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

ES 2 372 633 T3

DESCRIPTION

Procedure to avoid virus condensation: cells by inhibiting the function of the condensation initiation region in RNA viruses that have class I fusogenic membrane envelope proteins

The present application claims the benefit of United States Provisional Application Serial Number 60 / 169,066 filed on November 4, 2003.

Field of the invention

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

Introduction

All viruses must bind to, and invade, their target cells in order to multiply. For enveloped animal viruses, including RNA viruses having Class I membrane proteins (Type I viruses), the procedure involves (a) binding of the virion to the target cell, (b) condensation of the virus membrane with the plasma membrane or with an inner cell membrane, (c) destabilization of the viral envelope and cell membrane in the condensed zone to create a condensation pore, (d) transfer of viral RNA through the pore and (e) modification of cell function by viral RNA.

The fusion of the viral membrane and the cell envelope, steps (b) and (c) above, is mediated by the interaction of a viral transmembrane glycoprotein (fusion protein) with 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 peptide into the membrane of the target cell. This insertion is followed by additional conformational changes in the fusion protein that bring the cell envelope and cell membranes into close proximity and that result in the condensation of the two membranous bilayers.

A virus is unable to disperse and spread within its host if this condensation process is disturbed. Intentional alteration of this fusion procedure can be achieved by targeting homologous peptides and peptidomimetics to fusion protein sequences, antibodies that recognize the fusion protein, and other factors that act against the fusion protein.

Background of the invention

Structural similarities between RNA virus Class I fusion proteins.

Hemagglutinin 2 (HA2) of influenza virus, an orthomyxovirus, is the prototypical Class I RNA virus fusion protein and contains an amino-terminal hydrophobic domain, referred to as the fusion peptide, that is exposed during cleavage of the precursor protein of hemagglutinin. The membrane fusion proteins of RNA viruses of various families including arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses, share several common structural features with HA2 and have been referred to as Class I viral fusion proteins. The HIV-1 fusion protein, the transmembrane glycoprotein, and other retroviral transmembrane proteins such as those of orthomyxoviruses and paramyxoviruses 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 configurations, it has been suggested (Gallaher et al., 1989) that the outer part (ectodomain, amino terminus) of the HIV-1 transmembrane protein and the transmembrane proteins of other retroviruses, could all fit the HA2 framework as determined by X-ray crystallography (Wilson, Skehel, & Wiley, 1981).

Based on these observations, retroviral transmembrane proteins are expected to contain several structural features in addition to the fusion peptide in common with the known HA2 structure, including an expanded amino-terminal helix (N-helix, typically a repetitive heptad or leucine zipper), a carboxy-terminal helix (helix C) and an aromatic residue proximal 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 pattern of retroviral transmembrane proteins was subsequently confirmed by structural determinations and mutational analyzes (Chan et al., 1997; Kowalski et al .., 1991; Weissenhorn et al .., 1997). Common structural residues are present not only in 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 condensation protein (GP2) has also been confirmed by x-ray crystallographic procedures (Malashkevich et al., 1999; Weissenhom et al., 1998). WO 94/17826 describes polypeptides for use in preparing influenza A and influenza B vaccines, including the HA protein. Synthetic hemagglutinin peptide 16-unit polymers have been reported (Gelder et al., 1995). Hemagglutinin protein peptides from del

ES 2 372 633 T3 influenza viruses are also described in document 2003/0180328.

Figure 1 shows the five domains, previously described, of the fusion proteins of the six families of Type I viruses. The fusion proteins originate in a hydrophobic fusion peptide, terminate in an anchor peptide and incorporate an amino-terminal alpha helix. expanded (N helix, usually a repetitive heptad or a leucine zipper) a carboxy-terminal alpha helix (C helix) (Carr and Kim, 1993; Suárez et al. , 2000; Wilson, Skehel, & Wiley, 1981), and sometimes an aromatic residue proximal to the virion envelope. Also shown is the sixth domain, the Condensation Initiation Region (FIR), discovered by the present inventors.

Inhibition of condensation in Type I viruses

Previous attempts by the present inventors (Garry) and others to design peptides and peptidomimetics, antibodies, and other factors that inhibit condensation in type viruses have focused on the fusion peptide, the N-helix, and the C-helix of proteins. condensation. In the case of condensation peptides, it has been found that orthomyxovirus and paramyxovirus analogues (Richardson, Scheid, and Choppin, 1980) and condensation peptide domains (Gallaher et al., 1992; Owens et al .., 1990; Silburn et al. 1998), block viral infection, presumably forming inactive heteroaggregates. Peptides corresponding to parts of the N helix and the C helix have 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 carboxy-terminal part 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 peptide drug- 1 Propeller C DP178 (T-20 or FUZEON®). DP178 overlaps the C helix and the proximal aromatic anchor domain and inhibits HIV-1 virion: cell condensation at very low concentrations (50% inhibition at 1.7 nM) in vivo after injection. In a clinical trial, 100 mg / day of DP178 caused an approximately 100-fold reduction in the plasma load of HIV-1 from infected individuals (Kilby et al., 1998). This result has widely motivated the search for other HIV-1 inhibitory peptides based on the transmembrane protein structure (Pozniak, 2001; Sodroski, 1999). Peptide inhibitors of paramyxoviruses 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 approach of targeting the EboV GP2 N helix and C helix may also lead to useful inhibitors (Watanabe et al., 2000). Neutralizing antibodies directed against parts of the fusion protein domains have also been shown to inhibit virion: cell fusion.

Observations on HIV-1

A large study agreement has been devoted to the inhibition of the condensation of the human immunodeficiency virus HIV-1, one of the Type I RNA viruses. Bolognesi et al. (5,464,933) and the present inventors (Garry, USPN 5,567,805) teach that HIV-mediated killing of cells can be inhibited by introducing peptides that bind to parts of the HIV-1 virion transmembrane fusion protein. The DP178 binding region of Bolognesi, FUZEON ® in Figure 7, is mainly found in helix C and is outside of what is described in the present application in the condensation initiation region (FIR). Bolognesi demonstrates inhibition but does not teach any inhibition procedure. The present inventors (Garry) previously demonstrated inhibition in the CS3 region of HIV-1TM, labeled CS3 in Figure 7, but did not identify any inhibition procedure, suggesting only that the CS3: CS3 receptor interaction is inhibited. . The unexpected discovery of the FIR by the present inventors (as currently described herein) and the fact that the CS3 sequences are within the FIR indicates that the CS3: CS3 receptor binding described in the document USPN 5,567,805 is in fact binding that takes place between the CS3 part of the FIR and parts of the cell membrane for which the CS3 part of the FIR has an affinity. Furthermore, although Melikyan, Watanabe, Bewley, and others have described condensation inhibition with introduced peptides, they have not explained the mechanisms by which the inhibition occurs. Correspondingly, the location of the FUZEON® peptide is distant from the FIR, strongly suggesting that other elements of the condensation process operate in the FUZEON® region.

In view of the foregoing, it is clear that there is a need in the art for a more effective 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 herein provides an effective solution to these needs.

Summary of the invention

The present invention provides an isolated peptide consisting of a sequence of SEQ ID NO: 4 or a segment of 8 to 40 contiguous amino acids thereof in which said peptide inhibits viral infection by influenza virus. The peptides of the invention are also provided for use as a medicament. The invention includes the use of a peptide of the invention for the manufacture of a medicament for treating or preventing influenza in a patient. The peptides of the invention can therefore be used to treat or prevent influenza in a patient.

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The present invention also provides a composition comprising a recombinant DNA molecule encoding a peptide of the invention that enables or stimulates a 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 a patient to produce the peptide of the invention for the manufacture of a medicament to treat or prevent influenza in the patient. A further aspect of the invention provides a composition comprising a recombinant DNA molecule that enables or stimulates a patient to produce the peptide of the invention to treat or prevent influenza in a patient.

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

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

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

(b) introducing said antigen into an animal such as to facilitate an immune response thereto;

(c) collecting antibodies from said animal; and (d) identifying those antibodies that specifically recognize a peptide of SEQ ID NO: 4.

According to this procedure the antigen can comprise a peptide analog; a peptide derivative; or a peptidomimetic of a peptide having a sequence of SEQ ID NO: 4 or an antigenic fragment thereof.

The invention also provides a viral condensation inhibiting agent comprising a peptide having an amino acid sequence consisting of 8 to 50 amino acid residues, wherein the peptide comprises a peptide consisting of 8 to 40 contiguous amino acid residues of SEQ ID. NO: 4. The viral condensation inhibiting agent of this aspect is also provided for use as a medicament. This aspect also extends to the use of a viral condensation inhibitor agent as defined above for the preparation of a medicament for treating or preventing influenza in a patient. The viral condensation inhibiting agent of this aspect of the invention can be used to treat or prevent influenza in a patient.

Illustrative embodiments of the invention

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

The 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 fusion proteins of these Type I viruses have previously been shown by the present inventors (Garry) and others to incorporate five conserved residues, or domains (Carr and Kim, 1993; Gallaher et al .., 1989; Suárez et al .. , 2000; Wilson, Skehel, and Wiley, 1981). These domains comprise a condensation peptide, an N helix, a C helix, and an aromatic residue, all of which are ectodomains, and an anchor peptide that is an endodomain.

Using computational analysis, secondary structure modeling, interface hydrophobicity calculations, and other techniques, the present inventors have made the surprising discovery of a conserved sixth domain that is present in the fusion proteins of a wide variety of viruses. (the sixth domain is described in this document). Viruses possessing 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 that is an ectodomain, the domain is referred to herein as the fusion initiation region (FIR) of viruses.

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

Other embodiments stipulate factors that the present inventors have unexpectedly found to be effective in preventing or inhibiting viral infection and / or virus: cell fusion.

As used herein the term "factors" includes, but is not limited to, isolated peptides or functional peptide segments (or peptide analogs thereof) of the just-described initiation region domains (FIRs), peptidomimetics (peptidomimetic refers to any compound or substance that can 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 (eg, idiotypic or anti-idiotypic antibodies) and other molecular compounds that interfere with virus: cell binding or virus: cell 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 stimulating an antibody capable of recognizing and specifically join the FIR and / or

ES 2 372 633 T3 interfere with FIR-mediated cell infection.

