International Journal of Medical Microbiology 304 (2014) 894–901

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Mini Review

Influenza, a One Health paradigm—Novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential Stephan Ludwig a,∗ , Roland Zell b , Martin Schwemmle c , Susanne Herold d a Institute of Molecular Virology (IMV), Centre for Molecular Biology of Inflammation (ZMBE), University of Muenster, Von-Esmarch-Str. 56, D-48149 Muenster, Germany b Department of Virology and Antiviral Therapy, Jena University Hospital, Friedrich Schiller University Jena, Hans Knoell Str. 2, D-07745 Jena, Germany c Institute for Virology, University Medical Center Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany d Universities Giessen & Marburg Lung Center (UGMLC), Department of Internal Medicine II, Section of Infectious Diseases, Klinikstr. 33, D-35392 Giessen, Germany

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Keywords: Influenza virus Antiviral therapy Signal transduction Immune modulation Polymerase complex One Health

a b s t r a c t Influenza virus is a paradigm for a pathogen that frequently crosses the species barrier from animals to humans, causing severe disease in the human population. This ranges from frequent epidemics to occasional pandemic outbreaks with millions of death. All previous pandemics in humans were caused by animal viruses or virus reassortants carrying animal virus genes, underlining that the fight against influenza requires a One Health approach integrating human and veterinary medicine. Furthermore, the fundamental question of what enables a flu pathogen to jump from animals to humans can only be tackled in a transdisciplinary approach between virologists, immunologists and cell biologists. To address this need the German FluResearchNet was established as a first nationwide influenza research network that virtually integrates all national expertise in the field of influenza to unravel viral and host determinants of pathogenicity and species transmission and to explore novel avenues of antiviral intervention. Here we focus on the various novel anti-flu approaches that were developed as part of the FluResearchNet activities. © 2014 Elsevier GmbH. All rights reserved.

Introduction Influenza is a prime example of a zoonotic disease and highlights the relevance of a One Health approach, where experts in animal and human health-care and research combine their efforts to solve interrelated problems. Human disease due to pandemic (H1N1) 2009 influenza and avian to human transmission of influenza A/H5N1or A/H7N9 viruses are only recent examples of new zoonoses with significant global impact. Management, prevention and treatment of influenza requires the expansion and continuing support of collaborations between human and animal health experts at the clinical, diagnostic laboratory, public health, research and training levels. Influenza A/H5N1, first isolated in 1996 from a goose in Guangdong province in China, caused severe poultry losses and occasional human infections in Hong Kong in 1997 (Watanabe et al., 2012). The main human public health response that controlled this outbreak was an aggressive poultry cull. However, from 2003 the

∗ Corresponding author. Tel.: +49 251 835 7791; fax: +49 251 835 7793. E-mail address: [email protected] (S. Ludwig). http://dx.doi.org/10.1016/j.ijmm.2014.08.016 1438-4221/© 2014 Elsevier GmbH. All rights reserved.

virus continued to spread to different other parts of the world. Since then, more than 600 sporadic cases of human infection with influenza A/H5N1 viruses with high lethality have been reported, primarily by 15 countries in Asia, Africa, the Pacific, Europe and the Near East (Watanabe et al., 2012). In January 2014, the first case of a human infection with H5N1 in the Americas was reported from Canada. Fortunately, human-to-human transmission of H5N1 was and is still rare, so far preventing pandemic spread of this potentially devastating pathogen. Human infections with a new avian H7N9 virus were first reported in China in March 2013 (Liu et al., 2014). Most of these infections are believed to result from exposure to infected poultry or contaminated environments, as H7N9 viruses have also been found in poultry in China. Most patients have had severe respiratory illness, with about one-third resulting in death. The first case outside of China occurred in Malaysia and was reported in February 2014. The frightening feature of this virus is, that it does not belong to the group of highly pathogenic avian influenza viruses according to the structure of its surface glycoprotein hemagglutinin. This not only hampers proper surveillance in poultry but also challenges the general concept of avian virus pathogenicity for humans (Liu et al., 2014). Fortunately also here no evidence of sustained

