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

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DESCRIPTION

Procedure for preventing cell virus fusion by inhibiting the function of the fusion initiation region in RNA viruses having class I fusogenic membrane envelope proteins

This application claims the benefit of United States Provisional Application No. 60/517181 filed on November 4, 2003.

Field of the invention

The present invention relates to peptides for use in preventing or inhibiting viral infection by an arenavirus of a cell (thereby preventing the release of the viral genome into the cell cytoplasm, a step required for viral infection). The present invention provides compositions and uses thereof for preventing infection by an arenavirus by interfering with its fusion initiation region (FIR).

Introduction

All viruses must bind to and invade their target cells to replicate. For enveloped animal viruses, including RNA viruses that have class I membrane fusion proteins (type I viruses), the process involves:

a) binding of the virion to the target cell, b) fusion of the virus envelope with the plasma membrane or with an inner cell membrane, c) destabilization of the virus envelope and the cell membrane in the fusion area to create a pore fusion, d) transfer of viral RNA through the pore and e) modification of cell function by viral RNA.

The fusion of the viral membrane and cell envelope, steps b) and c) above, is mediated by the interaction of a viral transmembrane glycoprotein (fusion protein) with the surface proteins and membranes of the target cell. These interactions cause conformational changes in the fusion protein that result in the insertion of a viral fusion peptide into the membrane of the target cell. This insertion is followed by additional conformational changes within the fusion protein that bring the viral envelope and cell membranes closer together and result in the fusion of the two membrane bilayers.

A virus is unable to spread and spread within its host if this fusion process is disrupted. Intentional disruption of this fusion process can be achieved by targeting peptides and homologs that mimic peptides to sequences of the fusion protein, antibodies that recognize the fusion protein, and other factors that act against the fusion protein.

Background of the invention

Structural similarities between the fusion proteins of class I RNA viruses.

Influenza virus hemagglutinin 2 (HA2), an orthomyxovirus, is the prototype fusion protein of class I RNA viruses and contains an amino-terminal hydrophobic domain referred to as a fusion peptide, which is exposed during cleavage of the hemagglutinin precursor protein. The membrane fusion proteins of RNA viruses of various families, such as arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses, share some common structural features with HA2 and have been termed class I viral fusion proteins. The HIV-1 fusion protein, transmembrane glycoprotein, and other retroviral transmembrane proteins, such as orthomyxovirus and paramyxovirus, have been shown to possess a hydrophobic fusion peptide domain exposed during cleavage of a precursor (gp160) ( Gallaher, 1987; Gonzalez-Scarano et al., 1987). Based on these similarities and on computer algorithms that predict protein configuration, it has been suggested (Gallaher et al., 1989) that the outer portion (ectodomain, amino terminal end) of the HIV-1 transmembrane protein and the proteins transmembrane cells of other retroviruses, may all match the backbone of the HA2 structure as determined by X-ray crystallography (Wilson, Skehal, & Wiley, 1981).

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 extended amino-terminal helix (N-helix usually a repeating heptad). or leucine zipper), a helix at the carboxyl terminal end (helix C), and an aromatic motif close to the transmembrane domain. The presence of at least four of these five domains defines a viral envelope protein as a class I fusion protein. This retroviral transmembrane protein model was confirmed by subsequent structural determinations and mutational analysis (Chan et al., 1997 ; Kowalski et al., 1991; Weissenhorn et al., 1997). Common structural motifs are present not only in the fusion proteins of orthomyxovirus and retrovirus but also in those of paramyxovirus, filovirus (such as Ebola virus, EboV) (Gallaher, 1996) and arenavirus (Gallaher, DiSimone and Buchmeier, 2001) . Gallaher's structural model of the EboV fusion protein (GP2) has also been confirmed by X-ray crystallography procedures.

ES 2 392 891 T3 (Malashkevich et al., 1999; Weissenhorn et al., 1998).

Figure 1 shows the five previously described domains of the fusion proteins of the six families of type 1 viruses. Fusion proteins originate from a hydrophobic fusion peptide, terminate on an anchor peptide, and incorporate an extended amino-terminal alpha helix (N-helix, usually a “repeating heptad or a“ leucine zipper ”), an alpha helix at the carboxyl terminal end (helix C) (Carr and Kim, 1993; Suarez et al., 2000; Wilson, Skehel and Wiley, 1981) and sometimes an aromatic motif close to the virion envelope. Also shown is the sixth domain, the fusion initiation region (FIR), described by the present inventors.

Inhibition of fusion in type I viruses

Previous attempts by the present inventors (Garry) and other laboratories to design peptides and peptide mimetics, antibodies, and other factors that inhibit fusion in type I viruses have focused on the fusion peptide, the N-helix, and the helix. C of fusion proteins. In the case of fusion peptides, it has been found that analogs of the fusion peptide domains of orthomyxovirus and paramyxovirus (Richardson, Scheid and Choppin, 1980) and HIV-1 (Gallaher et al., 1992; Owens et al. , 1990; Silburn et al., 1998) block viral infection, presumably forming inactive heteroaggregates. Peptides corresponding to the N-helix and C-helix portions have also been found to be effective in inhibiting viral infection both in vitro and in vivo. For example, a 17 amino acid peptide corresponding to the carboxyl 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 inhibitor peptides have been developed based on the fusion protein structural model (Wild, Greenwell and Matthews, 1993; Wild et al., 1992), including the anti-helix peptide drug. C of HIV-1 DP178 (T-20 or FUZEON®). DP178 overlaps the C helix and the proximal aromatic anchor domain, and inhibits HIV-1 virion: cell fusion at very low concentrations (50% inhibition at 1.7 nM) achievable in vivo after injection. In a clinical trial, 100 mg / day of DP178 produced an approximately 100-fold reduction in the load of HIV-1 in the plasma of infected individuals (Kilby et al., 1998). This result has largely motivated the search for other HIV-1 inhibitory peptides based on the structure of the transmembrane protein (Pozniak, 2001; Sodroski, 1999). Peptide inhibitors of paramyxovirus have also been shown to inhibit viral replication (Lambert et al., 1996, Young et al., 1999). Studies by Watanabe et al. Suggest that a similar strategy targeting the EboV GP2 N helix and C helix may also lead to the discovery of useful inhibitors (Watanabe et al., 2000). Neutralizing antibodies directed against fusion protein domains have also been shown to inhibit virion: cell fusion.