As used herein, an analogous peptide or a modified peptide is preferably defined as an FIR peptide modified to contain an amino group, an acetyl group, a hydrophobic group (eg, carbobenzoxyl, dansyl, or t-butyloxycarbonyl) or a macromolecular carrier group (eg conjugated lipid, polyethylene glycol, a carbohydrate, or a protein) at the amino terminal end. A further class of FIR peptide analogs contains a carboxyl group, an amido group, a hydrophobic group, or a group of macromolecular transporters at the carboxy terminal end. Other peptide analogs are defined as FIR peptides in which at least one bond that joins adjacent amino acid residues is a non-peptide bond (for example an imido, ester, hydrazine, semicarbazoid, or azo bond), a peptide in which at least an amino acid residue is in D-isomer configurations or a peptide in which the order of the amino acids is reversed. Additional peptide analogs are FIR peptides that commit at least one amino acid substitution in which a first amino acid residue is substituted with 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 amino acid sequences in which the analogous sequences contain a majority of identical or chemically similar amino acids in the same order as the primary sequences.

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

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

Other molecules, such as idiotypic or anti-idiotypic antibodies or proteins selected by means of phage display procedures, that bind to the peptides, peptide analogs or peptidomimetics described in the present application can also function as inhibitors of viral infection and / or of the condensation virus: cell. Also contemplated by the present invention are plasmids, or recombinant viruses, or other molecules or compounds that enable or stimulate the patient to produce an analog of the inhibitory compounds. For example, a recombinant protein, produced in an engineered bacterial, fungal, or mammalian cell, can be used to produce an immunogenic FIR analog of a viral fusion protein. Similarly, an anti-idiotypic response could be induced in the individual using an engineered protein comprising a sequence corresponding to the binding site of an FIR-specific antibody.

As used herein the term "fusion peptide" preferably refers to a hydrophobic sequence at or near the amino terminal end 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 more than about 80% pure. More preferably, "substantially purified" refers to a peptide or peptide analog that is more than about 90% pure or more than about 95% pure. Most preferably this refers to a peptide or peptide analog that is more pure than 96%, 97%, 98% or 99%. Functionally, "substantially purified" means that it is free of contaminants to a degree that makes it suitable for the purposes provided herein. Procedures for evaluating purity are well known to those of skill in the art. Suitable procedures include, but are not limited to, gas chromatography (GC) linked mass spectrometry analysis, high performance liquid chromatography (HPLC) analysis, and functional assays in cell culture systems that, among other things, evaluate cytotoxicity. .

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

As used herein the term "peptide derivative" refers to a peptide having substituted amino acids different from those in the FIR sequence of a viral fusion protein. Wherein the substitutions do not render the peptide useful for the current invention.

According to various aspects of the present embodiment of the invention the peptides, isolated nucleic acid sequences, or antibodies can be produced by any methods known in the art, including, but not limited to, chemical syntheses, recombinant DNA methods, and combinations thereof.

As defined herein, the present invention provides compositions and uses for treating or preventing influenza virus infection. One of the possible mechanisms by which the current invention can prevent and / or inhibit infection is by interfering with FIR-mediated virus: cell fusion.

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Brief description of the figures

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

Figures 2 to 7 show the amino acid sequences of these fusion proteins (corresponding to SEQ ID NO: 16-21, respectively) and a schematic representation of their ectopic structures. The five domains described above are specifically shown as the fusion peptide, ie, the N-helix, the C-helix, the aromatic moiety (if present), and the anchor peptide. The newly discovered sixth domain, the condensation initiation region, or FIR, is also identified. Each FIR is indicated by a polygon in Figures 2 to 7.

The circled area behind the fusion proteins in each of Figures 2-7 represents the virus: major cell binding protein (VCBP) of the virus. VCBP usually interacts with the part of the fusion protein that is most distal to the viral membrane and thus is shown to be so positioned in the Figures. Unlike the highly conserved fusion protein, the VCBP of each family of viruses is more divergent. It is usually the VCBP that dictates the host range of viruses and determines which types of host cells are targeted for infection. VCBP acts in this capacity by recognizing and binding specific cell surface proteins. Binding of VCBP to targeted cell proteins occurs prior to 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 murine hepatitis virus strain A59 or SARS coronavirus Urbani strain were pre-incubated with or without the indicated peptides (~ 100 μΜ) in serum-free DMEM for 1 hour. Cells were then exposed to peptide treated inoculum or a vehicle control (no peptide). After 1 hr of adsorption, the inoculum was removed, the cells were washed twice with 1X phosphate buffered saline, and the cells were coated with DMEM containing 10% FBS and 0.5% agarose. Forty-eight hours after infection, the infected monolayers were fixed and stained with crystal violet to determine plaque numbers.

Figure 9: Inhibition of Lassa virus infectivity by fusion initiation region peptides. Between 50 and 100 CFU of Lassa virus were pre-incubated with or without the indicated peptides (~ 100 µM) in serum-free BME for 1 h. The cells were then exposed to the peptide treated inoculum or vehicle control (no peptide). After 1 hr of adsorption, the inoculum was removed, the cells were washed twice with 1X phosphate buffered saline, and the cells were coated with BME containing 5% FBS, 10 mM HEPES, and 0.5% agarose. Four days after infection a second coating containing 5% neutral red was applied, and plates were counted 24 hours later.

The six families of RNA viruses now known to have Class I fusion proteins (Type I viruses) and the representative members of each family are as follows:

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

<td>Family</td><td>Representative Virus</td><td>Shown in Figures</td>

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

<td></td><td>Lymphocytic Coriomeningis Virus (LCMV)</td><td>Do not</td>

<td></td><td>Junin virus</td><td>Do not</td>

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

<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>

ES 2 372 633 T3 (CONT'D)

Canine Distemper Virus No

Newcastle Disease Virus No

Retrovirus Human Immunodeficiency Virus 1 (HIV-1) Yes

Human Immunodeficiency Virus 2 (HIV-2) No

Human T Cell Lymphotrophic Virus (HTLV-1) No

Human T Cell Lymphotrophic Virus 2 (HTLV-2) No

Human Intracisternal Type A Particle 1 (HIAP-1) No

Human Intracisternal Type A Particle 1 2 (HIAP-2) No

The viruses shown in the Figures are as follows:

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

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

<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>

LASSA GP2 Class I (Type I Virus) Membrane Condensation Protein Sequence Listing (World Genetic Bank Accession Number: A43492, amino acids 257-490)

LLGT NEKHDEEFCD KNHLRDIMGI DIEQQADNMI IPTHRHIVGK

FTWTLSDSEG MLRLFDFNKQ PYCNYSRYWY TEMLQKEYID PCPKPHRLNH

NETPGGYCLT AIRRLKTEAQ LNHTSTGKTS RQGKTPLGLV MGICSCGLYK

RWMLIEAELK MSIQLINKAV LPRCWLISNG DLFVFSTSFY QPGVPVRWKR

CFGNTAVAKC NALINDQLIM SYLNETKFSD LISIFLHLVK (SEQ ID NO: 16)

S for SARS (World Genetic Bank Accession Number: AAQ9406, amino acids 864-1256)

WTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL TTTSTALGKL QDWNQNAQA LNTLVKQLSS NFGAISSVLN DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH GWFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASWN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV KLHYT (SEQ ID NO: 17)

EBOLA GP2 (World Genetic Bank Accession Number: AAM76034, amino acids 502-676)

EAIVNAQPK CNPNLHYWTT QDEGAAIGLA WIPYFGPAAE GIYTEGLMHN

QDGLICGLRQ LANETTQALQ LFLRATTELR TFSILNRKAI DFLLQRWGGT

CHILGPDCCI EPHDWTKNIT DKIDQIIHDF VDKTLPDQGD NDNWWTGWRQ

WIPAGIGVTG VIIAVIALFC ICKFVF (SEQ ID NO: 18)

INFLUENZA HA2 (World Genetic Bank Accession Number: P03437, amino acids 346-566)

ES 2 372 633 T3

GLFGA IAGFIENGWE GMIDGWYGFR HQNSEGTGQA ADLKSTQAAI DQINGKLNRV IEKTNEKFHQ IEKEFSEVEG RIQDLEKYVE DTKIDLWSYN AELLVALENQ HTIDLTDSEM NKLFEKIRGNIKEM NKLFEKTRRGN GCFNIKEM NKNGI DKTRRQ LRENAENACT NKLFEKIRGNIKD NKNGI NKIMRIKNIKEM VDNIKEM VDLINKTRRQ NKGFNIKEM NKGNIKEM VDLINKTRRQ NKGFNIKEMNIKD

MEASLES F1 (World Genetic Bank Accession Number: VGNZMV, amino acids 116-553)

FAGW LAGAALGVAT AAQITAGIAL HQSMLNSQAI DNLRASLETT NQAIEAIRQA GQEMXDAVQG VQDYXNNELX PSMNQLSCDL IGQKLGLKLL RYYTEILSLF GPSLRDPISA EISIQALSYA ΕβΘΟΙΝΚILSEVRY KLGYSIGYVYLSEVYLSIGYVYLSEVYLSIGIAVYLSEVGIAVYLSEVILSIGIAVYLSEVGIAVYLSEVGIAVYLSILSIGIAVYLSEVYLSIGYVILSEVRDISA EISIQALSYA ΕβΘΟΙΝΚILSIGSIGIVYLSEVYLSEVYLSEVYLSEVYLSIGYVILSEVRDISA EISIQALSYA.

SQEWYTTVPK YVATQGYLIS NFDESSCTFM PEGTVCSQNA LYPMSPLLQE CLRGSTKSCA RTLVSGSFGN RFILSQGNLX ANCASILCKC YTTGTIINQD PDKILTYXAA DHCPWEVNG VTIQVGSRRY PDAVYLHRID LGPPISLERL DVGTNLGNAI AKLEDAKELL · ESSDQILRSM KGLSSTSIVY ILIAVCLCGL IGIPALICCC RGRCNKKGEQ VGMSRPGLKP DLTGTSKSYV RSX, (SEQ ID NO: 20)

HIV TM (World Genetic Bank Accession Number: AAB50262, amino acids 512-710)

AVGIGALFL GFLGAAGSTM AQQHLLQLTV WGIKQLQARI NASWSNKSLE QIWNHTTWME LELDKWASLW NWFNITNWLW NO: 21)

GAASMTLTVQ LAVERYLKDQ

ARQLDSGIVQ QLLGIWGCSG

WDREXNNYTS LIHSLIEESQ

YXKDFIMIVG GLVGLRIVFA

QQNNLLRAXE KLICTTAVPW NQQEKNEQEL

VLSIVNRVRQ (SEQ ID

Procedure to Identify the FIR

For reference purposes, the following procedure is described to identify within the fusion proteins of viruses a conserved residue. The conserved remainder of the FIR regions of different viruses will have similar structure and function. Additionally, the FIR regions of related viruses may have, but do not necessarily have, highly similar amino acid sequences.

