CN1829736A - The severe acute respiratory syndrome coronavirus - Google Patents

Abstract

An outbreak of a virulent respiratory virus, now known as Severe Acute Respiratory Syndrome (SARS), was identified in Hong Kong, China and a growing number of countries around the world in 2003. The invention relates to nucleic acids and proteins from the SARS coronavirus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations, diagnostic reagents, kits, etc. The invention also provides methods for treating SARS by administering small molecule antiviral compounds, as well as methods of identifying potent small molecules for the treatment of SARS.

Description

All documents cited herein for severe acute respiratory syndrome coronavirus are incorporated herein by reference.

Priority-requiring related application

The following applications are incorporated herein by reference in their entirety: U.S. provisional patent application 60/462,218, attorney docket No. PP20474.001, filed by the united states post office in express mail on 10/4/2003; U.S. provisional patent application 60/462,465, attorney docket No. PP20480.001, filed by the united states post office in express mail on 11/4/2003; U.S. provisional patent application 60/462,418, attorney docket No. PP20480.002, filed by the united states post office in courier mail on 12 days 4 months 2003; U.S. provisional patent application 60/462,748, attorney docket No. PP20480.003, filed by the U.S. post office in courier mail on 13/4/2003; U.S. provisional patent application 60/463,109, attorney docket No. PP20480.004, filed by the united states post office in express mail on 14 days 4 months 2003; U.S. provisional patent application 60/463,460, attorney docket No. PP20480.005, filed by the united states post office in courier mail on day 4, 15 of 2003; U.S. provisional patent application 60/463,668, attorney docket No. PP20480.006, filed by the united states post office in express mail on day 16/4/2003; U.S. provisional patent application 60/463,983, attorney docket No. PP20480.007, filed by the united states post office in express mail on 17 th month 4 2003; U.S. provisional patent application 60/463,971, attorney docket No. PP20480.008, filed by the united states post office in express mail on 18/4/2003; U.S. provisional patent application 60/464,899, attorney docket No. PP20480.009, filed by the united states post office in express mail on day 22 of month 4 2003; U.S. provisional patent application 60/464,838, attorney docket No. PP20507.001, filed by the united states post office in express mail on day 22 of month 4 2003; U.S. provisional patent application 60/465,273, attorney docket No. PP20518.001, filed by the united states post office in express mail on 23/4/2003; U.S. provisional patent application 60/465,535, attorney docket No. PP20518.002, filed by the united states post office in express mail on 24/4/2003; U.S. provisional patent application 60/468,312, attorney docket No. PP20480.010, filed by the united states post office in express mail on 5/2003; U.S. provisional patent application 60/473,144, attorney docket No. PP20480.011, 22.5.2003, U.S. provisional patent application 60/495,024, attorney docket No. PP20480.012, submitted by the united states post office in courier mail at 14.8.2003; U.S. provisional patent application 60/505,652, attorney docket No. PP20480.013, filed by the united states post office in express mail on 23/9/2003; U.S. provisional patent application 60/510,781, attorney docket No. PP20480.014, filed by the united states post office in courier mail on 11/10/2003; U.S. provisional patent application 60/529,464, attorney docket No. PP20480.015, filed by the united states post office in courier mail on 11/12/2003; U.S. provisional patent application 60/536,177, attorney docket No. PP20480.016, filed by the united states post office in express mail on 12 days 1 month 2004; and U.S. provisional patent application 60/, attorney docket number PP20480.017, was filed in courier mail by the U.S. post office on 4/7 of 2004.

Technical Field

The present invention relates to nucleic acids and proteins of the Severe Acute Respiratory Syndrome (SARS) virus. These nucleic acids and proteins are useful in the preparation and manufacture of vaccine formulations for the treatment or prevention of SARS. The invention also relates to diagnostic reagents, kits (comprising such reagents) and methods for diagnosing or identifying the presence or absence of SARS virus in a biological sample. The invention also relates to methods of treating or preventing SARS using combinations of small molecule viral inhibitors and kits for treating SARS.

Background

Outbreaks of virulent respiratory virus, now known as Severe Acute Respiratory Syndrome (SARS), have been identified in hong kong, china and many countries of the world in 2003. Symptoms of a patient typically include fever, dry cough, dyspnea, headache, and hypoxemia. Isolates of the SARS virus show homology to at least several known RNA polymers of coronaviruses. Phylogenetic analysis of this homology is seen in Peiris et al, "coronaviruses are the likely causes of severe acute respiratory syndrome" (corona as a competent house of segment access respiratory syndrome), Lancet, published in http at 4/8/2003: thelavcet. com/extra/03 art3477web.pdf, incorporated herein by reference. Other sequenced fragments of the SARS virus genome were shown to overlap with the open reading frame 1b of the coronavirus. See Drost et al, "Identification of New Corona viruses in Patients with Severe acid reactivity Syndrome" in Patients with Severe Acute Respiratory Syndrome, New England Journal of Medicine, published in http 4/10/2003: // www.nejm.org, incorporated herein by reference.

The Genome Science Center (Genome Science Center) located in the great Britain Columbia province, Canada publishes a name TOR at its website (http:// www.bcgsc.ca/biolnifo/SARS /)₂The isolate is believed to be a genome assembly draft of 29738 base pairs of the SARS virus. SEQ ID NO: 1 gives a draft of the genome assembly.

Disease control center (CDC) at its website

(http. www. cdc. gov/ncidod/SARS/pdf/nucleoseq. pdf) discloses the nucleotide sequence of SARS-CoV strain (SEQ ID NO: 2). CDC also published a phylogenetic tree of predicted N, S and M proteins (see fig. 6). The phylogenetic tree places the SARS virus outside of any previously known coronavirus type.

There is an increasing need for prophylactic or therapeutic vaccines against SARS virus, as well as diagnostic, screening methods and compositions for identifying the presence of the virus in, for example, mammalian tissue or serum.

Summary of The Invention

The present invention relates to nucleic acids and proteins of the Severe Acute Respiratory Syndrome (SARS) virus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations for the treatment or prevention of SARS. Such vaccine formulations may include inactivated (or killed) SARS virus, attenuated SARS virus, isolated SARS virus preparations, and recombinant or purified preparations of one or more SARS virus antigen subunits. Expression and delivery of the polynucleotides of the invention may be facilitated by viral vectors and/or viral particles.

The invention also relates to diagnostic agents, kits (comprising such agents) and methods for diagnosing or identifying the presence or absence of SARS virus in a biological sample. The invention also includes polynucleotide sequences that do not encode SARS virus, SARS virus sequences that encode non-immunogenic proteins, and conserved and variant SARS virus polynucleotide sequences for use in such diagnostic compositions and methods.

The invention also relates to vaccine formulations comprising one or more SARS virus antigens and one or more other respiratory virus antigens. Other respiratory virus antigens suitable for use in the present invention include: influenza virus, Human Rhinovirus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus (RSV), adenovirus, metapneumovirus (metapneumovirus) and rhinovirus. Other respiratory virus antigens may also be of coronaviruses other than SARS coronavirus. Preferably the other respiratory virus antigen is an influenza virus antigen.

The compositions of the invention also comprise one or more adjuvants. Adjuvants suitable for use in the present invention include mucosal, transdermal or parenteral adjuvants. Mucosal adjuvants suitable for use in the present invention include the detoxified bacterial ADP-ribosylating toxins such as e.coli heat labile toxoids (e.g. LTK63), chitosan and derivatives thereof, and the non-toxic dual mutant forms of the Bordetella pertussis toxoid. Parenteral adjuvants suitable for use in the present invention include MF59 and aluminium or aluminium salts.

The invention also provides methods of treating SARS by administering small molecule compounds, and methods of identifying small molecules effective in treating SARS.

In one aspect of the invention, there is provided a method of identifying a therapeutically active agent, the method comprising: (a) contacting a therapeutically active agent with cells infected with SARS virus; (b) assaying for a decrease in activity of an enzyme associated with SARS.

In a more specific embodiment, the therapeutically active agent is a small molecule. In another more specific embodiment, the therapeutically active agent is a nucleoside analog. In another more specific embodiment, the therapeutically active agent is a peptoid, an oligopeptide or a polypeptide. In another embodiment, the SARS-associated enzyme is SARS protease. In another embodiment, the SARS-associated enzyme is SARS polymerase. In yet another embodiment, the SARS-associated enzyme is a kinase. Section V below further discusses methods of identifying therapeutically active agents for treating SARS virus infection.

In another aspect of the invention, a method of treating a human infected with SARS is provided comprising administering to a patient in need thereof a small molecule. In one embodiment, the small molecule is a SARS protease inhibitor. In another embodiment, the small molecule is a SARS polymerase inhibitor. In another embodiment, the SARS-associated enzyme is a kinase. In yet another embodiment, the small molecule is administered orally or parenterally.

The invention also provides the use of such small molecules in the manufacture of a medicament for the treatment of severe acute respiratory syndrome.

The small molecule compounds of the present invention include those molecules less than 1000g/mol, preferably containing an aromatic moiety and one or more heteroatoms selected from O, S or N.

Preferred small molecules include, but are not limited to: acyclovir, ganciclovir, vidarabidine, foscamet, cidofovir, amantidine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, and combinations thereof. Interferons may also be used to treat patients, including interferon- α and interferon- β. Interferon therapy has shown promise in monkeys for the treatment of SARS (Enserink (2004) Science 303: 1273-.

One aspect of the invention relates to methods of identifying individuals exposed to SARS virus (SARSV) and biological samples containing SARSV, and kits for performing such methods. Such methods may use nucleic acid detection techniques such as PCR, RT-PCR (Coronaviridae is an RNA virus), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), branched DNA signal amplification assays, nucleic acid sequence based isothermal amplification (NASBA), other self-sustained sequence replication assays, self-returning DNA amplification, strand displacement activation, cycling probe techniques or combinations of these amplification methods. As is well known in the art, these nucleic acid detection techniques employ oligonucleotides containing nucleotide sequences similar to or complementary to the SARS virus genome as primers (e.g., for amplification) and probes (e.g., for capture or detection).

Alternatively, or in addition to the nucleic acid detection methods described above, the methods of the invention can utilize various immunoassay techniques to detect SARSV antigens and/or antibodies.

Accordingly, the present invention relates to methods for identifying an individual exposed to SARSV or a biological sample containing SARSV by detecting the presence of an SARSV antigen with an antibody that specifically binds to the SARSV antigen. The antibody is preferably a monoclonal antibody. The amount of viral antigen present in a sample from an individual can be used to determine the prognosis of the infected individual. The preferred SARSV antigen to be detected is typically a structural protein, especially those present on the surface of viral particles, including, for example, spike glycoprotein (S), also known as E2; envelope (small membrane) protein (E), also known as sM; membrane glycoprotein (M), also known as E1; hemagglutinin-esterase glycoprotein (HE); also known as E3; and nucleocapsid phosphoprotein (N). In a preferred embodiment, the antigens to be detected are S, E and the M protein, which can be detected using their antibodies.

The present invention relates to kits for identifying individual SARSV and reagents for use in such kits. The kit contains a first container comprising an antibody that specifically binds to an antigen of SARSV and a second container comprising an antigen of SARSV. The antibody is preferably a monoclonal antibody. The kit can be used for quantitatively determining the amount of antigen in a sample of an individual. This information can be used to determine the prognosis of an infected individual.

The present invention relates to methods for identifying individuals exposed to SARS virus or biological samples containing SARSV by detecting the presence or absence of antibodies against SARS virus antigens in a sample using SARS antigens. Quantitative determination of the amount of anti-SARS protein, i.e., SARS antibodies, present in a sample from an individual can be used to determine the prognosis of the infected individual. Antibodies to SARSV can be detected using one or more viral proteins (structural or non-structural) as antigens; conserved SARSV antigens in SARSV isolates are preferred. In this regard, non-structural proteins (e.g., Pol, Hel, 3CLp, MP, PLP1, PLP2) may be particularly useful.

The present invention relates to kits for identifying individuals who have been exposed to SARS and reagents for use therein. The kit contains a first container of antibodies produced in response to exposure to the SARS virus antigen and a second container containing the SARS antigen. The kit is suitable for quantitatively determining the amount of anti-SARS antibody present in an individual sample. This information can be used to determine the prognosis of an infected individual.

The present invention relates to methods for identifying individuals exposed to SARS virus or biological samples containing SARSV by detecting the presence of SARS virus nucleic acid. Quantitative determination of the amount of SARS nucleic acid present in a sample from an individual can be used to determine the prognosis of the infected individual. The method employs oligonucleotide probes and/or primers that are similar or complementary to the SARSV genome or transcript or replication product sequences. Preferred probes and primers are described herein. The invention also includes kits for performing the methods used for nucleic acid detection of SARSV.

The present invention also includes methods of treating and/or preventing SARS by administering a therapeutically effective amount of at least one of those antiviral compounds described in the U.S. patents and published international patent applications listed in Table 1 and Table 2. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In another embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. In another embodiment, the antiviral compound is an RNA-dependent RNA polymerase inhibitor. In another embodiment, the first antiviral compound is a protease inhibitor that is administered with a second antiviral compound that is an inhibitor of RNA-dependent RNA polymerase. The present invention also provides the use of a steroidal anti-inflammatory drug in combination with at least one antiviral compound, for example as described in the documents listed in tables 1 and 2.

The present invention also provides methods for treating and/or preventing SARS by administering by inhalation a therapeutically effective amount of at least one of the antiviral compounds described in the U.S. patents and published international patent applications listed in tables 1 and 2. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In another embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. In another embodiment, the antiviral compound is an inhibitor of RNA-dependent RNA polymerase. In another embodiment, the first antiviral compound is a protease inhibitor that is administered with a second antiviral compound that is an inhibitor of RNA-dependent RNA polymerase. The present invention also provides for the administration of a steroidal anti-inflammatory drug in combination with at least one antiviral compound, such as the antiviral compounds described in the documents listed in tables 1 and 2, by inhalation. The steroidal anti-inflammatory drugs may be administered by inhalation to achieve a local effect, or may be administered by, for example, oral or intravenous routes to achieve systemic absorption.

The invention also provides the use of the antiviral compound in the manufacture of a medicament for the treatment of severe acute respiratory syndrome.

The invention also provides kits for use by a consumer for treating and/or preventing SARS. Such a kit contains: (a) a pharmaceutical composition comprising a therapeutically effective amount of at least one of the antiviral compounds described in the U.S. patents and published international patent applications listed in tables 1 and 2, and a pharmaceutically acceptable carrier or diluent; (b) a container containing the pharmaceutical composition; and, optionally; (c) instructions for using the pharmaceutical composition to treat and/or prevent SARS are described. The kit may optionally contain a plurality of antiviral compounds for the treatment of SARS, wherein the antiviral compounds are selected from the group consisting of a 3C-like protease inhibitor and a papain-like protease inhibitor. In another embodiment, the kit contains an antiviral compound that is an RNA-dependent RNA polymerase inhibitor. When the kit contains multiple antiviral compounds, the antiviral compounds in the kit can optionally be combined with the same pharmaceutical composition.

In another aspect of the invention, there is provided the use of at least one antiviral compound as described in the U.S. patents and published international patent applications listed in tables 1 and 2 in the manufacture of a medicament for the treatment or prevention of SARS.

Brief Description of Drawings

FIG. 1: schematic representation of the coronavirus genome structure.

FIG. 2: schematic representation of the coronavirus ORF1a/ORF1b gene products.

FIG. 3 (A-C): comparison of coronavirus genes selected from the group consisting of nucleocapsid (N), matrix (M) and hemagglutinin-esterase (HE) genes.

FIG. 4 (A-F): alignment of coronavirus polypeptide sequences (including ORF1a/ORF1b, Nucleocapsid (NP), hemagglutinin-esterase (HE), envelope (Sm or E), matrix (M) and spike (S).

FIG. 5: alignment of the junction regions of the spike (S) polypeptide sequences S1 and S2 domains taken from FIG. 4 and the selected coronavirus protease cleavage sites.

FIG. 6: CDC phylogenetic tree of SARS-CoV strain (Clustalx 1.82, adjacent junction tree).

Fig. 6A shows the result of analysis of coronavirus N protein, fig. 6B shows the result of analysis of coronavirus S protein, and fig. 6C shows the result of analysis of coronavirus M protein.

FIG. 7: the conserved and unique sequences of SARS virus. FIGS. 7A-7D show multiple sequence alignments of SARS virus genome structural proteins (CLUSTAL W1.82) (7A: PEP4 spike protein; 7B: PEP7 small membrane protein; 7C: PEP8 matrix glycoprotein; 7D: PEP13 nucleocapsid protein) with their counterparts in all or part of other known coronaviruses. FIGS. 7E-7H show dendrograms of protein distances in the aligned 7A-7D sequences. Marker 229E: a human coronavirus; and (4) MEV: murine hepatitis virus; TGV: transmissible gastroenteritis virus; AIBV: avian infectious bronchitis virus; BOVINE: bovine coronavirus; PEDV: porcine epidemic diarrhea virus.

FIG. 8: comparison of the 5 'UTRs of several coronaviruses shows the consensus nucleotide sequence of the 5' UTRs.

FIG. 9: sequences of preferred primers for amplification of the 5' UTR. F and R represent forward and reverse PCR primers, and the numbers represent the positions of nucleotides in FIG. 8.

FIG. 10: comparison of the 3 'UTRs of several coronaviruses shows the consensus nucleotide sequence of the 3' UTRs.

FIG. 11: preferred primer sequences for amplification of the 3' UTR. F and R represent forward and reverse PCR primers, and the numbers represent the positions of nucleotides in FIG. 10.

FIG. 12: SEQ ID NO: 6042 coiled coil prediction, using the Coils program (FIG. 12A) or LearrCoil (FIG. 12B).

FIG. 13: an example of inserting a reporter gene of interest between existing SARS virus genes at a site. Small non-structural gene products are not shown.

FIG. 14: schematic representation of a representative example of a SARS virus replicon. Small non-structural gene products are not shown.

FIG. 15: the nsp2 protease (3CLp) of SARS virus and the identification of catalytic and substrate sites.

FIG. 16: comparison of avian IBV, MHV and BcoV SARS virus nsp2 protease (3 CLp). Residues in the dashed box are critical residues for the substrate site (F, Y and H); the residues in the solid box are catalytic cysteine (C) and histidine (H) residues.

FIG. 17: genome structure of SARS coronavirus. Replicase regions and structural regions are shown, as well as predicted cleavage products within ORF1a and ORF1 b. The positions of the 5 'RNA leader (L), 3' poly A channel and the ribosomal frameshift consensus sequence between ORF1a and ORF1b are also indicated. Each box represents a protein product. They are shaded for their level of amino acid identity with the corresponding proteins of other coronaviruses (see also Table 2). The SARS-specific gene is white. The positions of the 9 SARS-specific six-base IG sequences (5 '-ACGAAC-3'; SEQ ID NO 7293) are indicated by arrows.

FIG. 18: representative groups of coronaviruses 1(HCoV-229E, accession No. AF304460), 2 (mouse hepatitis virus MHV, accession No. NC-001846), 3 (avian infectious bronchitis Virus AIBV, accession No. NC001451) and the genome structure of SARS coronavirus. Other fully sequenced coronaviruses used in this study were obtained from the following accession numbers: porcine Epidemic Diarrhea Virus (PEDV), AF 353511; transmissible Gastroenteritis Virus (TGV), NC 002306; bovine coronavirus (BCoV): AF 220295. The red boxes represent group-specific genes. The positions of the leader RNA sequence and the poly A channel are also indicated in the genome. The position of the specific IG sequence is indicated by circles of different shades. In the SARS genome, we also found three IG sequences characteristic of group 2 coronaviruses.

FIG. 19: the predicted spike protein anchors to the topological model of the viral membrane. The domains and predicted functional domains are indicated. The N-terminal region (S1) is expected to contain a receptor binding domain. The S2 domain, which is partially superimposed with the leucine zipper motif, has two coiled coil regions that are presumed to be involved in oligomerization. The hydrophobic domain is responsible for membrane anchoring.

FIG. 20: a phylogenetic tree was obtained by multiple sequence alignment of the 922bp internal region of the pol gene of 12 coronaviruses and SARS. The number of nodes represents the result of the bootstrap analysis, strongly supporting the branches. Sequences not present in the complete coronavirus genome have been retrieved from GenBank under accession numbers: porcine Hemagglutinating Encephalomyelitis Virus (PHEV), AF124988, human OC43 virus (OC43), AF124989, Canine Coronavirus (CCV), AF124986, Feline Infectious Peritonitis Virus (FIPV), AF124987, Turkey Coronavirus (TCV), AF124991, rat sialorrhiza adenitis virus (SDAV), AF 124990.

FIG. 21:21Arootless phylogenetic trees obtained by comparing the consensus sequences of the group I and group II spike protein S1 domains (G1_ cons and G2_ cons) and the group 3 spike protein (AIBV) with the spike protein of SARS virus. The numbers represent the results of the bootstrap analysis. The sequences used to generate the group 1 consensus sequence features are: HcoV-229E, accession number P15423; porcine Epidemic Diarrhea Virus (PEDV), accession number: NP 598310; transmissible Gastroenteritis Virus (TGV), accession No.: NP 058424; canine Coronavirus (CCV), accession number: s41453; porcine Respiratory Virus (PRV), accession no: s24284; feline Infectious Peritonitis Virus (FIPV), accession number: VGIH 79. The sequences used to generate the group 2 consensus sequences were: mouse Hepatitis Virus (MHV), accession number: NP 045300; bovine coronavirus (BCoV), accession No.: NP _ 150077; human coronavirus OC43, accession number: p36334; porcine Hemagglutinating Encephalomyelitis Virus (PHEV), accession no: AAL 80031; group 3 used only the spike protein sequence of Avian Infectious Bronchitis Virus (AIBV), accession No.: AA 034396. 21B: schematic representation showing the position of the cysteine in the S1 domain of groups 1, 2 and 3 compared to the SARS spike protein. Horizontal bands indicate the S1 amino acid sequence (SARS and AIBV) or consensus sequence features (generated by group 1, G1_ cons, and group 2, G2_ cons). The length of the stub is not exaggerated. The relevant cysteine positions are indicated by rectangular bands. Only cysteines are completely conserved among the respective consensus sequences. The line-linked cysteines are shown to be conserved between the SARS S1 domain and the consensus sequence.

FIG. 22: neisseria adhesin A protein (NadA).

FIG. 23: original translation of the SARS coronavirus genome (reading frame + 1).

FIG. 24: original translation of the SARS coronavirus genome (reading frame + 3).

FIG. 25: 1b and the open reading frame of the spike, separated by x.

FIG. 26: SARS grows in vero cells.

FIG. 27 is a schematic view showing: chromatogram of SARS coronavirus capture step on Matrix Cellufine Sulfate SuperPerformance 150/10. 100ml coronavirus harvest was analyzed. The left Y-axis shows the absorbance at 280 nm. The right Y-axis represents the gradient (% B). The X-axis represents volume (ml).

FIG. 28: silver stained MCS chromatography component. Lanes are: (1) a label; (2) coronavirus vero cell harvest; (3)0.65 μm post-filtration coronavirus vero cell harvest; (4) flowing through the liquid; (5) washing liquid; (6) 20% peak (viral peak). Mu.g of test protein was loaded on each lane.

FIG. 29: western blot of MCS chromatography fractions. Lanes are as described in FIG. 28.

FIG. 30: linear density gradient ultracentrifugation, 15-60% sucrose (SW28, 2 hours, 20000 rpm). The graph shows the protein concentration (■) and the sucrose concentration (. diamond-solid.).

FIG. 31: silver staining density gradient fractions on NuPage 4-12% Bis-Tris-Ge (Novex), reduction conditions, heating for 10 min at 70 ℃. Lanes are: (1) a label; (2) 20% peak MCS; (3) a density gradient component 11; (4) a density gradient component 12; (5) a density gradient component 13; (6) a density gradient component 14; (7) a density gradient component 15; (8) a density gradient component 16; (9) density gradient component 17. The fractions 15-17 have a large amount of protein. Lanes 2, 8 and 9 were loaded with 1. mu.g protein.

FIG. 32: chromatogram of SARS coronavirus capture step on MCS. The details are as in FIG. 27, but 200ml harvest was used.

FIG. 33: silver staining of the chromatographic fractions (left) and Western blot (right) results. Lanes are as described in FIGS. 28 and 29, but lane (6) is a 5% peak. The samples were treated for 30 min at room temperature before SDS-PAGE.

FIG. 34: density gradient ultracentrifugation, 15-40% sucrose (SW28, 2 hours, 20000 rpm). The graph shows the protein concentration (■) and the sucrose concentration (. diamond-solid.).

FIG. 35: results of silver staining (left) and Western blotting (right) of NuPage 4-12% Bis-Tris-Ge (Novex) ultracentrifugation fractions on density gradient, reducing conditions. Each lane is: (1) a label; (2) a density gradient component 6; (3) a density gradient component 7; (4) a density gradient component 8; (5) a density gradient component 9; (6) a density gradient component 10; (7) density gradient component 15. Fractions 7-10 (lanes 3-6) contained pure coronavirus proteins. Fraction 15 (lane 7) contains a number of impurities. Lanes 2, 8 and 9 were loaded with 1. mu.g protein. The samples were treated at room temperature for 30 minutes before SDS-PAGE.

FIG. 36: EM plots of density gradient fractions 8-10. FIG. 36A shows component 8; FIG. 36B shows component 9; fig. 36C shows component 10.

FIG. 37: spike/NadA fusion constructs.

Fig. 38 and 39: s1_L、S1_LNadA and SlL-NadA_{Delta anchor}Results of expression in E.coli. FIG. 38 shows the results for BL21(DE3)/pET, BL21(DE3)/pET-S1L and BL21(DE3)/pET-S1L-NadA_{Delta anchor}SDS-PAGE analysis of whole lysates. The bands are indicated by arrows, and from left to right the three lanes are: BL21(DE 3)/pET; BL21(DE3)/pET-S1_L；BL21(DE3)/pET-S1_L-NadA_{Delta anchor}. FIG. 39 shows BL21(DE3)/pET, BL21(DE3)/pET-S1_LNadA (cultured under non-inducing conditions) and BL21(DE3)/pET-S1 _LResults of SDS-PAGE (39A) and Western blot analysis (39B) of NadA (cultured under induction conditions) whole lysates. The bands are indicated by arrows, and from left to right the three lanes are: BL21(DE 3)/pET; BL21(DE3)/pET-S1_L-NadA；BL21(DE3)/pET-S1_L-NadA. Western blots showed the presence of oligomeric forms of the protein.

FIG. 40: schematic representation of SARS spike cloning.

FIG. 41: transient expression of SARS spike protein (Western blot result of COS7 cell lysate). Lanes of 4-20% TG SDS gels were loaded with 20. mu.g of cell lysate (total 1.2 mg). Labeled antibodies are shown.

FIG. 42: western blot analysis of COS7 cell lysates on 4% TG SDS gels showed the oligomeric state of intracellular S molecules.

FIG. 43: western blot analysis of COS7 cell lysates on 4-20% TG SDS gels showed transient expression of SARS spike protein. Each lane is: (1) simulation, AF; (2) simulation, DF; (3) nSh, AF; (4) nSh, DF; (5) nSh Δ TC, AF; (6) nSh Δ TC, DF. Each lane was loaded with 5. mu.1 of each sample, for a total of 400. mu.l. The blot was labeled with an anti-His-tagged protein.

FIG. 44: western blot analysis of COS7 cell lysates on 4-20% TG SDS gels showed transient expression of SARS spike protein. The truncated spike protein is secreted. The spike proteins were purified from the culture medium (10cm plate) first by passing through a ConA column and finally through His-tagged magnetic beads. The 1/3 substance was loaded on each lane.

FIG. 45: western blot analysis of COS7 cell lysates on 4-20% TG SDS gels showed transient expression of SARS spike protein. In the two left-hand blots (lanes 1-5), the samples were boiled in SDS and beta-mercaptoethanol; in the two blots on the right hand side (lanes 6-11), the samples were boiled in SDS only. Lanes 1-8 were labeled with monoclonal antibodies against His-tagged protein; lanes 9-11 were labeled with rabbit anti-SARS antibody.

FIG. 46: effect of SARS spike protein expression on cell viability.

FIG. 47: western blot analysis of COS7 cell lysates on 4% TG SDS gels showed the oligomeric state of intracellular spike molecules. The blot was labeled with anti-His-tagged mAb. The membrane fraction of the COS7 cell lysate was fractionated by exclusion column and then loaded onto the lanes. Fractions 7-14 showed band kDa values of: 71000. 1400, 898, 572, 365, 232, 148, and 99.

FIG. 48: the cells were divided into an aqueous phase fraction and a detergent fraction.

FIG. 49: schematic representation of the use of the constructs in OMV preparations.

FIG. 50: HR1 and HR of SARS₂And (3) constructing the structure.

FIG. 51: the vaccine protects Balb/c mouse model from SARS.

FIG. 52: expression of spike protein in transfected 293 cell lysate (52A) or COS7 cell culture supernatant (52B). Proteins were separated on 4-20% TG SDS gels. The tag was anti-His-tag, but the three lanes on the right hand side of 52B were tagged with rabbit anti-SARS serum. In FIG. 52A, the left three lanes are treated with DTT and boiled, but the right three lanes are untreated. In FIG. 52B, DTT treatment was not used, but all lanes (samples thereof) were heated at 80 ℃ for 5 minutes.

FIG. 53: western blot of spike protein expressed in COS7 cells. Proteins were incubated at Room Temperature (RT), 80 ℃ or 10 ℃ to examine how they affected molecular weight. FIG. 54 shows the results of a similar experiment performed on SARS virus particles.

FIG. 55: the results of the pulse-chase experiment show the expression and processing of SARS spike protein following infection with alphavirus replicon particles. As shown, cells were treated with or without Endo H.

FIG. 56: effect of heating on spike protein trimer.

FIG. 57: coomassie blue stained gel of yeast expressed protein. Each lane is: 1-visible blue standard (10. mu.l); 2-pAB24gbl (20. mu.g); 3-SARS spike S1c.1gbl (20. mu.g); 4-SARS spike S1c.2gbl (20. mu.g); 5-visible blue standard (10. mu.l); 6-pAB24ip (5. mu.l); 7-SARS spike S1c.l (5. mu.l); 8-SARS spike S1c.2 (5. mu.l).

FIGS. 58-64: schematic illustration of the preparation of yeast expression constructs.

FIGS. 65-66: a yeast-expressed spike sequence.

FIG. 67: western blot showing expression of SARS spike protein in alphavirus replicon particles and replicon RNA expression. Figure 67A was performed under non-reducing conditions and at room temperature (i.e., without heating), with lanes: (1) VEE/SIN-spike infection; (2) VEE/SIN-GFP infection; (3) replicon-spike RNA transfection; (4) replicon-GFP RNA transfection. FIG. 67B was performed with SARS virus particles at different temperatures as indicated.

FIG. 68: antibody responses were induced in mice. Each vaccine group was: (1) inactivating SARS virus; (2) a truncated recombinant spike protein; (3) full-length spikes: plg + alphavirus; (4) full-length spikes: only alphavirus particles.

FIG. 69: human monoclonal antibody S3.2 binds to purified truncated spike protein. The X-axis represents antibody concentration and the Y-axis represents ELISA absorbance. The interpolation result is 2158.13.

FIG. 70: ELISA titers of antibody induced by SARS-CoV spike protein delivered by different vaccines (left to right: inactivated virus; 3. mu.g truncated spike protein; 75. mu.g DNA encoding truncated spike protein).

FIG. 71: neutralizing titers after immunization with (left) nSd Δ TC protein or (right) DNA encoding nSd Δ TC delivered with PLG.

FIG. 72: spike antigen binding antibodies and neutralizing antibodies.

FIG. 73: western blot of CHO cell lines expressing either the full-length (left) or truncated (right) forms of the spike protein. Proteins were separated by 4-12% SDS-PAGE, boiled in DTT and stained with polyclonal serum.

FIG. 74: the SARS-CoV spike glycoprotein and expression constructs. L represents the leader peptide (residues 1-13), TM the transmembrane region, and Cy the cytoplasmic tail segment. The 6-His tag is not shown.

FIG. 75: western blot analysis of SARS spike protein expressed in COS7 cells. In FIG. 75A, COS7 cells were transfected with the indicated plasmid constructs and the expressed proteins in cell lysates were analyzed 48 hours post-transfection using SDS-PAGE (4-20% polyacrylamide) under reducing and denaturing conditions, with anti-histidine MAbs displaying the proteins. In fig. 75B, proteins in cell culture media were collected 48 hours after transfection and purified using ConA columns followed by His-tagged magnetic beads. The purified protein was analyzed by SDS-PAGE (4-20% polyacrylamide) and visualized with anti-SARS rabbit serum.

FIG. 76: endo H sensitivity of the C-terminally truncated spike protein (SA) was found in cell lysates (lanes 1, 2) and culture media (lanes 3, 4). The positions of intracellular and secreted S.DELTA.proteins are indicated by arrows.

FIG. 77: oligomeric state of SARS spike protein. Recombinant S protein oligomers in COS7 cells were transfected with the full-length spike construct (nSh). As indicated on each lane, the cell lysate was treated with DTT and/or heat. The different forms of S protein in the treated and untreated samples were analyzed by SDS-PAGE (4% polyacrylamide) and Western blotting, shown with anti-histidine MAb.

FIG. 78: effect of thermal denaturation on the oligomeric state of recombinant S proteins in the absence of DTT. COS7 cell lysates were heated prior to electrophoresis as indicated and the S proteins were visualized as described in fig. 77.

FIG. 79: effect of thermal denaturation on the oligomeric state of spike proteins in SARS virus particles. SARS-CoV was grown in Vero cells, purified and solubilized from the virus particles with SDS, heat denatured as described above and shown as described in FIG. 77, but using rabbit antisera against the purified virus as a probe.

FIG. 80: the oligomeric state of the spike protein of SARS virus particles was analyzed by a cross-linking assay. Solubilized SARS virion protein is treated with DMS. Untreated (-) and DMS-treated (+) virion proteins were heat denatured in the absence of DTT and visualized by 4% PAGE and silver staining.

Fig. 81 and 82: the oligomeric state of the truncated spike protein was analyzed by thermal denaturation. Truncated spike protein in COS7 cell lysate (81) or truncated spike protein secreted into the culture medium (82) was denatured with heat as indicated in the absence of DTT and shown by Western blot analysis.