Observations made on HIV-1

A large part of the study has been devoted to the inhibition of fusion in the human immunodeficiency virus HIV-1, one of the type I RNA viruses. Bolognesi et al. (5,464,933) and the current inventors (Garry, USPN 5,567,805) show us how HIV-mediated cell death can be inhibited by introducing peptides that bind to portions of the HIV-1 virion transmembrane fusion protein. . The DP178 binding region of Bolognesi, marked FUZEON® in Figure 7, is primarily within the C helix and is outside what is described in the present application as the fusion initiation region (FIR). Bolognesi demonstrates inhibition but does not present any inhibition procedure. The current inventors (Garry) previously showed inhibition in the CS3 region of HIV-1 TM, labeled CS3 in Figure 7, but did not identify inhibition procedure, suggesting that only the CS3: CS3 receptor interaction is inhibited. . The unexpected discovery of the FIR region by the current inventors (as currently described herein) and the fact that CS3 sequences are within FIR indicates that the CS3: CS3 receptor binding described in USPN 5,567,805 is in fact a binding that occurs between the CS3 portion of the FIR and portions of the cell membrane for which the CS3 portion of FIR has an affinity. Furthermore, although Melikyan, Watanabe, Bewley and other authors have described fusion inhibition with the introduced peptides, they have not explained the mechanisms by which the inhibition occurs. Consequently, the location of the FUZEON® peptide is away from the FIR region, strongly suggesting that other elements of the fusion process function in the FUZEON® region.

With reference to the foregoing, it is clear that there is a need in the art for a more efficient means of identifying those regions of viruses that are involved in the infection process and for compositions effective to prevent or inhibit viral infection. The invention described and disclosed in this document provides an effective solution for these needs.

Summary of the invention

According to a first aspect of the invention, there is provided an isolated viral fusion inhibitor peptide selected from the group consisting of:

a) a peptide having an amino acid sequence composed of SEQ ID No. 1,

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b) a peptide having an amino acid sequence composed of 8 or more contiguous amino acid residues of SEQ ID No. 1,

c) a peptide analog having an amino acid sequence composed of SEQ ID No. 1 that includes one or more conservative amino acid substitutions and in which most of the residues of the analog are identical to those in the sequence of SEQ ID # 1, and

d) a peptide analog having an amino acid sequence composed of 8 or more contiguous amino acid residues of SEQ ID NO: 1 that includes one or more conservative amino acid substitutions and in which the majority of the analog residues are identical to those of the sequence of the 8 or more contiguous amino acid residues of SEQ ID NO: 1, for use in treating an arenavirus infection.

The peptide can be an analogous peptide having the amino acid sequence of SEQ ID No. 30. These peptides can include an acetyl group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, a hydrophobic group, or a macromolecular group. at the amino terminal end of the peptide. Additionally, these peptides can include an amido group, a hydrophobic group, or a macromolecular group at the carboxyl terminal end of the peptide. Arenavirus infection can be Lassa virus infection.

According to a second aspect of the invention, there is provided the use of a peptide as defined in the first aspect of the invention for the preparation of a medicament for treating an arenavirus infection in a patient. Arenavirus infection can be Lassa virus infection.

According to a third aspect of the invention, there is provided a recombinant DNA molecule that enables, or stimulates, the production in a patient of the peptide defined in the first aspect above for the treatment of an arenavirus infection in a patient. This aspect includes the use of a recombinant DNA molecule that enables, or stimulates, the production in a patient of the peptide defined in the first aspect above for the preparation of a medicament for treating an arenavirus infection in a patient.

According to a fourth aspect of the invention, there is provided an agent that inhibits viral fusion comprising a peptide with an amino acid sequence composed of 8 to 50 amino acid residues for use in treating an arenavirus infection in which the peptide it comprises an amino acid sequence of a peptide of the first aspect above. The viral fusion inhibiting agent may include an acetyl group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, a hydrophobic group, or a macromolecular group at the amino terminal end thereof. Additionally, the viral fusion inhibiting agent may include an amido group, a hydrophobic group, or a macromolecular group at the carboxyl terminal end thereof. This aspect includes the use of a viral fusion inhibitor agent of this aspect for the preparation of a medicament for treating an arenavirus infection in a patient.

Illustrative embodiments of the invention

The sixth domain of RNA viruses that have class I membrane fusion proteins

Arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses are the six families of RNA viruses currently identified that have class I membrane fusion envelope proteins. The current inventors (Garry) as well as other researchers have previously shown that the fusion proteins of these type I viruses incorporate five conserved motifs or domains (Carr and Kim, 1993; Gallaher et al., 1989; Suarez et al., 2000; Wilson, Skehel, and Wiley, 1981). These domains comprise a fusion peptide, an H helix, a C helix, and an aromatic motif, all of which are ectodomains, and an anchor peptide that is an endodomain.

Using computer analysis, secondary structure modeling, interfacial hydrophobicity calculations, and other techniques, the present inventors have made the surprising discovery of a highly conserved sixth domain that is present in the fusion proteins of a wide variety of viruses. this sixth domain is described). Viruses possessing this domain are included in the list above, although it is not necessarily limited to these six classes of RNA viruses. To highlight the critical function of this newly identified domain, which is an ectodomain, the domain is referred to herein as the fusion initiation region (FIR) of viruses.

As used herein, the term "extended" alpha helix refers to an alpha helix that has more than four turns of an alpha helix (especially, more than 14 amino acids).

In other embodiments, "factors" are provided that the inventors have unexpectedly found to be effective in preventing and inhibiting viral infection and / or virus: cell fusion.

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As used herein, the term "factors includes, but is not limited to, isolated peptides or functional peptide segments (or peptides analogs thereof) of the recently described fusion initiation region (FIR) domains, mimetic peptides ( "Peptide mimetic" refers to any compound or substance that could serve as a substitute for a peptide that interacts with the FIR, which is any compound that mimics the properties of a functional segment of the FIR), antibodies specific for functional FIR domains (i.e., idiotype and anti-idiotype antibodies), and other molecular compounds that interfere with virus: cell binding and / or fusion .

As used herein the term "functional segment" or "functional fragment" of a fusion initiation region (FIR) refers to a fragment capable of inhibiting virus: cell fusion, inhibiting viral infectivity, capable of inducing a antibody capable of specifically recognizing and binding to FIR and / or interfering with FIR-mediated cellular infection.

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 (eg, carbobenzoxyl, dansyl, or t- butyloxycarbonyl) and a macromolecular carrier group (eg, lipid conjugate, polyethylene glycol, a carbohydrate, or a protein) at the amino terminus. A further class of FIR peptide analogs contains a carboxyl group, an amido group, a hydrophobic group, or a macromolecular carrier group at the carboxyl terminus. Other peptide analogs are defined as FIR peptides in which at least one bond that binds to adjacent amino acid residues is a non-peptide bond (e.g., an imido, ester, hydrazine, semicarbazoide, or azo bond), a peptide in the that at least one amino acid residue is in the D-isomer configuration or a peptide in which the order of the amino acids has been reversed. Additional peptide analogs are FIR peptides that comprise at least one amino acid substitution in which a first amino acid residue is replaced by a different second amino acid residue (the amino acid substitution can be a conserved substitution or a non-conserved substitution). As used herein, such peptide analogs can comprise analogous amino acid sequences in which the analogous sequences contain a majority of identical or chemically similar amino acids in the same order as the major sequences.

