Targeted mutations in IFNα2 improve its antiviral activity against various viruses.

During viral infections, type I interferons (IFN) are induced and play a key role in counteracting initial viral spread. Twelve different human IFNα subtypes exist that bind the same receptor; however, they elicit unique host responses and display distinct potencies of antiviral activities. Our previous studies on human immunodeficiency virus (HIV) and hepatitis B virus (HBV) demonstrated that the clinically used IFNα2 is not the most effective one among the IFNα subtypes. By sequence modeling, we identified a region in helix B with mainly conserved residues at the outside facing IFNAR1, but variable residues at the inside facing the core of IFNα, potentially representing a putative tunable anchor to tune pleiotropic IFN responses. Using site-directed mutagenesis, various mutations were introduced into the IFNα2b backbone targeting sites which are important for binding to IFNAR1 and IFNAR2, the putative tunable anchor, or outside these three regions. Selected mutations were based on sequence differences to high antiviral subtypes IFNα6 and IFNα14. Treatment assays against HBV and HIV identified several critical residues for the antiviral activity of IFNα mainly in the IFNAR1 binding region. Combined mutations of the IFNα2 IFNAR1/2 binding sites or the IFNAR1 binding region plus the putative tunable anchor by those of IFNα14 further augmented activation of different downstream signaling cascades providing a molecular correlate for the enhanced antiviral activity. We describe here important functional residues within IFNα subtype molecules, which enabled us to design novel and innovative drugs that may have the potential to be used in clinical trials against a variety of different viral infections.IMPORTANCEThe potency of interferon (IFN)α to restrict viruses was already discovered in 1957. However, until today, only IFNα2 out of the 12 distinct human IFNα subtypes has been therapeutically used against chronic viral infections. There is convincing evidence that other IFNα subtypes are far more efficient than IFNα2 against many viruses. In order to identify critical antiviral residues within the IFNα subtype sequence, we designed hybrid molecules based on the IFNα2 backbone with individual sequence motifs from the more potent subtypes IFNα6 and IFNα14. In different antiviral assays with HIV or HBV, residues binding to IFNAR1 as well as combinations of residues in the IFNAR1 binding region, the putative tunable anchor, and residues outside these regions were identified to be crucial for the antiviral activity of IFNα. Thus, we designed artificial IFNα molecules, based on the clinically approved IFNα2 backbone, but with highly improved antiviral activity against several viruses.

Immunotherapy With Interferon α11, But Not Interferon Beta, Controls Persistent Retroviral Infection.

Type I Interferons (IFNs), including numerous IFNα subtypes and IFNß, are key molecules during innate and adaptive immune responses against viral infections. These cytokines exert various non-redundant biological activities, although binding to the same receptor. Persistent viral infections are often characterized by increased IFN signatures implicating a potential role of type I IFNs in disease pathogenesis. Using the well-established Friend retrovirus (FV) mouse model, we compared the therapeutic efficacy of IFNα11 and IFNß in acute and chronic retroviral infection. We observed a strong antiviral activity of both IFNs during acute FV infection, whereas only IFNα11 and not IFNß could also control persistent FV infection. The therapeutic treatment with IFNα11 induced the expression of antiviral IFN-stimulated genes (ISG) and improved cytotoxic T cell responses. Finally, dysfunctional CD8+ T cells solely regained cytotoxicity after IFNα11 treatment. Our data provide evidence for opposing activities of type I IFNs during chronic retroviral infections. IFNß was shown to be involved in immune dysfunction in chronic infections, whereas IFNα11 had a strong antiviral potential and reactivated exhausted T cells during persistent retroviral infection. In contrast, during acute infection, both type I IFNs were able to efficiently suppress FV replication.

HIV infection does not alter interferon α/ß receptor 2 expression on mucosal immune cells.

