NF-κB Pathway as a Potential Target for Treatment of Critical Stage COVID-19 Patients.

Patients infected with SARS-CoV-2 show a wide spectrum of clinical manifestations ranging from mild febrile illness and cough up to acute respiratory distress syndrome, multiple organ failure, and death. Data from patients with severe clinical manifestations compared to patients with mild symptoms indicate that highly dysregulated exuberant inflammatory responses correlate with severity of disease and lethality. Epithelial-immune cell interactions and elevated cytokine and chemokine levels, i.e. cytokine storm, seem to play a central role in severity and lethality in COVID-19. The present perspective places a central cellular pro-inflammatory signal pathway, NF-κB, in the context of recently published data for COVID-19 and provides a hypothesis for a therapeutic approach aiming at the simultaneous inhibition of whole cascades of pro-inflammatory cytokines and chemokines. The simultaneous inhibition of multiple cytokines/chemokines is expected to have much higher therapeutic potential as compared to single target approaches to prevent cascade (i.e. redundant, triggering, amplifying, and synergistic) effects of multiple induced cytokines and chemokines in critical stage COVID-19 patients.

Pharmacodynamics, Pharmacokinetics, and Antiviral Activity of BAY 81-8781, a Novel NF-κB Inhibiting Anti-influenza Drug.

Influenza is a respiratory disease that causes annual epidemics. Antiviral treatment options targeting the virus exist, but their efficiency is limited and influenza virus strains easily develop resistance. Thus, new treatment strategies are urgently needed. In the present study, we investigated the anti-influenza virus properties of D,L-lysine acetylsalicylate ⋅ glycine (BAY 81-8781; LASAG) that is approved as Aspirin i.v. for intravenous application. Instead of targeting the virus directly BAY 81-8781 inhibits the activation of the NF-κB pathway, which is required for efficient influenza virus propagation. Using highly pathogenic avian influenza virus strains we could demonstrate that BAY 81-8781 was able to control influenza virus infection in vitro. In the mouse infection model, inhalation of BAY 81-8781 resulted in reduced lung virus titers and protection of mice from lethal infection. Pharmacological studies demonstrated that the oral route of administration was not suitable to reach the sufficient concentrations of BAY 81-8781 for a successful antiviral effect in the lung. BAY 81-8781 treatment of mice infected with influenza virus started as late as 48 h after infection was still effective in protecting 50% of the animals from death. In summary, the data represent a successful proof of the novel innovative antiviral concept of targeting a host cell signaling pathway that is required for viral propagation instead of viral structures.

Phenol-Soluble Modulin Peptides Contribute to Influenza A Virus-Associated Staphylococcus aureus Pneumonia.

Influenza A virus (IAV) infection is often followed by secondary bacterial lung infection, which is a major reason for severe, often fatal pneumonia. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains such as USA300 cause particularly severe and difficult-to-treat cases of IAV-associated pneumonia. CA-MRSA strains are known to produce extraordinarily large amounts of phenol-soluble modulin (PSM) peptides, which are important cytotoxins and proinflammatory molecules that contribute to several types of S. aureus infection. However, their potential role in pneumonia has remained elusive. We determined the impact of PSMs on human lung epithelial cells and found that PSMs are cytotoxic and induce the secretion of the proinflammatory cytokine interleukin-8 (IL-8) in these cells. Both effects were boosted by previous infection with the 2009 swine flu pandemic IAV H1N1 strain, suggesting that PSMs may contribute to lung inflammation and damage in IAV-associated S. aureus pneumonia. Notably, the PSM-producing USA300 strain caused a higher mortality rate than did an isogenic PSM-deficient mutant in a mouse IAV-S. aureus pneumonia coinfection model, indicating that PSMs are major virulence factors in IAV-associated S. aureus pneumonia and may represent important targets for future anti-infective therapies.

The MEK-inhibitor CI-1040 displays a broad anti-influenza virus activity in vitro and provides a prolonged treatment window compared to standard of care in vivo.

Influenza viruses (IV) continue to pose an imminent threat to human welfare. Yearly re-occurring seasonal epidemic outbreaks and pandemics with high mortality can occur. Besides vaccination against a limited number of viral strains only a few antiviral drugs are available, which are losing their effectiveness as more and more IV strains become resistant. Thus, new antiviral approaches that omit IV resistance are urgently needed. Here, the dependency on the cellular Raf/MEK/ERK signaling pathway for IV replication opens a new perspective. In consequence, we studied the antiviral potential of the MEK inhibitor Cl-1040 (PD184352). We show that Cl-1040 significantly reduces virus titers in vitro via retention of viral RNP complexes in the cell nucleus. Furthermore, Cl-1040 is effective against a broad range of IV strains, including highly pathogenic avian IV, as well as against a Tamiflu®-resistant IV strain. Using a mouse model, we demonstrate that Cl-1040 can reduce IV lung titers in vivo. Importantly, the treatment window for Cl-1040 expands up to 48 h post infection when Tamiflu® treatment has no effect. In conclusion, Cl-1040 offers an interesting perspective for anti-IV approaches.

