FIELD OF THE INVENTION

The present invention relates to the modulation of influenza virus replication. Inventors in particular herein provide FPR2 modulators as well as compositions, kits and uses thereof. Also herein described are methods for preventing and/or treating influenza infections and methods for producing a vaccine composition.

BACKGROUND OF THE INVENTION

Influenza (or flu) is a highly contagious respiratory infection caused by the influenza virus and is responsible for seasonal epidemics and sporadic pandemic outbreaks, leading to significant mortalities in humans ^l ' ¹ . According to the WHO, epidemics result to 3 to 5 million severe cases of influenza each year and 250 000 to 500 000 deaths from the virus. In addition to the epidemic outbreaks, a virus of animal origin (usually avian) can also be transmitted to humans and cause a pandemic, which can range from mild (250,000 deaths) to severe impacts in the population (40 million deaths for the Spanish 1918 pandemic). Thus, influenza is of great concern for human health. Recurrent transmission of highly pathogenic H5N1 avian influenza directly to humans, underscore the scope and severity of the consequences associated to such infections. The most severe complication is acute pneumonia, which develops rapidly and may result in respiratory failure and death. The influenza virus, which belongs to the orthomyxoviridae family, is enveloped and contains 8 single-stranded RNA segments coding for several proteins. While there are three types of influenza viruses (A, B and C), only types A and B cause significant illnesses in humans, type A viruses being the most problematic because of its continuous antigenic evolution. Type A influenza viruses are responsible for most of the seasonal epidemics severity and are further subtyped according to surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). The influenza virus evades the host immune system by undergoing a continuous antigenic evolution through the processes called antigenic "drift". Antigenic drift is the evolution of viral strains through frequent mutations among antibody binding sites of surface antigens leading to the emergence of new variants not adequately recognized by the host immune system. Antigenic drift is the reason why, every season, it is necessary to identify and predict the most probable strains that will circulate in order to produce the most appropriate vaccines for annual vaccinations. Antigenic shift results from the reassortment of the genetic material of co-circulating strains, leading to the replacement of surface glycoproteins HA, and less frequently NA, which in turn leads to the emergence of pandemics.

Two classes of anti-influenza drugs are currently available: inhibitors of the viral M2 channel (e.g., amantadine and rimantadine) and inhibitors of the viral neuraminidase (e.g., zanamivir and oseltamivir). Inhibitors of the viral M2 channel interact directly with the viral M2 ionic channel which participates in the acidification and the decapsulation of the virus in cellular endosomes, and the viral neuraminidase allow the detachment of nascent virions. Targeting viral proteins has proven to be an effective strategy. However, because of the mutational characteristics of the influenza virus and the widespread use of antiviral drugs, resistance has become an important problem. As a result, inhibitors of the M2 ion channel are no longer recommended for the prophylactic treatment of influenza. In addition, neuraminidases inhibitors have no effect if administered 48 hours following infection (too late when patients are hospitalized). Furthermore, according to the Center for Disease Control, almost all currently circulating strains of H3N2 influenza A are resistant to amantadine and almost all circulating H1N1 influenza A seasonal strains are resistant to oseltamivir (Tamiflu™). Fortunately, most H1N1 influenza A strains stemming from the 2009 pandemic are susceptible to the drug, although several resistant strains of this virus have been isolated.

Available treatments induce the emergence of viral resistance, are strain dependent and are likely not to act against new pandemic viruses. Thus, there is a need for new and improved anti-influenza virus medicinal agents. Targeting host cellular mechanisms that are crucial for influenza viral entry, protein synthesis, maturation, and replication, provides an exciting alternative strategy potentially obviating the resistance problem. The present invention meets the existing need and provides such an alternative strategy.

SUMMARY OF THE INVENTION:

The present invention is based on the discovery by inventors that Formyl Peptide Receptor 2 (FPR2), the role of which is well known in the context of bacterial infections, surprisingly plays also an important role in modulating influenza virus replication and lung inflammation.

The invention in particular relates to the use of a modulator of FPR2 (herein defined as an activator or as an inhibitor of FPR2) for modulating influenza virus replication.

A particular embodiment relates to an inhibitor of FPR2 for use as an anti-influenza virus agent, for use for preventing or treating influenza virus infection in a subject, and/or for use for preventing, decreasing or suppressing influenza virus replication and/or lung inflammation in a subject infected by an influenza virus.

Another embodiment relates to the use of an inhibitor of FPR2 for preparing a composition for preventing or treating influenza virus infection in a subject in need thereof.

Also herein described is a composition, typically a pharmaceutical composition, for use for preventing or treating influenza virus infection in a subject in need thereof comprising an inhibitor of FPR2 present in a therapeutically effective amount and a pharmaceutically acceptable support.

Further herein described is a kit, in particular an anti-influenza virus prophylactic or therapeutic kit, which comprises the following components:

- an effective amount of an inhibitor of FPR2 or of composition comprising said inhibitor, and

- a means or device for administering said inhibitor of FPR2 to a subject in need thereof, and optionally,

- instructions for using the kit for preventing or treating influenza virus infection in a subject. A method for treating a subject infected by an influenza virus or for preventing an influenza virus infection in a subject at risk of being infected by such a virus is also provided. The method comprises the administration to the subject of an inhibitor of FPR2.

Another particular embodiment relates to an activator of FPR2 for use for inducing or stimulating influenza virus replication.

Also herein described is a method for producing a vaccine composition comprising an inactivated or attenuated influenza virus or a portion thereof from cells infected with the wild- type influenza virus, said method comprising exposing the infected cells to an activator of FPR2.

In another aspect, the present invention relates to the use of an inhibitor of FPR2 or composition of the present invention comprising such an inhibitor for research assays.

DETAILED DESCRIPTION OF THE INVENTION Influenza A virus (IAV) pathogenesis is a multifactorial process, involving increased viral replication competence and dysregulation of the immune system. Lipid mediators lipoxins, protectins and resolvins that play a key and active role in the resolution of acute inflammation have received a particular interest in infectious disease lately ³ ' ⁴ . Surprisingly, the lipid mediator protectin Dl does not affect inflammatory processes during influenza but inhibits IAV replication and protects mice from severe infection ⁵ . To date, the contribution of inflammatory pro-resolving receptors that mediate the lipid signaling cascade to the pathogenesis of IAV infections remains to be determined.

Inventors are the first to describe the role of the Formyl Peptide Receptor 2 (FPR2), also commonly identified as FPRLl or LXA4 receptor or FPR2/LXA4R, in the pathogenesis and in the inflammatory process of influenza viruses' infections.

FPR2 belongs to the seven transmembrane domain of G protein-coupled receptors. FPR2 initiates an active resolving response during acute inflammation by binding resolving fatty acid lipid mediators or cellular proteins such as the most prominent lipoxin A4 (LXA4) or the glucocorticoid-modulated protein Annexin-Al (ANXA1). Although the involvement of FPR2/LXA4R in the resolution of inflammatory responses is now well-recognized both in vitro and in vivo ^6~8 , a distinct function of FPR2 includes the detection of bacterial formyl peptides and induction of pro-inflammatory responses ^9~n .

In the studies herein described, the inventors have shown that, in vitro, FPR2 expressed on A549 cells was activated by IAV which harbor its ligand Annexin-Al in their envelope. Using a pharmacological approach and silencing gene expression experiments, they have found that FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)-dependent pathway. In vivo, activating FPR2 by administering the agonist VKYMVm- NH ₂ 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 the deleterious role of FPR2 also occurs through an ERK-dependent pathway, in vivo. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections.

The term "FPR2" includes naturally occurring FPR2 and variants and modified form thereof. The FPR2 can be from any source and is typically an animal FPR2, preferably a bird FPR2or mammal FPR2, even more preferably a human FPR2. Nucleic acid sequence (SEQ ID NO:l) and amino acid sequence of (SEQ ID NO:2) of human FPR2 in particular are described in the art.

The present invention relates to the use of a modulator of FPR2 (herein defined as an activator or as an inhibitor of FPR2) for modulating influenza virus replication, typically in an eukaryote cell or cell line or in a subject.

As used herein, the term "modulator" refers to any molecule, agent or compound that increases or decreases FPR2 activity, including a molecule that changes FPR2 downstream signaling activities, said modulator being an activator or an inhibitor as defined herein below. As used herein, the term "influenza virus" refers to any influenza virus without consideration of type (type A, B, C or D) or serotype based on hemagglutinin (HI to at least HI 8) and neuraminidase (Nl to at least N9) expression. The virus is selected from influenza type A, B C, and D virus, and is preferably influenza type A virus or influenza type B virus.

In a particular embodiment of the invention, the present invention thus relates to the use of a modulator of FPR2 for modulating influenza virus type A replication.

Exemplary influenza virus type A that are contemplated by the invention include but are not limited to H1N1, H2N2, H3N2, H5N1, H6N2, H7N7, H7N9, H1N2, H9N2, H7N2, H7N3, and H10N7. In a preferred embodiment influenza virus type A according to the present invention is selected from H1N1, H3N2, H5N1, H6N2 and H7N9, preferably from H1N1, H3N2, H5N1, H6N2 and H7N9. In a particular embodiment, influenza virus type A is for example H1N1 [for example A/California/7/2009 (H1N1) pdm09 strain or A/PR/8/34 strain], H3N2 [for example A/Hong Kong/4801/2014(H3N2) strain or A/HK/68 strain] or H6N2 [for example A/Turkey/Massachussets/65].

In another particular embodiment of the invention, the present invention relates to the use of a modulator of FPR2 for modulating influenza virus type B or type C replication, preferably type B, for example B/70 strain.

A first embodiment of the invention relates to an inhibitor of FPR2, as a modulator of FPR2, for use for preventing, decreasing or suppressing influenza virus replication.

Herein described in particular is the use of a FPR2 inhibitor, typically in vitro or ex vivo, for preventing, decreasing or suppressing influenza virus replication.

In a particular embodiment, the invention relates to an inhibitor of FPR2 for use as an anti- influenza or anti-flu virus agent, for use for preventing or treating influenza virus infection and/or any associated symptoms or complications (as herein below defined), in a subject, and/or for use for preventing, decreasing or suppressing influenza virus replication and/or lung inflammation in a subject infected by an influenza virus, preferably for preventing, decreasing or suppressing both influenza virus replication and lung inflammation in a subject infected by an influenza virus. In a preferred aspect, the inhibitor of FPR2 is for use for preventing or treating influenza virus infection and in addition for use for preventing or treating lung inflammation in a subject infected by an influenza virus.

As used herein, the term "influenza virus infection" refers to any infection caused by an influenza virus. In a specific embodiment, the influenza infection is caused by a type A virus. In another specific embodiment, the influenza infection is caused by a type B, type C, or type D virus, more preferably by a type B.

The term "inhibitor" as used herein, refers to an agent that is capable of specifically binding and inhibiting signaling through a receptor to fully block, as does an antagonist, or detectably inhibit a response mediated by the receptor. For example, as used herein the term "FPR2 inhibitor" is a natural or synthetic compound which binds and inactivates fully or partially FPR2 for initiating or participating to a pathway signaling (such as the ERK signaling pathway) and further biological processes. In the context of the invention the FPR2 inhibitor in particular prevents, decreases or suppresses influenza virus replication. The influenza virus replication decrease observed can be by at least about 1%, 2%, 5%, 10%, e.g. by 20%, 30%, 40%), 50%), 60%), 70%o, 80%), or 90%>, as compared to the replication observed in a referenced cell.

Typically, the FPR2 inhibitor inhibits/abolished the binding of/activation by the influenza virus to/of FPR2, typically the binding of ANXA1 to FPR2, and/or binding of any other ligands/agonists to FPR2 such as LPXA4 (or LXA4) or a formylated protein/peptide. The FPR2 inhibitor may also or otherwise influence the downstream signaling activities in a way independent of the binding or not of the influenza virus on FPR2. In fine said inhibitor inhibits or abolishes influenza virus replication.