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

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

(1) The sequence of the fusion protein is first matched to the framework of the HIV transmembrane fusion protein, comprising the N-helix, the C-helix, and other domains described above in order to identify the N-helix and the C-helix. on the target fusion protein. This matching procedure is facilitated by searching the primary amino acid sequence of the protein for two or more cysteines that have a propensity to form at least one covalently linked loop, which 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 for other amino acids that have the propensity to form an alpha helix (eg, glutamine (Q), alanine (A) , tryptophan (P), lysine (K) and leucine (L)).

(2) The amino terminal end of the FIR is then identified at the N helix. This end will usually be within the final 10 to 20 amino acids of the N helix and will have a core that will typically comprise three or four 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 carboxy-terminal end of the FIR is then identified. In the case of all families except coronaviruses and paramyxoviruses, this end is the carboxy terminal end of the first peptide sequence with positive interface hydrophobicity that lies beyond the N helix. This end is usually located beyond the cysteine ​​loop , if the loop is present and sometimes overlaps with the C helix or is located on the C helix.

ES 2 372 633 T3 positive interface hydrophobicity sequences have a high percentage of aromatic amino acids (such as tryptophan (P), phenylalanine (F) and tyrosine (Y)) and small hydrophobic amino acids (such as glycine (G)). The degree of interface hydrophobicity of these sequences can be determined using the Wimley-White interface hydrophobicity scale, preferably with a computer program such as the MPEX program that incorporates this scale. (Interface hydrophobicity is a measure of the ability of a peptide to transfer from an aqueous solution to the membrane bilayer interface and is based on the hydrophobicity scale of the entire Wimley-White residue determined (Jaysinghe, Hristova, and White, 2000). Computer programs using this scale can identify a peptide sequence from a peptide chain that has positive interface hydrophobicity scores and is therefore the most likely to associate with the surface of membranes). See Example 1, as an example of the application of this procedure to the identification of the FIR in the Ebola virus.

In the case of coronaviruses, which have longer alpha helices and a generally larger scale, and paramyxoviruses, in which the FIR is discontinuous due to an insert of a non-FIR sequence, the carboxy-terminal end of the FIR is the extreme carboxy-terminal of the second peptide sequence with positive interface 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 paramyxovirus, which has a receptor-binding function , is correspondingly shorter. Upon inspection of computer models, it is obvious to those skilled in the art that the F1 protein contains a sequence insert between the N helix and the C helix. Consequently, the paramyxovirus FIR contains two cysteine ​​loops and two high-interface hydrophobicity sequences and is discontinuous because additional amino acids that are characteristic only of paramyxoviruses and appear between the N-helix and the high-interface hydrophobicity sequence are excluded from the FIR.

FIR sequences

The sequence of the fusion protein and FIR for each of the six representative viruses shown in the figures from Figure 2 to Figure 7 is given 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 NO: 7 provide the respective FIRs). Although there is less sequence variation between sister viruses among each of these six families, the FIR in any Type I virus can be easily identified using the representative sequence given in the appropriate figure.

Procedures to inhibit condensation in these viruses

The present invention provides compositions as defined in the claims that inhibit virus: cell condensation by interfering with FIR function. Various aspects of these embodiments include targeting the FIR with peptides, - isolated nucleic acid sequences as defined in order to interfere with virus: cell fusion. In the present invention the peptides consist of a sequence of SEQ ID NO: 4 or a segment of 8 to 40 contiguous amino acids thereof and are of such length as is necessary to provide effective inhibition of viral infection by influenza virus. . As used herein the term "such length as necessary to provide effective inhibition of the virus" preferably refers to a length sufficient to provide a 5-fold or greater reduction in viral infectivity, when used in accordance with the invention. current. Procedures for quantifying 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, binding inhibition, titer reduction assay, or by animal testing studies.

The FIR peptide of SEQ ID NO: 4 or fragments thereof contemplated as being part of the current invention are as defined in the claims. The following sequences are shown for the purposes of comparison with SEQ ID NO: 4.

LASSA

<img file="ES2372633T3_D0001.tif" />

SARS

X-LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPH

GVVMHViyVPSQERNFITAPAICHEGKAYFPRBGVFVFNGTSWECrQRjNFPS-Z (SEQ ID

NO: 2)

EBOLA

<img file="ES2372633T3_D0002.tif" />

ES 2 372 633 T3

INFLUENZA

<img file="ES2372633T3_D0003.tif" />

MEASLES

X-LGLKLLRYYTEILSLFG-Z (SEQ ID N0: 5)

X-WYTTVPKYVATQGYUSNTOESSCTFMPEGTVCSQNALYPMSPLLQE

CLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTn-Z (SEQ ID NO: 6) (The - indicates that the measles FIR is discontinuous).

HIV X-LQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLE QIWNHTTWMEWD-Z (SEQ ID NO: 7)

In each of the preceding sequences, X and Z respectively designate either the amino-terminal end or the carboxy-terminal end, respectively, of peptide or of an additional residue, as described below.

The peptide of SEQ ID NO: 4 provided by the current invention has the sequence of an FIR region. The FIR region of the peptide of SEQ ID NO: 4 is from the influenza virus that belongs to the viral family of orthomyxoviruses, which include Influenza A virus, Influenza B virus, and Influenza C virus.

Other aspects of this embodiment of the invention provide a contiguous 8 to 40 amino acid segment of the peptide of SEQ ID NO: 4 comprising a functional fragment of the FIR sequence of influenza virus SEQ ID NO: 4. belonging to the viral family of orthomyxoviruses that includes influenza A viruses, influenza B viruses and influenza C viruses.

Derived peptides may comprise altered sequences in which functionally equivalent amino acid residues are substituted in the sequence resulting in a silent change. For example, one or more amino acid residues in the sequence can be substituted for another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration (eg, substitution of isoleucine for leucine). Substitutes for an amino acid within the sequence can be selected from other members of the class to which said amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged amino acids (acids) include aspartic acid and glutamic acid. By way of further example, and not by way of limitation, such peptides may also comprise D amino acids, and / or may comprise an ineffective carrier protein, or no carrier protein at all.

FIR peptides can comprise peptides in which X comprises an amino group, an acetyl group, a hydrophobic group, or a macromolecular transporter; and / or Z comprises a carboxyl group, an amido group, a hydrophobic group, or a macromolecular carrier group.

The residue X can also be selected from the group comprising: a hydrophobic residue, a carbobenzoxyl residue, a dansyl residue, or a t-butyloxycarbonyl residue. -The Z moiety may be selected from the group comprising: a hydrophobic moiety, a t-butyloxycarbonyl moiety.

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

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

The peptide may comprise one or more amino acid residues that is / are in a D-isomer amino acid.

Peptides may comprise a substitution of one or more amino acids 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. Still other aspects of this embodiment of the invention stipulate peptides as

ES 2 372 633 T3 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 current invention comprise at least 8 contiguous residues of an FIR. As used herein the term FIR inhibitory peptide (s) preferably refers to a peptide or peptides having the sequence of an FIR (or a functional segment thereof) and to such functional peptides or segments in those in which one or more amino acids is / are substituted with functionally equivalent or chemically similar amino acids (see below). This also refers to derivatives of these peptides, including, but not limited to, benzylated derivatives, glycosylated derivatives, and peptides that include naturally occurring enantiomers of amino acids.

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 current invention contemplates molecules that comprise any combination of residues X and Z and / or other peptide modifications described above.

Peptides according to the present invention can be produced from naturally occurring or recombinant viral proteins. They can also be produced using standard recombinant DNA techniques (for example the expression of peptide by a microorganism containing recombinant nucleic acid molecules encoding the desired peptide, expressed under the control of a suitable transcriptional promoter and the collection of the desired peptide 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, solid phase Merrifield synthesis (Clark-Lewis et al., 1986, Science 231: 134139).

Embodiments of the present invention also provide antibodies as defined in the claims useful for treating or preventing infection of a cell by a virus. Antibodies include active segments thereof, significant parts of antibodies capable of specifically recognizing an FIR region or a functional segment thereof. The antibodies specifically recognize an FIR peptide of SEQ ID NO: 4, or an antigenic fragment thereof to prevent or reduce infection of the cell by the virus. The antibodies according to these claims of the invention can be monoclonal or polyclonal.

The invention provides a method as defined in the claims of producing antibodies capable of specifically recognizing an FIR peptide of SEQ ID NO: 4 useful for preventing or reducing infection of the cell by the virus. 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 peptide of SEQ ID NO:. 4;

(ii) exposes an animal's immune system to antigen such as to induce an immune response;

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

In accordance with various aspects of the current invention, the peptides and / or antibodies of the current invention useful for treating or preventing viral infection of a cell can target the surrounding amino acids and within the cysteine ​​loop of FIR, the distal portion of the N-helix of FIR, any of the hydrophobicity regions of the FIR, other areas of the FIR, or any combination thereof. These peptides (collectively composed) can be used individually; alternatively they can be used in combinations of two or more to prevent or inhibit infection of the cell by the virus. Methods of preventing or inhibiting viral infection of the cell by interfering with the function of the FIR provided by the current invention also include the use of neutralizing antibodies, exogenously or endogenously produced, against the entire FIR or against parts of the FIR. The purpose of such use is to interfere with the function of the FIR, thereby inhibiting viral infection of cells and / or the condensation of virus: cell membranes.

Other embodiments of the current invention provide compositions, including pharmaceutical compositions, comprising any and all of the peptides of the current invention. This includes, but is not limited to, compositions containing any molecule that comprises, consists essentially of, or consists of an FIR peptide of SEQ ID NO: 4 or a functional segment of the FIR. This further includes, but is not limited to, compositions comprising any compound that specifically recognizes, binds to, or interferes with the function of a viral FIR. As used herein, the phrase "interfering 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 such as to prevent or reduce infection of the cell. by the virus. Additionally, it is contemplated that the compositions may comprise either one of the disclosed molecules or mixtures of two or more of the molecules.

Other embodiments of the current invention provide uses of compounds of the invention in treating or preventing influenza virus infection of a cell. Various aspects of this embodiment of the invention stipulate an effective amount of any of the pharmaceutical compositions described herein for use in administration to a patient suspected of being exposed to influenza virus (or having the potential to be

ES 2 372 633 T3 exposed to influenza virus).