Antiviral activity of Ladania067, an extract from wild black currant leaves against influenza A virus in vitro and in vivo.

Influenza, a respiratory disease caused by influenza viruses, still represents a major threat to humans and several animal species. Besides vaccination, only two classes of drugs are available for antiviral treatment against this pathogen. Thus, there is a strong need for new effective antivirals against influenza viruses. Here, we tested Ladania067, an extract from the leaves of the wild black currant (Ribes nigrum folium) for potential antiviral activity against influenza A virus in vitro and in vivo. In the range of 0-1 mg/ml the extract showed no cytotoxic effect on three cell lines and a CC50 of 0.5 ± 0.3 mg/ml, on peripheral blood mononuclear cells. Furthermore, the extract did not influence the proliferative status of human lymphocytes. In contrast, Ladania067 was highly effective (EC50 value: 49.3 ± 1.1 ng/ml) against the human pandemic influenza virus strain A/Regensburg/D6/09 (H1N1). The extract exhibited an antiviral effect when the virus was pre-incubated prior to infection or when added directly after infection. No antiviral effect was found when infected cells were treated 2, 4, or 8 h after infection, indicating that Ladania067 blocks a very early step in the virus infection cycle. In the mouse infection model we were able to demonstrate that an intranasal application of 500 µg Ladania067 inhibits progeny virus titers in the lung up to 85% after 24 h. We conclude that the extract from the leaves of the wild black currant may be a promising source for the identification of new molecules with antiviral functions against influenza virus.

The NF-kappaB inhibitor SC75741 protects mice against highly pathogenic avian influenza A virus.

The appearance of pandemic H1N1 and highly pathogenic avian H5N1 viruses in humans as well as the emergence of seasonal H1N1 variants resistant against neuraminidase inhibitors highlight the urgent need for new and amply available antiviral drugs. We and others have demonstrated that influenza virus misuses the cellular IKK/NF-kappaB signaling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here, we show that the novel NF-kappaB inhibitor SC75741 significantly protects mice against infection with highly pathogenic avian influenza A viruses of the H5N1 and H7N7 subtypes. Treatment was efficient when SC75741 was given intravenously in a concentration of 5mg/kg/day. In addition, application of SC75741 via the intraperitoneal route resulted in a high bioavailability and was also efficient against influenza when given 15 mg/kg/day or 7.5 mg/kg/twice a day. Protection was achieved when SC75741 was given for seven consecutive days either prior to infection or as late as four days after infection. SC75741 treatment showed no adverse effects in the concentrations required to protect mice against influenza virus infection. Although more pre-clinical studies are needed SC75741 might be a promising candidate for a novel antiviral drug against influenza viruses that targets the host cell rather than the virus itself.

A plant extract of Ribes nigrum folium possesses anti-influenza virus activity in vitro and in vivo by preventing virus entry to host cells.

Infections with influenza A viruses (IAV) are still amongst the major causes of highly contagious severe respiratory diseases not only bearing a devastating effect to human health, but also significantly impact the economy. Besides vaccination that represents the best option to protect from IAV infections, only two classes of anti-influenza drugs, inhibitors of the M2 ion channel and the neuraminidase, often causing resistant IAV variants have been approved. That is why the need for effective and amply available antivirals against IAV is of high priority. Here we introduce LADANIA067 from the leaves of the wild black currant (Ribes nigrum folium) as a potent compound against IAV infections in vitro and in vivo. LADANIA067 treatment resulted in a reduction of progeny virus titers in cell cultures infected with prototype avian and human influenza virus strains of different subtypes. At the effective dose of 100 µg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation. Further, viruses showed no tendency to develop resistance to LADANIA067 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of LADANIA067 appears to be mainly due to interference with virus internalisation. In the mouse infection model LADANIA067 treatment reduces progeny virus titers in the lung upon intranasal application. In conclusion, an extract from the leaves of the wild black currant might be a promising source for the development of new antiviral compounds to fight IAV infections.

Combination of MEK inhibitors and oseltamivir leads to synergistic antiviral effects after influenza A virus infection in vitro.

