FEVER

FIELD OF THE INVENTION

The invention relates to the treatment of haemorrhage in viral infections.

The invention relates to methods and compounds inhibiting IFN-lambda signalling.

BACKGROUND OF THE INVENTION

Yellow fever virus (YFV) is an arthropod-borne flavivirus, which causes highly lethal viral haemorrhagic fever and viscerotropic disease, particularly liver damage, in humans. Despite the availability of efficacious live-attenuated YF17D vaccine, YFV continues to pose a significant threat to human health, causing ~200,000 infections and 30,000 deaths worldwide annually. Due to our limited understanding of the underlying mechanisms controlling YFV pathogenesis, the progress to develop antiviral therapies against YFV has been greatly hampered. Type I Interferons (IFN- a/p) are key in controlling replication and dissemination of wild-type (wt) YFV and YF17D vaccine. More recently, the activity of Type III Interferon (IFN-A) has been shown to complement this antiviral activity of IFN-a/P, and IFN-A mediated signalling has been demonstrated to restrict the neurotropism of live-attenuated YF17D at least in IFN-a/P deficient mice. In contrast to the ubiquitously expressed IFN-a/p receptors, IFN-A receptors (INFLR) are preferentially expressed on epithelial surfaces and thought to play an important role in protecting these tissue barriers. However, the potential role of Type III Interferon signalling for YFV pathogenesis in humans and hamsters as relevant animal model remains uncharacterized.

Yellow fever (YF) is a highly lethal haemorrhagic infection (>30% fatality) caused by the yellow fever virus (YFV). Severe YF is a sepsis-like disease that manifests as multiorgan interflammatory syndrome with liver and kidney most effected, and fulminant hepatitis and kidney insufficiency leading causes of death. Management of YF relies entirely on life supporting intensive care. Experimental therapies (mostly preclinical) include passive transfer of virus-neutralizing antibodies, small-molecule antiviral inhibitors and antiviral cytokines (interferons).

SUMMARY OF THE INVENTION

The present invention illustrates that IFN-A drives YFV pathogenesis and severe liver damage during YFV infection in vivo. Whereas live-attenuated YF17D vaccine is well tolerated in both WT and KO hamsters, almost all developed severe liver disease and needed to be euthanized around 5 days after YFV infection.

Surprisingly, IFN-A KO hamsters suffered with limited weight loss and fully survived lethal YFV infection. Intriguingly, IFN-A signalling has been previously described to control YFV infection in mice in which IFN-A was required to protect susceptible mice from lethal virus-induced encephalitis (Douam et al. (2017) mBio, 8, e00819-17).

IFN-A does not significantly impact antibody responses (adaptive immune responses; in contrast to what has been reported e.g. in a malaria infection model in mice (O'Hahn et al. (2020) virulence 11, 594-606), nor viral replication and dissemination in hamsters after YFV infection (in contrast to what has been reported e.g. for neurotropism in a mouse model (Douam et al. cited above).

There were no significant differences in neutralizing antibody titers after YFV infection in WT and IFN-A KO hamsters.

YF17D vaccination is equally efficacious and safe in WT and IFN-A KO hamsters.

There were no significant differences in viremia and viral loads in different organs after YFV infection in WT and IFN-A KO hamsters

YFV infection causes overshooting expression Interferon-Stimulated Genes (ISGs) in WT hamsters compared to KO hamsters.

Hyperinflammatory, sepsis-like response leads to lethal liver damage, liver failure and hemorrhage in YFV-infected WT animals. Ablation of IFN-A signalling ameliorates such symptoms and presents a therapeutic against opportunity.

The present has been exemplified by an IL-28 knockout, providing basis for inhibiting IL 28 signalling. Consequently, other cytokines of the type III interferon group can be equally targeted.

The present invention demonstrates that:

Blocking of IFN-A signalling prevents lethal YFV infection.

