of monoclonal anti-IFN-α antibodies have been generally disappointing possibly due to high specificity to limited IFN-α subtypes and low affinity. We investigated a novel IFN-α inhibitor, B18R, in an HIVE/severe combined immunodeficiency (SCID) mouse model. Immunostaining for B18R in systemically treated HIVE/SCID mice suggested the ability of B18R to cross the blood-brain barrier (BBB). Real-time PCR indicated that B18R treatment resulted in a decrease in gene expression associated with IFN-α signaling in the brain. Mice treated with B18R were found to have decreased mouse mononuclear phagocytes and significant retention of neuronal arborization compared to untreated HIVE/SCID mice. Increased mononuclear phagocytes and decreased neuronal arborization are key features of HIVE. These results suggest that B18R crosses the BBB, blocks IFN-α signaling, and it prevents key features of HIVE pathology. These data suggest that the high affinity and broad IFN-α subtype specificity of B18R make it a viable alternative to monoclonal antibodies for the inhibition of IFN-α in the immune-suppressed environment.

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+T cells

Human type I interferons (IFNs) include IFN-β and 12 subtypes of IFN-α. During viral infection, infiltrating memory CD4(+) T cells are exposed to IFNs, but their impact on memory T-cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN-α8 and -α10 most potently enhanced expression of IFN-γ, IL-2, and IL-4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL-2 and IL-4 correlated with the time of preincubation with type I IFN, IFN-γ production was enhanced best when IFN-α was added immediately preceding or simultaneously with T-cell stimulation. Comparison of T-cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN-α best enhanced cytokine expression when CD4(+) T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T-cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4(+) T-cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.

75 : Interferon Regulatory Factor 4 (IRF4) mediates IFN-β and λ1 expression upon TLR4 and MR challenge in human alternatively activated macrophages

Alternative pathway of macrophage activation (AAM) sustains or enhances the inflammation associated with chronic asthma. Using the THP-1 cell line, we have successfully established a model of “classically” and “alternatively” activated macrophages (THP-CAM and THP-AAM, respectively), each of which expresses the appropriate signature cytokines and chemokines. We demonstrate that in response to TLR4 challenge by LPS, THP-AAM had attenuated IFN α / β pathway genes including STAT1/2, MyD88, Mx1, CXCL10, OAS1, and ISG1. In addition, THP-AAM poorly expressed IFN- β , IFN- λ 1, and specific subtypes of IFN- α coupled with reduced ability to drive NF- κ B driven luciferase reporter, compared to their CAM counterparts. On the other hand, THP-AAM preferentially expressed both mannose receptor (MR) and IRF4. THP-AAM exhibit an MR-dependent expression of IFN- β , λ 1, and IL-10 upon binding to house dust mite protein Dermatophagoides pteronyssinus (Derp1). This binding and subsequent cytokine signature expression is compromised upon MR silencing or blocking MR with mannan. IRF4 is critical for expression of key AAM genes including MR and STAT6, and for AAM reduced phagocytosis of Staphylococcus aureus. IRF4 can regulate expression of IFN- β and IFN- λ 1 in response to TLR4 and MR challenge by LPS or Derp1 respectively, pointing to a potential role of IRF4 in mediating the AAM phenotype. Taken together, our data demonstrate that THP-AAM is a critical tool to investigate cytokines and chemokines expression profile, in particular IFN α / β pathway genes and type I and III IFN subtypes, in this phenotype. In addition, we demonstrate that IRF4 plays a critical role in mediating AAM response to bacterial and allergen challenge. These findings may provide the basis of a novel IRF4 or MR targeted therapy to decrease allergic.