Still other aspects of this embodiment of the invention provide for methods comprising an effective amount of a composition comprising at least one recombinant DNA or RNA molecule; wherein the RNA or DNA encodes an FIR peptide of SEQ ID NO: 4 (or functional segment thereof), such as to prevent or reduce influenza virus infection. In a preferred aspect of this embodiment the recombinant RNA or recombinant DNA molecule and / or the pharmaceutical composition further comprises the elements necessary to allow the protein encoded by the RNA or 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 recombinant RNA or 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 crystallographic procedures (Malashkevich et al., 1999). The terminal amino acids of the N-helix contain the sequence ILNRKAIDF (SEQ ID NO: 8) which agrees with the consensus 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 FIR is the sequence FLLQRWGGTCH-ILGPDCCI (SEQ ID NO: 10), which has a WimleyWhite interface hydrophobicity score of 2.59 as determined by the MPEX program (Jaysinghe et al. ., 2002). Thus, the FIR of EboIa virus GP2 extends from amino acid 579 to amino acid 610.

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. The N and C helices of measles virus F1 can be identified by examining the primary sequence of amino acids with the propensity to form helices. Alignment of the primary sequence of measles virus F1 with the primary amino acid sequence of the F1 protein of another paramyxovirus, Newcastle disease virus F1, may also aid in the identification of helix boundaries. The structure of Newcastle disease virus F1 has been determined by x-ray crystallographic procedures (Chen et al., 2001). It can thus be predicted that the boundaries of the N and C helices are amino acids 131-217 and 455-491 respectively. In contrast to Ebola virus GP2 and most other viral class I fusion proteins, the primary sequence between the N and C helices in measles viruses is longer than 100 amino acids. The FIR region of measles virus F1 contains an insert that, after inspection of computer models, is obvious to those skilled in the art and thus the structure of FIR is made up of a secondary arrangement that brings together two parts of the primary sequence. The inserted sequence forms a loop outside the FIR. The terminal amino acids of the N-helix contain the sequence LKLLRYYTE (SEQ ID NO: 11) which agrees with the consensus 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. There are eight cysteine ​​residues in the F1 measles virus F1 between the N and C helices. On the basis of the F1 alignment of the Newcastle disease virus it can be determined that the first two cysteines and the second two cysteines form disulfide-linked loops: The first pair of cysteines in the sequence, CTFMPEGTVC (SEQ ID NO: 12), it is part of the FIR because it is linked by a sequence WYTTVPKYVATQGYLISNF (SEQ ID NO: 13) with a Wimley-White interface hydrophobicity score of 3.36, as determined by the MPEX program., The second cysteine ​​pair in the sequence, CLRGSTKSC (SEQ ID NO: 14), is also part of the FIR because it is adjacent to a TLVSGSFGNRFILSQGNLIANCASILCKCYTTGTII (SEQ ID NO: 15) sequence with a Wimley-White interface hydrophobicity score of 2.54, as determined by the MPEX program. Thus, the FIR of measles virus F1 extends from amino acid 205 to amino acid 407, with amino acids 221 to 314 representing an insert that does not participate in FIR function.

Example 3: Identification of coronavirus condensation inhibitor peptides.

Background

Severe acute respiratory syndrome (SARS) is a recently recognized disease that spread from southern China in late 2002 / early 2003 to various countries in Asia, Europe, and North America (Guan et al., 2004). SARS usually begins with a fever above 38 ° C. Initial symptoms may also include headache, malaise, and mild respiratory symptoms. Within two days to a week, SARS patients can develop a dry cough and have respiratory problems. Patients in the more advanced stages of SARS develop either pneumonia or respiratory distress syndrome. In the initial outbreak there were 8,098 worldwide, with an overall mortality of 9.6%. A previously unrecognized 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.

ES 2 372 633 T3 cols., 2003; Mara et al., 2003). Public health interventions, such as surveillance, travel restrictions and quarantines, contained the original spread of SARS CoV in 2003 and appear to stop the spread of SARS again after the emergence of a few new cases in 2004. Unknown, no However, if draconian containment measures can be maintained with each occurrence of the SARS CoV in humans. Furthermore, the potential for this new and sometimes deadly CoV as a bioterrorism threat is obvious.

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

Materials and procedures

L2 cells or Vero E6 cells were maintained as monolayers in complete Dulbecco's Modified Eagle's Medium (DMEM) containing 0.15% HCO3 supplemented with 10% Fetal Bovine Serum (FBS), Penicillin G (100 U / ml) , streptomycin (100 mg / ml) and 2 mM L-glutamine at 37 ° C in a 5% CO2 incubator. The murine hepatitis virus (MHV) strain A59 or the SARS CoV strain Urbani were propagated in L2 cells. For plaque assays, L2 cells or E6 cells were seeded at a density of 1 x 10⁶ cells in each 6-well plate. Fifty to 100 plaque-forming units (pfu) of SARS MHV or CoV were preincubated with or without approximately 100 pg / ml serum-free DMEM epitium for 1 hr. Cells were then infected with peptide treated inoculum or vehicle control inoculum. After adsorption for 1 hour, the inoculum was removed, the cells were washed twice with 1X phosphate buffered saline and the cells were coated with 10% FBS / DMEM containing 0.5% SEAPLAQUE® agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME). Monolayers were fixed with 3.7% formalin and stained with crystal violet 1X 2 days after infection and the numbers of plaques were determined by light microscopy.

Results and Discussion

Synthetic peptides corresponding to the FIR domains of the MHV or SARS CoV protein S were tested for their ability to inhibit infection by these coronaviruses. The ability to inhibit plaque formation in cell monolayers is the most stringent in vitro test of a potential infection inhibitor drug. Two peptides (GNHILSLVQNAPYGLYFIHFSW, SEQ ID NO: 22 and GYFVQDDGEWKFTGSSYYY, SEQ ID NO: 23) from MVH FIR can inhibit plaque formation by MHV, although the first MHV FIR peptide is more efficient (see Fig 8A). Two peptides from the CoV FIR, from SARS (GYHLMS-FPQAAPHGVVFLHVTY, SEQ ID NO: 24 and GVFVFNGTSWFITQRNFFS, SEQ ID NO: 25) inhibited plaque formation by this coronavirus (see Fig. 8B). There was also a significant reduction (~ 50%) in the mean diameter of the residual plates. These results suggest that this peptide inhibits both entry and dispersal 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 non-specific cytotoxic effects of peptides. Except for the plates, the cells in the monolayers were intact and viable. The low number of plaques that grew were similar in size to the control plaques. Peptides from other regions also inhibited infection by these viruses, but to a lesser extent than the more active FIR peptides (Fig. 8). For example, peptides from the peptide condensation region and the carboxyl-terminal helix (helix C) of the S of MHV and of the S of CoV of SARS exhibited some inhibition (MHV S condensation peptide = MFPPWSAAAGVPFSLSVQY, SEQ ID NO. : 26; MHV S helix C = QDAIKKLNESYINLKEVGTYEMYVKW, SEQ ID NO: 27; SARS CoV S condensation peptide = MYKTPTLKY-FGGFNFSQIL, SEQ ID NO: 28; SARS CoV S helix = AACEVAKNLNESLIDLQELGKYEQYIKW, SEQ ID NO: 29. Inhibitory activities in the pM range with coronavirus C-helix peptides were recently reported by Bosch et al., (2003) and others (Bosch et al., 2004; Lui et al., 2004; Yuan et al., 2004). ; Zhu et al., 2004). However, coronavirus inhibitory peptides have not been reported in FIRs. However, in view of the current invention, the references cited collectively provide support for the tremendous advantages of the presently described and claimed inventions. That is, these references are consistent with the present inventors' assertion that the methods of the present invention can be used to advantage in identifying synthetic peptides that inhibit condensation / infectivity by members of the Coronaviridae family.

Example 4: Identification of arenavirus condensation inhibitor peptides.

Background

Lassa fever is an often fatal hemorrhagic disease named after the city in the Yedseram River Valley of Nigeria in which the first reported cases occurred in 1969 (Buckley and Casals, 1970). Parts of Guinea, Sierra Leone, Nigeria, and Liberia are endemic for the etiologic agent, Lassa virus (LasV). The public health impact of LasV in endemic areas is immense. The Centers for Disease Control and Prevention (CDC)

ES 2 372 633 T3 have estimated that there are 100,000-300,000 cases of Lassa per year in West Africa and 5,000 deaths. In some parts of Sierra Leone, 10-15% of all hospital patients have Lassa fever. Disease mortalities for Lassa fever are typically 15% to 20%, although in epidemics the overall mortality can be as high as 45%. The mortality rate for women in the last month of pregnancy is always high, ~ 90%, and infection with LasV causes high rates of fetal death in all stages of gestation. Mortality rates for Lassa appear to be higher in non-Africans, which is of concern because Lassa is the most commonly exported hemorrhagic fever. Due to the high case fatality rate and the ability to spread easily by human-human contact, LasV is classified as a Biosafety Level 4 agent and as a NIAID Biodefense Category A agent.

LasV is a member of the Arenaviridae family. The arenavirus genome is made up of two single-stranded RNA segments, in both directions. When viewed 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). Hence the use for the Latin family name arena which means sandy. In addition to LasV, other arenaviruses that cause disease in humans include Junin 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 species of rodents (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 occurs 1-3 weeks after exposure to the virus, are highly variable, but may include fever, retrosternal, back or abdominal pain, sore throat, cough, vomiting, diarrhea , conjunctival injection and facial swelling. Vs infect endothelial cells, resulting in increased capillary permeability, decreased effective circulating volume, shock, and multi-organ system failure. Frank hemorrhage, usually mucosal (gums, etc.), occurs in less than a third of cases, but confers a poor prognosis. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Surviving patients begin to lower their fever 2-3 weeks after onset of illness. The most common complication of Lassa fever is deafness. Unilateral or bilateral temporary or permanent deafness occurs in ~ 30% of Lassa fever patients during convalescence and is not associated with the severity of the acute illness. The antiviral drug ribavirin is effective in treating Lassa fever, but only if it is administered early (up to six 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 Junin virus, Machupo virus, Guanarito virus, or Sabia virus. No LasV vaccine is currently available.

Materials and procedures

Vero cells were maintained as monolayers in Basal Eagle's Medium (BME) containing 10 mM HEPES and 5% FBS. Lassa virus (Josiah strain) was propagated on Vero cells. For plaque assays, Vero cells were seeded at a density of 1 x 10⁶ cells in each well of 6-well plate. Fifty to 100 pfu of LasV were pre-incubated with or without peptide in serum-free BME for 1 h. Cells were then infected with peptide treated inoculum or vehicle control inoculum. After adsorption for 1 h, the inoculum was removed, the cells were washed twice with 1X phosphate buffered saline and the cells were coated with 2 ml of 0.5% agarose in BME containing 10 mM HEPES and 5 FBS. % and incubated for 4 days. A second coating containing 5% neutral red was applied and the plates were counted 24 hours later.

