Method of preventing virus: cell fusion by inhibiting the function of the fusion initiation region in rna viruses having class i membrane fusogenic envelope proteins

[0001] This application claims the benefit of United States Provisional Provisional Application Serial No. 60/517181, November 4, 2003.

FIELD OF THE INVENTION [0002] The present invention relates to peptides for use in preventing or inhibiting viral infection of an influenza virus in a cell (and thereby preventing delivery of the viral genome into the cytoplasm of the cell, the stage required for viral infection). The present invention provides compositions and their uses for preventing influenza virus infection by interfering with its fusion initiation region (FIR).

INTRODUCTION [0003] All viruses must bind to and enter their target cells in order for them to replicate. For enveloped animal viruses, including RNA viruses containing Class I membrane fusion proteins (Type I viruses), this process involves (a) binding of the virion to the target cell, (b) fusion of the viral envelope with the plasma membrane or the internal cell membrane, ( c) destabilization of the virus envelope and cell membrane in the fused area, resulting in the creation of a fusion pore, (d) the transfer of viral RNA through this pore, and (e) modification of cellular functions by viral RNA. [0004] The fusion of the viral envelope and cell membrane, steps (b) and (c) above, occurs through the interaction of the viral transmembrane glycoprotein (fusion protein) with surface proteins and membranes of the target cell. These interactions cause conformational changes in the fusion protein that lead to the insertion of the viral fusion peptide into the membrane of the target cell. This insertion is followed by further conformational changes in the fusion protein that bring the viral envelope and cell membranes closer together and fuse the two membrane bilayers.

[0005] The virus is not able to spread and multiply in the host organism if the fusion process is interrupted. The intended disruption of this fusion process can be achieved by directing peptides and peptide mimetics homologous to the fusion protein sequence, fusion protein recognizing antibodies, and other anti-fusion protein factors.

BACKGROUND OF THE INVENTION

Structural similarities between RNA virus Class I fusion proteins.

[0006] Hemagglutinin 2 (HA2) of influenza virus, orthomixovirus, is a prototype RNA virus Class I fusion protein and contains an N-terminal hydrophobic domain, called a fusion peptide, which is exposed during cleavage of the hemagglutinin precursor protein. Membrane fusion proteins of RNA viruses from several different families, including areaviruses, coronaviruses, filoviruses, orthomixoviruses, paramyxoviruses and retroviruses, share several common structural features with HA2, they are referred to as Class I viral fusion proteins. It has been observed that the HIV-1 fusion protein, transmembrane glycoprotein and other retroviral transmembrane proteins, such as those of orthomixovirus and paramyxovirus, have a hydrophobic fusion peptide domain exposed during precursor cleavage (gp160) (Gallaher, 1987; Gonzalez-Scarano. ). Based on these similarities and computer algorithms predicting protein configurations, it has been suggested (Gallaher et al., 1989) that the outer part (ectodomain, N-terminus) of the HIV-1 transmembrane protein and the transmembrane proteins of other retroviruses may match the scaffold structure of the designated HA2 using X-ray crystallography (Wilson, Skehel and Wiley, 1981).

[0007] Based on these observations, retroviral transmembrane proteins have been predicted to contain several structural features, in addition to the fusion peptide, in common with the known HA2 structure, including an elongated N-terminal helix (N-helix, usually called "heptad repeat" or " leucine zipper "), C-terminal helix (C-helix) and aromatic motif proximal to the transmembrane domain. The presence of at least four of these five domains defines the viral envelope protein as a Class I fusion protein. This retroviral transmembrane protein model was then confirmed by structural assays and mutational analyzes (Chan et al., 1997; Kowalski et al., 1991; Weissenhorn and et al., 1997). Common structural motifs are present not only in orthomixovirus and retrovirus fusion proteins, but also paramyxoviruses, filoviruses (such as Ebola virus, EboV) (Gallaher, 1996) and arenaviruses (Gallaher, DiSimone and Buchmeier, 2001). The structural model of the EboV (GP2) fusion protein according to Gallaher was also confirmed by X-ray crystallography (Malashkevich et al., 1999; Weissenhom et al., 1998). WO 94/17826 describes polypeptides for use in the preparation of influenza A and influenza B virus vaccines containing HA protein. 16-mer synthetic hemagglutinin peptides have been described (Gelder et al., 1995). Flu hemagglutinin protein peptides are also described in US 2003/0180328.

[0008] Figure 1 shows the five previously described fusion protein domains from six Type I virus families. Fusion proteins are derived from a hydrophobic fusion peptide, end with an anchor peptide and contain an elongated N-terminal alpha helix (N-helix, usually called "heptad repeat" or "leucine zipper"), C-terminal alpha helix (C-helix) (Carr and Kim, 1993; Suarez et al., 2000; Wilson, Skehel and Wiley, 1981) and sometimes an aromatic motif proximal to the virion envelope. Also shown is the sixth domain, the fusion initiation region (FIR), discovered by the present inventors.

Inhibition of Type I Virus Fusion [0009] Earlier attempts by the inventors of the present invention (Garry) and other researchers to design peptides and peptide mimetics, antibodies and other factors that inhibit Type I virus fusion have focused on fusion peptide, N-helix and C - helix fusion proteins. In the case of fusion peptides, it has been found that analogues of the fusion peptide domains of orthomixovirus and paramyxovirus (Richardson, Scheid and Choppin, 1980) and HIV-1 (Gallaher et al., 1992; Owens et al., 1990; Silburn et al., 1998) block virus infection, probably by forming inactive heteroaggregates. Peptides corresponding to parts of the N-helix and C-helix have also been found to be effective in inhibiting virus infection both in vitro and in vivo. For example, a 17-amino acid peptide corresponding to the C-terminal portion of the N-helix of the HIV-1 fusion protein, defined as the CS3 region, blocked HIV infection (Qureshi et al., 1990). In addition, other N-helix and C-helix inhibitory peptides were developed based on the fusion protein structural model (Wild, Greenwell and Matthews, 1993; Wild et al., 1992), including the anti-HIV-1 DP178 C-helix peptide drug ( T-20 or FUZEON®). DP178 coincides with the C-helix and the aromatic anchoring-proximal domain and inhibits the fusion of HIV-1 virion: cells at very low concentrations (50% inhibition of 1.7 nM) that can be obtained in vivo after injection. In the 100 mg / day clinical trial, DP178 resulted in an approximately 100-fold reduction in HIV-1 load in the plasma of infected patients (Kilby et al., 1998). This result strongly motivated the search for other HIV-1 inhibitory peptides based on transmembrane protein structure (Pozniak, 2001; Sodroski, 1999). Paramyxovirus peptide inhibitors have been shown to inhibit viral replication (Lambert et al., 1996; Young et al., 1999). Studies by Watanabe and colleagues suggest that a similar approach to targeting the EboV GP2 N-helix and C-helix may also lead to useful inhibitors (Watanabe et al., 2000). Neutralizing antibodies directed against portions of the fusion protein domain have also been shown to inhibit virion: cell fusion.

Observations with HIV-1 [0010] A large part of the research was devoted to inhibiting the fusion of the human immunodeficiency virus HIV-1, one of the Type I RNA viruses. Bolognesi et al. (5464933) and the present inventors (Garry, USPN 5567805) described that the killing of cells via HIV can be inhibited by the introduction of peptides binding portions of the HIV-1 virion transmembrane fusion protein. The Bolognesi DP178 binding region, designated FUZEON® in Figure 7, is located mainly in the C-helix and outside the fusion initiation region (FIR) described in this application. Bolognesi shows inhibition, but does not describe how to inhibit. The present inventors (Garry) had previously demonstrated inhibition in the HIV-1 CS3 TM region, designated CS3 in Figure

7, but no identified inhibition method, only suggesting that CS3: receptor-CS3 interaction is inhibited. The unexpected discovery of FIR by the inventors of the present invention (as described herein) and the fact that the CS3 sequences are in FIR means that the CS3: receptor-CS3 binding described in USPN 5567805 is actually a binding that occurs between the CS3 part of FIR and parts of the cell membrane, to which part

CS3 FIR has affinity. In addition, although Melikyan, Watanabe, Bewley and others described inhibition of fusion by introduced peptides, they did not explain the mechanisms by which inhibition occurs. Accordingly, the location of FUZEON® peptide is distant from FIR, strongly suggesting that other elements of the fusion process are active in the region

FUZEON.

[0011] In light of the above, it is clear that there is a need in the art for more effective agents for identifying those regions of viruses that are involved in the infection process and compositions effective in preventing or inhibiting viral infection. The invention described and disclosed herein provides an effective solution to these needs.

SUMMARY OF THE INVENTION [0012] The present invention provides an isolated peptide comprising the sequence SEQ ID NO: 4 or a segment thereof of 8 to 40 consecutive amino acids, said peptide inhibiting influenza viral infection. Also provided are peptides of the invention for use as a medicament. The invention includes the use of a peptide of the invention in the manufacture of a medicament for treating or preventing influenza in a patient. Thus, the peptides of the invention can be used to treat or prevent influenza in a patient.

[0013] The present invention also provides a composition comprising a recombinant DNA molecule encoding a peptide of the invention that enables or stimulates the patient to produce the peptide of claim 1 for use as a medicament. The invention includes the use of a composition comprising a recombinant DNA molecule that enables or stimulates the patient to produce a peptide of the invention, for the manufacture of a medicament for treating or preventing influenza in a patient. In a further aspect, the invention provides a composition comprising a recombinant DNA molecule that enables or stimulates the patient to produce a peptide of the invention for treating or preventing influenza in a patient.

[0014] The present invention also provides an isolated nucleic acid sequence encoding a polypeptide of the invention.

[0015] The invention further provides a method of producing an antibody comprising:

(a) providing a peptide antigen having the sequence SEQ ID NO: 4;

(b) introducing said antigen into an animal so as to elicit an immune response against it;

(c) collecting antibodies from this animal; and (d) identifying those antibodies that specifically recognize the SEQ peptide

ID NO: 4.

[0016] According to this method, the antigen may comprise a peptide analog; peptide derivative; or a peptide mimetic of a peptide having the sequence SEQ ID NO: 4 or an antigenic fragment thereof.