MEK inhibitors are very potent and promising compounds in cancer therapy. Earlier investigations have demonstrated that they also possess antiviral properties against influenza virus. This is due to the fact that activation of the Raf/MEK/ERK signaling pathway is a prerequisite for influenza virus replication. As an alternative to vaccination, antiviral therapy is a means to control influenza. The appearance of influenza virus strains that are resistant to current treatment options demonstrates the need for new antiviral strategies. The aim of the presented study was to investigate whether the combination of MEK inhibitors with oseltamivir, an inhibitor of viral neuraminidase activity, would result in a synergistic antiviral effect against pandemic influenza A/Regensburg/D6/2009 (H1N1pdm09) virus. Here we show that four different MEK inhibitors, PD-0325901, AZD-6244, AZD-8330 and RDEA-119 that are orally available and at least in a phase I clinical trial against cancer demonstrate antiviral activity as single agents or in combination with oseltamivir. Combination treatment increased the antiviral activity of oseltamivir significantly and resulted in a synergistic antiviral effect as determined by the Chou-Talalay method. Taken together, the results demonstrate increased antiviral activity of oseltamivir after combination with MEK inhibitors. These data are promising for further preclinical in vitro and in vivo investigations on the way to developing new antiviral regimens against influenza.

The NF-κB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance.

Ongoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine-origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. The recently identified virus-supportive function of the cellular IKK/NF-κB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF-κB inhibitor SC75741 as a broad and efficient blocker of IV replication in non-toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF-κB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro-apoptotic factors, subsequent inhibition of caspase activation and block of caspase-mediated nuclear export of viralribonucleoproteins. SC75741 reduces viral replication and H5N1-induced IL-6 and IP-10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus-induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741-derived drugs may serve as broadly non-toxic anti-influenza agents.

The NS1 protein of influenza A virus blocks RIG-I-mediated activation of the noncanonical NF-κB pathway and p52/RelB-dependent gene expression in lung epithelial cells.

Influenza A virus (IAV) infection of epithelial cells activates NF-κB transcription factors via the canonical NF-κB signaling pathway, which modulates both the antiviral immune response and viral replication. Since almost nothing is known so far about a function of noncanonical NF-κB signaling after IAV infection, we tested infected cells for activation of p52 and RelB. We show that the viral NS1 protein strongly inhibits RIG-I-mediated noncanonical NF-κB activation and expression of the noncanonical target gene CCL19.

The adaptor protein FHL2 enhances the cellular innate immune response to influenza A virus infection.

The innate immune response of influenza A virus-infected cells is predominantly mediated by type I interferon-induced proteins. Expression of the interferon ß (IFNß) itself is initiated by accumulating viral RNA and is transmitted by different signalling cascades that feed into activation of the three transcriptional elements located in the IFNß promoter, AP-1, IRF-3 and NF-κB. FHL2 (four-and-a-half LIM domain protein 2) is an adaptor molecule that shuttles between membrane and nucleus regulating signalling cascades and gene transcription. Here we describe FHL2 as a novel regulator of influenza A virus propagation. Using mouse FHL2 wild-type, knockout and rescued cells and human epithelial cells with different expression levels of FHL2 we showed that FHL2 decreases influenza A virus propagation by regulating the intrinsic cellular antiviral immune response. On virus infection FHL2 translocates into the nucleus, potentiating the IRF-3-dependent transcription of the IFNß gene.

Antiviral activity of the proteasome inhibitor VL-01 against influenza A viruses.

The appearance of highly pathogenic avian influenza A viruses of the H5N1 subtype being able to infect humans and the 2009 H1N1 pandemic reveals the urgent need for new and efficient countermeasures against these viruses. The long-term efficacy of current antivirals is often limited, because of the emergence of drug-resistant virus mutants. A growing understanding of the virus-host interaction raises the possibility to explore alternative targets involved in the viral replication. In the present study we show that the proteasome inhibitor VL-01 leads to reduction of influenza virus replication in human lung adenocarcinoma epithelial cells (A549) as demonstrated with three different influenza virus strains, A/Puerto Rico/8/34 (H1N1) (EC50 value of 1.7 µM), A/Regensburg/D6/09 (H1N1v) (EC50 value of 2.4 µM) and A/Mallard/Bavaria/1/2006 (H5N1) (EC50 value of 0.8 µM). In in vivo experiments we could demonstrate that VL-01-aerosol-treatment of BALB/c mice with 14.1 mg/kg results in no toxic side effects, reduced progeny virus titers in the lung (1.1 ± 0.3 log10 pfu) and enhanced survival of mice after infection with a 5-fold MLD50 of the human influenza A virus strain A/Puerto Rico/8/34 (H1N1) up to 50%. Furthermore, treatment of mice with VL-01 reduced the cytokine release of IL-α/ß, IL-6, MIP-1ß, RANTES and TNF-α induced by LPS or highly pathogen avian H5N1 influenza A virus. The present data demonstrates an antiviral effect of VL-01 in vitro and in vivo and the ability to reduce influenza virus induced cytokines and chemokines.

Low-dose interferon Type I treatment is effective against H5N1 and swine-origin H1N1 influenza A viruses in vitro and in vivo.