FPR2 inhibitory/antagonistic activity may be assessed by various known methods. A control FPR2 can be exposed to no antibody or antigen binding molecule, an antibody or antigen binding molecule that specifically binds to another antigen, or an anti-FPR2 antibody or antigen binding molecule known not to function as an inhibitor, for example as an antagonist. In a particular embodiment, a FPR2 inhibitor according to the invention can be a molecule selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a metabolic lipid, a pepducin, a polynucleotide and a compound comprising such a molecule or a combination thereof. A FPR2 inhibitor for use in the context of the present invention can be selected from WRW4 (i.e. WRWWWW, in particular the WRWWWW-NH ₂ peptide); PBP10 (QRLFQVKGRR- rhodamine-B as described in Forsman et al (2012) "Structural characterization and inhibitory profile of formyl peptide receptor 2 selective peptides descending fir om a PIP2-binding domain of gelsolin". J.Immunol. 189-629.); BOC-2 (Boc-Phe-Leu-Phe-Leu-Phe-OH); Isopropylureido-FLFLF; the FPRL1 -inhibitory protein (as described in Prat C, Bestebroer J, de Haas CJ, van Strijp JA, van Kessel KP. (2006) "A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like /". J. Immunol., 177 (11): 8017-26.), Pam-(Lys- NSpe)6-NH ₂ (as described in Skovbakke SL, Heegaard PM, Larsen CJ, Franzyk H, Forsman H, Dahlgren C (2015) "The proteolytically stable peptidomimetic Pam-(Lys-fJNSpe) _< ¾- NH ₂ selectively inhibits human neutrophil activation via formyl peptide receptor 2" Biochem. Pharmacol. Jan 15;93(2): 182-95); and a synthetic molecule such as Quin-C7 (also known as 4- butoxy-N-[2-(4-hydroxyphenyl)-4-oxo-l,2-dihydroquinazolin-3- yl]benzamide) and described in Zhou C, Zhang S, Nanamori M, Zhang Y, Liu Q, Li N, Sun M, Tian J, Ye PP, Cheng N, Ye RD, Wang MW. (2007) "Pharmacological characterization of a novel non peptide antagonist for formyl peptide receptor-like Γ. Mol Pharmacol, 72: 976-983.); BB-V-115 (Young SM, Bologa CM, Fara D, Bryant BK, Strouse JJ, Arterburn JB, Ye RD, Oprea TI, Prossnitz ER, Sklar LA, Edwards BS. (2009) "Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes". Cytometry A 75: 253-263); compound 796276 (Young SM, Bologa CM, Fara D, Bryant BK, Strouse JJ, Arterburn JB, Ye RD, Oprea TI, Prossnitz ER, Sklar LA, Edwards BS. (2009) "Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes" . Cytometry A 75: 253-263); compound 1754-20 [((R)-4-(2-([ 1 , 19-biphenyl] -4-yl)ethyl)-5-(4-hydroxybenzyl)- 1 -((R)- 1 -(4- hydroxyphenyl)-3-((S)-2-(((S)-6-isopropyl-2,3-dioxopiperazin -l-yl)methyl)pyrrolidin-l- yl)propan-2-yl)piperazine-2,3-dione] or compound 1754-31 [(R)-4-(cyclohexylmethyl)-5-(4- hydroxybenzyl)- 1 -((R)- 1 -((S)-2-(((S)-6-isopropyl-2,3-dioxopiperazin- 1 -yl)methyl)pyrrolidin- l-yl)-3-(naphthalene-2-yl)propan-2-yl)piperazine-2,3-dione] both described in Pinilla et al. ("Selective agonists and agonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries." Molecular Pharmacology 84:314-324, 2013). Other FPR2 inhibitors can be found in patent application WO2011/073918. A preferred FPR2 inhibitor is selected from WRW4, PBP10 and BOC-2. WRW4 is a particularly preferred FPR2 inhibitor.

As indicated previously the FPR2 inhibitor can be a peptide or peptide molecule comprising amino acid residues. As used herein the term "amino acid residue" refers to any natural/standard and non-natural/non-standard amino acid residue in (L) or (D) configuration, and includes alpha or alpha-disubstituted amino acids. It refers to isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, serine, tyrosine. It also includes beta-alanine, 3 -amino-propionic acid, 2,3-diamino propionic acid, alpha- aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, cyclopentylalanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, cyclopentylglycine, cyclobutylglycine, cyclopropylglycine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3- benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4- fluorophenylalanine, penicillamine, l,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2- thienylalanine, methionine sulfoxide, L-homoarginine (hArg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, selenocysteine, homocysteine, homoserine (HoSer), cysteic acid, epsilon- amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry.

In a particular embodiment, the peptide used as a FPR2 inhibitor is WRW4.

In another particular embodiment, the peptide used as a FPR2 inhibitor is PBP10 (QRLFQVKGRR-rhodamine-B).

In a further particular embodiment, the peptide used as a FPR2 inhibitor is BOC-2 or Isopropylureido-FLFLF.

Compounds of the present invention which include peptides may comprise replacement of at least one of the peptide bonds with an isosteric modification. Compounds of the present invention which include peptides may be peptidomimetics. A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent, but wherein one or more of the peptide bonds/linkages have been replaced, often by proteolytically more stable linkages. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many or all of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, potential for hydrogen bonding, etc. Typical peptide bond replacements include esters, polyamines and derivatives thereof as well as substituted alkanes and alkenes, such as aminomethyl and ketomethylene. For example, the peptide may have one or more peptide linkages replaced by linkages such as -CH ₂ NH-, - CH ₂ S-, -CH ₂ -CH ₂ -, -CH=CH- (cis or trans), -CH(OH)CH ₂ -, or -COCH ₂ -, -N-NH-, - CH ₂ NHNH-, or peptoid linkages in which the side chain is connected to the nitrogen atom instead of the carbon atom. Such peptidomimetics may have greater chemical stability, enhanced biological/pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.) and/or reduced antigenicity relative its peptide equivalent. The FPR2 inhibitor can also be a small organic molecule. The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Examples of small organic molecule are compound 1754-20, compound 1754-31, Quin-C7, BB-V-115 and compound 796276. In a particular embodiment, the FPR2 antagonist is a small organic molecule which can be selected from compound 1754-20, compound 1754-31, Quin-C7, BB-V-115 and compound 796276. The FPR2 inhibitor can also be an antibody or an antigen-binding molecule. In an embodiment, the antibody specifically recognize/bind FPR2 (e.g. FPR2 of SEQ ID NO:2) or an epitope thereof involved in the activation/stimulation of the ERK-pathway. In another preferred embodiment, the antibody is a monoclonal antibody. In an even more preferred embodiment the antibody is the antibody obtained with the clone FN-1D6-AI described in De Santo et al., Nat. Immunol. ²⁷ (available for sale from Genovac AG, Freiburg, Germany).

The term "antibody" is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies and humanized antibodies, so long as they exhibit the desired biological activity (e.g., as indicated previously, inhibiting the binding of/activation by the influenza virus of FPR2, typically the binding of ANXAl to FPR2, and/or of any other ligands/agonists to FPR2 such as LPXA4 (or LXA4) or formylated proteins/peptides). Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, V H regions (V H, V H-V H), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Antibodies according to the present invention can be of any class, such as IgG, IgA, IgDl IgEl IgMl or IgYl although IgG antibodies are typically preferred. Antibodies can be of any mammalian or avian origin, including human, murine (mouse or rat), donkey, sheep, goat, rabbit, camel, horse, or chicken. The antibodies can be modified by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or other modifications known in the art.

In general, techniques for preparing antibodies (including polyclonal antibodies, monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art, see .g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); and Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, 1985. Additionally, antibodies according to the present invention can be fused to marker sequences, such as a peptide tag to facilitate purification; a suitable tag is a hexahistidine tag. The antibodies can also be conjugated to a diagnostic or therapeutic agent by methods known in the art. Techniques for preparing such conjugates are well known in the art. Other methods of preparing these monoclonal antibodies, as well as chimeric antibodies, humanized antibodies, and single-chain antibodies, are known in the art.

The FPR2 inhibitor can also be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Also within the scope of the invention is a metabolic lipid as a FPR2 inhibitor. Disorders in endogenous lipid metabolism have been associated with infectious disease including influenza ⁵ . Lipid metabolites (such as Lipoxin A4) can act on FPR2 and modulate inflammation. Targeting metabolism of lipids (lipoxygenase 12/154, lipoxygenase 5) or use of specific active lipid metabolites such as lipoxin A4 have the following advantages: it overcomes the traditional challenge of resistance and should have a broad antiviral activity against circulating and future influenza virus strains by targeting the host instead of the virus. An example of metabolic lipid usable in the context of the invention is lipoxin A4.

The FPR2 inhibitor can also be a pepducin. Pepducins are cell-penetrating peptides that act as intracellular modulators of signal transference from receptors to G proteins (cf. Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A (January 2002). "Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides". Proc. Natl. Acad. Sci. U.S.A. 99 (2): 643-8). Pepducins utilize lipidated fragments of intracellular G protein- coupled receptor loops to modulate GPCR action in targeted cell-signaling pathways. A pepducin molecule comprises a short peptide derived from a GPCR intracellular loop tethered to a hydrophobic moiety. This structure allows pepducin lipopeptides to anchor in the cell membrane lipid bilayer and target the GPCR/G protein interface via a unique intracellular allosteric mechanism.

An example of pepducin usable in the context of the invention is for example the FlPali ₆ pepducin described in Winther et al. {"A neutrophil inhibitory pepducin derived from FPR1 expected to target FPR1 signaling hijacks the closely related FPR2 instead." FEBS Letters 589 (2015) 1832-1839).

The FPR2 inhibitor can also be a polynucleotide, typically an inhibitory nucleotide. These include short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, complementary DNA (cDNA) or guide RNA (gRNA usable in the context of a CRISPR/Cas system). In some preferred embodiments, a siRNA targeting FPR2 expression is used. Interference with the function and expression of endogenous genes by double-stranded RNA such as siRNA has been shown in various organisms. See, e.g., A. Fire et al., "Potent and Specific Genetic Interference by Double- Stranded RNA in Caenorhabditis elegans" Nature 391 :806-811 (1998); J. R. Kennerdell & R. W. Carthew, "Use of dsDNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway," CeJ 95:1017-1026 (1998); F. Wianni & M. Zernicka- Goetz, "Specific Interference with Gene Function by Double-Stranded RNA in Early Mouse Development," Nat. Cell Biol. 2:70-75 (2000). siRNAs can include hairpin loops comprising self-complementary sequences or double stranded sequences. siRNAs typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. Such double-stranded RNA can be synthesized by in vitro transcription of single- stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands. Double-stranded RNA targeting FPR2 can also be synthesized from a cDNA vector construct in which a FPR2 gene (e.g., human FPR2 gene) is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal. Double-stranded RNA targeting the FPR2 gene can be introduced into a cell (e.g., a tumor cell) by transfection of an appropriate construct.

Typically, RNA interference mediated by siRNA, miRNA, or shRNA is mediated at the level of translation; in other words, these interfering RNA molecules prevent translation of the corresponding mRNA molecules and lead to their degradation. It is also possible that RNA interference may also operate at the level of transcription, blocking transcription of the regions of the genome corresponding to these interfering RNA molecules.

The structure and function of these interfering RNA molecules are well known in the art and are described, for example, in R. F. Gesteland et al., eds, "The RNA World" (3 ^rd , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2006), pp. 535-565, incorporated herein by this reference. For these approaches, cloning into vectors and transfection methods are also well known in the art and are described, for example, in J. Sambrook & D. R. Russell, "Molecular Cloning: A Laboratory Manual" (3 ^rd , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001), incorporated herein by this reference.

In addition to double stranded RNAs, other nucleic acid agents targeting FPR2 can also be employed in the practice of the present invention, e.g., antisense nucleic acids. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific target mRNA molecule. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the translation of mRNA into protein, and, thus, with the expression of a gene that is transcribed into that mRNA. Antisense methods have been used to inhibit the expression of many genes in vitro. See, e.g., C J. Marcus- Sekura, "Techniques for Using Antisense Oligodeoxy ribonucleotides to Study Gene Expression," Anal. Biochem. 172:289-295 (1988); J. E. Hambor et al., "Use of an Epstein-Ban Virus Episomal Replicon for Anti-Sense RNA-Mediated Gene Inhibition in a Human Cytotoxic T-Cell Clone," Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014 (1988); H Arima et al., "Specific inhibition of lnterleukin-10 Production in Murine Macrophage-Like Cells by Phosphorothioate Antisense Oligonucleotides," Antisense Nucl. Acid Drug Dev. 8:319-327 (1998); and W.-F. Hou et al., "Effect of Antisense Oligodeoxynucleotides Directed to Individual Calmodulin Gene Transcripts on the Proliferation and Differentiation of PC 12 Cells," Antisense Nucl. Acid Drug Dev. 8:295-308 (1998), all incorporated herein by this reference. Antisense technology is described further in C. Lichtenstein & W. Nellen, eds., "Antisense Technology: A Practical Approach" (IRL Press, Oxford, 1997), incorporated herein by this reference. FPR2 polynucleotide sequences from human and many other animals in particular mammals have all been delineated in the art. Based on the known sequences, inhibitory nucleotides (e.g., siRNA, miRNA, or shRNA) targeting FPR2 can be readily synthesized using methods well known in the art.

Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integral number of base pairs between these numbers. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.) and Ambion, Inc. (Austin, Tex).

Another aspect of the invention relates to the use of an inhibitor of FPR2 for preparing a composition, typically a pharmaceutical composition, for preventing or treating influenza virus infection, in particular an influenza infection caused by a type A virus, and/or any associated symptoms or complications in a subject in need thereof.

