From Naproxen Repurposing to Naproxen Analogues and Their Antiviral Activity against Influenza A Virus.

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.

The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus.

The Formyl Peptide Receptor 2 (FPR2) is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.

Protease-activated receptor 1 inhibition protects mice against thrombin-dependent respiratory syncytial virus and human metapneumovirus infections.

BACKGROUND AND PURPOSE: Protease-activated receptor 1 (PAR1) has been demonstrated to be involved in the pathogenesis of viral diseases. However, its role remains controversial. The goal of our study was to investigate the contribution of PAR1 to respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections. EXPERIMENTAL APPROACH: Pharmacological approaches were used to investigate the role of PAR1 during RSV and hMPV infection, in vitro using epithelial A549 cells and in vivo using a mouse model of virus infection. KEY RESULTS: In vitro, the PAR1 antagonist RWJ-56110 reduced the replication of RSV and hMPV in A549 cells. In agreement with these results, RWJ-56110-treated mice were protected against RSV and hMPV infections, as indicated by less weight loss and mortality. This protective effect in mice correlated with decreased lung viral replication and inflammation. In contrast, hMPV-infected mice treated with the PAR1 agonist TFLLR-NH2 showed increased mortality, as compared to infected mice, which were left untreated. Thrombin generation was shown to occur downstream of PAR1 activation in infected mice via tissue factor exposure as part of the inflammatory response, and thrombin inhibition by argatroban reduced the pathogenicity of the infection with no additive effect to that induced by PAR1 inhibition. CONCLUSION AND IMPLICATIONS: These data show that PAR1 plays a detrimental role during RSV and hMPV infections in mice via, at least, a thrombin-dependent mechanism. Thus, the use of PAR1 antagonists and thrombin inhibitors may have potential as a novel approach for the treatment of RSV and hMPV infections.

FPR2: A Novel Promising Target for the Treatment of Influenza.

The Formyl-peptide receptor-2 (FPR2) is a seven transmembrane G protein-coupled receptor, which plays an important role in sensing of bacteria and modulation of immune responses. FPR2 is also used by viruses for their own profit. Annexin A1, one of the multiple ligands of FPR2, is incorporated in the budding virus membrane of influenza A viruses (IAV). Thereby, once IAV infect a host cell, FPR2 is activated. FPR2-signaling leads to an increase in viral replication, a dysregulation of the host immune response and a severe disease. Conversely, experiments using FPR2 antagonists in a preclinical model of IAV infections in mice showed that blocking FPR2 protects animals from lethal infections. Thus, FPR2 represents a very attractive host target against influenza. In this review we will give an overview on the pathogenesis of influenza with a focus on the role of FPR2 and we will discuss the advantages of using FPR2 antagonists to treat the flu.

Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.

Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza.

Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections.

BACKGROUND: The pathogenesis of influenza A virus (IAV) infections is a multifactorial process that includes the replication capacity of the virus and a harmful inflammatory response to infection. Formyl peptide receptor 2 (FPR2) emerges as a central receptor in inflammatory processes controlling resolution of acute inflammation. Its role in virus pathogenesis has not been investigated yet. METHODS: We used pharmacologic approaches to investigate the role of FPR2 during IAV infection in vitro and in vivo. RESULTS: In vitro, FPR2 expressed on A549 cells was activated by IAV, which harbors its ligand, annexin A1, in its envelope. FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)-dependent pathway. In vivo, activating FPR2 by administering the agonist WKYMVm-NH2 decreased survival and increased viral replication and inflammation after IAV infection. This effect was abolished by treating the mice with U0126, a specific ERK pathway inhibitor, showing that, in vivo, the deleterious role of FPR2 also occurs through an ERK-dependent pathway. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections. CONCLUSIONS: These data show that viral replication and IAV pathogenesis depend on FPR2 signaling and suggest that FPR2 may be a promising novel strategy to treat influenza.

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis.

RATIONALE: The hallmark of severe influenza virus infection is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. OBJECTIVES: To determine the role of platelets during influenza A virus infections and propose new therapeutics against influenza. METHODS: We used targeted gene deletion approaches and pharmacologic interventions to investigate the role of platelets during influenza virus infection in mice. MEASUREMENTS AND MAIN RESULTS: Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted influenza A virus pathogenesis. Activating protease-activated receptor 4, a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein IIIa protected mice from death caused by influenza viruses, and treating the mice with a specific glycoprotein IIb/IIIa antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other antiplatelet compounds (antagonists of protease-activated receptor 4, MRS 2179, and clopidogrel) were also protected from severe lung injury and lethal infections induced by several influenza strains. CONCLUSIONS: The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and antiplatelet drugs might be explored to develop new antiinflammatory treatment against influenza virus infections.