FIG. 83: reactivity of deglycosylated full-length spike oligomers with conformers or non-conformers antibodies. Full-length recombinant spike oligomers were partially deglycosylated with PNG enzyme under non-denaturing conditions, as shown by Western blot analysis with anti-histidine Mab ( lanes 1, 2, 3) or rabbit antisera against purified SARS CoV ( lanes 4, 5, 6).

FIG. 84: western blot showed the localization of the expressed SARS spike protein in the fractions of COS7 cell lysate. Cells were transfected with the indicated plasmids and 48 hours after transfection were lysed with a Dounce homogenizer in hypotonic buffer. The cell lysate was centrifuged to obtain a soluble cytosol fraction and an insoluble membrane fraction, which was further solubilized with 4% TritonX-100. Proteins were analyzed by SDS-PAGE (4-20% polyacrylamide) under reducing conditions after heating with SDS at 80 ℃. Proteins were visualized with anti-histidine mabs. The cytosol fraction was loaded into lanes 1, 3 and 5 and the membrane fraction was loaded into lanes 2, 4 and 6.

FIG. 85: expression of recombinant full-length (A, D) or truncated (B, E) spike proteins in and on COS7 cells. Cells were fixed 48 hours after transfection and either treated with detergent (Cytofix/perm, BD Biosciences) for intracellular immunofluorescence assay (A, B, C) or treated with 2% paraformaldehyde (40-fold magnification) for cell surface immunofluorescence (D, E, F). Transformed cells (C, F) were mock-transformed as controls.

FIGS. 86-105: SDS-PAGE of E.coli expressed proteins. Tot ═ total protein; sol ═ soluble protein fraction. The tags are the protein names (Table 26-30).

FIG. 106: immunofluorescence following administration of vector encoding optimized N antigen.

FIG. 107: (A) native M sequence and (B) codon-optimized M sequence immunofluorescence.

FIG. 108: (A) the native E sequence and (B) the codon-optimized E sequence.

FIG. 109-111: western blotting of Vero cells, rabbit antibodies obtained after immunization with E.coli-expressed spike protein.

FIG. 112: expression of spike protein in 293 cells. Lanes: (M) a label; (1) a transfected mimic; (2, 6) cells expressing the nS protein, lysate; (3, 7) cells expressing nSdTC protein, lysate; (4, 8) nS protein-expressing cells, supernatant; (5, 9) (4) cells expressing nSdTC protein, supernatant. Staining antibodies: (2-5) mouse serum obtained after DNA immunization; (6-9) Rabbit serum obtained after immunization with killed whole virus.

FIG. 113: SEQ ID NO: 9968.

FIG. 114: SEQ ID NO: 10033.

FIG. 115: bovine coronavirus pol lab (top row; SEQ ID NO: 10068), avian infectious bronchitis pol lab (second row; SEQ ID NO: 10069), mouse hepatitis virus pol lab (third row; SEQ ID NO: 10070), SEQ ID Nos: 9997/9998 (fourth row) and the consensus sequence (bottom row; SEQ ID NO: 10071).

FIG. 116: schematic representation of coronavirus genome structure.

Fig. 117: schematic representation of the coronavirus ORF1a/ORF1b gene products, including the "×" region.

FIG. 118: and (6) comparing.

FIG. 119: SEQ ID NO: 10080, an alternative initiation codon.

FIG. 120: SEQ ID NO: 10084.

FIG. 121: SEQ ID NO: 10033 and SEQ ID NO: comparison of 10084.

FIG. 122: SEQ ID NO: 10084 reading frame.

FIG. 123: SEQ ID NO: initiation codon analysis of 10084.

FIG. 124: SEQ ID NO: BLAST analysis of 10210.

FIG. 125: the amino acid sequence of SEQ ID NO: 10210 epitope analysis is performed.

FIG. 126: SEQ ID NO: 10299.

FIG. 127: SEQ ID NO: 10505 reading frame.

FIG. 128: SEQ ID NO: 11563.

FIG. 129: SEQ ID NO: 10033.

FIG. 130: SEQ ID NO: 9997 and SEQ ID NO: comparison of 10033.

FIG. 131: SEQ ID NO: 10299.

FIG. 132: SEQ ID NO: 10505 reading frame.

FIG. 133: western blot of SARS protease purified fractions.

FIG. 134: DABCYL-EDANS (a fluorescently labeled peptide containing a SARS protease cleavage site) was cleaved with various concentrations of SARS protease. The figure shows the activity/concentration relationship without protease (. diamond-solid.), 0.95uM protease (■) and 2.86uM protease (●).

When the sequences in the sequence table and the sequences in the figure are in, the sequences in the figure are taken as the standard.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional chemical, biochemical, molecular biological, immunological and pharmacological methods known to those skilled in the art. These techniques have been explained in detail in the literature. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19 th edition (1995); methods In Enzymology (s.colwick and n.kaplan eds., academic press, Inc.); and Handbook of Experimental Immunology, volumes I-IV (ed.m.weir and c.c.blackwell, eds., 1986, Blackwell Scientific Publications); sambrook et al, Molecular Cloning: a Laboratory Manual (second edition, 1989); handbook of surface and polar Chemistry (Birdi, K.S. ed., CRC Press, 1997); short Protocols in molecular Biology, fourth edition (eds. Ausubel et al, 1999, John Wiley & Sons); molecular biology Techniques: an intense Laboratory Course, (Ream et al, eds., 1998, academic Press); PCR (introduction to Biotechniques series), second edition, (edited by Newton & Graham, 1997, Springer Verlag); peters and Dalrymple, Fields Virology (second edition), Fields et al (ed.), b.n. raven Press, New York, NY.

The contents of all publications, patents and patent applications cited herein are hereby incorporated by reference.

Severe Acute Respiratory Syndrome (SARS) virus has recently been identified as a new viral species. The SARS virus species includes the following isolates.

Two viral isolates described by Peiris et al, "coronaviruses are the likely causes of severe acute respiratory syndrome" (corona as a reactive cause of segment access respiratory syndrome), Lancet is online published in http at 4/8/2003: com/extra/03 art3477web.pdf, the sequence of which is deposited in GenBank under accession number AY268070, incorporated herein by reference.

The isolates and viral sequences described by Drosten et al, "Identification of Novel coronaviruses in Patients with Severe acute respiratory Syndrome" (Identification of a Novel Coronavir in Patients with Severe Acuterepristeration Syndrome), New England and Journal of Medicine, online published in http at 4/10/2003: // www.nejm.org.

Isolates and viral sequences described on the WHO website on 3 months 25 and 24 of 2003.

Isolates and viral sequences described by Tsang et al, "a class of causes of Severe Acute Respiratory Syndrome in Hong Kong" (A Cluster of Cases of Severe acid Respiratory Syndrome in Hong Kong), New England Journal of Medicine, published online on http.// www.nejm.org on 31.3.2003.

The isolates and viral sequences described by Poutanen et al, "Identification of Severe Acute Respiratory Syndrome in Canada", New England journal of Medicine, published online on http.// www.nejm.org on 31.3.2003.

As described in the Lanet article, the 646 base pair polynucleotide of SARS virus has weak homology to viruses of the coronavirus family. The Lanet article also reports that the amino acid sequence deduced from this sequence (215 amino acids) has about 57% sequence homology with the RNA polymerases of bovine coronavirus and murine hepatitis virus. The Lanet article also provides a phylogenetic analysis of the protein sequence, showing that this polymerase sequence is most closely related to the group II coronaviruses.

Virologists skilled in the art can identify, isolate and/or sequence other SARS virus isolates. Virologists can easily identify whether a new viral isolate is the SARS virus. Criteria that virologists can use to identify new SARS isolates include: sequence homology of the new isolate to known SARS virus isolates; similarity of the genomic structure of the novel virus isolates to known SARS virus isolates; immunological (serological) similarity or identity to known SARS virus isolates; pathologically similar to the morphology of viral particles of known SARS virus isolates; and morphological similarity to known SARS virus isolates resulting in infected cells (e.g., by electron microscopy).

Methods for isolating and sequencing isolates of SARS virus include those described by Peiris et al in the Lancet paper. The Lancet article states that RNA from clinical samples can be reverse transcribed with random hexamers and in the presence of 2.5mmol/L magnesium chloride with a DNA sequence comprising the sequence SEQ ID NOS: 6584 & 6585 (94 ℃ for 1min, 50 ℃ for 1min, 72 ℃ for 1 min).

Retroviral isolates with random hexamers can be performed in RT-PCR assays as follows. The viral isolates are propagated in mammalian cells, particularly rhesus fetal kidney cells. All RNA from virus-infected and virus-uninfected rhesus fetal kidney cells was then isolated. Using a nucleic acid comprising SEQ ID NO: 6586 primers reverse transcribe the RNA sample. Using a nucleic acid comprising SEQ ID NO: 6587 the primers amplify cDNA. Unique PCR products (determined by size) from preparations of infected cells were then cloned and sequenced, comparing the sequence for genetic homology with sequences in GenBank.

Those skilled in the art will be able to identify and clone additional genomic regions based on the sequences provided above using various standard cloning techniques, e.g., RT-PCR using random primers and detecting sequences that overlap with one or more of the above sequences, and/or using oligonucleotide primers (e.g., degenerate primers) (see FIGS. 1-5, FIGS. 8-11, SEQ ID NOS: 3-20).

The cloning, sequencing and identification of SARS virus by those skilled in the art can be further facilitated by the use of polynucleotide sequences, particularly those related to the RNA polymerase of coronaviruses.

Sequence homology of the novel viral isolates with the above-described known SARS isolates can readily be determined by those skilled in the art. The percent homology of the viral nucleotide sequence can be used to identify novel SARS isolates, which may be 99%, 95%, 92%, 90%, 85% or 80% homologous to the known SARS virus polynucleotide sequence. Novel SARS isolates can also be identified using percent homology of polypeptides, which are encoded by novel viral polynucleotides that may be 99%, 95%, 92%, 90%, 85% or 80% homologous to polypeptides encoded by known SARS viruses.

Novel SARS isolates can also be identified by percent homology of the genomic polynucleotide sequence, which may be 99%, 95%, 92%, 90%, 85% or 80% homologous to the polynucleotide sequence of a specific genomic region of a known SARS virus. In addition, the percent polypeptide sequence homology can be used to identify novel SARS isolates, and the polypeptide sequence encoded by the polynucleotide of the novel SARS virus-specific genomic region can be 99%, 95%, 92%, 90%, 85%, or 80% homologous to the polypeptide sequence encoded by the polynucleotide of a known SARS virus-specific region. These genomic regions may include regions that are common to most coronaviruses (e.g., gene products), as well as group-specific regions (e.g., antigen groups), such as any of the following genomic regions, which are readily identified by virologists in the field: 5 'untranslated region (UTR), leader sequence, ORF1a, ORF1b, nonstructural protein 2(NS2), hemagglutinin-esterase glycoprotein (HE) (also known as E3), spike glycoprotein (S) (also known as E2), ORF3a, ORF3b, ORF3x, nonstructural protein 4(NS4), envelope (envelope) protein (E) (also known as sM), membrane glycoprotein (M) (also known as E1), ORF5a, ORF5b, nucleocapsid phosphoprotein (N), ORF7a, ORF7b, intergenic sequence, 3' UTR or RNA-dependent RNA polymerase (pol). The SARS virus can have an identifiable genomic region comprising one or more of the above genomic regions. The SARS virus antigen includes proteins encoded by any of these genomic regions. The SARS virus antigen may be a protein or fragment specific for SARS virus (as compared to known coronaviruses). (see FIGS. 1-5, FIGS. 8-11, SEQ ID NOS: 3-20).

One skilled in the art will also appreciate electron micrographs of mammalian cells infected with SARS virus. Electron micrographs of SARS-infected cells are found in Lancet paper. As described in this paper, electron micrographs of ultracentrifuged cell culture extracts of kidney cells of rhesus monkey foetus infected with SARS negatively stained (3% potassium phosphotungstate, pH 7.0) revealed the presence of polymorphous, enveloped virus particles with diameters of about 80-90nm (in the range 70-130nm) with a surface morphology consistent with that of coronaviruses (see Lancet's paper, FIG. 1). Electron micrographs of thin sections of infected cells revealed that the virus particles, which are 55-90nm in diameter, are located within smooth walled vesicles in the cytoplasm (see Lancet's paper, FIG. 2B). The electron microscope photograph can also be used to observe virus particles on the cell surface. Electron micrographs of human lung biopsies show similar virus morphology. See Lancet's paper FIG. 2A.

SARS polypeptides and polynucleotides

The present invention relates to nucleic acids and proteins from the SARS virus. Such polynucleotides and polypeptides are further exemplified below.

In one embodiment, the polynucleotide of the invention does not include http: the following 5 primers were disclosed in// content.nejfn.org/cgi/reprint/NEJMoa030781v2. pdf: SEQ ID NOS: 6034-38.

The invention includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes for use in methods of diagnosing or identifying the presence or absence of SARS virus in a biological sample. The invention includes a polypeptide comprising SEQ id no: 21-1020, or a polynucleotide sequence of one or more primer sequences identified in any one of seq id nos. The invention also includes nucleic acid sequences comprising sequences substantially identical to SEQ ID NOS: 21-1020, and a polynucleotide sequence complementary to the sequence of one or more of the primers identified in any one of claims 21-1020.

The invention includes polypeptides comprising an amino acid sequence derived from the sequence shown in figure 23. This amino acid sequence is seq id NOS: 6588-6809. The invention includes polypeptides comprising amino acid sequences having sequence identity to these sequences, as well as fragments of polypeptides comprising one of these sequences.

The invention includes a polypeptide sequence comprising an amino acid sequence derived from the sequence shown in figure 24. This amino acid sequence is SEQ ID NOS: 6810-7179. The invention encompasses proteins comprising amino acid sequences having sequence identity to these sequences, as well as fragments of proteins comprising one of these sequences.

The invention includes a polypeptide comprising SEQ ID NO: 6039. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 6039 a polypeptide having an amino acid sequence having sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 6039. The invention includes a diagnostic kit comprising a nucleic acid sequence comprising SEQ ID NO: 6039 or a fragment thereof. The invention includes a diagnostic kit containing a nucleic acid encoding SEQ ID NO: 6039 or a fragment thereof. The invention encompasses immunogenic compositions comprising SEQ ID NO: 6039 or a fragment thereof. The invention includes a polypeptide capable of recognizing a polypeptide comprising SEQ ID NO: 6039 or a fragment thereof. SEQ ID NO: 6039 was demonstrated to be functionally homologous to ORF1a of coronaviruses.

The following SEQ ID NOs: 6039 predicted transmembrane or hydrophobic region. Although the polyprotein of coronaviruses can be proteolytically cleaved into many small proteins, the hydrophobic domains in the polyprotein are known to mediate binding of the replication complex to the membrane and can significantly alter the structure of the host cell membrane. Thus, the hydrophobic domain of this polyprotein is a target for genetic mutations in the development of attenuated SARS virus vaccines. These hydrophobic domains are also targets for small molecule inhibitors of the SARS virus. These hydrophobic domains can also be used to generate antibodies specific for those regions to treat or prevent SARS virus infection.

Predicted SEQ ID NO: transmembrane helix in 6039

Sequence positions in parentheses indicate the core region.

Only if the score exceeds 500 is considered meaningful.

Inward-outward-screwing: 43 outward-inward spirals were found: found 43

Beginning at end of scoring center and beginning at end of scoring center

100(100)118(116) 103 107 94(97)118(112) 291 104

473(473)488(488) 1003 481 400(400)418(415) 243 407

529(532)549(549) 541 539 473(473)488(488) 1113 481

584(584)606(601) 1049 594 523(528)548(548) 285 538

773(773)791(789) 514 782 583(583)606(601) 662 593

1071(1071)1089(1086) 243 1078 776(776)791(791) 1435 783

1121(1121)1137(1137) 459 1130 1068(1071)1089(1086) 370 1078

1679(1679)1696(1696) 404 1686 1121(1121)1137(1137) 455 1130

2098(2102)2119(2116) 509 2109 1679(1679)1696(1694) 340 1686

2145(2145)2160(2160) 797 2153 2098(2098)2119(2116) 678 2109

2206(2209)2224(2224) 2686 2216 2148(2148)2163(2163) 434 2155

2316(2316)2332(2332) 2123 2325 2208(2210)2231(2226) 2389 2219

2335(2339)2358(2354) 2101 2346 2309(2309)2332(2326) 1773 2318

2373(2373)2390(2390) 532 2380 2342(2342)2368(2360) 1666 2353

2597(2600)2615(2615) 307 2607 2373(2373)2390(2390) 254 2380

2753(2753)2770(2768) 2242 2760 2753(2755)2770(2770) 2119 2763

2831(2833)2854(2851) 759 2841 2832(2835)2854(2851) 687 2844

2879(2882)2900(2897) 526 2889 2858(2858)2873(2873) 253 2866

2990(2996)3012(3010) 1289 3003 2879(2882)2899(2899) 400 2889

3024(3024)3042(3039) 2281 3032 2990(2990)3005(3005) 875 2998

3054(3058)3075(3072) 2536 3065 3020(3024)3042(3042) 2795 3032

3105(3109)3127(3123) 2010 3116 3059(3059)3075(3075) 2137 3067

3143(3143)3163(3159) 184 3152 3105(3108)3127(3123) 1902 3115

3253(3255)3272(3272) 319 3262 3142(3145)3162(3162) 540 3152

3346(3346)3366(3366) 203 3356 3343(3351)3366(3366) 496 3358

3375(3375)3392(3392) 305 3384 3437(3437)3453(3453) 848 3444

3438(3438)3455(3453) 1021 3445 3489(3491)3508(3505) 302 3498

3559(3567)3584(3581) 1885 3574 3560(3560)3577(3577) 1460 3569

3589(3589)3606(3604) 2018 3596 3591(3591)3606(3606) 2193 3598

3611(3611)3629(3629) 2304 3621 3610(3610)3627(3627) 1484 3620

3659(3659)3674(3674) 1561 3667 3656(3658)3678(3675) 1240 3668

3756(3758)3777(3774) 2352 3767 3681(3684)3701(3699) 590 3691

3890(3890)3904(3904) 485 3897 3710(3713)3738(3728) 1696 3721

3916(3919)3934(3934) 241 3926 3723(3723)3738(3738) 1670 3730

4035(4035)4051(4051) 335 4044 3760(3760)3777(3775) 2367 3767

4217(4217)4232(4232) 272 4224 3881(3884)3902(3900) 249 3892

4239(4239)4257(4254) 402 4247 4099(4099)4114(4114) 389 4106

4234(4234)4254(4249) 325 4241

4338(4341)4360(4360) 505 4348

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6039, wherein the fragment comprises an amino acid sequence comprising one or more of the hydrophobic transmembrane sequences identified above. The invention includes a polypeptide comprising SEQ ID NO: 6039, wherein the fragment comprises one or more of SEQ ID NOs: 6039 polypeptide sequence: 473-488, 529-549, 584-606, 773-791, 2098-2119, 2145-2160, 2206-2224, 2316-2332, 2335-2358, 2373-2390, 2753-2770, 2831-2854, 2879-2900, 2990-3012, 3024-3042, 3054-3075, 3105-3127, 3438-3455, 3559-3584, 3589-3606, 3611-3629, 3659-3674, 3756-3777, 473-488, 583-606, 776-791, 2098-2119, 2208-2231, 2309-2332, 2342-2368, 2753-2770, 2832-2854, 2990-3005, 3020-3042, 3059-3075, 3105-3127, 3142-3162, 3437-3453, 3560-3577, 3591-3606, 3610-3627, 3656-3678, 3710-3738, 3723-. Preferably the fragment comprises one or more of SEQ ID NOs: 6039 polypeptide sequence: 2206-. Preferably the fragment comprises one or more of SEQ ID NOs: 6039 polypeptide sequence: 2206-2224 and 3020-3042. The invention also includes polynucleotides encoding each of the polypeptide fragments identified above.

The present invention includes an attenuated SARS virus, wherein said attenuated SARS virus comprises an addition, deletion or substitution in a polynucleotide encoding one of the hydrophobic domains identified above. The invention also includes a method of making an attenuated SARS virus comprising altering the expression of SEQ ID N0: 6039 the coding of one or more hydrophobic domains mutates the SARS virus.

The invention includes methods of specifically recognizing SEQ ID NO: 6039 antibodies to one or more hydrophobic domains. The invention includes polypeptides capable of binding to SEQ ID NOs: 6039, small molecules that interfere with or disrupt their hydrophobicity.

Predicted SEQ ID NO: the N-glycosylation site of 6039 is identified in the following table.

Positional likelihood agreement with Nglyc outcomes

48 NGTC SEQ ID NO：7180 0.6371 (7/9) +

389 NHSN SEQ ID NO：7181 0.6132 (6/9) +

916 NFSS SEQ ID NO：7182 0.5807 (7/9) +

1628 NHTK SEQ ID NO：7183 0.5610 (7/9) +

1696 NKTV SEQ ID NO：7184 0.5297 (5/9) +

2031 NPTI SEQ ID NO：9764 0.5299 (5/9) + WARNING：PRO-X1.

2249 NSSN SEQ ID NO：7185 0.6329 (9/9) ++

2459 NPTD SEQ ID NO：9765 0.5599 (6/9) + WARNING：PRO-X1.

2685 NVSL SEQ ID NO：7186 0.6071 (8/9) +

4233 NATE SEQ ID NO：7187 0.6144 (7/9) +

Thus, the invention includes SEQ ID NO: 6039, wherein the fragment comprises an amino acid sequence comprising one or more of the N-glycosylation sites identified above. Preferably the fragment comprises one or more sequences selected from the group consisting of: SEQ ID NOS: 7180 7187 and 9764 9765. Preferably, the fragment comprises the amino acid sequence NSSN (SEQ ID NO: 7185).

The invention includes a polypeptide comprising SEQ ID NO: 6039, wherein said fragment does not contain one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

SEQ ID NO: t-epitope of 6039. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 7400-7639; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 7400-7639, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus.

The invention provides the use of a polypeptide as defined above in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, preferably a CTL response. The immune response is preferably protective or therapeutic.

The ORF1a and ORF1b sequences of coronaviruses are usually translated into one ORF1ab polyprotein. Slippage of the ribosome during translation produces an a-1 frameshift. One area of such sliding is as follows:

gggttttacacttagaaacacagtctgtaccgtctgcggaatgtggaaaggttatggctgtagttgtga

+1 G F T L R N T V C T V C G M W K G Y G C S C D

+3 G F Y T - K H S L Y R L R N V E R L W L - L -

ccaactccgcgaacccttgatgcagtctgcggatgcatcaacgtttttaaacgggtttgcggtgtaagt

+1 Q L R E P L M Q S A D A S T F L N G F A V - V

+3 P T P R T L D A V C G C I N V F K R V C G V S

gcagcccgtcttacaccgtgcggcacaggcactagtactg(SEQ ID NO：7224)

+1 Q P V L H R A A Q A L V L(SEQ ID NOS：7225-7226)

+3 A A R L T P C G T G T S T(SEQ ID NOS：7227-7229)

it will produce the following translational translations (SEQ ID NOS: 7230-7231):

ccaactccgcgaacccttgatgcagtctgcggatgcatcaacgtttttaaacgggtttgcggtgtaagt

Q L R E P L M Q S A D A S T F L N R V C G V S

accordingly, the invention includes a polypeptide comprising SEQ ID NO: 7232. The invention includes polypeptides comprising a sequence identical to SEQ DNO: 7232 and has a sequence identity with amino acid sequence. The invention includes a polypeptide comprising SEQ ID NO: 7232A fragment of a polypeptide. The invention includes a diagnostic kit comprising a nucleic acid sequence comprising SEQ ID NO: 7232 or a fragment thereof. The invention includes a diagnostic kit, in which a nucleic acid sequence comprising the nucleic acid sequence encoding SEQ ID NO: 7232 or a fragment thereof. The invention includes immunogenic compositions comprising SEQ ID NO: 7232 or a fragment thereof. The invention includes methods for identifying a polypeptide comprising SEQ ID NO: 7232 or a fragment thereof.

The invention also includes compositions comprising amino acid sequence X₁-X₂-X₃Wherein, X₁Is SEQ ID NO: 7233, X₂Is 1-10 amino acids, X₃Is SEQ ID NO: 7234. x₂Any sequence of 1-10 amino acids can be included (SEQ ID NOS: 7235-7244), but in a preferred embodiment, X₂Selected from: F. FL, FLN, FLNR (SEQ ID NO: 7245), FLNRV (SEQ ID NO: 7246) and FLNRVC (SEQ ID NO: 7247). Preferably X₂Is SEQ ID NO: 7247. these preferred embodiments are shown in SEQ ID NOS: 7248-7253.

The polypeptide comprises the amino acid sequence X₁-X₂-X₃Amino acid sequences having sequence identity. The invention includes the amino acid sequence X₁-X₂-X₃A fragment of the polypeptide of (1). The invention includes a diagnostic kit, in which the kit contains the amino acid sequence X₁-X₂-X₃Or a fragment thereof. The invention includes diagnostic kits comprising a nucleic acid encoding said amino acid sequence X₁-X₂-X₃Or a fragment thereof. The immunogenic composition comprising the amino acid sequence X₁-X₂-X₃Or a fragment thereof. The invention includes the ability to recognize sequences X containing said amino acid sequence₁-X₂-X₃Or a fragment thereof.

Amino acid sequence X₁-X₂-X₃(i.e., SEQ ID NOS: 7235-7244) was confirmed It is functionally homologous with the polyprotein of the mouse hepatitis virus. The polyprotein is cleaved to produce a variety of proteins. Can be composed of X₁-X₂-X₃Polyprotein of which X₂Is 6 amino acids (SEQ ID NO: 7240), and the resulting protein is as follows.

Mouse viral proteins	Coordinates in mouse virus	In SEQ ID NO: 7240 of the coordinates
			Nsp2	3334-3636	3241-3546
Nsp3	3637-3923	3547-3836
			Nsp4	3924 and 4015 (or 4012)	3837-3919
Nsp5	4016 (or 4013) -4209	3920-4117
			Nsp6	4210-4319	4118-4230
Nsp7	4320-4456	4231-4369
			Nsp9	4457-5384	4370-5301
Nsp10	5385-5984	5302-5902
			Nsp11	5985-6505	5903-6429
Nsp12	6506-6879	6430-6775
			Nsp13	6880-7178	6776-7073

The invention includes amino acid sequence X₁-X₂-X₃(i.e., SEQ ID NOS: 7235-7244), wherein said fragment comprises one of the polypeptide sequences identified in the above table. The invention also includes amino acid sequence X₁-X₂-X₃Wherein said fragment comprises a polypeptide sequence having a serine at its N-terminus and a glutamine at its C-terminus. The invention also includes amino acid sequence X₁-X₂-X₃The fragment of (1), wherein said fragment comprises a polypeptide sequence having an alanine at its N-terminus and a glutamine at its C-terminus. The invention also includes amino acid sequence X₁-X₂-X₃Wherein said fragment comprises a polypeptide sequence having an asparagine at its N-terminus and a glutamine at its C-terminus. The invention also includes amino acid sequence X₁-X₂-X₃Wherein said fragment comprises a polypeptide sequence having a cysteine at its N-terminus and a glutamine at its C-terminus. Each of the above fragments can be used in fusion proteins.

The invention includes diagnostic kits comprising at least one amino acid sequence X identified in the preceding paragraph₁-X₂-X₃(i.e., SEQ ID NOS: 7235-7244). The invention includes diagnostic kits comprising a nucleic acid sequence encoding at least one of the amino acid sequences identified in the preceding paragraph₁-X₂-X₃The polynucleotide sequence of a fragment of (a). The invention includes immunogenic compositions comprising an immunogenic composition comprising an amino acid sequence X identified in the preceding paragraph₁-X₂-X₃The polypeptide of a fragment of (1). The invention includes the ability to recognize sequences containing one of the amino acids identified in the preceding paragraph₁-X₂-X₃An antibody to the polypeptide of fragment(s) of (a).

Amino acid sequence X₁-X₂-X₃(wherein X₂Is 6 amino acids) is identified as asparagine at the following amino acid position: 48; 389; 556; 916; 1628, mixing the above powders with water; 1696; 1899; 2079; 2249; 2252; 2507 of a paper base; 2685; 3303, respectively; 3373; 3382; 3720 and (b); 4150; 4233; 4240; 5016; 5280; 5403; 5558; 5650; 5905; 6031; 6130 of; 6474; 6918; 6973. thus, the invention includes seq id NO: 7239, wherein said fragment has at least 10 amino acids, and wherein said fragment comprises one or more of the amino acid sequences of SEQ ID NOs: 7239 asparagine at the following amino acid position: 8; 389; 556; 916; 1628, mixing the above powders with water; 1696; 1899; 2079; 2249; 2252; 2507 of a paper base; 2685; 3303, respectively; 3373; 3382; 3720 and (b); 4150; 4233; 4240; 5016; 5280; 5403; 5558; 5650; 5905; 6031; 6130 of; 6474; 6918; and 6973.

SEQ ID NOS: 7235-7244 the zinc binding region 2 site was identified at amino acid residue 2102-2112(SEQ ID NO: 7254 HGIAAINSVPW). SEQ ID NOS: 7235 the polypeptide of 7244 can be processed into several peptides by SARS virus. The zinc binding region falls within the nsp1 region of the polypeptide. SEQ ID NO: 7254 it is a target for screening chemical inhibitor of SARS virus. The invention includes a polypeptide comprising SEQ ID NO: 7254. The invention includes encoding SEQ ID NO: 7254. The invention includes screening for SEQ ID NO: 7254 and a method for using the same as an inhibitor. The invention includes recombinant expression of SEQ ID NO: 7254. the invention includes SEQ ID NOS: 7235-7244, wherein said fragment comprises the sequence of SEQ ID NO: 7254. the invention includes a polypeptide comprising SEQ ID NO: 7254, wherein the polypeptide is complexed with a zinc atom. The invention includes a zinc ion inhibitor that can prevent zinc ions from reacting with SEQ ID NO: 7254 a small molecule to which the polypeptide binds. The invention includes fusion proteins, wherein the fusion protein comprises SEQ ID NO: 7254.

the SARS virus encoded polyprotein will contain at least two protease domains: papain-like cysteine (PLP) protease and chymotrypsin-picornavirus 3C-like protease (3 CLp). (more than one copy of the PLP domain may be present). The function of these proteases is to cleave the polyprotein into smaller proteins. The 3C-like protease, also known as "main protease" or Mpro, is itself cleaved from the polyprotein by its own protease (autoprotease) activity. See generally Fields Virology (Fields Virology) by Fields et al (second edition, b.n. raven Press, New York, NY) chapter 35, and Anand et al, EMBO Journal (2002)21 (13): 3213-3224. The 3CLp generally corresponds to the Nsp2 region identified above.

The SARS virus 3CLp protein is also expressed as SEQ ID NO: 6569 (and also SEQ ID NO: 9769), as shown in FIG. 15.

FIG. 16 also shows the results of SARS virus 3CLp in comparison with 3CLp of avian infectious bronchitis (IBV; SEQ ID NO: 6570), mouse hepatitis virus (MHV; SEQ ID NO: 6571) and bovine coronavirus (BCoV; SEQ ID NO: 6572). Accordingly, the invention includes polypeptide sequences comprising SEQ ID NO: 6569 or a fragment thereof or a polypeptide sequence having sequence identity thereto. The invention also includes nucleic acids encoding SEQ ID NO: 6569 or a fragment thereof. The invention includes a polypeptide encoding a polypeptide corresponding to SEQ ID NO: 6569 polynucleotide sequence having a polypeptide sequence with sequence identity.

The invention also includes a method for screening SARS virus 3CLp protein inhibitor. In one embodiment, the invention comprises screening for SEQ ID NO: 6569 method of inhibiting agent. The present invention includes a method for recombinant expression of SARS virus 3CLp protein in a host cell. The invention includes recombinant expression of a polypeptide comprising SEQ ID NO: 6569 polypeptide sequence or its enzymolysis active fragment or polypeptide sequence with sequence identity. The invention includes small molecules that inhibit or reduce the proteolytic activity of the SARS virus 3CLp protein. The invention includes inhibiting or reducing the expression of a polypeptide comprising SEQ id no: 6569 small molecule of proteolytic activity of the polypeptide.

The catalytic residues of SARS virus 3CLp are identified in figures 15 and 16. Specifically, catalytic histidine and catalytic cysteine were identified. Such catalytic sites are targets for small molecules that can inhibit or reduce the activity of 3CLp protease. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6569 wherein said fragment comprises at least one catalytic site. Preferably the catalytic site is selected from the group consisting of catalytic histidine and catalytic cysteine in figures 15 and 16. The present invention includes polynucleotides encoding polypeptides, wherein the polypeptides comprise SEQ ID NOs: 6569 wherein said fragment comprises at least one catalytic site. Preferably the catalytic site is selected from the group consisting of catalytic histidine and catalytic cysteine in figures 15 and 16.

The invention also includes methods of screening compound libraries to identify small molecules that inhibit the catalytic site of SARS virus 3 CLp. Preferably, the 3CLp comprises SEQ ID NO: 6569 or a fragment thereof, or a sequence having sequence identity thereto. The catalytic site is preferably selected from the group consisting of catalytic histidine and catalytic cysteine as shown in figures 15 and 16.

The invention includes small molecules that inhibit the catalytic site of SARS virus 3 CLp. Preferably, the 3CLp comprises SEQ id no: 6569 or a fragment thereof, or a sequence having sequence identity thereto. The catalytic site is preferably selected from the group consisting of catalytic histidine and catalytic cysteine as shown in figures 15 and 16.

The residues of the SARS virus 3CLp substrate site are identified in FIGS. 15 and 16. In particular, the substrate sites are located on phenylalanine, tyrosine and histidine. This substrate site is a target for small molecules that inhibit or reduce the activity of 3CLp protease. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6569 wherein said fragment comprises at least one substrate site. Preferably the substrate site is selected from the substrates phenylalanine, tyrosine and histidine in figures 15 and 16. The present invention includes polynucleotides encoding polypeptides, wherein the polypeptides comprise SEQ ID NOs: 6569 said fragment comprising at least one substrate site. Preferably the substrate site is selected from the substrates phenylalanine, tyrosine and histidine in figures 15 and 16.

The invention also includes methods of screening compound libraries to identify small molecules that block the SARS virus 3CLp substrate site. Preferably, the 3CLp comprises SEQ ID NO: 6569 or a fragment thereof, or a sequence having sequence identity thereto. The substrate site is preferably selected from the substrates phenylalanine, tyrosine and histidine as shown in figures 15 and 16.