The terms "therapeutic treatment" and "treating" refer to therapeutic treatment or measures able to reverse, alleviate, cure or inhibit the progress of influenza virus infection or associated symptoms or complications, typically inhibit the influenza virus type A proliferation, preferably avoid the appearance of any complications.

The terms "prevention", "preventive treatment", "prophylactic treatment" and "preventing" refer to prophylactic or preventive measures comprising the administration of a FPR2 antagonist or composition comprising said antagonist that prevents the symptoms of influenza infection, enhances the subject's resistance or retard the progression of an influenza infection. Such treatments are intended for a animal subject, typically a mammal subject as herein identified below, preferably a human subject in need thereof. Are considered as such, any subject that will benefit or that is likely to benefit from the inhibitor of the present invention, typically the subjects suffering from a flu/influenza virus infection, or those who are not already suffering of influenza infection but are considered "at risk of, or as having a predisposition to, developing such an infection or associated complications (in whom this has to be prevented).

Most common symptoms of flu include chills, high fever, runny nose, sore throat, muscle pains, headache, chest congestion, head congestion, coughing, weakness, exhaustion, loss of appetite and general discomfort. Nausea and vomiting can also occur. Complications of influenza (also herein identified as "flu complications") may include viral pneumonia, in particular acute pneumonia, secondary bacterial pneumonia, sinus infection, encephalitis, septicemia, hemostasis disorders and worsening of previous health problems such as asthma, diabete or heart failure. A composition, typically pharmaceutical composition, for use for preventing or treating influenza virus infection in a subject in need thereof is also herein described. It comprises an inhibitor of FPR2 present in a therapeutically effective amount, in particular as the sole therapeutic agent, typically in a pharmaceutically acceptable support or excipient. The inhibitor of FPR2 or composition comprising said inhibitor advantageously inhibits viral replication and propagation. It typically reduces viral load/titre in an infected subject's blood, cells or in a nasopharingeal swab of that subject. In an embodiment, the cells are epithelial cells of the respiratory system (e.g., nasal, of the pharynx, tracheal, bronchial, bronchiolar, alveolar and lung epithelial cell).

The inhibitor of FPR2, composition comprising said inhibitor and corresponding therapeutic treatment are particularly efficient and advantageous when compared to the existing as it is efficient even when administered more than two days following influenza infection, which means that it can be used in particular in subjects suffering of flu complications.

The composition may further comprise at least one distinct additional therapeutic agent. This additional therapeutic agent is typically an agent (also herein identified as anti- influenza virus agent) used in the art for preventing or treating an influenza infection and/or associated symptoms or complications as herein described, such as a viral M2 ion channel inhibitor or a neuraminidase inhibitor. Known therapeutic agents for the prevention and treatment of flu infections includes Tamiflu™ (oseltamivir), Relenza™ (zanamivir), laninamivir, peramivir, amantadine, rimantadine, ribavirin, vitamin C, Cold Fx™, echinacea, ginseng, etc..

The distinct additional therapeutic agent can be selected for example from any known anti- influenza virus agent, vaccine, antibody, immunomodulatory molecule and nucleic acid. In the context of the invention, the terms "immunomodulatory molecule" cover any antiinflammatory molecule or drug as well as any cytokine such as interferon (IFN). In a particular embodiment, the selected distinct additional therapeutic agent(s) may act by very differet biochemical pathways to provide particularly beneficial results. The distinct additional therapeutic agent can also be selected for example from an antitussive, expectorant, antiinflammatory analgesic, and decongestant.

The at least one or more therapeutic agent may be delivered as either co-administered monotherapy formulation or as a single co-formulation. The composition may further comprise an adjuvant such as for example an aluminum salts.

A particular pharmaceutical composition for use for preventing or treating influenza virus infection in a subject in need thereof according to the invention comprises an inhibitor of FPR2 present in a therapeutically effective amount, at least one distinct compound selected from a therapeutic agent, an adjuvant, and a combination thereof, and a pharmaceutically acceptable support or excipient. In a preferred embodiment, a composition for use for preventing or treating influenza virus infection in a subject in need thereof according to the invention comprises an inhibitor of FPR2 and oseltamivir both present in a therapeutically effective amount, more particularly a FPR2 inhibitor selected from WRW4, BOC-2 and PBP10 in combination with oseltamivir.

The FPR2 inhibitor or composition comprising said inhibitor can be combined with a pharmaceutically acceptable support or excipient, and optionally sustained-release matrice, such as biodegradable polymer, to form therapeutic composition. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. The terms "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. The term can refer to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. The pharmaceutical compositions of the present invention may therefore contain non-limiting pharmaceutically acceptable excipients/carriers such as solubilizing/diluting agents, antioxidants, enteric coatings, absorption enhancers, pH adjusting agents and buffers, dispersing agents, coatings, antibacterial, antifungal agents, absorption delaying agents (controlled time-release), osmolarity adjusters, isotonic agents, preservative agents, stabilizers, surfactants, emulsifiers, sweeteners, thickening agents, solvents, emollients, coloring agents, wetting agents, as well as colors and flavors and salts for the variation of osmotic pressure. The carrier/excipient is selected for administration by the selected route of administration.

Another object of the invention relates to a method for preventing or treating a influenza/flu infection in a subject in need thereof with a FPR2 antagonist as herein described. A method for treating a subject infected by an influenza virus or for preventing an influenza virus infection in a subject at risk of being infected by such a virus is thus herein provided. The method comprises a step of administering an inhibitor of FPR2 in a therapeutically effective amount, or a composition as herein described comprising said inhibitor, to the subject. By a "therapeutically effective amount" is meant a sufficient amount the FPR2 antagonist according to the invention to treat or prevent influenza virus infections at a reasonable benefit/risk ratio applicable to any medical treatment. An effective amount can be administered in one or more doses. It will be understood that the total daily usage and frequency of administration of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the deficit being treated and the severity of the deficit; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The daily dosage of the inhibitor of FPR2 may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15,0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the inhibitor of FPR2 is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The effective amount may be given daily in a single or several doses (e.g., twice daily, three times per day or 4 times per day). It may also be given every 2 days, every 3 days or once a week, as prescribed. Preferably, the effective amount is given once daily.

When the composition comprises several therapeutic agents, those may be administered simultaneously, separately or sequentially.

FPR2 inhibitors may be administered in a pharmaceutical composition. Pharmaceutical compositions may be administered in unit dosage form. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals, and particulars about the subject. Any appropriate route of administration may be employed, for example, nasal, transdermal (topical), parenteral, subcutaneous, intramuscular, intramammary, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraarticular, intraspinal, intracisternal, intraperitoneal, or oral administration. Examples of specific routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramammary; oral; transdermal (topical); transmucosal, and rectal administration

Therapeutic formulations may be for example in the form of tablets, capsules, troches, dragees, hard or soft gelatin capsules, solutions (e.g; syrops), aerosols, emulsions or suspensions, for oral administration; in the form of ointments, powders, nasal drops, sprays/aerosols or suppositories for transmucosal (e.g., rectal, oral, buccal, sub-lingual, intranasal) or transdermal/percutaneous administration; in the form of ointments, creams, gels or solutions for topical administration; or in the form of an injectable solution for parenteral administration (e.g., intraperitoneally, intrathecally, intravenously, intramuscularly, intradermally, transdermally, or subcutaneously). Intranasal or oral administration is a preferred form of use. The inhibitor of FPR2 or composition comprising said inhibitor is preferably administered in an oral or intranasal administration form with a nebulizer, inhaler, spray or aerosol. Inhaler and nebulizer allow a direct administration to the lung of the subject to be treated.

Also herein described is a kit, in particular an anti-influenza virus prophylactic or therapeutic kit, which comprises the following components:

- an effective amount of an inhibitor of FPR2 or of composition comprising said inhibitor, and

- a means or device for administering said inhibitor of FPR2 to a subject in need thereof.

The means or device can be selected for example from a nebulizer, inhaler, spray or aerosol. The inhibitor of FPR2 or composition comprising said inhibitor can be packaged in a suitable container. The kit can further comprise instructions for using the kit for preventing or treating influenza virus infection in a subject.

In the context of the present invention, the patient or subject is an animal (wild or domestic), typically a mammal or bird. The mammal can be selected for example from a primate, for example a monkey or a human being, a rodent, in particular a mouse, rat or bat, a cat, a dog, a cow, a pig, a rabbit and a horse. The bird can be selected for example from a duck, goose and pigeon. In a particular embodiment, the mammal is a primate or non-primate mammal. Preferably the subject is a human being, whatever its age or sex.

In a particular embodiment, the selected patient is suffering of flu complications and is typically hospitalized. As explained previously, flu complications include viral pneumonia, in particular acute pneumonia, secondary bacterial pneumonia, sinus infection, encephalitis, septicemia and worsening of previous health problems such as asthma, diabete or heart failure. The patient suffering of a complication may be pregnant women, elderly, young children or immune deficient subjects. The likelihood of contracting an influenza infection can be determined for instance with the prevalence of the disease in the subject's environment including close members of the family (sisters, brothers, parents, grandparents, uncles and aunts, spouse, colleagues, friends, etc.). In a typical embodiment, a subject in need thereof is a subject suffering from the influenza infection or any associated symptoms. Another embodiment of the invention relates to an activator of FPR2, as a modulator of FPR2, for use for stimulating influenza virus replication, in particular in a method of producing an anti- influenza virus vaccine.

The term "activator" as used herein, refers to an agent that is capable of specifically binding and activating FPR2 for initiating a pathway signaling and further biological processes through a receptor to fully activate, as does an agonist, or detectably induce or stimulate a response mediated by the receptor. For example, as used herein the term "FPR2 activator" is a natural or synthetic compound which binds and activates fully or partially FPR2 for initiating or participating to a pathway signaling (such as the ERK signaling pathway) and further biological processes. In the context of the invention the FPR2 activator in particular induces or stimulates influenza virus replication. FPR2 agonistic activity may be assessed by various methods known by the skilled person.

In a particular embodiment, a FPR2 activator according to the invention can be selected from a peptide, typically a formylated peptide, a peptide, a peptide mimetic (or peptidomimetic), a protein, a small organic molecule, a metabolic lipid, and a pepducin as herein defined.

Preferred formylated peptides have a mitochondrial or bacterial origin.

In a particular embodiment, the peptide used as a FPR2 activator is the WKYMVM peptide, in particular the WKYMVm-NH ₂ peptide; fMLP (formyl-Met-Leu-Phe); TIPMFVPESTSKLQKFTSWFM-amide (also known as CGEN-855A, described in Hecht I, Rong J, Sampaio AL, Hermesh C, Rutledge C, Shemesh R, Toporik A, Beiman M, Dassa L, Niv H, Cojocaru G, Zauberman A, Rotman G, Perretti M, Vinten-Johansen J, Cohen Y "A novel peptide agonist of formyl-peptide receptor-like 1 (ALX) displays anti-inflammatory and cardioprotective effects" J Pharmacol Exp Ther. 2009 Feb;328(2):426-34); sCKbeta8-l (as described in Elagoz Al, Henderson D, Babu PS, Salter S, Grahames C, Bowers L, Roy MO, Laplante P, Grazzini E, Ahmad S, Lembo PM, "A truncated form of CKbeta8-l is a potent agonist for human formyl peptide-receptor-like 1 receptof Br J Pharmacol. 2004 Jan;141(l):37-46); PSMa3 (i.e. MEFVAKLFKFFKDLLGKFLGN described in Kretschmer D, Gleske AK, Rautenberg M, Wang R, Koberle M, Bohn E, Schoneberg T, Rabiet MJ, Boulay F, Klebanoff SJ et al. (2010) "Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus", Cell Host Microbe, 7 (6): 463-73); PSMa2 described in Forsman, H. et al. (2015 "Structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a formylmethionyl group ", Biochim. Biophys. Acta 1853, 192-200); MMK1 (for example the LESIFRSLLFRVM-NH ₂ peptide described in Hu JY, Le Y, Gong W, Dunlop NM, Gao JL, Murphy PM, Wang JM. (2001) Synthetic peptide MMK-1 is a highly specific chemotactic agonist for leukocyte FPRL1. J. Leukoc. Biol., 70 (1): 155-61.). In a particular embodiment, the metabolic lipid used a FPR2 activator is lipoxin A4 (LXA4).

In a particular embodiment, the small organic molecule used as FPR2 activator is Quin-Cl (i.e. 4-butoxy-N-[2-(4-methoxyphenyl)-4-oxo-l,2-dihydroquinazolin- 3-yl]benzamide, described in Nanamori M, Cheng X, Mei J, Sang H, Xuan Y, Zhou C, Wang MW, Ye RD. (2004), "A novel nonpeptide ligand for formyl peptide receptor-like 1. Mol. Pharmacol., 66 (5): 1213-22).