[0017] The invention also provides a virus fusion inhibiting agent comprising a peptide having an amino acid sequence of 8 to 50 amino acid residues, which peptide comprises a peptide consisting of 8 to 40 consecutive amino acid residues of SEQ ID NO: 4. The viral fusion inhibiting agent of this aspect is also provided for use as a medicament. This aspect also extends to the use of a fusion inhibiting agent as defined above in the manufacture of a medicament for treating or preventing influenza in a patient. The viral fusion inhibiting agent of this aspect of the invention may be used to treat or prevent influenza in a patient.

ILLUSTRATIVE FORMS OF THE INVENTION

The sixth domain of RNA viruses containing Class I membrane fusion proteins. [0018] Arenaviruses, coronaviruses, filoviruses, orthomixoviruses, paramyxoviruses and retroviruses are six families of RNA viruses currently identified as containing Class I membrane fusion proteins. The present inventors (Garry) and others researchers have shown that the fusion proteins of these Type I viruses contain five conserved motifs or domains (Carr and Kim, 1993; Gallaher et al., 1989; Suarez et al., 2000;

Wilson, Skehel and Wiley, 1981). These domains include the fusion peptide, N-helix, C-helix and aromatic motif, which are all ectodomains, and an anchor peptide, which is an endodomain.

[0019] Using computational analysis, secondary structure models, interfacial hydrophobicity calculations, and other techniques, the present inventors made the surprising discovery that a highly conserved sixth domain is present in many different viral fusion proteins (this sixth domain is described herein). Viruses having this domain include, but are not necessarily limited to, the six classes of RNA viruses listed above. To emphasize the critical function of this newly identified domain, which is an ectodomain, this domain was called the fusion initiation region (FIR) of viruses.

[0020] As used herein, the term "elongated" alpha helix refers to an alpha helix containing more than four "alpha helix turns" (in particular, more than 14 amino acids).

[0021] Other forms provide "factors" that the inventors have unexpectedly identified as effective in preventing or inhibiting viral infection and / or virus: cell fusion.

[0022] As used herein, the term "factors" includes, but is not limited to, isolated peptides or functional peptide segments (or analogs of these peptides) of the newly described fusion initiation domain (FIR) domain, peptide mimetics (the term "peptide mimetic" refers to any compound or substances that can be used to replace the peptide interacting with FIR, i.e. any compound that mimics the properties of a functional FIR segment), antibodies specific to functional FIR domains (e.g., idiotype or anti-idiotype antibodies), and other molecular compounds that interfere with binding and / or virus: cell fusion.

[0023] As used herein, the term "functional segment" or "functional fragment" of the fusion initiation region (FIR) refers to a fragment capable of inhibiting the virus: cell fusion, inhibiting the infectivity of the virus, capable of eliciting an antibody response capable of recognizing and specifically binding to FIR and / or interfering with cell infection via FIR.

[0024] As used herein, a "peptide analog" or "modified peptide" is preferably defined as a FIR peptide modified to contain an amino group, an acetyl group, a hydrophobic group (for example a carbobenzoxy, dansyl or t-butyloxycarbonyl group) or a macromolecular carrier group (for example lipid conjugate, polyethylene glycol, carbohydrate or protein) at the N-terminus. An additional class of FIR peptide logs contains a carboxyl group, an amide group, a hydrophobic group, or a macromolecular carrier group at the C-terminus. Other peptide analogs are defined as FIR peptides, wherein at least one bond connecting adjacent amino acid residues is a non-peptide bond (e.g., imide, ester, hydrazine, semicarbazoid or azo linkage), a peptide in which at least one amino acid residue is in the isomer configuration D and a peptide in which the amino acid binding has been reversed. Additional peptide analogs are FIR peptides containing at least one amino acid substitution, wherein the first amino acid residue is substituted with a second other amino acid residue (amino acid substitution may be a conservative or non-conservative substitution). As used herein, such peptide analogs may contain analogous amino acid sequences in which the analogous sequences contain the majority of identical or chemically similar amino acids in the same order as in the original sequences.

[0025] As used herein, the term "fusion initiation region" (FIR) generally refers to a viral region of a fusion protein associated with the initial stage or steps of virus infection and / or fusion with a host cell.

[0026] As used herein, the term "peptide mimetic" includes, but is not limited to, organic compounds or other chemical compounds that mimic the structure or activity of a FIR peptide. Examples of peptide mimetics include, but are not limited to, organic compounds containing functional amino acid or peptide side groups but lacking a carbon / nitrogen backbone or peptide linkages. The peptide mimetic also relates to compounds that mimic the action of these functional side groups with other residues.

[0027] Other molecules such as idiotype or anti-idiotype antibodies or proteins selected by phage display methods that bind peptides, peptide analogs or peptide mimetics described in this application may also act as inhibitors of virus infection and / or virus fusion: cell . Also included in the present invention are plasmids or recombinant viruses or other molecules or compounds that enable or stimulate the patient to produce an analog of inhibitory compounds. For example, a recombinant protein produced in a modified bacterial, fungal or mammalian cell can be used to produce the immunogenic FIR analogue of a viral fusion protein. Similarly, an anti-idiotypic response can be induced in a subject using a modified protein containing a sequence corresponding to the binding site of an FIR-specific antibody.

[0028] As used herein, the term "fusion protein" preferably refers to a hydrophobic sequence at or near the N-terminus of a class I viral fusion protein (see,

Gallaher et al., 1987; 1992).

[0029] As used herein, the term "substantially purified" peptide or peptide analogue preferably refers to a peptide or peptide analogue that is more than about 80% pure. More preferably the term "substantially purified" refers to a peptide or peptide analogue that is more than about 90% or more than about 95% pure. More preferably it relates to a peptide or peptide analogue that is more than 96%, 97%, 98% or 99% pure. Functionally "substantially purified" means that it is free of impurities to the extent that it is suitable for the purposes provided herein. Methods for assessing purity are well known to those skilled in the art. Suitable methods include, but are not limited to, gas chromatography coupled mass spectrometry (GC), high performance liquid chromatography (HPLC) analysis, and functional tests in cell culture systems that, among other things, assess cytotoxicity.

[0030] As used herein, the term "stable analog" refers to a peptide that exhibits a half-life with pharmacological activity in biological systems. Biological half-lives of over 60 minutes are considered.

[0031] As used herein, the term "peptide derivative" refers to a peptide that contains substituted amino acids other than those in the FIR sequence of a viral fusion protein. However, substitutions do not render the peptide useless for the present invention.

[0032] In accordance with various aspects of the present embodiment, the peptides, isolated nucleic acid sequences or antibodies can be produced using any methods known in the art, including, but not limited to, chemical synthesis, recombinant DNA methods, and combinations thereof.

[0033] As defined herein, the present invention provides compositions and uses for treating or preventing influenza virus infection. One possible mechanism by which the present invention can prevent and / or inhibit infection is by interfering with the virus: cell fusion through FIR.

BRIEF DESCRIPTION OF THE FIGURES [0034] Figure 1 shows the domains of fusion proteins of one member of each of these six virus families (namely, arenaviruses, coronaviruses, filoviruses, orthomixoviruses, paramyxoviruses and retroviruses). The circles in Figure 1 show the approximate location of FIR in each virus shown.

[0035] Figures 2 to 7 show the amino acid sequences of these fusion proteins (from 9 corresponding to SEQ ID NO 16-21 respectively) and schematically show their ectopic structure. In particular, the five domains described above are a fusion peptide, i.e., N-helix, C-helix, aromatic motif (if present), and anchor peptide. The newly discovered sixth domain, the fusion initiation region, or FIR, was also identified. Each FIR is indicated using a polygon in Figures 2 to 7.

[0036] The area circled behind the fusion proteins in each of Figures 2-7 shows the primary virus: cell (VCBP) virus binding protein. VCBP usually interacts with the portion of the fusion protein that is most distant from the viral membrane, and is therefore depicted in the Figures as so located. Unlike the highly conserved fusion protein, the VCBP of each virus family is more variable. Typically, it is VCBP that dictates the range of virus hosts and determines which types of host cells are targeted. VCBP works in this respect by recognizing and binding to specific cell surface proteins. Binding of VCBP to target cell proteins occurs before and is usually a prerequisite for virus: cell fusion.

[0037] Figure 8: Inhibition of coronavirus infectivity by fusion initiation region peptides. Between 50 and 100 PFU of the murine hepatitis A59 strain or SARS strain of the Urbani coronavirus pre-incubated with or without the indicated peptides (~ 100 μΜ) in serum free DMEM for 1 hour. The cells were then exposed to a peptide-treated inoculum or control vehicle (without peptide). After 1 hour inoculum adsorption was removed, the cells were washed twice with 1X aqueous phosphate buffered saline and the cells were covered with DMEM containing 10% FBS and 0.5% agarose. Forty-eight hours after infection, single layers of infected cells were fixed and stained with crystal violet to determine plaque number.

[0038] Figure 9: Inhibition of Lassa virus infectivity by fusion initiation region peptides. Between 50 and 100 PFU of Lassa virus pre-incubated with or without the indicated peptides (~ 100 μΜ) in serum-free BME for 1 hour. The cells were then exposed to a peptide-treated inoculum or control vehicle (without peptide). After 1 hour inoculum adsorption was removed, the cells were washed twice with 1X aqueous phosphate buffered saline and the cells were covered with BME containing 5% FBS, 10 mM HEPES and 0.5% agarose. Four days after infection, a second layer containing 5% neutral red was applied and plaques were counted after 24 hours.