As used herein, the term "fusion initiation region" (FIR) generally refers to a region of a viral fusion protein involved in the initial stage or stages of viral infection and / or fusion with a host cell. .

As used herein, the term peptide mimetic includes, but is not limited to, organic compounds or other chemical compounds that mimic the structure or function of the FIR peptide. Examples of mimetic peptides include, but are not limited to, organic compounds that comprise the functional side groups of an amino acid or peptide but lack the carbon / nitrogen structure or peptide bonds. Peptide mimetic also refers to compounds that mimic the action of these functional side groups with other moieties.

Other molecules, such as idiotype or anti-idiotype antibodies or proteins selected by phage display procedures, which bind to the peptides, peptide analogs or peptide mimetics described in the present application can also function with inhibitors of viral infection and / or fusion. virus: cell. The present invention also contemplates plasmids, or recombinant viruses, or other molecules or compounds that allow or stimulate the patient to produce an analog of the inhibitory compounds. For example, a recombinant protein, produced in a genetically modified bacterium, fungus, or mammalian cell can be used to produce an immunogenic FIR analog of a viral fusion protein. Similarly, an anti-idiotype response could be induced in the individual using a genetically modified protein comprising a sequence that corresponds to the binding site of a FIR-specific antibody.

As used herein, the term "fusion peptide" preferably refers to a hydrophobic sequence at the amino terminus, or proximal, of a class I viral fusion protein (see, Gallaher et al., 1987; 1992 ).

As used herein the term "substantially purified peptide or peptide analog" preferably refers to a peptide or peptide analog that is greater than 80% pure. More preferably, "substantially purified" refers to a peptide or peptide analog that is greater than about 90% pure or greater than 95% pure. More preferably, it refers to a peptide or peptide analog that is greater than 96%, 97%, 98%, or 99% pure. Functionally, "substantially purified" means that it is free of contaminants to a level that makes it suitable for the purposes provided herein. Procedures for evaluating purity are well known to those of skill in the art. Appropriate procedures include, but are not limited to, gas chromatography (GC) coupled with mass spectrophotometry, analysis by high performance liquid chromatography (HPLC) and functional assays in cell culture systems in which, among others, it is evaluated cytotoxicity.

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As used herein the term "stable analog" refers to a peptide that has a pharmacologically active half-life in biological systems. Biological half-lives greater than 60 minutes are contemplated.

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

According to various aspects of the present embodiment of the invention, peptides, isolated nucleic acid sequences, or antibodies can be produced by any system known in the art, but without limitations, of chemical synthesis, recombinant DNA procedures, and combinations thereof. .

As defined herein, the present invention provides compositions and uses for treating or preventing infection with an arenavirus. One possible mechanism by which the present invention may prevent and / or inhibit infection is through interference with FIR-mediated virus: cell fusion.

Brief description of the figures

Figure 1 shows the domains of the fusion proteins of a member of each of these six viral families (specifically, arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses). The circles in Figure 1 show the approximate location of the FIR in each virus shown.

Figures 2 to 7 show the amino acid sequences of these fusion proteins (corresponding to SEQ ID Nos. 16-21, respectively) and a schematic representation of their ectopic structure. Specifically, the five previously described domains that make up the fusion peptide are shown, ie, the N-helix, the C-helix, the aromatic motif (if present), and the anchor peptide. The newly discovered sixth domain, the fusion initiation region or FIR, is also identified. Each FIR is incised with a polygon in Figures 2 through 7.

The circled area behind the fusion proteins in each of Figures 2-7 represents the major virus: cell binding protein (PUVC) of the virus. PUVC normally interacts with the portion of the fusion protein that is furthest from the virus membrane and is therefore shown in this position in the figures. Unlike the highly conserved fusion protein, the PUVC of each family of viruses is more divergent. Usually it is the PUVC that dictates the host range of the virus and determines which types of host cells are targeted for infection. PUVC acts in its ability to recognize and bind with specific cell surface proteins. Binding of PUVC to cellular target proteins occurs earlier and is typically a prerequisite for virus: cell fusion.

Figure 8: Inhibition of coronavirus infectivity by fusion initiation region peptides. Between 50 and 100 PFU of mouse hepatitis virus strain A59 or SARS coronavirus strain Urbani were previously incubated with or without the indicated peptide (~ 100 μΜ) in serum-free DMEM for 1 h. The cells were then exposed to the peptide treated inoculum or a vehicle control (non-peptide). After 1 hr of adsorption, the inoculum was removed, the cells were washed twice with 1x phosphate buffered saline and these were coated with DMEM containing 10% SFT and 0.5% agarose. At 48 hours after infection, the infected monolayers were fixed and stained with crystal violet to determine the number of plaques.

Figure 9: Inhibition of Lassa virus infectivity by fusion initiation region peptides. Between 50 and 100 CFU of Lassa virus with or without the indicated peptides (~ 100 µM) were previously incubated in BME without serum for 1 h. The cells were then exposed to the peptide-treated inoculum or the vehicle control (no peptide). After 1 h of adsorption, the inoculum was removed, the cells were washed twice with 1x phosphate buffered saline and these were coated with BME containing 5% SFT, 10 Mm HEPES and 0.5% agarose.

Four days after infection, a second coating containing 5% neutral red was applied and the plaques were counted 24 hours later.

The six families of RNA viruses that are known today to have class I membrane fusion proteins (type I viruses) and representative members of each family are as follows:

Representative RNA Viruses Having Class I Membrane Fusion Proteins (Type I Viruses)

Representative Virus Family Shown in Figures

<td>Arenavirus</td><td>Lassa virus</td><td>Yes</td>

<td></td><td>Lymphocytic choriomeningitis virus (LCMV)</td><td>Do not</td>

<td></td><td>Junín virus</td><td>Do not</td>

<td></td><td>Machupo virus</td><td>Do not</td>

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<td></td><td>Guanarito virus</td><td>Do not</td>

<td></td><td>Sabia virus</td><td>Do not</td>

<td>Coronavirus</td><td>Severe acute respiratory syndrome virus (SARS)</td><td>Yes</td>

<td></td><td>Murine hepatitis virus (MHV)</td><td>Do not</td>

<td></td><td>Bovine coronavirus</td><td>Do not</td>

<td></td><td>Canine coronavirus</td><td>Do not</td>

<td></td><td>Feline infectious peritonitis virus</td><td>Do not</td>

<td>Filovirus</td><td>Ebola virus</td><td>Yes</td>

<td></td><td>Marburg virus</td><td>Do not</td>

<td>Orthomyxovirus</td><td>Influenza A virus</td><td>Yes</td>

<td></td><td>Influenza B virus</td><td>Do not</td>

<td></td><td>Influenza C virus</td><td>Do not</td>

<td>Paramyxovirus</td><td>Measles virus</td><td>Yes</td>

<td></td><td>Mumps virus</td><td>Do not</td>

<td></td><td>Distemper virus</td><td>Do not</td>

<td></td><td>Newcastle disease virus</td><td>Do not</td>

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

<td></td><td>Human immunodeficiency virus 2 (HIV-2)</td><td>Do not</td>

<td></td><td>Human T-cell lymphotrophic virus 1 (HTLV-1)</td><td>Do not</td>

<td></td><td>Human T-cell lymphotrophic virus 2 (HTLV-2)</td><td>Do not</td>

<td></td><td>Human intracisternal type A particle 1 (HIAP-1)</td><td>Do not</td>

<td></td><td>Human intracisternal type A particle 2 (HIAP-2)</td><td>Do not</td>

The viruses shown in the figures are as follows:

Examples of RNA Viruses Having Class I Membrane Fusion Proteins (Type I Viruses)

Figure Virus Family shown Protein shown

<td>Figure 2</td><td>Arenavirus</td><td>Lassa virus</td><td>GP2</td>

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

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

<td>Figure 5</td><td>Orthomyxovirus</td><td>Influenza A virus</td><td>HA2</td>

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

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

Sequence Listing of Class I Membrane Fusion Protein Examples (Type I Viruses)

LASSA GP2 (Genbank Accession Number: A43492, amino acids 257-490) 10

LLGT £ JEKHDESFCD KNHLREIHGT DIÍQQATWÍX IPTHRH1VGK

FTMTLSDSEG MTiTrbFDfWQ PYCHYSRYWY TEMLQKEYZD PCPKPHRIiNH

NEDPtMWCDT AlftWCTEAQ LWTSTGKTS RQCKTPLGLV MGICSCGLYK

RHMLTEAELK MSIQLINKAV KPRCWLISNG DLFVFSTSFY

NMjINDQLIM

LTSIPLHLVK (SEQ ID NO: 16)

SARS S (Genbank Accession Number: AAQ9406, amino acids 864-1256)

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KAISQIQESL DIUSRLDKVE MEECVLGQSK PAICHEGKAY VIGIINNTVY iokeidrine MVTTEiIJCCMT

GAGAAl £ I? P amOHaYHFNG TTTSTALGKIr QDVVNQNAQA AEVQIDRLIT GRL ^ SLCTYV PVDFGGKGYE LHSFPQAAPH FPRBGVFVFH GTSWFITQRN DPLQFELDSF KZELDKELYFKYF KZELDKELYFKIDE VAQFELDSF KZELDKELYFKIDE VAQFELDSF KZELDKELYFKIF VAQFKfi

IQVTQHVLYE NQKQIAI5QFN Ltm&gt; VKQLEG NFGAISSVLU TOOLIRAAEI RASANIiAATK GWFliHVTYV PSQEHMFFEA FPSPQIITTD MEFVffeíHCnV ETSBDVDU3D IBGIMASFWVYW IDIVIKPWGIA IDBGIMASVYT

NO: 17)

EBOLA GP2 (Genbank Accession Number: AAM76034, amino acids 502-676)

EAIVNAQPK CNFNLHYWTT QDEGAAIGLA WIPYPíGFAAE QIYTBGLMHN gDGLlCGUtQ L1ANETTQALÜ EFLRATTELR TFSILNRKAI DFLIíQBNGGT CHXLGPDCFCCI EPHDWTKNTJWTBGLMHN gDGLlCGUtQ L1ANETTQALÜ EFLRATTELR TFSILNRKAI DFLIíQBNGGT CHXLGPDCCI EPHDWTKNTJWTDQVDLI DKPVITDQVI DKPVITDQVDVG 16

FLU HA2 (Genbank Accession Number: P03437, amino acids 346-566)

GL-FGA IAGFI ^ igWE GMIDGWYGra HQN £ EGTGQA ADLKSTQAAI dqdjgklnrv iektnekfhq iekefSevtc biqdlekyvz dtkidlwsyn AEIiI ^^ AXtENQ üit-LSJkÍ!

NAClESlHira TYDHDVYRDH ALHMfLFQIKG VELKSOYKDW EtCHICl (SEQ U&gt; NO: 19)

MEASLES F1 (Genbank Accession Number: VGNZMV, amino acids 116-553),

FAGW LAGAALGVAT AAQITAGIAL NQAJSÁIPQA GQEtaLAVQG VQDYIMNEliI P.YYTEILSLF GFSUU3PISA The STOALLA QILESR3IKA RlTHVTKtESif PIVESIAI sqewwtvpk ^ P ^ yvatqqyeis nfeessct ™ CGRaS FSCA RTLVSGSFGM RF1LSQGNLI PDKUZTYIAA DHÜPWEVMG VTIQWGSHRY DVGTMLGKAI AKLEDAKELL BSSDQILRSK XGipALlÚOC NGrOHKKOSQ VGMSRPGLKF hqsmlhsqai: wlrasiiEtt

PL Scnj (l&gt; 1 E

LGGDIWUTiE KLGYSGGDLL LSEIKOVTVH EE &amp; 3VSVN1O PEGTVGSQNA LYFTÍSFLLQB ANCASILCKC YITGTllNQD PÜAVYLBRrD LGPPISIiEBLKGLSGTSIVY ILIAVEDCLGGLQYDTfTS

HIV TM (Genbank Accession Number: AAB50262, amino acids 512-710)

AVGIGñLFE GFLGAAGSW GAASMTIiTVQ AEQIIiSGIVQ QQWRLUIAIE AaOHELQLTV WIKQLOMLI IAVEHYIiKdQ OÍiLGTNQCSG KElCTTJWPN NASflSNKSLE QTWMHTTWME WÜREINNYTS LTH3EXEESQ NCQEEDÍIEQEL t £ fr3KMLGW ΝΗΡΝΤΤΝΒΪΕΗ YIKLFIMIVG GLVGLRIVFA VLfilVNRVHQ (SEQ ID NO: 21)

FIR region identification procedure

For reference purposes, the following procedure is described to identify within virus fusion proteins a conserved motif. A conserved motif from the FIR regions of different viruses will have a similar structure and function. Additionally, the FIR regions of related viruses may, although not necessarily, have very similar primary amino acid sequences.

As described above, the present invention provides compositions useful for preventing or inhibiting viral infection by arenavirus using isolated peptides, antibodies, and nucleic acids directed against the arenavirus-specific FIR and that interfere with the function of said FIR.

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The FIR of a viral function protein can be identified by a procedure such as the following which comprises the following steps:

1) The sequence of the fusion protein is first matched to the structure of the HIV transmembrane fusion protein, comprising the N-helix, the C-helix, and other previously described domains, to identify the N-helix and the C-helix. on the fusion protein in question. This matching process is facilitated by searching the protein's primary amino acid sequence for two or more cysteines that are prone to form at least one covalently linked loop, which will be present in most of these sequences, but not all. The N helix can then be identified in the region preceding this cysteine ​​loop by examining in that region for the presence of charged amino acids and other amino acids that have the propensity to form an alpha helix (e.g., glutamine (Q), alanine ( A), tryptophan (W), lysine (K) and leucine (L)).