In another particular embodiment, the protein used as a FPR2 activator is Annexin-1 (ANXA1).

Other FPR2 activators can be found in WO2011073918.

Also herein described is a method for producing a vaccine composition comprising an inactivated or attenuated influenza virus or a portion thereof from cells infected with the wild- type influenza virus, said method comprising exposing the infected cells to an activator of FPR2.

The most common way that flu vaccines are made is using an egg-based manufacturing process that has been in existence for more than 70 years. Egg-based vaccine manufacturing is used to make both inactivated (killed) vaccine and live attenuated (weakened) vaccine. The cells to be infected may thus be a chicken egg classically used to produce a vaccine in the conventional egg-based flu vaccine production technique well known by the skilled person. The cell can otherwise be an animal cell, typically a mammalian cell, for use for cell-based vaccine production. In the cell-based production process, the viruses are mixed with cultured mammalian cells (instead of being incubating in eggs) and allowed to replicate for a few days. Then the virus-containing fluid is collected from the cells and the virus antigen is purified. The manufacturing process continues with purification and testing.

In another aspect, the present invention relates to the use of an inhibitor of FPR2 or composition of the present invention comprising such an inhibitor for research assays (e.g., biochemical assay, enzymatic assay, in vitro or in vivo assays on cellular or animal models).

Other characteristics and advantages of the invention are given in the following experimental section (with reference to figures 1 to 15), which should be regarded as illustrative and not limiting the scope of the present application.

FIGURES

Figure 1. Cell surface expression and function of FPR2 upon IAV infection in A549 epithelial cells.

(A, left panel) A549 cells were either left uninfected or infected with A/PR/8/34, A/Udorn/72 or A/WSN/33 viruses (MOI of 1). At 24 hours post-infection, flow cytometry analysis was performed using an anti-FPR2 antibody (open histograms) or an isotype control (closed histograms). The viral protein M2 was used as a marker for viral infection. Results are representative of two independent experiments. (A, right panel) A549 epithelial cells were treated or not with different concentrations of FPR2-AP WKYMVm-NH2 (as indicated) for 5 minutes at 37°C. Cells were then lysed and ERK phosphorylation was analyzed by western blotting using an anti-phospho ERK antibody (p-ERK). Total ERK protein was used as a loading control. (B, left panel) A549 cells were left untreated or treated with the indicated concentrations of FPR2-AP WKYMVm-NH2 for 24 hours. Cell viability was estimated by trypan blue staining. Results show the mean values ± standard deviations from three independent experiments. (B, right panel) A549 epithelial cells were infected or not with IAV A/WSN/33 and treated or not with the indicated concentration of FPR2-AP WKYMVm-NH2. After 16 hours, infectious virus titers were determined by plaque assay. (C, left panel) A549 epithelial cells were pre-treated with various concentrations of the WRW4 FPR2-antagonist as indicated, and infected with IAV A/WSN/33 (MOI 1). Thirty-six hours post-infection, infectious virus titers were determined by plaque assay. (C, right panel) A549 cells were pre- treated with 10 μΜ of the WRW4 for 20 minutes and infected with A/WSN/33 virus at an MOI of 1. After the indicated times post infection, the infectious virus titers in the culture supernatants were determined by plaque assay. (D, left panel) Treatment of A549 cells with WRW4 blocked ERK activation (p-ERK) by 1 μΜ FPR2-AP but not 200 μΜ PAR4-AP. (D, right panel) A549 cells were left untreated or treated with the indicated concentrations of WRW4 FPR2-antagonist for 24 hours. Cell viability was estimated by trypan blue staining. Results show the mean values ± standard deviations from three independent experiments. Figure 2. Influenza virus activates FPR2-induced ERK activation and virus replication. (A) A549 cells were incubated with purified A/WSN/33 particles (MOI 10). Then cells were lysed and ERK phosphorylation was analysed by western blotting at the indicated times postinfection. (B) A549 cells were first pre- incubated 20 minutes with the indicated concentration of WRW4 FPR2-antagonist and then incubated with A/WSN/33 or A/Udorn/72 purified particles (MOI 10) for 5 minutes in presence of vehicle or U0126 ERK signalling-pathway inhibitor (60 μΜ). Cells were then lysed and ERK activation was evaluated by western blotting. (C) A549 cells were first pre- incubated 20 minutes with the WRW4 FPR2-antagonist (10 μΜ) and then infected with A/WSN/33 or A/Udorn/72 particles (MOI 10) in presence of vehicle or U0126 (60 μΜ). Infectious virus titers were determined by plaque assay thirty-six hours post-infection.

Figure 3. ANXA1 incorporation into IAV particles.

(A) Proteins from purified A/PR/8/34, A/Udorn/72 and A/WSN/33 viruses were analysed by western blotting using anti-ANXAl, anti-M2 and anti-ERK antibodies. Aliquots of total proteins from uninfected or A/PR/8/34 virus infected MDCK cells were used as controls. Protein molecular weight was presented in kDa. Results are representative of three independent experiments. (B) Electron microscopic immunogold labeling was performed on purified virions using anti-ANXAl and anti-HA antibodies. Results are representative of two independent experiments.

Figure 4. ANXA1 expression at the cell surface and in the lipid rafts after IAV infection. (A) Detection of cell surface ANXA1 in A549 (upper panels) or MDCK cells (lower panels) after infection with A/Udorn/72, A/PR/8/34 or A/WSN/33 virus. A549 and MDCK cells were infected with A/Udorn/72, A/PR/8/34 or A/WSN/33 virus (MOI 1) for 24 hours. Then flow cytometry analysis was performed using an anti-ANXAl antibody (open histograms) or an isotype control (closed histograms). The viral protein M2 was used as a marker of viral infection. Results are representative of two independent experiments. (B) A549 cells were either left uninfected or were infected with A/WSN/33 virus for 16 hours at a MOI of 1. Cells were then lysed, and fractions 1-10 were collected from the top of the tube, after sucrose gradient ultracentrifugation. Proteins within each fraction were then characterized by western blot analysis. Blots were probed with cholera-toxin B subunit (GM1) or anti-ERK (ERK), anti-HA (HA0-HA2), anti-M2 and anti-ANXAl (ANXA1) antibodies. Fractions 3-4 and 9-10 correspond to rafts and soluble fractions, respectively.

Figure 5. Effect of packaged ANXA1 on virus replication and involvement of the ERK pathway.

(A) A549 cells were transfected with siRNA targeting ANXA1 at the indicated concentration (siRNA) or control siRNA at 80 nM (-). Forty eight hours post-transfection, cells were lysed and proteins from the lysates were analysed by western blotting using an anti-ANXAl or anti- A5 antibodies. (B) Proteins from A/WSN/33 or A/Udorn/72 viruses harvested from supernatants of infected A549 cells transfected with siRNA targeting ANXA1 (Al-KD virus) or control siRNA (WT virus) were characterized by western blot analysis. Blots were probed with anti-ANXAl, anti-M2 and anti-ERK antibodies. Lysates from uninfected or A/PR/8/34 virus infected A549 cells were used as controls. (C) A549 cells were incubated with Al-KD virus or WT virus (MOI 1) for 5 minutes in presence of vehicle or U0126 ERK signalling- pathway inhibitor (60 μΜ). Cells were then lysed and proteins were analysed by western blotting using the anti p-ERK or anti ERK antibodies. (D) A549 cells were infected with WT virus or Al-KD virus (MOI 1) for 16 hours in presence of vehicle or U0126 (60 μΜ). Then, infectious virus titers were determined by plaque assay.

Figure 6. Effect of FPR2 activation on IAV pathogenicity.

(A) Time course of IAV-induced pathogenesis and death in mice in response to FPR2 stimulation. Mice were inoculated intranasally with H1N1 (500 PFU, n = 13-15/group) and treated with either vehicle or 8 mg/kg FPR2-AP (left panel). Time course of uninfected mice treated or not with FPR2-AP (n = 8-12/group, right panel). Results are average percent survival from 2 experiments. (B) infectious virus titers in the BAL of infected mice treated or not with 8 mg/kg of FPR2-AP. Data are average ± SD from 6 individual animals per group. (C) IFN-β and IL-6 analysis in the BAL of H1N1 A/PR/8/34 infected mice treated with FPR2- AP WKYMVm-NH2 (8mg/kg) or vehicle (DMSO 1%); n= 3-6/group. (D) Mice were inoculated intranasally with H1N1 (250 PFU, n = 6/group) and treated with either vehicle or 8 mg/kg FPR2-AP in presence of U0126 (8mg/kg) or vehicle (Untreated). Mice were then followed for survival. Figure 7. Antiviral effect of FPR2 antagonist.

(A) Survival of mice treated with FPR2 antagonists at days 0, 2 and 4 post-infection (n=7/group) or vehicle (n=7/group) after infection with IAV A/PR/8/34 (left panel) or uninfected mice (right panel, n=12/group). (B) Infectious A/PR/8/34 lung virus titers in vehicle or FPR2 treated mice. Data represent mean ± s.e.m of 5-6 individual mice per group. (C) IFN-β and IL-6 analysis in the BAL of H1N1 A/PR/8/34 infected mice (n= 3-5/group) treated with FPR2 antagonist (8mg/kg) or vehicle (DMSO 1%). (D) Mice were inoculated with IAV A/PR/8/34 (n= 18/group) or A/HK/68 (n=6/group), as indicated. FPR2 treatment was initiated two days post-inoculation.

Figure 8: Cell viability and inhibition of viral genome replication by PBP10 and BOC2

(A) A549 cells were treated with 1.25-320 μΜ of PBP10 (white panels) or BOC2 (grey panels) and cell viability was estimated by trypan blue staining 72 hours onwards. (B) A549 cells were pre- incubated with 5 μΜ of either BOC2 or PBP10 (or vehicle) and then infected with IAV A/PR/8/34 virus (MOI 1). RNA was extracted and real-time qPCRs were performed with specific primers to quantify gene expression of viral NS 1 protein (mRNA or vRNA) at the indicated time point post-infection. Data are represented as means ± SEM, n = 3-6 replicates.

Figure 9: Antiviral activity of PBP10 and BOC2

(A) A549 cells were pretreated with 5 μΜ of PBP10 (upper panel) or BOC2 (lower panel) and infected with IAV A/PR/8/34 virus at a MOI of 1. At the indicated time points after infection, infectious virus titres were determined by plaque assay. (B) A549 cells were pretreated with different concentrations of PBP10 (upper panel) or BOC2 (lower panel) and infected with A/PR/8/34 virus at a MOI of 1. Twenty- four hours post- infection, virus titres were determined by plaque assay. NI: Non infected; I: Infected. All experiments are representative of at least two independent assays.

Figure 10: PBP10 and BOC2 inhibit IAV-induced ERK activation

(A) A549 cells were incubated with A/PR/8/34 viruses (MOI 10) for the indicated time point (minutes). Cells were then lysed and ERK phosphorylation was analysed by western blotting.

(B) A549 cells were first pre- incubated or not with the FPR2-antagonist PBP10 or BOC2 and then incubated with A/PR/8/34 virus (MOI 1) for 5 minutes. Cells were then lysed and ERK activation was evaluated by western blotting. (C) A549 cells were first pre-incubated or not with PBP10 or BOC2 and then infected with A/PR/8/34 virus in presence or absence of U0126. Infectious virus titers were determined by plaque assay 24 hours post-infection. Figure 11: Co-treatment of FPR2 antagonists (PBP10 or BOC2) plus oseltamivir

The antiviral effect of FPR2 antagonist alone, oseltamivir alone or a combination of FPR2 antagonist and oseltamivir was evaluated on A/PR/8/34-infected A549 cells. Infectious virus titers were determined by plaque assay 24 hours post-infection.

Figure 12: Antiviral activity of FPR2 antagonists is independent on virus strain

A549 cells were first pretreated or not with 5 μΜ of FPR2 antagonists PBP10, BOC2 or WRW4. Cells were then infected with influenza virus A/HK/68 (IAV, H3N2), A/Turkey/Massachusetts/65 (IAV, H6N2) or B/NL/076/06 and 24 hours post-infection, infectious virus titers were determined by plaque assay.

Figure 13: FPR2 antagonists protect from influenza virus pathogenesis

(A) Survival of mice treated with BOC2 or vehicle (n=8/group) and infected the same day with IAV A/PR/8/34 (500 PFU/mouse). (B) Survival of mice treated with BOC2 or vehicle (n=5/group) and infected the next day with IAV A/PR/8/34 (750 PFU/mouse). (C) Survival of mice treated with WRW4 or vehicle (n=5/group) and infected the next day with IAV A/PR/8/34 (750 PFU/mouse). (D, E) Mice were treated with WRW4 or vehicle (n=5/group) and then subsequently infected on the next day with IAV A/PR/8/34 (750 PFU/mouse). Infectious lung virus titers (D) or total proteins in the BAL of vehicle or WRW4-treated mice were evaluated at day 6 post-infection. Data represent mean ± s.e.m of 3 individual mice per group.