[0039] It is now known that six families of RNA viruses contain Class I membrane fusion proteins (Type I viruses), and representative members of each of these families are as follows:

Representative RNA viruses containing membrane fusion proteins of Class I (Type I viruses)

<td>Family</td><td>Example virus</td><td>Shown in the figures</td>

<td>arenaviruses</td><td>Lass virus</td><td>Yes</td>

<td></td><td>Lymphocytic meningitis virus (LCMV)</td><td>No</td>

<td></td><td>Junin virus</td><td>No</td>

<td></td><td>Machupo virus</td><td>No</td>

<td></td><td>Guanarito virus</td><td>No</td>

<td></td><td>Sabia virus</td><td>No</td>

<td>coronaviruses</td><td>Acute severe respiratory distress syndrome (SARS) virus</td><td>Yes</td>

<td></td><td>Mouse Hepatitis Virus (MHV)</td><td>No</td>

<td></td><td>Bovine coronavirus</td><td>No</td>

<td></td><td>Dog coronavirus</td><td>No</td>

<td></td><td>Feline infectious peritonitis virus</td><td>No</td>

<td>filoviruses</td><td>Ebola virus</td><td>Yes</td>

<td></td><td>Marburg virus</td><td>No</td>

<td>orthomyxoviruses</td><td>Influenza virus</td><td>Yes</td>

<td></td><td>Influenza B virus</td><td>No</td>

<td></td><td>C flu virus</td><td>No</td>

<td>paramyxoviruses</td><td>Measles virus</td><td>Yes</td>

<td></td><td>Mumps virus</td><td>No</td>

<td></td><td>Canine nasal virus</td><td>No</td>

<td></td><td>Newcastle disease virus</td><td>No</td>

<td>retroviruses</td><td>Human acquired immunodeficiency virus 1 (HIV-1)</td><td>Yes</td>

<td></td><td>Human acquired immunodeficiency virus 2 (HIV-2)</td><td>No</td>

<td></td><td>Human T-lymphotropic virus 1 (HTLV-1)</td><td>No</td>

<td></td><td>Human T-lymphotropic virus 2 (HTLV-2)</td><td>No</td>

<td></td><td>Human Intracisternal A-type Particle 1 (HIAP-1)</td><td>No</td>

<td></td><td>Human Intracisternal A-type Particle 2 (HIAP-2)</td><td>No</td>

The viruses shown in the Figures are as follows:

RNA viruses shown containing Class I membrane fusion proteins (Type I viruses)

<td>Figure</td><td>Family</td><td>Virus shown</td><td>Protein shown</td>

<td>Figure 2</td><td>arenaviruses</td><td>Lass virus</td><td>GP2</td>

<td>Figure 3</td><td>coronaviruses</td><td>SARS virus</td><td>S</td>

<td>Figure 4</td><td>filoviruses</td><td>Ebola virus</td><td>GP2</td>

<td>Figure 5</td><td>orthomyxoviruses</td><td>Influenza virus</td><td>HA2</td>

<td>Figure 6</td><td>paramyxoviruses</td><td>Measles virus</td><td>F1</td>

<td>Figure 7</td><td>retroviruses</td><td>HIV-1</td><td>TM</td>

List of sequences of presented fusion proteins with Class I membrane (Type I viruses) GP2 LASSA (Genbank accession no .: A43492, amino acids 257-490)

LLGT FTWTLSDSEG NETPGGYCLT RWMLIEAELK NEKHDEEFCD MLRLFDFNKQ AIRRLKTEAQ MSIQLINKAV KNHLRDIMGI PYCNYSRYWY LNHTSTGKTS LPRCWLISNG DIEQQADNMI TEMLQKKGPKFQQGQKGLQKLQKGKLQKLQKQKKLQTQQKKLY

CFGNTAYAKC NALINDQLIM SYLNETKFSD LISIFLHLYK (SEQ ID NO: 16)

S SARS (Genbank accession no .: AAQ9406, amino acids 864-1256)

WTF GAGAALQIPF KAISQIQESL TTTSTALGKL DILSRLDKVE AEVQIDRLIT MSECVLGQSK RYDFCGKGYH PAICHEGKAY FPREGVFVFN VIGIINNTVY DPLQPELDSF IQKEIDRLNE VAKNLNESKAC MYT

AMQMAYRFNG IGVTQNVLYE QDVVNQNAQA LNTLVKQLSS GRLQSLQTYV TQQLIRAAEI LMSFPQAAPH GVVFLHVTYV GTSWFITQRN FFSPQIITTD KEELDKYFKN HTSPQLYYKLY

NQKQIANQFN

NFGAISSYLN

RASANLAATK

PSQERNFTTA

NTFVSGNCDV

ISGINASWN

GFIAGLIAIV

KLHYT (SEQ ID NO: 17)

GP2 EBOLA (Genbank accession no .: AAM76034, amino acids 502-676)

EAIVNAQPK CNPNLHYWTT QDEGAAIGLA WIPYFGPAAE GIYTEGLMHN QDGLICGLRQ LANETTQALQ LFLRATTELR TFSILNRKAI DFLLQRWGGT CHILGPDCCI EPHDWTKNIT DKIDQIIQWFFGGY

HA2 FLU (Access no. In Genbank: P03437, amino acids 346-566)

GLFGA IAGFIENGWE GMIDGWYGFR HQNSEGTGQA ADLKSTQAAI DQINGKLNRV IEKTNEKFHQ IEKEFSEVEG RIQDLEKYVE DTKIDLWSYN AELLVALENQ HTIDLTDSEM NKLFEKTRRQ LRENAEKNGKSKG

F1 ODRY (Access no. In Genbank: VGNZMV, amino acids 116-553)

FAGW LAGAALGVAT AAQITAGIAL NQAIEAIRQA GQEMILAVQG VQDYINNELI RYYTEILSLF GPSLRDPISA EISIQALSYA GILESRGIKA RITHVDTESY FIVLSIAYPT SQEWYTTVPK YVATQGYLIS NFDESSCTFM CLRGSTKSCA RTLVSGSFGN RFILSQGNLI PDKILTYIAA DHCPWEVNG VTIQVGSRRY DVGTNLGNAI AKLEDAKELL ESSDQILRSM IGIPALICCC RGRCNKKGEQ VGMSRPGLKP

HQSMLNSQAI DNLRASLETT

PSMNQLSCDL IGQKLGLKLL

LGGDINKVLE KLGYSGGDLL

LSEIKGVTVH RLEGVSYNIG.

PEGTVCSQNA LYPMSPLLQE ANCASILCKC YTTGTIINQD PDAVYLHRID LGPPISLERL KGLSSTSIVY ILIAVCLGGL DLTGTSKSYV RSI »{SEQ ID NO: 20)

TM HIV (Genbank accession no .: AAB50262, amino acids 512-710)

AVGIGALFL GFLGAAGSTM GAASMTLTVQ ARQLLSGIVQ QQNNLLRAIE AQQHLLQLTV WGIKQLQARI LAVERYLKDQ QLLGIWGCSG KLICTTAVPW NASWSNKSLE QIWNHTTWME WDREINNYTS LIHSLIEESQ NQQEKNEQEL LELDKWASLW NWFNITNWLW YIKLFIMIVG GLVGLRIVFA VLSIVNRVRQ (SEQ ID NO: 21)

Identification method of FIR [0040] For reference purposes, the following method is described to identify a conserved motif in virus fusion proteins. The conservative themes of FIR regions from different viruses will have similar structure and operation. In addition, FIR regions of related viruses may or may not have highly similar amino acid sequences.

[0041] As described above, the present invention provides compositions useful in preventing or inhibiting viral infection with influenza virus using peptides or an isolated nucleic acid that are targeted to the specific FIR of the influenza virus and interfere with the operation of this FIR.

[0042] FIR of a viral fusion protein can be identified by the following method, which includes the following steps:

(1) The fusion protein sequence is first matched to the scaffold of the HIV fusion transmembrane protein, which contains the N-helix, C-helix and previously described domains, to identify the N-helix and C-helix in the subject fusion protein. This alignment process is facilitated by screening the primary amino acid sequence of the protein for two or more cysteines that have the ability to form at least one loop with covalent bonds that will be present in most but not all of these sequences. The N-helix can then be identified in the region preceding this cysteine ​​loop by examining the region for charged amino acids and other amino acids that have the ability to form an alpha helix (e.g.

glutamine (Q), alanine (A), tryptophan (W), lysine (K) and leucine (L)).

(2) Then the N-terminus of the FIR is identified on the N-helix. This end will usually lie in the final 10 to 20 N-helix amino acids and will contain a core usually containing three or four hydrophobic amino acids (such as leucine (L) or alanine (A)), a positively charged amino acid (such as lysine (K) or arginine (R)), a negatively charged amino acid (such as glutamate (E)) and an aromatic amino acid (such as tyrosine (Y)).

(3) The FIR C-terminus is then identified. For all families except coronaviruses and paramyxoviruses, this end is the C-terminus of the first peptide sequence with positive interfacial hydrophobicity which is downstream of the N-helix. This end is usually located after the cysteine ​​loop if this loop is present, and sometimes overlaps the C-helix or is located on Chelis. Sequences with positive interfacial hydrophobicity contain a high percentage of aromatic amino acids (such as tryptophan (W), phenylalanine (F) and tyrosine (Y)) and small hydrophobic amino acids (such as glycine (G)). The degree of interphase hydrophobicity of these sequences can be determined using a Wimley-White hydrophobicity scale, preferably using a computer program such as the MPEX program that includes this scale. ("Interphase Hydrophobicity" is a measure of the ability of a peptide to move from an aqueous solution to the interlayer of the membrane bilayer and is based on the experimentally established scale of the hydrophobicity of entire Wimley-White residues (Jaysinghe, Hristova and White, 2000). Computer programs using this scale can identify the peptide chain peptide sequence with a positive assessment of interfacial hydrophobicity, and therefore are most likely associated with membrane surfaces.) See Example 1 as an example of using this method to identify FIR in Ebola virus.

[0043] For coronaviruses that contain longer alpha helices and generally a larger scale, and paramyxoviruses in which FIR is discontinuous due to insertion from a non-FIR sequence, the FIR end of the FIR is the C-terminus of the second peptide sequence with positive hydrophobicity interphase, which is located behind the N-helix. The N-helix and C-helix sequence in the F1 paramyxovirus protein is longer than the inter-helical sequences of other Class I viral fusion proteins. The paramyxovirus F2 protein, which is used for receptor binding functions, is correspondingly shorter. After examining computer models for those skilled in the art, it is clear that the F1 protein contains a sequence insert between the N-helix and the C-helix. Consequently, FIR paramyxovir14 contains two cysteine ​​loops and two sequences with high interfacial hydrophobicity and is discontinuous due to the fact that amino acids that are characteristic only of paramyxoviruses and occur between the N-helix and the first sequence with high interfacial hydrophobicity, are excluded from FIR.

FIR SEQUENCES [0044] The fusion protein and FIR sequences for each of the six representative viruses shown in Figure 2 to Figure 7 are shown in the respective Figure and in the Sequence Listing below (SEQ ID NO: 16 to SEQ ID NO: 21 provide the corresponding proteins fusion; and SEQ ID NO: 1 to SEQ NO: 7 provide the corresponding

FIR). Although there is some sequence variation between the sister viruses in these six virus families, FIR in any Type I virus can be easily identified using the exemplary sequence given in the corresponding figure.