18: Characterization of human interferon-alpha subtypes and mutants

IFN- α subtypes and mutants have been cloned in our laboratory in a yeast expression system. The mutants were constructed with point mutations at the C-termini working under the hypothesis that so doing would increase binding affinity of the IFN to its receptor and hence its activity. The following subtypes, IFN- α 1a, IFN- α 2c, IFN- α 6, IFN- α 8b and IFN- α 14c were purified by HPLC and characterized. Their antiproliferative activities were tested on Daudi (Burkitt’s lymphoma) cells and their antiviral activities were tested on A549 (human adenocarcinoma epithelial) cells, OVCAR-3 (ovarian cancer) cells and HUH7 (hepatocellular carcinoma) cells. IFN- α 8b and IFN- α 2c had the highest antiproliferative activities. IFN- α 8b had the highest antiviral activity while IFN- α 6 had the lowest on the three cell lines tested. In addition, in both A549 and HUH7 cells, the antiviral activities of IFN- α 8b were seen to be a log greater than that of IFN- α 2c. To determine relationships of these IFNs to the interferon-alpha receptor 2 (IFNAR2), soluble, extracellular IFNAR2 (IFNAR2-EC) neutralization assays were done in which the IFNs were exposed to IFNAR2-EC in the antiviral assays. Addition of the soluble IFNAR2-EC decreased the antiviral activities of all of the IFN subtypes ten-fold except for IFN- α 1a whose antiviral activity was decreased two fold. Western blot analyses of STAT pathways in A549 cells did not show any apparent correlation with phosphorylation of STAT 1, 2 or 3 (1 h post IFN treatment) and either antiviral or antiproliferative activities. Western blot analyses done to detect selected Interferon Stimulated Genes (ISGs), at both 6 and 15 h timepoints, show that the ISGs RIG-G, UBE2l6 and Mx1 were most up regulated in cells treated with IFN- α 1a, - α 2c and - α 14c, while PKR and IRF9 were equally up regulated by all of the IFN subtypes tested. We plan to more broadly characterize both the subtypes and mutants.

CD141+ dendritic cells produce prominent amounts of IFN-α after dsRNA recognition and can be targeted via DEC-205 in humanized mice

AbstractFunctional differences between human dendritic cell (DC) subsets and the potential benefits of targeting them with vaccines remain poorly defined. Here we describe that mice with reconstituted human immune system components (huNSG mice) develop all human conventional and plasmacytoid DC compartments in lymphoid organs. Testing different Toll-like receptor agonists for DC maturation in vivo, we found that IL-12p70 and interferon (IFN)-α production correlated with the maturation of CD141+ (BDCA3+) conventional DCs in huNSG mice. Furthermore, depletion of CD141+ DCs before stimulation significantly reduced IFN-α levels in vivo. This DC subset produced similar total amounts but different subtypes of IFN-α in response to synthetic double-stranded RNA compared with plasmacytoid DCs in response to a single-stranded RNA equivalent. Moreover, synthetic double-stranded RNA as adjuvant and antigen targeting to the endocytic receptor DEC-205, a combination that focuses antigen presentation for T-cell priming on CD141+ DCs, stimulated antigen-specific human CD4+ T-cell responses. Thus, the human CD141+ DC subset is a prominent source of IFN-α and interleukin-12 production and should be further evaluated for vaccine development.

Genomic Analysis and mRNA Expression of Equine Type I Interferon Genes

This study aimed at identifying all of the type I interferon (IFN) genes of the horse and at monitoring their expression in equine cells on in vitro induction. We identified 32 putative type I IFN loci on horse chromosome 23 and an unplaced genomic scaffold. A phylogentic analysis characterized these into 8 different type I IFN classes, that is, putative functional genes for 6 IFN-α, 4 IFN-β, 8 IFN-ω (plus 4 pseudogenes), 3 IFN-δ (plus 1 pseudogene), 1 IFN-κ and 1 IFN-ε, plus 1 IFN-ν pseudogene, and 3 loci belonging to what has previously been called IFN-αω. Our analyses indicate that the IFN-αω genes are quite distinct from both IFN-α and IFN-ω, and we refer to this type I IFN as IFN-μ. Results from cell cultures showed that leukocytes readily expressed IFN-α, IFN-β, IFN-δ, IFN-μ, and IFN-ω mRNA on induction with, for example, live virus; while fibroblasts only expressed IFN-β mRNA on stimulation. IFN-κ or IFN-ε expression was not consistently induced in these cell cultures. Thus, the equine type I IFN family comprised 8 classes, 7 of which had putative functional genes, and mRNA expression of 5 was induced in vitro. Moreover, a relatively low number of IFN-α subtypes was found in the horse compared with other eutherian mammals.

SAT0193 Results of a randomized placebo controlled phase ia study of AGS-009, a humanized anti-interferon-α monoclonal antibody in subjects with systemic lupus erythematosus

BackgroundUnabated Type I interferon expression is implicated in the pathogenesis of systemic lupus erythematosus (SLE). A monoclonal anti-IFN-α antibody, AGS-009, which neutralizes a broad range of IFN-α subtypes was developed...