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person-to-person spread of H7N9 has been found, though some evidence points to limited person-to-person spread in rare circumstances. Nevertheless, because avian influenza A viruses have the potential to change and gain the ability to spread easily between people, monitoring for human infection and person-to-person transmission of A/H5N1 and A/H7N9 is extremely important for public health. While most experts were concerned about a pandemic threat due to flu activity in Asia, the first pandemic influenza virus of the 21st century came from a completely different and unexpected region of the world. The first descriptions of pandemic (H1N1) 2009 influenza virus infection occurred in the southwestern United States and Mexico in April 2009 (York and Donis, 2013). This virus was identified to have animal origins, with reassortment of influenza gene segments from North American and Eurasian swine, avian and human viruses (reviewed in Zell et al., 2013). Although seasonal A/H1N1 viruses had been circulating for many years, this novel reassortant A/H1N1 virus was not covered by current seasonal influenza vaccines and could spread very quickly all over the world. Fortunately, this pig borne virus in general caused only relatively mild disease symptoms in humans and did not acquire higher pathogenicity over time. The emergence of the panH1N1 virus from a yet unidentified pig source is not a unique event. There is a plethora of reports indicating mutual transspecies infections of humans and pigs. In Europe, the prevalent lineages of swine influenza viruses presently comprise avian-like H1N1, human-like H1N2 and human-like H3N2 serotypes (Zell et al., 2013). The avian-like H1N1 lineage emerged in 1979 after transmission of an Eurasian avian H1N1 virus to pigs. This virus strain rapidly spread to pigs in many European countries and replaced the circulating classical swine H1N1 strains. In the following years, numerous reassortants of avian-like H1N1 and the human seasonal H3N2 could be detected but only one achieved to establish a persistent lineage. This human-like swine H3N2 lineage has strain A/Port Chalmers/1/73-like hemagglutinin and neuraminidase genes and six internal genes of the avian-like H1N1. In the UK, seasonal H3N2 and H1N1 reassorted with avian-like H1N1 to yield a human-like swine H1N2 virus which spread to many other European countries. In the following years, the avian-like H1N1 as well as the human-like H3N2 and H1N2 developed distinctive genetic lineages and became prevalent in the major swine-producing countries in Europe but also diffused to several Asian countries (Thailand, China, Hong Kong). It is obscure where and when an avian-like swine H1N1 virus recombined with a North American triple reassortant swine influenza virus to yield the novel swine-origin H1N1 (panH1N1) that caused the 2009 pandemic. During the ongoing pandemic, panH1N1 was repeatedly transmitted to swine as shown by numerous successful isolations from pigs. However panH1N1 failed to establish a stable infection chain in the European pig population. This failure was attributed to a pronounced one-sided antigenic cross-reactivity of the wide-spread avian-like H1N1: panH1N1 refused to propagate in pig herds presenting with antibodies against avian-like H1N1 as shown by the FluResearchNet laboratory Dürrwald (Dessau-Rosslau; Dürrwald et al., 2010). Later, the FluResearchNet laboratory Zell (Jena) demonstrated that a 7 + 1 reassortant of panH1N1 with the neuraminidase gene of human-like H1N2 exhibited additional six substitutions in the antigenic sites of the hemagglutinin gene (Lange et al., 2013). This reassortant showed no or only little cross-reaction with avian-like H1N1 post infection sera and hence was able to establish a persistent infection chain. Due to the wide distribution of swine influenza viruses with prevalences (on the herd level) of each subtype of 40–50% in Germany (FluResearchNet laboratory Dürrwald, unpublished data), vaccination is recommended to limit the economical