Figure 14: Cell viability

A549 cells were treated with 10 μg/ml of monoclonal antibody FN-1D6-AI (grey panel) and cell viability was estimated by trypan blue staining hours onwards.

Figure 15: Antiviral activity of monoclonal antibody FN-1D6-AI

A549 cells were pre-treated with WRW4 (middle panel) or monoclonal antibody FN-1D6-AI (right panel) at concentration of 5μΜ and 10μg/ml respectively; and then infected with A/PR/8/34 virus at a MOI of 1. After overnight incubation, virus titers were determined by plaque assay.

EXPERIMENTAL PART

EXAMPLE 1 - Formyl peptide receptor 2 plays a deleterious role during influenza A virus infections

In example 1, the inventors show that FPR2 play a crucial role during IAV replication. In particular they show that a peptide antagonist of FPR2, WRW4, inhibits viral replication of H1N1 and H3N1 subtype of IAV. WRW4 acts as an anti-influenza virus agent and protects against IAV infection.

METHODS

Ethics statement

Experiments were performed according to recommendations of the "National Commission of Animal Experiment (CNEA)" and the "National Committee on the Ethic Reflexion of Animal Experiments (CNREEA)". The protocol was approved by the committee of animal experiments of the Faculty of Marseille la Timone (Permit number: Gl 30555). All animal experiments were also carried out under the authority of license issued by "la Direction des Services Veterinaires" (accreditation number 693881479).

Viruses and reagents

The following reagents were used: A/PR/8/34 (H1N1), A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) IAV, Protease- Activated-Receptor 4 (PAR4) agonist AYPGKF-NH2 (Bachem, Bubendorf Switzerland), antagonist of FPR2 WRW4 and agonist of FPR2 WKYMVm-NH2 (R&D Systems, Lille, France), MEK inhibitor U0126 (Promega, Charbonnieres, France), siRNA targeting ANXA1, monoclonal anti-ANXAl, polyclonal anti-A5, monoclonal anti-M2 and monoclonal anti-HA (Santa Cruz Biotechnology, Heidelberg, Germany), cholera toxin B subunit and monoclonal anti-tubulin (Sigma Aldrich, Lyon, France), polyclonal anti-ERK and anti-p-ERK (Cell Signalling, Saint Quentin, France), ELISA kits for mouse IL-6 (R&D Systems) and IFN-β (Invitrogen, Cery Pontoise, France).

Cell culture

The human alveolar A549 and the Madin-Darby canine kidney (MDCK) cell lines were obtained from the American Type Culture Collection (ATCC). MDCK cells were maintained in EMEM (Lonza, Levallois Perret, France) supplemented with 10% Fetal Bovine Serum (Lonza, France), 2 mM L-glutamine, and penicillin-streptomycin (PS). A549 cells were grown in DMEM (Lonza, France) supplemented with 10% FBS, 2 mM L-glutamine and PS.

Virus production, titration, purification and immunogold analysis

Viruses were produced and titrated as previously described ¹² . Purified virus particles were obtained from MDCK cells supernatants as done previously ¹³ . Immunogold labeling of ANXA1 and HA was performed on gradient-purified virus particles as previously described ¹² . Flow cytometry, ELISA and western blot analysis

A549 or MDCK cells were infected or not with A/PR/8/34, A/Udorn/72 or A/WSN/33 (MOI of 1) for 24 hours, and the expression of FPR2 was assessed using flow-cytometry analysis as previously ¹⁵ . ELISA was performed according to the manufacturers' instructions. For western blot analysis, purified virions or cells were lysed and proteins were analyzed, as previsouly described ¹⁶ .

ERK activation experiments

Before cells lysis for western blot analysis, A549 cells were stimulated with the indicated concentration of FPR2 agonist WKYMVm-NH2 for 5 minutes at 37°C. Regarding the specificity of the WRW4 antagonist (10 μΜ), cells were first pretreated for 20 minutes at 37°C with FPR2 antagonist WRW4 and stimulated with either 1 μΜ FPR2 agonist WKYMVm-NH2 or 200 μΜ PAR4 agonist AYPGKF for 5 minutes at 37°C. For the kinetic of virus-induced ERK phosphorylation, A549 cells were stimulated or not with purified A/WSN/33 virus (MOI 10) for the indicated time point before cell lysis. The effect of FPR2 blockade was assessed as followed: A549 cells were pre- incubated with the indicated concentration of FPR2 antagonist WRW4 for 20 minutes at 37°C. Cells were then stimulated or not with purified A/WSN/33 or A/Udorn/72 viruses (MOI 10) for 5 minutes in the presence of 60 μΜ U0126 or vehicle. Stimulation of A549 cells with WT or Al-KD viruses (MOI of 1) was performed for 5 minutes at 37°C in the presence of 60 μΜ U0126 or vehicle.

Viral replication experiments

A549 cells were infected with IAV A/WSN/33 (MOI 1) and stimulated with the indicated concentration of FPR2 agonist, WKYMVm. For FPR2 blockade experiments, A549 cells were first pre- incubated for 20 minutes with 10 μΜ otherwise indicated of FPR2 antagonist WRW4 for 20 minutes at 37°C before infection. In some experiments, assays were performed in presence of 60 μΜ U0126 or vehicle. Regarding experiments with IAV harboring KD ANXAl, cells were infected with WT or ANXAl KO virus at a MOI of 1 in presence of U0126 (60 μΜ) or vehicle. In all conditions, virus titers were evaluated by plaque assay in the supernatant 16 hours post-inoculation, otherwise indicated. Cell viability in presence of agonist or antagonist of FPR2 was assessed by trypan blue staining 24 hours post-treatment. siRNA experiments

Specific siRNA targeting ANXAl was used to knock-down protein expression, in A549 cells. Non-targeted siRNA was used as a control, as previously described ¹² . Western blot analysis was performed to control the transfection efficiency, 48 hours post-transfection. At this step, control siRNA or targeting ANXAl siRNA transfected cells were infected with IAV (A/WSN/33 or A/Udorn/72, MOI 1) and supernatants containing the virus particles were harvested 24 hours post-infection and used in experiments. Reduced expression of packaged ANXAl in the virions released in the supernatant of ANXAl -specific siRNA-treated cells (referred to as Al-KD virus) compared to control viruses (referred to as WT virus) was confirmed by loading 20μ1 of the corresponding supernatants on a gel followed by western blot analysis.

Mouse infection and treatment

C57/BL/6 mice were anesthetized with Ketamine/Xylazine (43/5 mg/kg) and inoculated intranasally with 20 μΐ of a solution containing A/PR/8/34 virus. Inoculation was made with 500 PFU and 5000 PFU of A/PR/8/34 virus regarding stimulation experiments with the agonist and antagonist of FPR2, respectively. 8 mg/kg FPR2 agonists, FPR2 antagonist or U0126 were administered intraperitoneally. Mice were subjected to treatment either at days 0, 2 and 4 post-infection or at days 2 and 4 post-infection. For assessing virus replication, broncho-alveolar lavages (BAL) were harvested from scarified mice, and infectious virus titers were determined by plaque assay, as previously described ¹² .

Statistical analysis

The Mann-Whitney test was used for statistical analysis, regarding viral replication and cytokine production. Kaplan-Meir method was used to calculate the survival fractions in in vivo experiments. Two survival curves were compared by the log-rank test (Mantel-Cox test). Results were considered statistically significant at p < 0.05 (*).

RESULTS

Formyl Peptide Receptor 2 promotes IAV replication

Flow cytometry analysis showed increased FPR2 cell surface expression after infection of the cells with A/Udorn/72, A/PR/8/34, or A/WSN/33 viruses (Figure 1 A, left panel). Viral protein M2 was only detected after virus infection. Despite low expression of FPR2 at the surface of uninfected A549 cells, addition of the FPR2 agonist peptide WKYMVm-NH2 (FPR2-AP) to A549 cells triggered ERK phosphorylation (Figure 1A, right panel). Maximal effect was observed at about 1 μΜ, while the percentage of cell viability was not affected (Figure IB, left panel). Because ERK phosphorylation is essential for IAV infectivity ¹⁷ , A549 cells were infected with IAV and stimulated or not with the FPR2-AP. When exposed to the FPR2-AP, infectious virus titers were significantly increased in a dose-dependent manner in the supernatant of these cells compared to vehicle-treated cells (Figure IB, right panel). Thus, FPR2-signaling inhibition blocks viral production in A549-infected cells. FPR2 antagonist inhibits viral replication

Treatment of IAV- infected A549 cells with FPR2 antagonist WRW4 reduced viral production in a dose- and time course-dependent manner (Figure 1C). WRW4 inhibited the FPR2-AP- induced ERK activation but not the one mediated by PAR4 agonist peptide (Figure ID, left panel), suggesting that WRW4 blocks FPR2 signalling specifically. Furthermore, WRW4 had no effect on A549 cell viability (Figure ID, right panel). Thus, FPR2 activation promotes viral replication during IAV infections, in vitro.

FPR2 activation by IAV increases viral replication through the ERK pathway

Binding of purified virions to A549 cells for 5 or 10 minutes induced ERK activation (Figure 2A) that was prevented in a dose-dependent manner when cells were pre-incubated with FPR2 antagonist WRW4 (Figure 2B, Vehicle). Similar results were observed using IAV A/Udorn/72. Thus, IAV-FPR2 recognition activated ERK. Then, A/WSN/33 or A/Udorn/72 virus-infected cells were exposed to the FPR2 antagonist in the presence or absence of the ERK signaling-pathway inhibitor U0126. Control experiments showed that U0126 efficiently blocked ERK phosphorylation mediated by IAV recognition of FPR2 on A549 cells (Figure 2B, U0126). As expected, WRW4 treatment decreased virus production by IAV-infected cells (Figures 2C). U0126 also showed antiviral activity in cell culture against IAV. In the presence of U0126 and WRW4, no additional antiviral effect was observed showing that U0126 treatment abolished the difference in viral replication between untreated and WRW4-treated cells. Thus, FPR2 promotes IAV replication through an ERK-dependent pathway.

Annexin 1 is incorporated into IAV particles

The ligand on IAV particles that mediated FPR2 activation upon binding to A549 cells was investigated. Proteins from purified IAV A/PR/8/34, A/Udorn/72, and A/WSN/33 were analyzed by Western blot. Results indicated that all purified virions contained the M2 viral protein and ANXAl (Figure 3A). ERK was only detectable in uninfected and virus infected lysates. Microscopic immunogold analysis also showed a specific immunogold staining of ANXAl and viral HA on all IAV particles (Figure 3B). Thus, ANXAl is incorporated into IAV particles. Consistently, ANXAl was increased at the surface of infected cells and was enriched in lipid rafts, the site of virus budding (4A-B). Thus, IAV particles harbor in their enveloppe the endogenous FPR2 ligand, ANXAl .

Annexin 1 incorporated into IAV promotes viral replication

Wild-type (WT) virions or virions for which ANXAl was knock-down by silencing gene expression (A/WSN/33 and A/Udorn/72 strains) were generated. Western blot analysis confirmed that A549 cells transfected with the siRNA targeting ANXAl, express fewer ANXAl but not A5 proteins, compared to A549 cells transfected with a siRNA control (Figure 5 A). Viruses released by these cells were knock-down for ANXA1 levels (Al-KD virus) compared to control viruses (WT virus) (Figure 5B). Binding of WT virus to A549 cells induced ERK activation (Figure 5C, Vehicle). This activation was strongly impaired in the presence of Al-KD virus. Also, WT virus replicated more efficiently compared to Al-KD virus (Figure D, Vehicle). In the conditions where U0126 efficiently blocked virus-induced ERK activation (Figure 5D, U0126), this difference in virus fitness was abolished. Thus, ANXA1 incorporated in IAV is responsible for ERK activation upon IAV binding to the cells and increases virus replication.

FPR2 contributes to the pathogenesis of IAV infection

Mice were infected with IAV A/PR/8/34 and treated or not with FPR2-AP, VKYM Vm-NH2. Infected mice treated with the FPR2-AP displayed significant increased mortality rates, compared to control-treated mice (Figure 6 A, left panel). FPR2-AP had not effect on uninfected mice (Figure 6A, right panel). At days 3 or 6 after inoculation, lung virus titers and cytokine production were significantly increased after FPR2-AP treatment (Figure 6B-C). Thus, FPR2-induced IAV pathogenesis which correlated with increase viral titers and inflammation in the lungs.