The invention includes small molecules that inhibit the SARS virus 3CLp substrate site. Preferably, the 3CLp comprises SEQ id no: 6569 or a fragment thereof, or a sequence having sequence identity thereto. The substrate site is preferably selected from the substrates phenylalanine, tyrosine and histidine as shown in figures 15 and 16.

The invention also includes a diagnostic kit comprising a polynucleotide encoding SARS virus 3CLp or a fragment thereof. Preferably, the 3CLp of the SARS virus comprises SEQ ID NO: 6569 or a fragment thereof, or a polypeptide sequence having sequence identity thereto. Preferably the fragment comprises one or more sites selected from the group consisting of a catalytic site and a substrate site. Preferably the catalytic site is selected from one or more of the sites identified in figures 15 and 16. Preferably the substrate site is selected from one or more of the sites identified in figures 15 and 16.

The invention also includes a diagnostic kit comprising SARS virus 3CLp specific antibodies or fragments thereof. Preferably, the antibody is a polypeptide comprising SEQ ID NO: 6569 or a fragment thereof, or a polypeptide sequence having sequence identity thereto. Preferably the antibody is specific for one or more SARS virus 3CLp sites selected from the catalytic site and the substrate site. Preferably the catalytic site is selected from one or more of the sites identified in figures 15 and 16. Preferably the substrate site is selected from one or more of the sites identified in figures 15 and 16.

The invention includes a polypeptide comprising the amino acid sequence of the sequence shown in figure 25. The amino acid sequence in figure 25 separated by x is SEQ ID NOS: 7188 and 7189. The present invention includes polypeptides comprising amino acid sequences having sequence identity to the translation of figure 25. The invention includes fragments of a polypeptide comprising the sequence of figure 25. The invention includes a diagnostic kit comprising a polypeptide comprising the translation product of figure 25 or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding the translation product of figure 25 or a fragment thereof. The invention includes a polypeptide comprising the translation of figure 25 or a fragment thereof in an immunogenic composition. The invention includes an antibody which recognizes a polypeptide comprising the sequence shown in figure 25 or a fragment thereof. The sequence of FIG. 25 was shown to be functionally homologous to ORF1b of coronavirus.

SEQ ID NO: 7188 is the open reading frame of figure 25. The invention includes a polypeptide comprising SEQ ID NO: 7188. The invention includes polypeptides comprising a polypeptide having an amino acid sequence substantially identical to SEQ ID NO: 7188 it has amino acid sequence with sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 7188. The invention includes a diagnostic kit, in which a kit containing a nucleic acid sequence comprising SEQ ID NO: 7188 or a fragment thereof. The invention includes a polypeptide comprising a nucleotide sequence encoding SEQ ID NO: 7188 or fragments thereof. The present invention includes immunogenic compositions comprising a polypeptide comprising SEQ ID NO: 7188 or a fragment thereof. The invention includes methods for identifying a polypeptide comprising SEQ ID NO: 7188 or a fragment thereof.

SEQ ID NO: 7190 is SEQ ID NO: 7188 open reading frame. The invention includes a polypeptide comprising SEQ ID NO: 7190, a fragment thereof, or a polypeptide having sequence identity thereto. The invention also includes nucleic acids encoding SEQ ID NO: 7190. fragments thereof, or polynucleotides having a polypeptide having sequence identity thereto. Encoding the amino acid sequence of SEQ ID NO: 7190 is the polynucleotide of SEQ ID NO: 7191.

SEQ ID NO: 7188 also contains a peptide comprising SEQ ID NO: 6042 open reading frame. The invention includes a polypeptide comprising SEQ id no: 6042. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 6042 and a polypeptide having an amino acid sequence having sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 6042. The invention includes a diagnostic kit comprising a nucleic acid sequence comprising SEQ ID NO: 6042 or a fragment thereof. The invention includes a polypeptide comprising a nucleotide sequence encoding SEQ ID NO: 6042 or a fragment thereof. The present invention includes immunogenic compositions comprising a polypeptide comprising SEQ ID NO: 6042 or a fragment thereof. The invention includes a polypeptide capable of recognizing a polypeptide comprising SEQ ID NO: 6042 or a fragment thereof. SEQ ID NO: 6042 was shown to be functionally homologous to the coronavirus spike protein.

Predicted SEQ ID NO: the transmembrane region of 6042 was identified as follows.

Predicted SEQ ID NO: 6042 transmembrane helix

Sequence positions in parentheses indicate the core region.

Only if the score exceeds 500 is considered meaningful.

Inward-outward-screwing: 18 outward-inward spirals were found: find 13

Beginning at end of scoring center and beginning at end of scoring center

1(1)16(16) 959 9 1(1)17(17) 684 10

233(237)257(252) 905 244 222(222)240(237) 238 229

345(347)364(361) 490 354 244(247)264(264) 613 254

345(354)369(369) 420 362 349(355)369(369) 314 362

497(497)513(513) 239 506 496(496)511(511) 488 503

573(573)588(588) 811 580 573(573)591(591) 712 581

645(648)666(663) 302 656 650(652)666(666) 474 659

690(696)714(711) 428 704 674(679)702(696) 190 686

857(860)882(874) 1508 867 691(696)713(711) 210 704

1031(1031)1046(1046) 446 1039 866(868)886(886) 1172 876

1199(1203)1219(1217) 2667 1210 1198(1201)1215(1215) 3221 1208

Spike protein SEQ IID NO: 6042 is a surface exposed polypeptide. The hydrophobic transmembrane region may prevent recombinant expression of the protein. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042, wherein one or more of the hydrophobic regions identified above are removed. The present invention also includes polynucleotides encoding such polypeptides. The invention includes recombinant expression of the protein in a host cell. Primers and fragments thereof for amplifying spike protein genes, such as fragments encoding soluble ectodomains, including the sequences set forth in SEQ ID NOS: 9753-9763(Xiao et al (2003) Biochem Biophys Res Comm 312: 1159-1164).

SEQ ID NO: 6042 other characteristics are identified below.

PSORT-PROTEIN LOCATION SITE PREDICTION

Version 6.4(WWW)

SEQ ID NO: 6042-1255 residue

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 2

Most N-terminal position TMS: 496, i ═ 2

MTOP: membrane surface structure (Hartmann et al)

I (middle): 503 charge difference (C-N): 1.0

McG: detection Signal peptide sequence (McGeoch)

UR length: 13

UR peak value: 3.28

CR net charge: 0

Discrimination scoring: 8.66

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): 5.94

Possible cleavage sites: 13

> > appear to contain a cleavable N-terminal signal sequence

Predicted amino acid composition of the mature form:

from 14 bit calculation

ALOM new count: 1-domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 0B

ALOM: transmembrane region was found (Klein et al)

Counting: 1 value: -12.26 threshold value: -2.0

Internal probability-12.26 transmembrane 1202-1218(1194-1228)

Peripheral probability of 0.16

Modified ALOM score: 2.55

> > appear to contain type Ia membrane proteins

Cytoplasmic tails 1219 to 1255(37 residues)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

(14) Or not cuttable?

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 14

Is it uncut? Ipos is set as: 24

Identification of targeted mitochondrial sequences:

positive electricity (2.18)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

Inference step: 2

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 24) from: 1 to: and (3) scoring 10: 8.0

SKL motif (peroxisome signal peptide):

position: 964(1255), count: 1SRL

SKL score (peroxisome): 0.1

Peroxisome-prone amino acid composition: 1.37

AAC, modified Scoring not counted from N-terminal

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.079

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 0.39 state: it is unclear

The GY motif in the type Ia tail? (lysosome)

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

Type Ia plasma membrane proteins

Verification of the NPXY motif.

The YXRF motif was verified.

N-myristoylation was verified.

-end result-

Plasma membrane-certainty 0.460 (affirmative) < success >

Microbody (peroxisome) - -, certainty ═ 0.171 (affirmative) < success >

Endoplasmic reticulum (membrane) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -certainty of 0.100 (affirmative) < success >

Endoplasmic reticulum (lumen) - - -, certainty 0.100 (affirmative) < success >

SEQ ID NO: 6042 shows a N-terminal signal region followed by a surface exposed region, a transmembrane region, and a C-terminal cytoplasmic domain. Thus, the invention includes SEQ ID NO: 6042 and surface exposed fragments. Preferably the fragment does not contain SEQ ID NO: 6042 amino acid sequence of the last 50 amino acids of the C-terminus. Preferably the fragment comprises a fragment not comprising SEQ ID NO: 6042 amino acid sequence of the last 70 amino acids of the C-terminus. Preferably the fragment does not contain SEQ ID NO: 6042 (translomain region). Preferably the fragment does not contain SEQ ID NO: the C-terminal cytoplasmic domain of 6042. Preferably, the fragment does not contain an N-terminal signal sequence. Preferably the fragment does not contain SEQ ID NO: 6042 amino acids 1-10 of the N-terminus. Preferably the fragment does not contain SEQ ID NO: 6042 amino acids 1-14 of the N-terminus. SEQ id no: 6042 are SEQ ID NOS: 7398 and 7399, as described by Xiao et al (with an additional C-terminal cysteine), 2003, Biochem Biophys Res Comm 312: 1159-1164. Yang et al (2004) describe a truncated C-terminus of the spike protein, which truncation removes a portion of the cytoplasmic domain, or also the transmembrane region (Nature 428: 561-564).

The SEQ ID NOs: 6042 is a variant of SEQ ID NO: 9962. and SEQ ID NO: 6042 the sequence has Ser instead of Ala at residue 581 and Phe instead of Leu at residue 1152.

The spike protein of coronaviruses can be cleaved into two separate strands S1 and S2. These two chains can still be joined together to form a dimer or trimer. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the polypeptide is cleaved into S1 and S2 domains. The invention also includes a polypeptide comprising SEQ ID NO: 6042, wherein amino acids 1-10, preferably amino acids 1-14, of the N-terminus are removed, and wherein the polypeptide of SEQ ID NO: 6042 is cleaved into the S1 and S2 domains. Preferably the polypeptide is in the form of a trimer.

The spike protein appears to form an alpha-helical structure in the transmembrane region of the protein, preferably in the S2 domain. This alpha-helical structure is thought to associate with at least two other spike proteins to form a trimer. The helical or coiled-coil region of the spike protein is identified below. The prediction of SEQ ID NO: the coiled-coil of 6042 (spike protein) is located at amino acids 900-1005 and 1151-1185 (see FIG. 12).

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the fragment comprises the polypeptide sequence of SEQ ID NO: 6042 crimp zone. The fragment preferably comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: amino acids 900-1005 and amino acids 1151-1185 of 6042. The invention includes a polypeptide comprising SEQ id no: 6042, wherein the fragment does not comprise the polypeptide sequence of SEQ ID NO: 6042 crimp zone. The fragment preferably comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: amino acids 900-1005 and amino acids 1151-1185 of 6042.

The spike protein is thought to play a central role in coronavirus fusion and infection with mammalian host cells. At least two structural motifs associated with this fusion were identified by analysis of the coronavirus spike protein and similar surface proteins in other viruses: heptad Repeat (HR) and membrane fusion peptides, these two motifs are usually located in the S2 domain.

At least two 4, 3 hydrophobic Heptad Repeat (HR) domains are typically found in the ectodomain of the coronavirus S2 domain. One heptad repeat region (HR1) is typically located adjacent to the fusion peptide, while the second heptad repeat region (HR1)₂) Typically located near the C-terminus of the S2 domain, adjacent to the transmembrane anchor. Heptad repeats are characterized by coiled-coil structures, and heptad repeats found in viral surface proteins (e.g., coronavirus spike proteins) are believed to form bundled helical structures associated with viral entry. See Bosch et al, j.virology (2003) 77: 8801-8811 (FIG. 1B of this reference shows SARS and 5 coronavirus HR1 and BR₂Comparison of regions, denoted "HCov-SARS").

Heptad repeats typically contain a 7 amino acid repeat structure, denoted as a-b-c-d-e-f-g, in which the hydrophobic side chains of residues a and d typically form a non-polar stripe (stripe), while electrostatic interactions are found in residues e and g. Position a is most often Leu, Ile or Ala, and position d is often Leu or Ala. Residues e and g are often Glu or Gln, while Arg and Lys also occur predominantly at position g. Charged residues are common at positions b, c and f, and these residues may be contacted with a solvent. Exceptions to these conventional parameters are known. For example, Pro residues are sometimes found in these 7 amino acids.

The HR1 and HR of the MHV strain have been postulated₂The sequences can be assembled into thermostable oligomeric alpha helical rod-like complexes in which HR1 and HR are present₂The spirals run in an anti-parallel manner. Also, in this study it was proposed that HR is₂Are strong inhibitors of viral entry into cells and cell-cell fusion.

HR1 and HR have been identified in the SARS virus genome₂And (4) sequencing. The SARS virus HR1 region comprises substantially SEQ id no: amino acids 879-1005 of 6042, or a fragment thereof capable of forming at least one alpha-helical rotation. Preferably, the fragment comprises at least 7 (e.g., at least 14, 21, 28, 35, 42, 49, or 56) amino acid residues. SEQ ID NO: 7192 includes SEQ ID NO: amino acid 879-1005 of 6042.

A preferred HR1 fragment comprises SEQ ID NO: amino acid residue 879-980 of 6042. This preferred fragment is seq id NO: 7193.

another preferred HR1 fragment comprises SEQ ID NO: amino acid residue 901 of 6042 and 1005. The preferred fragment is SEQ ID NO: 7194.

HR of SARS virus₂The region substantially comprises SEQ ID NO: amino acids 1144 of 6042-1201, or a fragment thereof capable of forming at least one alpha-helical turn. Preferably, the fragment comprises at least 7 (e.g., at least 14, 21, 28, 35, 42, 49, or 56) amino acid residues. SEQ ID NO: 7195 includes amino acids 1144-. Preferred HR ₂The fragment comprises SEQ ID NO: amino acids 1144-1195 of 6042. The preferred fragment is SEQ id no: 7196。

The membrane fusion peptide sequence in the spike protein is also thought to be involved in viral fusion (and infection) with host cells. Fusion peptides typically contain about 16-26 amino acid residues that are conserved within the virus family. These membrane fusion peptides are relatively hydrophobic and typically exhibit an asymmetric hydrophobic profile when they form an alpha helix. They are also generally rich in alanine and glycine.

At least three hydrophobic membrane fusion peptide regions were identified in coronaviruses (PEP1, PEP2, and PEP 3). See Luo et al, "role of Two Conserved Hydrophobic Regions in murine coronavirus Spike proteins in Cell-Cell Fusion" (circles in Cell-Cell Fusion of Two Conserved Hydrophobic Regions in the MurinCooronavirus Spike Protein), Virology (1998) 244: 483-494. FIG. 1 of the paper shows an alignment of the membrane fusion peptide sequences of mouse hepatitis virus, bovine coronavirus, feline infectious peritonitis virus, transmissible gastroenteritis virus and infectious bronchitis virus. See also Bosch et al, "coronavirus spike proteins are class I viral fusion proteins: the structure and function of The Fusion Core Complex "(The Coronavir Spike Protein isa Class I Virus Fusion Protein: Structural and Functional characterization of The Fusion Core Complex) Journal of Virology (2003)77 (16): 8801-8811.

PEP1(SEQ ID NO: 7197), PEP2(SEQ ID NO: 7198) and PEP3(SEQ ID NO: 7199) sequences within the SARS spike protein were also identified.

The coronavirus spike protein (and other similar surface viral proteins) is believed to undergo a conformational change upon receptor binding to the target cell membrane. It is believed that the result of this conformational change is that one or more hydrophobic membrane fusion peptides are exposed and inserted into the target cell membrane. The free energy released when the spike protein subsequently refolds into its most stable conformation is believed to play an important role in the fusion of the virus with the cell membrane.

One or more SARS HR sequences, preferably HR, may be used₂Or fragments thereof, to inhibit viral entry andmammalian host target cell membrane fusion. The present invention provides a method of inhibiting viral infection comprising administering a composition comprising one or more SARS HR polypeptides or fragments thereof. Preferably, the composition comprises SARS HR₂And (4) sequencing.

In another embodiment, the invention includes a composition comprising the SARS HR1 sequence or a fragment thereof and SARS HR₂A composition of sequences or fragments thereof. The HR1 and HR₂The sequences may optionally be joined to each other to form an oligomer. The composition may comprise HR1 and HR ₂Intermediate domain sequences between the domains. The use of such an intermediate sequence may promote oligomerization or other structural interactions between the HR regions.

The HR sequences useful in the present invention may be produced recombinantly by methods known in the art. The SARS HR sequence may be modified to facilitate bacterial expression. In particular, the HR sequence may be modified to facilitate delivery of the recombinant protein to the surface of a bacterial host cell. For example, a leader sequence of a bacterial membrane protein may be added at the N-terminus of the recombinant HR sequence. The HR sequences useful in the present invention may also be generated by chemical synthesis using methods known in the art (see below).

The applicants have identified structural similarities between the SARS spike protein and the surface protein NadA of Neisseria meningitidis (as well as other similar bacterial adhesion proteins), and this specification will be discussed in more detail below. Another method for facilitating bacterial expression of HR sequences involves the addition of a stalk (talk) and/or anchor sequence of the NadA-like protein at the C-terminus of the recombinant HR sequence. Recombinant sequences containing bacterial anchor sequences are preferably prepared in outer membrane vesicles (the preparation of which is described in more detail later in the application). Recombinant sequences lacking the bacterial anchor sequence can be secreted and isolated from the supernatant.

The polypeptide sequences encompassed by the present invention comprise a first sequence comprising the leader sequence of the bacterial membrane protein and a second sequence comprising the HR sequence of the coronavirus. Preferably, the first sequence comprises a leader sequence of a bacterial adhesin protein. More preferably the bacteriumThe adhesive protein is NadA. Preferably said second sequence comprises HR1, HR₂Or both. In one embodiment, the second sequence comprises HR1, HR₂And the intermediate domain sequences found in naturally occurring spike proteins. For example, the second sequence may comprise a fragment of the coronavirus spike protein comprising an N-terminus beginning at the HR1 region and ending at the HR₂The C-terminal amino acid of the region.

The invention also includes a polypeptide sequence comprising a first, second, third and fourth sequence, wherein the first sequence comprises a leader sequence of a bacterial membrane protein; wherein the second sequence comprises the HR sequence of a coronavirus; wherein the third sequence comprises a stalk region of a bacterial adhesion protein; and wherein the fourth sequence comprises an anchor region of a bacterial adhesion protein. In one embodiment, the first sequence comprising the leader peptide sequence is removed. In another embodiment, the third sequence comprising the handle region is removed. In another embodiment, the fourth sequence containing the anchor region is removed.

The polypeptide sequences of the above constructs may be joined together by methods known in the art, for example, by glycine linkers.

An example of a construct that can be used in such a bacterial expression system is shown in FIG. 50. The polypeptide sequences of each construct shown in fig. 50 are given in SEQ ID NOS: 7200-.

7200 leader domain NadA (2-29) -HR1(879-980) -6Xgly-HR₂(1144-1195) -handle + Anchor NadA (88-405)

7201 leader NadA (1-29) -HR1(879-980) -6Xgly-HR₂(1144-1196) -handle NadA (88-351)

7202 leader NadA (1-29) -HR1-HR₂(879-1196) -Stem + Anchor NadA (88-405)

7203 leader domain NadA (1-29) -HR1-HR₂(879-1196) -Stem NadA (88-351)

7204 HR1-HR₂(879-1196) -Stem NadA (88-351) -6 his

7205 leader domain NadA (1-29) -HR1-HR₂(879-1196) -Anchor NadA (351-405)

7206 leader NadA (1-29) -HR1-HR₂(879-1196)

Administration of one or more of these membrane fusion sequences may also interfere with the ability of the coronavirus to fuse with the host cell membrane. Accordingly, the present invention includes isolated polypeptides comprising the amino acid sequence: SEQ ID NO: 7197. SEQ ID NO: 7198 and SEQ ID NO: 7199. the invention also includes isolated polypeptides comprising an amino acid sequence having sequence homology to the amino acid sequence: SEQ ID NO: 7197. SEQ ID NO: 7198 and SEQ ID NO: 7199.

Two or more of these SARS membrane fusion peptides can be bound together. The present invention includes a composition comprising two SARS membrane fusion peptides, wherein the peptides are selected from at least two amino acids selected from the group consisting of: SEQ ID NO: 7197. SEQ ID NO: 7198 and SEQ ID NO: 7199 or a sequence having sequence identity thereto.

Two or more SARS membrane fusion peptides can be linked together. Accordingly, the present invention includes a polypeptide comprising a first amino acid sequence and a second amino acid sequence, wherein the first and second amino acid sequences are selected from the group consisting of: SEQ ID NO: 7197. SEQ ID NO: 7198 and SEQ ID NO: 7199 or a sequence having sequence identity thereto. Preferably, the first amino acid sequence and the second amino acid sequence are different SARS membrane fusion peptides, i.e. they are not identical.

The invention also includes methods of treating or preventing SARS virus infection comprising administering one or more of the above-described SARS membrane fusion peptide compositions.

As described above, the spike protein is capable of forming a trimer. The invention also includes compositions comprising trimeric forms of SEQ id no: 6042. The invention includes compositions comprising at least polypeptides, wherein each polypeptide comprises at least the alpha-helical coiled coil region of the spike protein of SARS virus. Preferably, the spike protein comprises SEQ ID NO: 6042 or a fragment thereof.

The invention also includes compositions comprising a SARS virus spike protein or fragment thereof, wherein the protein is associated with a transmembrane, and wherein the fragment comprises an alpha-helical region of the SARS virus spike protein. Preferably, the composition comprises at least three SARS virus spike proteins or fragments thereof, wherein said fragments comprise the alpha-helical region of the SARS virus spike protein.

The invention also includes antibodies that specifically bind to the trimeric form of the spike protein of SARS virus. Preferably, the spike protein comprises SEQ ID NO: 6042 or a fragment thereof. The invention includes antibodies that specifically bind to trimeric forms of the spike protein of the SARS virus, wherein the protein is associated with a transmembrane.

The invention also includes antibodies that specifically bind to monomeric forms of the spike protein of SARS virus or fragments thereof. Preferably the antibody specifically binds to SEQ ID NO: 6042 or a fragment thereof.

The invention also includes small molecules that interfere with or disrupt the frizzled spike protein trimer of the SARS virus.

The invention also includes an attenuated SARS virus for use as a vaccine, wherein the attenuated virus comprises a polynucleotide insertion, deletion or substitution that does not disrupt the trimeric conformation of the spike protein of the SARS virus. The invention also includes an attenuated SARS virus for use as a vaccine, wherein the attenuated virus comprises a polynucleotide insertion, deletion or substitution that does not disrupt the formation of the spike protein alpha-helix of the SARS virus.

The spike protein may be produced by recombinant methods. In one embodiment, the spike protein is expressed in a virus-like particle such that the protein is attached to the cell membrane. This attachment is useful for presenting immunogenic epitopes of the spike protein. Preferably, the alpha-helical portion of the spike protein binds to the cell membrane. Preferably, the spike protein forms a trimer within the bound transmembrane region.

Predicted SEQ ID NO: the N-glycosylation site of 6042 was identified as follows:

location likelihood agreement NGlyc outcome

29 NYTQ SEQ ID NO：7207 0.7751 (9/9) +++

65 NVTG SEQ ID NO：7208 0.8090 (9/9) +++

109 NKSQ SEQ ID NO：7209 0.6081 (7/9) +

119 NSTN SEQ ID NO：7210 0.7039 (9/9) ++

158 NCTF SEQ ID NO：7211 0.5808 (7/9) +

227 NITN SEQ ID NO：7212 0.7518 (9/9) +++

269 NGTI SEQ ID NO：7213 0.6910 (9/9) ++

318 NITN SEQ ID NO：7214 0.6414 (9/9) ++

330 NATK SEQ ID NO：7215 0.6063 (8/9) +

357 NSTF SEQ ID NO：7216 0.5746 (8/9) +

589 NASS SEQ ID NO：7217 0.5778 (6/9) +

602 NCTD SEQ ID NO：7218 0.6882 (9/9) ++

699 NFSI SEQ ID NO：7219 0.5357 (7/9) +

783 NFSQ SEQ ID NO：7220 0.6348 (9/9) ++

1080 NGTS SEQ ID NO：7221 0.5806 (7/9) +

1116 NNTV SEQ ID NO：7222 0.5106 (5/9) +

1176 NESL SEQ ID NO：7223 0.6796 (9/9) ++

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the fragment comprises one or more of the glycosylation sites identified above (SEQ ID NOS: 7207-7223). The invention also includes polynucleotides encoding one or more of the above fragments. Such glycosylation sites can be covalently attached to the sugar. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the fragment comprises one or more of the glycosylation sites identified above, and wherein the polypeptide is glycosylated at one or more of the above sites.

The predicted O-glycosylation sites were identified as follows:

Threshold opinion for residue numbering probability

Thr 698 0.8922 0.7696 T

Thr 706 0.9598 0.7870 T

Thr 922 0.9141 0.7338 T

Ser 36 0.8906 0.7264 S

Ser 703 0.8412 0.7676 S

The invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the fragment comprises one or more O-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the above fragments. The invention includes a polypeptide comprising SEQ ID NO: 6042, wherein said fragment comprises one or more O-glycosylation sites identified above, and wherein said polypeptide is covalently attached to a sugar at one or more of the contained glycosylation sites.

The invention also includes a polypeptide comprising SEQ ID NO: 6042, wherein said fragment comprises one or more of the N-glycosylation sites identified above, and wherein said fragment comprises one or more of the O-glycosylation sites identified above.

The invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the fragment does not contain one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

Predicted SEQ ID NO: 6042 the phosphorylation sites are Ser-346, Tyr-195 and Tyr-723. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042, wherein the fragment comprises at least 10 amino acid residues, and wherein the fragment comprises one or more amino acids selected from the group consisting of: ser-346, Tyr-195 and Tyr-723. In one embodiment, one or more amino acids selected from the group consisting of Ser-346, Tyr-195, and Tyr-723 is phosphorylated.

Xiao et al (2003) Biochem Biophys Res Comm 312: 1159-1164 have described the expression and functional characteristics of spike glycoproteins.

SEQ ID NO: t-epitope of 6042. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group consisting of the nucleic acids identified as SEQ ID NOS: 8041-8280 amino acid sequence of the T-epitope sequence; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes the polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising two or more of the nucleic acids identified as SEQ ID NOS: 8041-8280, or a polynucleotide encoding such a polypeptide.

The following antigenic uses are preferred: (1) as a T-cell antigen; (2) for generating a complex between an MHC class I protein (e.g., HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) antigens that elicit a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal (particularly a human) which method comprises administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6040. The polypeptide comprises a polypeptide having a sequence identical to that of SEQ ID NO: 6040 and amino acid sequences having sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 6040. The invention includes encoding SEQ ID NO: 6040 or a fragment thereof. The invention includes a polypeptide comprising SEQ id no: 6040 or a fragment thereof. The invention includes a polypeptide comprising a nucleotide sequence encoding SEQ ID NO: 6040 or fragments thereof. The invention includes methods for identifying a polypeptide comprising SEQ ID NO: 6040 or a fragment thereof.

SEQ ID NO: 6040 was demonstrated to be functionally homologous to the membrane proteins of coronaviruses. Predicted SEQ ID NO: the transmembrane helix of 6040 was identified as follows:

predicted transmembrane helices

Sequence positions in parentheses indicate the core region.

Only if the score exceeds 500 is considered meaningful.

Inward-outward-screwing: find 3

From to the scoring center

27(30) 48(45) 1138 38

137(139) 153(153) 486 146

Outward internal screw: find 3

From to the scoring center

28(31) 45(45) 819 38

71(73) 90(90) 210 81

136(142) 156(156) 272 149

The region of amino acids with the highest predicted transmembrane helical region is represented by SEQ ID NO: 6040 amino acid 27 to 48. Such transmembrane regions are often difficult to express recombinantly. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6040, wherein the fragment does not comprise the amino acid sequence between positions 27-48. The invention includes a polypeptide comprising SEQ ID NO: 6040, wherein the fragment does not comprise the amino acid sequence between positions 28-45. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6040 is a hypothetical SARS virus protein. For SEQ ID NO: 6040 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6040 is located in one of the following locations: mitochondrial matrix space, microbodies (peroxisomes), nuclei, and the inner mitochondrial membrane. The prediction of SEQ ID NO: 6040 binding to organelles in the infected cell.

Thus, SEQ ID NO: 6040 it is the target for screening the SARS virus chemical inhibitor. The invention includes a polypeptide comprising SEQ ID NO: 6040 or a fragment thereof. The invention includes encoding SEQ ID NO: 6040 or a fragment thereof. The invention includes screening for SEQ ID NO: 6040. The invention includes recombinant expression of SEQ ID NO: 6040. the invention includes methods for preventing the expression of SEQ ID NO: 6040 small molecules that bind to organelles within infected cells. The invention includes a polypeptide comprising SEQ ID NO: 6040.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

SEQ ID NO: 6040163 residue

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 0

McG: detection Signal peptide sequence (McGeoch)

UR length: 9

UR peak value: 1.75

CR net charge: 1

Discrimination scoring: -2.56

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): 1.94

Possible cleavage sites: 53

> > there appears to be no N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 0 x domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 0B

ALOM: transmembrane region was found (K1ein et al)

Counting: value 0: 1.32 threshold value: -2.0

Peripheral probability of 1.32

Modified ALOM score: -1.16

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 156

HRSVTI

Identification of targeted mitochondrial sequences:

unclear (0.88)

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

Inference step: 2

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 156) is selected from: 27 to: and 44, grading: 5.0

Mitochondrial matrix? And (3) scoring: 0.36

SKL motif (peroxisome signal peptide):

position: 99(163), count: 1 SKL

SKL score (peroxisome): 0.3

Peroxisome-prone amino acid composition: -4.28

Peroxisome proteins? The state is as follows: it is unclear

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 0.02 state: it is unclear

Modification scoring of lysosomes: 0.152

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

The following are found: position: 132(5) KRKR

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear modification scoring: 0.60

Nuclear signal peptide status: unclear (0.30)

The CaaX motif was verified.

N-myristoylation was verified.

The CaaX motif was verified.

-end result-

Mitochondrial matrix gap-certainty 0.480 (affirmative) < success >

Microbody (peroxisome) - - -, certainty ═ 0.300 (affirmative) < success >

Kernel-certainty 0.300 (affirmative) < success >

Inner mitochondrial membrane-certainty 0.188 (affirmative) < success >

Predicted SEQ ID NO: the N-glycosylation site of 6040 was identified as follows.

Location likelihood agreement NGlyc outcome

2NKTG(SEQ ID NO：7255) 0.7804 (9/9) +++

106NLTL(SEQ ID NO：7256) 0.6123 (7/9) +

Thus, the invention includes SEQ ID NO: 6040, wherein the fragment has at least 10 amino acids, and wherein the fragment has at least one amino acid sequence selected from SEQ id NO: 6040 amino acid positions 2-106 contain one or more asparagines. The invention includes SEQ ID NO: 6040, wherein the fragment comprises one or more sequences selected from SEQ ID NOs: 7255 and SEQ ID NO: 7256. Preferably, the fragment comprises the amino acid sequence NKTG (SEQ ID NO: 7255).

The invention includes a polypeptide comprising SEQ ID NO: 6040, wherein the fragment does not comprise one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

Table 14 identifies SEQ ID NOs: t-epitope of 6040. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group consisting of the nucleic acids identified as SEQ ID NOS: 7640 amino acid sequence of T-epitope sequence of 7800; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising two or more of the nucleic acids identified as SEQ ID NOS: 7640 7800, or a polynucleotide encoding such a polypeptide.

The following antigenic uses are preferred: (1) as a T-cell antigen; (2) for generating a complex between an MHC class I protein (e.g., HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) antigens that elicit a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus.

The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal (particularly a human) which method comprises administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6041. SEQ ID NO: 6041 has been shown to be functionally homologous to a portion of the ORF1ab polyprotein. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 6040 and a polypeptide having an amino acid sequence having sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 6041. The invention includes a method of encoding a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 6041 and a polypeptide having an amino acid sequence having sequence identity. The invention includes encoding a polypeptide comprising SEQ ID NO: 6041 polypeptide fragments.

The invention includes a polypeptide comprising SEQ ID NO: 6041 or a fragment thereof. The invention includes a polypeptide comprising a nucleotide sequence encoding SEQ ID NO: 6041 or a fragment thereof. The invention encompasses immunogenic compositions comprising SEQ ID NO: 6041 polypeptide or a fragment thereof. The invention includes the ability to recognize nucleic acids comprising SEQ id no: 6041 or a fragment thereof.

The polyprotein of coronaviruses is associated with enzymatic activity. Thus, SEQ ID NO: 6041 is the target for screening chemical inhibitor of SARS virus. The invention includes a polypeptide comprising SEQ ID NO: 6041 or a fragment thereof. The invention includes encoding SEQ ID NO: 6041 polypeptide sequence or fragment thereof. The invention includes screening for SEQ ID NO: 6041 inhibitor. The invention includes recombinant expression of SEQ ID NO: 6041. the invention includes methods for preventing the expression of SEQ ID NO: 6041 small molecules where the polypeptide exerts a coactivation activity. The invention includes a polypeptide comprising SEQ ID NO: 6041.

The following SEQ ID NOs: 6041 predicted transmembrane or hydrophobic regions. Although the polyprotein of coronaviruses is proteolytically cleaved into many smaller proteins, hydrophobic domains within the polyprotein are known to mediate membrane-to-replication complex binding and to significantly alter the structure of the host cell membrane. Thus, the hydrophobic domain of the polyprotein is the target for genetic mutation to generate an attenuated SARS virus vaccine. The hydrophobic structure domain is also the target of SARS virus small molecule inhibitor. The hydrophobic domains can also be used to generate antibodies that specifically bind to those regions to treat or prevent SARS virus infection.

SEQ ID NO: 6041 possible transmembrane helices

Sequence positions in parentheses indicate the core region.

Only if the score exceeds 500 is considered meaningful.