Results and Discussion

Synthetic peptides corresponding to the FIR domains of LasV glycoprotein 2 (GP2) were tested for their ability to inhibit infection by this arenavirus. A peptide (NYSKYWYLNHTtGr, SEQ ID NO: 30) analogous to the sequence NYSRYWYLNHTSTGK from SEQ ID NO: 1 (LASsA FIR) can inhibit plaque formation by LasV (Fig. 9). A peptide analogous to another GP2 region, the fusion peptide, (GTFTWTLSDSEGKDTPGGY, SEQ ID NO: 31) also inhibited LasV infection, but to a lesser degree (Fig. 9). Arenavirus inhibitory peptides have not been reported. Collectively, these results suggest that our approaches can identify synthetic peptides that inhibit condensation / infectivity by members of the Arenaviruses. These results, in combination with our results with coronavirus FIR inhibitor peptides, establish proof of the principle that peptides from the FIR regions can function as viral inhibitors.

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SEQUENCE LISTING &lt;110&gt; Garry, Jr., Robert F.

Wilson, Russell B.

&lt;120&gt; PROCEDURE TO AVOID VIRUS CONDENSATION: CELL INHIBITING THE FUNCTION OF THE CONDENSATION INITIATION REGION IN RNA VIRUSES THAT HAVE CLASS I FUSION-GENERIC MEMBRANE ENVELOPE PROTEINS &lt;130&gt; 12920.0013.00 USA 60PC00 &lt;150&gt; 12920.0013.00 USA 60PC00 &lt;150&gt; &lt;151&gt; 4-11-2003 &lt;160&gt; 31 &lt;170&gt; PatentIn version 3.3 &lt;210&gt; 1 &lt;211&gt; 39 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

<td>Leu 1</td><td>lie</td><td>Met</td><td>Lys</td><td>Asn 5</td><td>His</td><td>Leu</td><td>Arg</td><td>Asp</td><td>lie 10</td><td>Met</td><td>Gly</td><td>lie</td><td>Pro</td><td>Tyr fifteen</td><td>Cys</td>

<td>Asn</td><td>Tyr</td><td>To be</td><td>Arg twenty</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Leu</td><td>Asn 25</td><td>His</td><td>Thr</td><td>To be</td><td>Thr</td><td>Gly 30</td><td>Lys</td><td>Thr</td>

<td>Leu</td><td>Pro</td><td>Arg 35</td><td>Cys</td><td>Trp</td><td>Leu</td><td>lie</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 2

ES 2 372 633 T3 &lt;211&gt; 100 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

<td rowspan="2">Leu 1</td><td colspan="3" rowspan="2">lie Arg Wing</td><td colspan="6">Wing Glu lie Arg Wing Ser</td><td colspan="3" rowspan="2">Wing Asn Leu</td><td rowspan="2">To</td><td colspan="2" rowspan="2">Wing Thr fifteen</td>

<td> 5</td><td colspan="5"> 10</td>

<td>Lys</td><td>Met</td><td>To be</td><td>Glu</td><td>Cys</td><td>Val</td><td>Leu</td><td>Gly</td><td>Gln</td><td>To be</td><td>Lys</td><td>Arg</td><td>Val</td><td>Asp</td><td>Phe</td><td>Cys</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>Gly</td><td>Lys</td><td>Gly</td><td>Tyr</td><td>His</td><td>Leu</td><td>Met</td><td>To be</td><td>Phe</td><td>Pro</td><td>Gln</td><td>To</td><td>To</td><td>Pro</td><td>His</td><td>Gly</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>Val</td><td>Val</td><td>Phe</td><td>Leu</td><td>His</td><td>Val</td><td>Thr</td><td>Tyr</td><td>Val</td><td>Pro</td><td>To be</td><td>Gln</td><td>Glu</td><td>Arg</td><td>Asn</td><td>Phe</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> 60</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Thr</td><td>To</td><td>Pro</td><td>To</td><td>lie</td><td>Cys</td><td>His</td><td>Glu</td><td>Gly</td><td>Lys</td><td>To</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Arg</td>

70 75 80

Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp Phe lie Thr Gln Arg 85 90 95

Asn Phe Phe Ser

100 &lt;210&gt; 3 &lt;211&gt; 32 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

Leu Arg Thr Phe Ser lie Leu Asn Arg Lys Ala lie Asp Phe Leu Leu 15 10 15

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

ES 2 372 633 T3 &lt;223&gt; Synthetic peptide &lt;400&gt; 4

<td>lie</td><td>Gln</td><td>Asp</td><td>Leu</td><td>Glu</td><td>Lys</td><td>Tyr</td><td>Val</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Lys</td><td>lie</td><td>Asp</td><td>Leu</td><td>Trp</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>To be</td><td>Tyr</td><td>Asn</td><td>To</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Val</td><td>To</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Gln</td><td>His</td><td>Thr</td><td>lie</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>Asp</td><td>Leu</td><td>Thr</td><td>Asp</td><td>To be</td><td>Glu</td><td>Met</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Phe</td><td></td><td></td><td></td><td></td><td></td>

40 &lt;210&gt; 5 &lt;211&gt; 17 &lt;212&gt; PROTEIN &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 lie Leu Ser Leu Phe 15 10 15

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

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

ES 2 372 633 T3

<td>Trp 1</td><td>Tyr</td><td>Thr</td><td>Thr</td><td>Val 5</td><td>Pro</td><td>Lys</td><td>Tyr</td><td>Val</td><td>To 10</td><td>Thr</td><td>Gln</td>

<td>To be</td><td>Asn</td><td>Phe</td><td>Asp twenty</td><td>Glu</td><td>To be</td><td>To be</td><td>Cys</td><td>Thr 25</td><td>Phe</td><td>Met</td><td>Pro</td>

<td>Cys</td><td>To be</td><td>Gln 35</td><td>Asn</td><td>To</td><td>Leu</td><td>Tyr</td><td>Pro 40</td><td>Met</td><td>To be</td><td>Pro</td><td>Leu</td>

<td>Leu</td><td>Arg fifty</td><td>Gly</td><td>To be</td><td>Thr</td><td>Lys</td><td>To be 55</td><td>Cys</td><td>To</td><td>Arg</td><td>Thr</td><td>Leu 60</td>

<td>Phe 65</td><td>Gly</td><td>Asn</td><td>Arg</td><td>Phe</td><td>lie 70</td><td>Leu</td><td>To be</td><td>Gln</td><td>Gly</td><td>Asn 75</td><td>Leu</td>

<td>To</td><td>To be</td><td>lie</td><td>Leu</td><td>1 Cys</td><td>Lys</td><td>Cys</td><td>Tyr</td><td>Thr</td><td>Thr</td><td>Gly</td><td>Thr</td>

90

<td>Gly</td><td>Tyr</td><td>Leu fifteen</td><td>lie</td>

<td>Glu</td><td>Gly 30</td><td>Thr</td><td>Val</td>

<td>Leu Four. Five</td><td>Gln</td><td>Glu</td><td>Cys</td>

<td>Val</td><td>To be</td><td>Gly</td><td>To be</td>

<td>lie</td><td>To</td><td>Asn</td><td>Cys 80</td>

<td>lie</td><td>lie</td><td></td><td></td>

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

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

<td>Leu</td><td>Gln</td><td>To</td><td>Arg</td><td>lie</td><td>Leu</td><td>To</td><td>Val</td><td>Glu</td><td>Arg</td><td>Tyr</td><td>Leu</td><td>Lys</td><td>Asp</td><td>Gln</td><td>Gln</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>dy</td><td>lie</td><td>Trp</td><td>Gly</td><td>Cys</td><td>To be</td><td>Gly</td><td>Lys</td><td>Leu</td><td>lie</td><td>Cys</td><td>Thr</td><td>Thr</td><td>To</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>Asn</td><td>To</td><td>To be</td><td>Trp</td><td>To be</td><td>Asn</td><td>Lys</td><td>To be</td><td>Leu</td><td>Glu</td><td>Gln</td><td>lie</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>Asn</td><td>His</td><td>Thr</td><td>Thr</td><td>Trp</td><td>Met</td><td>Glu</td><td>Trp</td><td>Asp</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

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

&lt;223&gt; Synthetic peptide &lt;400&gt; 8 lie Leu Asn Arg Lys Ala lie Asp Phe 1 5 &lt;210&gt; 9

ES 2 372 633 T3 &lt;211&gt; 8 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

Cys His lie Leu Gly Pro Asp Cys

5 &lt;210&gt; 10 &lt;211&gt; 19 &lt;212&gt; PROTEIN &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 lie Leu Gly Pro Asp

1,5 10 15

Cys Cys lie &lt;210&gt; 11 &lt;211&gt; 9 &lt;212&gt; PROTEIN &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

5 &lt;210&gt; 12 &lt;211&gt; 10 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

ES 2 372 633 T3

Cys Thr Phe Met Pro Glu Gly Thr Val Cys 15 10 &lt;210&gt; 13 &lt;211&gt; 19 &lt;212&gt; PROTEIN &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 lie 15 10 15

Ser Asn Phe &lt;210&gt; 14 &lt;211&gt; 9 &lt;212&gt; PROTEIN &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

5 &lt;210&gt; 15 &lt;211&gt; 36 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

<img file="ES2372633T3_D0004.tif" />

<td rowspan="2">Thr Leu Val 1</td><td colspan="3">Ser Gly Ser Phe Gly Asn Arg Phe lie Leu Ser Gln Gly</td>

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

<td>Asn Leu lie</td><td>Wing Asn Cys Wing</td><td>Be lie leu</td><td>Cys Lys Cys Tyr Thr Thr</td>

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

<td>Gly Thr lie</td><td>lie</td><td></td><td></td>

<td> 35</td><td></td><td></td><td></td>

&lt;210&gt; 16

ES 2 372 633 T3 &lt;211&gt; 234 &lt;213&gt; VIRUS LASSA &lt;400&gt; 16

<td>Leu 1</td><td>Leu</td><td>Gly</td><td>Thr</td><td>Phe 5</td><td>Thr</td><td>Trp</td><td>Thr</td><td>Leu</td><td>To be 10</td><td>Asp</td><td>To be</td><td>Glu</td><td>Gly</td><td>Asn fifteen</td><td>Glu</td>

<td>Thr</td><td>Pro</td><td>Gly</td><td>Gly twenty</td><td>Tyr</td><td>Cys</td><td>Leu</td><td>Thr</td><td>Arg 25</td><td>Trp</td><td>Met</td><td>Leu</td><td>lie</td><td>Glu 30</td><td>To</td><td>Glu</td>