Pharmaceutical ablation of IFN-A or its cognate receptor, e.g. by means of neutralizing or blocking antibodies or specific small molecule inhibitors, can serve as therapy in severe YFV infection.

Ablation of IFN-A signalling does not pose a risk for YF vaccination using live- attenuated YF17D vaccines.

Lethal YFV infection can be treated by blocking of IFN-A signalling without imparing adaptive immune responses. YFV infection can be treated without directly targeting virus replication, whereby blocking of IFN-A signalling prevents lethal YFV infection without the need of directly lowering viral loads.

The invention is further summarised in the following statements.

1. An Interferon-lambda inhibitor for use in the treatment of a viral infection, wherein the Interferon-lambda inhibitor is an inhibitor against interferon-lambda itself or against its receptor.

2. The Interferon-lambda inhibitor for use in the treatment according to statement 1, wherein the viral infection is a filovirus, a flavivirus, an arenavirus, or a bunyavirus.

3. The Interferon-lambda inhibitor for use in the treatment according to statement 1 or 2, wherein the viral infection is Yellow Fever virus.

4. An Interferon-lambda inhibitor for use according to any one of statements 1 to 3, wherein Interferon-lambda is IL28.

5. An Interferon-lambda inhibitor for use according to any one of statement 1 or 4, in the treatment of haemorrhagic fever and/or multiorgan inflammatory syndrome in patients with Yellow Fever.

6. An Interferon-lambda inhibitor for use according to statement 4, wherein the inhibitor is an IL28 binding antibody, or an IL28 receptor binding antibody.

DETAILED DESCRIPTION

Figure 1 shows experimental scheme of example 1.

Figure 2 shows weight change until day 21.

Figure 3 shows survival until day 21.

Figure 4 shows severe viscerotropic yellow fever-like disease at day 5.

Figure 5 shows YFV-specific nAbs titres at day 5 and day 21 after infection.

Figure 6 shows viremia after YFV infection.

Figure 7 shows viral RNA copies in visceral organs after YFV infection.

Figure 8 shows viral RNA copies in brains after YFV infection.

Figure 9 shows the experimental scheme of example 2.

Figure 10 shows heat maps (generated with median) showing the expression profile at day 5.

Figure 11 shows gene expression kinetics of selected cytokine genes in whole blood cells from day 3 to 8. Figure 12 shows prothrombin time (PT) in WT and IFN-A KO hamsters after YFV infection.

Figure 13 shows elative activity of LDH (lactate dehydrogenase) to the healthy controls in WT and KO hamsters after YFV infection.

Figure 14 shows elative activity of Alanine Transaminase (ALT) to the healthy controls in WT and KO hamsters after YFV infection.

"Fulminant yellow fever" is a severe form of yellow fever that progresses rapidly and is often fatal.

The symptoms of fulminant yellow fever usually start 3 to 6 days after the bite of an infected mosquito. They include fever, headache, muscle pain, nausea and vomiting, jaundice (yellowing of the skin and eyes), bleeding from the gums, nose, or other orifices, shock, multiorgan failure (in particular liver and kidney failure) The case fatality rate of fulminant yellow fever is 50% or higher.

Three types of interferons (IFNs) are distinguished (Type 1, 2 and 3). Type 1 and Type 3 are considered antiviral cytokines. Type 1 (INF alpha, beta and omicron) are required to control YFV replication. The role of Type 3 IFN is restricted to barrier tissues (epithelia, lung, etc.). Its role in Yellow Fever is not known. In mouse models of YF vaccination using the live-attenuated YF17D viral vaccine, a double KO in both Type 1 IFN receptor (IFNAR) and IL28R lead to more pronounced neuro-invasive replication of the vaccine virus (Douam et al. (2017) mBio, e00819-17). A similar phenotype for YF17D is seen in hamsters with a KO in the STAT2 gene that is a common signalling molecule downstream of IFNAR and IL28R (Sanchez-Felipe et al. (2021) Nature 590, 320-325). In humans IFN deficiency is very rare and should thus generally not increase the risk of anti-IL28 treatment, also considering the very high fatality rate of severe YF, and the thus favourable risk-benefit ratio for any YF treatment.