The innate immune response induced by type I interferons (IFNs) plays a critical role in the establishment of HIV infection. IFNs are induced early in HIV infection and trigger an antiviral defense program by signaling through the IFNα/ß receptor (IFNAR), which consists of two subunits, IFNAR1 and IFNAR2. Changes in IFNAR expression in HIV target cells, as well as other immune cells, could therefore have important consequences for initial HIV spread. It was previously reported that IFNAR2 expression is increased in peripheral blood CD4+ CXCR4+ T cells of HIV+ patients compared to HIV uninfected controls, suggesting that HIV infection may alter the IFN responsiveness of target cells. However, the earliest immune cells affected by HIV in vivo reside in the gut-associated lymphoid tissue (GALT). To date, it remains unknown if IFNAR expression is altered in GALT immune cells in the context of HIV infection and exposure to IFNs, including the 12 IFNα subtypes. Here, we analyzed the expression of surface bound and soluble IFNAR2 on Lamina propria mononuclear cells (LPMCs) isolated from the GALT of HIV- individuals and in plasma samples of HIV+ patients. IFNAR2 expression varied between different T cells, B cells and natural killer cells, but was not altered following HIV infection. Furthermore, expression of the soluble IFNAR2a isoform was not changed in HIV+ patients compared to healthy donors, nor in LPMCs after HIV-1 infection ex vivo. Even though the 12 human IFNα subtypes trigger different biological responses and vary in their affinity to both receptor subunits, stimulation of LPMCs with different recombinant IFNα subtypes did not result in any significant changes in IFNAR2 surface expression. Our data suggests that potential changes in the IFN responsiveness of mucosal immune cells during HIV infection are unlikely dictated by changes in IFNAR2 expression.

Diverse Immunomodulatory Effects of Individual IFNα Subtypes on Virus-Specific CD8+ T Cell Responses.

Clinical administration of Interferon α (IFNα) resulted in limited therapeutic success against some viral infections. Immune modulation of CD8+ T cell responses during IFNα therapy is believed to play a pivotal role in promoting viral clearance. However, these clinical studies primarily focused on IFNα subtype 2. To date, the immunomodulatory roles of the remaining 10-13 IFNα subtypes remains poorly understood, thereby precluding assessments of their potential for more effective treatments. Here, we report that virus-specific CD8+ T cell responses were influenced to various extents by individual IFNα subtypes. IFNα4, 6, and 9 had the strongest effects on CD8+ T cells, including antiproliferative effects, improved cytokine production and cytotoxicity. Interestingly, augmented cytokine responses were dependent on IFNα subtype stimulation of dendritic cells (DCs), while antiproliferative effects and cytotoxicity were mediated by IFNAR signaling in either CD8+ T cells or DCs. Thus, precise modulation of virus-specific CD8+ T cell responses may be feasible for specific antiviral immunotherapies through careful selection and administration of individual IFNα subtypes.

Different antiviral effects of IFNα subtypes in a mouse model of HBV infection.

Interferon alpha (IFNα) is commonly used for the treatment of chronic hepatitis B (CHB) patients. There are 13 different IFNα subtypes in humans, but only the subtype IFNα2 is used for clinical treatment. The antiviral activities of all other IFNα subtypes against HBV have not been studied. To obtain basic knowledge about the direct antiviral as well as the immunomodulatory effects of IFNα subtypes, we used the HBV hydrodynamic injection (HI) mouse model. Application of most IFNα subtype proteins inhibited HBV replication in vivo, with IFNα4 and IFNα5 being the most effective subtypes. Decreased viral loads after therapeutic application of IFNα4 and IFNα5 correlated with expanded effector cell populations of NK cells and T cells in both liver and spleen. Hydrodynamic injection of plasmids encoding for the effective IFNα subtypes (pIFNα) was even more potent against HBV than injecting IFNα proteins. The combination of pIFNα4 and pIFNα5 showed a synergistic antiviral effect on HBV replication, with a strong increase in NK cell and T cell activity. The results demonstrate distinct anti-HBV effects of different IFNα subtypes against HBV in the mouse model, which may be relevant for new therapeutic approaches.

Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo.

UNLABELLED: Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8(+) T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL(+) NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. IMPORTANCE: The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

Activated regulatory T cells suppress effector NK cell responses by an IL-2-mediated mechanism during an acute retroviral infection.

BACKGROUND: It is well established that effector T cell responses are crucial for the control of most virus infections, but they are often tightly controlled by regulatory T cells (Treg) to minimize immunopathology. NK cells also contribute to virus control but it is not known if their antiviral effect is influenced by virus-induced Tregs as well. We therefore analyzed whether antiretroviral NK cell functions are inhibited by Tregs during an acute Friend retrovirus infection of mice. RESULTS: Selective depletion of Tregs by using the transgenic DEREG mouse model resulted in improved NK cell proliferation, maturation and effector cell differentiation. Suppression of NK cell functions depended on IL-2 consumption by Tregs, which could be overcome by specific NK cell stimulation with an IL-2/anti-IL-2 mAb complex. CONCLUSIONS: The current study demonstrates that virus-induced Tregs indeed inhibit antiviral NK cell responses and describes a targeted immunotherapy that can abrogate the suppression of NK cells by Tregs.

Friend retrovirus drives cytotoxic effectors through Toll-like receptor 3.

BACKGROUND: Pathogen recognition drives host defense towards viral infections. Specific groups rather than single members of the protein family of pattern recognition receptors (PRRs) such as membrane spanning Toll-like receptors (TLRs) and cytosolic helicases might mediate sensing of replication intermediates of a specific virus species. TLR7 mediates host sensing of retroviruses and could significantly influence retrovirus-specific antibody responses. However, the origin of efficient cell-mediated immunity towards retroviruses is unknown. Double-stranded RNA intermediates produced during retroviral replication are good candidates for immune stimulatory viral products. Thus, we considered TLR3 as primer of cell-mediated immunity against retroviruses in vivo. RESULTS: Infection of mice deficient in TLR3 (TLR3(-/-)) with Friend retrovirus (FV) complex revealed higher viral loads during acute retroviral infection compared to wild type mice. TLR3(-/-) mice exhibited significantly lower expression levels of type I interferons (IFNs) and IFN-stimulated genes like Pkr or Ifi44, as well as reduced numbers of activated myeloid dendritic cells (DCs) (CD86(+) and MHC-II(+)). DCs generated from FV-infected TLR3(-/-) mice were less capable of priming virus-specific CD8(+) T cell proliferation. Moreover, cytotoxicity of natural killer (NK) cells as well as CD8(+) T cells were reduced in vitro and in vivo, respectively, in FV-infected TLR3(-/-) mice. CONCLUSIONS: TLR3 mediates antiretroviral cytotoxic NK cell and CD8(+) T cell activity in vivo. Our findings qualify TLR3 as target of immune therapy against retroviral infections.

Expanded regulatory T cells in chronically friend retrovirus-infected mice suppress immunity to a murine cytomegalovirus superinfection.

It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically Friend virus (FV)-infected mice decreased murine cytomegalovirus (mCMV)-specific CD8(+) T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was found only in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.

Distinct roles of NK cells in viral immunity during different phases of acute Friend retrovirus infection.