2) The amino terminal end of the FIR at the N helix is ​​then identified. This end will typically be within the final 10 to 20 amino acids of the N helix and will have a core typically comprised of 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 carboxyl terminal end of the FIR is identified below. In the case of all families except coronaviruses and paramyxoviruses, this terminal end is the carboxyl terminal end of the first peptide sequence with positive interfacial hydrophobicity that lies beyond the N helix. This terminal end is usually located beyond of the cysteine ​​loop, if the latter is present and sometimes overlaps with the C helix or is positioned on the C helix. Positive interfacial hydrophobicity sequences have 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 interfacial hydrophobicity of these sequences can be determined using the Wimley-White interfacial hydrophobicity scale, preferably with a computer program such as the MPEX program that incorporates this scale ("interfacial hydrophobicity" is a measure of the peptide's ability to transfer from an aqueous solution to the membrane bilayer interface and is based on the hydrophobicity scale of the complete Wimley-White residue determined experimentally (Jaysinghe, Hristova and White, 2000)). Computer programs using this scale can identify a peptide sequence of a peptide chain that has positive interfacial hydrophobicity scores and, therefore, the one most likely associated with the surface of the membranes). See example 1 for an example of the application of this procedure to the identification of FIR in the Ebola virus.

For coronaviruses, which have longer alpha helices and a generally larger scale, and for paramyxoviruses in which the FIR is discontinuous due to non-FIR sequence insertion, the carboxyl terminal end of the FIR is the carboxyl terminal end of the second peptide sequence with positive interfacial hydrophobicity that lies beyond the N helix. The sequence between the N helix and the C helix in the F1 protein of paramyxoviruses is longer than the interhelical sequences of other viruses with class I viral fusion proteins. The F2 protein of paramyxoviruses, which has a binding function to receiver is therefore shorter. Upon inspection of computer models, it is obvious to those of skill in the art that the F1 protein contains a sequence inserted between the N helix and the C helix. Consequently, the paramyxovirus FIR contains two cysteine ​​loops and two sequences of high interfacial hydrophobicity, and is discontinuous due to additional amino acids that are characteristic only of paramyxoviruses and appear between the N helix and the first high interfacial hydrophobicity sequence is excluded. from the FIR region.

FIR SEQUENCES

The sequence of the fusion protein and FIR for each of the six representative viruses shown in Figures 2 to 7 is collected in the respective figure and in the sequence listing provided below (SEQ ID NO: 16 to SEQ ID No. 21 provide the respective fusion proteins and SEQ ID No. 1 to SEQ ID No. 7 provide the respective FIR). Although there are some minor sequence variations among sister viruses within each of these six families, the FIR of any type I virus can easily be identified using the representative sequence provided in the appropriate figure.

Fusion inhibition procedures in these viruses

The present invention provides compositions as defined in the claims that inhibit virus: cell fusion by interfering with FIR function. Various aspects of these embodiments include targeting the FIR regions with isolated peptides, antibodies, and nucleic acid sequences as defined herein to interfere with virus: cell fusion. In the present invention the peptides and peptide analogs consist of a sequence SEQ ID No. 1 or 8 amino acids contiguous thereto, and are of this length as it is necessary to provide effective inhibition of viral infection by arenavirus. As used herein, the term of a length necessary to provide effective virus inhibition preferably refers to a length sufficient to provide a 5-fold or greater reduction in viral infectivity when used in accordance with

ES 2 392 891 T3 present invention. Procedures for quantifying the reduction in viral infectivity are well known to those of skill in the art. For example, reductions in viral activity can be determined by plaque reduction, inhibition of binding, titer reduction assays, or by stimulation studies in animals.

The FIR peptides of SEQ ID No. 1, peptides of analogous sequences of SEQ ID No. 1 or fragments thereof, contemplated as part of the present invention are as defined in the claims. The following sequences are shown for the purpose of comparison with SEQ ID No. 1.

LASSA

X-LIMKNHLRDIMGIPYCNYSRYWYLNHTSTGKTLPRCWLI-Z (SEQ ID No. 1)

SARS

Χ-ΉΚΑΑΉΠ1ΑΝΑΤΠ ^ ΑΑΤΚΜ $ ΕΉ9λ3 ^

GVVM ^ VTYVPSQffiUMFTTAPJUCHBGKAYEPBEaVFVENCJrSWErrQRNH'iJ-Z (SEQ

NOÍO

EBOLA

X-LRTFSILNRKAIDFLLQRWGGTCHILGPDCCI-Z (SEQ ID No. 3)

FLU

X-IQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLF-Z (SEQ ID No. 4)

MEASLES

X-iGLKIJ ^ Iri ^ TEIIjSLFG-Z (SEQ ID NO: 5)

X-WYTB^FPKYVATQGYI2SNFDESSCTFNÍPIX7rVCSQNAL) c'FMSPJXQE cr ARTT, vsR3FnNRiYijscjGm; iANC A ^ nxnccYTTGTii-g (SEQid nclq ("-" indicates that the FIR is discontinuous for measles virus).

HIV

X-LQAKtAVERYraQQl ^ IWGÍSGmCITAWWASWSNKSl ^ QIWNETTWMEWM (SEQ ID NO 7)

In each of the above sequences the X and Z respectively designate the amino or carboxyl terminal ends of the peptide or an additional residue, as described below.

The peptides of SEQ ID No. 1 provided by the present invention have the sequence of an FIR region. The FIR region is from a virus that belongs to the viral family of arenaviruses, which includes Lassa virus, lymphocytic choriomeningitis virus (LCMV), Junín virus, Machupo virus, Guanarito virus. and the Sabia virus.

Other aspects of this embodiment of the invention is to provide the sequence SEQ ID No. 30 comprising a functional fragment of an FIR sequence or sequences analogous to the same in a virus belonging to the viral family of arenaviruses in which they are included Lassa virus, lymphocytic choriomeningitis virus (LCMV), Junín virus, Machupo virus, Guanarito virus and Sabia virus.

Derived peptides can comprise altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a synonymous change. For example, one or more amino acid residues may be substituted in the sequence for any other amino acid of

ES 2 392 891 T3 similar polarity, which act as a functional equivalent, giving rise to a synonymous alteration (for example substitution of leucine for isoleucine). Substitutes for an amino acid in the sequence can be selected from other members of the class of amino acids to which it belongs. For example, nonpolar (hydrophobic) amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids are arginine, lysine, and histidine. Negatively charged amino acids (acids) include aspartic acid and glutamic acid. By way of further example, and not limitation, these peptides may also comprise D amino acids and / or may comprise an ineffective carrier protein or any kind of carrier protein.

