Optimization of naked DNA delivery for interferon subtype immunotherapy in cytomegalovirus infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200μgIFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMVgB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases. Published: February 17, 2003     

Type I IFN-beta gene therapy suppresses cardiac CD8+ T-cell infiltration during autoimmune myocarditis

Gene therapy using DNA encoding type I IFN subtypes IFNA6, IFNA9 and IFNB suppresses murine cytomegalovirus (MCMV)-myocarditis, a predominantly cell-mediated disease in BALB/c mice. CD8(+) T cells are the principal cell type within the inflamed myocardium. As such, we investigated the effects of IFN subtype treatment on this T-cell subset and other cell types in the cardiac infiltrate. In the acute phase of disease, IFNA6 and IFNA9 treatments significantly reduced the number of CD8(+) T cells within the foci of cellular infiltration in the heart. During the chronic phase, which is primarily autoimmune in nature, IFNB treatment significantly reduced CD8(+) T cells. B-cell and neutrophil numbers in the cardiac infiltrate were also reduced following IFNB immunotherapy. Although early inflammatory responses are important for resolution of virus infection, high numbers of lymphocytes persisting in the myocardium may lead to exacerbation of disease. Our data suggests that type I IFN DNA therapy regulates cardiac cellular infiltration. Thus, treatment with IFN-beta administered prophylactically to high-risk patients in acquiring CMV infection may reduce the development of chronic autoimmune myocarditis.     

Direct application of plasmid DNA containing type I interferon transgenes to vaginal mucosa inhibits HSV-2 mediated mortality

The application of naked DNA containing type I interferon (IFN) transgenes is a promising potential therapeutic approach for controlling chronic viral infections. Herein, we detail the application of this approach that has been extensively used to restrain ocular HSV-1 infection, for antagonizing vaginal HSV-2 infection. We show that application of IFN-α1, -α 5, and -β transgenes to vaginal mouse lumen 24 hours prior to HSV-2 infection reduces HSV-2 mediated mortality by 2.5 to 3-fold. However, other type I IFN transgenes (IFN- α 4, -α 5, -α 6, and -α 9) are non effectual against HSV-2. We further show that the efficacy of IFN-1 transgene treatment is independent of CD4+ T lymphocytes. However, in mice depleted of CD8+ T lymphocytes, the ability of IFN-α 1 transgene treatment to antagonize HSV-2 was lost.     

The POU transcription factor Oct-1 represses virus-induced interferon A gene expression

Alpha interferon (IFN-alpha) and IFN-beta are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation.     

Combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model

The essential role of multispecific immune responses for the control of hepatitis B virus (HBV) infection implies the need of multimodal therapeutic strategies for chronic HBV infection, including antiviral chemotherapy and immunomodulation. This hypothesis was tested in the woodchuck model by a combination of lamivudine pretreatment and subsequent immunizations of woodchucks chronically infected with woodchuck hepatitis virus. The immunizations were performed with DNA vaccines or antigen-antibody immune complexes (IC)/DNA vaccines. Immunizations with IC/DNA vaccines led to an anti-woodchuck hepatitis virus surface antibody response and significant reductions of viral load and antigenemia, suggesting that such a strategy may be effective against chronic HBV infection.     

TRIF is a critical survival factor in viral cardiomyopathy

TRIF is a member of the innate immune system known to be involved in viral recognition and type I IFN activation. Because IFNs are thought to play an important role in viral myocarditis, we investigated the role of TRIF in induced myocarditis in mice. Whereas C57BL/6 (wild-type) mice showed only mild myocarditis, including normal survival postinfection with coxsackievirus group B serotype 3 (CVB3), infection of TRIF(-/-) mice led to the induction of cardiac remodeling, severe heart failure, and 100% mortality (p < 0.0001). These mice showed markedly reduced virus control in cardiac tissues and cardiomyocytes. This was accompained with dynamic cardiac cytokine activation in the heart, including a suppression of the antiviral cytokine IFN-β in the early viremic phase. TRIF(-/-) myocytes displayed a TLR4-dependent suppression of IFN-β, and pharmacological treatment of CVB3-infected TRIF(-/-) mice with murine IFN-β led to improved virus control and reduced cardiac inflammation. Additionally, this treatment within the viremic phase of myocarditis showed a significant long-term outcome indexed by reduced mortality (20 versus 100%; p < 0.001). TRIF is essential toward a cardioprotection against CVB3 infection.     