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losses of pig farmers. However, the virus isolation program and the genetic characterization of the isolates from pigs that were conducted in the recent years by the FluResearchNet laboratories Dürrwald (Dessau-Rosslau) and Zell (Jena) revealed additional novel, persisting virus lineages which complicate both diagnostics and vaccine development (Dürrwald and Zell, unpublished data). Beside human-to-swine transmission, there is also evidence of swine-to-human infections. In contrast to zoonotic infections with the avian subtypes H5N1, H7N2, H7N3, H7N7, and H9N2 which are often associated with severe diseases and high case fatality rates, swine-to-human infections are believed to be mostly mild. Since 1993, several case reports from the Netherlands, Switzerland, Spain, Germany, Hong Kong and China indicate that close occupational contact to pigs increases the risk of zoonotic infections with the European lineages of swine influenza viruses in exposed pig farm workers and their family members. As most zoonotic swine-to-human infections do not result in severe clinical symptoms and thus may not be diagnosed, seroprevalence studies are considered a suitable approach to describe the general risk of infection. Consistently, serological evidence of frequent zoonotic infections in Germany was demonstrated by the FluResearchNet laboratories Zell (Jena) and Dürrwald (Dessau-Rosslau) in two studies (Krumbholz et al., 2010, 2013). The data demonstrate that direct contact to pigs is associated with higher risk of seroreactivity to swine influenza viruses. This risk is more obvious in the younger participants. Comparable results were obtained in serosurveys from Luxembourg and Italy (Gerloff et al., 2009; De Marco et al., 2013). The frequent and unpredictable transmissions of influenza from birds and pigs to humans highlight the urgent need of a joint One Health approach integrating human and veterinary medicine in surveillance, communication, pathogen characterization and intervention strategies. Especially the latter point is of major importance. Considering that vaccination is not an option in the very early phase of a pandemic and that there is only a very limited arsenal of licensed antiviral drugs, we urgently need novel therapeutic intervention approaches. In Germany, three Federal Ministries, the Ministry of Health (BMG), the Ministry of Education and Research (BMBF), and the Ministry of Food, Agriculture and Consumer Protection (BMELV, since 2013 BMEL) have jointly raised a funding scheme for network actions to fight zoonotic diseases in 2006. As part of this action the aforementioned FluResearchNet, (www.fluresearchnet.de) was installed in 2007, which is a first nationwide research network on zoonotic influenza, that integrates virtually all national expertise to meet the One Health challenge. While the network covers several different lines of research activities, a unique strength of the FluResearchNet was and still is the exploration of various novel anti-influenza approaches directed to both viral and host factors, which will be summarized in the following.

State-of the art: currently licensed antiviral drugs There are two classes of clinically approved antiviral agents against influenza: M2 channel inhibitors (rimantadine and amantadine) and neuraminidase (NA) inhibitors (zanamivir and oseltamivir). M2 inhibitors specifically block an ion channel in the viral envelope formed by the viral M2 protein that is derived from a spliced mRNA from RNA segment 7. These inhibitors are however limited in clinical practice due to their toxicity, lack of activity against influenza B and rapid emergence of drug resistance (Pinto and Lamb, 2006, 2007). High frequency of resistance in clinical isolates in the US have led to the conclusion that M2 inhibitors should not be used for the treatment and prophylaxis of influenza until susceptibility to these drugs has been reestablished among

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circulating influenza A isolates (Bright et al., 2006). As most of the recently emerged virus subtypes, including panH1N1, H5N1 and H7N9 exhibit genotypic resistance to M2 inhibitors, promising attempts with participation of FluResearchNet laboratory Zell (Jena) were made to develop adamantane derivatives that efficiently block M2 proton channels with S31N substitutions (Kolocouris et al., 2014). Activity against amantadine-resistent M2 proton channels were also obtained with isoxazole- and 1,2,4-oxadiazole-containing amantadine derivatives and pyrrolidine derivatives (Rey-Carrizo et al., 2014; Wang et al., 2013). Regarding the neuraminidase as a target, several highly selective competitive inhibitors have been developed that bind tightly to the active site of the enzyme (reviewed in Crusat and de Jong, 2007; Oxford et al., 2002). Zanamivir, a dehydrated sialic acid derivative, and oseltamivir, the oral prodrug of the active oseltamivir carboxylate, both entered clinical practice in 1999. During clinical trials of oseltamivir in seasonal influenza only a low percentage of resistance has been reported (Aoki et al., 2007). However, more worrying rates of resistance have been first detected in a smaller study in Japanese children where 18% of all isolates were resistant (Kiso et al., 2004). Since than, the number of reports on viral resistance to oseltamivir has rapidly increased, including findings on the emergence of influenza B viruses as well as A/H5N1, A/H7N9 and pandemic A/H1N1 type viruses that are insensitive against the compound. During the 2007–2008 influenza season oseltamivir-resistant variants of saisonal influenza H1N1 emerged, leading to a global subtype wide resistance in the following years (Lackenby et al., 2008; Thorlund et al., 2011). Besides the problem of resistance, recent studies also questioned the clinical benefit of oseltamivir based on a critical evaluation of clinical trial data (Jefferson et al., 2009, 2014). Thus we are in urgent need for novel anti-influenza drugs. The search for those drugs should also include new concepts to overcome the problem of resistance.