FIR peptides can comprise peptides in which "X" comprises an amino group, an acetyl group, a hydrophobic group or a macromolecular carrier group and / or "Z" comprises a carboxyl group, an amido group, a hydrophobic group, a carrier group macromolecular. The residue X can also be selected from the group consisting of a hydrophobic residue, a carbobenzoxyl residue, a dansyl residue, and a t-butyloxycarbonyl residue. The Z moiety can be selected from the group consisting of a hydrophobic moiety and a t-butyloxycarbonyl moiety.

The "X" moiety may comprise a macromolecular carrier group. This macromolecular carrier group can be selected from the group consisting of, but not limited to, a lipid conjugate, a polyethylene glycol moiety, or a carbohydrate moiety. Similarly, "Z" may also comprise a macromolecular transporter group, wherein said macromolecular transporter is selected from the group consisting of, but not limited to, a lipid conjugate, a polyethylene glycol moiety, or a carbohydrate moiety.

One or more of the molecular bonds that join adjacent amino acid residues can be non-peptide bonds. These non-peptide linkages include, but are not limited to, imido, ester, hydrazine, semicarbazoid, and azo linkages.

The peptide can comprise one or more amino acid residues that are in a D isomeric amino acid.

The peptides may comprise one or more amino acid substitutions in which a first amino acid residue is replaced by a different second amino acid residue in the sequences provided above (or a functional segment thereof). 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. In still other aspects of this embodiment of the invention, peptides are provided as described above except that one or more amino acid residues have been removed.

In various preferred aspects of the current embodiments, the FIR peptides of the invention comprise at least 8 contiguous residues of an FIR. As used herein, the term "FIR inhibitory peptides preferably refers to a peptide or peptides having the sequence of an FIR (or functional segment thereof) and to said FIR peptides or functional segments in which one or more amino acids are substituted by functionally equivalent or chemically similar amino acids (see below). It also refers to derivatives of these peptides including but not limited to benzylated derivatives, glycosylated derivatives, and peptides that include enantiomers of natural amino acids. In a preferred aspect of this embodiment, the peptides have the sequence of SEQ ID NO: 30.

In still other aspects of this embodiment of the invention, the FIR peptides can be attached to a carrier molecule such as a protein including, but not limited to, human serum albumin (HSA).

Furthermore, the present invention contemplates molecules that comprise any combination of residues X and Z and / or the modifications of other peptides described above.

The peptides according to the present invention can be produced from natural or recombinant viral proteins. They can also be produced using conventional recombinant DNA techniques (for example, expression of the peptide by a microorganism containing the recombinant nucleic acid molecule encoding the desired peptide, expressed under the control of a suitable transcriptional promoter and harvesting of the desired peptide at starting from said microorganism). In a preferred aspect of the invention, any of the peptides of the invention can be prepared using any chemical synthesis methodology known in the art including, but not limited to, Merrifield's solid phase synthesis (Clark-Lewis et al., 1986, Science 231: 134-139).

Embodiments of the present invention also provide antibodies useful for treating or preventing infection of a cell by a virus. Antibodies include active fragments thereof, which means portions of antibodies capable of specifically recognizing an FIR region or a functional segment thereof. Antibodies specifically recognize an FIR, or antigenic fragment thereof, to prevent or reduce infection of the cell by the virus. Antibodies according to these embodiments of the invention can be monoclonal or polyclonal.

ES 2 392 891 T3

General procedures for producing antibodies are well known to those of skill in the art. Methods for producing antibodies according to the present invention comprise the steps of (i) providing an antigen comprising an FIR or an antigenic fragment thereof (this antigen can be an unmodified peptide, an analogous peptide or a peptide derivative); (ii) expose an animal's immune system to antigen so as to induce an immune response, (iii) collect antibodies from the animal and identify those antibodies that specifically recognize an FIR (or functional fragment thereof) and / or are capable of inhibiting or reducing virus: cell infection in a dose-response manner in assays in which viral infectivity is measured.

According to various aspects of the present invention, the peptides and / or antibodies of the present invention useful for treating or preventing viral infection of a cell may be directed against the amino acids around or within the cysteine ​​loop of FIR, the portion distal to the N-helix of FIR, any of the interfacial hydrophobicity regions of the FIR, other areas of the FIR, or any combination thereof. These antibodies, peptides or peptide analogs (collectively compounded) can be used individually; alternatively combinations of two or more can be used to prevent or inhibit infection of the cell by the virus. Methods for preventing or inhibiting viral infection of the cell by interfering with the function of the FIRs provided in the present invention also include the use of neutralizing antibodies, exogenously or endogenously produced, against all or parts of the FIR region. The objective of this use is to interfere with the function of the FIR, thus inhibiting viral infection of the cell and / or virus: cell membrane fusion.

Other embodiments of the present invention provide compositions, including pharmaceutical compositions comprising any and all peptides (including analogs or antibodies). This includes, but is not limited to, compositions containing any molecule that comprises, essentially consists of, or consists of an FIR of SEQ ID NO: 1 or a functional segment of an FIR. This further includes, but is not limited to, compositions comprising any compound that specifically recognizes, binds, or interferes with the function of a viral FIR. As used herein, the phrase "that interferes with the function of the FIR" means that a compound interacts with the FIR or with the cellular protein that serves as the receptor that recognizes the FIR, thereby preventing or reducing infection. of the cell by the virus. Additionally, it is contemplated that the compositions may comprise any of the disclosed molecules or mixtures of one or more of these molecules.

Additional embodiments of the present invention provide uses of compounds of the invention to treat or prevent infection of a cell by an arenavirus. Various aspects of this embodiment of the invention provide an effective amount of any of the pharmaceutical compositions described herein for use in administration to a patient suspected of having been exposed to an arenavirus (or who has the potential to be exposed to an arenavirus). In various aspects of the invention, the pharmaceutical composition comprises an antibody that specifically recognizes and binds to an FIR peptide of SEQ ID NO: 1 (or a functional segment of an FIR) or a fragment of said antibody that specifically recognizes and joins a FIR, or a functional segment of a FIR.

Still other aspects of this embodiment of the invention as defined in the claims provide an effective amount of a composition comprising at least one recombinant DNA molecule, wherein the DNA encodes an FIR of sEc ID No. 1 (or a functional segment thereof) so that infection by an arenavirus is prevented or reduced. In a preferred aspect of this embodiment the recombinant DNA molecule and / or the other pharmaceutical composition comprise the elements necessary for the protein encoded by the DNA molecule to be expressed in a human cell. By way of non-exclusive example, in certain aspects of this embodiment of the invention, the DNA molecule is part of a recombinant plasmid or recombinant virus.