The effect of FPR2 activation occurs through an ERK dependent pathway

Infected mice were treated or not with the ERK inhibitor U0126 in presence or absence of FPR2-AP treatment. In contrast to untreated mice, treatment of mice with U0126 abrogated the effect of FPR2-AP on mortality rates after IAV infection (Figure 6D). Thus, the role of FPR2 in IAV pathogenesis occurs through an ERK signaling pathway in vivo.

FPR2 antagonist protects against IAV infection

Mice treated with FPR2 antagonist WRW4 were more resistant to A/PR/8/34 infection than vehicle-treated mice (Figure 7A). No effect was observed on uninfected mice. Protection mediated by WRW4 correlated with reduced lung virus titers and inhibition of cytokines IL6 and INFP production (Figure 7B and 7C). Interestingly, when WRW4 was administered from day 2 post inoculation onward, mice were also significantly protected from A/PR/8/34 and A/Hong-Kong/68 virus infections (Figure 7D). Thus, inhibition of FPR2-signaling protect mice from IAV replication in the lungs, inflammation and severe disease development.

DISCUSSION

The present experiments showed that FPR2 plays an important role during IAV infections. In vitro, stimulation of FPR2 using specific agonists increased viral replication while blocking FPR2 with a specific FPR2 inhibitor did the opposite, indicating that FPR2-signaling plays a pro-viral effect during IAV infections. Consistent with inventors' in vitro studies, FPR2-AP promoted virus replication and exacerbated the effects of IAV infection in infected mice. Moreover, FPR2-antagonist-treated mice were protected from IAV infection showing that FPR2 activation contributes to the pathogenesis of IAV infection.

FPR2 activation did not exacerbate the effects of IAV infection in mice treated with an inhibitor of ERK activation. Thus, ERK is playing a permissive role for the effect of FPR2 activation in IAV infection. Inventors' observation that FPR2 promoted an ERK-dependent proviral effect in lung epithelial cultures and in the lungs of infected mice demonstrate a link between FPR2 signaling, ERK activation and the ability of IAV to replicate. Interestingly, inventors' results showed that FPR2 inhibition induces the late production of protectins in the lungs of infected mice, suggesting that FPR2 signaling blocks the production of protectin generation. This observation is of particular interest since protectin attenuates IAV replication though inhibition of virus RNA export ⁵ , a step which requires signalling through the ERK cascade ¹⁷ . Thus, it is possible that FPR2 signaling inhibits protectin generation, leading to RNA export and virus replication.

The endogenous activators of FPR2 in the airways are not well characterized. FPR2 expressed by respiratory epithelial cells as well as leucocytes is susceptible to be activated by various ligands of diverse classes and from different sources. The observation that ANXA1 is incorporated into IAV particles and that its shut down abolished the effect of FPR2 suggests that the first ligand that maybe involved at an early stage during IAV infection could be ANXA1 which was incorporated into IAV particles. Interestingly, FPR2 -binding to peptides derived from the envelope protein gp41 of human immunodeficiency viruses (HIV) acts as an efficient coreceptor for virus entry ¹⁸ ' ¹⁹ . Thus, several strategies seems to be developed by different viruses to activate FPR2 for efficient replication, highlighting an emerging role for FPR2 during viral infections. Possibly, the extent of ANXA1 incorporation into IAV particles in a strain-dependent manner may explain differences in pathogenicity of IAV strains through activation FPR2.

Formylated peptides are the prototypical ligands for FPR2 and to inventors' knowledge, bacterial and mitochondrial proteins are the only source of N-formyl peptides ¹⁰ ' ²⁰ . However, a broad range of non-N formyl and protein ligands have also been identified, including lipid metabolites such as LXA4, in addition to cellular ANXA1. In this context, activation of FPR2 rather elicits anti-inflammatory and pro-resolving reactions in several models of acute inflammation ²¹ . The fact that ANXA1 was incorporated into IAV suggests that as soon as IAV infects a cell, FPR2 is activated. Thus, it is most likely that IAV developed mechanisms to escape immune surveillance by inducing a proresolution pathway through ANXA1/FPR2 before acute inflammation occurs. This dampened early immune response together with an increase virus replication might be responsible for a subsequent dysregulated and harmfull inflammation of the lungs. Indeed, dysregulated inflammation is a well-known contributor of lung damage during severe influenza, a process that limits respiratory capacity and may account for IAV pathogenesis in humans ^l ' ¹¹ . Consistently, along with increased viral replication, FPR2 exacerbated lung inflammation during IAV infection, in mice. Increase inflammation mediated by FPR2 might also be the consequence of a direct activation of the ERK pathway, a known signaling mediator of inflammation ²³ . FPR2 controls platelet/neutrophil aggregates leading to the rapid generation of circulating LXA4 that subsequently further activates FPR2 ²⁴ . Thus, the involvement of a dysregulated platelet activation, known to promote acute lung injury during influenza cannot also be ruled out in the deleterious role of FPR2 ²⁵ ' ²⁶ .

Altogether the herein described results show that FPR2 is an important receptor involved in IAV pathogenesis, acting both at the level of IAV replication and inflammation. Inventors' results also indicate that inhibitors of FPR2 should be explored as a novel strategy for the treatment of IAV infections.

EXAMPLE 2 - Antiviral activity of BOC2, PBP10 against influenza viruses The aim of example 2 is to go further into the identification of molecules targeting FPR2 in order to foster the development of FPR2 antagonists as antiviral molecules against influenza. The present example shows that the FPR2 antagonists PBP10 and BOC2 (Ortiz-Munoz et al., 2014 ²⁸ ; Skovbakke et al., 2015 ²⁹ ) are two potent antiviral inhibitors of both influenza A and B viruses. Thus, FPR2 is a tractable target for treating a broad range of influenza viruses. The present example also shows that FPR2 antagonist can be useful to protect a subject from influenza viruses when used with both a prophylactic and curative intent.

MATERIALS AND METHODS

Viruses, cells and reagents

IAV A/PR/8/34 (H1N1), A/HK/68 (H3N2) and influenza B virus B/70 were a gift from GF. Rimmelzwaan (Erasmus Medical Center, Rotterdam, the Netherlands). A/Turkey/Massachussets/65 (H6N2) was a gift from V. Jestin (Agence nationale de securite sanitaire de Γ alimentation, de l'environnement et du travail, Maisons-Alfort, France). The human alveolar A549 and the Madin-Darby canine kidney (MDCK) cell lines used in this study were a gift from GF. Rimmelzwaan. Cells were cultured as previously described (Berri et al., 2014 ¹² ). The following reagents were used in the study: monoclonal anti-tubulin antibody (Sigma Aldrich), polyclonal anti-p-ERK antibody (Cell Signaling Technology), antiviral M2 protein (Santa Cruz), and oseltamivir (Sigma-Aldrich). The ERK inhibitor pathway U0126 was obtained from Sigma-Aldrich.

It is reminded that WRW4 is a selective antagonist of FPR2. It inhibits the binding of WKYMVm (FPR2 agonist) to FPR2, resulting in the complete inhibition of ERK signalling as well as intracellular calcium increase (Bae et al., 2004 ³¹ ). WRW4 was obtained from Alomone Labs. PBP10 is a 10-aa-long rhodamine-linked and membrane-permeable peptide inhibitor. It adopts a phosphatidylinositol 4,5-bisphosphate-binding sequence in the cytoskeletal protein gelsolin. Its activity might depend on its ability to pass membrane, disassemble actin filament structures and FPR2 -mediated cellular response (Cunningham et al., 2001 ³¹ ). It is highly specific to FPR2 and has no inhibitory function on FPR1. PBP10 was obtained from Tocris. BOC2 (Boc-Phe-Leu-Phe-Leu-Phe-OH) is a competitive antagonist of the binding of formyl peptides to FPR. BOC2 blocks both human and murine FPR2 (Verriere et al., 2012 ³² ; Vital et al., 2016 ³³ ). It also inhibits FPR1 signalling. BOC2 was obtained from CliniSciences.

Infection experiments and cell viability

A549 cells were pre-incubated for 20 minutes with or without the indicated concentration of FPR2 agonist WRW4, PBP10 or BOC2 before being infected with the indicated influenza virus (MOI 1). In some experiments, assays were performed in the presence of 0.25 μΜ U0126 or vehicle. After one hour, the virus was removed and medium containing the above- mentioned FPR2 inhibitors was added in the presence or absence of the indicated concentrations of oseltamivir to let virus replication proceed. At the indicated time point postinfection, infectious virus titres were assessed in the supernatant or RNA was extracted. Cell viability in the presence of FPR2 antagonists was assessed by trypan blue staining 24 hours post-treatment. Real-Time quantitative PCR analyses

Total RNA was extracted for each experimental condition from A549 cells using QIAzol reagent (Qiagen) according to the manufacturer's protocol. 5 μg of the resulting RNA was then reverse transcribed using the M-MLV Reverse Transcriptase kit (Invitrogen). Regarding vRNA (viral RNA) reverse transcription, Unil2 primer was used as previously described (Hoffmann et al., 2001 ³⁴ ). A specific primer for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for reverse transcription (Baier et al., 1993 ) as housekeeping gene. Real-Time qPCRs were then performed with the 5X HOT Pol EvaGreen qPCR Mix Plus (Invitrogen). Amplification plots were generated using the LightCycler 480 software (Roche), and fold induction was calculated using the threshold cycle method (2-AACt), GAPDH was used for normalization.

Primer sequences were the following:

Gene Primer sequence ^a Viral NS 1 Forward:

5 '-CTGTGTCAAGCTTTCAGGTAGA-3 ' Reverse:

5 '-GGTACAGAGGCCATGGTCAT-3 '

Human GADPH Forward:

5 '-GAAGGTGAAGGTCGGAGT-3 '

Reverse:

5 '-GAAGATGGTGATGGGATTTC-3 '

Unil2 5 '-AGCAAAAGCAGG-3 ' GAPDH RT 5*-GAGATGATGACCCTTTTGGC-3 ' Virus production and titration

Virus production was performed on MDCK cells that were seeded at 15 x 10 ⁶ cells per 75 cm ² tissue culture flask and incubated at 37°C overnight. The next day, cells were infected with IAV at a multiplicity of infection (MOI) of 10 ^~3 in medium containing 1 μg/ml of trypsin. Two days post-infection, the supernatant was harvested, purified by centrifugation and subsequently the virus particles were frozen at -80°C. For viral titration, MDCK cells were grown in 6 well culture plates and infected with serial dilutions of the supernatant containing the infectious viruses for one hour, at 37°C. After adsorption, cells were overlaid with medium containing 2% agarose and ^g/ml of trypsin and incubated for 3 days, at 37°C. Viral plaques were then visualized by crystal violet staining. Fluorescence Microscopy Experiments

Fluorescence microscopy was performed as previously described (Berri et al., 2014 ¹² ). Briefly, A549 cells were seeded and cultured on glass coverslips in a multiwell plate. The next day, infection experiments in the presence or absence of PBP10 or BOC2 were performed as described above. Cells were then fixed and permeabilized with 4% paraformaldehyde containing 0.2% Triton-XlOO. Cells were then extensively washed with phosphate-buffered saline and were incubated with a viral anti-M2 primary antibody for 1 hour at 37°C. Subsequently, an Alexa Fluor (Life Technologies) secondary antibody was used for 1 hour at 37°C. Cells were counterstained with DAPI for 15 minutes. Images were analyzed using a Zeiss IMAGER.Ml.

ERK activation experiments

Regarding the kinetic of virus-induced ERK phosphorylation, A549 cells were incubated with IAV A/PR/8/34 (MOI 10 or 1) at the indicated time point before cell lysis. Regarding the effect of PBP10 or BOC2, A549 cells were first pretreated for 20 minutes at 37°C with FPR2 antagonists PBP10 or BOC2. Cells were then incubated with A/PR/8/34 virus (MOI 10) for 5 minutes and then lysed in ice-cold lysis buffer (1% Triton X-100, 100 mM Tris-HCl [pH 7.4], 1.5 M NaCl, 5 mM EDTA, in the presence of a complete proteinase inhibitor mixture). Proteins from the lysates were then analyzed by western blot, as previously described (Riteau et al., 2003 ¹⁶ ).

In vivo experiments

Five- to six-week-old female C57BL/6 mice (Charles River) were anesthetized with Ketamine/Xylazine (43/5 mg/kg) and inoculated intranasally with 20 μΐ of a solution containing A/PR/8/34 virus, as previously described (Berri et al., 2013 ³⁶ ; Le et al., 2015 ²⁵ ). Prophylactic treatment with WRW4 (8 mg/kg) was achieved by treating mice once, one day before virus inoculation (750 PFU). Regarding BOC2 treatment (4 mg/kg), mice were treated once the day of virus inoculation (500 PFU). WRW4 and BOC2 were both administered intraperitoneally. Upon virus inoculation, survival rates and loss of body weight was scored daily. At the end of the experiment, mice were sacrificed by cervical dislocation. For assessing virus replication, BAL was harvested from sacrificed mice, and infectious virus titres were determined by plaque assay. Total protein was evaluated by using the Coomassie Bradford Protein assay kit (Thermo Scientific, Franklin, MA). The protocol was approved by the committee of animal experiments of the Faculty of Marseille la Timone (number: Gl 30555). All animal experiments were also carried out under the authority of a license issued by "la direction des services Veterinaires" (accreditation 693881479).