The viral polymerase complex as a target for antiviral intervention The viral replication machinery is an attractive target for the development of virus-specific antivirals. Replication and transcription of influenza viruses occurs in the nucleus by the heterotrimeric viral polymerase complex consisting of the PB1, PB2, and PA subunits. This includes the synthesis of the genomic RNA (vRNA) from an intermediate copy RNA (cRNA). Both vRNAs and cRNAs are encapsidated by the viral nucleoprotein (NP) and form together with trimeric polymerase the ribonucleoprotein complexes designated vRNPs and cRNPs. PB1 possesses the RNA polymerization activity, PB2 is known to bind capped mRNA and the N-terminal part of PA harbors an RNase activity, required to cleave the primer off the remaining host mRNA. Antivirally active compounds were either identified by screening approaches, or designed based on available structures of specific components of the viral replication machinery. Screening of large compound libraries (for overview Beyleveld et al., 2013) identified several small molecules targeting polymerase complex components. For example Nucleozin is known to bind to a defined pocket in NP and seems to tether NP to an inactive high order structure (Gerritz et al., 2011; Kao et al., 2010), thereby preventing viral replication and intracellular transport of vRNPs (Amorim et al., 2013). Although very efficient, Nucleozin fails to block all influenza A virus strains, including currently circulating H1N1 viruses due to a mutation in NP (Kao et al., 2010). Favipiravir (T-705), a nucleoside type of inhibitor and probably the most advanced novel anti-influenza compound to date (De Clercq, 2007; Furuta et al., 2013), efficiently blocks RNA

synthesizing activity of PB1 and is currently in phase III trials. In addition, this compound also exhibits a broad antiviral activity against other RNA viruses, including alpha-, arena, bunya and flaviviruses (Furuta et al., 2013). The availability of detailed structures of NP (Chenavas et al., 2013) the Cap-binding domain of PB2 (Guilligay et al., 2008) and the endonuclease domain of PA (Dias et al., 2009; Yuan et al., 2009) allowed the targeted development of specific small molecules with antiviral activity (Kowalinski et al., 2012). Besides targeting the polymerase subunits, protein-protein interaction surfaces also represent attractive antiviral targets. This includes the NP-NP oligomerization, which is required for efficient transcription and replication (Turrell et al., 2013). Guided by the available structure of NP dimers, small molecules were successfully designed to target the oligomerization domain of NP (Shen et al., 2011). Similarly, the formation of the heterotrimeric polymerase complex is also essential for polymerase function. While PB1 can bind PB2 and PA, the latter two proteins do not efficiently interact with each other. PB1 binds to PA via a 310-helix at its N-terminus comprised of amino acids (aa) 5–15 (He et al., 2008; Obayashi et al., 2008) whereas the C-terminus of PB1 binds to the N-terminus of PB2 (Sugiyama et al., 2009). The FluResearchNet laboratory Schwemmle (Freiburg) has shown that a peptide derived from the first 25 amino acids of PB1 of an influenza A virus strain (PB11-25A) inhibits both the polymerase activity and viral spread of influenza A viruses (Ghanem et al., 2007), providing proof of principle that targeting the polymerase subunit protein-protein interaction sites is achievable. The 310-helix is highly conserved amongst all influenza A virus strains, but differs significantly from influenza B viruses. Indeed, the PB1125A peptide fails to bind to PA of influenza B viruses (Wunderlich et al., 2010), reflecting virus-type specific differences of the PAbinding domain. However, a peptide-based screening of PB11-25A variants harboring individual amino acid substitution resulted in the identification of a chimeric peptide that binds efficiently PA of both influenza A and B viruses and exhibits antiviral activity against both virus types (Wunderlich et al., 2009, 2011). Finally, in silico screening using the available high resolution structures of the protein-protein interface between the PB1 and PA subunits revealed antiviral small molecule compounds that specifically targeted both influenza A and B viruses by disrupting the formation of PB1/PA complexes (Muratore et al., 2012). Dimer formation between PB1 and PB2 can be prevented by PB2-derived peptides harboring the PB1-binding site (Reuther et al., 2011), however, it remains to be shown whether this interaction is a suitable target for the development of antivirally active small molecules.