<td>Leu</td><td>Lys</td><td>Cys 35</td><td>Phe</td><td>Gly</td><td>Asn</td><td>Thr</td><td>To 40</td><td>Val</td><td>To</td><td>Lys</td><td>cys</td><td>Asn Four. Five</td><td>Glu</td><td>Lys</td><td>His</td>

<td>Asp</td><td>Glu fifty</td><td>Glu</td><td>Phe</td><td>Cys</td><td>Asp</td><td>Met 55</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Phe</td><td>Asp 60</td><td>Phe</td><td>Asn</td><td>Lys</td><td>Gln</td>

<td>To 65</td><td>lie</td><td>Arg</td><td>Arg</td><td>Leu</td><td>Lys 70</td><td>Thr</td><td>Glu</td><td>To</td><td>Gln</td><td>Met 75</td><td>To be</td><td>lie</td><td>Gln</td><td>Leu</td><td>lie 80</td>

<td>Asn</td><td>Lys</td><td>To</td><td>Val</td><td>Asn 85</td><td>To</td><td>Leu</td><td>lie</td><td>Asn</td><td>Asp 90</td><td>Gln</td><td>Leu</td><td>lie</td><td>Met</td><td>Lys 95</td><td>Asn</td>

<td>His</td><td>Leu</td><td>Arg</td><td>Asp 100</td><td>lie</td><td>Met</td><td>Gly</td><td>lie</td><td>Pro 105</td><td>Tyr</td><td>Cys</td><td>Asn</td><td>Tyr</td><td>To be 110</td><td>Arg</td><td>Tyr</td>

<td>Trp</td><td>Tyr</td><td>Leu</td><td>Asn</td><td>His</td><td>Thr</td><td>To be</td><td>Thr</td><td>Gly</td><td>Lys</td><td>Thr</td><td>To be</td><td>Leu</td><td>Pro</td><td>Arg</td><td>Cys</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Trp</td><td>Leu</td><td>lie</td><td>To be</td><td>Asn</td><td>Gly</td><td>To be</td><td>Tyr</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Thr</td><td>Lys</td><td>Phe</td><td>To be</td><td>Asp</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>lie</td><td>Glu</td><td>Gln</td><td>Gln</td><td>To</td><td>Asp</td><td>Asn</td><td>Met</td><td>lie</td><td>Thr</td><td>Glu</td><td>Met</td><td>Leu</td><td>Gln</td><td>Lys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Glu</td><td>Tyr</td><td>lie</td><td>Asp</td><td>Arg</td><td>Gln</td><td>Gly</td><td>Lys</td><td>Thr</td><td>Pro</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Val</td><td>Asp</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Phe</td><td>Val</td><td>Phe</td><td>To be</td><td>Thr</td><td>To be</td><td>Phe</td><td>Tyr</td><td>Leu</td><td>lie</td><td>To be</td><td>lie</td><td>Phe</td><td>Leu</td><td>His</td><td>Leu</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Val</td><td>Lys</td><td>lie</td><td>Pro</td><td>Thr</td><td>His</td><td>Arg</td><td>His</td><td>lie</td><td>Val</td><td>Gly</td><td>Lys</td><td>Pro</td><td>Cys</td><td>Pro</td><td>Lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Pro</td><td>His</td><td>Arg</td><td>Leu</td><td>Asn</td><td>His</td><td>Met</td><td>Gly</td><td>lie</td><td>Cys</td><td>To be</td><td>Cys</td><td>Gly</td><td>Leu</td><td>Tyr</td><td>Lys</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gln</td><td>Pro</td><td>Gly</td><td>Val</td><td>Pro</td><td>Val</td><td>Arg</td><td>Trp</td><td>Lys</td><td>Arg</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 17 &lt;211&gt; 388 &lt;212&gt; PROTEIN &lt;213&gt; SARS VIRUS

ES 2 372 633 T3 &lt;400&gt; 17

<td>Trp 1</td><td>Thr</td><td>Phe</td><td>Gly</td><td>To 5</td><td>Gly</td><td>To</td><td>To</td><td>Leu</td><td>Gln 10</td><td>lie</td><td>Pro</td><td>Phe</td><td>To</td><td>Met fifteen</td><td>Gln</td>

<td>Met</td><td>To</td><td>Tyr</td><td>Arg twenty</td><td>Phe</td><td>Asn</td><td>Gly</td><td>lie</td><td>Gly 25</td><td>Val</td><td>Thr</td><td>Gln</td><td>Asn</td><td>Val 30</td><td>Leu</td><td>Tyr</td>

<td>Glu</td><td>Asn</td><td>Gln 35</td><td>Lys</td><td>Gln</td><td>lie</td><td>To</td><td>Asn 40</td><td>Gln</td><td>Phe</td><td>Asn</td><td>Lys</td><td>To Four. Five</td><td>lie</td><td>To be</td><td>Gln</td>

<td>lie</td><td>Gln fifty</td><td>Glu</td><td>To be</td><td>Leu</td><td>Thr</td><td>Thr 55</td><td>Thr</td><td>To be</td><td>Thr</td><td>To</td><td>Leu 60</td><td>Gly</td><td>Lys</td><td>Leu</td><td>Gln</td>

<td>Asp 65</td><td>Val</td><td>Val</td><td>Asn</td><td>Gln</td><td>Asn 70</td><td>To</td><td>Gln</td><td>To</td><td>Leu</td><td>Asn 75</td><td>Thr</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gln 80 '</td>

<td>Leu</td><td>To be</td><td>To be</td><td>Asn</td><td>Phe 85</td><td>Gly</td><td>To</td><td>lie</td><td>To be</td><td>To be 90</td><td>Val</td><td>Leu</td><td>Asn</td><td>Asp</td><td>lie 95</td><td>Leu</td>

<td>To be</td><td>Arg</td><td>Leu</td><td>Asp 100</td><td>Lys</td><td>Val</td><td>Glu</td><td>To</td><td>Glu 105</td><td>Val</td><td>Gln</td><td>lie</td><td>Asp</td><td>Arg 110</td><td>Leu</td><td>lie</td>

<td>Thr</td><td>Gly</td><td>Arg 115</td><td>Leu</td><td>Gln</td><td>To be</td><td>Leu</td><td>Gln 12 0</td><td>Thr</td><td>Tyr</td><td>Val</td><td>Thr</td><td>Gln 125</td><td>Gln</td><td>Leu</td><td>lie</td>

<td>Arg</td><td>To 130</td><td>To</td><td>Glu</td><td>lie</td><td>Arg</td><td>To 135</td><td>To be</td><td>To</td><td>Asn</td><td>Leu</td><td>To 140</td><td>To</td><td>Thr</td><td>Lys</td><td>Met</td>

<td>To be 145</td><td>Glu</td><td>Cys</td><td>Val</td><td>Leu</td><td>Gly 150</td><td>Gln</td><td>To be</td><td>Lys</td><td>Arg</td><td>Val 155</td><td>Asp</td><td>Phe</td><td>Cys</td><td>Gly</td><td>Lys 160</td>

<td>Gly</td><td>Tyr</td><td>His</td><td>Leu</td><td>Met 165</td><td>To be</td><td>Phe</td><td>Pro</td><td>Gln</td><td>To 170</td><td>To</td><td>Pro</td><td>His</td><td>Gly</td><td>Val 175</td><td>Val</td>

ES 2 372 633 T3

<td colspan="4">Phe Leu His Val</td><td colspan="3" rowspan="2">Thr Tyr Val</td><td colspan="3" rowspan="2">Pro Ser Gln 185</td><td colspan="3" rowspan="2">Glu Arg Asn</td><td colspan="2" rowspan="2">Phe Thr 190</td><td rowspan="2">Thr</td>

<td colspan="3"></td><td> 180</td>

<td>To</td><td>Pro</td><td>To</td><td>lie</td><td>Cys</td><td>His</td><td>Glu</td><td>Gly</td><td>Lys</td><td>To</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Gly</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Val</td><td>Phe</td><td>Val</td><td>Phe</td><td>Asn</td><td>Gly</td><td>Thr</td><td>To be</td><td>Trp</td><td>Phe</td><td>lie</td><td>Thr</td><td>Gln</td><td>Arg</td><td>Asn</td><td>Phe</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>To be</td><td>Pro</td><td>Gln</td><td>lie</td><td>lie</td><td>Thr</td><td>Thr</td><td>Asp</td><td>Asn</td><td>Thr</td><td>Phe</td><td>Val</td><td>To be</td><td>Gly</td><td>Asn</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Cys</td><td>Asp</td><td>Val</td><td>Val</td><td>lie</td><td>Gly</td><td>lie</td><td>lie</td><td>Asn</td><td>Asn</td><td>Thr</td><td>Val</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Gln</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Asp</td><td>To be</td><td>Phe</td><td>Lys</td><td>Glu</td><td>Glu</td><td>Leu</td><td>Asp</td><td>Lys</td><td>Tyr</td><td>Phe</td><td>Lys</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Asn</td><td>His</td><td>Thr</td><td>To be</td><td>Pro</td><td>Asp</td><td>Val</td><td>Asp</td><td>Leu</td><td>Gly</td><td>Asp</td><td>lie</td><td>To be</td><td>Gly</td><td>lie</td><td>Asn</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>To</td><td>To be</td><td>Val</td><td>Val</td><td>Asn</td><td>lie</td><td>Gln</td><td>Lys</td><td>Glu</td><td>lie</td><td>Asp</td><td>Arg</td><td>Leu</td><td>Asn</td><td>Glu</td><td>Val</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>To</td><td>Lys</td><td>Asn</td><td>Leu</td><td>Asn</td><td>Glu</td><td>To be</td><td>Leu</td><td>lie</td><td>Asp</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Leu</td><td>Gly</td><td>Lys</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>Tyr</td><td>Glu</td><td>Gln</td><td>Tyr</td><td>lie</td><td>Lys</td><td>Trp</td><td>Pro</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Trp</td><td>Leu</td><td>Gly</td><td>Phe</td><td>lie</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>To</td><td>Gly</td><td>Leu</td><td>lie</td><td>To</td><td>lie</td><td>Val</td><td>Met</td><td>Val</td><td>Thr</td><td>lie</td><td>Leu</td><td>Leu</td><td>Cys</td><td>Cys</td><td>Met</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Thr</td><td>To be</td><td>Cys</td><td>Cys</td><td>To be</td><td>Cys</td><td>Leu</td><td>Lys</td><td>Gly</td><td>To</td><td>Cys</td><td>To be</td><td>Cys</td><td>Gly</td><td>To be</td><td>Cys</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Cys</td><td>Lys</td><td>Phe</td><td>Asp</td><td>Glu</td><td>Asp</td><td>Asp</td><td>To be</td><td>Glu</td><td>Pro</td><td>Val</td><td>Leu</td><td>Lys</td><td>dy</td><td>Val</td><td>Lys</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>His</td><td>Tyr</td><td>Thr</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