Methods for inhibiting the fusion of these viruses [0045] The present invention provides compositions as defined in the claims that inhibit virus: cell fusion by interfering with FIR. Various aspects of these forms include FIR-directed peptides and isolated nucleic acid sequences as defined herein to disrupt virus: cell fusion. In the present invention, the peptides contain the sequence SEQ ID NO: 4 or a segment thereof with 8 to 40 consecutive amino acids and are of such length as is required to provide effective inhibition of viral influenza infection. As used herein, the term "such length as is required to provide effective inhibition" of a virus preferably refers to a length sufficient to provide a 5-fold or greater reduction in virus infectivity when used in accordance with the present invention. Methods for quantifying the reduction of virus infectivity are well known to the person skilled in the art. For example, a reduction in viral activity can be determined by reducing the number of plaques, in binding inhibition assays, a lowering of titer or in animal challenge studies.

[0046] The FIR peptide of SEQ ID NO: 4 or fragments thereof considered to be part of the present invention are as defined in the claims. The following sequences are presented for comparison with SEQ ID NO: 4.

LASSA

X-LIMKNHLRDIMGIPYCNYSRYWYLNHTSTCKTLPRCWLI-Z (SEQ ID NO: 1).

SARS

X-LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPH GWFLHVTYVPSQERNFTTAPAICHEGKAYKPREGVFVFNGTSWFITQRNFFS-Z (SEQ ID N0: 2)

EBOLA

X-LRTFSILNRKAIDFLLQRWGGTCHILGPDCCI-Z (SEQ ID NO: 3)

FLU

X-IQDLEKYVEDTKIDLWSYNAELLVALENQHTIDSEMNKLF-Z (SEQ ID NO: 4)

MEASLES

X-LGLKLLRYYTEILSLFG-Z (SEQ ID NO: 5)

X-WYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQE CLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTn-Z (SEQ ID NO: 6) ("-" means that measles is not continuous).

HIV X-LQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLE

QIWNHTTWMEWD-Z (SEQ ID NO: 7)

In each of the above sequences, "X" and "Z" represent the amino or carboxy terminus of the peptide or an additional moiety, respectively, as described below.

[0047] The peptide of SEQ ID NO: 4 provided by the present invention comprises the sequence of the FIR region. The FIR region of the peptide of SEQ ID NO: 4 is derived from an influenza virus belonging to the viral orthomixovirus family, which includes influenza A virus, influenza B virus and influenza C virus.

[0048] Other aspects of this embodiment of the invention provide a segment of 8 to 40 consecutive amino20 acids of a peptide of SEQ ID NO: 4 containing a functional fragment of the FIR SEQ ID NO: 4 sequence from an influenza virus belonging to the orthomixovirus viral family, which include influenza A virus, influenza virus B and influenza C virus.

[0049] Peptide derivatives may contain altered sequences in which functionally equivalent amino acid residues are substituted by residues in the sequence, which causes a constant change. For example, one or more amino acid residues in the sequence may be substituted by another amino acid of the same polarity that acts as a functional equivalent, resulting in a silent change (e.g., leucine substitution with isoleucine). The amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In addition, for example, but not limited to, such peptides may also contain D-amino acids and / or may contain an inefficient carrier protein or no carrier protein at all.

[0050] FIR peptides may contain peptides in which "X" includes an amino group, an acetyl group, a hydrophobic group, or a macromolecular carrier group; and / or "Z" includes a carboxy group, an amide group, a hydrophobic group, or a macromolecular carrier group.

The "X" moiety may also be selected from the group consisting of: a hydrophobic moiety, a carbobenzoxy moiety, a dansyl moiety or a t-butyloxycarbonyl moiety. The "Z" moiety may be selected from the group consisting of: a hydrophobic moiety, a t-butylcarbonyl moiety.

[0051] The "X" moiety of the invention may contain a macromolecular carrier group. Such macromolecular carrier group may be selected from the group consisting of, but not limited to: lipid conjugate, polyethylene glycol moiety or carbohydrate moiety. Similarly, "Z" may also contain a macromolecular carrier group; wherein the macromolecular carrier is selected from the group consisting of, but not limited to: lipid conjugate, polyethylene glycol moiety or carbohydrate moiety.

[0052] One or more molecular bonds connecting adjacent amino acid residues may be a non-peptide bond. Such peptide bonds include, but are not limited to, imide, ester, hydrazine, semicarbazoid or azo linkages.

[0053] The peptide may further comprise one or more amino acid residues in the form of the D-isomer (s) of the amino acid.

[0054] The peptides may contain one or more amino acid substitutions, wherein the first amino acid residue is substituted with a second other amino acid residue, in the sequences provided above (or in their functional segment). In various aspects of this embodiment, the amino acid substitution is a conservative substitution. In other aspects of this embodiment, the amino acid substitution is a non-conservative substitution. Still other aspects of this embodiment of the invention provide the peptides described above, except that one or more residues have been deleted.

[0055] In various preferred aspects of the present embodiments, the FIR peptides of the invention contain at least 8 consecutive FIR residues. As used herein, the term "inhibitory FIR peptide (s)" preferably refers to a peptide or peptides containing the FIR sequence (or functional segment thereof) and such FIR peptides or functional segments in which one or more amino acids are / are substituted a functionally equivalent or chemically similar amino acid (see below). This also applies to derivatives of these peptides, including, but not limited to, benzylated derivatives, glycosylated derivatives and peptides that contain enantiomers of naturally occurring amino acids.

[0056] In yet other aspects of this embodiment of the invention, FIR peptides can be combined with a carrier molecule such as a protein, including but not limited to human serum albumin (HSA).

[0057] In addition, the present invention contemplates molecules comprising any combination of X and Z moieties and / or other peptide modifications described above.

[0058] The peptides of the present invention can be produced from naturally occurring or recombinant viral proteins. They can also be produced using standard recombinant DNA techniques (e.g., peptide expression by a microorganism containing a recombinant nucleic acid molecule encoding the desired peptide, expressed under the control of a suitable transcriptional promoter, and collecting the desired peptide from that microorganism). In a preferred aspect of the invention, any of the peptides of the invention may be prepared using chemical synthesis methods known in the art including, but not limited to, solid phase Merrifield synthesis (Clark-Lewis et al., 1986, Science 231: 134-139).

[0059] Embodiments of the present invention also provide antibodies as defined in the claims useful for treating or preventing a virus from infecting a cell. Antibodies contain their active segments, i.e. parts of the antibodies capable of specifically recognizing the FIR region or its functional segment. Antibodies specifically recognize the FIR peptide of SEQ ID NO: 4 or an antigenic fragment thereof to prevent or reduce cell infection by a virus. Antibodies of these embodiments of the invention may be monoclonal or polyclonal.

[0060] The invention provides a method as defined in the claims, for producing antibodies capable of specifically recognizing a FIR peptide of SEQ ID NO: 4, useful in preventing or reducing viral cell infection. General methods for making antibodies are well known to those skilled in the art. Methods for making the antibodies of the present invention include the steps of (i) providing an antigen comprising the FIR peptide of SEQ ID NO: 4;

(ii) exposing the animal's immune system to the antigen so as to elicit an immune response;

(iii) collecting antibodies from the animal and identifying those antibodies that specifically recognize the FIR (or functional segment thereof).

[0061] According to various aspects of the present invention, the peptides and / or antibodies of the present invention useful in treating or preventing infection of a cell by a virus can be targeted to the amino acids surrounding and in the FIR cysteine ​​loop in the distal portion of the FIR N-helix, any of the regions with FIR-phase hydrophobicity, in other FIR areas or in any of their combinations. These peptides (collectively compounds) can be used alone; otherwise they can be used in combinations of two or more compounds to prevent or inhibit viral cell infection. The methods of preventing or inhibiting viral cell infection by interfering with the FIR operation, provided by the present invention also include the use of neutralizing antibodies, exogenously or endogenously produced, against all or part of the FIR. The purpose of this application is to interfere with FIR, thereby inhibiting viral cell infection and / or virus: cell membrane fusion. [0062] Other embodiments of the present invention provide compositions, including pharmaceutical compositions, containing any or all of the peptides of the present invention. This includes, but is not limited to, compositions containing any molecule that contains, essentially consists of, or consists of a FIR peptide of SEQ ID NO: 4, or a functional FIR segment. This further includes, but is not limited to, compositions containing any compound that specifically recognizes, binds to or interferes with the activity of the viral FIR. As used herein, the term "interfering with FIR" means that the compound interacts with FIR or a cellular protein that serves as the FIR recognition receptor so as to prevent or reduce infection of the cell with the virus. In addition, it is contemplated that the compositions may contain one of the described molecules or a mixture of two or more of these molecules.

[0063] Further embodiments of the present invention provide uses of the compounds of the invention in treating or preventing infection of a cell by an influenza virus. Various aspects of this embodiment of the invention provide an effective amount of any of the pharmaceutical compositions described herein for use in administering to a patient suspected of being exposed to influenza virus (or who is likely to be exposed to influenza virus).

[0064] Still other aspects of this embodiment of the invention provide methods that comprise an effective amount of a composition comprising at least one recombinant DNA or RNA molecule; wherein the RNA or DNA encodes the FIR peptide of SEQ ID NO: 4 (or a functional segment thereof) to prevent or reduce influenza virus infection. In a preferred aspect of this embodiment, the recombinant RNA or DNA molecule and the pharmaceutical composition further comprise the elements necessary to allow expression of the protein encoded by the RNA or DNA molecule in a human cell. For example, but without limiting the scope of the invention, in certain aspects of this embodiment of the invention, the recombinant RNA or DNA molecule is part of a recombinant plasmid or recombinant virus.

EXAMPLES

Example 1: FIR Identification in Ebola Virus [0065] A method for identifying FIR FIR Class I fusion proteins can be illustrated by two examples. The first example is the identification of FIR in the minimal fusion protein Class I glycoprotein 2 (GP2) of the Ebola virus, filovirus. The N-helix and Chelisa GP2 limits of the Ebola virus were determined by X-ray crystallographic methods (Malashkevich et al., 1999). The terminal amino acids in the N-helix contain the ILNRKAIDF sequence (SEQ ID NO: 8), which fits into a consensus core containing three to four hydrophobic amino acids, a positively charged amino acid, a negatively charged amino acid, and an aromatic amino acid. Between these two helixes there are two cysteines in the CHILGPDC sequence (SEQ ID NO: 9). The FIR GP2 ends of the Ebola virus are defined by the FLLQRWGGTCHILGPDCCI sequence (SEQ ID NO: 10), assessing the Wimley-White 2.59 interphase hydrophobicity as determined by the MPEX program (Jaysinghe et al., 2002). Thus, FIR GP2 of Ebola virus includes amino acids 579 to 610.