Inward-outward-screwing: found 18

Beginning with the end of the scoring center

234(234)254(250) 1046 241

256(256)272(270) 252 263

319(319)334(334) 227 327

503(505)522(519) 405 512

613(615)633(629) 619 622

677(679)703(696) 467 689

849(851)869(865) 229 858

1080(1080)1097(1094) 306 1087

1147(1149)1163(1163) 354 1156

1557(1557)1581(1577) 817 1567

1954(1954)1971(1971) 832 1964

2369(2372)2395(2387) 300 2379

2513(2513)2532(2529) 690 2522

Outward internal screw: find 14

Beginning with the end of the scoring center

239(239)254(254) 924 247

239(248)272(263) 468 256

311(314)334(328) 267 321

499(503)522(519) 485 512

617(617)634(631) 425 624

849(853)872(872) 572 864

1147(1147)1162(1162) 765 1155

1564(1564)1581(1579) 883 1572

1951(1951)1968(1966) 657 1958

2513(2522)2539(2537) 711 2529

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6041, wherein the fragment comprises an amino acid sequence comprising one or more of the hydrophobic transmembrane sequences identified above. The invention includes a polypeptide comprising SEQ ID NO: 6041, wherein the fragment comprises the polypeptide of SEQ ID NO: 6041 one or more of the following polypeptide sequences: 234-. Preferably, the fragment comprises SEQ ID NO: 6041 one or more of the following polypeptide sequences: 234-. The invention also includes polynucleotides encoding each of the polypeptide fragments identified above.

The present invention includes an attenuated SARS virus, wherein the attenuated SARS virus has an addition, deletion or substitution in a polynucleotide encoding one of the hydrophobic domains identified above. The invention also includes a method of making an attenuated SARS virus, the method comprising mutating the SARS virus by addition, deletion or substitution to the genome of the SARS virus, thereby altering the sequence of the above identified SEQ ID NO: 6041 encoding one or more hydrophobic domains.

The invention includes methods of specifically recognizing SEQ ID NO: 6041 antibodies to one or more hydrophobic domains. The invention includes binding to the SEQ ID NO: 6041 one or more hydrophobic domains, small molecules that interfere with their hydrophobicity or disrupt them.

Predicted SEQ ID NO: the N-glycosylation site of 6041 is identified in the table below.

Location likelihood agreement NGlyc outcome

571NLSH(SEQ ID NO：7257) 0.6598 (8/9) +

835NTSR(SEQ ID NO：7258) 0.5762 (7/9) +

958NVTD(SEQ ID NO：7259) 0.7494 (9/9) ++

1113NISD(SEQ ID NO：7260) 0.7259 (8/9) +

1205NSTL(SEQ ID NO：7261) 0.6296 (9/9) ++

1460NVTG(SEQ ID NO：7262) 0.6844 (9/9) ++

1685NHSV(SEQ ID NO：7263) 0.5181 (5/9) +

2029NKTT(SEQ ID NO：7264) 0.5423 (5/9) +

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6041, wherein said fragment contains one or more of the N-glycosylation sites identified above. The invention includes a polypeptide comprising SEQ ID NO: 6041, wherein the fragment comprises SEQ ID NOS: 7257-. Preferably the fragment comprises one or more of the following sequences: SEQ ID NOS: 7257. 7259, 7260, 7261 and 7262. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising SEQ ID NO: 6041, wherein the fragment does not contain one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

SEQ ID NO: t-epitope of 6041. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 7801-8040; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising the nucleic acid sequences identified as SEQ ID NOS: 7801 and 8040, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus.

The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes polypeptide sequence SEQ ID NO: 6043 or a fragment thereof. The invention includes a polypeptide comprising a nucleotide sequence substantially identical to SEQ id no: 6043 and an amino acid sequence having sequence identity. The invention includes encoding SEQ ID NO: 6043, or a fragment thereof.

Predicted SEQ ID NO: the transmembrane region of 6043 is as follows.

Inward-outward-screwing: find 4

From to the scoring center

41(41) 56(56) 1789 49

76(79) 99(99) 2142 89

105(105) 125(125) 1250 115

Outward internal screw: find 3

From to the scoring center

41(41) 59(56) 2053 49

76(82) 98(96) 1580 89

103(105) 125(123) 1257 115

The region of amino acids containing the most predicted transmembrane helical region is represented by SEQ ID NO: 6043 amino acid 27 to 99. Such transmembrane regions are often difficult to express recombinantly. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6043, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include amino acids between positions 27-48. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6043 is a hypothetical SARS virus protein. For SEQ ID NO: 6043 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6043 located in one of the following locations: the inner mitochondrial membrane, the cytoplasmic membrane, the golgi apparatus and the inner mitochondrial membrane space. The prediction of SEQ ID NO: 6043 bind to organelles within the infected cell.

Thus, SEQ ID NO: 6043 it is the target for screening the SARS virus chemical inhibitor. The invention includes a polypeptide comprising SEQ ID NO: 6043 or a fragment thereof. The invention includes encoding SEQ ID NO: 6043 or a fragment thereof. The invention includes screening for SEQ ID NO: 6043 inhibitor. The invention includes recombinant expression of SEQ ID NO: 6043. the invention includes methods for preventing the expression of SEQ ID NO: 6043 and the organelles in the infected cell. The invention includes a polypeptide comprising SEQ ID NO: 6043.

PSORT- -protein localization site prediction, SEQ ID NO: 6043

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 3

Most N-terminal position TMS: 40, i-2

MTOP: membrane surface structure (Hartmann et al)

I (middle): 47 charge differential (C-N): 3.5

McG: detection Signal peptide sequence (McGeoch)

UR length: 12

UR peak value: 1.41

CR net charge: 0

Discrimination scoring: -4.67

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): 3.44

Possible cleavage sites: 15

> > there appears to be no N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 2-domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 0B

ALOM: transmembrane region was found (Klein et al)

Counting: 2 value: -6.90 threshold: -2.0

Internal possibility-6.90 transmembrane 83-99(78-101)

Internal possibility-5.04 transmembrane 40-56(37-60)

Peripheral probability-0.32

Modified ALOM score: 1.48

Probably type IIIb membrane protein (Nexo Ccyt)

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 128

MRCWLC

Identification of targeted mitochondrial sequences:

unclear (0.76)

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

Inference step: 2

Type IIIa or IIIb is endoplasmic reticulum membrane protein

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 128) is selected from: 39 to: rating 56: 11.5

> > appears to contain an intramitochondrial signal peptide

Inner mitochondrial membrane? And (3) scoring: 0.59

Mitochondrial inner membrane void? And (3) scoring: 0.22

SKL motif (peroxisome signal peptide):

position: 92(274), count: 1SHL

SKL score (peroxisome): 0.3

Peroxisome-prone amino acid composition: 4.78

Peroxisome proteins? The state is as follows: positive for

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 1.16 State: it is unclear

Type III proteins may be localized to the Golgi apparatus

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

Verification of type III TMS number (plasma Membrane)

N-myristoylation was verified.

-end result-

Inner mitochondrial membrane — certainty 0.664 (affirmative) < success >

Plasma membrane-certainty 0.600 (affirmative) < success >

Golgi-certainty 0.400 (affirmative) < success >

Mitochondrial inner membrane void-certainty 0.362 (affirmative) < success >

Predicted SEQ ID NO: the N-and O-glycosylation sites of 6043 were identified as follows.

Location likelihood agreement NGlyc outcome

227NATF(SEQ ID NO：7265) 0.6328 (7/9) +

Threshold opinion for residue numbering probability

Thr 28 0.9095 0.6280T

Thr 32 0.8740 0.6595 T

Thr 34 0.9058 0.6655 T

Thr 170 0.6816 0.6600 T

Thr 267 0.9240 0.5779 T

Thr 268 0.7313 0.5708 T

Thr 269 0.9859 0.5583 T

Thr 270 0.8023 0.5492 T

Ser 27 0.6930 0.6091 S

Ser 252 0.6457 0.5977 S

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6043, wherein said fragment comprises an N-glycosylation site or an O-glycosylation site identified above. The invention includes a polypeptide comprising SEQ ID NO: 6043, wherein the fragment comprises one or more of the N-glycosylation sites or O-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising SEQ ID NO: 6043, wherein the fragment does not contain one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

SEQ ID NO: t-epitope of 6043. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 8281-8486; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 8281-8486, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6044. The invention includes a polypeptide comprising SEQ ID NO: 6044 or a fragment comprising an amino acid sequence substantially identical to SEQ ID NO: 206, or a polypeptide having a sequence with sequence identity. The invention includes encoding SEQ id no: 6044.

SEQ ID NO: 6044 was identified as a hypothetical protein. The predicted SEQ ID NO: hydrophobic or transmembrane region of 6044:

Inward-outward-screwing: find 3

From to the scoring center

1(1) 17(15) 891 8

47(47) 66(63) 221 56

Outward internal screw: find 4

From to the scoring center

1(4) 21(19) 599 11

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6044, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include amino acids between positions 1-19. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6044 is a hypothetical SARS virus protein. For SEQ ID NO: 6044 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6044 is located in one of the following locations: nuclei, mitochondrial matrix, lysosomes (cavities) and microbodies (peroxisomes). The prediction of SEQ ID NO: 6044 binding to organelles in the infected cell.

Thus, SEQ ID NO: 6044 is the target for screening chemical inhibitors of SARS virus. The invention includes a polypeptide comprising SEQ id no: 6044 or a fragment thereof. The invention includes encoding SEQ ID NO: 6044 or a fragment thereof. The invention includes screening for SEQ ID NO: 6044 inhibitor. The invention includes recombinant expression of SEQ ID NO: 6044. the invention includes methods for preventing the expression of SEQ ID NO: 6044 and small molecules that are associated with organelles within the infected cell. The invention includes a polypeptide comprising SEQ ID NO: 6044.

PSORT- -protein localization site prediction, SEQ ID NO: 6044

Version 6.4(WWW)

154 residue

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 0

McG: detection Signal peptide sequence (McGeoch)

UR length: 7

UR peak value: 1.06

CR net charge: 1

Discrimination scoring: -7.97

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): -3.28

Possible cleavage sites: 34

> > there appears to be no N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 0 x domain charge to-2

Is a cleavable signal peptide detected in ALOM? : OB

ALOM: transmembrane region was found (Klein et al)

Counting: value 0: 1.43 threshold value: -2.0

Peripheral probability of 1.43

Modified ALOM score: -1.19

Gavel: detecting boundaries of targeted mitochondrial sequences

Motif position: 151

FRKKQV

Identification of targeted mitochondrial sequences:

unclear (-0.46)

Inference step: 2

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 151) from: 46 to: and (3) scoring: 5.0

Mitochondrial matrix? And (3) scoring: 0.36

SKL motif (peroxisome signal peptide):

position: -1(154), count: 0

Peroxisome-prone amino acid composition: 0.61

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.149

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 0.81 State: it is unclear

Modification scoring of lysosomes: 0.231

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

The following are found: position: 134(3) KHKK

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

The following are found: position: 136(3) KK VSTNLCTHSF RKKQV

Final Robbins score (nuclei): 0.60

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear modification scoring: 0.90

Nuclear signal state: positive electricity (0.70)

The CaaX motif was verified.

N-myristoylation was verified.

The CaaX motif was verified.

-end result-

Core-certainty 0.880 (affirmative) < success >

Mitochondrial matrix gap-certainty 0.360 (affirmative) < success >

Lysosome (luminal) - - -, certainty 0.231 (affirmative) < success >

Microbody (peroxisome) - -, certainty ═ 0.149 (affirmative) < success >

SEQ ID NO: 6044 one predicted O-glycosylation site.

Threshold opinion for residue numbering probability

Thr 4 0.6839 0.6484 T

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6044, wherein said fragment comprises an O-glycosylation site identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising SEQ ID NO: 6044, wherein the fragment does not contain one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

SEQ ID NO: t-epitope of 6044. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 8487 and 8665; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes nucleic acid molecules comprising SEQ ID NOS: 8487-8665 was identified as one or more

A T-epitope sequence, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6045. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 6045 and amino acid sequence with sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 6045. The present invention includes polynucleotide sequences encoding any of these polypeptides.

SEQ ID NO: 6045 was demonstrated to be functionally homologous to envelope or small membrane proteins of coronaviruses. The invention includes a diagnostic kit comprising a nucleic acid sequence comprising SEQ ID NO: 6045 or a fragment thereof. The invention includes a diagnostic kit containing a nucleic acid encoding SEQ ID NO: 6045 or a fragment thereof. The invention includes a polypeptide comprising SEQ id no: 6045 or a fragment thereof. The invention includes methods for specifically recognizing a polypeptide comprising SEQ ID NO: 6045 or a fragment thereof.

The predicted SEQ ID NO: transmembrane region 6045:

inward-outward-screwing: find 1

From to the scoring center

17(19) 33(33) 2881 26

Outward internal screw: find 1

From to the scoring center

17(17) 34(34) 2981 27

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6045, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include amino acids between positions 17-34. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above. In one embodiment, the invention includes a polypeptide comprising SEQ ID NO: 6045, wherein the fragment does not comprise SEQ ID NO: 6045 amino acid residues 1-34.

Predicted SEQ ID NO: the protein localization sites of 6045 are as follows.

PSORT- -protein localization site prediction, SEQ ID NO: 6045

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 2

Most N-terminal position TMS: 17, i ═ 1

MTOP: membrane surface structure (Hartmann et al)

I (middle): 24 charge difference (C-N): 2.0

McG: detection Signal peptide sequence (McGeoch)

UR length: 29

UR peak value: 3.40

CR net charge: -2

Discrimination scoring: 13.07

GvH: detection Signal peptide sequence (yon Heijne)

Signal peptide score (-3.5): 4.37

Possible cleavage sites: 32

… mtop Positive value …

> > there appears to be a non-cleavable N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 1-domain charge to-2

Is a cleavable signal peptide detected in ALOM? : OB

ALOM: transmembrane region was found (Klein et al)

Counting: 1 value: 15.12 threshold values: -2.0

Internal possibility-15.12 transmembrane 17-33(8-44)

Peripheral probability of 0.47

Modified ALOM score: 3.12

> > appear to be type Ib (Nexo Ccty) membrane proteins

Cytoplasmic tails from 34 to 76(44 residues)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

(6) Or not cuttable?

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 6

Is it uncut? Ipos is set as: 16

Identification of targeted mitochondrial sequences:

unclear (0.19)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

Inference step: 2

Relative position of the tail end: 44 percent of

Membrane proteins typically have a non-cleavable signal peptide in the ER

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 16) from: 70 to: 99 scoring: 21.5

> > appears to contain an intramitochondrial signal peptide

SKL motif (peroxisome signal peptide):

position: -1(76), count: 0

Peroxisome-prone amino acid composition: -4.11

Peroxisome proteins? The state is as follows: negative of

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 0.68 state: it is unclear

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

Verification of type Ib cytoplasmic Tail (plasma membrane)

Verification of the NPXY motif.

The YXRF motif was verified.

N-myristoylation was verified.

-end result-

Plasma membrane-certainty 0.730 (affirmative) < success >

Endoplasmic reticulum (membrane) - - -, certainty ═ 0.640 (affirmative) < success >

Endoplasmic reticulum (lumen) - - -, certainty 0.100 (affirmative) < success >

Outer-certainty 0.100 (affirmative) < success >

In SEQ ID NO: predicted N-glycosylation sites were identified at residues 48 and 66 of 6045.

Location likelihood agreement NGlyc outcome

48NVSL 0.6514 (9/9) ++ (SEQ ID NO：7266)

66NSSE 0.5880 (7/9) + (SEQ ID NO：7267)

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6045, wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention includes a polypeptide comprising SEQ ID NO: 6045, wherein the fragment does not contain one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising SEQ ID NO: 6045, wherein the fragment does not comprise one or more of the glycosylation sites identified above. The present invention also includes polynucleotides encoding such polypeptides.

SEQ ID NO: t-epitope of 6045. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 8666 amino acid sequence of the T-epitope sequence of 8820; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 8666-8820, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6046. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 6046 and amino acid sequence with sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 6046. The present invention includes polynucleotides encoding any of these polypeptides.

SEQ ID NO: 6046 it is functionally homologous to matrix proteins of coronaviruses. The invention includes a diagnostic kit comprising a nucleic acid sequence comprising SEQ ID NO: 6046 or a fragment thereof. The invention includes a diagnostic kit containing a nucleic acid encoding SEQ ID NO: 6046 or a fragment thereof. The invention includes a polypeptide comprising SEQ ID NO: 6046 or a fragment thereof. The invention includes methods for specifically recognizing a polypeptide comprising SEQ ID NO: 6046 or a fragment thereof.

The predicted SEQ ID NO: the transmembrane regions of 6046 are as follows:

Inward-outward-screwing: find 3

From to the scoring center

21(21) 38(36) 2412 29

51(53) 69(69) 2645 60

74(82) 96(96) 2464 89

Outward internal screw: find 3

From to the scoring center

18(21) 38(38) 2363 28

52(52) 67(67) 2363 60

76(76) 95(92) 2605 84

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6046, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. Preferably the fragment does not comprise an amino acid selected between the following positions: 18-38, 52-67 and 76-95. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicted SEQ ID NO: 6046 the protein is localized as follows.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 3

Most N-terminal position TMS: 21, i ═ 1

MTOP: membrane surface structure (Hartmann et al)

I (middle): 28 charge difference (C-N): 6.0

McG: detection Signal peptide sequence (McGeoch)

UR length: 1

UR peak value: 3.16

CR net charge: -3

Discrimination scoring: 2.21

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): 4.29

Possible cleavage sites: 39

… mtop Positive value …

> > there appears to be a non-cleavable N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

Is a cleavable signal peptide detected in ALOM? : OB

ALOM: transmembrane region was found (Klein et al)

Counting: 3 value: -7.64 threshold: 0.5

Internal possibility-7.64 transmembrane 21-37(18-39)

Internal possibility-7.59 transmembrane 50-66(43-72)

Internal possibility-5.04 transmembrane 79-95(72-99)

Peripheral probability of 2.38

Modified ALOM score: 2.13

Probably type IIIb membrane protein (Nexo Ccyt)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

(2) Or not cuttable?

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 2

Is it uncut? Ipos is set as: 12

Identification of targeted mitochondrial sequences:

negatives (-4.16)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

Inference step: 2

Type IIIa or IIIb is endoplasmic reticulum membrane protein

Membrane proteins typically have a non-cleavable signal peptide in the ER

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

SKL motif (peroxisome signal peptide):

position: -1(221), count: 0

Peroxisome-prone amino acid composition: 5.01

Peroxisome proteins? The state is as follows: it is unclear

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 2.30 State: positive for

Type III proteins may be localized to the Golgi apparatus

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

Verification of type III TMS number (plasma Membrane)

N-myristoylation was verified.

-end result-end

Endoplasmic reticulum (membrane) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [ 0.685 (affirmative) <

Plasma membrane-certainty 0.640 (affirmative) < success >

Golgi-certainty 0.460 (affirmative) < success >

Endoplasmic reticulum (lumen) - - -, certainty 0.100 (affirmative) < success >

In SEQ ID NO: 6046 a predicted N-glycosylation site was identified at residue 4:

prediction of N-glycosylation sites

Location likelihood agreement NGlyc outcome

4NGTI 0.8430 (9/9) +++ (SEQ ID NO：7268)

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6046, wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention also includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6046, wherein the fragment does not contain one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

SEQ ID NO: 6046 is a variant of SEQ ID NO: 9963. and SEQ ID NO: 6046 the sequence has Val instead of Ala at residue 72.

SEQ ID NO: t-epitope of 6046. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 8821-9018; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 8821-9018, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6047 or a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted SEQ ID NO: the transmembrane region of 6047 was identified as follows.

Inward-outward-screwing: find 2

From to the scoring center

7(10) 29(27) 729 17

21(24) 41(41) 640 34

Outward internal screw: find 2

From to the scoring center

4(4) 22(19) 874 2

22(24) 41(41) 499 31

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6047, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. Preferably the fragment does not comprise an amino acid selected between the following positions: 4-22 and 22-41. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6047 is a hypothetical SARS virus protein. For SEQ ID NO: 6047 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6047 located in one of the following locations: plasma membrane, endoplasmic reticulum, golgi apparatus and microbodies (peroxisomes). The prediction of SEQ ID NO: 6047 binding to organelles in the infected cell or associated with the entry of the virus into the host cell.

Thus, SEQ ID NO: 6047 is the target for screening chemical inhibitors of SARS virus. The invention includes a polypeptide comprising SEQ id no: 6047 or a fragment thereof. The invention includes encoding SEQ ID NO: 6047 or a fragment thereof. The invention includes screening for SEQ ID NO: 6047 inhibitor. The invention includes recombinant expression of SEQ ID NO: 6047. the invention includes methods for preventing the expression of SEQ ID NO: 6047 and small molecules that bind to organelles within the infected cell or interact with the host cell membrane. The invention includes a polypeptide comprising SEQ id no: 6047. Predicted SEQ ID NO: 6047 the protein was located as follows.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 1

Most N-terminal position TMS: 2, i ═ 1

MTOP: membrane surface structure (Hartmann et al)

I (middle): 9 charge difference (C-N): 0.5

McG: detection Signal peptide sequence (McGeoch)

UR length: 6

UR peak value: 3.08

CR net charge: 0

Discrimination scoring: 5.12

GvH: detection Signal peptide sequence (von Hei jne)

Signal peptide score (-3.5): -4.45

Possible cleavage sites: 34

> > there appears to be a non-cleavable N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 1-domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 0B

ALOM: transmembrane region was found (Klein et al)

Counting: 1 value: 2.44 threshold value: -2.0

Internal possibility-2.44 transmembrane 2-18(1-20)

Peripheral probability of 1.22

Modified ALOM score: 0.59

> > appear to be type II (Ncty Cexo) membrane proteins

Cytoplasmic tail from 1 to 1(1 residue)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

(5) Or not cuttable?

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 5

Is it uncut? Ipos is set as: 15

Identification of targeted mitochondrial sequences:

unclear (1.48)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

Inference step: 2

Relative position of cytoplasmic tail: 1 percent of

ER membrane proteins are preferably of large value (> 30%)

Membrane proteins typically have a non-cleavable signal peptide in the ER

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 15) from: 64 to: 93 scoring: 30.0

> > appears to contain an intramitochondrial signal peptide

SKL motif (peroxisome signal peptide):

position: -1(63), count: 0

Peroxisome-prone amino acid composition: 1.91

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.161

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 0.04 state: it is unclear

Verification of Golgi consensus sequences

Verification of Golgi consensus sequences

Verification of cytoplasmic tails of type II (Golgi)

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

Verification of mitochondrial Signal peptide (plasma Membrane) of type II

Type II is plasma membrane protein preferred

Verification of the NPXY motif.

The YXRF motif was verified.

N-myristoylation was verified.

-end result-

Plasma membrane-certainty 0.685 (affirmative) < success >

Endoplasmic reticulum (membrane) - - -, certainty ═ 0.640 (affirmative) < success >

Golgi-certainty 0.370 (affirmative) < success >

Microbody (peroxisome) - -, certainty ═ 0.161 (affirmative) < success >

SEQ ID NO: t-epitope of 6047. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 9019-9131; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 9019-9131, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6048 or a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted SEQ ID NO: the transmembrane region of 6048 was identified as follows.

Inward-outward-screwing: find 2

From to the scoring center

3(3) 18(18) 1857 10

100(100) 117(115) 2904 107

Outward internal screw: find 2

From to the scoring center

1(1) 15(15) 1299 8

100(100) 117(115) 3009 107

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6048, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. Preferably the fragment does not comprise an amino acid at a position selected from: 1-15 and 100-. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6048 is a hypothetical SARS virus protein. For SEQ ID NO: 6048 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6048 is located in one of the following locations: cytoplasmic membranes, lysosomes (membranes), microbodies (peroxisomes), and endoplasmic reticulum (membranes). SEQ ID NO: 6048 it may be associated with organelles within the infected cell or interact with the host cell cytoplasmic membrane when the virus enters the host cell.

Thus, SEQ ID NO: 6048 it is the target for screening the chemical inhibitor of SARS virus. The invention includes a polypeptide comprising SEQ ID NO: 6048 or a fragment thereof. The invention includes encoding SEQ ID NO: 6048 or a fragment thereof. The invention includes screening for SEQ ID NO: 6048. The invention includes recombinant expression of SEQ ID NO: 6048. the invention includes methods for preventing the expression of SEQ ID NO: 6048 small molecules that bind to organelles within the infected cell or interact with the host cell membrane. The invention includes a polypeptide comprising SEQ id no: 6048. Predicted SEQ ID NO: 6048 the protein is located as follows.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 2

Most N-terminal position TMS: 3, i-2

MTOP: membrane surface structure (Hartmann et al)

I (middle): 10 charge difference (C-N): -2.5

McG: detection Signal peptide sequence (McGeoch)

UR length: 13

UR peak value: 3.38

CR net charge: 1

Discrimination scoring: 10.02

GvH: detection Signal peptide sequence (yon Heijne)

Signal peptide score (-3.5): 2.56

Possible cleavage sites: 15

> > appear to contain a cleavable N-terminal signal sequence

Predicted amino acid composition of the mature form:

slave bit calculation 16

ALOM new count: 2-domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 1B

ALOM: transmembrane region was found (Klein et al)

Counting: 1 value: -14.75 threshold: -2.0

Internal probability-14.75 transmembrane 101-

Peripheral probability of 6.63

Modified ALOM score: 3.05

> > appear to contain type Ia membrane proteins

Cytoplasmic tails 118 to 122(5 residues)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

(15) Or not cuttable?

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 15

Is it uncut? Ipos is set as: 25

Identification of targeted mitochondrial sequences:

unclear (0.73)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

Inference step: 2

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 25) from: 3 to: 12, grading: 8.5

SKL motif (peroxisome signal peptide):

position: -1(122), count: 0

Peroxisome-prone amino acid composition: 2.46

AAC, modified Scoring not counted from N-terminal

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.115

Preferential amino acid composition of lysosomal proteins

And (3) scoring: -0.40 state: negative of

The GY motif in the type Ia tail? (lysosome)

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

Type Ia plasma membrane proteins

Verification of the NPXY motif.

The YXRF motif was verified.

N-myristoylation was verified.

The GPI anchor was verified.

> > seems to be the GPI-anchor (0.85)

-end result-

Plasma membrane-certainty 0.919 (affirmative) < success >

Lysosome (membrane) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - [ 0.200 (affirmative) < success >

Microbody (peroxisome) - - -, certainty ═ 0.115 (affirmative) < success >

Endoplasmic reticulum (membrane) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -certainty of 0.100 (affirmative) < success >

SEQ ID NO: t-epitope of 6048. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 9132-9308 amino acid sequence; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 9132-9308, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6049 or a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted SEQ ID NO: the transmembrane or hydrophobic region of 6049 was identified as follows.

Inward-outward-screwing: find 1

From to the scoring center

13(13) 30(28) 3532 20

Outward internal screw: find 1

From to the scoring center

9(11) 29(26) 3395 19

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6049, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6049 is a hypothetical SARS virus protein. For SEQ ID NO: 6049 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6049 located in one of the following locations: lateral, microbody (peroxisome), endoplasmic reticulum (membrane) and endoplasmic reticulum (lumen). The highest localization level indicates SEQ ID NO: 6049 it is located outside the cell. Thus, SEQ ID NO: 6049 may be surface exposed proteins.

Thus, SEQ ID NO: 6049 can be used in immunogenic compositions for eliciting an immune response against SARS virus. It can also be used to generate antibodies specific for SARS virus. The antibodies are useful in methods for treating or preventing SARS virus infection. Such antibodies can also be used in diagnostic assays to identify the presence of SARS virus in a biological sample.

The invention includes a polypeptide comprising SEQ ID NO: 6049 or a fragment thereof. The invention includes encoding SEQ id no: 6049 or a fragment thereof. The invention includes screening for SEQ ID NO: 6049. The invention includes recombinant expression of SEQ ID NO: 6049. the invention includes a polypeptide comprising SEQ ID NO: 6049. Predicted SEQ ID NO: 6049 the protein was localized as follows.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 1

Most N-terminal position TMS: 11, i ═ 1

MTOP: membrane surface structure (Hartmann et al)

I (middle): 18 charge difference (C-N): -2.0

McG: detection Signal peptide sequence (McGeoch)

UR length: 24

UR peak value: 3.69

CR net charge: -2

Discrimination scoring: 13.56

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): 0.52

Possible cleavage sites: 25

> > appear to contain a cleavable N-terminal signal sequence

Predicted amino acid composition of the mature form:

slave bit calculation 26

ALOM new count: 1-domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 1B

ALOM: transmembrane region was found (Klein et al)

Counting: value 0: 14.80 threshold value: -2.0

Peripheral probability of 14.80

Modified ALOM score: -3.86

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

(2) Or not cuttable?

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 2

Is it uncut? Ipos is set as: 12

Identification of targeted mitochondrial sequences:

unclear (1.42)

The function is as follows: vesicle passage

The function is as follows: vesicle passage

The function is as follows: vesicle passage

Inference step: 2

KDEL counting: 0

Number of potential N-glycosylation sites: 0

And (3) outside: score 0.800

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 12) from: 44 to: 73, scoring: 30.0

> > appears to contain an intramitochondrial signal peptide

SKL motif (peroxisome signal peptide):

position: -1(44), count: 0

Peroxisome-prone amino acid composition: 9.47

AAC, modified Scoring not counted from N-terminal

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.320

Preferential amino acid composition of lysosomal proteins

And (3) scoring: -6.47 state: negative of

NX (S/T) number of motifs: 0

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

The CaaX motif was verified.

N-myristoylation was verified.

The CaaX motif was verified.

-end result-

Outer-certainty 0.820 (affirmative) < success >

Microbody (peroxisome) - - -, certainty ═ 0.320 (affirmative) < success >

Endoplasmic reticulum (membrane) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -certainty of 0.100 (affirmative) < success >

Endoplasmic reticulum (lumen) - - -, certainty 0.100 (affirmative) < success >

SEQ ID NO: t-epitope of 6049. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 9309-9437; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 9309-9437, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6050 or a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted SEQ ID NO: the transmembrane or hydrophobic region of 6050 was identified as follows.

Inward-outward-screwing: find 1

From to the scoring center

13 (15)32(30) 558 23

Outward internal screw: find 1

From to the scoring center

16(16) 30(30) 364 23

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6050, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicting the expression of SEQ ID NO: 6050 is a hypothetical SARS virus protein. For SEQ ID NO: 6050 prediction of protein localization is as follows. Predicting the expression of SEQ ID NO: 6050 it is located in one of the following places: lysosomes (lumen), mitochondrial matrix space, inner mitochondrial membrane and intermitochondrial membrane space. SEQ ID NO: 6050 may bind to organelles within infected cells during the viral replication cycle.

Thus, SEQ ID NO: 6050 is the target for screening chemical inhibitors of SARS virus. The invention includes a polypeptide comprising SEQ id no: 6050 or a fragment thereof. The invention includes encoding SEQ ID NO: 6050 or a fragment thereof. The invention includes screening for SEQ ID NO: 6050. The invention includes recombinant expression of SEQ ID NO: 6050. the invention includes methods for preventing the expression of SEQ ID NO: 6050 small molecules that bind to organelles within the infected cell or interact with the host cell membrane. The invention includes a polypeptide comprising SEQ ID NO: 6050. Predicted SEQ ID NO: 6050 the protein was localized as follows.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

MYSEQ 84 residue

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 0

McG: detection Signal peptide sequence (McGeoch)

UR length: 3

UR peak value: 1.46

CR net charge: 2

Discrimination scoring: -5.73

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): -0.12

Possible cleavage sites: 29

> > there appears to be no N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 0 x domain charge to-2

Is a cleavable signal peptide detected in ALOM? : 0B

ALOM: transmembrane region was found (Klein et al)

Counting: value 0: 8.43 threshold value: -2.0

Peripheral probability of 8.43

Modified ALOM score: -2.59

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 61

ARCWYL

Identification of targeted mitochondrial sequences:

positive electricity (1.66)

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

The function is as follows: mitochondrial proteins

Inference step: 2

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

(Gavel position 61) from: 52 to: score 58: 6.0

Mitochondrial matrix? And (3) scoring: 0.38

SKL motif (peroxisome signal peptide):

position: -1(84), count: 0

Peroxisome-prone amino acid composition: 1.47

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.263

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 2.86 State: positive for

Modification scoring of lysosomes: 0.850

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear signal state: negatives (0.00)

The CaaX motif was verified.

N-myristoylation was verified.

The CaaX motif was verified.

-end result-

Lysosome (luminal) -certainty 0.850 (affirmative) < success >

Mitochondrial matrix gap-certainty 0.544 (affirmative) < success >

Inner mitochondrial membrane-certainty 0.266 (affirmative) < success >

Mitochondrial inner membrane void-certainty 0.266 (affirmative) < success >

SEQ ID NO: 6050 one predicted N-glycosylation site:

Location likelihood agreement NGlyc outcome

43NVTI 0.6713 (9/9) ++ (SEQ ID NO：7269)

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6050, wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention also includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6050, wherein said fragment does not contain one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding such fragments.

SEQ ID NO: t-epitope of 6050. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 9438-9538; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 9438-9538, or a polynucleotide encoding such a polypeptide.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6051 or a fragment thereof or an amino acid sequence having sequence identity thereto. The invention includes a polypeptide comprising SEQ ID NO: 6052 or a fragment thereof or an amino acid sequence having sequence identity thereto.

SEQ ID NO: 6051 and SEQ ID NO: 6052 was demonstrated to be functionally homologous to the nucleocapsid protein of coronaviruses. The invention includes a diagnostic kit comprising a nucleic acid sequence comprising SEQ ID NO: 6051. SEQ ID NO: 6052 or a fragment thereof. The invention includes a diagnostic kit containing a nucleic acid encoding SEQ ID NO: 6051. SEQ ID NO: 6052 or a fragment thereof. The invention includes a polypeptide comprising SEQ ID NO: 6051. SEQ ID NO: 6052 or a fragment thereof. The invention includes a polypeptide capable of recognizing a polypeptide comprising SEQ ID NO: 6051. SEQ ID NO: 6052 or a fragment thereof.

Predicting the expression of SEQ ID NO: 6051 at Ser-79; thr-92; ser-106; thr-116; thr-142; ser-184; ser-188; ser-202; ser-236; thr-248; ser-251; ser-256; is phosphorylated on Thr-377. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6051, wherein the fragment comprises the polypeptide of SEQ id no: 6051 one or more of the following amino acid residues: ser-79; thr-92; ser-106; thr-116; thr-142; ser-184; ser-188; ser-202; ser-236; thr-248; ser-251; ser-256; thr-377. The invention also includes a polypeptide comprising SEQ ID NO: 6051, wherein the fragment does not comprise SEQ id no: 6051 one or more of the following amino acid residues: ser-79; thr-92; ser-106; thr-116; thr-142; ser-184; ser-188; ser-202; ser-236; thr-248; ser-251; ser-256; thr-377. Two other relevant fragments of the N protein (e.g. for immunoprecipitation) are SEQ ID NOS: 9783&9784, which are rich in lysine and can be used to distinguish SARS virus from other coronaviruses.