385 &lt;210&gt; 18 &lt;211&gt; 175 &lt;212&gt; PROTEIN &lt;213&gt; EBOLA VIRUS &lt;400&gt; 18

ES 2 372 633 T3

<td>Glu 1</td><td>To</td><td>lie</td><td>Val</td><td>Asn 5</td>

<td>Trp</td><td>Thr</td><td>Thr</td><td>Gln twenty</td><td>Asp</td>

<td>Tyr</td><td>Phe</td><td>Gly 35</td><td>Pro</td><td>To</td>

<td>Asn</td><td>Gln fifty</td><td>Asp</td><td>Gly</td><td>Leu</td>

<td>Thr 65</td><td>Gln</td><td>To</td><td>Leu</td><td>Gln</td>

<td>To</td><td>Gln</td><td>Pro</td><td>Lys</td><td>Cys 10</td><td>Asn</td><td>Pro</td><td>Asn</td><td>Leu</td><td>His fifteen</td><td>Tyr</td>

<td>Glu</td><td>Gly</td><td>To</td><td>To 25</td><td>lie</td><td>Gly</td><td>Leu</td><td>To</td><td>Trp 30</td><td>lie</td><td>Pro</td>

<td>To</td><td>Glu</td><td>Gly 40</td><td>lie</td><td>Tyr</td><td>Thr</td><td>Glu</td><td>Gly Four. Five</td><td>Leu</td><td>Met</td><td>His</td>

<td>lie</td><td>Cys 55</td><td>Gly</td><td>Leu</td><td>Arg</td><td>Gln</td><td>Leu 60</td><td>To</td><td>Asn</td><td>Glu</td><td>Thr</td>

<td>Leu 70</td><td>Phe</td><td>Leu</td><td>Arg</td><td>To</td><td>Thr 75</td><td>Thr</td><td>Glu</td><td>Leu</td><td>Arg</td><td>Thr 80</td>

<td>Phe</td><td>To be</td><td>lie</td><td>Leu</td><td>Asn 85</td><td>Arg</td><td>Lys</td><td>To</td>

<td>Gly</td><td>Gly</td><td>Thr</td><td>Cys 100</td><td>His</td><td>lie</td><td>Leu</td><td>Gly</td>

<td>ASP</td><td>Trp</td><td>Thr 115</td><td>Lys</td><td>Asn</td><td>lie</td><td>Thr</td><td>Asp 120</td>

<td>Phe</td><td>Val 130</td><td>Asp</td><td>Lys</td><td>Thr</td><td>Leu</td><td>Pro 135</td><td>Asp</td>

<td>Thr 145</td><td>Gly</td><td>Trp</td><td>Arg</td><td>Gln</td><td>Trp 150</td><td>lie</td><td>Pro</td>

<td>lie</td><td>lie</td><td>To</td><td>Val</td><td>lie 165</td><td>To</td><td>Leu</td><td>Phe</td>

<td>I have</td><td>Asp SO</td><td>Phe</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Arg 95</td><td>Trp</td>

<td>Pro 105</td><td>Asp</td><td>Cys</td><td>Cys</td><td>lie</td><td>Glu 110</td><td>Pro</td><td>His</td>

<td>Lys</td><td>lie</td><td>Asp</td><td>Gln</td><td>lie 125</td><td>lie</td><td>His</td><td>Asp</td>

<td>Gln</td><td>Gly</td><td>Asp</td><td>Asn 140</td><td>Asp</td><td>Asn</td><td>Trp</td><td>Trp</td>

<td>To</td><td>Gly</td><td>lie 155</td><td>Gly</td><td>Val</td><td>Thr</td><td>Gly</td><td>Val 160</td>

<td>Cys</td><td>lie 170</td><td>Cys</td><td>Lys</td><td>Phe</td><td>Val</td><td>Phe 175</td><td></td>

&lt;210&gt; 19 &lt;211&gt; 191 &lt;212&gt; PROTEIN &lt;213&gt; INFLUENZA VIRUS &lt;400&gt; 19

ES 2 372 633 T3

<td colspan="7">Gly Leu Phe Gly Wing lie Wing</td><td rowspan="2">Gly</td><td colspan="8">Phe lie Glu Asn Gly Trp Glu Gly</td>

<td colspan="2"> 1</td><td colspan="5"> 5</td><td colspan="2"> 10</td><td colspan="6"> 15</td>

<td>Met</td><td>lie</td><td>Asp</td><td>Gly</td><td>Trp</td><td>Tyr</td><td>Gly</td><td>Phe</td><td>Arg</td><td>His</td><td>Gln</td><td>Asn</td><td>To be</td><td>Glu</td><td>Gly</td><td>Thr</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>Gly</td><td>Gln</td><td>To</td><td>To</td><td>Asp</td><td>Leu</td><td>Lys</td><td>To be</td><td>Thr</td><td>Gln</td><td>To</td><td>To</td><td>lie</td><td>Asp</td><td>Gln</td><td>lie</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>Asn</td><td>Gly</td><td>Lys</td><td>Leu</td><td>Asn</td><td>Arg</td><td>Val</td><td>lie</td><td>Glu</td><td>Lys</td><td>Thr</td><td>Asn</td><td>Glu</td><td>Lys</td><td>Phe</td><td>His</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> 60</td><td></td><td></td><td></td><td></td>

<td>Gln</td><td>lie</td><td>Glu</td><td>Lys</td><td>Glu</td><td>Phe</td><td>To be</td><td>Glu</td><td>Val</td><td>Glu</td><td>Gly</td><td>Arg</td><td>lie</td><td>Gln</td><td>Asp</td><td>Leu</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>Glu</td><td>Lys</td><td>Tyr</td><td>Val</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Lys</td><td>lie</td><td>Asp</td><td>Leu</td><td>Trp</td><td>To be</td><td>Tyr</td><td>Asn</td><td>To</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> 95</td><td></td>

<td>Glu</td><td>Leu</td><td>Leu</td><td>Val</td><td>To</td><td>Leu</td><td>Glu</td><td>Asn</td><td>Gln</td><td>His</td><td>Thr</td><td>lie</td><td>Asp</td><td>Leu</td><td>Thr</td><td>Asp</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>To be</td><td>Glu</td><td>Met</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Phe</td><td>Glu</td><td>Lys</td><td>Thr</td><td>Arg</td><td>Arg</td><td>Gln</td><td>Leu</td><td>Arg</td><td>Glu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Asn</td><td>To</td><td>Glu</td><td>Glu</td><td>Met</td><td>Gly</td><td>Asn</td><td>Gly</td><td>Cys</td><td>Phe</td><td>Lys</td><td>lie</td><td>Tyr</td><td>His</td><td>Lys</td><td>Cys</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Asn</td><td>To</td><td>Cys</td><td>lie</td><td>Glu</td><td>To be</td><td>lie</td><td>Arg</td><td>Asn</td><td>Gly</td><td>Thr</td><td>Tyr</td><td>Asp</td><td>His</td><td>Asp</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Val</td><td>Tyr</td><td>Arg</td><td>Asp</td><td>Glu</td><td>To</td><td>Leu</td><td>Asn</td><td>Asn</td><td>Arg</td><td>Phe</td><td>Gln</td><td>lie</td><td>Lys</td><td>Gly</td><td>Val</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Glu</td><td>Leu</td><td>Lys</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Lys</td><td>Asp</td><td>Trp</td><td>Arg</td><td>Cys</td><td>Asn</td><td>lie</td><td>Cys</td><td>lie</td><td></td>

180 185 190 &lt;210&gt; 20 &lt;211&gt; 438 &lt;212&gt; PROTEIN &lt;213&gt; MEASLES VIRUS &lt;400&gt; 20

ES 2 372 633 T3

<td>Phe</td><td>To</td><td>Gly</td><td>Val</td><td>Val</td><td>Leu</td><td>To</td><td>Gly</td><td>To</td><td>To</td><td>Leu</td><td>Gly</td><td>Val</td><td>To</td><td>Thr</td><td>To</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>To</td><td>Gln</td><td>lie</td><td>Thr</td><td>To</td><td>Gly</td><td>lie</td><td>To</td><td>Leu</td><td>His</td><td>Gln</td><td>To be</td><td>Met</td><td>Leu</td><td>Asn</td><td>To be</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>Gln</td><td>To</td><td>lie</td><td>Asp</td><td>Asn</td><td>Leu</td><td>Arg</td><td>To</td><td>To be</td><td>Leu</td><td>Glu</td><td>Thr</td><td>Thr</td><td>Asn</td><td>Gln</td><td>To</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>lie</td><td>Glu</td><td>To</td><td>lie</td><td>Arg</td><td>Gln</td><td>To</td><td>Gly</td><td>Gln</td><td>Glu</td><td>Met</td><td>lie</td><td>Leu</td><td>To</td><td>Val</td><td>Gln</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> 60</td><td></td><td></td><td></td><td></td>

<td colspan="2">Gly'Val</td><td>Gln</td><td>Asp</td><td>Tyr</td><td>lie</td><td>Asn</td><td>Asn</td><td>Glu</td><td>Leu</td><td>lie</td><td>Pro</td><td>To be</td><td>Met</td><td>Asn</td><td>Gln</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>Leu</td><td>To be</td><td>cys</td><td>Asp</td><td>Leu</td><td>lie</td><td>Gly</td><td>Gln</td><td>Lys</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Lys</td><td>Leu</td><td>Leu</td><td>Arg</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> 95</td><td></td>