FRI0031 Naturally occurring antibodies against different IFN-alpha subtypes are observed in some SLE patients, and may impact the IFN gene signature and disease activity

BackgroundUpregulation of mRNA IFN-alpha activity (IFN signature) has been demonstrated in some patients with systemic lupus erythematosus (SLE). Based on this observation anti-IFN-alpha mAbs are currently being developed for the...

Reactive oxygen species and IRF1 stimulate IFNα production by proximal tubules during ischemic AKI.

We previously reported that expression of the transcription factor interferon regulatory factor 1 (IRF1) is an early, critical maladaptive signal expressed by renal tubules during murine ischemic acute kidney injury (AKI). We now show that IRF1 mediates signals from reactive oxygen species (ROS) generated during ischemic AKI and that these signals ultimately result in production of α-subtypes of type I interferons (IFNαs). We found that genetic knockout of the common type I IFN receptor (IFNARI-/-) improved kidney function and histology during AKI. There are major differences in the spatial-temporal production of the two major IFN subtypes, IFNβ and IFNαs: IFNβ expression peaks at 4 h, earlier than IFNαs, and continues at the same level at 24 h; expression of IFNαs also increases at 4 h but continues to increase through 24 h. The magnitude of the increase in IFNαs relative to baseline is much greater than that of IFNβ. We show by immunohistology and study of isolated cells that IFNβ is produced by renal leukocytes and IFNαs are produced by renal tubules. IRF1, IFNαs, and IFNARI were found on the same renal tubules during ischemic AKI. Furthermore, we found that ROS induced IFNα expression by renal tubules in vitro. This expression was inhibited by small interfering RNA knockdown of IRF1. Overexpression of IRF1 resulted in the production of IFNαs. Furthermore, we found that IFNα stimulated production of maladaptive proinflammatory CXCL2 by renal tubular cells. Altogether our data support the following autocrine pathway in renal tubular cells: ROS > IRF1 > IFNα > IFNARI > CXCL2.

Influence of cytokine auto antibodies on serum IFNα activity in SLE (P6287)

Abstract The type-I interferon (IFN-I) family consists of 12 IFN-α subtypes as well as IFN-β and IFN-ω. IFN-α subtypes are thought to play a pivotal role in the pathogenesis of autoimmune diseases such as lupus by enhancing the production of anti-nuclear autoantibodies and driving end organ damage in patients with lupus nephritis. Despite the putative role of IFN-αs in disease, serum IFN-α activity levels are often low or undetectable in lupus patients. Auto antibodies (AAbs) against a single IFNα subtype (IFNα4) have been detected in lupus patients suggesting they may alter serum IFNα activity and disease severity. To better understand this relationship, we have measured total IFNα and IFNα subtype-specific AAbs levels in lupus patient serum. Our results thus far reveal an excellent correlation between IFNα activity and AAb levels, suggesting AAbs may provide an indirect marker of cytokine activity in some patients. Toward this end, we have determined the AAb profile for eleven cytokines implicated in lupus to understand their relationship to IFNα levels and overall disease severity. The resulting cytokine profile will be presented towards the goal of determining the best targets for anti- cytokine therapy in lupus.

IFN-α subtypes: distinct biological activities in anti-viral therapy.

During most viral infections, the immediate host response is characterized by an induction of type I IFN. These cytokines have various biological activities, including anti-viral, anti-proliferative and immunomodulatory effects. After induction, they bind to their IFN-α/β receptor, which leads to downstream signalling resulting in the expression of numerous different IFN-stimulated genes. These genes encode anti-viral proteins that directly inhibit viral replication as well as modulate immune function. Thus, the induction of type I IFN is a very powerful tool for the host to fight virus infections. Many viruses evade this response by various strategies like the direct suppression of IFN induction or inhibition of the IFN signalling pathway. Therefore, the therapeutic application of exogenous type I IFN or molecules that induce strong IFN responses should be of great potential for future immunotherapies against viral infections. Type I IFN is currently used as a treatment in chronic hepatitis B and C virus infection, but as yet is not widely utilized for other viral infections. One reason for this restricted clinical use is that type I IFN belongs to a multigene family that includes 13 different IFN-α subtypes and IFN-β, whose individual anti-viral and immunomodulatory properties have so far not been investigated in detail to improve IFN therapy against viral infections in humans. In this review, we summarize the recent achievements in defining the distinct biological functions of type I IFN subtypes in cell culture and in animal models of viral infection as well as their clinical usage in chronic hepatitis virus infections.