Example 2: FIR identification in measles virus [0066] The second example is a complex Class I fusion protein, measles virus virus paramyxovirus F1. The measles virus N- and C-helix F1 can be identified by examining the primary sequence for amino acids prone to helix formation. Aligning the primary sequence of the measles virus F1 with the primary amino acid sequence of another paramyxovirus F1 protein, Newcastle disease virus F1, can also help identify helix boundaries. The structure of Newcastle disease virus F1 protein was determined by X-ray crystallographic methods (Chen et al., 2001). Thus, the boundaries of N- and C-helices can be predicted within amino acids 131 217 and 455-491, respectively. Unlike Ebola GP2 and most other Class I fusion proteins, the primary sequence between N and C helices in the measles virus is longer than 100 amino acids. The FIR F1 region of the measles virus contains an insert that, when examined using computer models, has become apparent to a person skilled in the art, and therefore the FIR structure is formed by a secondary arrangement that connects two parts of the primary sequence together. The inserted sequence forms an outer loop relative to FIR. N-helix terminal amino acids contain the sequence

LKLLRYYTE (SEQ ID NO: 11), which matches the core consensus containing three or four hydrophobic amino acids, a positively charged amino acid, a negatively charged amino acid, and an aromatic amino acid. There are eight cysteine ​​residues between the N and C helices in measles virus F1. Based on the match with Newcastle disease virus F1, it can be determined that the first two cysteines and the second two cysteines form loops connected by disulfide bridges: The first pair of cysteines in the sequence,

CTFMPEGTVC (SEQ ID NO: 12), is part of the FIR because it is bound by the sequence WYTTVPKYVATQGYLISNF (SEQ ID NO: 13) with an assessment of Wimley-White interphase hydrophobicity 3.36, which was determined using the MPEX program. The second pair of cysteines in the sequence, CLRGSTKSC (SEQ ID NO: 14), is also part of the FIR because it adheres to the sequence

TLVSGSFGNRFILSQGNLIANCASILCKCYTTGTII (SEQ ID NO: 15) with the assessment of Wimley-White 2.54 interphase hydrophobicity, which was determined using the MPEX program. Thus FIR FIR of the measles virus extends from amino acid 205 to 407, with amino acids 221 to 314 being an insert that does not participate in FIR.

Example 3: Identification of peptides that inhibit coronavirus fusion.

Background [0067] Acute severe respiratory distress syndrome (SARS) is a newly diagnosed disease spreading from southern China in late 2002 / early 2003 to several Asian, European and North American countries (Guan et al., 2004). SARS usually begins with a fever higher than 38 ° C. Initial symptoms may also include headache, malaise and mild breathing symptoms. Within two days to a week, patients with SARS may develop a dry cough and breathing problems. Patients in the more advanced stages of SARS develop pneumonia or respiratory distress syndrome. At the initial stage of the outbreak, 8098 cases were reported worldwide, with an overall mortality rate of 9.6%. It has been shown that the cause of the new disease is previously unrecognized coronavirus (CoV) (Poutanen et al., 2003; Peiris et al., 2003; Drosten et al., 2003; Rota et al., 2003; Mara et al., 2003). Public healthcare interventions, such as surveillance supervision, travel restrictions and quarantine, included the initial spread of SARS CoV in 2003 and it was again found that the spread of SARS stopped after several new cases in 2004. However, it is not known if these draconian security measures can be maintained whenever SARS CoV appears in humans. In addition, the potential of this new and sometimes deadly CoV as a bioterror threat is obvious.

[0068] Coronaviruses are large RNA viruses with a positive polarization strand with a wide range of hosts. Like other CoV enveloped viruses, they enter target cells by fusing between viral and cell membranes, in which viral spike (S) protein participates. It turns out that the CoV proteins characterized so far contain two non-covalently bound subunits, S1 and S2. Using computer analysis, Garry and Gallaher (2003) first proposed that the portion of the SARS-CoV S protein corresponding to the S2 subunit fits the prototype model of a Class I viral fusion protein based on the presence of two predicted alpha helical regions in the N- and C-terminal S2 regions (N-helix, C-helix) and the region rich in aromatic amino acids immediately preceding the membrane anchoring domain.

Materials and methods [0069] L2 cells or Vero E6 cells were maintained as single layers in Dulbecco's complete Eagle Modified Medium (DMEM) containing 0.15% HCO3^- supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U / ml), streptomycin (100 mg / ml) and 2mM L-glutamine at 37 ° C in a 5% CO2 incubator. The murine hepatitis A (MHV) A59 strain or SARS CoV Urbani or HK strain was grown on L2 cells. For plaque tests, L2 cells or Vero E6 cells were plated at a density of 1x10⁶ in each well of a 6-well plate.

Fifty to 100 plaque forming units (pfu) MHV or SARS CoV were preincubated with or without approximately 100 μg / ml peptide in serum free DMEM for 1 hour. The cells were then infected with the inoculum treated with the peptide or vehicle as a control inoculum. After 1 hour inoculum adsorption was removed and the cells washed twice with 1X phosphate buffered saline and the cells covered with 10%

FBS / DMEM containing 0.5% SEAPLAQUE® agarose (Cambrex Bio Science Rockland inc., Rockland, ME). Single layers were fixed with 3.7% formalin and stained 1X with crystal violet 2 days after infection and plaque numbers were determined by optical microscopy.

Results and discussion [0070] Synthetic peptides corresponding to the FIR domains of MHV S protein or SARS CoV were tested for their ability to inhibit infection with these coronaviruses. The ability to inhibit plaque formation in single cell layers is the most stringent in vitro test for a potential infection-inhibiting drug. Two peptides (GNHILSLVQNAPYGLYFIHFSW, SEQ ID NO: 22 and

GYFVQDDGEWKFTGSSYYY, SEQ ID NO: 23) from FIR MHV can inhibit MHV plaque formation, although the first MHV FIR peptide is more effective (see Fig. 8A). Two peptides from FIR SARS, CoV (GYHLMSFPQAAPHGVVFLHVTY, SEQ ID NO: 24 and GVFVFNGTSWFITQRNFFS, SEQ ID NO: 25) inhibited plaque formation by this coronavirus (see Fig. 8B). A significant reduction (~ 50%) in the average diameter of the other plaques was also observed. These results suggest that this peptide inhibits both entry and spread of MHV. Similar results with these inhibitory peptides were obtained in independent experiments in which 50% inhibition of plaque formation was observed at concentrations &lt;5 μΜ. These results cannot be explained by the nonspecific cytotoxic effects of these peptides. With the exception of plaques, the cells in single layers were intact and viable. The small number of plaques growing was very similar in size to control plaques. Peptides from other regions also inhibited infection with these viruses, but to a lesser extent than those most active FIR peptides (Fig. 8). For example, peptides from the fusion peptide region and the C-terminal helix (C-helix) of S MHV and S SARS CoV provided some inhibition (S MHV fusion peptide = MFPPWSAAAGVPFSLSVQY, SEQ ID NO: 26; C-helix S MHV = QDAIKKLNESYINLKEVWTYQ NO: 27; S SARS CoV fusion peptide = MYKTPTLKYFGGFNFSQIL, SEQ ID NO: 28; C-helix S SARS CoV = AACEVAKNLNESLIDLQELGKYEQYIKW, SEQ ID NO: 29. Inhibitory activities in the μΜ range have been recently described for coronavirus C-helix peptides by Bosch et al. (2003) and others (Bosch et al., 2004; Lui et al., 2004; Yuan et al., 2004; Zhu and et al., 2004). However, inhibitory FIR coronavirus peptides have not been described. However, from the point of view of the present invention, the cited references collectively provide confirmation of the enormous advantages of the disclosed and claimed inventions herein.

That is, these references are in line with the inventors' assertion that the methods of the present invention can be advantageously used to identify synthetic peptides that inhibit fusion / infectivity of members of the Coronaviridae family. Example 4: Identification of peptides that inhibit arenavirus fusion.

Background [0071] Lassa fever is often a deadly hemorrhagic disease, which was named after the city in the Yedseram River Valley in Nigeria, in which the first described cases occurred in 1969 (Buckley and Casals, 1970). Parts of Guinea, Sierra Leone, Nigeria and Liberia are endemic to this etiological factor, the Lassa virus (LasV). The impact of Las V on the health status of society in endemic areas is enormous. Centers for disease control and prevention ( Centers for Diseases Control, and Prevention; CDC) estimated that there are 100,000300,000 Lass cases and 5,000 deaths annually in West Africa. In some parts of Sierra Leone, 1015% of all patients admitted to hospitals suffer from Lassa fever. The mortality rate for Lassa fever is usually 15% to 20%, however, in the case of an epidemic, the overall mortality rate can be as high as 45%. The mortality rate for women in the last month of pregnancy is always high, ~ 90%, and Las V infection causes a high percentage of fetal death at all stages of pregnancy. It turns out that the mortality rate for Lass is higher in non-Africans, which is worrying because Lassa is the most commonly exported hemorrhagic fever. Due to the high mortality rate and the ability to easily transfer through human-human contact, Las V has been classified as a factor of Biosafety Level 4 and NIAID Biodefense category.

[0072] LasV is a member of the Arenaviridae family. The genome of arenaviruses consists of two segments of single-stranded, ambisense RNA. When analyzed by transmission electron microscopy, enveloped spherical virions (diameter: 110-130 nm) show granular particles that are ribosomes acquired from host cells (Murphy and Whitfield, 1975). Thus for this family of viruses the Latin name "arena" was used, which means "sandy". In addition to LasV, other arenaviruses that cause human disease include the Junin virus (Argentine hemorrhagic fever), Machupo virus (Bolivian hemorrhagic fever), Guanarito virus (Venezuelan hemorrhagic fever) and the Sabia virus (Brazilian hemorrhagic fever). Arenaviruses are zoonotic; each virus is associated with a specific rodent species (Bowen, Peters and Nichol, 1997). The "Mastonays" multi-mammal rat is the reservoir for LasV (Monath et al., 1974). The widespread occurrence of Mastomys in Africa makes extinction of this rodent as a reservoir impractical and ecologically undesirable.