<td>Tyr</td><td>Tyr</td><td>Thr</td><td>Glu</td><td>lie</td><td>Leu</td><td>To be</td><td>Leu</td><td>Phe</td><td>Gly</td><td>Pro</td><td>To be</td><td>Leu</td><td>Arg</td><td>Asp</td><td>Pro</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>lie</td><td>To be</td><td>To</td><td>Glu</td><td>lie</td><td>To be</td><td>lie</td><td>Gln</td><td>To</td><td>Leu</td><td>To be</td><td>Tyr</td><td>To</td><td>Leu</td><td>Gly</td><td>Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Asp</td><td>I have</td><td>Asn</td><td>Lys</td><td>Val</td><td>Leu</td><td>Glu</td><td>Lys</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>To be</td><td>Gly</td><td>Gly</td><td>Asp</td><td>Leu</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Gly</td><td>lie</td><td>Leu</td><td>Glu</td><td>To be</td><td>Arg</td><td>Gly</td><td>lie</td><td>Lys</td><td>To</td><td>Arg</td><td>lie</td><td>Thr</td><td>His</td><td>Val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Asp</td><td>Thr</td><td>Glu</td><td>To be</td><td>Tyr</td><td>Phe</td><td>lie</td><td>Val</td><td>Leu</td><td>To be</td><td>lie</td><td>To</td><td>Tyr</td><td>Pro</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>To be</td><td>Glu</td><td>lie</td><td>Lys</td><td>Gly</td><td>Val</td><td>lie</td><td>Val</td><td>His</td><td>Arg</td><td>Leu</td><td>Glu</td><td>Gly</td><td>Val</td><td>To be</td><td>Tyr</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Asn</td><td>lie</td><td>Gly</td><td>To be</td><td>Gln</td><td>Glu</td><td>Trp</td><td>Tyr</td><td>Thr</td><td>Thr</td><td>Val</td><td>Pro</td><td>Lys</td><td>Tyr</td><td>Val</td><td>To</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Thr</td><td>Gln</td><td>Gly</td><td>Tyr</td><td>Leu</td><td>lie</td><td>To be</td><td>Asn</td><td>Phe</td><td>Asp</td><td>Glu</td><td>To be</td><td>To be</td><td>Cys</td><td>Thr</td><td>Phe</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Met</td><td>Pro</td><td>Glu</td><td>Gly</td><td>Thr</td><td>Val</td><td>Cys</td><td>To be</td><td>Gln</td><td>Asn</td><td>To</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Met</td><td>To be</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Pro</td><td>Leu</td><td>Leu</td><td>Gln</td><td>Glu</td><td>Cys</td><td>Leu</td><td>Arg</td><td>Gly</td><td>To be</td><td>Thr</td><td>Lys</td><td>To be</td><td>Cys</td><td>To</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Thr</td><td>Leu</td><td>Val</td><td>To be</td><td>Gly</td><td>To be</td><td>Phe</td><td>Gly</td><td>Asn</td><td>Arg</td><td>Phe</td><td>lie</td><td>Leu</td><td>To be</td><td>Gln</td><td>Gly</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Asn</td><td>Leu</td><td>lie</td><td>To</td><td>Asn</td><td>Cys</td><td>To</td><td>To be</td><td>lie</td><td>Leu</td><td>Cys</td><td>Lys</td><td>Cys</td><td>Tyr</td><td>Thr</td><td>Thr</td>

<td></td><td></td><td>27S</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>lie</td><td>lie</td><td>Asn</td><td>Gln</td><td>Asp</td><td>Pro</td><td>Asp</td><td>Lys</td><td>lie</td><td>Leu</td><td>Thr</td><td>Tyr</td><td>lie</td><td>To</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

ES 2 372 633 T3

<td>To 305</td><td>Asp</td><td>His</td><td>Cys</td><td>Pro</td><td>Val 310</td><td>Val</td><td>Glu</td><td>Val</td><td>Asn</td><td>Gly 315</td><td>Val</td><td>Thr</td><td>lie</td><td>Gln</td><td>Val 320</td>

<td>Gly</td><td>To be</td><td>Arg</td><td>Arg</td><td>Tyr 325</td><td>Pro</td><td>Asp</td><td>To</td><td>Val</td><td>Tyr 330</td><td>Leu</td><td>His</td><td>Arg</td><td>lie</td><td>Asp 335</td><td>Leu</td>

<td>Gly</td><td>Pro</td><td>Pro</td><td>lie 340</td><td>To be</td><td>Leu</td><td>Glu</td><td>Arg</td><td>Leu 3. 4. 5</td><td>Asp</td><td>Val</td><td>Gly</td><td>Thr</td><td>Asn 350</td><td>Leu</td><td>Gly</td>

<td>Asn</td><td>To</td><td>lie 355</td><td>To</td><td>Lys</td><td>Leu</td><td>Glu</td><td>Asp 360</td><td>To</td><td>Lys</td><td>Glu</td><td>Leu</td><td>Leu 365</td><td>Glu</td><td>To be</td><td>To be</td>

<td>Asp</td><td>Gln 370</td><td>lie</td><td>Leu</td><td>Arg</td><td>To be</td><td>Met 375</td><td>Lys</td><td>Gly</td><td>Leu</td><td>To be</td><td>To be 380</td><td>Thr</td><td>To be</td><td>lie</td><td>Val</td>

<td>Tyr 385</td><td>lie</td><td>Leu</td><td>lie</td><td>To</td><td>Val 390</td><td>Cys</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Leu 395</td><td>lie</td><td>Gly</td><td>lie</td><td>Pro</td><td>To 400</td>

<td>Leu</td><td>lie</td><td>Cys</td><td>Cys</td><td>Cys 405</td><td>Arg</td><td>Gly</td><td>Arg</td><td>Cys</td><td>Asn 410</td><td>Lys</td><td>Lys</td><td>Gly</td><td>Glu</td><td>Gln 415</td><td>Val</td>

<td>Gly</td><td>Met</td><td>To be</td><td>Arg 420</td><td>Pro</td><td>Gly</td><td>Leu</td><td>Lys</td><td>Pro 425</td><td>Asp</td><td>Leu</td><td>Thr</td><td>Gly</td><td>Thr 430</td><td>To be</td><td>Lys</td>

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

ES 2 372 633 T3

<td colspan="4" rowspan="2">Wing Val Gly He 1</td><td colspan="12">Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly</td>

<td> 5</td><td colspan="6"> 10</td><td colspan="5"> 15</td>

<td>To be</td><td>Thr</td><td>Met</td><td>Gly twenty</td><td>To</td><td>To</td><td>To be</td><td>Met</td><td>Thr 25</td><td>Leu</td><td>Thr</td><td>Val</td><td>Gln</td><td>To 30</td><td>Arg</td><td>Gln</td>

<td>Leu</td><td>Leu</td><td>To be 35</td><td>Gly</td><td>lie</td><td>Val</td><td>Gln</td><td>Gln 40</td><td>Gln</td><td>Asn</td><td>Asn</td><td>Leu</td><td>Leu Four. Five</td><td>Arg</td><td>To</td><td>lie</td>

<td>Glu</td><td>To fifty</td><td>Gln</td><td>Gln</td><td>His</td><td>Leu</td><td>Leu 55</td><td>Gln</td><td>Leu</td><td>Thr</td><td>Val</td><td>Trp 60</td><td>Gly</td><td>I have</td><td>Lys</td><td>Gln</td>

<td>Leu 65</td><td>Gln</td><td>To</td><td>Arg</td><td>I have</td><td>Leu 70</td><td>To</td><td>Val</td><td>Glu</td><td>Arg</td><td>Tyr 75</td><td>Leu</td><td>Lys</td><td>Asp</td><td>Gln</td><td>Gln 80</td>

<td>Leu</td><td>Leu</td><td>dy</td><td>I have</td><td>Trp 85</td><td>Gly</td><td>Cys</td><td>To be</td><td>Gly</td><td>Lys 90</td><td>Leu</td><td>I have</td><td>Cys</td><td>Thr</td><td>Thr 95</td><td>To</td>

<td>Val</td><td>Pro</td><td>Trp</td><td>Asn 100</td><td>To</td><td>To be</td><td>Trp</td><td>To be</td><td>Asn 105</td><td>Lys</td><td>To be</td><td>Leu</td><td>Glu</td><td>Gln 110</td><td>I have</td><td>Trp</td>

<td>Asn</td><td>His</td><td>Thr 115</td><td>Thr</td><td>Trp</td><td>Met</td><td>Glu</td><td>Trp 120</td><td>Asp</td><td>Arg</td><td>Glu</td><td>I have</td><td>Asn 125</td><td>Asn</td><td>Tyr</td><td>Thr</td>

<td>To be</td><td>Leu 130</td><td>lie</td><td>His</td><td>To be</td><td>Leu</td><td>I have 135</td><td>Glu</td><td>Glu</td><td>To be</td><td>Gln</td><td>Asn 140</td><td>Gln</td><td>Gln</td><td>Glu</td><td>Lys</td>

<td>Asn</td><td>Glu</td><td>Gln</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Glu</td><td>Leu</td><td>Asp</td><td>Lys</td><td>Trp</td><td>To</td><td>To be</td><td>Leu</td><td>Trp</td><td>Asn</td>

145 150 155 160

Trp Phe Asn lie Thr Asn Trp Leu Trp Tyr He Lys Leu Phe lie Met

165 170 175

He Val Gly Gly Leu Val Gly Leu Arg He Val Phe Ala Val Leu Ser

180 185 190 lie Val

Asn Arg Val Arg Gln

195 &lt;210&gt; 22 &lt;211&gt; 22 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

ES 2 372 633 T3

Gly Asn His lie Leu Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu Tyr 15 10 15

Phe lie His Phe Ser Trp &lt;210&gt; 23 &lt;211&gt; 19 &lt;212&gt; PROTEIN &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 Ser 15 10 15

Tyr Tyr Tyr &lt;210&gt; 24 &lt;211&gt; 22 &lt;212&gt; PROTEIN &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 &lt;210&gt; 25 &lt;211&gt; 19 &lt;212&gt; PROTEIN &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 lie Thr Gln Arg Asn 15 10 15

Phe Phe Ser

ES 2 372 633 T3 &lt;210&gt; 26 &lt;211&gt; 19 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence &lt;220&gt;

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

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

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

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

Gln Asp Ala lie Lys Lys Leu Asn Glu Ser Tyr lie Asn Leu Lys Glu 15 10 15

Val Gly Thr Tyr Glu Met Tyr Val Lys Trp

25 '&lt;210&gt; 28 &lt;211&gt; 19 &lt;212&gt; PROTEIN &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 lie Leu &lt;210&gt; 29 &lt;211&gt; 28 &lt;212&gt; PROTEIN &lt;213&gt; Artificial sequence

ES 2 372 633 T3 &lt;220&gt;

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

<td colspan="4">Wing Wing Cys Glu Val Wing Lys Asn Leu Asn Glu Ser Leu lie Asp Leu</td>

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

<td>Gln Glu Leu</td><td>Gly Lys Tyr twenty</td><td>Glu Gln Tyr lie 25</td><td>Lys Trp</td>

&lt;210&gt; 30 &lt;211&gt; 15 &lt;212&gt; PROTEIN &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; PROTEIN &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