"Xnterferon-lambda" or"IFNL" relates to cytokines of the type III interferon group, which is a group of anti-viral cytokines that consists in humans of four IFN-A (lambda) molecules called IFN-A1 (IL29), IFN-A2 (IL28A), IFN-A3 (IL28B) and IFN-A4.

"Xnterferon-lambda inhibitor" is a compound inhibiting fully or partially the IFN- lambda signalling. Inhibitors can bind or inactivate the ligand and/or the receptor, and can be organic compounds, parts of the ligand or of the receptor disrupting signalling, or antibodies binding to the ligand or the receptor and disrupting signalling.

Examples of Filoviruses which cause haemorrhagic fever are Marburg and Ebola virus.

Examples of flavivirus which cause haemorrhagic fever are Yellow fever, Dengue viruses (types 1-4), Omsk virus, Kyasanur virus and Alkhumra virus.

The invention is in particular suitable in Y infections.

Examples of arenaviruses which cause haemorrhagic fever are arenavirus, Junin virus A, Machupo virus, Guanarito virus, Sabia virus, Lassa virus, Lujo virus and Chapare Virus.

Examples of bunyaviruses which cause haemorrhagic fever are Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Hantaan, Puumala, Seoul, and Dobrava viruses, Sin Nombre virus, Andes virus, Choclo virus, and other Hantaviruses.

Syrian golden hamsters are a pathophysiological relevant model of human YFV infection, replicating hallmarks of human YF including fulminant hepatitis and haemorrhage. We infected wild-type (WT) hamsters and hamsters with a knock-out (KO) in the interleukin 28 receptor (IL28R) with hamster-adapted YFV-Asibi strain Ap7. As expected, WT hamsters succumb to Ap7 infection, whereas, unexpectedly, L28R-KO hamsters fully recover. This illustrates that (1) fulminant YF is driven by an overshooting innate immune response (likely IL28 driven), and (2) that YF can be treated by drugs that ablate IL28 signalling. Such drugs could be small IL28R antagonists (e.g. IL28R blocking antibodies), or IL28 neutralizing antibodies.

The present invention shows the role of Type III Interferon signalling in both wt and IFN-A knockout (ifnlr-/-) hamsters by using a hamster-adapted YFV strain, which induces mortality and liver disease in hamsters, alike clinical YFV infection in humans. In contrast to the protective role of IFN-A against YF17D infection in mice, the results herein demonstrate that Type III Interferon signalling is a critical factor that drives severe disease in hamsters following YFV infection. As compared to the rapid weight loss, high morality (as early as 5 days post infection) and severe liver inflammation and damage in wt hamsters, ifnlr-/- hamsters suffered only limited weight loss and recovered fully from infection without any signs of liver damage observed. These results demonstrate IFN-A as driver of YFV-induced immunopathology and likewise potential target for therapeutic intervention. The efficacy of IFN-A monoclonal antibodies to mitigate overshooting inflammatory responses and fulminant liver disease in yellow fever is evaluated in the hamster model towards clinical translation.

Example 1. Virulent YFV vs. live-attenuated YF17D infection in WT and in IFN-lambda deficient hamsters

Figure 1 discloses the experimental scheme of embodiments of the present invention. Groups of wild-type (WT) and IFN-A (IFNL) receptor knockout (KO) hamsters were injected with an infectious dose of 104 plaque-forming units (PFU) of either live- attenuated YF17D vaccine (YF17D), or virulent hamster-adapted YFV-Asibi (hamster- adapted YFV) strain (for infection model see Ma et al. (2022) EBioMedicine 83, 104240).

Weight change (Figure 2) and survival (Figure 3) were monitored until day 21.