[0073] Signs and symptoms of Lassa fever that occur 1-3 weeks after exposure to the virus are highly variable, but may include fever, retrosternal, back and abdominal pain, sore throat, coughing, vomiting, diarrhea, conjunctival hyperemia and swelling of the face. Forest V infects endothelial cells, resulting in increased capillary capacity, reduced effective circulating blood volume, shock and systemic multiorgan failure. Overt bleeding, usually mucosal (gums, etc.) occurs in less than a third of cases, but it carries a poor prognosis. Neurological problems have also been described, including hearing loss, tremor and encephalitis. Patients who survive the fever begin to subside 2-3 weeks after the onset of the disease. Deafness is the most common complication associated with Lassa fever. Transient or permanent one-sided or double-sided deafness occurs in ~ 30% of patients with Lassa fever during convalescence and is not associated with the severity of the acute illness. The ribavirin antiviral drug is effective in the treatment of Lassa fever, but only when given early (up to six days) in the course of the disease (Johnson et al., 1987; McCormick et al., 1986). It is unknown whether ribavirin is effective against other arenaviruses such as Junin, Machupo, Guanarito or Sabiά viruses. No LasV vaccine is currently available.

Materials and methods [0074] Vero cells were maintained as single layers in basic Eagel medium (BME) containing 10 mM HEPES and 5% FBS. Lassa virus (Josiah strain) was propagated on Vero cells. For plaque tests, Vero cells were plated at a density of 1x10⁶ cells for each well of a 6-well plate. Fifty to 100 pfu La20 sV pre-incubated with or without peptide in serum-free BME for 1 hour. The cells were then infected with the inoculum treated with the peptide or vehicle as a control inoculum. After 1 hour inoculum adsorption was removed and the cells were washed twice with 1X aqueous phosphate buffered saline and the cells were covered with 2 ml 0.5% agarose in BME containing 10 mM HEPES and 5% FBS and incubated for 4 days. A second layer containing 5% neutral red was applied and the plaques were counted 24 hours later.

Results and discussion [0075] The synthetic peptides corresponding to the FIR domains of glycoprotein 2 (GP2) Las V were tested for their ability to inhibit infection with this arenavirus. Peptide (NYSKYWYLNHTTGR, SEQ ID NO: 30) analogous to the sequence

NYSRYWYLNHTSTGK with SEQ ID NO: 1 (FIR LASSA) can inhibit forest V plaque formation (Fig. 9). An peptide analogous to another GP2 region, a fusion peptide, (GTFTWTLSDSEGKDTPGGY, SEQ ID NO: 31) also inhibited LasV infection, but to a lesser extent (Fig. 9). So far, inhibitory p Arenaviruses have not been described. Collectively, these results suggest that the inventors' approach identifies synthetic peptides that inhibit fusion / infectivity of members of the Arenaviridae family. These results, in conjunction with the inventors' results regarding coronavirus inhibitory FIR peptides, establish the principle that FIR region peptides can act as virus inhibitors.

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SEQUENCE LIST [0077] &lt;110&gt; Garry, Jr., Robert F.

Wilson, Russell B.

&lt;120&gt; HOW TO PREVENT VIRUS FUSION: A CELL BY INHIBITION OF THE ACTION OF FUSION INITIATION IN RNA VIRUSES CONTAINING FUZOGENIC MEMBRANE PROTEINS OF CLASS I &lt;130&gt; 12920.0013.00PC00 &lt;15&gt; US 60-115 &lt;170&gt; PatentIn version 3.3 &lt;210&gt; 1 &lt;211&gt; 39 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 1

Leu Ile Met Lys Asn His Leu Arg Asp Ile Met Gly Ile Pro Tyr Cys 15 10 15

Asn Tyr Ser Arg Tyr Trp Tyr Leu Asn His Thr Ser Thr Gly Lys Thr 20 25 30

Leu Pro Arg Cys Trp Leu Ile 35 &lt;210&gt; 2 &lt;211&gt; 100 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 2

<td>Leu 1</td><td>How much</td><td>Arg</td><td>ala</td><td>ala 5</td><td>Glu</td><td>How much</td><td>Arg</td><td>ala</td><td>Cheese 10</td><td>ala</td><td>own</td><td>Leu</td><td>ala</td><td>ala 15</td><td>Thr</td>

<td>lys</td><td>Underworld</td><td>Cheese</td><td>Glu twenty</td><td>Cys</td><td>val</td><td>Leu</td><td>Gly</td><td>Gin 25</td><td>Cheese</td><td>lys</td><td>Arg</td><td>val</td><td>asp thirty</td><td>phe</td><td>Cys</td>

<td>Gly</td><td>lys</td><td>Gly 35</td><td>Tyr</td><td>His</td><td>Leu</td><td>Underworld</td><td>Cheese 40</td><td>phe</td><td>Pro</td><td>Gin</td><td>ala</td><td>ala 45</td><td>Pro</td><td>His</td><td>Gly</td>

<td>val</td><td>val 50</td><td>phe</td><td>Leu</td><td>His</td><td>val</td><td>Thr 55</td><td>Tyr</td><td>val</td><td>Pro</td><td>Cheese</td><td>Gin 60</td><td>Glu</td><td>Arg</td><td>own</td><td>phe</td>

<td>Thr 65</td><td>Thr</td><td>ala</td><td>Pro</td><td>ala</td><td>How much 70</td><td>Cys</td><td>His</td><td>Glu</td><td>Gly</td><td>lys 75</td><td>ala</td><td>Tyr</td><td>phe</td><td>Pro</td><td>Arg 80</td>

Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gin Arg 85 90 95

Asn Phe Phe Ser 100 &lt;210&gt; 3 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 3

Leu Arg Thr Phe Cheese Ile Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu 15 10 15

Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys Cys Ile 20 25 30 &lt;210&gt; 4 &lt;211&gt; 43 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 4

<td>How much 1</td><td>Gln</td><td>asp</td><td>Leu</td><td>Glu 5</td><td>lys</td><td>Tyr</td><td>val</td><td>Glu</td><td>asp 10</td><td>Thr</td><td>lys</td><td>How much</td><td>asp</td><td>Leu 15</td><td>Trp</td>

<td>Cheese</td><td>Tyr</td><td>own</td><td>ala twenty</td><td>Glu</td><td>Leu</td><td>Leu</td><td>val</td><td>ala 25</td><td>Leu</td><td>Glu</td><td>own</td><td>Gln</td><td>His thirty</td><td>Thr</td><td>How much</td>

<td>asp</td><td>Leu</td><td>Thr 35</td><td>asp</td><td>Cheese</td><td>Glu</td><td>Underworld</td><td>own 40</td><td>lys</td><td>Leu</td><td>phe</td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 5 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 5

Leu Gly Leu Lys Leu Leu Arg Tyr Tyr Thr Glu Ile Leu Ser Leu Phe 15 10 15

Gly &lt;210&gt; 6 &lt;211&gt; 94 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 6

<td colspan="5">Trp Tyr Thr Thr Val</td><td colspan="2" rowspan="2">Pro Lys</td><td colspan="9">Tyr Val Ala Thr Gin Gly Tyr Leu Ile</td>

<td colspan="2"> 1</td><td colspan="3"> 5</td><td colspan="4"> 10</td><td colspan="5"> 15</td>

<td>Cheese</td><td>own</td><td>phe</td><td>asp</td><td>Glu</td><td>Cheese</td><td>Cheese</td><td>Cys</td><td>Thr</td><td>phe</td><td>Underworld</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Thr</td><td>val</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Cys</td><td>Cheese</td><td>Gin</td><td>own</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Underworld</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Leu</td><td>Gin</td><td>Glu</td><td>Cys</td>

40 45

<td colspan="2">Leu Arg</td><td colspan="2" rowspan="2">Gly Cheese</td><td colspan="2" rowspan="2">Thr Lys</td><td colspan="3" rowspan="2">Cys Ala 55 cheese</td><td rowspan="2">Arg</td><td colspan="3" rowspan="2">Thr Leu Val 60</td><td rowspan="2">Cheese</td><td colspan="2" rowspan="2">Gly Cheese</td>

<td></td><td> 50</td>

<td>phe</td><td>Gly</td><td>own</td><td>Arg</td><td>phe</td><td>How much</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>Gly</td><td>own</td><td>Leu</td><td>How much</td><td>ala</td><td>own</td><td>Cys</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>ala</td><td>Cheese</td><td>How much</td><td>Leu</td><td>l Cys</td><td>lys</td><td>Cys</td><td>Tyr</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Thr</td><td>How much</td><td>How much</td><td></td><td></td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 7 &lt;211&gt; 57 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 7

<td>Leu</td><td>Gin</td><td>ala</td><td>Arg</td><td>How much</td><td>Leu</td><td>ala</td><td>val</td><td>Glu</td><td>Arg</td><td>Tyr</td><td>Leu</td><td>lys</td><td>asp</td><td>Gin</td><td>Gin</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Leu</td><td>Leu</td><td>Gly</td><td>How much</td><td>Trp</td><td>Gly</td><td>Cys</td><td>Cheese</td><td>Gly</td><td>lys</td><td>Leu</td><td>How much</td><td>Cys</td><td>Thr</td><td>Thr</td><td>ala</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>val</td><td>Pro</td><td>Trp</td><td>own</td><td>ala</td><td>Cheese</td><td>Trp</td><td>Cheese</td><td>own</td><td>lys</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Gin</td><td>How much</td><td>Trp</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>own</td><td>His</td><td>Thr</td><td>Thr</td><td>Trp</td><td>Underworld</td><td>Glu</td><td>Trp</td><td>asp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 8 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 8

Ile Leu Asn Arg Lys Ala Ile Asp Phe

5 &lt;210&gt; 9 &lt;211&gt; 8 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 9

Cys His Ile Leu Gly Pro Asp Cys 1 5 &lt;210&gt; 10 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 10

Phe Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp 15 10 15

Cys Cys Ile &lt;210&gt; 11 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 11

Leu Lys Leu Leu Arg Tyr Tyr Thr Glu 1 5 &lt;210&gt; 12 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 12

Cys Thr Phe Met Pro Glu Gly Thr Val Cys 15 10 &lt;210&gt; 13 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 13

Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala Thr Gln Gly Tyr Leu Ile 15 10 15