Lack of IL-6 during Coxsackievirus Infection Heightens the Early Immune Response Resulting in Increased Severity of Chronic Autoimmune Myocarditis

Background

Chronic myocarditis is often initiated by viral infection, the most common of which is coxsackievirus infection. The precise mechanism by which viral infection leads to chronic autoimmune pathology is poorly understood, however it is clear that the early immune response plays a critical role. Previous results have shown that the inflammatory cytokine interleukin (IL)-6 is integral to the development of experimental-induced autoimmune myocarditis. However, the function of IL-6 during viral-mediated autoimmunity has yet to be elucidated.

Methods and Results

To address the requirement of IL-6 during disease induction, IL-6 deficient mice were infected with coxsackievirus B3 (CB3). Following infection, mice lacking IL-6 developed increased chronic autoimmune disease pathology compared to wild type controls without a corresponding change in the level of viral replication in the heart. This increase in disease severity was accompanied by elevated levels of TNF-α, MCP-1, IL-10, activated T cells and cardiac infiltrating macrophage/monocytes. Injection of recombinant IL-6 early following infection in the IL-6 deficient mice was sufficient to lower the serum cytokines TNF-α and IL-10 as well as the serum chemokines MCP-1, MIP-1β, RANTES and MIG with a corresponding decrease in the chronic disease pathology strongly suggests an important regulatory role for IL-6 during the early response.

Conclusions

While IL-6 plays a pathogenic role in experimental-induced autoimmune disease, its function following viral-induced autoimmunity is not reprised. By regulating the early immune response and thereby controlling the severity of chronic disease, IL-6 directs the outcome of chronic autoimmune myocarditis.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

TLR ligands act directly upon T cells to restore proliferation in the absence of protein kinase C-theta signaling and promote autoimmune myocarditis

The serine/threonine kinase, protein kinase C-theta (PKC-theta), plays a central role in the activation and differentiation of Th2 cells while being redundant in CD4+ and CD8+ antiviral responses. Recent evidence indicates that PKC-theta may however be required for some T cell-driven autoimmune responses. We have investigated the role of PKC-theta in the induction of autoimmune myocarditis induced by either Coxsackie B3 virus infection or immunization with alpha-myosin/CFA (experimental autoimmune myocarditis (EAM)). PKC-theta-deficient mice did not develop EAM as shown by impaired inflammatory cell infiltration into the heart, reduced CD4+ T cell IL-17 production, and the absence of a myosin-specific Ab response. Comparatively, PKC-theta was not essential for both early and late-phase Coxsackie virus-induced myocarditis. We sought to find alternate pathways of immune stimulation that might reconcile the differential requirements for PKC-theta in these two disease models. We found systemic administration of the TLR ligand CpG restored EAM in PKC-theta-deficient mice. CpG could act directly upon TLR9-expressing T cells to restore proliferation and up-regulation of Bcl-x(L), but exogenous IL-6 and TGF-beta was required for Th17 cell differentiation. Taken together, these results indicate that TLR-mediated activation of T cells can directly overcome the requirement for PKC-theta signaling and, combined with the dendritic cell-derived cytokine milieu, can promote the development of autoimmunity.     

Effects of type I interferons on Friend retrovirus infection

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-alpha and IFN-beta in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-alpha and IFN-beta could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR(-/-)) or IFN-beta (IFN-beta(-/-)) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-alpha and IFN-beta and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-beta enhanced viral loads during acute and chronic FV infection. Exogenous IFN-alpha could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.     

Inhibition of coxsackievirus B3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosome entry site

Coxsackievirus B3 (CVB3) is a primary cause of viral myocarditis, yet no effective therapeutic against CVB3 is available. Nucleic acid-based interventional strategies against various viruses, including CVB3, have shown promise experimentally, but limited stability and inefficient delivery in vivo remain as obstacles to their potential as therapeutics. We employed phosphorodiamidate morpholino oligomers (PMO) conjugated to a cell-penetrating arginine-rich peptide, P007 (to form PPMO), to address these issues. Eight CVB3-specific PPMO were evaluated with HeLa cells and HL-1 cardiomyocytes in culture and in a murine infection model. One of the PPMO (PPMO-6), designed to target a sequence in the 3' portion of the CVB3 internal ribosomal entry site, was found to be especially potent against CVB3. Treatment of cells with PPMO-6 prior to CVB3 infection produced an approximately 3-log(10) decrease in viral titer and largely protected cells from a virus-induced cytopathic effect. A similar antiviral effect was observed when PPMO-6 treatment began shortly after the virus infection period. A/J mice receiving intravenous administration of PPMO-6 once prior to and once after CVB3 infection showed an approximately 2-log(10)-decreased viral titer in the myocardium at 7 days postinfection and a significantly decreased level of cardiac tissue damage, compared to the controls. Thus, PPMO-6 provided potent inhibition of CVB3 amplification both in cell cultures and in vivo and appears worthy of further evaluation as a candidate for clinical development.     

