Interferon-α mediated immunotherapy in Friend retroviral infection

Die Expression von Typ I Interferon (IFN) ist in der Regel die erste Antwort des Wirtes auf eine Virusinfektion. Typ I IFN hat eine direkte antivirale Aktivität, welche durch antivirale Enzyme wie Proteinkinase R und Oligoadenylatsynthetase vermittelt wird. Zusätzlich kann Typ I IFN Einfluss auf Zellen des Immunsystem wie z.B. dendritische Zellen, NK-Zellen, T- und B-Zellen, nehmen und deren Funktion modulieren. Viele Viren haben darum verschiedenste Wege gefunden die initiale Produktion von IFN-α/β zu unterdrücken. In retroviralen Infektionen ist die Expression von IFN-α/β sehr schwach, auch wenn der Mechanismus der Suppression der IFNα-Antwort bisher unbekannt ist. Daher ist eine Möglichkeit der Immuntherapie von Virusinfektionen die Verstärkung der Immunantwort durch die Erhöhung der IFN-α/β-Antwort. Dies kann zum Einen über eine exogene Applikation von rekombinantem Interferon-α geschehen, oder zum Anderen durch die Injektion von Liganden für Toll-ähnliche Rezeptoren (TLR), wie z.B. Poly I:C, das eine starke Expression von Interferon-α induziert. Poly I:C, ein artifizieller Ligand für die zytoplasmatische Helikase MDA5 und den in den Endosomen lokalisierten TLR3, wurde bereits für die Behandlung diverser Virusinfektionen verwendet. Allerdings wurde Poly I:C nicht in retroviralen Infektionen untersucht und vor allem wurde der genaue immunologische Mechanismus, welcher einer erfolgreichen Therapie mit Poly I:C zugrunde liegt, nicht aufgeklärt. In der vorliegenden Arbeit sollte mit Hilfe des Friend Retrovirus-Mausmodell (FV) die Wirkung von Poly I:C in einer antiretroviralen Immuntherapie untersucht werden. Die Behandlung mit Poly I:C in FV-infizierten Mäusen führte zu einer signifikanten Reduktion der Viruslast und verhinderte die Entstehung einer letalen Erythroleukämie. Dieser Effekt von Poly I:C war abhängig von Typ I IFN, denn eine Poly I:C-Therapie in Mäusen, die keinen Interferon-α/β-Rezeptor besitzen, resultierte in keiner Reduktion der Viruslast. Durch die Behandlung mit Poly I:C wurden antivirale Enzyme exprimiert, welche direkt die FV-Replikation inhibierten. Des Weiteren wurde eine Aktivierung und eine verbesserte Effektorfunktion von T-Zellen nach der Therapie mit Poly I:C beobachtet. Die Behandlung induzierte dabei keine Expansion von virus-spezifischen CD4+ und CD8+ T-Zellen, sondern verbesserte die Effektorfunktion (z.B. Produktion von zytotoxischen Molekülen und Zytokinen) dieser Zellen. Typ I IFN gehört zu einer Multigenfamilie, die aus 14 verschiedenen IFNα Subtyp-Genen und einem IFNβ-Gen besteht. All diese Subtypen binden an den gleichen Rezeptor, allerdings besitzen sie sehr unterschiedliche biologische Aktivitäten. Es konnte gezeigt werden, dass Poly I:C in vivo die Expression aller IFNα Subtypen induzierte. Im Weiteren wurden 3 Subtypen (IFNα2, 5 und 11) auf ihre antivirale Aktivität gegen FV genauer untersucht. IFNα2 und 5 besaßen in vivo keine antiviralen Effekte gegen das FV, obwohl IFNα5 besonders stark durch Poly I:C induziert wurde. Eine Therapie mit dem Subtyp IFNα11, welcher allerdings nur sehr moderat durch Poly I:C induziert wurde, reduzierte die Viruslast signifikant und aktivierte NK-, B- und T-Zellen. Die Ergebnisse zeigen einen direkten antiviralen und immunomodulatorischen Effekt durch die Poly I:C-induzierte IFNα-Antwort und die Therapie mit einem einzelnen Subtyp. Folglich ist der klinische Einsatz von Poly I:C oder einem bestimmten IFNα Subtyp vielversprechend für die Therapie von retroviralen Infektionen.The induction of type I Interferon (IFN) is the most immediate host response to viral infections. Type I IFN has a direct antiviral activity mediated by antiviral enzymes like proteinkinase R or oligoadenylatesynthetase. It also modulates the function of cells of the adaptive immune system e.g. dendritic cells, NK cells, T or B cells. Many viruses can suppress type I IFN production. In retroviral infections the initial type I IFN is weak, but the mechanism of the suppressed IFNα response is unclear. Thus, one strategy of immunotherapy in viral infection is the improvement of the host immune response by the induction of type I IFN. On the one hand, this can be done by exogenous application of recombinant IFNα, or on the other hand, by infection of Toll-like receptor (TLR) ligands, like Poly I:C which induces a strong IFNα expression. Poly I:C is an artificial ligand of the cytoplasmic helicase MDA5 and the endosomal TLR3, which has already been used to treat viral infections. However, Poly I:C has not been investigated in retroviral infections so far and the immunological mechanisms underlying a successful therapy have not been defined until now. In this study the Friend Retrovirus (FV) mouse model was used to investigate the mode of action of Poly I:C in anti-retroviral immunotherapy. Post exposure Poly I:C treatment of FV-infected mice resulted in a significant reduction in viral loads and protection from virus-induced leukemia. This effect was IFNα dependent since type I IFN receptor deficient mice could not be protected by Poly I:C. The Poly I:C induced IFN response resulted in the expression of antiviral enzymes, which directly suppressed FV replication. Also the virus-specific T-cell response was augmented. Interestingly, it did not enhance the number of virus-specific CD4+ and CD8+ T cells but rather the functional properties of these cells, like cytokine production and cytotoxic activity. Type I IFN belongs to a multigene family that includes 14 different IFNα subtypes and only one IFNβ subtype. All these subtypes bind to same receptor, but they have various biological activities. It was shown that Poly I:C induced the expression of all IFNα subtypes in vivo. The antiviral activity against FV of three IFNα subtypes (IFNα2, 5 and 11) was further analyzed. IFNα2 and 5 had no antiviral effects against FV in vivo, although IFNα5 was strongly induced by Poly I:C. A therapy with IFNα11 , which was moderately expressed by Poly I:C, reduced the viral loads significantly and activated NK, B and T cells. The results demonstrate a direct antiviral and immunomodulatory effect of the Poly I:C induced IFNα response and the therapy with a single IFNα subtype. Therefore, the clinical treatment with Poly I:C or a certain IFNα subtype is promising for the therapy of retroviral infections