Predicted SEQ ID NO: the transmembrane region of 6051 was identified as follows.

Inward-outward-screwing: find 1

From to the scoring center

304(304) 323(319) 495 312

Outward internal screw: find 1

From to the scoring center

304(304) 319(319) 597 312

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6051, wherein the fragment does not comprise one or more of the hydrophobic amino acid sequences identified above. The invention also includes polynucleotide sequences encoding any of the polypeptides identified above.

Predicted SEQ ID NO: the protein localization of 6051 is shown below. The prediction of SEQ ID NO: 6051 is located near the nucleus, lysosomes (lumen), mitochondrial matrix space and microbodies (peroxisomes). The highest localization level is located near the nucleus. The nucleocapsid protein of coronaviruses is known to bind to viral RNA. The coronavirus nucleocapsid protein is also believed to be important for cell-mediated immunity. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6051. The invention also includes viral vectors or particles suitable for in vivo delivery of a polynucleotide sequence comprising a SARS virus nucleocapsid polynucleotide sequence or a fragment thereof. In one embodiment, the polynucleotide comprises SEQ ID N0: 6051 or a fragment thereof. The invention also includes a method of eliciting a cell-mediated immune response comprising delivering to a mammal a polynucleotide encoding a SARS virus nucleocapsid protein or a fragment thereof.

The invention includes screening for SEQ ID NO: 6051. The invention includes recombinant expression of SEQ ID NO: 6051. the invention includes the nucleic acid sequences of SEQ ID NO: 6051 Small molecules that bind to SARS virus RNA. The invention includes a polypeptide comprising SEQ ID NO: 6051. Predicted SEQ id no: 6051 the protein was localized as follows.

PSORT- -prediction of protein localization sites

Version 6.4(WWW)

Species classification: 4

Inference step: 1

Initial calculation ALOM (threshold: 0.5)

Counting: 0

McG: detection Signal peptide sequence (McGeoch)

UR length: 3

UR peak value: 0.19

CR net charge: 0

Discrimination scoring: -15.98

GvH: detection Signal peptide sequence (von Heijne)

Signal peptide score (-3.5): -6.36

Possible cleavage sites: 58

> > there appears to be no N-terminal signal sequence

Predicted amino acid composition of the mature form:

calculating 1 from the bit

ALOM new count: 0 x domain charge to-2

Is a cleavable signal peptide detected in ALOM? : OB

ALOM: transmembrane region was found (Klein et al)

Counting: value 0: 5.04 threshold value: -2.0

Peripheral probability of 5.04

Modified ALOM score: -1.91

Gavel: detecting boundaries of targeted mitochondrial sequences

The motifs are located: 17

PRITFG

Identification of targeted mitochondrial sequences:

negatives (-3.97)

Inference step: 2

KDEL counting: 0

Non-polar signal peptides for checking sorting in mitochondria

Mitochondrial matrix? And (3) scoring: 0.10

SKL motif (peroxisome signal peptide):

position: -1(399), count: 0

Peroxisome-prone amino acid composition: 0.04

Peroxisome proteins? The state is as follows: it is unclear

AAC score (peroxisome): 0.072

Preferential amino acid composition of lysosomal proteins

And (3) scoring: 0.96 state: it is unclear

Modification scoring of lysosomes: 0.246

Verification of the amount of basic residues (nucleus)

Verification of a 4-residue model of a targeted nucleus

The following are found: position: 256(4) KKPR

The following are found: position: 372(5) KKKKKKK

Verification of nuclear-targeted 7-residue patterns

Verification of Robbins & Dingwall consensus sequence (core)

The following are found: position: 372(3) KK KKTDEAQPLP QRQKK

The following are found: position: 373(3) KK KTDEAQPLPQ RQKKQ

Final Robbins score (nuclei): 0.80

Verification of RNA binding motifs (Nuclear or cytoplasmic)

Nuclear modification scoring: 0.90

Nuclear signal state: positive (0.90)

The CaaX motif was verified.

N-myristoylation was verified.

The CaaX motif was verified.

-end result-

Kernel-certainty 0.980 (affirmative) < success >

Lysosome (lumen) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - (affirmative) < success

Mitochondrial matrix gap-certainty 0.100 (affirmative) < success >

Microbody (peroxisome) - -, certainty ═ 0.072 (affirmative) < success >

Predicted SEQ ID NO: the N-glycosylation site of 6051 was identified as follows.

Location likelihood agreement NGlyc outcome

48NNTA 0.6879 (9/9) ++ (SEQ ID NO：7270)

270NVTQ 0.7684 (9/9) +++ (SEQ ID NO：7271)

Threshold opinion for residue numbering probability

Thr 166 0.8547 0.6439 T

Thr 367 0.5575 0.5403 T

Thr 394 0.8217 0.5821 T

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6051, wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding one or more of the polypeptides identified above.

The invention also includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6051, wherein the fragment does not contain one or more of the N-glycosylation sites identified above. The invention also includes polynucleotides encoding such fragments.

SEQ ID NO: t-epitope of 6052. The present invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) selected from the group identified as SEQ ID NOS: 9539 and 9752; (b) an amino acid sequence having sequence identity to the amino acid sequence of (a). The present invention also includes a polynucleotide sequence encoding the polypeptide of (a) or (b). The invention also includes methods of expressing or delivering such polynucleotides by viral vectors and/or viral particles. The invention also includes compositions comprising one or more of the nucleic acids identified as SEQ ID NOS: 9539-9752, or a polynucleotide encoding such a polypeptide.

SEQ ID NO: one variant of 6052 is SEQ ID NO: 9964. and SEQ ID NO: 6052 comparison, Ile replaces Thr at residue 54 of the sequence.

Antigens are preferably used for: (1) as a T-cell antigen; (2) forming a complex between an MHC class I protein (e.g. HLA class I) and the antigen fragment; (3) as an antigen that elicits a cell-mediated immune response; and/or (4) as an antigen that elicits a CTL response. The use is preferably for the prevention or treatment of a disease and/or infection caused by SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal, particularly a human, against infection by the SARS virus, wherein the polypeptide is as defined above.

The present invention provides a method of eliciting an immune response in a mammal, particularly a human, comprising the step of administering to said mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response, and preferably a CTL response. The immune response is preferably protective or therapeutic.

The present invention includes compositions comprising a SARS virus nucleocapsid protein or fragment thereof and further comprising a SARS virus membrane protein or fragment thereof. The composition may also include one or more of the adjuvants described below.

The invention also includes a composition comprising a polypeptide comprising SEQ ID NO: 6051 or a fragment thereof or a sequence having sequence identity thereto, and further comprising a polypeptide comprising SEQ ID NO: 6040 or a fragment thereof or a polypeptide having a sequence identity thereto. Such compositions are useful, for example, as vaccines. Such compositions may also include one or more of the adjuvants described below.

The present invention includes compositions comprising a SARS virus nucleocapsid protein or fragment thereof and a SARS virus spike protein or fragment thereof. In one embodiment, the nucleocapsid protein comprises a polypeptide comprising SEQ ID NO: 6051 or a fragment thereof or a sequence having sequence identity thereto. In one embodiment, the spike protein comprises a polypeptide comprising SEQ id no: 6042 or a fragment thereof or a sequence having sequence identity thereto. The composition may also include one or more of the adjuvants described below.

The invention also includes compositions comprising antibodies specific for the nucleocapsid protein of the SARS virus and antibodies specific for the spike protein of the SARS virus. In one embodiment, the nucleocapsid protein-specific antibody comprises a heavy chain variable region comprising SEQ id no: 6051 or a fragment thereof or a sequence having sequence identity thereto. In one embodiment, the spike protein specific antibody comprises a heavy chain variable region comprising SEQ ID NO: 6042 or a fragment thereof or a sequence having sequence identity thereto.

The present invention also includes the conserved polynucleotide sequence of SARS virus in coronavirus and its fragment and the polypeptide encoded by it. Such conserved sequences are identified in the alignment shown in FIG. 7. Such conserved sequences may be used in the vaccines of the present invention or in the diagnostic agents, kits and methods of the present invention.

The invention also includes SARS virus specific polynucleotide sequences and fragments thereof not shared by coronaviruses. This SARS-specific sequence was identified as SEQ ID NOS: 6040. 6043, 6044, 6047, 6048, 6049 and 6050. Such SARS-specific sequences can be used in the vaccines of the present invention or in the diagnostic agents, kits and methods of the present invention.

The invention also includes polynucleotide sequences useful as probes or primers in diagnostic agents, kits (including such agents), and methods for diagnosing or identifying the presence of SARS virus in a biological sample. The polynucleotide sequence included in the invention comprises SEQ ID NOS: 6076-6265 was identified as one or more primer sequences (Table 5). The invention also includes nucleic acid molecules comprising SEQ ID NOS: 6076-6265 polynucleotide sequences identified as complements of one or more primer sequences.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes or primers for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The polynucleotide sequence included in the invention comprises SEQ ID NOS: 6266 one or more primer sequences were identified in 6343 (Table 6). The invention also includes nucleic acid molecules comprising SEQ ID NOS: 6266 Polynucleotide sequences identified as complements of one or more primer sequences in-.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes or primers for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The polynucleotide sequence included in the invention comprises SEQ ID NOS: 6344-6392 were identified as one or more primer sequences (Table 7). The invention also includes nucleic acid molecules comprising SEQ ID NOS: 6344-.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes or primers for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The polynucleotide sequence included in the invention comprises SEQ ID NOS: 6393-6559 were identified as one or more primer sequences (tables 8 and 9). The invention also includes nucleic acid molecules comprising SEQ ID NOS: 6393-6559 the polynucleotide sequence identified as the complement of the one or more primer sequences.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes or primers for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The polynucleotide sequence included in the invention comprises SEQ ID NOS: 6560 and 6568 are identified as one or more primer and probe sequences. The invention also includes nucleic acid molecules comprising SEQ ID NOS: 6560-6568 polynucleotide sequences identified as complements of the one or more primer sequences.

The invention includes nucleic acid sequences comprising SEQ ID NOS: 7272-7290 or a fragment thereof or a sequence having sequence identity thereto. The invention also includes nucleic acids encoding SEQ ID NOS: 7272-7290 or a fragment thereof or a sequence polynucleotide sequence having sequence identity thereto. Examples of such polynucleotide sequences are SEQ ID NOS: 7273-the odd numbered sequences in 7291.

The present invention includes polynucleotide sequences comprising intergenic sequences common to the individual open reading frames of the SARS virus. The SARS virus is thought to utilize this sequence as a signal to translate the open reading frame. The intergenic sequence includes 10 mer SEQ ID NO: 7292, or optionally comprises a hexamer of SEQ ID NO: 7293. when a virus transcribes its plus (+) RNA strand into a minus (-) RNA strand, the replicating viral construct uses the minus (-) strand template to transcribe the nucleotide at the 5' end before the first intergenic sequence, then transcribe the intergenic sequence, and then transcribe the selected open reading frame. The virus then produces multiple mrnas containing the 5' end, intergenic sequence, and coding sequence. For more details on replication of nidovirus (including coronavirus) see, e.g., Ziebuhr et al, "virally encoded proteases and proteolytic processes in nidovirus" (viruses-encoded proteases and proteolytic processes in the Nidoviruses), Journal of General Virology 81: 853-879(2000), the contents of which are incorporated herein by reference.

The invention includes a polypeptide comprising SEQ ID NO: 7292 or the complement thereof. The invention includes a polypeptide comprising SEQ ID NO: 7293 or the complement thereof. The invention also includes polynucleotide sequences comprising the 5' terminal nucleotide of the SARS virus genome or the reverse complement thereof, and further comprising intergenic sequences or the reverse complement thereof. The polynucleotide may also comprise one or more SARS virus open reading frames. Examples of polynucleotide sequences comprising nucleotides of the SARS virus genome 5' end followed by intergenic sequences are SEQ ID NOS: 7294-7301. The present invention includes polynucleotide sequences comprising a sequence selected from the group consisting of: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301 or a fragment thereof, or a sequence having sequence identity thereto. In one embodiment, the polynucleotide does not consist entirely of the known SARS virus sequence.

The SARS virus intergenic sequence can be used to generate RNAi molecules. The SARS virus specific RNAi molecule can be used for treating SARS virus infection. The invention includes RNAi molecules comprising a double-stranded RNA molecule, one RNA strand of the double strand comprising a sequence selected from the group consisting of: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301 or a fragment thereof. Preferably, the RNA strand comprises a sequence selected from SEQ id nos: 7292 and SEQ ID NO: 7293. Preferably, the other RNA strand comprises the reverse complement of the first strand or a polynucleotide sequence that hybridizes to the first strand.

In the method of the present invention for treating SARS virus infection by RNAi, an effective amount of an siRNA molecule is administered to a mammal. Preferably the RNAi molecule comprises a molecule as described above. Section IV of this specification further discusses the use of intergenic sequences for RNAi.

The invention also includes the use of an antisense nucleotide sequence of SARS virus, preferably an antisense sequence of an intergenic sequence of SARS virus. Such antisense sequences can be used to treat individuals infected with SARS virus. The antisense sequence of the intergenic sequence of the SARS virus that binds to the SARS virus polynucleotide can be designed to prevent the viral replication machinery from entering the intergenic sequence. Such antisense sequences can also be used to identify the presence of SARS virus in a biological sample. The antisense sequence can be labeled itself or the antisense sequence that binds to the viral polynucleotide can be detected by methods known in the art. Antisense nucleic acids are designed to bind specifically to RNA, which results in the formation of RNA-DNA or RNA-RNA hybrids, thereby preventing DNA replication, reverse transcription, or translation of messenger RNA. Antisense polynucleotides based on the selected sequence may interfere with the expression of the corresponding gene. The antisense polynucleotide will bind to and/or interfere with translation of the corresponding mRNA.

The invention also includes the use of intergenic regions with ribozymes.

Trans-cleaving catalytic RNA (ribozymes) are RNA molecules with endoribonuclease activity. Ribozymes are specifically designed for a particular target, and the target messenger RNA must contain a specific nucleotide sequence. They can be genetically modified to specifically cleave any kind of RNA site-especially sites in the RNA backbone of cells. This cleavage destabilizes the mRNA and prevents protein expression. Importantly, ribozymes can be used to inhibit the expression of a gene of unknown function, so that the function of the gene in vitro or in vivo can be determined by detecting phenotypic effects.

One commonly used ribozyme motif is the hammerhead ribozyme, which has minimal requirements for substrate sequence. The design of hammerhead ribozymes is described in Usman et al, Current Opifz.struct.biol. (1996) 6: 527-533. Usman also discusses the therapeutic use of ribozymes. Ribozymes can also be prepared and used as described below: long et al, FASEBJ, (1993) 7: 25; symons, ann.rev.biochem. (1992) 61: 641; perrotta et al, Biochem, (1992) 31: 16-17; ojwang et al, Proc.Natl.Acad.Sci. (USA) (1992) 89: 10802-10806; and us patent 5254678. U.S. Pat. No. 8, 5144019 describes the cleavage of HIV-I RNA by ribozymes; U.S. Pat. No. 5116742 describes a method of cleaving RNA using ribozymes; U.S. Pat. No. 5, 5225337 and Koizumi et al, nucleic Acid Res (1989) 17: 7059-7071 describes methods for increasing the specificity of ribozymes. Koizumi et al, nucleic Acid Res (1989) 17: 7059-7071 also describes the preparation and use of hammerhead-structured ribozyme fragments. Chowrira and Burke, nucleic Acid Res. (1992) 20: 2835 describes the preparation and use of hairpin-structured ribozyme fragments. Ribozymes can also be made by rolling circle transcription, such as Daubendiek & Kool, nat. biotechnol. (1997)15 (3): 273 and 277.

The ribozyme's hybridizing region can be modified or made into a branched structure, such as horns & Urdea, nucleic acid Res (1989) 17: 6959-67. The basic structure of ribozymes can also be altered by chemical processes using methods familiar to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified with monomeric units. Liposome-mediated delivery of ribozymes improves cellular uptake in therapy, as described by Birikh et al, eur.j.biochem. (1997) 245: 1-16.

The therapeutic and functional genomic use of ribozymes begins with an understanding of the portion of the gene coding sequence to be inhibited. In the present invention, the target sequence preferably includes intergenic sequences of SARS virus. Preferably the sequence is selected from SEQ id no: 7292 and SEQ ID NO: 7293. the target cleavage site is selected from the group consisting of target sequences, and ribozymes are constructed based on the 5 'and 3' nucleotide sequences flanking the cleavage site. Preferably, the 5 'nucleotide sequence includes the 5' untranslated region of the SARS virus. Ribozymes can then be constructed from one or more polynucleotide sequences selected from the group consisting of: SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301.

Antisense treatment of HIV infection is described in the following references, which are incorporated by reference in their entirety. (antisense RNA complementary to the mRNA of gag, tat, rev, env) (Sezakiel et al, 1991, J.Virol.65: 468) -472; Chatterjee et al, 1992, Science 258: 1485-1488; Rhodes et al, 1990, J.Gen.Virol.71: 1965; Rhodes et al, 1991, AIDS 5: 145-151; Sezakiel et al, 1992, J.Virol.66: 5576-5581; Joshi et al, 1991, J.Virol.65: 5524-5530).

The present invention involves the use of decay RNA to disrupt the replication and life cycle of SARS virus. Methods of making and using such decay RNA to treat viral infections are known in the art. The invention includes the delivery of genes encoding, for example, intergenic sequences of the SARS virus to infected cells. Preferably, the sequence comprises one or more sequences selected from the group consisting of: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301. preferably, the sequence comprises one or more sequences selected from the group consisting of: SEQ ID NO: 7292 and SEQ ID NO: 7293. preferably the sequence comprises SEQ ID NO: 7293.

In the present invention, delivery of intergenic sequences not linked to the SARS virus open reading frame disrupts the translation process of the viral RNA and reduces viral protein production. Similar methods for treating HIV viral infections have been described. The following references describe the use of decay RNA from HIV TAR or RRE to treat HIV infection. The contents of these references are incorporated herein by reference. (Sullenger et al, 1990, Cell 63: 601-608; Sullenger et al, 1991, J.Virol.65: 6811-6816; Lisziewicz et al, 1993, New biol.3: 82-89; Lee et al, 1994, J.Virol.68: 8254-8264), ribozymes (Sarver et al, 1990, Sci 247: 1222-1225; Wecraschenghe et al, 1991, J.Virol.65: 5531-5534; Dropulic et al, 1992, J.Virol.66: 1432-1441; Ojwang et al, 1992, Proc.Natl.Acad.Sci.USA.89: 10802-10806; Yu et al, 1993, Proc.Natl.Acad.Sci.6390: Yac.Natl.USA.44-1995, Nat.92: Nat-69da et al, 1995: USA-6992; Nat-35, USA-1994, USA, 1995, USA, 35, J.92, J.V.V.V.V.V.V.V.V.V. 35, USA, 35.

The invention includes the use of the intergenic sequence of the SARS virus in diagnostic reagents, kits (containing such reagents) and methods for diagnosing or identifying the presence of SARS virus in a biological sample. Such diagnostic agents, kits and methods are further described in section II of the specification.

The present invention includes a primer pair for amplifying a SARS polynucleotide sequence comprising (i) a first primer comprising a sequence substantially identical to a sequence portion selected from the group consisting of seq id nos: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301, and (ii) a second primer comprising a primer that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1 and the sequence SEQ ID NO: 2 such that primer pairs (i) and (ii) are substantially complementary to each other in the sequence of SEQ ID NO: 1 and the sequence SEQ ID NO: 2, a template sequence is determined. Preferably (i) the first primer comprises a primer that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 7292 and SEQ ID NO: 7293 a part of the sequence of which is substantially the same. Preferably (i) the first primer comprises a primer identical to SEQ ID NO: 7293 a part of the sequence of which is substantially the same. The length of the amplicon defined by the first and second primers is preferably between 50 and 250 nucleotides. These primers may optionally be labeled to facilitate their detection. Methods and compositions for labeling primers are further discussed in section III of this application.

The invention also includes a primer pair for amplifying a SARS polynucleotide sequence comprising (i) a first primer comprising a sequence substantially identical to a complement portion of a sequence portion selected from the group consisting of seq id nos: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301, and (ii) a second primer comprising a primer that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1 and the sequence SEQ ID NO: 2 such that the primer pair is substantially complementary to a portion of the complement of SEQ ID NO: 1 and the sequence SEQ ID NO: 2, a template sequence is determined. The length of the amplicon determined by the first and second primers is preferably between 50 and 250 nucleotides. The primers may optionally be labeled to facilitate detection. Methods and compositions for labeling primers are further discussed in section III of this application.

The invention includes a kit comprising (i) a first primer comprising a sequence substantially identical to a sequence portion selected from the group consisting of: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301, and (ii) a second primer comprising a primer that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 1 and the sequence SEQ ID NO: 2, such that primer pairs (i) and (ii) are substantially complementary to each other in a sequence defined by the sequence SEQ id no: 1 and the sequence SEQ ID NO: 2, a template sequence is determined. Preferably (i) the first primer comprises a primer that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 7292 and SEQ ID NO: 7293 a portion of the sequence of SEQ ID NO. Preferably (i) the first primer comprises a primer identical to SEQ ID NO: 7293 a portion of the sequence of SEQ ID NO. The primers may optionally be labeled to facilitate detection. Methods and compositions for labeling primers are further discussed in section III of this application.

Other preferred kits comprise (i) a first primer comprising a sequence substantially identical to a complement portion of a sequence selected from the group consisting of seq id no: SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301, and (ii) a second primer comprising a primer that hybridizes to a sequence selected from the group consisting of SEQ id nos: 1 and the sequence SEQ ID NO: 2 such that the primer pair is substantially complementary to the primer pair in a sequence consisting of the complement of sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2, a template sequence is determined.

The invention also includes attenuated SARS viruses for use as vaccines in which the intergenic region has been mutated such that expression of structural or non-structural proteins of the virus is reduced. Such an attenuated SARS virus may have one or more additions, deletions or insertions within one or more intergenic regions of the viral genome. Preferably, the attenuated SARS virus is expressed in a nucleic acid sequence selected from seq id NO: 7292 and SEQ ID NO: 7293 has one or more additions, deletions or insertions in the sequence. Preferably in SEQ ID NO: 7293 there are one or more additions, deletions or insertions.

The invention also includes small molecules that inhibit the binding or association of the SARS virus replication machinery, such as ribonucleoproteins, with intergenic regions of the viral genome. Preferably, the small molecule is capable of inhibiting the association of the SARS virus machinery with a sequence selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293 to or associated with a sequence. Preferably, the small molecule can inhibit the SARS virus mechanism and the sequence SEQ ID NO: 7293 binding or associating. The invention also includes a method of screening for small molecules for use in the treatment of SARS virus infection, the method comprising using an assay to identify small molecules that interfere with the binding of the SARS virus replication machinery to the intergenic region of the SARS virus genome.

The present invention also provides a new SARS polynucleotide sequence SEQ ID NO: 9968. all 6 reading frames of the 690mer sequence are shown in FIG. 113. Figure 113 has at least 4 amino acids in a constituent amino acid sequence that is set forth as SEQ ID NOS: 9969 + 10032.

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 9968. The invention also provides a polypeptide having the sequence shown in SEQ ID NO: 9968 has a polynucleotide sequence with sequence identity. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 80%, 85%, 88%, 90%, 92%, 95%, 99% or higher).

The invention includes a polypeptide consisting of SEQ ID NO: 9968, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9969-10032. Preferably the amino acid sequence comprises SEQ ID NO: 9997 or comprises SEQ ID NO: 9998.

the invention also provides a polypeptide corresponding to SEQ ID NO: 9968 has a sequence identity to the amino acid sequence. The present invention provides a polypeptide linked to a nucleic acid sequence selected from SEQ ID NOS: 9969-10032 has sequence identity with the amino acid sequence. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 80%, 85%, 88%, 90%, 92%, 95%, 99% or higher).

SEQ ID NO: 9968 matches, at about 98% identity, a previously published SARS polynucleotide sequence commonly referred to as "BNI-1" (SEQ ID NO: 10033). BNI-1 has been sequenced in the National Center for Tropical Infectious Diseases Reference (National Reference Center for Tropical Infectious Diseases) research institute of Tropical medicine Bernhard Nocht, Hamburg, Germany. BNI-1 sequence was published on WHO website http 4 months 4 in 2003: // www.who.int/csr/sars/primers/en, and published in Dorsten et al, "Identification of New Corona Virus in Patients with Severe Acute Respiratory Syndrome" (Identification of a novel Coronavir in Patients with Server Acerty Respiratory Syndrome Syndrome), New England journal of Medicine, published online at 10/4/2003 in http: // www.nejm.org. Both references are incorporated herein by reference in their entirety. The 6 reading frames of the 302mer sequence are shown in FIG. 114 (see also FIG. 129). At least 4 amino acids in the constituent amino acid sequence of fig. 114, which are identified as SEQ id nos: 10034 and 10065. SEQ ID NO: 10034 and SEQ ID NO: 9997 comparison is shown in FIG. 130.

The present invention provides a polypeptide comprising SEQ ID NO: 9968 fragment. In one embodiment, the fragment does not consist entirely of SEQ ID NO: 10033 or a known coronavirus sequence.

The present invention provides a polypeptide comprising a sequence defined by SEQ ID NO: 9968 in a fragment thereof. In one embodiment, the fragment does not consist entirely of SEQ ID NO: 10033 or a sequence of a known coronavirus.

The present invention provides a polypeptide comprising a sequence selected from SEQ ID NOS: 9969-10032. In one embodiment, the fragment does not consist entirely of SEQ ID NO: 10033 or a sequence of a known coronavirus.

SEQ ID NO: 9968 does not match any portion of the BNI-1 polynucleotide sequence (SEQ ID NO: 10033) by about 100 nucleotides at the 5' terminus. This mismatched portion is set forth in SEQ ID NO: 10066. Accordingly, the present invention also provides a polypeptide comprising a nucleotide sequence comprising SEQ ID NO: 10066, a polynucleotide having the sequence of SEQ ID NO: 10066 or a polynucleotide sequence having sequence identity thereto comprising SEQ ID NO: 10066.

The invention also includes a polypeptide consisting of SEQ ID NO: 10066, and the amino acid sequence encoded by SEQ ID NO: 10066 or an amino acid sequence comprising the amino acid sequence encoded by SEQ ID NO: 10066 and amino acid sequence of a fragment of the amino acid sequence encoded thereby. Preferably the amino acid sequence comprises SEQ ID NO: 10067.

SEQ ID NO: 9997/9998 were shown to be homologous to the pol1ab region of several coronaviruses. Fig. 115 shows SEQ ID NOS: 9997/9998 and bovine coronavirus pol1ab (SEQ ID NO: 10068). Alignment of the amino acid sequence of avian infectious bronchitis virus pol1ab (SEQ ID NO: 10069) and the amino acid sequence of murine hepatitis virus pol1ab (SEQ ID NO: 10070). SEQ ID NOS: 9997/9998, SEQ ID NO: 10068. SEQ ID NO: 10069 and SEQ ID NO: the consensus amino acid sequence of 10070 is shown in the last line of the alignment in FIG. 115 (e.g., SEQ ID NO: 10071).

As shown in figure 113, the encoded SEQ ID NO: 9997 following codon 205, SEQ ID NOS: 9997 and 9998 has a stop codon between them. The stop codon can optionally be removed and the amino acid sequence made continuous (SEQ ID NO: 10072). Accordingly, the present invention provides a polypeptide comprising SEQ ID NO: 9997 and/or SEQ ID NO: 9998, or SEQ ID NO: 10072, and further comprises an amino acid sequence encoding the amino acid sequence of the C-terminus of the coronavirus pol1ab gene or fragment thereof.

As shown in fig. 115, SEQ ID NOS: 10068. 10069, 10070, and 10071 are set forth in SEQ ID NOs: 9997 contains an amino acid before the N-terminus. The amino acid sequence provided by the invention also comprises SEQ ID NO: 9997 and further comprises an amino acid sequence encoding the N-terminus of the coronavirus pol1ab protein or fragment thereof.

The pol1ab sequence of fig. 115 contains a coding region, which is denoted by an "+" in fig. 117. In fig. 115, the sequences are shown with SEQ ID NOs: the arrow preceding 10071 amino acid 6080 indicates the start of the genomic region. The end of the genomic region is indicated by the arrow before crossing the consensus amino acid 6604. The amino acid sequence provided by the invention contains SEQ ID NO: 9997 and/or SEQ IID NO: 9998, or SEQ ID NO: 10072, and further comprising said SEQ ID NO: 9997 and/or SEQ ID NO: 9998, or SEQ ID NO: 10072, wherein said first amino acid sequence has homology to the N-terminal sequence of a known coronavirus pol1ab "protein or fragment thereof.

The invention also provides a polypeptide comprising SEQ ID NO: 9997 and SEQ ID NO: 9998, wherein, SEQ ID NO: 9971 (i.e., SEQ ID NO: 10072) and further comprises the amino acid sequence of SEQ ID NO: 9998C-terminal, wherein said second amino acid sequence is homologous to the C-terminal of a known coronavirus pol1ab "protein or fragment thereof.

Examples of such proteins are shown in comparison in fig. 118, which are SEQ ID NOS: 10073-10077. SEQ ID NO: 10073 comprises SEQ ID NO: 9997 and further comprises amino acids before and after the N-terminus of the avian infectious bronchitis virus pol1ab "" protein. SEQ ID NO: 10074 comprises SEQ ID NO: 9997 and further comprises amino acids before and after the N-terminus of the bovine coronavirus pol1ab "" protein. SEQ ID NO: 10075 comprises SEQ ID NO: 9997 and further comprises amino acids before and after the N-terminus of the murine hepatitis virus pol1ab "" protein. SEQ ID NO: 10076 comprises SEQ ID NO: 9997 and further comprises amino acids before and after the N-terminus of the consensus sequence of proteins avian infectious bronchitis virus, bovine coronavirus and murine hepatitis virus pol1ab "" (fig. 115). SEQ ID NO: 10077 comprises SEQ ID NOS: 10073 and 10076.

The invention includes nucleic acid sequences selected from SEQ ID NOS: 10073. the amino acid sequences of 10074, 10075, 10076, and 10077. The invention also includes compositions comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 10073. the amino acid sequence of the amino acid sequence fragments of 10074, 10075, 10076 and 10077. The invention also includes polypeptides related to a nucleic acid sequence selected from SEQ ID NOS: 10073. the amino acid sequences of 10074, 10075, 10076, and 10077 have sequence identity.

The invention includes nucleic acids encoding a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 10073. polynucleotides of the amino acid sequences of 10074, 10075, 10076, and 10077. The invention includes nucleic acid sequences encoding nucleic acids selected from the group consisting of SEQ ID NOS: 10073. polynucleotides of the amino acid sequences of 10074, 10075, 10076, and 10077 have sequence identity. The invention includes encoding SEQID NOS: 10073. fragments of the polynucleotides 10074, 10075, 10076, and 10077.

As shown in fig. 113, SEQ ID NO: 9968 comprises a nucleic acid sequence encoding SEQ ID NO: 10020, followed by a stop codon, given a C-terminal threonine (Thr) residue. The corresponding sequence of the amino acid sequence encoded by BNI-1 is SEQ ID N0: 10078, which continues until SEQ ID NO: 10020C-terminal end. Accordingly, the invention includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10020 or a variant of SEQ ID NO: 10020 or an amino acid sequence having sequence identity or comprising SEQ ID NO: 10020, wherein the C-terminal residue of said protein is threonine. Preferably, the C-terminus of the protein is-ST. More preferably, the C-terminus of the protein is an-EST. The invention also includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10078 or a variant of SEQ ID NO: 10078 or an amino acid sequence comprising SEQ ID NO: 10078, wherein the C-terminal residue of said protein is Thr. Preferably, the C-terminus of the protein is-ST. More preferably, the C-terminus of the protein is an-EST.

SEQ ID NO: 9968 also encodes a 54mer amino acid sequence of SEQ ID NO: 10015. this code SEQ id no: the polynucleotide of 10015 has two stop codons at its C-terminus (FIG. 113). The corresponding region of the BNI-1 sequence does not contain this 54mer sequence. Accordingly, the invention includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10015 or a sequence identical to SEQ ID NO: 100015 or an amino acid sequence comprising SEQ ID NO: 10015, or a fragment thereof. The invention also includes a polypeptide comprising SEQ ID NO: 10015 and further comprising SEQ id no: 10015N-terminal front first amino acid sequence.

SEQ ID NO: 9968 encodes the amino acid sequence of SEQ ID NO: 9969. the polynucleotide sequence is shown in SEQ ID NO: 9969 contains a stop codon at the C-terminus. Accordingly, the invention includes a polypeptide comprising the amino acid sequence of SEQ id no: 9969 or a variant of SEQ ID NO: 9969 has an amino acid sequence having sequence identity. The invention also includes a polypeptide comprising SEQ ID NO: 9969 and further comprises SEQ ID NO: 9969 before the N-terminus. The invention also includes a polypeptide comprising the sequence of SEQ ID NO: 10079.

SEQ ID NO: 9968 encodes the amino acid sequence QRT (FIG. 113), followed by a stop codon. Thus, the invention encompasses proteins comprising the amino acid sequence QRT. The invention also includes polypeptides comprising the amino acid sequence QRT and further comprising the first amino acid sequence preceding the N-terminus of the sequence QRT.

SEQ ID NO: 9968 encodes the amino acid sequence of SEQ ID NO: 10022, followed by a stop codon at its C-terminus. Accordingly, the invention includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10022 or a sequence identical to SEQ ID NO: 10022 a protein having an amino acid sequence having sequence identity. The invention also includes a polypeptide comprising SEQ ID NO: 10022 and further comprising SEQ ID NO: 10022N-terminal of the first amino acid sequence.

SEQ ID NO: 9968 encodes the amino acid sequence of SEQ ID NO: 10027. SEQ ID NO: within the 10027 coding sequence are at least 3 initiation codons, underlined in figure 119. The open reading frame indicated by the first start codon is SEQ ID NO: 10081. the open reading frame indicated by the second start codon is SEQ ID NO: 10082. the third start codon indicates an open reading frame of SEQ ID NO: 10083.

the present invention provides a new SARS polynucleotide sequence SEQ ID NO: 10084. all 6 reading frames of the 1463mer sequence are shown in FIG. 120 (see also FIG. 122). Figure 120 has at least 4 amino acids in the constituent amino acid sequence that are set forth as SEQ ID NOS: 10085-10209 (see FIGS. 120A-120F).