Examples

Example 1: Identification of the FIR in the Ebola virus

The procedure for identifying the FIR of class I viral fusion proteins can be illustrated by two examples. The first example is the identification of the FIR in the minimal class I fusion protein glycoprotein 2 (GP2) of the Ebola virus, a filovirus. Ebola virus GP2 N helix and C helix boundaries have been determined by X-ray crystallography procedures (Malashkevich et al., 1999). The terminal amino acids of the N helix contain the sequence ILNRKAIDF (SEQ ID NO: 8) that matches the consensus sequence of a nucleus comprising three or four hydrophobic amino acids, one positively charged amino acid, one negatively charged amino acid, and one aromatic amino acid. Between these two helices are two cysteines in the sequence CHILGPDC (SEQ ID No. 9). Defining the ends of the Ebola virus GP2 FIR is the sequence FLLQRWGGTCHILGPDCCI (SEQ ID NO: 10), which has a Wimley-White interfacial hydrophobicity score of 2.59 as determined by the MPEX program (Jaysinghe et al., 2002). Thus, the Ebola virus GP2 FIR spans amino acids 579 to 610.

ES 2 392 891 T3

Example 2: Identification of the FIR in measles virus

The second example is a complex class I fusion protein, the F1 protein of measles virus, a paramyxovirus. Measles virus F1 N and C helices can be identified by examining the primary sequence for amino acids with a propensity to form helices. Alignment of the measles virus F1 main sequence with the primary amino acid sequence of the F1 protein from another paramyxovirus, Newcastle disease virus F1, can also aid in the identification of helix boundaries. The structure of the F1 protein of Newcastle disease virus has been determined by X-ray crystallography procedures (Chen et al., 2001). Thus, it can be predicted that the boundaries of the H and C helices are amino acids 131-217 and 455-491, respectively. Unlike Ebola virus GP2 and most class I viral fusion proteins, the primary sequence between the N and C helices in measles virus is greater than 100 amino acids. The FIR region of measles virus F1 contains an insert that, upon inspection of computer models, is obvious to those of skill in the art and, therefore, the structure of the FIR region is formed by a secondary order linking two parts of the primary sequence. The inserted sequence forms an external loop to the FIR. The terminal amino acids of the N-helix contain the sequence LKLLRYYTE (SEQ ID NO: 11) that matches the consensus sequence of a nucleus comprising three or four hydrophobic amino acids, one positively charged amino acid, one negatively charged amino acid, and one aromatic amino acid. In measles virus F1 there are eight cysteine ​​residues between the N and C helices. Based on the F1 alignment of the Newcastle disease virus, it can be determined that the first two cysteines and the next two form disulfide-linked bonds. The first pair of cysteines in the sequence, CTFNIPEGTVC (SEQ ID No. 12), is part of the FIR as it is linked by a WYTTVPKYVATQGYLISNF sequence (SEQ ID No. 13) with a Wimley-White interfacial hydrophobicity score. 3.36 as determined by the MPEX program. The second pair of cysteines in the sequence, CLRGSTKSC (SEQ ID No. 14), is also part of the FIR as it is adjacent to the sequence

TLVSGSFGNRFILSQGNLIANCASILCKCYTTGTII (SEQ ID NO: 15) with a Wimley-White interfacial hydrophobicity score of 2.54 as determined by the MPEX program. Thus, the FIR of measles virus F1 ranges from amino acids 205 to 407, with amino acids 221 to 314 representing an insert that does not participate in FIR function.

Example 3: Identification of coronavirus fusion inhibitor peptides.

Background

Severe acute respiratory syndrome (SARS) is a newly recognized disease that spread from southern China between late 2002 and early 2003 to various countries in Asia, Europe, and North America (Guan et al., 2004), SARS typically starts with a fever over 38 ° C. Initial symptoms may also include headache, malaise, and mild respiratory symptoms. Between two days and a week, SARS patients can develop a dry cough and have trouble breathing. Patients in more advanced stages of SARS develop pneumonia or respiratory distress syndrome. In the initial outbreak, 8098 cases were detected worldwide, with an overall mortality of 9.6%. A previously unidentified coronavirus (CoV) has been shown to be the cause of the new disease (Poutanen et al., 2003; Peiris et al., 2003; Drosten et al., 2003; Rota et al., 2003; Mara et al. col., 2003). Public health interventions, such as surveillance, travel restrictions, and quarantines, contained the original spread of SARS CoV in 2003, and the spread of SARS was halted again following the emergence of some new cases in 2004. However, it is unknown if these draconian containment measures can sustain me at each occurrence of the SARS CoV in humans. Additionally, the potential of this new and sometimes lethal CoV as a bioterrorist threat is obvious.

Coronaviruses are long positive chain RNA viruses typically with a wide host range. Like other enveloped viruses, CoV enters target cells by fusion between the virus and cell membranes, a process mediated by the spicular viral protein (S). The CoV S proteins characterized to date appear to consist of two non-covalently associated subunits, S1 and S2. Using computer analysis, Garry and Gallaher (2003) first proposed that the portion of the SARS-CoV protein S that corresponds to the S2 subunit matches the prototypical model of a class I viral fusion protein based on the presence of two predicted alpha helix regions in the terminal N and C regions of S2 (N helix and C helix) and a region rich in aromatic amino acids just before the transmembrane anchoring domain.

Materials and procedures

L2 cells or Vero E6 cells were maintained as monolayers in Dulbecco's Modified Eagle's Complete Medium (DMEM) containing 0.15% HCO3 'supplemented with 10% Fetal Calf Serum (SFT), Penicillin G (100 U / ml), streptomycin (100 mg / ml) and 2 mM L-glutamine at 37 ° C in a 5% CO2 incubator. Murine hepatitis virus (MHV) strain A59, Urbani or HK strain of SARS CoV or HK were propagated in L2 cells. For plaque assays, L2 cells or Vero E6 cells were seeded at a density of 1x10⁶ cells in each well of a 6-well plate. 50 to 100 plaque-forming units (pfu) of SARS VHM or CoV were pre-incubated with or without approximately 100 pg / ml of peptides in serum-free DMEM for 1 h. The cells were then infected with peptide treated inoculum or vehicle control inoculum. After 1 h of

ES 2 392 891 T3 adsorption, inoculum was removed, cells were washed twice with 1x phosphate buffered saline and coated with 10% DMEM / SFT containing 0.5% SEAPLAQUE® agarose (Cambrex Bio Science Rockland, Inc. ., Rockland, ME). The monolayers were fixed with 3.7% formalin and stained with 1X crystal violet 2 days after infection and the number of plaques was determined by light microscopy.