Statistical analysis

All statistical analyses were performed using GraphPad Prism software. The Mann-Whitney test was used for statistical analysis, regarding viral replication. Results were analysed using a Wilcoxon test or one-way analysis of variance (ANOVA) for real-time PCR analysis. Differences in survival rates were analyzed using a Log-Rank (Mantel-Cox) test. Results were considered statistically significant at p < 0.05 (*),p < 0.01 (**), P < 0,0001 (****).

RESULTS

Treatment of A549 cells with PBP10 or BOC2 inhibits A/PR/8/34 virus replication

To examine the antiviral properties of PBP10 and BOC2 against IAV, the inventors first evaluated the cytotoxic effects of A549 cell treatment with different concentrations of PBP10 or BOC2 (1.25-320 μΜ). After an incubation period of 72 hours, they observed that 10 μΜ- 320 μΜ of PBP10 and 320 μΜ of BOC2 were cytotoxic to the cells, as measured by blue trypan staining (Figure 8A). The inventors thus determined that 5 μΜ will be used in the subsequent experiments for both PBP10 and BOC2, a concentration also used by others (Fu et al., 2004 ³⁷ ). Next, to examine the antiviral properties of PBP10 and BOC2 against IAV, they first evaluated the mRNA and vRNA levels of the viral NS1 (nonstructural 1) protein in A/PR/8/34 (HlNl) infected A549 alveolar epithelial cells, pretreated or not with 5 μΜ of PBP10 or BOC2. Results showed that compared to infected untreated cells, cell pretreatment with PBP10 or BOC2 significantly reduced the NS1 mRNA and vRNA expression level in infected cells (Figure 8B). Thus, these results show that BOC2 and PBP10 inhibit viral genome replication.

Then, to investigate whether PB10 and BOC2 treatment would affect the release of infectious viral particles, A549 cells were infected with A/PR/8/34 virus and pretreated or not with the indicated dose of FPR2 antagonist PBP10 or BOC2. At different time points post-infection, infectious viral titres were then assessed by classical plaque assays. Treatment of IAV- infected cells with FPR2 antagonist significantly reduced viral production in a time course and dose- dependent manner (Figure 9A-B). To confirm and visualize the antiviral effect of PBP10 and BOC2, immunofluorescence staining was performed. A549 cells were pretreated with each inhibitor and infected with IAV for 24 hours. The expression of the viral M2 protein was then assessed by immunofluorescence microscopy, using a specific anti-M2 antibody. Results showed that in untreated infected cells, M2 was highly expressed and distributed in the cytoplasm and at the cell membrane of all infected cells. In contrast, in the presence of PBPIO or BOC2, not only the intensity of fluorescence was reduced but also a large percentage of cells showed no significant M2 expression. In these assays, nuclei were stained with DAPI. As controls, uninfected cells displayed undetectable M2 proteins. Altogether, these results indicated that cell treatment with PBPIO or BOC2 leads to decreased A/PR/8/34 virus production in infected cells. PBPIO and BOC2 inhibits A/PR/8/34 virus replication through ERK activation

The inventors previously reported in example 1 that IAV promoted its own replication through binding to FPR2. Subsequently, this leads to ERK activation, a signaling pathway required for virus life cycle. Thus, the inventors next tested whether the antiviral effect of PBPIO and BOC2 occurred through blocking influenza-virus induced ERK activation. First, they confirmed that binding of A/PR/8/34 virus to A549 cells promoted ERK phosphorylation (Figure 10A). More importantly, A549 cell pre-treatment with FPR2 antagonist PBPIO or BOC2 prevented IAV-induced ERK activation (Figure 10B). Thus, PBPIO and BOC2 blocked IAV-FPR2 recognition, leading to impaired ERK activation. To evaluate the role of this signaling pathway in the antiviral activity of PBPIO and BOC2, A549 cells were pre-treated with either FPR2 antagonist in the presence or absence of the ERK pathway inhibitor, U0126. Afterwards, infectious virus titres were evaluated by plaque assays. In absence of U0126, cell treatment with PBPIO alone decreased virus production by A/PR/8/34 virus-infected cells (Figure IOC, left panel). As expected, cell treatment with U0126 alone also showed antiviral activity. However, in the presence of U0126, the difference in viral replication between untreated and PBPlO-treated cells was abrogated. Similar results were also observed using BOC2 to inhibit FPR2 (Figure IOC, right panel). Thus, PBPIO and BOC2 blocked viral replication through FPR2 induced-ERK activation.

Combined treatment of FPR2 antagonists with Oseltamivir

The inventors next assessed the antiviral efficacy of combined treatment of FPR2 antagonists with oseltamivir. Infectious virus titres in the supernatant of IAV-infected A549 cells treated or not with PBPIO, oseltamivir or a combination of PBPIO and oseltamivir were evaluated. As expected, cell treatment with either oseltamivir or PBPIO alone showed antiviral activity against A/PR/8/34 virus (Figure 11 A, upper panel). The combination of PBPIO with oseltamivir led to a potent inhibitory effect. Similar results were observed when BOC2 was used as the FPR2 antagonist (Figure 11 A, lower panel). Thus, FPR2 antagonist used in combination with oseltamivir boosts the antiviral activity.

Antiviral effect of FPR2 antagonists on several influenza A virus strains and B viruses The antiviral activity of the FPR2 antagonists was then evaluated against other subtypes of IAV strains as well as influenza B viruses. A549 cells were left untreated or were pre-treated with either PBP10, BOC2 or WRW4 (5 μΜ). Cells were then infected with B/NL/076/06 influenza B viruses, A/HK/68 IAV (H3N2) or A/Turkey/Massachusetts/65. After 24 hours post-infection, the supernatant was collected and infectious virus titres were analyzed by classical plaque assays. Results showed that all viruses replicated efficiently in untreated cells. However, cell treatment with WRW4, PBP10 or BOC2 (Figure 12A-C) significantly inhibited virus replication. Thus, it can be concluded that FPR2 antagonists have an antiviral effect on different influenza viruses. Protective effect of FPR2 antagonists in vivo

Example 1 shows that mice treatment with WRW4 protected them from IAV-induced death. Example 2 shows that other antagonists of FPR2 have the same effect. As a matter of fact, results showed that mice treated with BOC2 were significantly more resistant to A/PR/8/34 infection than vehicle-treated mice (Figure 13 A). In contrast, treatment of uninfected mice with BOC2 did not affect their survival rates, which suggests that FPR2 antagonists do not cause side effects. Finally, the inventors showed that blocking FPR2 in a prophylactic manner was still protective. In fact, when BOC2 or WRW4 FPR2 antagonists were administered once and one day before inoculation, mice were also significantly protected from A/PR/8/34 virus infections (Figure 13B-C). In addition, virus replication and total proteins in BAL of WRW4- treated mice were significantly decreased compared to the ones of vehicle-treated mice (Figure 13 D-E). Thus, inhibition of FPR2 signaling protected mice from IAV replication, inflammation in the lungs and severe disease development when used with a prophylactic or curative intent. DISCUSSION

The present example showed that the FPR2 antagonists PBP10 and BOC2 are potent antiviral molecules in vitro against a broad range of IAV and B viruses. Consistently, example 1 showed that FPR2 plays a deleterious role during IAV infections and that another FPR2 antagonist WRW4 inhibits IAV replication in vitro and in vivo. Mechanistically, the effect of PBP10 and BOC2 was abolished by treating the cells with U0126, a specific ERK pathway inhibitor. The antiviral role of these molecules occurs through ERK activation, a pathway necessary for the viral life cycle in vitro and in vivo (Droebner et al., 2011 ^3B ; Pleschka et al., 2001 ⁿ ).

In vivo, administration of BOC2 to infected mice protected them from lethal IAV infections. These results confirm results of example 1 showing that another inhibitor of FPR2, WRW4, efficiently protected mice against lethal IAV infections. Protein sequence alignment shows 76 % amino acid identity between human and mouse FPR2 (85% when considering similar residues) and 97% amino acid identity between human and macaque FPR2 (98% when considering similar residues). The conservation of the FPR2 gene between species is likely to explain the effect of WRW4 and BOC2 on both human and mouse FPR2.

It is noteworthy that WRW4 or BOC2 administration in a prophylactic manner also had a protective effect. Thus, FPR2 antagonism might be explored not only as a new treatment for influenza but also to prevent the disease. This effect would be particularly valuable in case of a pandemic. Indeed, although preventive vaccination exists, based on the inventors 'knowledge of previous pandemic plans, the delay of 6-12 months to produce a pandemic vaccine cannot fit with a required rapid response (Webby and Webster, 2003 ³⁹ ). Vaccines are reduced to specific viral strains that should first be identified, produced in large amount and their inactivation controlled. In addition, they are accessible only to a small, privileged fraction of the world population. Finally, the increasing skepticism towards vaccination has led to a drop in immunization coverage rates. The development of new antiviral drugs thus appears as a relevant strategy. Regarding the advantage of FPR2 antagonists acting in a therapeutic or prophylactic manner, it would not only prevent virus from spreading from human to human but also protect the population before infection occurs. In contrast to the current antiviral drugs, FPR2 antagonists could most likely be used without the emergence of resistant viruses. Indeed, the current commercialized antivirals target viral proteins which are highly subjected to mutations. In contrast, blocking a cellular receptor will slow down viral growth and at the same time diminish the probability of the virus escaping from mutation pressure since the virus is unable to modify the host genome.

It is also noteworthy that mice lacking FPR2 develop normally, and their lifespan in a pathogen- free facility is equivalent to wild-type mice (Chen et al., 2010 ⁴⁰ ). This suggests that FPR2 is not a crucial receptor for cellular functions.

It is also noteworthy that FPR2 belongs to the family of G-protein coupled receptors (GPCRs). GPCRs have been one of the most popular targets for drug developers. According to a recent publication, 30-50% of commercialized drugs exert their effect through GPCRs and from 2005-2014, 25 % of novel approved drug from US Food and Drug Administration target GPCRs (Fang et al., 2015 ⁴¹ ). FPR2 plays a key role in inflammatory processes and thus is also a major target for drug developers. However, to the inventors' knowledge FPR2 inhibitors did not go through clinical trials yet. The reasons might be multiple. First, in comparison to other receptors, the precise role of FPR2 (pro-inflammatory versus resolution of inflammation) is only emerging. Then, FPR2 also belongs to the FPR family, in which two other FPRs were described in humans (FPR1 and 3) and all FPRs have similarities in their amino acid sequences. While WRW4 and PBP10 are highly specific inhibitors of FPR2, BOC2 is less specific and also inhibits FPR1. Thus, a limitation in the use of FPR2 antagonists is to develop very specific small molecules against FPR2. Unfortunately, the discovery of specific molecules targeting GPCRs is very intractable and currently, bio-therapeutic has been demonstrated to be a better approach (Mujic-Delic et al., 2014 ⁴² ). Antibodies to GPCRs have been difficult to develop since they are very unstable when purified.

Regarding IAV, 18 types of hemagglutinin and 11 types of neuraminidase were described and none of the commercialized antiviral drugs are susceptible to protect against all strains that will emerge from the animal reservoir (Webby and Webster, 2003 ³⁹ ). The strong dependencies of influenza viruses on well-known specific cellular functions appear particularly relevant for the development of universal antivirals. As shown here, antagonists of FPR2 inhibited replication of several strains of influenza A and B viruses. FPR2 antagonists act through ERK inhibition, a signaling pathway required for viral ribonucleoprotein (vRNP) translocation from the nucleus to the cytoplasm and viral replication (Droebner et al., 2011 ³⁸ ; Pleschka et al., 2001 ⁿ ). Since vRNP translocation is required for all strains of influenza virus life cycle, FPR2 should most likely protect against any novel influenza strain that could emerge from the animal reservoir and cause a pandemic. Very interestingly, FPR2 antagonists showed a potent effect with oseltamivir regarding inhibition of virus replication. Altogether, this report suggests that the use of FPR2 antagonists in combination with current antiviral drugs could be an interesting strategy to develop novel antiviral drugs.

EXAMPLE 3 - Antiviral activity of a monoclonal antibody against influenza viruses

In example 3, the inventors showed that a monoclonal antibody anti-FPR2 can be used as anti- influenza virus agent. METHODS

Viruses and reagents

The following reagents were used: A/PR/8/34 (H1N1) IAV, antagonist of FPR2 WRW4, PBP10 and monoclonal antibody FN-1D6-AI (as described in Carmela De Santo et al. Nat Immunol ²⁷ and Lucy V. Norling et al. Arterioscler Thromb Vase Biol. 2012;32: 1970-1978 ⁴³ ), human alveolar A549 and MDCK cell lines, and monoclonal anti-M2.