Asn Phe cheese &lt;210&gt; 14 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 14

Cys Leu Arg Gly Ser Thr Lys Ser Cys 1 5 &lt;210&gt; 15 &lt;211&gt; 36 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 15

<td>Thr</td><td>Leu</td><td>val</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td>phe</td><td>Gly Asn</td><td>Arg</td><td>phe</td><td>How much</td><td>Leu</td><td>Cheese</td><td>Gln</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>own</td><td>Leu</td><td>How much</td><td>ala</td><td>own</td><td>Cys</td><td>ala</td><td>Ile cheese</td><td>Leu</td><td>Cys</td><td>lys</td><td>Cys</td><td>Tyr</td><td>Thr</td><td>Thr</td>

25 30

Gly Thr Ile Ile 35 &lt;210&gt; 16 &lt;211&gt; 234 &lt;212&gt; PRT &lt;213&gt; WIRUS LASSA &lt;400&gt; 16

Leu Leu Gly Thr Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly Asn Glu 15 10 15

Thr Pro Gly Gly Tyr Cys Leu Thr Arg Trp Met Leu Ile Glu Ala Glu 20 25 30

Leu Lys Cys Phe Gly Asn Thr Ala Val Ala Lys Cys Asn Glu Lys His 35 40 45

<td>asp</td><td>Glu 50</td><td>Glu</td><td>phe</td><td>Cys</td><td>asp</td><td>Underworld 55</td><td>Leu</td><td>Arg</td><td>Leu</td><td>phe</td><td>asp 60</td><td>phe</td><td>own</td><td>lys</td><td>Gln</td>

<td>ala 65</td><td>How much</td><td>Arg</td><td>Arg</td><td>Leu</td><td>lys 70</td><td>Thr</td><td>Glu</td><td>ala</td><td>Gln</td><td>Underworld 75</td><td>Cheese</td><td>How much</td><td>Gln</td><td>Leu</td><td>How much 80</td>

<td>own</td><td>lys</td><td>ala</td><td>val</td><td>own</td><td>ala</td><td>Leu</td><td>How much</td><td>own</td><td>asp</td><td>Gln</td><td>Leu</td><td>How much</td><td>Underworld</td><td>lys</td><td>own</td>

90 95

His Leu Arg Asp Ile Met Gly Ile Pro Tyr Cys Asn Tyr Ser Arg Tyr 100 105 110

Trp Tyr Leu Asn His Thr Ser Thr Gly Lys Thr Ser Leu Pro Arg Cys

115 120 125

Trp Leu Ile Ser Asn Gly Cheese Tyr Leu Asn Glu Thr Lys Phe Cheese Asp 130 135 140

Asp Ile Glu Gln Gln Ala Asp Asn Met Ile Thr Glu Met Leu Gln Lys

145 150 155 160

Glu Tyr Ile Asp Arg Gln Gly Lys Thr Pro Leu Gly Leu Val Asp Leu

165 170 175

Phe Val Phe Ser Thr Ser Phe Tyr Leu Ile Ser Ile Phe Leu His Leu 180 185 190

Val Lys Ile Pro Thr His Arg His Ile Val Gly Lys Pro Cys Pro Lys 195 200. 205

Pro His Arg Leu Asn His Met Gly Ile Cys Ser Cys Gly Leu Tyr Lys 210 215 220

Gln Pro Gly Val Pro Val Arg Trp Lys Arg

225 230 &lt;210&gt; 17 &lt;211&gt; 388 &lt;212&gt; PRT &lt;213&gt; WIRUS SARS &lt;400&gt; 17

Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln

5 10 15

Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr

25 30

Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Cheese Gln 35 40 45

Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln 50 55 60

Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln

70 75 80 '

Leu Cheese Cheese Asn Phe Gly Ala Ile Cheese Cheese Val Leu Asn Asp Ile Leu

90 95

Cheese Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile 100 105 110

Thr Gly Arg Leu Gln Cheese Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile 115 120 125

Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met 130 135 140

Cheese Glu Cys Val Leu Gly Gln Cheese Lys Arg Val Asp Phe Cys Gly Lys

145 150 155 160

Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val

165 170 175

<td>phe</td><td>Leu</td><td>His</td><td>val 180</td><td>Thr</td><td>Tyr</td><td>val</td><td>Pro</td><td>Cheese 185</td><td>Gln</td><td>Glu</td><td>Arg</td><td>own</td><td>phe 190</td><td>Thr</td><td>Thr</td>

<td>ala</td><td>Pro</td><td>ala 195</td><td>How much</td><td>Cys</td><td>His</td><td>Glu</td><td>Gly 200</td><td>lys</td><td>ala</td><td>Tyr</td><td>phe</td><td>Pro 205</td><td>Arg</td><td>Glu</td><td>Gly</td>

<td>val</td><td>phe 210</td><td>val</td><td>phe</td><td>own</td><td>Gly</td><td>Thr 215</td><td>Cheese</td><td>Trp</td><td>phe</td><td>How much</td><td>Thr 220</td><td>Gln</td><td>Arg</td><td>own</td><td>phe</td>

<td>phe 225</td><td>Cheese</td><td>Pro</td><td>Gln</td><td>How much</td><td>How much 230</td><td>Thr</td><td>Thr</td><td>asp</td><td>own</td><td>Thr 235</td><td>phe</td><td>val</td><td>Cheese</td><td>Gly</td><td>own 240</td>

<td>Cys</td><td>asp</td><td>val</td><td>val</td><td>How much 245</td><td>Gly</td><td>How much</td><td>How much</td><td>own</td><td>own 250</td><td>Thr</td><td>val</td><td>Tyr</td><td>asp</td><td>Pro 255</td><td>Leu</td>

<td>Gln</td><td>Pro</td><td>Glu</td><td>Leu 260</td><td>asp</td><td>Cheese</td><td>phe</td><td>lys</td><td>Glu 265</td><td>Glu</td><td>Leu</td><td>asp</td><td>lys</td><td>Tyr 270</td><td>phe</td><td>lys</td>

<td>own</td><td>His</td><td>Thr 275</td><td>Cheese</td><td>Pro</td><td>asp</td><td>val</td><td>asp 280</td><td>Leu</td><td>Gly</td><td>asp</td><td>How much</td><td>Cheese 285</td><td>Gly</td><td>How much</td><td>own</td>

<td>ala</td><td>Cheese 290</td><td>val</td><td>val</td><td>own</td><td>How much</td><td>Gln 295</td><td>lys</td><td>Glu</td><td>How much</td><td>asp</td><td>Arg 300</td><td>Leu</td><td>own</td><td>Glu</td><td>val</td>

<td>ala 305</td><td>lys</td><td>own</td><td>Leu</td><td>own</td><td>Glu 310</td><td>Cheese</td><td>Leu</td><td>How much</td><td>asp</td><td>Leu 315</td><td>Gln</td><td>Glu</td><td>Leu</td><td>Gly</td><td>lys 320</td>

<td>Tyr</td><td>Glu</td><td>Gln</td><td>Tyr</td><td>How much 325</td><td>lys</td><td>Trp</td><td>Pro</td><td>Trp</td><td>Tyr 330</td><td>val</td><td>Trp</td><td>Leu</td><td>Gly</td><td>phe 335</td><td>How much</td>

<td>ala</td><td>Gly</td><td>Leu</td><td>How much 340</td><td>ala</td><td>How much</td><td>val</td><td>Underworld</td><td>val 345</td><td>Thr</td><td>How much</td><td>Leu</td><td>Leu</td><td>Cys 350</td><td>Cys</td><td>Underworld</td>

<td>Thr</td><td>Cheese</td><td>Cys 355</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>Leu</td><td>lys 360</td><td>Gly</td><td>ala</td><td>Cys</td><td>Cheese</td><td>Cys 365</td><td>Gly</td><td>Cheese</td><td>Cys</td>

<td>Cys</td><td>lys 370</td><td>phe</td><td>asp</td><td>Glu</td><td>asp</td><td>asp 375</td><td>Cheese</td><td>Glu</td><td>Pro</td><td>val</td><td>Leu 380</td><td>lys</td><td>Gly</td><td>val</td><td>lys</td>

Leu His Tyr Thr 385 &lt;210&gt; 18 &lt;211&gt; 175 &lt;212&gt; PRT &lt;213&gt; WIRUS EBOLA &lt;400&gt; 18

Glu Ala Ile Val Asn Ala Gln Pro Lys Cys Asn Pro Asn Leu His Tyr 15 10 15

Trp Thr Thr Gln Asp Glu Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro 20 25 30

Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Thr Glu Gly Leu Met His 35 40 45

Asn Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln Leu Ala Asn Glu Thr 50 55 60

Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr Thr Glu Leu Arg Thr 65 70 75 80

Phe Cheese Ile Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu Gln Arg Trp 85 90 95

Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys Cys Ile Glu Pro His 100 105 110

Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile His Asp 115 120 125

Phe Val Asp Lys Thr Leu Pro Asp Gln Gly Asp Asn Asp Asn Trp Trp 130 135 140

Thr Gly Trp Arg Gln Trp Ile Pro Ala Gly Ile Gly Val Thr Gly Val 145 150 155 160

Ile Ile Ala Val Ile Ala Leu Phe Cys Ile Cys Lys Phe Val Phe 165 170 175 &lt;210&gt; 19 &lt;211&gt; 191 &lt;212&gt; PRT &lt;213&gt; FLU VIRUS &lt;400&gt; 19

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

25 30

Gly Gln Ala Ala Asp Leu Lys Cheese Thr Gln Ala Ala Ile Asp Gln Ile 35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55. 60

Gln Ile Glu Lys Glu Phe Cheese Glu Val Glu Gly Arg Ile Gln Asp Leu

70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp 100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu 115 120 125

Asn Ala Glu Glu Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 130 135 140

Asp Asn Ala Cys Ile Glu Cheese Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Cheese Gly Tyr Lys Asp Trp Arg Cys Asn Ile Cys Ile 180 185 190 &lt;210&gt; 20 &lt;211&gt; 438 &lt;212&gt; PRT &lt;213&gt; ODRA VIRUS &lt;400&gt; 20

<td>phe 1</td><td>ala</td><td>Gly</td><td>val</td><td>val 5</td><td>Leu</td><td>ala</td><td>Gly</td><td>ala</td><td>ala 10</td><td>Leu</td><td>Gly</td><td>val</td><td>ala</td><td>Thr 15</td><td>ala</td>