Whereas, as anticipated, only 1/11 (<10%) WT hamsters survived (YFV-WT) following YFV-Asibi infection (death or euthanasia with as humane endpoint >20% weight loss associated with severe disease with no perspective for recovery), fully unexpectedly 10/10 (100%) KO hamsters lacking functional IFNL signalling (YFV- IFN-A KO) survive the same vigorous virus exposure (for reference on IFNL receptor KO hamsters, i.e. IL28R-/- hamsters see Boudewijns et al. (2020) Nat Commun 11, 5838).

Blood and organs were collected at day 5 and day 21, respectively, for further analysis.

Figure 4 shows severe viscerotropic yellow fever-like disease at day 5, the timepoint for peak of pathology in hamsters infected with hamster-adapted YFV-Asibi. Compared to uninfected controls, YFV-Asibi infected WT hamsters show a discoloration of their livers and haemorrhage in their intestines and spleens, indicative for severe yellow fever pathology (viscerotropic disease). No haemorrhage was observed in YFV-Asibi infected KO hamsters, and already macroscopically their liver was markedly less affected than YFV-Asibi infected WT hamsters; clearly demonstrating a surprising yet obvious health benefit of an ablation of IFNL signalling in YFV infection.

Figure 5 shows YFV-specific nAbs titres at day 5 and day 21 after infection. Humoral immune responses are not affected by the hamster genotype, i.e. adaptive antiviral immunity appears to be largely independent of IFNL signalling. Viremia (Figure 6) and viral RNA copies in visceral organs (Figure 7) and brains (Figure 8) after YFV infection. Dashed line represents lower limit of quantification (LLOQ). Unexpectedly, also viral loads are not affected by the hamster genotype, i.e. they are largely independent of IFNL signalling. Hence, differences in the susceptibility for YFV infection (entry, replication and dissemination) and the respective resulting viral loads within the infected host are independent form the host genotype (WT or KO). Hence also subsequent immune pathogenesis of YFV cannot be associated with a particular viral load. By contrast and much to our surprise, the severity and outcome of YFV infection is inversely correlated with the activity of a signalling pathway that is generally considered to be involved in the protection from virus infection.

Severe yellow fever disease can be prevented by ablating the activity of IFNL, for instance by neutralizing the activity of IFNL directly, or by antagonising its cellular receptor; hereby YFV infection also serves as prototype and pathophysiologic paradigm for other viral haemorrhagic fever syndromes and viral sepsis.

EXAMPLE-2. YFV disease kinetics in WT and IFN-lambda deficient hamsters The Experimental scheme is shown in figure 9. Groups of WT and IFN-A KO hamsters were injected with an infectious dose of 10 ⁴ PFU of hamster-adapted YFV. Gene expression in the whole blood cell RNA extract.

Figure 10 shows heat maps (generated with median) with expression profiles at day 5.

Figure 11 shows gene expression kinetics of selected cytokine genes in whole blood cells from day 3 to 8.

Figurer 12 shows Prothrombin time (PT) in WT and IFN-A KO hamsters after YFV infection.

Figure 13 and Figure 14 show relative activity of LDH (lactate dehydrogenase) and Alanine Transaminase (ALT) to the healthy controls in WT and KO hamsters after YFV infection

The example shows that IFN-A drives systemic pathology and sepsis like hyperinflammation during YFV infection.

YFV infection causes overshooting Interferon-Stimulated Genes (ISGs) expression as observed in peripheral blood of WT hamsters compared to KO hamsters. KO hamsters survive YFV infection, despite prolonged prothrombin time (PTH) and higher LDH and ALT serum levels as compared to WT hamsters.

Coagulation (measured as prothrombin time, PTH) and acute liver cell death (measured as elevation of liver enzymes, ALT and LDH) may not predict survival, as respective clinical parameters values in WT animals may be lower than in those without IFN-A signalling.