The invention includes a polypeptide comprising SEQ ID NO: 10084. The invention also provides a polypeptide similar to SEQ ID NO: 10084 polynucleotide sequences having sequence identity. The invention also provides a polypeptide comprising SEQ ID NO: the polynucleotide sequence of a fragment of 10084. In one embodiment, the polynucleotide fragment does not consist entirely of SEQ ID NO: 10033 or a known coronavirus polynucleotide sequence or a known SARS polynucleotide sequence.

The invention includes a polypeptide consisting of SEQ ID NO: the amino acid sequence encoded by the polynucleotide sequence of 10084, including the amino acid sequences of figures 120A-120F, e.g., a sequence selected from the group consisting of SEQ ID NOS: 10085-10209. Preferably the amino acid sequence comprises seq id NO: 10149.

the invention also provides a polypeptide corresponding to SEQ ID NO: 10084 encoding an amino acid sequence having sequence identity. The invention provides amino acids having sequence identity to the amino acid sequences of fig. 120A-120F, e.g., selected from SEQ ID NOS: 10085-10209.

The invention also provides SEQ ID NO: 10084. The invention also provides a nucleic acid sequence selected from SEQ ID NOS: a fragment of the amino acid sequence of 10085-10209. In one embodiment, the fragment does not consist entirely of SEQ ID NO: 10033 or the known coronavirus amino acid sequence or the known SARS virus amino acid sequence. SEQ ID NO: 10033 and SEQ ID NO: a comparison of the 10084 matching portions is shown in figure 121.

In one embodiment, the invention includes a polypeptide comprising SEQ ID NO: 10149. Polynucleotide sequence SEQ ID NO: 10084 and the encoded SEQ ID NO: 10149 comparison is shown in FIG. 122(5 '3' reading frame 3). Analysis of the 5 '3' reading frame 3 translation (fig. 123) by a computer program (NetStart 1.0) predicting the methionine start codon shows that SEQ ID NOS: 10210-10215.

The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10210. SEQ ID NO: 10211. SEQ ID NO: 10212. SEQ ID NO: 10213. SEQ ID NO: 10214 and SEQ ID NO: 10215 amino acid sequence. The invention includes a polypeptide that binds to a polypeptide selected from the group consisting of SEQ ID NO: 10210. SEQ ID NO: 10211. SEQ ID NO: 10212. SEQ ID NO: 10213. SEQ ID NO: 10214 and SEQ ID NO: 10215 amino acid sequence of a protein having sequence identity. In one embodiment, the protein does not consist entirely of the amino acid sequence of a known SARS virus or a known coronavirus.

The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1021O, SEQ ID NO: 10211. SEQ ID NO: 10212. SEQ ID NO: 10213. SEQ ID NO: 10214 and SEQ ID NO: 10215 amino acid sequence. In one embodiment, the fragment does not consist entirely of the amino acid sequence of a known SARS virus or a known coronavirus.

In one embodiment, the invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10210. SEQ ID NO: 10211 and SEQ ID NO: 10212. Fig. 124 includes the sequence of SEQ ID NO: 10210 BLAST algorithm. These results indicate that SEQ ID NOS: 10210. 10211 and 10212 are functionally similar to coronavirus RNA polymerases, particularly RNA polymerases of murine hepatitis virus, bovine coronavirus, and avian infectious bronchitis.

In one embodiment, the present invention relates to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10210. SEQ ID NO: 10211 and SEQ ID NO: 10212 and a second amino acid sequence C-terminal to the sequence of coronavirus ORF1 ab. Preferably, the second amino acid sequence is from bovine coronavirus. An example of this embodiment is SEQ id no: 10216. SEQ ID NO: 1-481 amino acids of 10216 are SEQ ID NO: 10210, amino acids 482 and 1152 are the second amino acid sequence (Gi 26008080) (NP-150073.2) at the C-terminus of the polyprotein of bovine coronavirus orf1ab (SEQ ID NO: 10217).

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 10216. The invention also includes a polypeptide comprising SEQ id no: 10210 and the first amino acid sequence of SEQ ID NO: 10217. The invention also includes compositions comprising a peptide having an amino acid sequence substantially identical to SEQ ID NO: 10210 greater than x% identity and a first amino acid sequence identical to SEQ id no: 10217 greater than y%, wherein x is greater than or equal to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and y is greater than or equal to 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more).

The invention also includes a polypeptide comprising SEQ ID NO: 10210, wherein the fragment contains an epitope. Predicted by computer using a 17mer window, SEQ ID NO: 10210 epitopes are shown in FIG. 125A (Hopp & Woods) and FIG. 125B (Kyte & Doolittle).

SEQ ID NO: 10210 also contains two predicted glycosylation sites at amino acids 81-84 (NNTE; SEQ ID NO: 10218) and 180-183 (NHSV; SEQ ID NO: 10219). Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 10210, wherein the fragment contains a glycosylation site. The invention also includes a polypeptide comprising SEQ ID NO: 10210, wherein the fragment has an Asn at position 81. Preferably, the Asn is glycosylated. The invention also includes a polypeptide comprising SEQ ID NO: 10210, wherein the fragment has an Asn at position 180. Preferably, the Asn is glycosylated.

In one embodiment, the invention includes a polypeptide comprising an amino acid sequence from figure 120D and/or a sequence from SEQ id no: 10150-10160 (e.g., from SEQ ID NOS: 10154, 10155, 10158 and 10160). SEQ ID NO: 10154 has been identified as containing the following amino acid sequence starting at Met and ending at a stop codon: SEQ ID NOS: 10220-10227.

Accordingly, the invention includes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 10220. SEQ ID NO: 10221. SEQ ID NO: 10222. SEQ ID NO: 10223. SEQ ID NO: 10224. SEQ ID NO: 10225. SEQ ID NO: 10226 and SEQ ID NO: 10227, or a fragment thereof, or an amino acid sequence having sequence identity thereto.

In one embodiment, the invention includes a polypeptide comprising the amino acid sequence in fig. 120E, e.g., a sequence from SEQ id no: 10161-10182, in particular SEQ ID NOS: 10171 and 10176. In seq id NOS: 10171-10176 the following amino acid sequence starting at Met and ending at a stop codon can be identified: SEQ ID NO: 10228 and SEQ ID NO: 10229.

accordingly, the invention includes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 10228 and SEQ ID NO: 10229, or a fragment thereof, or an amino acid sequence having sequence identity thereto.

In one embodiment, the invention includes a polypeptide comprising the amino acid sequence of FIG. 120F (e.g., SEQ ID NOS: 10183-10209). In FIG. 120F, the following amino acid sequence starting at Met and ending at a stop codon can be identified: SEQ ID NO: 10187. accordingly, the invention includes a polypeptide comprising SEQ ID NO: 10187 or a fragment thereof or an amino acid sequence having sequence identity thereto.

In one embodiment, the polynucleotide of the invention does not contain one of the following primers, which are disclosed in http: v/content. nejnz. org/cgi/print/NEJMoa030781v2. pdf:

5’GGGTTGGGACTATCCTAAGTGTGA3’(SEQ ID NO：10230)

5’TAACACACAACICCATCATCA3’(SEQ ID NO：10231)

5’CTAACATGCTTAGGATAATGG3’(SEQ ID NO：10232)

5’GCCTCTCTTGTTCTTGCTCGC3’(SEQ ID NO：10233)

5’CAGGTAAGCGTAAAACTCATC3’(SEQ ID NO：10234)

the invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The invention includes the polynucleotide primers identified in Table 31 (SEQ ID NOS: 10235-10258), the forward primers SEQ ID NOS: 10259-10281 and reverse primer SEQ ID NOS: 10282-10298. The invention also includes polynucleotide sequences complementary to any of the primer sequences disclosed herein.

The present invention provides SARS polynucleotide sequence SEQ ID NO: 10299. fig. 126 includes all 6 reading frames of the sequence (see also fig. 131). At least 4 amino acids in the constituent amino acid sequence of fig. 126, which are set forth as SEQ ID NOS: 10300 and 10337.

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 10299. The invention also provides a polypeptide having the sequence shown in SEQ ID NO: 10299A polynucleotide sequence having sequence identity. The invention also provides a polypeptide comprising SEQ ID NO: 10299 fragment. In one embodiment, the polynucleotide fragment does not consist entirely of the known polynucleotide sequence of the SARS virus or the known polynucleotide sequence of the coronavirus.

The invention includes a polypeptide consisting of SEQ ID NO: 10299 comprising the amino acid sequence shown in figure 126 and an amino acid sequence selected from the group consisting of SEQ ID NOS: 10300 and 10337. Preferably the amino acid sequence comprises SEQ ID NO: 10316.

the invention also provides a polypeptide consisting of SEQ ID NO: 10299 an amino acid sequence having sequence identity. The present invention provides a polypeptide substantially similar to a polypeptide selected from SEQ ID NO: 10300 and 10337 has a sequence identity.

The invention also provides a polypeptide consisting of SEQ ID NO: 10299 to a fragment of an amino acid sequence encoded thereby. The invention also provides a nucleic acid sequence selected from SEQ ID NOS: 10300 and 10337. In one embodiment, the fragment does not consist entirely of the known amino acid sequence of the SARS virus or the known amino acid sequence of the coronavirus.

In one embodiment, the invention includes a polypeptide comprising SEQ ID NO: 10316. SEQ ID NO: 10316 comprises the open reading frame of SEQ ID NO: 10338 and SEQ ID NO: 10339.

in one embodiment, the invention includes a polypeptide comprising a sequence from SEQ ID NO: 102995 '3' reading frame 1 translation. The following encoded open reading frames were found in this translation: SEQ ID NO: 10340.

In one embodiment, the invention includes a polypeptide comprising a sequence from SEQ ID NO: 102995 '3' reading frame 1 translation. The following encoded open reading frames were found in this translation: SEQ ID NO: 10341.

in one embodiment, the invention includes a polypeptide comprising a sequence from SEQ ID NO: 102995 '3' reading frame 1 translation. The following encoded open reading frames were found in this translation: SEQ ID NO: 10342.

the invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10338. the amino acid sequence of SEQ NO: 10339. SEQ NID NO: 10340. SEQ ID NO: 10341 and SEQ ID NO: 10342. The invention includes polypeptides that are substantially similar to those selected from SEQ ID NOs: 10338. the amino acid sequence of SEQ NO: 10339. SEQ NID NO: 10340. SEQ ID NO: 10341 and SEQ ID NO: 10342, and a polypeptide having sequence identity thereto. The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10338. SEQ NO: 10339. SEQ NID NO: 10340. SEQ ID NO: 10341 and SEQ ID NO: 10342. In one embodiment, the fragment does not consist entirely of the amino acid sequence of a known SARS virus or the amino acid sequence of a known coronavirus.

In one embodiment, SEQ ID NOS: 10338-10342 was used for the fusion protein. Thus, the start codon methionine is removed. The invention includes amino acid sequences selected from the group consisting of SEQ ID NO: 10343. SEQ ID NO: 10344. SEQ ID NO: 10345. SEQ ID NO: 10346 and SEQ ID NO: 10347.

in one embodiment, the invention includes a polypeptide selected from the group consisting of SEQ ID NOs: 10338 and SEQ ID NO: 10339. The following is the SEQ ID NO: partial BLAST results of 10338 search:

r i 133593 g sp P18457R RRPB _ CVPFS RNA-directed RNA polymerase (ORF1B)

gi |93934| pir | | A43489 RNA-directed RNA polymerase (Ec 2.7.7.48) -porcine transmissible gastroenteritis virus (fragment)

gi |833161| emb | CAA37284.1| polymerase [ transmissible gastroenteritis virus ]

Length 533

Score 131 bits (329), estimate 3e-30

Identity of 55/89 (61%), positive of 69/89 (77%), void of 1/89 (1%)

Inquiring: 1 MLWCKDGHVETFYPKLQASQAWQPGVAMPNLYKMQRMLLEKCDLQNYGENAVIPKGIMMN 60

MLWC++ H++TFYP+LQ+++ W PG +MP LYK+QRM LE+C+L NYG +P GI N

The target is as follows: 217 MLWCENSHIKTFYPQLQSAE-WNPGYSMPTLYKIQRMCLERCNLYNYGAQVKLPDGITTN 275

Inquiring: 61 VAKYTQLCQYLNTLTLAVPSNMRVIHFGA 89

V KYTQLCQYLNT TL VP MRV+H GA

The target is as follows: 276 VVKYTQLCQYLNTTTLCVPHKMRVLHLGA 304

These results indicate that SEQ ID NO: 10338 it is similar in function to RNA polymerase directed by porcine transmissible gastroenteritis virus RNA.

GenBank is performed on the sequence shown in SEQ ID NO: the partial BLAST results of the 10339 search are as follows:

' gb | AAL57305.1| replicase [ bovine coronavirus ]

Length 7094

Score 139 bits (351), estimate 7e-33

Identity 64/108 (59%), positive 78/108 (72%)

Inquiring: 1 MSVISKVVKVTIDYAEISFMLWCKDGHVETFYPKLQASQAWQPGVAMPNLYKMQRMLLEK 60

M++SKVV V +D+ + FMLWC D V TFYP+LQA+ W+PG +MP LYK +E+

The target is as follows: 6760 LNCVSKVVNVNVDFKDFQFMLWCNDEKVMTFYPRLQAASDWKPGYSMPVLYKYLNSPMER 6819

Inquiring: 61 CDLQNYGENAVIPKGIMMNVAKYTQLCQYLNTLTLAVPSNMRVIHFGA 108

L NYG+ +P G MMNVAKYTQLCQYLNT TLAVP NMRV+H GA

The target is as follows: 6820 VSLWNYGKPVTLPTGCMMNVAKYTQLCQYLNTTTLAVPVNMRVLHLGA 6867

These results indicate that SEQ ID NO: 10339 is functionally similar to the replicase of bovine coronavirus.

SARS virus is represented by SEQ ID NO: a polymorphism at residue Glu-20 of 10338. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 10338, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of ASQAW (SEQ ID NO: 10348) and ASRAW (SEQ ID NO: 10349). The invention includes a polypeptide comprising SEQ ID NO: 10338, wherein said fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 10348 and SEQ ID NO: 10349 amino acid sequence.

SARS virus is represented by SEQ ID NO: 10338 has a polymorphism at the Ser-80 residue. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 10338, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of VPSNM (SEQ ID NO: 10350) and VPTNM (SEQ ID NO: 10351). The invention includes a polypeptide comprising SEQ ID NO: 10338, wherein said fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 10350 and SEQ ID NO: 10351.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The invention includes polynucleotide sequences comprising one or more of the primer sequences identified in table 32. The invention also includes polynucleotide sequences comprising sequences complementary to one or more of the primer sequences identified in table 32.

The present invention provides SARS polynucleotide sequence SEQ ID NO: 10505. fig. 127 shows all 6 reading frames of the sequence (see also fig. 132). The component amino acid sequence of fig. 127 contains at least 4 amino acids that are set forth as SEQ ID NOS: 10506, 10570.

The invention includes a polypeptide comprising SEQ ID NO: 10505. The invention also provides a polypeptide similar to SEQ ID NO: 10505 polynucleotide sequence having sequence identity. The invention also provides a polypeptide comprising SEQ ID NO: the polynucleotide sequence of the 10505 fragment. In one embodiment, the polynucleotide fragment does not consist entirely of the known SARS virus polynucleotide sequence or the known coronavirus polynucleotide sequence.

The invention includes a polypeptide consisting of SEQ ID NO: 10505 comprising the amino acid sequence of figure 127, particularly those selected from the group consisting of SEQ ID NOS: 10506, 10570. Preferably the amino acid sequence comprises SEQ ID NO: 10532 and/or SEQ ID NO: 10533.

the invention also provides a polypeptide consisting of SEQ ID NO: 10505 encodes an amino acid sequence having sequence identity. The present invention provides a polypeptide having an amino acid sequence selected from the group consisting of the sequences shown in fig. 127, in particular SEQ id nos: 10506-10570 has an amino acid sequence with sequence identity.

The invention also provides a polypeptide consisting of SEQ ID NO: 10505, or a fragment of the amino acid sequence encoded by 10505. The invention also provides a nucleic acid sequence selected from SEQ ID NOS: 10506-10570. In one embodiment, the fragment does not consist entirely of the known amino acid sequence of the SARS virus or the known amino acid sequence of the coronavirus.

In one embodiment, the invention includes a polypeptide comprising the amino acid sequence of 5 '3' reading frame 3 from figure 127. Some of the encoded open reading frames in this translation are: SEQ ID NO: 10533; SEQ ID NO: 10571; SEQ ID NO: 10572, respectively; SEQ ID NO: 10573; SEQ ID NO: 10574.

the invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10533. SEQ ID NO: 10571. SEQ ID NO: 10572. SEQ ID NO: 10573 and SEQ ID NO: 10574, or a pharmaceutically acceptable salt thereof. The invention includes a polypeptide that binds to a polypeptide selected from the group consisting of SEQ ID NO: 10533. SEQ ID NO: 10571. SEQ ID NO: 10572. SEQ ID NO: 10573 and SEQ ID NO: 10574 has a sequence identity to the amino acid sequence of said polypeptide. The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10533. SEQ ID NO: 10571. SEQ ID NO: 10572. SEQ ID NO: 10573 and SEQ ID NO: 10574, or a fragment of a polypeptide sequence of the amino acid sequence of 10574.

GenBank is performed on the sequence shown in SEQ ID NO: the partial BLAST results of the 10533 search are as follows:

' gi |7739601| gb | from F68926.1| AF207902_11 nucleocapsid protein [ murine hepatitis virus ML-11 strain ]

Length 451 ═ 451

Score 147 bits (370), estimate 3e-34

Identity 102/252 (40%), positive 137/252 (54%), void 18/252 (7%)

Inquiring: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106

SWF+ +TQ K +E +F +GQGVPI + +Q GY +R RR + DG +K+L PRW

The target is as follows: 63 SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQLLPRW 122

Inquiring: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166

YFYYLGTGP A YG +EG +VWVA++ A + R+P+++ A + GT LP

The target is as follows: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTRFAPGTVLP 182

Inquiring: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLN 226

+GFY EGS + AS S N SS PA +A L+L +L

The target is as follows: 183 QGFYVEGSGRSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVLAKLG 242

Inquiring: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK- - -KPRQKRTATKQYNVTQAFGRRGPEQTQG 282

+ GQ+Q VTK+SA E + KPRQKRT KQ V Q FG+RGP Q

The target is as follows: 243K- - -DAQPKQ- - -VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPNQ- - -290

Inquiring: 283 NFGDQDLIRQGT 294

NFG ++++ GT

The target is as follows: 291NFGGSEMLKLGT 302

(> gi |3132999| gb | AAc16422.1| nucleocapsid protein [ murine hepatitis virus strain 2]

Length 451 ═ 451

Score 147 bits (370), estimate 3e-34

Identity 102/252 (40%), positive 137/252 (54%), void 18/252 (7%)

Inquiring: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106

SWF+ +TQ K +E +F +GQGVPI + +Q GY+ R RR + DG+K+L PRW

The target is as follows: 63 SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQLLPRW 122

Inquiring: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166

YFYYLGTGP A YG + EG+VWVA++ A + R+P+++ A + GT LP

The target is as follows: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTKFAPGTVLP 182

Inquiring: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLN 226

+GFY EGS + AS S N SS PA +A L+L +L

The target is as follows: 183 QGFYVEGSGKSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVLAKLG 242

Inquiring: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK- - -KPRQKRTATKQYNVTQAFGRRGPEQTQG 282

+ GQ +Q VTK+SA E + KPRQKRT KQ V Q FG+RGP Q

The target is as follows: 243K- - -DAQPKQ- - -VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPNQ- - -290

Inquiring: 283 NFGDQDLIRQGT 294

NFG ++++GT

The target is as follows: 291 NFGGSEMLKLGT 302

'gi' 127877| sp | P03417| NcAP _ CVMJH nucleocapsid protein

gi |74859| pir | VHIHMJ nucleocapsid protein-murine hepatitis virus (strain JHM)

gi |58973| emb | CAA25497.1| nucleocapsid protein [ murine hepatitis virus ]

455 length

Score 146 bits (369), estimate 4e-34

Identity 110/254 (43%), positive 142/254 (55%), void 22/254 (8%)

Inquiring: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106

SWF+ +TQ K +E +F +GQGVPI Q GY+ R RR + DG+ K+L PRW

The target is as follows: 67 SWFSGITQFQKGKEFQFAQGQGVPIANGIPASQQKGYWYRHNRRSFKTPDGQQKQLLPRW 126

Inquiring: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166

YFYYLGTGP A YG + EG+VWVA++ A I R+P+++ A + GT LP

The target is as follows: 127 YFYYLGTGPYAGAEYGDDIEGVVWVASQQAETRTSADIVERDPSSHEAIPTRFAPGTVLP 186

Inquiring: 167 KGFYAEGSRGGSQASSRSSSR-SRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDR 224

+GFY EGS G S +SRS SR SRG N SS PA +A L+L +

The target is as follows: 187 QGFYVEGS-GRSAPASRSGSRPQSRG-PNNRARSSSNQRQPASTVKPDMAEEIAALVLAK 244

Inquiring: 225 LNQLESKVSGKGQQQQGQTVTKKSAAEASK- - -KPRQKRTATKQYNVTQAFGRRGPEQT 280

L+ GQ +Q VTK+SA E + KPRQKRT KQ V Q FG+RGP Q

The target is as follows: 245 LGK- - -DAQPKQ- - -VTKQSAKEVRQKILNKPRQKRTPNKQCPVQQCFGKRGPNQ-294

Inquiring: 281 QGNFGDQDLIRQGT 294

NFG ++++ GT

The target is as follows: 295-NFGGPEMLKLGT 306

> gi |6625766| gb | AAF19389.1| AF201929_7 nucleocapsid protein [ murine hepatitis virus strain 2]

gi |7769348| gb | AAF69338.1| AF208066_11 nucleocapsid protein [ murine hepatitis virus ]

Length 451 ═ 451

Score 146 bits (368), estimate 5e-34

Identity 102/252 (40%), positive 137/252 (54%), void 18/252 (7%)

Inquiring: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106

SWF+ +TQ K +E +F +GQGVPI + +Q GY+ R RR + DG+ K+L PRW

The target is as follows: 63 SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQLLPRW 122

Inquiring: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166

YFYYLGTGP A YG + EG+VWVA++ A + R+P+++ A + GT LP

The target is as follows: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTRFAPGTVLP 182

Inquiring: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLN 226

+GFY EGS + AS S N SS PA +A L+L +L

The target is as follows: 183 QGFYVEGSGRSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVLAKLG 242

Inquiring: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK- - -KPRQKRTATKQYNVTQAFGRRGPEQTQG 282

+ GQ +Q VTK+SA E + KPRQKRT KQ V Q FG+RGP Q

The target is as follows: 243K- - -DAQPKQ- - -VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPNQ- - -290

Inquiring: 283 NFGDQDLIRQGT 294

NFG ++++ GT

The target is as follows: 291 NFGGSEMLKLGT 302

(> gi |21734854| gb | AAM77005.1| AF481863_7 phosphorylated nucleocapsid protein N [ porcine hemagglutinating encephalomyelitis virus ]

449 of length

Score 145 bits (366), estimate 8e-34

Identity 107/253 (42%), positive 145/253 (57%), void 18/253 (7%)

Inquiring: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106

SWF+ +TQ K +E F GQGVPI + GY+ R RR + DG ++L PRW

The target is as follows: 64 SWFSGITQFQKGKEFEFAEGQGVPIAPGVPATEAKGYWYRHNRRSFKTADGNQRQLLPRW 123

Inquiring: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGA-LNTPKDHIGTRNPNNNAATVLQLPQGTTL 165

YFYYLGTGP A YG + +G+ WVA+ A +NTP D I R+P+++ A + P GT L

The target is as follows: 124 YFYYLGTGPHAKHQYGTDIDGVFWVASNQADINTPAD-IVDRDPSSDEAIPTRFPPGTVL 182

Inquiring: 166 PKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRL 225

P+G+Y EGS G S +SRS+SR+ N S SR NS R ++ G +A D++

The target is as follows: 183 PQGYYIEGS-GRSAPNSRSTSRA-PNRAPSAGSRSRANSGNRTSTPGVTPDMA-DQI 236

Inquiring: 226 NQLESKVSGKGQQQQGQTVTKKSAAEASK- - -KPRQKRTATKQYNVTQAFGRRGPEQTQ 281

L GK + Q VTK++A E + KPRQKR+ KQ V Q FG+RGP Q

The target is as follows: 237 ASLVLAKLGK-DATKPQQVTKQTAKEVRQKILNKPRQKRSPNKQCTVQQCFGKRGPNQ- -293

Inquiring: 282 GNFGDQDLIRQGT 294

NFG ++++GT

The target is as follows: 294-NFGGGEMLKLGT 305

' gi |23295765| gb | AAL80036.1| nucleocapsid protein [ porcine hemagglutinating encephalomyelitis virus ]

449 of length

Score 145 bits (365), estimate 1e-33

Identity 107/253 (42%), positive 145/253 (57%), void 18/253 (7%)

Inquiring: 49 SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106

SWF+ +TQ K +E F GQGVPI + GY+ R RR + DG ++L PRW

The target is as follows: 64 SWFSGITQFQKGKEFEFAEGQGVPIAPGVPSTEAKGYWYRHNRRSFKTADGNQRQLLPRW 123

Inquiring: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGA-LNTPKDHIGTRNPNNNAATVLQLPQGTTL 165

YFYYLGTGP A YG + +G +WVA + A +NTP D I R+P+++ A + P GT L

The target is as follows: 124 YFYYLGTGPHAKDQYGTDIDGVFWVASNQADINTPAD-IVDRDPSSDEAIPTRFPPGTVL 182

Inquiring: 166 PKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRL 225

P+G+Y EGS G S +SRS+SR+ N S SR NS R++G +A D++

The target is as follows: 183 PQGYYIEGS-GRSAPNSRSTSRA-PNRAPSAGSRSRANSGNRTSTPGVTPDMA-DQI 236

Inquiring: 226 NQLESKVSGKGQQQQGQTVTKKSAAEASK- - -KPRQKRTATKQYNVTQAFGRRGPEQTQ 281

L GK + Q VTK++A E + KPRQKR+ KQ V Q FG+RGP Q

The target is as follows: 237 ASLVLAKLGK-DATKPQQVTKQTAKEVRQKILNKPRQKRSPNKQCTVQQCFGKRGPNQ- -293

Inquiring: 282 GNFGDQDLIRQGT 294

NFG ++++GT

The target is as follows: 294-NFGGGEMLKLGT 305

These results indicate that SEQ ID NO: 10533 are functionally similar to the coronavirus nucleocapsid protein.

In one embodiment, the invention includes the amino acid sequence of 5 '3' reading frame 3 of fig. 127, e.g., SEQ id nos: 10506-10514. Some of the open reading frames encoded within this region are SEQ ID NO: 10575-10578.

Accordingly, the invention includes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 10575. SEQ ID NO: 10576. SEQ ID NO: 10577 and SEQ ID NO: 10578. The invention includes a polypeptide that binds to a polypeptide selected from the group consisting of SEQ ID NO: 10575. SEQ ID NO: 10576. SEQ ID NO: 10577 and SEQ ID NO: 10578 having a sequence identity to the amino acid sequence of the polypeptide. The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10575. SEQ ID NO: 10576. SEQ ID NO: 10577 and SEQ ID NO: 10578, or a fragment of a polypeptide sequence of the amino acid sequence of seq id no.

In one embodiment, the invention includes an amino acid sequence comprising 3 '5' reading frame 2 of fig. 127, such as SEQ id nos: 10547-10559. One open reading frame within this region is SEQ ID NO: 10579.

The invention includes a polypeptide comprising SEQ ID NO: 10579 amino acid sequence. The invention includes a polypeptide comprising a nucleotide sequence substantially identical to SEQ id no: 10579 a polypeptide having an amino acid sequence having sequence identity. The invention includes a polypeptide comprising SEQ ID NO: 10579.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The invention includes polynucleotide sequences comprising one or more of the primer sequences identified in table 33. The invention also includes polynucleotide sequences comprising sequences complementary to one or more of the primer sequences identified in table 33.

The invention includes a polypeptide comprising SEQ ID NO: 11323. Consisting of SEQ ID NO: 11323 one polypeptide encoded by SEQ ID NO: 11324.

the invention includes a polypeptide comprising SEQ ID NO: 11324. and SEQ ID NO: 11324 and SEQ ID NO: 11324 and a polypeptide having a sequence having sequence identity. The invention includes SEQ ID NO: 11324, wherein the polypeptide fragment begins with methionine.

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 11323. The invention also provides a polypeptide corresponding to SEQ ID NO: 11323 and polynucleotide sequences having sequence identity. The invention also provides a polypeptide comprising SEQ ID NO: 11323 and the polynucleotide sequence of the fragment. In one embodiment, the polynucleotide fragment does not consist entirely of the known SARS virus polynucleotide sequence or the known coronavirus polynucleotide sequence.

The invention includes a polynucleotide consisting of the polynucleotide sequence of SEQ ID NO: 11323, comprising the amino acid sequence encoded by SEQ ID NO: 11324.

The invention also provides a polypeptide corresponding to SEQ ID NO: 11323 encodes an amino acid sequence having sequence identity. The invention provides a polypeptide having the sequence shown in SEQ ID NO: 11324 has amino acid sequence with sequence identity.

The present invention provides a polypeptide consisting of SEQ ID NO: 11323 and a fragment of the amino acid sequence encoded thereby. The invention also provides SEQ ID NO: 11324. In one embodiment, the fragment does not consist entirely of the amino acid sequence of a known SARS virus or the amino acid sequence of a known coronavirus.

The invention also includes polynucleotide sequences useful in diagnostic agents, kits (including such agents), and probes for use in methods of diagnosing or identifying the presence of SARS virus in a biological sample. The invention includes a polypeptide comprising the amino acid sequence identified as SEQ ID NO: 11325-11440 (left part) and SEQ ID NOS: 11441-11551 (right part). The invention also includes a polypeptide comprising the amino acid sequence identified as SEQ ID NO: 11325, 11551, and a polynucleotide sequence complementary to the sequence of one or more of the primer sequences.

The invention includes a polypeptide comprising SEQ ID NO: 11552. SARS virus is polymorphic at isoleucine residues Ile-324. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11552, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of seq id nos: YSYAI (SEQ ID NO: 11553), SYAIH (SEQ ID NO: 11554), YAIHH (SEQ ID NO: 11555), IHHDK (SEQ ID NO: 11556), SYAI (SEQ ID NO: 11557), YAIH (SEQ ID NO: 11558), AIHH (SEQ ID NO: 11559), IHHD (SEQ ID NO: 11560), YAI, AIH, and IHH. The invention includes a polypeptide comprising SEQ ID NO: 11552, wherein the fragment comprises an amino acid sequence selected from the group consisting of seq id nos: YSYAI (SEQ ID NO: 11553), SYAIH (SEQ ID NO: 11554), YAIHH (SEQ ID NO: 11555), IHHDK (SEQ ID NO: 11556), SYAI (SEQ ID NO: 11557), YAIH (SEQ ID NO: 11558), AIHH (SEQ ID NO: 11559), IHHD (SEQ ID NO: 11560), YAI, AIH, and IHH.

The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11561 and SEQ ID NO: 11562 in a pharmaceutically acceptable carrier. The invention includes a polypeptide selected from the group consisting of SEQ ID NO: 11561 and SEQ ID NO: 11562 in a pharmaceutically acceptable carrier.

The invention includes diagnostic kits comprising a nucleic acid sequence comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOS: 11561 and 11562. The present invention includes a diagnostic kit comprising a polynucleotide sequence encoding a polypeptide comprising at least one polypeptide selected from the group consisting of SEQ ID NOs: 11561 and 11562. The present invention includes an immunogenic composition comprising a polypeptide comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOS: 11561 and 11562. The present invention includes methods for identifying a polypeptide comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOS: 11561 and 11562.

The invention includes polynucleotide sequences of SEQ ID NO: 11563 or a fragment thereof or a sequence having sequence identity thereto. Can be derived from SEQ ID NO: 11563 the translated polypeptide sequence is shown in FIG. 128. Figure 128 has at least 4 amino acids in the constituent amino acid sequence that are set forth as SEQ ID NOS: 11564-.

The invention includes polypeptide sequences selected from the sequences of fig. 128, or fragments thereof or sequences having sequence identity thereto, such as SEQ ID NOS: 11563-11617.

SEQ ID NO: 11600 is a polypeptide sequence of SEQ ID NO: 11618. the invention includes a polypeptide comprising SEQ ID NO: 11618 or a fragment thereof or a sequence having sequence identity thereto.

SEQ ID NO: 11602 is a polypeptide sequence of SEQ ID NO: 11641. the invention includes a polypeptide comprising SEQ id no: 11641 or a fragment thereof or a sequence having sequence identity thereto.

SEQ ID NO: 11609 is SEQ ID NO: 11619.

the invention includes polynucleotides encoding the following sequences: (i) an amino acid sequence selected from the group consisting of: (1) the amino acid sequence of figure 128, particularly SEQ ID NOS: 11564-11617; (2) SEQ ID NO: 11618; and (3) SEQ ID NO: 11619, or (ii) a fragment thereof. The invention includes diagnostic kits comprising one or more of these proteins. The invention includes diagnostic kits comprising polynucleotide sequences encoding one or more of these polypeptide sequences. The invention includes antibodies that recognize one or more polypeptide sequences.

SARS virus is represented by SEQ ID NO: 11620(Chi-PEP3) may have a polymorphism at isoleucine residue Ile-326. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11620 a polypeptide having an amino acid sequence with sequence identity, wherein said polypeptide comprises an amino acid sequence selected from YAIHH (SEQ ID NO: 11621) and YATHH (SEQ ID NO: 11622). The invention includes a polypeptide comprising SEQ ID NO: 11620, wherein said fragment comprises a polypeptide selected from YA IHH (SEQ ID NO: 11621) and YATHH (SEQ ID NO: 11622).

SARS virus is represented by SEQ ID NO: 11620 may have a polymorphism at Gln-830. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11620 a polypeptide having an amino acid sequence with sequence identity, wherein said polypeptide comprises an AS selected from the group consisting ofQAW (SEQ ID NO: 11623) and ASRThe amino acid sequence of AW (SEQ ID NO: 11624). The invention includes a polypeptide comprising SEQ ID NO: 11620, wherein said fragment comprises an AS selected from the group consisting ofQAW (SEQ ID NO: 11623) and ASRThe amino acid sequence of AW (SEQ ID NO: 11624).