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication.

UNLABELLED: During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

Switch from protective to adverse inflammation during influenza: viral determinants and hemostasis are caught as culprits.

Influenza viruses cause acute respiratory infections, which are highly contagious and occur as seasonal epidemic and sporadic pandemic outbreaks. Innate immune response is activated shortly after infection with influenza A viruses (IAV), affording effective protection of the host. However, this response should be tightly regulated, as insufficient inflammation may result in virus escape from immunosurveillance. In contrast, excessive inflammation may result in bystander lung tissue damage, loss of respiratory capacity, and deterioration of the clinical outcome of IAV infections. In this review, we give a comprehensive overview of the innate immune response to IAV infection and summarize the most important findings on how the host can inappropriately respond to influenza.

Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model.

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.

PAR1 contributes to influenza A virus pathogenicity in mice.

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses.

The influenza fingerprints: NS1 and M1 proteins contribute to specific host cell ultrastructure signatures upon infection by different influenza A viruses.

Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus-host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

HLA-G1 and HLA-G5 active dimers are present in malignant cells and effusions: the influence of the tumor microenvironment.

Dimers of the nonclassical HLA-G class I molecule have recently been shown to be active structures that mediate inhibition of NK-cell cytotoxic activity through interaction with the immunoglobulin-like transcript (ILT)-2 inhibitory receptor. However, this has only been proven in trophoblasts and HLA-G transfectants. Here, we document for the first time the existence of HLA-G dimers in cancer. Indeed, we identified both surface and soluble HLA-G dimers in tumor cells and malignant ascites respectively. Interestingly, factors from the tumor microenvironment, such as interferons, enhanced the formation of HLA-G dimers and increased the protection of tumors from NK cell-mediated lysis. These data emphasize the impact of HLA-G conformation on its efficiency at inhibiting the antitumor response and thus favoring tumor progression. In view of these results, the effect of the tumor microenvironment on upregulation of HLA-G function deserves particular attention when designing cancer immunotherapy protocols.

Ex vivo and in vivo inhibition of human rhinovirus replication by a new pseudosubstrate of viral 2A protease.

Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2A(pro)) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

Role for proteases and HLA-G in the pathogenicity of influenza A viruses.

Influenza is one of the most common infectious diseases in humans occurring as seasonal epidemic and sporadic pandemic outbreaks. The ongoing infections of humans with avian H5N1 influenza A viruses (IAV) and the past 2009 pandemic caused by the quadruple human/avian/swine reassortant (H1N1) virus highlights the permanent threat caused by these viruses. This review aims to describe the interaction between the virus and the host, with a particular focus on the role of proteases and HLA-G in the pathogenicity of influenza viruses.

Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles.

Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness.

Plasminogen promotes influenza A virus replication through an annexin 2-dependent pathway in the absence of neuraminidase.

Proteolytic cleavage of haemagglutinin (HA) is essential for the infectivity of influenza A viruses (IAVs). This is usually mediated by trypsin-like proteases present in the respiratory tract. However, the ability to use plasminogen (PLG) as an alternative protease may contribute to pathogenesis of IAV infections and virus replication outside the respiratory tract. It was demonstrated previously that neuraminidase (NA) of the IAV strain A/WSN/33 can sequester PLG, allowing this virus to replicate in a PLG-dependent fashion. However, PLG also promotes replication of other IAVs, although its mode of action is poorly understood. Here, using NA-deficient viruses, we demonstrate that NA is not required for the binding of PLG and subsequent cleavage of HA. However, we demonstrate that the cellular protein annexin 2 (A2) can bind PLG and contributes to PLG-dependent cleavage of HA and subsequent IAV replication. Collectively, these results indicate that PLG promotes IAV replication in an A2-dependent fashion in the absence of NA.

Antimicrobial activity of mucosal-associated invariant T cells.

Mucosal-associated invariant T lymphocytes (MAIT lymphocytes) are characterized by two evolutionarily conserved features: an invariant T cell antigen receptor (TCR) alpha-chain and restriction by the major histocompatibility complex (MHC)-related protein MR1. Here we show that MAIT cells were activated by cells infected with various strains of bacteria and yeast, but not cells infected with virus, in both humans and mice. This activation required cognate interaction between the invariant TCR and MR1, which can present a bacteria-derived ligand. In humans, we observed considerably fewer MAIT cells in blood from patients with bacterial infections such as tuberculosis. In the mouse, MAIT cells protected against infection by Mycobacterium abscessus or Escherichia coli. Thus, MAIT cells are evolutionarily conserved innate-like lymphocytes that sense and help fight off microbial infection.