TRAF6 and IRF7 control HIV replication in macrophages

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.     

电转联合热休克蛋白佐剂疫苗引发抗HBV的免疫应答

针对HBV感染的治疗性DNA疫苗虽然具有很好的应用前景,但目前抗病毒效果并不高,表明在病毒长期感染过程中存在免疫抑制机制。以HBV的表面蛋白(HBsAg)和核心蛋白(HBcAg)为DNA疫苗抗原,采用gp96和HSP70作为佐剂联合电转以提高疫苗的活性。将gp96为佐剂的HBsAg/HBcAg DNA疫苗免疫HBV转基因鼠后引发抗原特异性的细胞免疫和体液免疫应答。使用gp96和HSP70佐剂引起Treg下调20%。与没有免疫的小鼠相比,以gp96和HSP70为佐剂的DNA疫苗显著降低血清中病毒S抗原水平和DNA拷贝数,大幅降低小鼠肝脏中HBc的表达。该研究为设计以gp96为佐剂的乙肝治疗性DNA疫苗提供了依据。     

Ifih1 gene dose effect reveals MDA5-mediated chronic type I IFN gene signature, viral resistance, and accelerated autoimmunity

Type I IFNs (IFN-I) are normally produced during antiviral responses, yet high levels of chronic IFN-I expression correlate with autoimmune disease. A variety of viral sensors generate IFN-I in their response, but other than TLRs, it is not fully known which pathways are directly involved in the development of spontaneous immune pathologies. To further explore the link between IFN-I induced by viral pathways and autoimmunity, we generated a new transgenic mouse line containing multiple copies of Ifih1, a gene encoding the cytoplasmic dsRNA sensor MDA5 with proven linkage to diabetes and lupus. We show that MDA5 overexpression led to a chronic IFN-I state characterized by resistance to a lethal viral infection through rapid clearance of virus in the absence of a CD8(+) or Ab response. Spontaneous MDA5 activation was not sufficient to initiate autoimmune or inflammatory pathology by itself, even though every immune cell population had signs of IFN activation. When combined with the lupus-susceptible background of the FcγR2B deficiency, MDA5 overexpression did accelerate the production of switched autoantibodies, the incidence of glomerulonephritis, and early lethality. Thus, MDA5 transgenic mice provide evidence that chronic elevated levels of IFN-I are not sufficient to initiate autoimmunity or inflammation although they might exacerbate an ongoing autoimmune pathology.     

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.     

Soluble Interleukin-6 Receptor-Mediated Innate Immune Response to DNA and RNA Viruses

The interleukin-6 (IL-6) receptor, which exists as membrane-bound and soluble forms, plays critical roles in the immune response. The soluble IL-6 receptor (sIL6R) has been identified as a potential therapeutic target for preventing coronary heart disease. However, little is known about the role of this receptor during viral infection. In this study, we show that sIL6R, but not IL-6, is induced by viral infection via the cyclooxygenase-2 pathway. Interestingly, sIL6R, but not IL-6, exhibited extensive antiviral activity against DNA and RNA viruses, including hepatitis B virus, influenza virus, human enterovirus 71, and vesicular stomatitis virus. No synergistic effects on antiviral action were observed by combining sIL6R and IL-6. Furthermore, sIL6R mediated antiviral action via the p28 pathway and induced alpha interferon (IFN-α) by promoting the nuclear translocation of IFN regulatory factor 3 (IRF3) and NF-κB, which led to the activation of downstream IFN effectors, including 2′,5′-oligoadenylate synthetase (OAS), double-stranded RNA-dependent protein kinase (PKR), and myxovirus resistance protein (Mx). Thus, our results demonstrate that sIL6R, but not IL-6, plays an important role in the host antiviral response.