SARS virus is represented by SEQ ID NO:11620 may have a polymorphism at aspartic acid residue Asp-935. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11620 a polypeptide having an amino acid sequence with sequence identity, wherein said polypeptide comprises a sequence selected from the group consisting of DADST (SEQ ID NO: 11625) and DAYThe amino acid sequence of ST (SEQ ID NO: 11626). The invention includes a polypeptide comprising SEQ ID NO: 11620, wherein said fragment comprises a polypeptide selected from the group consisting of DADST (SEQ ID NO: 11625) and DAYThe amino acid sequence of ST (SEQ ID NO: 11626).

SARS virus is represented by SEQ ID NO: 11627(Chi-PEP4) may have a polymorphism at serine residue Ser-577. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11627 polypeptide having an amino acid sequence with sequence identity, wherein said polypeptide comprises a sequence selected from the group consisting of PC SFG (SEQ m NO: 11628) and PCAFG (SEQ ID NO: 11629). The invention includes a polypeptide comprising SEQ ID NO: 11627, wherein said fragment comprises a polypeptide selected from the group consisting of PCSFG (SEQ ID NO: 11628) and PCAFG (SEQ ID NO: 11629).

SARS virus is represented by SEQ ID NO: 11630(Chi-PEP8) may have a polymorphism at valine residues Val-68. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11630, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of LAVVY (SEQ ID NO: 11631) and LAAThe amino acid sequence of VY (SEQ ID NO: 11632). The invention includes a polypeptide comprising SEQ ID NO: 11630, wherein the fragment comprises a polypeptide selected from the group consisting of LAVVY (SEQID NO: 11631) and LAAThe amino acid sequence of VY (SEQ ID NO: 11632).

SARS virus is represented by SEQ ID NO: 11633(Chi-PEP13) has a polymorphism at Ile-50 of isoleucine residue. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11633, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of NNIAS (SEQ ID NO: 11634) and NNTAmino acids of AS (SEQ ID NO: 11635)And (4) sequencing. The invention includes a polypeptide comprising SEQ ID NO: 11633, wherein the fragment comprises NN IAS (SEQ ID NO: 11634) and NNTThe amino acid sequence of AS (SEQ ID NO: 11635).

SARS virus is represented by SEQ ID NO: 11636 may have a polymorphism at Ser-943. The invention includes a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 11636 a polypeptide having an amino acid sequence with sequence identity, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of AVSAC (SEQ ID NO: 11637) and AVGThe amino acid sequence of AC (SEQ ID NO: 11638). The invention includes a polypeptide comprising SEQ ID NO: 11636, wherein said fragment comprises an AV selected fromSAC (SEQ ID NO: 11637) and AVGThe amino acid sequence of AC (SEQ ID NO: 11638).

The invention includes polynucleotides SEQ ID NO: 11639, or a fragment thereof or a sequence having sequence identity thereto. The invention includes a polypeptide consisting of SEQ ID NO: 11639, or a fragment thereof, or a polypeptide sequence having sequence identity thereto.

The invention includes SEQ ID NO: 11640, or a fragment thereof or a sequence having sequence identity thereto. The invention includes a polypeptide consisting of SEQ ID NO: 11640 or a fragment thereof or a polypeptide sequence having sequence identity thereto.

The invention includes each of the polynucleotides identified above. The invention includes each of the polynucleotides listed in the sequence listing. The invention also includes polynucleotides having sequence identity to each of the polynucleotides identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

The invention includes polynucleotide sequences comprising fragments of each of the polynucleotide sequences identified above. The fragment should contain the specific SEQ ID NO: at least n contiguous polynucleotides, n can be 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more), depending on the sequence.

The invention includes the individual amino acid sequences identified above that encode the individual polynucleotide sequences. The invention encompasses the respective amino acid sequences of the respective polynucleotide sequences listed in the sequence listing. The invention also includes amino acid sequences having sequence identity to the amino acid sequences encoded by each of the polynucleotide sequences identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). The invention also includes fragments of the amino acid sequences encoded by each of the polynucleotide sequences identified above. The fragment should contain the specific SEQ ID NO: at least n consecutive amino acids, n can be 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more), depending on the sequence.

The present invention includes the respective amino acid sequences identified above. The invention includes each amino acid sequence listed in the sequence listing. The invention also includes amino acid sequences having sequence identity to each of the amino acid sequences identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

The invention also includes fragments of the amino acid sequences identified above. The fragment should contain the specific SEQ ID NO: at least n consecutive amino acids, n can be 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more), depending on the sequence.

The present invention includes polynucleotides encoding the respective amino acid sequences identified above. The invention includes polynucleotides encoding each of the amino acid sequences listed in the sequence listing. The present invention also includes polynucleotides having sequence identity to polynucleotides encoding the respective amino acid sequences identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

The invention also includes fragments of the polynucleotides encoding the respective amino acid sequences identified above. The fragment should contain the specific SEQ ID NO: at least n consecutive polynucleotides, n can be 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more), depending on the sequence.

As described in more detail below, polynucleotides used as primers and/or probes may contain at least 4 or 8 contiguous nucleotides from a polynucleotide sequence of the invention, e.g., at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides, and may be up to about 50, 75, 100, 200 contiguous nucleotides or more. Since 6-8 nucleotides are of workable length, sequences of 10-12 nucleotides are preferred, with about 13, 14, 15, 16, 17, 18, 19, 20, or 21 or more nucleotides being optimal for hybridization.

In one embodiment, the invention relates to polynucleotides and amino acid sequences that are not entirely comprised of known SARS virus polynucleotides or amino acid sequences, or known coronavirus polynucleotides or amino acid sequences. In one embodiment, the polynucleotide and amino acid sequences of the invention do not consist entirely of the sequence SEQ ID NO: 1. In another embodiment, the polynucleotide and amino acid sequences of the invention do not consist entirely of the sequence SEQ ID NO: 2. SEQ ID NO: 9967 is the SARS genome sequence of Frankfurt (FRA) isolate (GenBank: AY 310120). And SEQ ID NO: 1 compared to it, it differs at nucleotides 2546, 2590, 11437, 18954, 19073, 20585, 20899, 23209, 24922, 26589 and 28257; and SEQ ID NO: 2 compared to it, it differs at nucleotides 2560, 7922, 11451, 16625, 18968 and 19067. Since the initial filing of this application, other genomic sequences have been available from GenBank under accession numbers: AY559097, AY559096, AY559095, AY559094, AY559093, AY559092, AY559091, AY559090, AY559089, AY559088, AY559087, AY323977, AY291315, AY502932, AY502931, AY 502502930, AY502929, AY502927, AY502926, AY502924, AY502923, AY 291923, AY291451, AY390556, AY395003, AY 39391, AY395000, AY 3939393999, AY559096, AY 493949397798, AY 397798, AY 39779, AY 397339779, AY 39773977397776, AY 3977397739779, AY 39779, AY 397324, AY 397739779, AY 397339779, AY 3973397339735639779, AY 39735639779, AY 39735639774939779, AY 397356324690, AY 39779, AY 3239735632469, AY 3239779, AY 3246493973563239735632397370, AY 323970, AY 32397370, AY 3239735632397370, AY 32397370, AY 3239735632397370, AY 397370, AY 32397370, AY 397370, AY 3239735630, AY 3973567, AY 39735648, AY 39735630, AY 39735648, AY 39735632397356323973563239735632397356323973567, AY 39735630, AY 397356324690, AY 39735630, AY 397356323973567, AY 39493973567, AY 3949394939493973567, AY 3973567, AY 39735630, AY 3973567, AY 397356.

In another embodiment, the invention relates to polynucleotides encoding proteins that do not immunologically cross-react with proteins of mouse hepatitis virus, bovine coronavirus, or avian infectious bronchitis virus. In another embodiment, the invention relates to proteins that do not immunologically cross-react with proteins of mouse hepatitis virus, bovine coronavirus, or avian infectious bronchitis virus.

Each of the polynucleotides identified above may be used to encode a portion of a fusion protein. Accordingly, the invention includes one or more of the polynucleotides identified above wherein the polynucleotide encoding the initiation codon has been removed. The invention also includes one or more of the above identified amino acids wherein the initial methionine is removed.

Any of the above polynucleotide or amino acid sequences can be used in a vaccine for treating or preventing SARS virus infection, including as a SARS virus antigen. In addition, any of the polynucleotide or amino acid sequences described above can be used in diagnostic reagents, kits (including such reagents), and methods for diagnosing or identifying the presence of SARS virus in a biological sample.

SARS virus antigens of the invention include polypeptides having 99%, 95%, 90%, 85% or 80% homology to one or more of the following proteins: nonstructural protein 2(NS 2); hemagglutinin-esterase glycoprotein (HE) (also known as E3), spike glycoprotein (S) (also known as E2), non-structural region 4(NS4), envelope (envelope) protein (E) (also known as sM), membrane glycoprotein (M) (also known as E1), nucleocapsid phosphoprotein (N) or RNA-dependent RNA polymerase (pol).

Details of coronavirus biology are found in Fields Virology (second edition), Fields et al (eds.), b.n. raven Press, New York, ny., chapter 35.

Other examples of SARS virus isolates are listed in example 1 below. The invention includes the individual polypeptide and polynucleotide sequences identified in example 1. In addition, the invention includes vaccine formulations comprising one or more of the polypeptide or polynucleotide sequences identified in example 1. The invention includes diagnostic agents, kits (including such agents) and methods for diagnosing or identifying the presence of SARS virus in a biological sample using one or more of the polypeptide or polynucleotide sequences identified in example 1. The invention includes methods of treating or preventing SARS virus infection with small molecule viral inhibitors and combinations of small molecule viral inhibitors and kits to treat SARS. The small molecule inhibitor can specifically target one or more of the polypeptides or polynucleotides identified in example 1.

The following is a further description of the terminology used in the present invention.

"respiratory virus" as used herein refers to a virus capable of infecting the human respiratory tract. Respiratory viral antigens suitable for use in the present invention include severe acute respiratory syndrome virus, coronavirus, influenza virus, Human Rhinovirus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus (RSV), adenovirus, supper pneumovirus and rhinovirus.

The terms "polypeptide", "protein" and "amino acid sequence" are used herein to refer generally to a polymer of amino acid residues, without limitation to the minimum product length. Thus, peptides, oligopeptides, dimers, multimers, and the like are included within the definition. Both full-length proteins and fragments thereof are encompassed within the definition. The smallest polypeptide fragment useful for the present invention may have at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or even 15 amino acids. In general, polypeptides useful in the invention can have a maximum length suitable for the desired application. Typically, the maximum length is not a strict criterion and is readily selected by one skilled in the art.

The polypeptides of the invention can be prepared by various methods, such as by chemical synthesis (at least in part), by digestion of longer polypeptides with proteases, by translation of RNA, by purification from cell culture (e.g., recombinant expression), from the organism itself (e.g., from viral culture or directly from the patient), from cell line sources, and the like. Preferred methods for making peptides less than 40 amino acids in length include in vitro chemical Synthesis (Bodanszky (1993) Principles of Peptide Synthesis (ISBN: 0387564314); Fields et al (1997) methods in enzymology 289: solid phase Peptide Synthesis. ISBN: 0121821900). Particular preference is given to solid-phase peptide synthesis, e.g.based on t-Boc or Fmoc (Chan) &White (2000) Fmoc Solid Phase peptide Synthesis (Fmoc Solid Phase peptide Synthesis) ISBN: 0199637245). Enzymatic Synthesis (Kullmann (1987) by Enzymatic Peptide Synthesis (ISBN: 0849368413) may also be used in part or in whole. In addition to chemical synthesis, biological synthesis may also be used, for example, by translation to produce a polypeptide. This can be done in vitro or in vivo. Biological methods are generally limited to the production of L-amino acid-based polypeptides, but various translation mechanisms (e.g., aminoacyl tRNA molecules) can be used to introduce D-amino acids (or other unnatural amino acids such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) (Ibba (1996) Biotechizol Geizet Eng Rev 13: 197-216). When a D-amino acid is included, chemical synthesis is preferably used. The C-terminal and/or N-terminal of the polypeptides of the invention may be covalently modified, especially when they are used for in vivo administration, for example as in Fuzeon^TMThe product is modified by the addition of acetyl or carbamoyl.

When referring to polypeptides, derivatives of the amino acid sequences of the invention are also included. Such derivatives may include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Amino acid derivatives may also comprise modifications to the native sequence, such as deletions, insertions and substitutions (generally conservative in nature), as long as the protein maintains the desired activity. These modifications may be made artificially, e.g., by site-directed mutagenesis, or may be accidental, e.g., by mutation of the host producing the protein or by error due to PCR amplification. In addition, the modification may cause one or more of the following effects: the toxicity is reduced; ease of cellular processing (e.g., secretion, antigen presentation, etc.); and ready to present B-cells and/or T-cells.

"fragment" or "protein" as used herein refers to a polypeptide consisting of only a portion of the entire full-length polypeptide sequence and structure as found in nature. For example, a fragment may include a C-terminal deletion and/or an N-terminal deletion of a protein.

A "recombinant" protein is a protein made by recombinant DNA techniques described herein. Typically, the gene of interest is cloned and then expressed in a transformed organism, as described below. The host organism expresses the foreign gene under expression conditions to produce the protein.

The term "polynucleotide" generally refers in the art to a nucleic acid molecule. "Polynucleotide" may include double-and single-stranded sequences, including, but not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, viral genomic RNA and DNA sequences (e.g., RNA viruses and DNA viruses, and retroviruses), or prokaryotic DNA, and in particular synthetic DNA sequences. The term also includes sequences containing any known base analogs of DNA and RNA, and includes modifications, such as deletions, additions, and substitutions (typically conserved in nature), of the native sequence, so long as the nucleic acid molecule encodes a therapeutic or antigenic protein. These modifications may be made manually, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the antigen. Modification of a polynucleotide can produce a variety of effects, including, for example, expression of the polypeptide product in a host cell.

Polynucleotides of the invention can be prepared by a variety of methods, e.g., by chemical synthesis, in whole or in part, from genomic or cDNA libraries (e.g., phosphoramidite synthesis of DNA), digestion of longer nucleic acids with nucleases (e.g., restriction enzymes), ligation of shorter nucleic acids or nucleotides (e.g., with ligases or polymerases), and the like.

The polynucleotide may encode a biologically active (e.g., immunogenic or therapeutic) protein or polypeptide. Depending on the nature of the polypeptide encoded by the polynucleotide, the polynucleotide may comprise as few as 10 nucleotides, for example a polynucleotide encoding an antigen.

"isolated" when used with respect to a polynucleotide or polypeptide means that the referenced molecule is isolated and dissociated from the intact organism in which it naturally occurs, or, when the polynucleotide or polypeptide is not naturally occurring, substantially free of other biological macromolecules so that the polynucleotide or polypeptide can be used for a desired purpose. The polynucleotides and polypeptides of the present invention are preferably isolated polynucleotides and isolated polypeptides.

As known in the art, an "antibody" comprises one or more biological moieties that chemically or physically bind to an epitope on a polypeptide of interest. Antibodies of the invention include antibodies that specifically bind to an antigen of the SARS virus. The term "antibody" includes antibodies obtained from polyclonal and monoclonal preparations, as well as: hybrid (chimeric) antibody molecules (see, e.g., Winter et al (1991) Nature 349: 293-299; and U.S. Pat. No. 4816567; F (ab') ₂And F (ab) a fragment; f_vMolecules (non-covalent heterodimers, see, e.g., Inbar et al (1972) Proc Natl Acad Sci USA 69: 2659-; single chain Fv molecules (sFv) (see, e.g., Huston et al (1988) Proc Natl Acad Sci USA 85: 5897-; dimeric and trimeric antibody fragment constructs; bodies (minibodies) (see, e.g., Pack et al (1992) Biochem 31: 1579-1584; Cumber et al (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, e.g., Riechmann et al (1988) Nature 332: 323-327; Verhoeyan et al (1988) Science 239: 1534-1536; and British patent publication No. 21/9/1994Application GB 2276169); and any functional fragment of these molecules, wherein such fragment retains the immunological binding properties of the parent antibody molecule. The term "antibody" also includes antibodies obtained by non-conventional methods, such as phage display.

The term "monoclonal antibody" as used herein refers to an antibody composition comprising a homogeneous population of antibodies. The term is not limited by the kind or source of the antibody, nor by the method of producing the antibody. Thus, the term includes antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained from human, rather than murine, hybridomas. See, e.g., Cote et al, "Monoclonal Antibodies and Cancer Therapy" (Monoclonal Antibodies and Cancer Therapy), alanr.

An "immunogenic composition" as used herein refers to a composition comprising an antigenic molecule, and the application of such a composition to a subject will result in the subject generating a humoral and/or cellular immune response to the antigenic molecule of interest. The immunogenic composition can be introduced directly into the subject, for example, by injection, inhalation, oral, intranasal or other parenteral route of administration, mucosal or transdermal (e.g., intrarectal or intravaginal) route of administration.

The term "derived from" is used to indicate the source of the molecule (e.g., the molecule may be derived from a polynucleotide, a polypeptide, an immortalized cell line may be derived from any tissue, etc.). A first polynucleotide is "derived from" a second polynucleotide if it contains a base pair sequence that is identical or substantially identical to a region of the second polynucleotide, its cDNA, or its complement, or if there is sequence identity as described above. Thus, a first polynucleotide sequence is "derived from" a second sequence if it has (i) a sequence that is identical or substantially identical to the second sequence, or (ii) sequence identity to a polypeptide of that sequence.

A first polypeptide is "derived from" a second polypeptide if it is (i) encoded by a first polynucleotide derived from the second polynucleotide, or (ii) exhibits the sequence identity described above for the second polypeptide. Thus, a polypeptide (protein) "derived from" a SARS virus is said to be a specific SARS virus if the polypeptide (protein) is (i) encoded by an open reading frame of a SARS virus polynucleotide or (ii) exhibits the above-described sequence identity to a SARS virus polypeptide.

Both polynucleotide and polypeptide molecules can be derived from the SARS virus by physical methods or, for example, can be produced recombinantly or synthetically based on known sequences.

A cultured cell or cell line is "derived" from another cell or tissue if it originally obtained its own cell or tissue. Non-limiting examples of tissues from which cells can be obtained include skin, retina, liver, kidney, heart, brain, muscle, intestine, ovary, breast, prostate, cancerous tissue, tissue infected with one or more pathogens (e.g., viruses, bacteria, etc.), and the like. The cells described herein may also be derived from other cells, including but not limited to primary cultures, existing immortalized cell lines, and/or other isolated cells.

An "antigen" refers to a molecule that contains one or more epitopes (which may be linear or conformational epitopes, or both) that will stimulate the host's immune system to mount a humoral and/or cellular antigen-specific response. The term may be used interchangeably with the term "immunogen". Typically, an epitope will comprise about 3-15, typically about 5-15 amino acids. B-cell epitopes usually contain about 5 amino acids, but may also contain fewer, e.g., 3-4 amino acids. T-cell epitopes, such as CTL epitopes, will contain at least 7-9 amino acids and helper T-cell epitopes contain at least about 10-20 amino acids. Typically, an epitope will contain about 7-15 amino acids, such as 9, 10, 12, or 15 amino acids. The term "antigen" refers to subunit antigens (i.e., antigens that are isolated and dissociated from the intact organism with which the antigen naturally binds), as well as killed, attenuated, or inactivated bacterial, viral, fungal, parasitic, or other microbial and tumor antigens, including the extracellular domains of cell surface receptors and the intracellular portions containing T-cell epitopes. Antibodies, such as anti-idiotypic antibodies, or fragments thereof, as well as synthetic peptide mimotopes (which can mimic an antigen or antigenic determinant) are also included within the definition of antigen as used herein. Similarly, oligonucleotides or polynucleotides capable of expressing an antigen or antigenic determinant in vivo (e.g., in gene therapy and DNA immunization) are also included within the definition of antigen herein.

An "immune response" to an antigen or composition is a subject's humoral and/or cellular immune response to the antigen in the composition of interest. For the purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a "cellular immune response" is T-lymphocyte and/or other self-cell mediated. An important aspect of cellular immunity relates to the antigen-specific response of cytolytic T-cells ("CTLs"). CTLs are specific for peptide antigens presented in association with proteins expressed on the cell surface encoded by the Major Histocompatibility Complex (MHC). CTLs help to induce and promote the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another approach to cellular immunity involves antigen-specific responses of helper T-cells. "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other leukocytes, including those derived from CD4+ and CD8+ T-cells. In addition, various leukocytes or endothelial cells can induce chemokine production in response to administered antigens.

Vaccine formulations

The present invention relates to vaccine formulations for the treatment or prevention of Severe Acute Respiratory Syndrome (SARS). The vaccine formulations of the present invention include inactivated (or killed) SARS virus, attenuated SARS virus, split SARS virus preparations, and recombinant or purified subunit preparations of one or more SARS virus antigens. The present invention includes polypeptides and polynucleotides encoding SARS virus antigens and immunogenic fragments thereof. Expression and delivery of the polynucleotides of the invention is facilitated by viral vectors and/or viral particles, including virus-like particles (VLPs).

A. Inactivated (or killed) SARS vaccine

The present invention includes compositions comprising inactivated (or killed) SARS virus and methods of making the same. The inactivated SARS virus composition can be used for preventive or therapeutic SARS virus vaccine. Preferably, the inactivated SARS virus vaccine composition comprises an amount of inactivated SARS virus corresponding to a viral titer of about 4-7log Plaque Forming Units (PFU) or 4-7log half of a Tissue Culture Infectious Dose (TCID) per milliliter prior to inactivation₅₀). More preferably the virus titer prior to inactivation is 4-11, 7-11 or 9-11PFU or TCID₅₀. More preferably, the inactivated SARS virus vaccine composition comprises an amount of inactivated SARS virus corresponding to a viral titer of about 5-9PFU or 5-9TCID per ml prior to inactivation ₅₀. In one embodiment, the PFU or TCID of SARS virus is cultured₅₀6-8 at harvest, more preferably about 7.5PFU or TCID per ml₅₀. In terms of concentration at harvest of virus, PFU or TCID₅₀Preferably 8 to 11, more preferably about 9PFU or TCID per ml₅₀. The vaccine composition contains a SARS virus antigen in an amount sufficient to generate an immune response in a primate.

Methods known in the art to inactivate or kill viruses are to destroy the ability of the virus to infect mammalian cells. Such methods include chemical methods or physical methods. Chemical methods for inactivating SARS virus include treating the virus with an effective amount of one or more of the following: detergent, formaldehyde, formalin, beta-propiolactone or UV light. Other chemical inactivation methods include treatment with methylene blue, psoralen, carboxyfullerene (C60), or combinations thereof. Other methods of viral inactivation are known in the art, such as binary ethylamine (binary ethyl amine), acetyl aziridine, or gamma irradiation.

For example, formaldehyde may be used at a concentration of, e.g., 0.1 to 0.02%, preferably 0.02 to 0.1%, more preferably 0.04 to 0.05%. An inactivating agent is added to the culture supernatant either before or after harvesting the culture supernatant from the vessel used to transmit the virus, and the cells can be disrupted or destroyed to release the virus bound to the cells prior to harvesting. In addition, the inactivating agent may be added after the culture supernatant has been cryopreserved and thawed, or after one or more purification steps to remove cellular contaminants. However, it is preferred to add formaldehyde after removal of cells and cell debris or after one or more purification steps. After the addition of formaldehyde, the virus-containing mixture is transferred to an incubation container and incubated for 12 hours to 7 days at refrigerated temperatures (e.g. +2 to 8 ℃) or at higher temperatures, such as room temperature of about 20-30 ℃ or 33-37 ℃, wherein the temperature chosen should be compatible with the incubation time. Preferred conditions are, for example, 3 to 7 days (preferably 3 to 7 days) at +2 to 8 ℃, 16 hours to 3 days (preferably 24 to 48 hours) at room temperature, or 12 to 36 hours at 35 to 37 ℃. If it is desired to remove excess formalin, sodium thiosulfate or sodium metabisulfite can be added in equimolar or 1.5-fold molar concentration (relative to formaldehyde) after the end of the inactivation process.

For example, beta-propiolactone may be used at a concentration of, for example, 0.01 to 0.5%, preferably 0.5% to 0.2%, more preferably 0.025 to 0.1%. The step of disrupting the cells to release the cell-bound virus may or may not be performed prior to harvesting the culture supernatant (viral material) containing the virus, with or without an inactivating agent added to the culture supernatant prior to or after harvesting the culture supernatant from the vessel used to transmit the virus. In addition, the inactivating agent may be added after the culture supernatant has been cryopreserved and thawed, or after one or more purification steps to remove cellular contaminants. Beta-propiolactone is added to the viral material and sodium hydroxide (e.g., 1N NaOH), Tris buffer or sodium bicarbonate solution is used to control the pH shift towards acidity which can be detrimental. After transferring the mixture to another inactivation vessel, the inactivator-viral material mixture is incubated at 4-37 ℃ for a period of time, preferably 24-72 hours.

Other inactivators that may be used are Binary Ethyleneimine (BEI). Equal volumes of 0.2 molar bromoethylamine hydrobromide solution and 0.4 molar sodium hydroxide solution were mixed and incubated at about 37 deg.C for 60 minutes. The cyclized inactivating agent is then binary aziridine, which is added to the viral material in a volume ratio of 0.5 to 4%, preferably 1 to 3%. The inactivated viral material is left at about 4-37 ℃ for 24-72 hours with agitation. At the end of the incubation, 20ml of a 1 molar sterile sodium thiosulfate solution was added to ensure that the BEI was neutralized.

In one embodiment, the invention includes an inactivation method designed to maximize viral exposure to the inactivating agent and minimize exposure of temperature sensitive SARS virus particles to elevated temperatures. The invention includes a method of inactivation comprising contacting the virus with an inactivating agent (e.g., BPL) at refrigeration temperatures for 12-24 hours, followed by an increase in temperature for 3 hours to hydrolyze any remaining inactivating agent. Preferably, the refrigeration temperature is from 0 to 8 deg.C, more preferably about 4 deg.C. Preferably the elevated temperature is from 33 to 41 deg.C, more preferably about 37 deg.C. Evaluation of residual infectious virus in each 10ml of the inactivated preparation revealed that this method was able to inactivate SARS-CoV in the primary cell culture harvest to below the theoretical value of 0.03 infectious units/ml.

Diluted and undiluted samples of inactivated virus material are added to a susceptible cell (tissue) culture (e.g., VERO) to detect any non-inactivated virus. The cultured cells are passaged multiple times and tested for the presence of SARS virus by any of a number of methods such as cytopathic effect (CPE) and antigen detection (e.g., by a fluorescein antibody conjugate specific for SARS virus). This test enables the determination of complete virus inactivation.

Before inactivation, SARS virus was cultured in mammalian cell culture. The cell culture may be adherently growing cells or cells growing in suspension. Preferably the cells are of mammalian origin, but may also be derived from birds (e.g. chicken cells such as chicken embryo cells (CEF cells)), amphibians, reptiles, insects or fish. Mammalian sources of cells include, but are not limited to, human or non-human primate cells (e.g., MRC-5 (ATCCCL-171), WI-38(ATCC CCL-75), HeLa cells, human diploid cells, rhesus fetal lung cells (e.g., ATCC CL-160), human embryonic kidney cells (293 cells, typically transformed with sheared adenovirus type 5 DNA), Vero cells (e.g., monkey kidney Vero cells), horses, cows (e.g., MDBK cells), sheep, dogs (e.g., dog kidney MDCK cells, ATCCCL 34MDCK (NBL2) or MDCK 33016, deposited under the accession number DSM 2219 as described in WO 97/37001), cats and mice (e.g., hamster cells, such as BHK21-F, HKCC cells or Chinese hamster ovary cells (CHO cells)), and can be obtained from various stages of development, including, for example, adult, neonatal, fetal or embryonic.

In some embodiments, the cell is immortalized (such as a perc.6 cell as described in, for example, WO 01/38362 and WO02/40665 and as deposited with ECACC under the reference number 96022940, both of which are incorporated herein by reference in their entirety), or any other cell type that is immortalized using the techniques described herein.

In a preferred embodiment mammalian cells are used, such cells may be selected from and/or derived from one or more of the following non-limiting cell types: fibroblasts (e.g., dermal cells, lung cells), endothelial cells (e.g., aortic cells, coronary cells, lung cells, vascular cells, skin microvascular cells, umbilical cord cells), hepatocytes, keratinocytes, immune cells (e.g., T cells, B cells, macrophages, NK cells, dendritic cells), breast cells (e.g., epithelial cells), smooth muscle cells (e.g., vascular cells, aortic cells, coronary cells, arterial cells, uterine cells, bronchial cells, cervical cells, periretinal cells), melanocytes, neural cells (e.g., astrocytes), prostate cells (e.g., prostate epithelium, smooth muscle cells), kidney cells (e.g., epithelial cells, glomerular cells, proximal tubule cells), skeletal cells (e.g., chondrocytes, osteoclasts, osteoblasts), muscle cells (e.g., myoblasts, skeletal cells, smooth cells, bronchial cells), hepatocytes, retinoblasts, and stromal cells. WO 97/37000 and WO 97/37001 (incorporated herein by reference in their entirety) describe the production of animal cells and cell lines that can be grown in suspension and serum-free media and that can be used to produce and replicate viruses.

Preferably, the SARS virus of the invention is cultured in VERO cells or rhesus fetal kidney cells.

Culture conditions for the above cell types have been described in detail in various publications, or alternative media, additives and culture conditions are commercially available, such as catalogues and other references of Cambrex Bioproducts (East Rutherford, N.J.).

In some embodiments, the host cells used in the methods described herein are cultured in serum and/or protein free media. Herein, a serum-free medium is referred to as a serum-free medium when the medium does not contain a human or animal-derived serum supplement. Protein-free refers to a culture in which cells proliferate without proteins, growth factors, or other protein supplements and non-serum proteins. The cells grown in this culture naturally contain proteins themselves.

Known serum-free media include Iscove's medium, Ultra-CHO medium (BioWhittaker), or EX-CELL (JRH bioscience). Conventional serum-containing media include Eagle minimal medium (BME) or Minimal Essential Medium (MEM) (Eagle, Science, 130, 432(1959)) or Dulbecco's modified Eagle medium (DMEM or EDM), which typically contains up to 10% fetal bovine serum or similar supplements. Optionally, Minimal Essential Medium (MEM) (Eagle, Science, 130, 432(1959)) or Dulbecco's modified Eagle medium (DMEM or EDM) without any serum supplements may be used. Protein-free media, such as PF-CHO (JHRBIOSCE), chemically defined media such as ProCHO 4CDM (BioWhittaker) or SMIF7(Gibco/BRL Life Technologies), and mitogenic peptides such as Primactone, Peptidase or HyPepTM (all from Quest International) or whey protein hydrolysate (Gibco among other manufacturers) are also known in the art. A particular advantage of using plant hydrolysate-based media supplements is that contamination by viruses, mycoplasma or unknown infectious agents can be excluded.

The cell culture conditions (temperature, cell density, pH, etc.) used for the desired purpose vary widely to suit the cell line used in the present invention and to suit the requirements of the SARS virus.

A method for propagating SARS virus in cultured cells (e.g., mammalian cells) comprises the steps ofThe method comprises the following steps: inoculating the cultured cells with the SARS virus, culturing the infected cells for a time necessary to allow the virus to proliferate, the culturing time will be determined by, for example, SARS virus titer or SARS virus antigen expression (e.g., about 24-168 hours after inoculation), and collecting the proliferated virus. By virus (through PFU or TCID)₅₀Determination) cell ratio of 1: 10000 to 1: 10 SARS virus was inoculated into the cultured cells. A lower ratio range may be used, for example from 1: 500 to 1: 1, preferably from 1: 100 to 1: 5, more preferably from 1: 50 to 1: 10. The SARS virus is added to a cell suspension or cell monolayer and the virus is adsorbed to the cells for at least 60 minutes, typically less than 300 minutes, preferably from 25-40 ℃ for 90-240 minutes, more preferably from 28-37 ℃ and most preferably 33 ℃. Infected cell cultures (e.g., monolayers) are treated by freeze-thawing or by enzymatic action to increase the virus concentration in the harvested culture supernatant. The harvested fluid may then be inactivated or stored frozen.

FIG. 26A is a comparison of SARS-infected Vero cells cultured in the presence and absence of fetal calf serum ("FCS"). Briefly, Vero cells were dispensed and cultured in T175 flasks the day before infection. The second was infected with SARS-SoV stock (FRA strain, passage 4, accession AY310120) with or without 3% FCS at 90% confluency with a Vero cell monolayer. Addition of FCS to the cell culture medium showed little effect on virus production.

The cultured cells may be infected at a multiplicity of infection ("m.o.i.") of about 0.0001-10, preferably 0.002-5, more preferably 0.001-2. More preferably, the cells are infected at an m.o.i. of about 0.01. A comparison of virus yields at different m.o.i. levels is shown in fig. 26B.

Infected cells were harvested 30-60 hours post infection. Preferably, the cells are harvested 34-48 hours post infection. More preferably, the cells are harvested 38-40 hours post infection. See fig. 26C.

Methods for purifying inactivated viruses are known in the art and include one or more of, for example, gradient centrifugation, ultracentrifugation, continuous flow ultracentrifugation, and chromatography, such as ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography. Other purification methods include ultrafiltration and diafiltration. See JP Gregersen "Herstellung von Virusssimpfstoffen aus Zellkulturen", Pharmazeutische Biotechology section 4.2 (O.Kayser and RH Mueller eds.) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000. See also O' Neil et al, "Virus harvesting and liquid affinity chromatography. Methods for concentrating and purifying viruses "(Virus Harvesting and Affinity Based Liquid chromatography. AMethod for Virus targeting and Purification), Biotechnology (1993) 11: 173-; prior et al, "improved method for making Inactivated HIV-1" (Process development for Manufacture of Inactivated HIV-1), Pharmaceutical Technology (1995) 30-52; and Majhdi et al, "Isolation and characterization of Coronavirus from diarrhea Elk Calves" (Isolation and characterization of a Coronavir from Elk Calves with direct) Journal of clinical Microbiology (1995)35 (11): 2937-2942.