Cellular targets: inhibitors of virus-supporting signaling processes Infection with influenza viruses results in the activation of a variety of intracellular signaling responses (Ludwig et al., 2006). Exploiting cellular signaling factors to support viral replication is very effective for the virus but also creates dependencies that may be used to develop novel antiviral strategies that disrupt signal transmission (Ludwig et al., 2003). The approach has four major advantages. First, the emergence of resistance may be greatly diminished since the virus cannot easily replace the missing cellular function. Second, the virus-supportive signaling pathways are only active in infected cells, thus, blockade occurs just in time when the pathway is active and needed for replication. Third, the risk of side effects may be reduced since the pathways are not active in uninfected resting epithelial cells in the neighboring lung tissue and consequently will not be affected by the blocking compound. Finally, several drugs against these signaling factors are already in clinical evaluation against other diseases, which may

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Fig. 1. Virus induced intracellular signaling pathways that drive vRNP export. Infection with influenza virus results in the activation of signaling pathways, such as the Raf/MEK/ERK kinase cascade and the NF-␬B module. Both pathways have been shown to drive nuclear export of vRNPs by different modes of action (see main text). Inhibition of the pathways with specific inhibitors, such as the MEK blockers U0126, PD-0325901, or AZD-8330 or the NF-␬B inhibiting agents ASA, LASAG or SC75741 leads to retention of vRNPs in the nucleus and to impaired production of novel virions. This occurs in the absence of major side effects or the tendency to induce viral resistance. Following this concept it could be shown that infected mice can be protected from lethal infection with these inhibiting agents.

speed up further development as an anti-flu drug. Interestingly, several of these drugs showed very little or well manageable side effects, even in the application against chronic diseases. Thus, although targeting the host, it is very likely that these drugs can be applied safely for a short term treatment of acute flu. Some of the most advanced approaches developed by members of the FluResearchNet are highlighted here: blockade of MAPK pathways (Ludwig, 2007) and inhibition of the IKK/NF-␬B module (Ludwig and Planz, 2008). Inhibition of virus-induced MAPK pathways The Raf/MEK/ERK kinase pathway belongs to the family of the so-called mitogen activated protein kinase (MAPK) cascades (reviewed in Widmann et al., 1999). The kinase pathway is activated upon infection with all influenza A and B viruses tested so far (Droebner et al., 2011; Ludwig et al., 2004; Planz, 2013; Pleschka et al., 2001). Strikingly, specific blockade of the pathway strongly impaired growth of avian and human influenza A as well as human B-type viruses in vitro (Ludwig et al., 2004; Pleschka et al., 2001) and in vivo (Droebner et al., 2011; Olschlager et al., 2004) indicating that activation of the Raf/MEK/ERK kinase cascade is required for efficient virus growth. The pathway controls the active nuclear export of vRNPs, most likely due to interference with the activity of the viral nuclear export protein NEP (Pleschka et al., 2001; Fig. 1). The timely activation of the pathway was shown to be achieved by membrane accumulation of the viral HA protein and its tight association with lipid-raft domains (Marjuki et al., 2006). This event triggered protein kinase C ␣ (PKC␣)-dependent activation of the Raf/MEK/ERK cascade late in the infection cycle and thereby induces vRNP export (Marjuki et al., 2006). The requirement of Raf/MEK/ERK activation for efficient influenza virus replication suggests that this pathway may be a

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promising cellular target for anti-influenza approaches. This has been jointly studied by FluResearchNet laboratories Ludwig (Münster), Planz (Tübingen) and Pleschka (Giessen). In their studies it could be shown that inhibitors of the cascade, including those that are in phase I to phase III clinical trials against cancer, exhibited a strong antiviral activity against different influenza viruses in infected mice (Droebner et al., 2011). Besides their direct antiviral effect, MEK inhibitors also reduced virus induced cytokine levels, which could prevent pathology due to an overshooting cytokine response (Pinto et al., 2011). The compounds showed surprisingly little toxicity and side effects in cell culture (Ludwig et al., 2004; Planz et al., 2001; Pleschka et al., 2001), in mouse models (Droebner et al., 2011; Sebolt-Leopold et al., 1999) and in clinical trials for the use as anti-cancer agents (reviewed in Cohen, 2002; Planz, 2013). Furthermore, MEK inhibitors showed no tendency to induce formation of resistant virus variants (Ludwig et al., 2004). Finally, it was demonstrated that cell-directed MEK inhibitors act synergistically with virus-directed oseltamivir to impair virus replication (Haasbach et al., 2013a) and to suppress the tendency of oseltamivir to cause viral resistance (Dierkes and Ludwig, unpublished observation), highlighting the opportunity of combination therapy. While studies on the role of the Raf/MEK/ERK pathway in influenza virus infection are most advanced, there are also indications of a functional involvement of other MAPKs, such as p38 MAPK and Jun-N-terminal kinase (JNK). Inhibitors of p38 MAPK did not significantly affect virus titers in cell culture or mouse lungs, but protected mice from lethal H5N1 infection most likely by suppression of the detrimental cytokine burst (Borgeling et al., 2014). Although JNK was previously regarded as an antiviral pathway that is inhibited by the viral non-structural protein 1 (NS1; Ludwig et al., 2002), a recent study showed that inhibitors of JNK block virus replication in cells and animals in a NS1 dependent fashion (Nacken et al., 2012). Thus, JNK may play a bivalent role in virus propagation and may be affected by NS1 in both directions (Nacken et al., 2014), similar as described for NS1 in phosphatidyl-inositol 3 kinase activation (reviewed in Ehrhardt and Ludwig, 2009).