Infection experiments and cell viability

A549 cells were pre-incubated for 20 minutes with or without the indicated concentration of WRW4, or monoclonal antibody anti-FPR2 FN-1D6-AI before being infected with the indicated influenza virus (MOI 1). After one hour, the virus was removed and medium containing the above- mentioned FPR2 inhibitors was added to let virus replication proceed. At the indicated time point post-infection, infectious virus titres were assessed in the supernatant. Cell viability in the presence of FN-1D6-AI antibody was assessed by trypan blue staining 24 hours post-treatment.

Virus production and titration

Virus production was performed on MDCK cells that were seeded at 15 x 10 ⁶ cells per 75 cm ² tissue culture flask and incubated at 37°C 24 hours. The next day, cells were infected with IAV at a multiplicity of infection (MOI) of 10 ^"3 in medium containing 1 μg/ml of trypsin. Two days post-infection, the supernatant was harvested, purified by centrifugation and subsequently the virus particles were frozen at -80°C. For viral titration, MDCK cells were grown in 6 well culture plates and infected with serial dilutions of the supernatant containing the infectious viruses for one hour, at 37°C. After adsorption, cells were overlaid with medium containing 2% agarose and ^g/ml of trypsin and incubated for 3 days, at 37°C. Viral plaques were then visualized by crystal violet staining.

Fluorescence Microscopy Experiments

Fluorescence microscopy was performed as previously described in example 2. Briefly, A549 cells were seeded and cultured on glass coverslips in a multiwell plate. The next day, infection experiments in the presence or absence of PBP10 or monoclonal antibody anti-FPR2 FN-1D6- Al were performed as described above. Cells were then fixed and permeabilized with 4% paraformaldehyde containing 0,2% Triton-XlOO. Cells were then extensively washed with phosphate-buffered saline and were incubated with a viral anti-M2 primary antibody for 1 hour at 37°C. Subsequently, an Alexa Fluor (Life Technologies) secondary antibody was used for 1 hour at 37°C. Cells were counterstained with DAPI for 15 minutes. Images were analyzed using a Zeiss IMAGER.M1. RESULTS

Treatment of A549 cells with monoclonal antibody anti-FPR2 inhibits A/PR/8/34 virus replication

To examine the antiviral properties of monoclonal antibody FN-1D6-AI against IAV, the inventors first evaluated the cytotoxic effects of A549 cell treatment a concentration of FN- 1D6-AI of lC^g/ml. After an incubation period of 72 hours, they observed that such concentration of antibody were not cytotoxic to the cells, as measured by blue trypan staining (Figure 14).

Then, to investigate whether a monoclonal antibody anti-FPR2 treatment would affect the release of infectious viral particles, A549 cells were infected with A/PR/8/34 virus and pre- treated or not with the indicated dose monoclonal antibody FN-1D6-AI (lC^g/ml) or WRW4 (as a comparison). At different time points post-infection, infectious viral titre were then assessed by classical plaque assays. Treatment of IAV- infected cells with FPR2 antagonist significantly reduced viral production (Figure 15). To confirm and visualize the antiviral effect of the monoclonal antibody FN-1D6-AI, immunofluorescence staining was performed. A549 cells were pre-treated with FN-1D6-AI and PBP10 (as a comparison); then infected with IAV for 24 hours. The expression of the viral M2 protein was then assessed by immunofluorescence microscopy, using a specific anti-M2 antibody. Results showed that in untreated infected cells, M2 was highly expressed and distributed in the cytoplasm and at the cell membrane of all infected cells. In contrast, in the presence of PBP10 or FN-1D6-AI, not only the intensity of fluorescence was reduced but also a large percentage of cells showed no significant M2 expression. In these assays, nuclei were stained with DAPI. As controls, uninfected cells displayed undetectable M2 proteins. Altogether, these results indicated that cell treatment with FN-1D6-AI antibody leads to decreased A/PR/8/34 virus production in infected cells.

CONCLUSIONS

Al together, example ,1 example 2, and example 3 show that FPR2 inhibitors are expected to (i) show efficacy to block virus replication, (ii) limit the emergence of virus resistance, (iii) have a broad spectrum of action, regardless of the strain of influenza virus, (iv) have limited adverse effects in humans, (v) be effective when administered early or late post-infection and (vi) boost the antiviral activity of current antiviral drugs such as oseltamivir when used in combination therapy. The inventors have also demonstrated that a wide variety of FPR2 inhibitors can be used as anti-influenza agent such as different peptides as well as monoclonal antibodies both in a prophylactic or curative approach. Moreover, such FPR2 inhibitors can be also used against different subtypes of Influenza (such as A, B, C and D subtypes).

REFERENCES

1. Kuiken, T., Riteau, B., Fouchier, R.A. & Rimmelzwaan, G.F. Pathogenesis of influenza virus infections: the good, the bad and the ugly. Curr Opin Virol 2, 276-286 (2012).

2. Fukuyama, S. & Kawaoka, Y. The pathogenesis of influenza virus infections: the contributions of virus and host factors. Current opinion in immunology 23, 481-486 (2011).

3. Serhan, C.N. Pro-resolving lipid mediators are leads for resolution physiology. Nature

510, 92-101 (2014).

4. Russell, CD. & Schwarze, J. The role of pro-resolution lipid mediators in infectious disease. Immunology 141, 166-173 (2014).

5. Morita, M. et al. The lipid mediator protectin Dl inhibits influenza virus replication and improves severe influenza. Cell 153, 112-125 (2013).

6. Perretti, M. et al. Endogenous lipid- and pepti de-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Nature medicine 8, 1296-1302 (2002).

7. Chiang, N. et al. The lipoxin receptor ALX: potent ligand-specific and stereoselective actions in vivo. Pharmacological reviews 58, 463-487 (2006).

8. Perretti, M. & DAcquisto, F. Annexin Al and glucocorticoids as effectors of the resolution of inflammation. Nat Rev Immunol 9, 62-70 (2009).

9. De, Y. et al. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor- like 1 (FPRLl) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192, 1069-1074 (2000).

10. Carp, H. Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils. J Exp Med 155, 264-275 (1982).

11. Liu, M. et al. Formylpeptide receptors are critical for rapid neutrophil mobilization in host defense against Listeria monocytogenes. Sci Rep 2, 786 (2012).

12. Berri, F. et al. Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication. J Virol 88, 11215-11228 (2014). 13. LeBouder, F. et al. Annexin II incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin. J Virol 82, 6820-6828 (2008).

14. LeBouder, F. et al. Immunosuppressive HLA-G molecule is upregulated in alveolar epithelial cells after influenza A virus infection. Hum Immunol 70, 1016-1019 (2009).

15. Riteau, B. et al. Characterization of HLA-Gl, -G2, -G3, and -G4 isoforms transfected in a human melanoma cell line. Transplant Proc 33, 2360-2364 (2001).

16. Riteau, B., Barber, D.F. & Long, E.O. Vavl phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization. J Exp Med 198, 469-474 (2003).

17. Pleschka, S. et al. Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade. Nature cell biology 3, 301-305 (2001).

18. Shimizu, N. et al. A formylpeptide receptor, FPRLl, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus. Retrovirology 5, 52 (2008).

19. Shimizu, N. et al. Broad usage spectrum of G protein-coupled receptors as coreceptors by primary isolates of HIV. AIDS 23, 761-769 (2009).

20. Schiffmann, E. et al. The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli. J Immunol 114, 1831-1837 (1975). 21. Oldekamp, S. et al. Lack of formyl peptide receptor 1 and 2 leads to more severe inflammation and higher mortality in mice with of pneumococcal meningitis. Immunology 143, 447-461 (2014).

22. de Jong, M.D. et al. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nature medicine 12, 1203-1207 (2006).

23. Kurosawa, M., Numazawa, S., Tani, Y. & Yoshida, T. ERK signaling mediates the induction of inflammatory cytokines by bufalin in human monocytic cells. American journal of physiology. Cell physiology 278, C500-508 (2000).

24. Brancaleone, V. et al. A vasculo-protective circuit centered on lipoxin A4 and aspirin- triggered 15-epi-lipoxin A4 operative in murine microcirculation. Blood 122, 608-617

(2013).

25. Le, V.B. et al. Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis. Am J Respir Crit Care Med 191, 804-819 (2015).

26. Sugiyama, M.G. et al. Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury. J Virol 90, 1812- 1823 (2015).

27. Carmela De Santo, Ramon Arscott, Sarah Booth, Ioannis Karydis, Margaret Jones, Ruth Asher, Mariolina Salio, Mark Middleton & Vincenzo Cerundolo. Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A. Nature Immunology.

28. Ortiz-Munoz, G., Mallavia, B., Bins, A., Headley, M., Krummel, M.F., Looney, M.R., 2014. Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice. Blood 124, 2625-2634.

29. Skovbakke, S.L., Heegaard, P.M., Larsen, C.J., Franzyk, H., Forsman, H., Dahlgren, C, 2015. The proteolytically stable peptidomimetic Pam-(Lys-betaNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

Biochemical pharmacology 93, 182-195.

30. Bae, Y.S., Lee, H.Y., Jo, E.J., Kim, J.I., Kang, H.K., Ye, R.D., Kwak, J.Y., Ryu, S.H., 2004. Identification of peptides that antagonize formyl peptide receptor- like 1 - mediated signaling. J Immunol 173, 607-614.

31. Cunningham, C.C., Vegners, R., Bucki, R., Funaki, M., Korde, N., Hartwig, J.H., Stossel, T.P., Janmey, P.A., 2001. Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly. J Biol Chem 276, 43390-43399.

32. Verriere, V., Higgins, G., Al-Alawi, M., Costello, R.W., McNally, P., Chiron, R., Harvey, B.J., Urbach, V., 2012. Lipoxin A4 stimulates calcium-activated chloride currents and increases airway surface liquid height in normal and cystic fibrosis airway epithelia. PLoS One 7, e37746.

33. Vital, S.A., Becker, F., Holloway, P.M., Russell, J., Perretti, M., Granger, D.N., Gavins, F.N., 2016. Formyl-Peptide Receptor 2/3/Lipoxin A4 Receptor Regulates Neutrophil-Platelet Aggregation and Attenuates Cerebral Inflammation: Impact for Therapy in Cardiovascular Disease. Circulation 133, 2169-2179.

34. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146, 2275- 2289.

35. Baier, G., Telford, D., Gulbins, E., Yamada, N., Kawakami, T., Altman, A., 1993.

Improved specificity of RT-PCR amplifications using nested cDNA primers. Nucleic

Acids Res 21, 1329-1330.

36. Berri, F., Rimmelzwaan, G.F., Hanss, M., Albina, E., Foucault-Grunenwald, M.L., Le, V.B., Vogelzang-van Trierum, S.E., Gil, P., Camerer, E., Martinez, D., Lina, B., Lijnen, R., Carmeliet, P., Riteau, B., 2013. Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis. PLoS Pathog 9, el 003229.

37. Fu, H., Bjorkman, L., Janmey, P., Karlsson, A., Karlsson, J., Movitz, C, Dahlgren, C, 2004. The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide. BMC Cell Biol 5, 50.

Droebner, K., Pleschka, S., Ludwig, S., Planz, O., 2011. Antiviral activity of the MEK-inhibitor U0126 against pandemic HINlv and highly pathogenic avian influenza virus in vitro and in vivo. Antiviral Res 92, 195-203.

Webby, R.J., Webster, R.G., 2003. Are we ready for pandemic influenza? Science 302, 1519-1522.

Chen, K., Le, Y., Liu, Y., Gong, W., Ying, G., Huang, J., Yoshimura, T., Tessarollo, L., Wang, J.M., 2010. A critical role for the g protein-coupled receptor mFPR2 in airway inflammation and immune responses. J Immunol 184, 3331-3335.

Fang, Y., Kenakin, T., Liu, C, 2015. Editorial: Orphan GPCRs As Emerging Drug Targets. Front Pharmacol 6, 295.

Mujic-Delic, A., de Wit, R.H., Verkaar, F., Smit, M.J., 2014. GPCR-targeting nanobodies: attractive research tools, diagnostics, and therapeutics. Trends Pharmacol Sci 35, 247-255.

Lucy V. Norling, Jesmond Dalli, Roderick J. Flower, Charles N. Serhan, Mauro Perretti. Resolvin Dl Limits Polymorphonuclear Leukocyte Recruitment to Inflammatory Loci Receptor-Dependent Actions. Arteriosclerosis, Thrombsis, and Vascular Biology.