Results and Discussion

The synthetic peptides corresponding to the FIR domains of the S protein of the VHM or the SARS CoV were analyzed for their ability to inhibit infection by these coronaviruses. The ability to inhibit plaque formation in cell monolayers is the most stringent evidence in vivo for a drug that inhibits possible infection. Two peptides (GNHILSLVQNAPYGLYFIHFSW, SEQ ID No. 22 and GYFVQDDGEWKFTGSSYYY, SEQ ID No. 23) from VHM FIR can inhibit VHM plaque formation, although the first VHM FIR peptide is more efficient (see Figure 8A ). Two peptides from the SARS CoV FIR (GYHLMSFPQAAPHGVVFLHVTY, SEQ ID No. 24 and GVFVFNGTSWFITQRNFFS, SEQ ID No. 25) inhibited plaque formation by this coronavirus (see Figure 8B). A significant reduction (~ 50%) in the mean diameter of the residual plaques was also observed. These results suggest that this peptide inhibits both entry and spread of MHV. Similar results were obtained with these inhibitory peptides in independent experiments, with 50% plaque inhibition observed at concentrations &lt;5 pM. These results are unlikely to be explained by the non-specific cytotoxic effects of the peptides. Except for the plates, the cells in the monolayers were intact and viable. The low number of grown plates was similar in size to the control plates. Peptides from other regions also inhibited infection by these viruses although to a lesser degree than the more active FIR peptides (Figure 8). For example, peptides in the fusion peptide region and the carboxyl terminal helix (helix C) of the S of VHM and the S of the CoV of SARS provided some inhibition (fusion peptide S of the VHM = MFPPWSAAAGVPFSLSVQY, SEQ ID # 26; VHM S C helix = QDAIKKLNESYINLKEVGTYEMYVKW, SEQ ID # 27; SARS CoV fusion peptide =

MYKTPTLKYFGGFNFSQIL, SEQ ID No. 28; SARS CoV S helix C =

AACEVAKNLNESLIDLQELGKYEQYIKW, SEQ ID NO: 29. Inhibitory activities in the pM range have recently been reported with coronavirus C-helix peptides by Bosch et al., (2003) et al. (Bosch et al., 2004; Lui et al. , 2004; Yuan et al., 2004; Zhu et al., 2004)). However, no inhibitory peptides of the coronavirus FIR region have been reported. However, in light of the present invention, the current references collectively provide support for the enormous advantages of the presently described and claimed inventions. That is, these references are consistent with the inventors' claim that the methods of the present invention can be used to advantage to identify synthetic peptides that inhibit fusion / infectivity by members of the Coronaviridae family.

Example 4: Identification of arenavirus fusion inhibitor peptides.

Background

Lassa fever is an often fatal hemorrhagic disease named for the people of the Yedseram River Valley of Nigeria where the first cases occurred in 1969 (Buckley and Casals, 1970). Areas of Guinea, Sierra Leone, Nigeria and Liberia are endemic for the etiological agent, Lassa virus (LasV). The public health impact of LasV in endemic areas is immense. The Centers for Disease Control and Prevention (CDC) have estimated that 100,000 - 300,000 cases of Lassa occur annually in West Africa and 5,000 deaths. In some areas of Sierra Leone, 10-15% of all patients admitted to hospitals have Lassa fever. The fatality rates for Lassa fever are typically 15% to 20%, although in epidemic cases the total mortality can be as high as 45%. The mortality rate in women during the last month of pregnancy is always high, ~ 90%, and LasV infection causes high rates of fetal death at all stages of gestation. Lassa mortality rates are higher in non-Africans, which is concerning as Lassa is the most frequently exported hemorrhagic fever. Due to its high fatality rate and the ability to spread easily through human-to-human contact, the LasV virus is classified as a Biosafety Level 4 and NIAID Biosafety Category A agent.

LasV is a member of the Arenaviridae family. The arenavirus genome consists of two segments of single-stranded ambisense RNA. When viewed under a transmission electron microscope, enveloped spherical virions (diameter: 110 nm - 130 nm) show granular particles that are ribosomes acquired from host cells (Murphy and Whitfield, 1975). Hence the use of the Latin word arena for the family name. In addition to LasV, other arenaviruses that cause disease in humans are Junín virus (Argentine hemorrhagic fever), Machupo virus (Bolivian hemorrhagic fever), Guanarito virus (Venezuelan hemorrhagic fever) and Sabia virus (Brazilian hemorrhagic fever). Arenaviruses are zoonotic; each virus is associated with a specific rodent species (Bowen, Peters, and Nichol, 1997). The reservoir for LasV is the "multi-mammary rat" of the genus Mastomys (Monath et al., 1974). The wide distribution of Mastomys in Africa makes eradication of this rodent reservoir impractical and ecologically undesirable.

The signs and symptoms of Lassa fever, which appear 1 - 3 weeks after exposure to the virus, are highly variable although they may include fever, chest, back or abdominal pain, sore throat,

ES 2 392 891 T3 cough, vomiting, diarrhea, conjunctival hyperemia and facial swelling. LasV infects endothelial cells, resulting in increased capillary permeability, decreased effective circulating volume, shock, and multi-organ failure. External bleeding, usually from the mucosa (gums, etc.) occurs in less than a third of cases, but confers a poor diagnosis. Neurological problems, such as hearing loss, tremors, and encephalitis, have also been described. Surviving patients begin defervescence 2-3 weeks after disease onset. The most common complication of Lassa fever is deafness. Temporary or permanent unilateral or bilateral deafness occurs in ~ 30% of patients with Lassa fever during convalescence and is not associated with the intensity of the acute illness. The antiviral drug ribavirin is effective in treating Lassa fever, although only if it is administered early (before 6 days) in the course of the disease (Johnson et al., 1987; McCormick et al., 1986). It is not known if ribavirin is effective against other arenaviruses, such as Junín, Machupo, Guanarito or Sabia viruses. There is currently no vaccine available for LasV.

Materials and procedures

Vero cells were maintained as monolayers in Eagle's basal medium (BME) containing 10mM HEPES and 5% SFT. Lassa virus (Josiah strain) was propagated in Vero cells. For plaque assays, Vero cells were seeded at a density of 1x10⁶ cells in each well of a 6-well plate. 50 to 100 pfu of LasV with or without peptide were previously incubated in BME without serum for 1 h. The cells were then infected with peptide treated inoculum or vehicle control inoculum. After 1 h of adsorption, the inoculum was removed, the cells were washed twice with 1x phosphate buffered saline and coated with 2 ml of 0.5% agarose in BME containing 10 Mm HEPES and 5% SFT, and incubated for 4 days. A second coating containing 5% neutral red was applied and the plates were counted 24 h later.

Results and Discussion

The ability of the synthetic peptides corresponding to the FIR domains of LasV glycoprotein 2 (GP2) to inhibit infection by this arenavirus was verified. A peptide (NYSKYWYLNHTTTGR, SEQ ID No. 30) analogous to the sequence NYSRYWYLNHTSTGK of SEQ ID No. 1 (LASSA FIR) can inhibit LasV plaque formation (Figure 9). A peptide analogous to another region of GP2, the fusion peptide (GTFtWtLSDSEGkDtPGGY, SEQ ID NO: 31) also inhibited LasV infection, although to a lesser extent (Figure 9). Arenavirus inhibitory peptides have not been reported. Collectively, these results suggest that our techniques can identify synthetic peptides that inhibit fusion / infectivity by members of the Arenaviridae family. These results, together with our results with coronavirus FIR inhibitor peptides, establish proof of principle that peptides from the FIR regions can function as viral inhibitors.

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