The IKK/NF-␬B module as a target for antiviral intervention Another important influenza virus induced signaling process is the activation of the transcription factor NF-␬B via the upstream activator kinase I␬B kinase (IKK) 2. Although in general regarded as an anti-pathogen pathway (Chu et al., 1999), two independent studies from 2004 showed that replication of influenza viruses is much more efficient in cells with pre-activated NF-␬B (Nimmerjahn et al., 2004; Wurzer et al., 2004). Conversely, progeny virus titers were reduced when grown in host cells in which NF-␬B signaling was impaired by specific inhibitors, such as BAY11-7085 or BAY11-7082, or by the use of dominant-negative mutants of IKK2 or the inhibitor of ␬B, I␬B␣ (Nimmerjahn et al., 2004; Wurzer et al., 2004). From these studies it could be concluded that influenza viruses have acquired the capability to turn the antiviral activity of NF-␬B into a virus-supportive action. Mechanistically, NF-␬B was shown to regulate virus-induced expression of proapoptotic factors, such as TNF-related apoptosis inducing ligand (TRAIL) or FasL (Wurzer et al., 2004) that lead to the auto- and paracrine activation of caspases. These caspases specifically cleave cellular proteins including those of the nuclear pores, resulting in an enhanced diffusion limit for protein transport in and out of the nucleus (Faleiro and Lazebnik, 2000; Kramer et al., 2008). Influenza viruses exploit this activity for nuclear export of vRNPs (Wurzer et al., 2003). This mechanism appears to be crucial for viral replication since in the presence of both, caspase- and NF-␬B-inhibitors efficient nuclear retention of vRNPs could be observed (Ehrhardt et al., 2013; Mazur et al., 2007; Wurzer et al., 2003; Fig. 1). This event most likely

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prevented formation of progeny virus particles. In the meantime still other virus-supportive functions of NF-␬B were described including regulation of the suppressor of cytokine signaling-3 (SOCS-3) gene (Pauli et al., 2008) or differential regulation of viral RNA synthesis (Kumar et al., 2008). NF-␬B may not only influence pathogenesis of influenza virus by direct effects. Excessive inflammation due to overabundant production of proinflammatory cytokines and chemokines (also known as cytokine burst) is considered an important factor in disease pathogenesis. The majority of cytokines/chemokines is regulated by NF-␬B (Pahl, 1999). Consequently, activation of NF-␬B in airway epithelia, endothelial cells or infiltrating monocytes may strongly influence the outcome of the cytokine burst after influenza A virus infection. This is supported by findings from genome wide gene expression array assays that demonstrated a major role of NF␬B for cytokine responses induced by highly pathogenic influenza viruses such as A/H5N1 (Schmolke et al., 2009; Viemann et al., 2011). Accordingly, it was shown that NF-␬B inhibition not only blocked viral replication in mice but also strongly suppressed cytokine synthesis in lungs (Pinto et al., 2011). The unexpected dependence of influenza viruses on NF-␬B activity led to the hypothesis that the signaling pathway could be a suitable target for antiviral intervention. The first proof-of-concept study was performed with acetylsalicylic acid (ASA), also known as aspirin, that has been previously shown to be an efficient and quite selective inhibitor of the NF-␬B activating kinase IKK2 in low millimolar concentration ranges (Yin et al., 1998). Via NF-␬B inhibition, ASA efficiently blocked replication of influenza viruses of all strains tested so far without toxicity or the tendency to induce resistance (Mazur et al., 2007). Application of the compound as an aerosol directly into the trachea of lethally infected mice reduced virus titers in the lung and significantly promoted survival (Mazur et al., 2007). On the basis of these data, an ASA-derived NF-␬B inhibiting compound is now used for the very first phase II clinical trial of a host-directed drug against severe influenza (www.clinicaltrialsregister.eu/ctr-search/trial/2012-00407219/DE). More recently, also other NF-␬B inhibitors, such as the compound SC75741 have been assessed for an anti-flu activity. FluResearchNet laboratories Ludwig (Münster) and Planz (Tübingen) jointly demonstrated that the compound exhibited strong anti-flu activity mediated by the caspase-dependent mechanism described above. This occurred in the absence of toxicity or induction of viral resistance in cell culture (Ehrhardt et al., 2013). Interestingly, in infected mice the drug still could protect infected animals when administered as late as four days post lethal infection with an H5N1 virus (Haasbach et al., 2013b).