BACKGROUND: In many virus infections natural killer (NK) cells are critical for the rapid containment of virus replication. Polymorphisms in NK cell receptors as well as viral escape from NK cell responses are associated with pathogenesis and viral loads in HIV-infected individuals, emphasizing their importance in retroviral immunity. In contrast, NK cells of LCMV-infected mice dampened virus-specific T cell responses resulting in impaired virus control. Thus, the exact role of NK cells during different phases of viral infections remains elusive. In this study we characterized the NK cell response at different time points of an acute retroviral infection by using the Friend retrovirus (FV) mouse model. FINDINGS: Depletion of NK1.1⁺ cells during the initial phase of FV infection (3 to 4 days post infection) resulted in increased viral loads, which correlated with enhanced target cell killing and elevated NK cell effector functions. At days 7 to 15 post infection, NK and NKT cells did not contribute to anti-retroviral immunity. In the transition phase between acute and chronic infection (30 days post infection), NK and NKT cells exhibited an inhibitory role and their depletion resulted in reduced viral loads and significantly improved FV-specific CD8⁺ T cell responses. CONCLUSIONS: Our results demonstrate an opposed activity of NK cells during retroviral infection. They were protective in the initial phase of infection, when adaptive T cell responses were not yet detectable, but were dispensable for viral immunity after T cell expansion. At later time points they exhibited regulatory functions in inhibiting virus-specific CD8⁺ T cell responses.

NK cells improve control of friend virus infection in mice persistently infected with murine cytomegalovirus.

BACKGROUND: Co-infection of HIV patients with cytomegalovirus (CMV) is associated with enhanced AIDS progression and CMV end-organ diseases. On the other hand, persistent CMV infection has recently been shown to decrease tumor relapse and protect against lethal bacterial infection. The influence of persistent CMV on the outcome of an acute retroviral superinfection is still unknown. RESULTS: Here we show that a persistent murine CMV (mCMV) infection surprisingly confers higher resistance to a primary Friend retrovirus infection (FV) of mice. Decreased FV titers and augmented FV-specific CD8 T-cell responses were found in mCMV infected mice during primary FV superinfection. NK cells produced higher amounts of IFNgamma after FV infection of persistently mCMV infected mice suggesting that these cells were involved in the 'protective' effect. Depletion of NK1.1+ cells or neutralization of IFNgamma during FV superinfection abrogated the mCMV-mediated effect. CONCLUSION: Our data demonstrate for the first time that a persistent CMV infection induces long-lasting NK cell responses that can enhance immunity to primary retroviral infections. To our knowledge, studies investigating primary HIV infection have not analyzed the role of the CMV seropositivity in these patients. Our observations suggest that NK cells in CMV seropositive individuals might contribute to the control of primary HIV infection.

Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection.

The innate immune response mediated by cells such as natural killer (NK) cells is critical for the rapid containment of virus replication and spread during acute infection. Here, we show that subtype 11 of the type I interferon (IFN) family greatly potentiates the antiviral activity of NK cells during retroviral infection. Treatment of mice with IFN-α11 during Friend retrovirus infection (FV) significantly reduced viral loads and resulted in long-term protection from virus-induced leukemia. The effect of IFN-α11 on NK cells was direct and signaled through the type I IFN receptor. Furthermore, IFN-α11-mediated activation of NK cells enabled cytolytic killing of FV-infected target cells via the exocytosis pathway. Depletion and adoptive transfer experiments illustrated that NK cells played a major role in successful IFN-α11 therapy. Additional experiments with Mouse Cytomegalovirus infections demonstrated that the therapeutic effect of IFN-α11 is not restricted to retroviruses. The type I IFN subtypes 2 and 5, which bind the same receptor as IFN-α11, did not elicit similar antiviral effects. These results demonstrate a unique and subtype-specific activation of NK cells by IFN-α11.

Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points.

Although chronic infections with viruses such as HIV and hepatitis C virus have been associated with regulatory T cell (Treg)-mediated suppression of virus-specific CD8(+) T-cell activity, no causal relationship between Tregs and chronic viral set points has been established. Using transgenic mice in which Tregs can be selectively ablated, we now show that transient depletion of Tregs during a chronic retroviral infection allows exhausted CD8(+) T cells to regain antiviral functions, including secretion of cytokines, production of cytotoxic molecules, and virus-specific cytolytic activity. Furthermore, short-term Treg ablation resulted in long-term reductions in chronic virus loads. These results demonstrate that Treg-mediated immunosuppression can be a significant factor in the maintenance of chronic viral infections and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic infectious diseases.