The recent emergence of pandemic swine-origin influenza virus (H1N1) and the severe outbreaks of highly pathogenic avian influenza virus of the H5N1 subtype leading to death in humans is a reminder that influenza remains a frightening foe throughout the world. Besides vaccination, there is an urgent need for new antiviral strategies to protect against influenza. The innate immune response to influenza viruses involves production of interferon alpha and beta (IFN-α/ß), which plays a crucial role in virus clearance during the initial stage of infection. We examined the effect of IFN-α on the replication of H5N1 and H1N1 in vitro and in vivo. A single pretreatment with low-dose IFN-α reduced lung virus titers up to 1.4 log(10) pfu. The antiviral effect increased after multiple pretreatments. Low-dose IFN-α protected mice against lethal H5N1 viral infection. Further, IFN-α was also effective against H1N1 in vitro and in the mouse model. These results indicate that low-dose IFN-α treatment leads to the induction of antiviral cytokines that are involved in the reduction of influenza virus titers in the lung. Moreover, it might be possible that a medical application during pandemic outbreak could help contain fulminant infections.

Influenza virus infection aggravates stroke outcome.

BACKGROUND AND PURPOSE: Stroke is triggered by several risk factors, including influenza and other respiratory tract infections. However, it is unknown how and in which way influenza infection affects stroke outcome. METHODS: We infected mice intranasally with human influenza A (H1N1) virus and occluded the middle cerebral artery to induce ischemic strokes. Infarct volume and intracerebral hemorrhage were determined by histology. To evaluate the integrity of the blood-brain barrier and inflammation, we measured various cytokines in vivo and in vitro and performed immunohistochemistry of leukocyte markers, collagen IV, immunoglobulins, and matrix metalloproteinase-9. RESULTS: Influenza virus infection increased infarct size. Whereas changes in cardiovascular parameters did not explain this effect, we found evidence for an inflammatory mechanism. In influenza virus infection, the respiratory tract released cytokines into the blood, such as RANTES that induced macrophage inflammatory protein-2 and other inflammatory mediators in the ischemic brain. In infected mice, there was an increased number of neutrophils expressing the matrix metalloproteinase-9 in the ischemic brain. This was accompanied by severe disruption of the blood-brain barrier and an increased rate of intracerebral hemorrhages after tissue plasminogen activator treatment. To investigate the role of cytokines, we blocked cytokine release by using GTS-21, a selective agonist of the α7 nicotinic acetylcholine receptor. GTS-21 ameliorated ischemic brain damage and improved survival. CONCLUSIONS: Influenza virus infection triggers a cytokine cascade that aggravates ischemic brain damage and increases the risk of intracerebral hemorrhage after tissue plasminogen activator treatment. Blockade of cytokine production by α7 nicotinic acetylcholine receptor agonists is a novel therapeutic option to treat stroke in a proinflammatory context.

Highly pathogenic influenza virus infection of the thymus interferes with T lymphocyte development.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in humans. Still, the basis for their increased pathogenesis remains unclear. Additionally, the high morbidity in the younger population stays inexplicable, and the recent pandemic H1N1v outbreak in 2009 demonstrated the urgent need for a better understanding about influenza virus infection. In the present study, we demonstrated that HPAIV infection of mice not only led to lung destruction but also to functional damage of the thymus. Moreover, respiratory dendritic cells in the lung functioned as targets for HPAIV infection being able to transport infectious virus from the lung into the thymus. The pandemic H1N1 influenza virus was able to infect respiratory dendritic cells without a proper transport to the thymus. The strong interference of HPAIV with the immune system is especially devastating for the host and can lead to lymphopenia. In summary, from our data, we conclude that highly pathogenic influenza viruses are able to reach the thymus via dendritic cells and to interfere with T lymphocyte development. Moreover, this exceptional mechanism might not only be found in influenza virus infection, but also might be the reason for the increased immune evasion of some new emerging pathogens.

Antibodies and CD4(+) T-cells mediate cross-protection against H5N1 influenza virus infection in mice after vaccination with a low pathogenic H5N2 strain.

A H5N2 low pathogenic avian influenza virus (LPAIV) was isolated from a natural reservoir in Bavaria during a routine screen and was used as a vaccine strain to scrutinize the immune response involved in cross-protection after challenge infection with a H5N1 highly pathogenic avian influenza virus (HPAIV). The challenge virus was also isolated from a natural reservoir in Bavaria. Wild type, antibody deficient (muMT), CD4(-/-) and CD8(-/-) mice were infected with the apathogenic H5N2 vaccine strain and challenge infection with a 100-fold MLD(50) of the H5N1 strain was performed 80 days later. While 100% of the wild type and 100% of the CD8(-/-) mice stayed healthy, only 50% of the CD4(-/-) and none of the antibody deficient mice were protected. These results support the view that the humoral immune response and to certain extends the CD4(+) T helper cells are a prerequisite for cross-protective immunity against H5 influenza virus.