<td>ala</td><td>Gln</td><td>How much</td><td>Thr twenty</td><td>ala</td><td>Gly</td><td>How much</td><td>ala</td><td>Leu 25</td><td>His</td><td>Gln</td><td>Cheese</td><td>Underworld</td><td>Leu thirty</td><td>own</td><td>Cheese</td>

<td>Gln</td><td>ala</td><td>How much 35</td><td>asp</td><td>own</td><td>Leu</td><td>Arg</td><td>ala 40</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Thr</td><td>Thr 45</td><td>own</td><td>Gln</td><td>ala</td>

<td>How much</td><td>Glu 50</td><td>ala</td><td>How much</td><td>Arg</td><td>Gln</td><td>ala 55</td><td>Gly</td><td>Gln</td><td>Glu</td><td>Underworld</td><td>How much 60</td><td>Leu</td><td>ala</td><td>val</td><td>Gln</td>

<td>Gly 65</td><td>val</td><td>Gln</td><td>asp</td><td>Tyr</td><td>How much 70</td><td>own</td><td>own</td><td>Glu</td><td>Leu</td><td>How much 75</td><td>Pro</td><td>Cheese</td><td>Underworld</td><td>own</td><td>Gln 80</td>

<td>Leu</td><td>Cheese</td><td>Cys</td><td>asp</td><td>Leu 85</td><td>How much</td><td>Gly</td><td>Gln</td><td>lys</td><td>Leu 90</td><td>Gly</td><td>Leu</td><td>lys</td><td>Leu</td><td>Leu 95</td><td>Arg</td>

<td>Tyr</td><td>Tyr</td><td>Thr</td><td>Glu 100</td><td>How much</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>phe 105</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Leu</td><td>Arg 110</td><td>asp</td><td>Pro</td>

<td>How much</td><td>Cheese</td><td>ala 115</td><td>Glu</td><td>How much</td><td>Cheese</td><td>How much</td><td>Gln 120</td><td>ala</td><td>Leu</td><td>Cheese</td><td>Tyr</td><td>ala 125</td><td>Leu</td><td>Gly</td><td>Gly</td>

<td>asp</td><td>How much 130</td><td>own</td><td>lys</td><td>val</td><td>Leu</td><td>Glu 135</td><td>lys</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Cheese 140</td><td>Gly</td><td>Gly</td><td>asp</td><td>Leu</td>

<td>Leu 145</td><td>Gly</td><td>How much</td><td>Leu</td><td>Glu</td><td>Cheese 150</td><td>Arg</td><td>Gly</td><td>How much</td><td>lys</td><td>ala 155</td><td>Arg</td><td>How much</td><td>Thr</td><td>His</td><td>val 160</td>

<td>asp</td><td>Thr</td><td>Glu</td><td>Cheese</td><td>Tyr 165</td><td>phe</td><td>How much</td><td>val</td><td>Leu</td><td>Cheese 170</td><td>How much</td><td>ala</td><td>Tyr</td><td>Pro</td><td>Thr 175</td><td>Leu</td>

<td>Cheese</td><td>Glu</td><td>How much</td><td>lys 180</td><td>Gly</td><td>val</td><td>How much</td><td>val</td><td>His 185</td><td>Arg</td><td>Leu</td><td>Glu</td><td>Gly</td><td>val 190</td><td>Cheese</td><td>Tyr</td>

<td>own</td><td>How much</td><td>Gly 195</td><td>Cheese</td><td>Gln</td><td>Glu</td><td>Trp</td><td>Tyr 200</td><td>Thr</td><td>Thr</td><td>val</td><td>Pro</td><td>lys 205</td><td>Tyr</td><td>val</td><td>ala</td>

<td>Thr</td><td>Gln 210</td><td>Gly</td><td>Tyr</td><td>Leu</td><td>How much</td><td>Cheese 215</td><td>own</td><td>phe</td><td>asp</td><td>Glu</td><td>Cheese 220</td><td>Cheese</td><td>Cys</td><td>Thr</td><td>phe</td>

<td>Underworld 225</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Thr</td><td>val 230</td><td>Cys</td><td>Cheese</td><td>Gln</td><td>own</td><td>ala 235</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Underworld</td><td>Cheese 240</td>

<td>Pro</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Glu 245</td><td>Cys</td><td>Leu</td><td>Arg</td><td>Gly</td><td>Cheese 250</td><td>Thr</td><td>lys</td><td>Cheese</td><td>Cys</td><td>ala 255</td><td>Arg</td>

<td>Thr</td><td>Leu</td><td>val</td><td>Cheese 260</td><td>Gly</td><td>Cheese</td><td>phe</td><td>Gly</td><td>own 265</td><td>Arg</td><td>phe</td><td>How much</td><td>Leu</td><td>Cheese 270</td><td>Gln</td><td>Gly</td>

<td>own</td><td>Leu</td><td>How much 275</td><td>ala</td><td>own</td><td>Cys</td><td>ala</td><td>Cheese 280</td><td>How much</td><td>Leu</td><td>Cys</td><td>lys</td><td>Cys 285</td><td>Tyr</td><td>Thr</td><td>Thr</td>

<td>Gly</td><td>Thr 290</td><td>How much</td><td>How much</td><td>own</td><td>Gln</td><td>asp 295</td><td>Pro</td><td>asp</td><td>lys</td><td>How much</td><td>Leu 300</td><td>Thr</td><td>Tyr</td><td>How much</td><td>ala</td>

<td>ala</td><td>asp</td><td>His</td><td>Cys</td><td>Pro</td><td>val</td><td>val</td><td>Glu</td><td>val</td><td>own</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>Gin</td><td>val</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Gly</td><td>Cheese</td><td>Arg</td><td>Arg</td><td>Tyr</td><td>Pro</td><td>asp</td><td>ala</td><td>val</td><td>Tyr</td><td>Leu</td><td>His</td><td>Arg</td><td>How much</td><td>asp</td><td>Leu</td>

325 330 335

Gly Pro Pro Ile Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly 340 345 350

Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu Cheese Cheese 355 360 365

Asp Gin Ile Leu Arg Ser Met Lys Gly Leu Ser Cheese Thr Ser Ile Val 370 375, 380

Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly Ile Pro Ala

385 390 395 400

Leu Ile Cys Cys Cys Arg Gly Arg Cys Asn Lys Lys Gly Glu Gin Val

405 410 415

Gly Met Ser Arg Pro Gly Leu Lys Pro Asp Leu Thr Gly Thr Ser Lys 420 425 430

Cheese Tyr Val Arg Cheese Leu 435 &lt;210&gt; 21 &lt;211&gt; 199 &lt;212&gt; PRT &lt;213&gt; HIV &lt;400&gt; 21

Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly 15 10 15

Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gin Ala Arg Gin 20 25 30

Leu Leu Cheese Gly Ile Val Gin Gin Gin Asn Asn Leu Leu Arg Ala Ile 35 40 45

Glu Ala Gin Gin His Leu Leu Gin Leu Thr Val Trp Gly Ile Lys Gin 50 55 60

Leu Gin Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gin Gin 65 70 75 '80

Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 85 90 95

Val Pro Trp Asn Ala Cheese Trp Cheese Asn Lys Cheese Leu Glu Gin Ile Trp 100 105 110

Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 115 120 125

Cheese Leu Ile His Cheese Leu Ile Glu Glu Cheese Gin Asn Gin Gin Glu Lys 130 135 140

Asn Glu Gin Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 145 150 155 160

<td>Trp</td><td>phe</td><td>own</td><td>How much</td><td>Thr 165</td><td>own</td><td>Trp</td><td>Leu</td><td>Trp</td><td>Tyr 170</td><td>How much</td><td>lys</td><td>Leu</td><td>phe</td><td>How much 175</td><td>Underworld</td>

<td>How much</td><td>val</td><td>Gly</td><td>Gly 180</td><td>Leu</td><td>val</td><td>Gly</td><td>Leu</td><td>Arg 185</td><td>How much</td><td>val</td><td>phe</td><td>ala</td><td>val 190</td><td>Leu</td><td>Cheese</td>

Ile Val Asn Arg Val Arg Gln 195 &lt;210&gt; 22 &lt;211&gt; 22 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 22

Gly Asn His Ile Leu Cheese Leu Val Gln Asn Ala Pro Tyr Gly Leu Tyr 15 10 15

Phe Ile His Phe Ser Trp 20 &lt;210&gt; 23 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 23

Gly Tyr Phe Val Gln Asp Asp Gly Glu Trp Lys Phe Thr Gly Ser Cheese 15 10 15

Tyr Tyr Tyr &lt;210&gt; 24 &lt;211&gt; 22 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 24

Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val 15 10 15

Phe Leu His Val Thr Tyr 20 &lt;210&gt; 25 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 25

Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn 15 10 15

Phe Phe Ser &lt;210&gt; 26 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 26

Met Phe Pro Pro Trp Cheese Ala Ala Ala Gly Val Pro Phe Cheese Leu Cheese 15 10 15

Val Gln Tyr &lt;210&gt; 27 &lt;211&gt; 26 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 27

Gln Asp Ala Ile Lys Lys Leu Asn Glu Cheese Tyr Ile Asn Leu Lys Glu 15 10 15

Val Gly Thr Tyr Glu Met Tyr Val Lys Trp 20 25 '&lt;210&gt; 28 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 28

Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe Ser 15 10 15

Gln Ile Leu &lt;210&gt; 29 &lt;211&gt; 28 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 29

<td rowspan="2">ala 1</td><td rowspan="2">ala</td><td rowspan="2">Cys</td><td rowspan="2">Glu Val 5</td><td colspan="2" rowspan="2">Ala Lys</td><td colspan="6">Asn Leu Asn Glu Cheese Leu Ile Asp Leu</td>

<td colspan="4"> 10</td><td colspan="2"> 15</td>

<td>Gln</td><td>Glu</td><td>Leu</td><td>Gly Lys</td><td>Tyr</td><td>Glu</td><td>Gln</td><td>Tyr</td><td>How much</td><td>lys</td><td>Trp</td><td></td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 30 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 30

Asn Tyr Ser Lys Tyr Trp Tyr Leu Asn His Thr Thr Thr Gly Arg 15 10 15 &lt;210&gt; 31 &lt;211&gt; 19 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic peptide &lt;400&gt; 31

Gly Thr Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly Lys Asp Thr Pro 15 10 15

Gly Gly Tyr