Immunomodulatory approaches Type I interferons for therapeutic and prophylactic intervention The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. IFN-␣ is in clinical use against other RNA viruses such as hepatitis C virus. This prompted FluResearchNet laboratories Haller and Staeheli (Freiburg) to assess the protective potential of exogenous IFN-␣ against seasonal and highly pathogenic influenza viruses in vivo (Kugel et al., 2009). They used the ferret model as the most representative model for human influenza disease. The study showed that intranasal application of IFN-␣ can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. While therapeutic IFN-␣ treatment may thus have limited potential because lack of broad activity

and potential side effects (Högner et al., 2013), a more intensive evaluation of the cytokine as an emergency or even prophylactic drug against pandemic influenza A may still be warranted. This is supported by a study of FluResearchNet laboratory Planz (Tübingen) who explored the effects of pre-treatment with low doses of IFN-␣ and showed that a single dose of the cytokine was sufficient to reduce mouse lung titers. The antiviral effect increased after multiple pretreatments and protected mice against lethal H5N1 and H1N1 infections. The use of non-toxic low-doses of IFN-␣ might even allow a prophylactic use, an idea that is now further driven forward by other laboratories in clinical trials (Bennett et al., 2013). The role of protease activated receptors PAR1 and PAR2 in influenza virus infection Protease-activated receptors (PAR) are a subfamily of related G protein-coupled receptors that are activated by cleavage of part of their extracellular domain by the action of serine proteases such as thrombin (acts on PARs 1, 3 and 4) and trypsin (PAR 2). PARs are highly expressed in the respiratory tract and can influence inflammation at mucosal surfaces. The role of these receptors in influenza virus infection was recently addressed by two independent studies that explored the role of PAR2 in epithelial cells and monocytes as well as in mice (Feld et al., 2008; Khoufache et al., 2009). In both studies it could be demonstrated that activation of PAR2 inhibited influenza virus replication through the production of IFN-␥. In vivo, stimulation of PAR2 using specific agonists protected mice from Influenza A virus-induced acute lung injury and death which was associated with increased IFN-␥ production (Khoufache et al., 2009). Accordingly, the protective effect of the PAR2 agonist was totally abrogated in IFN-␥-deficient mice. In turn, mice deficient for PAR2 were more susceptible to Influenza A virus infection and displayed more severe lung inflammation (Khoufache et al., 2009). Collectively, these results showed that PAR2 plays a protective role during IAV infection through IFN-␥ production and decreased excessive recruitment of inflammatory cells to lung alveoli. Thus PAR2 agonists may serve as drugs that confer antiinfluenza activity in an immune-enhancing manner. This approach may also be applicable to other infectious agents that are sensitive to IFN-␥ induced immune responses, such as Staphylococcus aureus bacteria (Shpacovitch et al., 2011) that do not only represent a major health burden alone, but also cause severe problems in influenza-S. aureus co-infections. In a corresponding study involving FluResearchNet laboratories Ludwig and Planz the role of PAR1 in pathogenesis of influenza virus infections was explored (Khoufache et al., 2013). Here it could surprisingly be demonstrated that PAR1, in contrast to PAR2, rather contributes to the deleterious inflammatory response after influenza virus infection in mice (Khoufache et al., 2013) a finding that could at least in part confirmed by another independent report (Antoniak et al., 2013). Administration of a PAR1 agonist decreased survival and increased severe lung inflammation after influenza infection. In turn, both, administration of a PAR1 antagonist or PAR1 deficiency protected mice from lethal infection with influenza virus. PAR1 antagonism might thus be explored as a treatment for severe influenza, an approach which may be facilitated by the fact that such compounds are already in human trials for other indications. GM-CSF as treatment for influenza virus-induced ARDS Influenza viruses may cause pneumonia in humans with progression to ARDS (acute respiratory distress syndrome). Dendritic cells (DC) are central orchestrators in the anti-viral immune response, which is crucial to restore alveolar barrier function. By use of mouse bone marrow chimeric and cell-specific