Susceptibility of different hepatitis B virus isolates to interferon-alpha in a mouse model based on hydrodynamic injection.

Interferon alpha (IFN-α) is commonly used for the treatment of chronic hepatitis B (CHB) patients. Many factors including viral genetics may determine the outcome of IFN-α therapy. In this study, we tested whether the expression of IFN-α directly in the liver inhibits HBV gene expression and replication using a HBV hydrodynamic injection (HI) mouse model. Two replication-competent clones from different HBV isolates that belonging to HBV genotype A and B based on a pAAV vector (pAAV-HBV-A and pAAV-HBV-B) were compared for their susceptibility to IFN-α. HBV clones were injected into mice either alone or in combination with a murine (m) IFN-α expression plasmid (pmIFN-α). HBsAg and HBeAg concentrations and HBV DNA levels in mice differed after injection of these two HBV clones. Co-application of pmIFN-α together with the two distinct isolates resulted in markedly different kinetics of decline of HBsAg, HBeAg, and HBV DNA levels in the mice. Immunohistochemical staining of liver sections with anti-HBc showed that mIFN-α application completely inhibited the expression of HBcAg in mice inoculated with pAAV-HBV-B, whereas the expression of HBcAg was only reduced in mice with pAAV-HBV-A. Consistently, mice injected with pAAV-HBV-B and pmIFN-α showed higher expression levels of the IFN-stimulated genes (ISGs) ISG15, OAS, PKR as well as proinflammatory cytokine IL-6 in the liver. In addition, expression levels of anti-inflammatory cytokine IL-10 was down-regulated significantly in liver of the mice injected with pAAV-HBV-B and pmIFN-α. Our data demonstrate that IFN-α exerts antiviral activity in HBV mouse model, but different HBV isolates may have diverse susceptibility to IFN-α

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

Additional file 4: of Activated regulatory T cells suppress effector NK cell responses by an IL-2-mediated mechanism during an acute retroviral infection

Influence of IL-2/anti-IL-2 mAb complex treatment on T cell subsets Mice were infected with 20.000 SFFU of FV and sacrificed at 12 dpi. If indicated, Tregs were depleted by repeated injections of DT and mice were stimulated by injections of IL-2/anti-IL-2 mAb complex. Control mice were inoculated with isotype control. Single cell suspensions of splenocytes were stained for characteristic T cell markers ((a) CD4+ T cells; (b) CD8+ T cells and (c) Tregs) as well as activation (CD43) was analyzed using flow cytometry. Tregs were also stimulated with IL-2/anti-IL-2 mAb complex targeting CD25 on cell surface of Tregs (JES6-1) (c). Dotted lines represent the mean activation in naive mice. At least four mice per group were analyzed. Statistically significant differences between FV and FV+IL2/anti-IL-2 (JES6-1) were analyzed by Mann-Whitney test and indicated by * for p < 0.05