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depletion approaches, FluResearchNet laboratory Herold (Giessen) demonstrated that alveolar epithelial cell-expressed GM-CSF (granulocyte-colony stimulating factor) promotes recovery from IV-induced lung injury, mainly by affecting lung DC functions (Unkel et al., 2012). Epithelial GM-CSF induced lung recruitment of different DC subsets and was required for the presence of CD103+ migratory DC in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLN) during IV infection. GM-CSF-dependently expanded and activated CD103+ DC were indispensable for sufficient clearance of IV by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, intratracheally applied GM-CSF activated CD103+ DC for increased migration to MLN and enhanced viral clearance, resulting in attenuated murine lung injury. Recently, several publications highlighted GM-CSF to be protective in different pre-clinical models of pneumonia-associated lung injury. Importantly, GM-CSF protects the host in both the early phase of acute lung infection, by acting on both macrophages and DC, and during the stage of repair of the injured lung epithelium (Cakarova et al., 2009; Huang et al., 2011; Standiford et al., 2012; Steinwede et al., 2011; Sturrock et al., 2012; Unkel et al., 2012). Of note, mice overexpressing GM-CSF in type II alveolar epithelial cells to high local levels are widely protected from infection and injury (Cakarova et al., 2009; Huang et al., 2011; Steinwede et al., 2011; Unkel et al., 2012), suggesting that high concentrations of GM-CSF in the alveoli or the alveolar lining fluid should be achieved for putative Acute Lung Injury (ALI)/ARDS treatment. GM-CSF levels in BAL fluid were correlated with improved outcome of ARDS patients (Matute-Bello et al., 2000), and early clinical studies suggested that it might improve respiratory function in septic patients (Matute-Bello et al., 2000; Presneill et al., 2002). In a translational approach by Herold et al. GM-CSF was applied per inhalation to 6 patients with pneumonia-associated ARDS (including influenza patients) as compassionate treatment. They observed a significant improvement in oxygenation and morbidity scores in patients who had received GM-CSF twice within 48 h compared to non-treated patients, during a 10 day period after the first GM-CSF application. Alveolar macrophages of non GM-CSF-treated patients displayed a progressive shift toward M2 polarization, however, GM-CSF inhalation promoted an M1 phenotype with increased CD80 and persistently low CD206 expression, suggesting that improved macrophage host defense capacity may underlie the beneficial effect of inhaled GM-CSF (Herold et al., 2014). Although the data are derived from a small number of patients and are not obtained under controlled trial conditions, these findings provide first evidence that GM-CSF inhalative treatment may represent an effective strategy to drive lung host defense and improve oxygenation and outcome in pneumonia-associated ARDS, including influenza-associated disease. A controlled clinical trial is currently in preparation.

Conclusions Continuing emergence of novel animal virus subtypes that infect humans and may have pandemic potential and increasing levels of resistance to licensed drugs highlights the requirement for a One Health approach and the need for novel strategies for anti-flu interventions that are broadly active and are not prone to induce resistance. One must clearly state that vaccination is not an option in a very early phase of a pandemic, since it still needs 4–6 month to produce sufficient amounts of a new vaccine. Thus we are left with novel therapeutic approaches and the exploration of new strategies has rapidly increased in the last couple of years. Although some of the novel concepts still target the virus particle itself (most importantly the polymerase complex), there is an increasing interest in

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cellular and immune modulatory targets. Especially approaches that help to repair the lung damage after infection attract more and more interest. Further development of these host directed drugs may be facilitated by the fact that some compounds with respective activities are already under clinical evaluation for other diseases and have been proven to exhibit a very favorable side effect profile. Finally, there is a revival of the idea of combinatory strategies combining drugs that collectively target the virus and the host cell. Following the One-Health idea, it will now also be crucial to demonstrate activity of the novel drug candidates in flu-sensitive animals such as pigs or horses. This would not only open new therapeutic options but also may help to prevent transmission of viruses to humans.

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