Examples of other Purification Methods suitable for use in the present invention include polyethylene glycol Precipitation or ammonium sulfate surface Precipitation (see Trepanier et al, "Concentration of human respiratory syncytial Virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration" (convention of human respiratory syncytial Virus (1981)3 (4): 201. sup. 211; Hagen et al, "Optimization of polyethylene glycol precipitated hepatitis Virus for the preparation of high purity inactivated vaccine VAQTA" (timing of Purification of hepatitis Virus) to preparation of vaccine VAQTA, 1994 a high purity Purified infectious Necrosis Virus infection vaccine, 12: 412 and "Purification of infectious Necrosis Virus by ion Exchange of protein anion 47" (see Trepanier et al: 36: Virus of infectious Necrosis Virus), and ultrafiltration and microfiltration (see Pay et al, development in Biological staging (1985) 60: 171- & 174; Tsouumi et al, "Structure and filtration Performance of modified cuprammonium regenerated cellulose hollow fiber (modified BMM hollow fiber) for Virus removal" (Structure and filtration and purification for use in enhanced cuprammonium regenerated cellulose hollow fiber) (enhanced BMM hollow fiber) Polymer Journal (1990) 22: 1085- & 1100; and Makino et al, "Concentration of liver retrovirus with regenerated cellulose hollow fiber M" (Concentration of liver retrovirus with a regenerated liver tissue) 139 (1-87).

Preferably, the virus is purified by chromatography, such as ion exchange chromatography. Chromatographic purification can produce large amounts of virus-containing suspension. The viral product of interest can interact with the chromatography medium by a simple adsorption/desorption mechanism, allowing for the processing of large volumes of sample in one operation. Impurities that do not have affinity for the adsorbent flow through the column. The viral material may be eluted in concentrated form.

Preferred anion exchange resins for use in the present invention include DEAE, EMI) TMAE. Preferred cation exchange resins may have sulfonic acid modified surfaces. In one embodiment, the first step is ion exchange chromatography using a column containing a strong anion exchange resin (e.g., EMD TMAE), and the second step is EMD-SO₃(cation exchange resin) purification of the virus. Further purification can optionally be achieved by metal binding affinity chromatography (see, e.g., WO 97/06243).

The preferred resin for use in the present invention is Fractogel^TMEMD. Such synthetic methacrylate resins have long linear polymer chains (so-called "tentacles") covalently bound. This "tentaclechemical" allows a large number of sterically accessible ligands to bind to biomolecules without steric hindrance. Such resins also have improved pressure stability.

Column-based liquid affinity chromatography is another preferred purification method for use in the present invention. An example of a resin for use in such a purification process is Matrex^TMCellufineTM Sulfate (MCS). The MCS consists of a rigid spherical (diameter about 45-105 μm) cellulose matrix, the exclusion poles of whichWith a limit of 3000 daltons (the largest molecule allowed by its pore structure) and a low concentration of sulfate functional groups at the 6-position of cellulose. Due to the relatively high dispersion of the functional ligand (sulfate), it provides insufficient cation exchange density for most soluble proteins to adsorb to the bead surface. Thus, the large amount of protein found in typical virus fluids (cell culture supernatants, such as pyrogens and most contaminating proteins, as well as nucleic acids and endotoxins) is washed off the column, resulting in some degree of purification of the bound virus.

Rigid high strength MCS beads have a tendency to resist compression. The pressure/flow characteristics of the MCS resin allow for high linear flow rates and thus high speed processing, even in large columns, which makes it easy to scale up the unit operation. The MCS chromatography purification step improves the safety and the sterility of the product, and also avoids excessive product treatment and safety problems. Since endotoxin does not bind to it, the MCS purification step enables rapid and contamination-free depyrogenation. Mild conjugate elution conditions provide high capacity and product yield. Thus, the MCS resin is a simple, fast, efficient and economical method of concentration, purification and depyrogenation. Furthermore, the MCS resin can be reused.

Inactivated virus may also be further purified by gradient centrifugation, preferably density gradient centrifugation. Continuous flow sucrose gradient centrifugation is preferred for commercial scale operations. This method is widely used for the purification of antiviral vaccines and is well known to the person skilled in the art (see JP Gregeren "Herstellung von Virussian fstoffsfen aus Zellkulturen", Pharmazeutische Bioteconologic 4.2 section (O.Kayser and RH Mueller eds.) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.)

The density gradient centrifugation step may be performed using a laboratory scale or commercial scale gradient centrifugation apparatus. For example, a float bowl, fixed angle, or vertical tube rotor is particularly suitable for laboratory scale virus production. Preferably, the gradient centrifugation step is performed using a floating bucket rotor. This type of brick has a channel length long enough to provide high quality separation, especially for multi-component samples. In addition, the pontoon-type rotor has a greatly reduced wall effect and the contents do not reorient during acceleration and deceleration. Due to their longer channel length, separation takes longer than with fixed angle or vertical pipe swivels. The sucrose concentration of the prepared sucrose solution can be controlled by a refractometer.

The sucrose gradient for density gradient centrifugation, such as a keg float centrifuge tube, can be formed (continuous/linear) by a gradiometer prior to centrifugation. The volume of sample added to the gradient liquid in the float bowl rotor tube is a function of the cross-sectional area of the gradient liquid contacting the sample. If the sample volume is too large, the centrifuge tube does not have sufficient radial length to effectively separate the components in a multi-component sample.

The sample volume for the float bowl SW 28 was about 1-5ml per tube (tube diameter 2.54 cm). The sample is applied to the top of the gradient by pipette. The flat end of the pipette is placed at an angle of 45-60 ° to the wall of the tube, about 2-3 mm above the gradient. The sample was slowly injected and allowed to flow along the walls of the tube onto the gradient. After centrifugation, the gradient fractions were recovered by carefully inserting a metering needle down to the bottom of the tube and pumping the liquid from the tube into a falcon tube to begin collection of 2ml of each fraction.

Sucrose density gradients suitable for use in the density gradient centrifugation purification step include 0-60%, 5-60%, 15-60%, 0-50%, 5-50%, 15-50%, 0-40%, 5-40%, and 15-40%. Preferably, the sucrose density gradient is 15-40%, 5-40% or 0-40%.

Alternatively, purification can be performed using a discontinuous sucrose density gradient. The discontinuous sucrose density method uses separate, overlapping layers of sucrose of different concentrations. In one example, the first layer is 50% sucrose, overlaid with the second layer 40% sucrose, the second layer is overlaid with the third layer 20% sucrose, the third layer is overlaid with the fourth layer 10% sucrose, and the fourth layer is overlaid with a solution containing the virus to be purified.

In one embodiment, the method of purification of inactivated virus comprises a first chromatographic purification and a second gradient centrifugation. Preferably, the first step comprises liquid affinity chromatography, such as MCS. Preferably the second step comprises density gradient centrifugation with a floating bucket rotor.

Other purification methods for the purification of inactivated SARS virus include the use of nucleic acid degrading agents, preferably nucleic acid degrading enzymes such as nucleases having DNase and RNAse activity, or endonucleases such as those of Serratia marcescens (Serratiamarcesses) under the trade name Benzonase^TMAnionic functional group-containing membrane adsorbents (e.g., Sartobind)^TM) Or other chromatography steps with anionic functional group containing fillers (e.g., DEAE or TMAE). Ultrafiltration/diafiltration and a final sterile filtration step may also be added to the purification process.

Preferably, the purification method comprises treating the SARS virus isolate with one or more nucleic acid degrading enzymes. These enzymes can be used to reduce the levels of host cell nucleic acids in virus purification processes. Nucleic acid degrading enzymes for use in cell culture are known in the art and include, for example, Benzonase^TM。

The treatment of the virus with the nucleic acid degrading enzyme and the inactivating agent may be performed sequentially or in a combined manner or simultaneously. Preferably, the nucleic acid degrading agent is added to the virus preparation prior to the addition of the inactivating agent.

The purified virus preparations of the invention are substantially free of contaminated proteins from cells or cell cultures, preferably contain less than about 1000, 500, 250, 150, 100 or 50pg cellular nucleic acid per microgram of viral antigen, preferably less than about 1000, 500, 250, 150, 100 or 50pg cellular nucleic acid per dose. More preferably, the purified virus preparation contains less than about 20pg, more preferably less than about 10pg, cellular nucleic acids. Methods for detecting host cell nucleic acid levels in a viral sample are known in the art. Standard methods approved or recommended by regulatory agencies such as the WHO or FDA are preferred.

The present invention includes an inactivated vaccine composition comprising a prophylactically effective amount of SARS virus antigen, preferably spike or immunogenic fragment thereof. The SARS virus antigen is preferably present in a concentration of 0.1 to 50. mu.g antigen/dose, more preferably 0.3 to 30mg antigen/dose. More preferably, the amount of antigen is about 15 μ g/dose.

In one embodiment, a lower concentration of SARS virus antigen is employed in the inactivated vaccine composition of the invention. Such lower concentration vaccines may optionally contain adjuvants to elicit an immune response in the host to the antigen. In such "low dose" vaccines, the SARS virus antigen is preferably present at a concentration of less than 15 μ g antigen/dose, i.e. less than 10, 7.5, 5 or 3 μ g antigen/dose.

The inactivated vaccine preparation of the present invention further comprises a stabilizer to maintain the integrity of the immunogenic proteins in the inactivated virus preparation. Stabilizers suitable for use in vaccines are known in the art and may include, for example, buffers, sugars, sugar alcohols, and amino acids. The stability buffer is preferably adjusted to a physiological pH range and may comprise phosphate buffer, Tris buffer, TE (Tris/EDTA), TEN (Tris/NaCl/EDTA) S and Earle' S salt solution. Stabilizing sugars may include, for example, sucrose, glucose, fructose, dextran, glucose sulfate, and trehalose. Stabilizing sugar alcohols may include, for example, xylitol, mannitol, sorbitol, and glycerol. Amino acids suitable for use in the present invention include, for example, L-glutamine, arginine, cysteine and lysine, and other stabilizers useful in the present invention include tartaric acid, Pluronic F68 and Tween 80.

SARS virus isolates useful in the inactivated virus preparations of the invention can be obtained and identified by any of the methods described above. For example, SARS isolates can be obtained from clinical samples and purified plaque. Such methods of isolating viruses are known in the art.

Other purification methods may be employed to ensure that the viral seeds used to prepare the vaccine are free of, for example, unwanted foreign agents. In one embodiment, viral RNA from a viral isolate can be isolated from the virus, purified (and optionally subsequently confirmed by PCR or other methods), and then introduced into an appropriate cell culture.

As an example of such a technique, clinical virus samples are plaque purified and propagated on vero cells to produce sufficient virus samples for analysis. Cell debris in the supernatant was removed by centrifugation. The virus can then be ultracentrifuged and the viral pellet resuspended in PBS. After further centrifugation purification, the virus-containing fraction is treated with DNase (and optionally RNase as well). Viral RNA is then isolated from this fraction and transfected into host cells.

Examples 2 and 3 illustrate a method for purifying inactivated whole SARS virus using MCS resin purification followed by density gradient ultracentrifugation.

The route and method of immunization with the vaccine of the present invention is described in detail in the following sections. Examples 4 and 5 provide examples of immunization of mice with the inactivated SARS virus of the present invention.

Attenuated SARS vaccine

The present invention includes a composition comprising an attenuated SARS virus. The composition can be used as preventive or therapeutic SARS virus vaccine. Methods of attenuating viruses are known in the art. These methods involve serial passage of SARS virus in cultured cells (e.g., mammalian cell culture, preferably rhesus fetal kidney cells or Vero cells-see section A for a description of cultured SARS virus) until attenuated SARS virus virulence is demonstrated. The virus growth temperature may be any temperature at which the tissue culture is capable of undergoing passaging attenuation. The degree of attenuation of the toxicity of SARS virus after one or more passages in cell culture can be detected by one skilled in the art. Attenuation is defined herein as a reduction in the virulence of the SARS virus in humans. Evidence of reduced virulence may be manifested as a reduced level of viral replication or reduced virulence in animal models.

Other methods of making attenuated SARS viruses include passaging the virus in cell culture at sub-optimal or "cold" temperatures and introducing mutations into the SARS virus genome that attenuate virulence by random mutagenesis (e.g., chemical mutagenesis) or site-directed mutagenesis. Methods for making and producing attenuated REV vaccines, which are also generally applicable to SARS viruses, are disclosed in, for example, EP 0640128, U.S. patent No.6284254, U.S. patent No.5922326, U.S. patent No. 5882651.

Attenuated derivatives of SARS virus can be made by several methods, for example, by introducing temperature sensitive mutations by passaging in culture at "cold" temperatures, with or without chemical mutagens (e.g., 5-fluorouracil). This cold acclimation process involves passaging at about 20-32 ℃, preferably at about 22-30 ℃, and most preferably at about 24-28 ℃. Cold adaptation or attenuation can be passaged at progressively lower temperatures to introduce additional growth-limiting mutations. The number of passages required to obtain a safe, immunoattenuated virus depends, at least in part, on the conditions employed. Periodic testing of the virulence and immunocompetence of SARS virus cultures in animals (e.g., mice, primates) can readily determine the parameters required for a particular combination of tissue culture and temperature. Attenuated vaccines are typically formulated to have a maximum potency of about 10 per dose ³-10⁶PFU or TCID₅₀Or more.

Attenuated virus vaccines for SARS-CoV can also be made by creating a virus chimera comprising sequences from at least two different coronaviruses, one of which is SARS-CoV. For example, a virus chimera is made that contains a gene encoding a non-structural protein from a first coronavirus (e.g., murine, bovine, porcine, canine, feline, avian coronavirus) and one or more genes encoding a structural protein from SARS-CoV (e.g., spike, E, M). Alternatively, such virus chimeras can contain sequences from a non-SARS-CoV human coronavirus (e.g., OC43, 229E) as well as sequences from SARS-CoV. Chimeric coronaviruses of the invention can be produced by a variety of methods, including, for example, recombination of native RNA in eukaryotic (e.g., mammalian) cells containing the RNA of the respective parental coronavirus (e.g., following infection), or engineering of the desired virus chimera (or portions thereof) as a cDNA clone using standard molecular biology techniques known to those skilled in the art, and then using them to produce infectious viruses (see, for example, US 6593111B 2; Yount et al, 2003, Proc. Natl. Acad. Sci. USA 100 (22): 12995-. The attenuated phenotype of the coronavirus chimeras described herein is readily detected by one of skill in the art.

Attenuated viruses may also be produced by deleting one or more Open Reading Frames (ORFs) that are not essential for viral replication. Preferably, these deletions occur in structural regions of the genome, such as ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, 9 b. See, e.g., Haijema BJ, Volders H, Rottier pj. j Virol (2004)78 (8): 3863-71; and de Haan, c.a., p.s. masters, x.shen, s.weiss and p.j.rotter, "group-specific murine coronavirus genes are not essential, but deletion thereof by reverse genetics can produce attenuation in The natural host" (The group-specific viral microorganism subunit genes, but by reverse genetics, is associating in The natural host) Virology (2002) 296: 177-189. Deletion of such regions within coronaviruses such as SARS can be achieved, for example, by reverse genetics methods or "targeted recombination", see, for example, Masters, p.s., "reverse genetics of maxi RNA viruses" adv.virus Res. (1999) 53: 245-264.

Methods for purifying attenuated viruses are known in the art and may include one or more of the following methods, such as gradient centrifugation and chromatography. See Gregersen "Herstellung von Virusssimpfstoffen aus Zellkulturen", Pharmazeutische Biotechology, section 4.2 (O.Kayser and RHMaeller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.

SARS split vaccine

The present invention includes a composition containing a split SARS virus preparation and a method for producing the same. Such compositions are useful as prophylactic or therapeutic SARS virus vaccines.

Methods for splitting enveloped viruses are known in the art. Methods for lysing enveloped viruses are described, for example, in WO02/28422, which is incorporated herein by reference, and specifically includes the lysing agents and methods described therein. Methods for splitting influenza viruses are described, for example, in WO 02/067983, WO 02/074336 and WO 01/21151, each of which is incorporated herein by reference in its entirety.

Lytic viruses are whole viruses that are disrupted or fragmented with a disrupting concentration of a lytic agent, which may be infectious (wild-type or attenuated) or non-infectious (e.g., inactivated) viruses. Fragmentation results in complete or partial solubilization of viral proteins, altering viral integrity.

Preferably the lysing agent is a non-ionic or ionic surfactant. Thus, the split SARS virus formulation of the present invention further comprises at least one non-ionic surfactant or detergent. Examples of lysing agents useful in the present invention include: bile acids and derivatives thereof, nonionic surfactants, alkyl or alkylthio glycosides and derivatives thereof, acyl sugars, sulphobetaines, betaines, polyoxyethylene alkyl ethers, N-dialkyl-glucamides (N, N-dialkyl-glucamides), Hecameg, alkylphenoxypolyethoxyethanol, quaternary ammonium compounds, sodium dodecylsarcosinate, CTAB (cetyltrimethylammonium bromide) or cetofuran (Cetavlon).

Preferably the ionic surfactant is a cationic detergent. Cationic detergents suitable for use in the present invention include detergents comprising compounds having the formula:

wherein,

R₁、R₂and R₃May be the same or different and each represents an alkyl group or an aryl group, or

R₁And R₂And the nitrogen atom to which they are bound together form a 5-or 6-membered heterocyclic ring, and

R₃represents alkyl or aryl, or

R₁、R₂And R₃Together with the nitrogen atom to which they are bound form a 5-or 6-membered heterocyclic ring which is unsaturated on the nitrogen atom,

R₄represents an alkyl or aryl group, and

x represents an anion

Examples of such cationic detergents are cetyltrimethylammonium salts, such as cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethylammonium salts.

Other cationic detergents suitable for use in the present invention include lipofectamine, and DOT-MA.

Nonionic surfactants suitable for use in the present invention include one or more of the following: octylphenoxy or nonylphenoxypolyols (polyoxyyethonols) (for example the commercially available Triton series), polyoxyethylene sorbitan esters (Tween series) and polyoxyethylene ethers or esters of the general formula:

O(CH₂CH₂O)n-A-R

wherein n is 1-50, A is a bond or-C (O) -, and R is C _1-50Alkyl or phenyl C_1-50An alkyl group; and combinations of two or more thereof.

The invention includes a method of making a SARS lytic virus, the method comprising contacting a SARS virus with a sufficient amount of a lytic agent to disrupt the viral envelope. After lysis the virus loses integrity rendering the virus non-infectious. Once the viral envelope proteins are disrupted and no longer bind to intact viral particles, other viral proteins are fully or partially solubilized and do not bind to only partially intact viral particles after lysis.

The method for producing SARS lytic virus may further comprise removing the lytic agent and a portion or a majority of the viral lipid material. The process may also include some differential filtration steps and/or other separation steps such as various combinations of ultracentrifugation, ultrafiltration, zonal centrifugation, and chromatography steps. The process may also optionally include an inactivation step (as described above), which may be performed before or after lysis. The lysis step may be carried out in a batch, continuous or semi-continuous manner.

The SARS split virus vaccine of the present invention may comprise structural proteins, membrane fragments and membrane envelope proteins. Preferably, the SARS split virus preparation of the invention comprises at least half of the viral structural proteins.

One example of a method for making a SARS split virus preparation includes the steps of:

(i) propagating SARS virus in cell cultures such as MRC-5 cells (ATCC CCL-171), WI-38 cells (ATCC CCL-75), rhesus fetal kidney cells, or vero cells (see section A above for SARS virus culture);

(ii) harvesting SARS virus-containing material from the cell culture;

(iii) clarifying the harvested material to remove non-SARS viral material;

(iv) concentrating the harvested SARS virus;

(v) separating intact SARS virus from non-viral material;

(vi) lysing intact SARS virus with a suitable lysing agent in a density gradient centrifugation step; and

(vii) filtering to remove unwanted material.

The above steps are preferably performed in order.

The clarification step is preferably accomplished by medium speed centrifugation. Alternatively, the filtration step may use, for example, a 0.2 μm membrane.

The concentration step is preferably carried out by adsorption, e.g. using CaHPO₄. Alternatively, filtration, such as ultrafiltration, may be employed.

Other separation steps may also be used in the process of the present invention. The other separation step is preferably zonal centrifugation and optionally a sucrose gradient may be used. Preservatives may also be included in the sucrose gradient to prevent microbial growth.

The lysis step may also be performed in a sucrose gradient, wherein the sucrose gradient contains a lysis agent.

The method may also include a sterile filtration step, which may optionally be performed at the end of the treatment. Preferably, the final filtration step is preceded by an inactivation step.

The method for preparing the SARS lytic virus preparation may further comprise treating the virus preparation with DNA digesting enzymes. These enzymes can be used to reduce the level of host cell DNA during the virus purification step. DNA digesting enzymes for use in cell culture are known in the art and include, for example, Benzonase^_。

The treatment of the SARS virus preparation with DNA digesting enzyme can be carried out at any time of the purification treatment and the cleavage treatment. However, it is preferred to treat the SARS virus preparation with DNA digesting enzymes prior to use of the detergent. More preferably, the SARS virus preparation is treated with a DNA digesting enzyme such as Benzonas prior to treatment with a cationic detergent such as CTAB.

Methods for purifying split viruses are known in the art. See JP Gregersen "Herstellung von Virusssimpfstoffen aus Zellkulturen", Pharmazeutische Biotechology section 4.2 (O.Kayser and RH Mueller eds.) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.

The present invention includes a split vaccine composition comprising a prophylactically effective amount of a SARS virus antigen, preferably a split or immunogenic fragment thereof. The concentration of the SARS virus antigen is preferably 0.1 to 50. mu.g antigen/dose, more preferably 0.3 to 30. mu.g antigen/dose. More preferably, the antigen is about 15 μ g/dose.

In one embodiment, lower concentrations of SARS virus antigen are used in the split vaccine compositions of the invention. Such lower concentration vaccines may optionally contain an adjuvant to enhance the host's immune response to the antigen. In such "low dose" vaccines, the concentration of SARS virus antigen is preferably less than 15 μ g antigen/dose, i.e. less than 10, 7.5, 5 or 3 μ g antigen/dose.

SARS subunit vaccine

The present invention includes compositions comprising an isolated purified SARS virus antigen or derivative thereof. Such compositions may also contain one or more adjuvants.

The SARS virus antigen can be isolated or purified from SARS virus grown in cell culture. Alternatively, the SARS virus antigen can be recombinantly produced using methods known in the art.

The SARS virus antigen for use in the present invention can be produced in a number of different expression systems, which are known in the art; for example, expression systems using mammalian cells, baculovirus, bacteria and yeast. Such expression systems typically employ polynucleotides encoding the viral antigens of the invention. Such sequences may be obtained using standard molecular biology techniques, including the translation of the amino acid sequences set forth herein. Accordingly, the invention includes polynucleotides encoding the viral antigens of the invention. In addition, the viral antigens of the invention may be produced (at least in part, preferably in bulk) by chemical synthesis.

Insect cell expression systems, such as baculovirus systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/inserted cell expression systems are available from commercially available kits including Invitrogen, San Diego CA. Similarly, bacterial and mammalian cell expression systems are also known in the art and are described, for example, in Yeast genetic engineering (Barr et al, 1989) butterworks, London.

Many suitable host cells are known for use in the above systems. For example, mammalian cell lines are known in the art, including immortalized cell lines obtained from the American Type Culture Collection (ATCC), such as, but not limited to, chinese hamster ovary (CH0) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney cells (e.g., HepG2), Madin-Darby bovine kidney ("MDBK") cells, and others. Mammalian sources of cells include, but are not limited to, human or non-human primate cells (e.g., MRC-5(ATCC CCL-171), WI-38(ATCC CCL-75), rhesus fetal lung cells (e.g., ATCC CL-160), human embryonic kidney cells (293 cells, typically transformed with sheared adenovirus type 5 DNA), Vero cells (e.g., monkey kidney Vero cells), horses, cows (e.g., MDBK cells), sheep, dogs (e.g., dog kidney MDCK cells, ATCC CCL34 MDCK (NBL2), or MDCK 33016, deposited as ACC DSM 352219 as described in WO 97/37001), cats and mice (e.g., hamster cells, such as BHK21-F, HKCC cells or Chinese hamster ovary cells (CHO cells)) and may be obtained from various stages of development, including, for example, humans, neonates, fetuses, or embryos.

Similarly, bacterial hosts such as E.coli (E.coli), Bacillus subtilis (Bacillus subtilis) and Streptococcus may also be used in the expression constructs of the invention. Yeast hosts useful in the present invention include Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), Candida maltosa (Candida maltosa), Hansenula polymorpha (Hansenula polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia gilularirimonii, Pichia pastoris (Pichia pastoris), Schizosaccharomyces pombe (Schizosaccharomyces pombe), and Yarrowia lipolytica (Yarrowia polylithica). Insect cells for baculovirus expression vectors include Aedes aegypti (Aedes aegypti), Autographa californica (Autographa californica), Bombyx mori (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), Spodoptera frugiperda (Spodoptera frugiperda), and Ectropis calis (Trichoplusia ni).

The nucleic acid molecules comprising the viral antigen or antibody encoding nucleotide sequences of the present invention can be stably integrated into the host cell genome or maintained as stable episomal elements in a suitable host cell using a variety of gene delivery techniques well known in the art. See, for example, U.S. Pat. No.5,399,346.

Depending on the expression system and host chosen, the host cell transformed with the expression vector is propagated under conditions for protein expression to produce the desired molecule. The expressed protein is then isolated from the host cell and purified. If the expression system secretes the protein into the growth medium, the product can be purified directly from the medium. If not secreted, can be isolated from cell lysates. The selection of appropriate culture conditions and recovery methods is known to those skilled in the art.

The present invention includes compositions comprising an isolated or purified SARS virus antigen or derivative thereof. The invention also includes compositions comprising at least two isolated or purified SARS virus antigens or derivatives thereof, which have been purified together or separately and then combined. In one embodiment, the SARS virus antigen is the spike (S) protein. In yet another embodiment, the SARS virus antigen is a nucleocapsid (N) protein, a membrane (M) glycoprotein, or an envelope (E) protein. Preferably, the SARS virus antigen in the composition is greater than 75% pure (e.g., 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98%).

The present invention includes a vaccine composition comprising a prophylactically effective amount of a SARS virus antigen, preferably spike or immunogenic fragment thereof. The concentration of the SARS virus antigen is preferably 0.1 to 50. mu.g antigen/dose, more preferably 0.3 to 30. mu.g antigen/dose. Even more preferably, the antigen is about 15 μ g/dose.

In one embodiment, lower concentrations of SARS virus antigen are used in the vaccine compositions of the present invention. Such lower concentration vaccines may optionally contain an adjuvant to elicit an immune response in the host to the antigen. In such "low dose" vaccines, the concentration of SARS virus antigen is preferably less than 15 μ g antigen/dose, i.e. less than 10, 7.5, 5 or 3 μ g antigen/dose.

The following example illustrates a method for preparing a SARS virus spike (S) protein subunit vaccine.

The SARS virus S antigen can be isolated and purified from a variety of sources by a variety of methods including, but not limited to, expression of the S antigen in cultured eukaryotic cells (e.g., mammalian cells such as VERO, CHO) or bacteria (e.g., E.coli). Expression can be achieved by a variety of methods, e.g., expression from a cell culture or cell culture supernatant infected with SARS virus, expression from a cultured cell stably transformed with a DNA expression cassette encoding the SARS virus S protein (e.g., an RNA polymerase II promoter operably linked to the SARS virus S gene), or expression from a cultured cell infected with an expression vector encoding a replication-competent or non-replication-competent virus of the SARS virus S protein (e.g., an adenoviral vector, a poxviral vector, an alphaviral vector, a retroviral vector), such that the use of infectious SARS virus is not required.

1. Production of SARS subunit vaccine from SARS virus culture

SARS virus can be grown in cultured mammalian cells, such as Vero cells, and then isolated from the cultured cells. The SARS virus antigen, e.g., S protein, can then be solubilized and separated from the SARS virus, and further separated and purified.

In one embodiment, the SARS virus can be produced according to the methods of the SARS inactivated vaccine embodiment, and the desired SARS antigen, e.g., spike protein, can then be further purified from the final product using techniques known in the art.

In another example, a SARS subunit vaccine can be produced as follows. The desired mammalian cell line on microcarrier beads in a large controlled fermentor was used to produce SARS virus. For example, the concentration is 10⁵cells/mL vaccine grade African Green monkey kidney cells (Vero cells) 60-75 liters of CMRL1969 medium, pH 7.2 were added in a 150 liter bioreactor containing 360 grams of Cytodex-1 microcarrier beads and stirred for 2 hours. CMRL1969 was added to a total volume of 150 liters. Fetal Bovine Serum (FBS) was added to a final concentration of 3.5%. Glucose was added to a final concentration of 3.0g/L and glutamine was added to a final concentration of 0.6 g/L. Dissolved oxygen, pH, agitation and temperature were controlled, and cell growth, glucose levels, lactate levels and glutamine levels were monitored. When the cells are in log phase (usually at days 3-4, densities of about 1.0-2.5X 10 are reached ⁶cells/mL), the medium in the fermentor was decanted and 120 liters of CMRL 1969, pH 7.2 (no FBS) was added, and the culture was stirred for 10 minutes. Emptying and filling are usually repeated once, but may be repeated up to 3 times. After washing the cells, the fermentor was emptied and 50 liters of CMRL 196 containing 0.1% (v/v) FBS was added9. The SARS virus inoculum was added at a multiplicity of infection (m.o.i) of 0.001-0.01. Trypsin may be added to promote effective infection. CMRL 1969 containing 0.1% FBS was added to a final volume of 150 liters. The culture was continued at 34 ℃. One fermentation can yield one viral harvest, usually 2-7 days after infection. Multiple harvests can also be obtained by one fermentation.

S protein can be isolated and purified by various methods as described below. For example, an S protein-containing effluent from ion exchange chromatography of solubilized SARS virus envelope proteins is collected; this effluent is applied to a hydroxyapatite matrix and the S protein is selectively eluted from the hydroxyapatite matrix. The selectively eluted S protein may be further concentrated by tangential flow ultrafiltration.

Alternatively, the separation and purification can be carried out by: collecting the S protein-containing effluent of ion exchange chromatography of dissolved SARS virus envelope protein; loading the effluent on a hydroxyapatite matrix and collecting the S protein containing effluent, and selectively removing the detergent used in the solubilization step from the hydroxyapatite matrix effluent to obtain an isolated and purified S protein. The isolated and purified S protein can then be concentrated by tangential flow ultrafiltration. The nucleic acid degradation agent described in the inactivation section above may be used to remove nucleic acid contaminants from the isolated and purified S protein. Preferably the nucleic acid degrading agent is a nuclease, such as Benzonase.

The separated and purified S protein may be used in a gel filtration medium and then collected therefrom to separate the S protein from contaminants of other molecular weights.

Alternatively, the isolation and purification may be accomplished by: loading the S protein onto a first ion exchange medium, allowing the contaminants to pass through the medium, eluting the S protein from the first ion exchange medium, and separating the S protein from contaminants of other molecular weights. The eluted S protein is loaded onto a second ion exchange medium and the contaminants are passed through the second ion exchange medium. From which the S protein is subsequently eluted to give an isolated and purified S protein. The eluted S protein may be concentrated by tangential flow ultrafiltration.

Alternatively, a substantially purified SARS virus S protein suitable for use as an immunogen in a subunit vaccine formulation can be prepared from infected cell lysates, for example, by lysing the infected cells using a non-denaturing detergent buffer containing 1% Triton X-100 and deoxycholate. The cell lysate was clarified by centrifugation and purified from the cell lysate by immunoaffinity purification to give the S protein. Monoclonal antibodies against the S protein were generated, coupled to beads and used to pack the column with these beads. SARS-infected cell lysate was applied to the column and the column was washed with PBS containing 0.1% Triton X-100. The bound proteins to the column were eluted with 0.1M glycine, pH 2.5, 0.1% Triton X-100. The eluted sample is buffered, for example with Trix and analyzed for the presence of protein therein. The protein-containing fractions were pooled and dialyzed against PBS.

As described above, the present invention includes the isolated and purified SARS virus S protein. In one embodiment, the virus is cultured on a vaccine grade cell line, such as Vero cells, and the cultured virus is harvested. The viral harvest was filtered and then concentrated by tangential flow ultrafiltration using diafiltration against a membrane with the desired molecular weight cut-off. The virus harvest concentrate may be centrifuged and the supernatant discarded. The solubilized S protein is then extracted from the centrifuged pellet with detergent, e.g., the pellet is resuspended in an extraction buffer containing a detergent (e.g., a non-ionic detergent, including TRITON X-100) to the volume of the initially harvested concentrate.

After centrifugation to remove undissolved protein, the S protein extract was purified by chromatography. The extract may be first applied to an ion exchange chromatography column, such as an equilibrated TMAE-or S-fraction column, to allow the S protein to flow through while the impurities remain on the column.

The effluent is then loaded onto an equilibrated hydroxyapatite column, binding the S protein to the matrix and allowing the contaminants to pass through the column. The bound S protein is then eluted from the column with a suitable eluent. The resulting purified S protein solution may be further processed to increase its purity. The eluate may first be concentrated by tangential flow ultrafiltration using a membrane with the desired molecular weight cut-off. The filtrate may be contacted with polyethylene glycol having a desired molecular weight (e.g., about 6000-8000) to precipitate the protein. After centrifugation and discarding of the supernatant, the pellet can be resuspended in PBS and dialyzed to remove polyethylene glycol. Finally, the dialyzed protein S solution can be sterile filtered. The sterile filtered liquid can be adsorbed onto alum. If necessary, in the purification operation of early stage polyethylene glycol precipitation and heavy suspension purification steps.

Alternatively, the SARS virus is recovered after culturing and harvesting the virus, e.g., by PEG precipitation or tangential flow filtration to give a concentrate. The virus is contacted with a detergent to solubilize the S protein. After centrifugation, the supernatant was recovered to further purify the S protein and to discard the insoluble protein.

The supernatant is applied to an ion exchange chromatography column, such as a TMAE-or S-fractional column, which has been suitably equilibrated to retain the S protein on the column. The S protein is eluted from the ion exchange column under suitable conditions. The eluate is then passed through a gel filtration column, such as a Sephacryl S-300 column, to separate the S protein from other molecular weight contaminants. The Sephacryl column can be replaced by hydroxyapatite column.

The S protein may be eluted from the column to provide a purified S protein solution. The eluate may be concentrated by tangential flow ultrafiltration using a membrane with the desired molecular weight cut-off. The concentrated S protein solution may then be sterile filtered.

Alternatively, the virus harvest may be concentrated by ultrafiltration, and the concentrated virus harvest may be subjected to an initial purification step, for example, by gel filtration chromatography, polyethylene glycol precipitation, or Cellufine sulfate chromatography. The purified virus may then be treated with detergent to solubilize the S protein. After solubilization of the S protein, the supernatant may be applied to an ion exchange column, such as a Cellufine sulfate column, which has been equilibrated to allow the protein to bind to the column and allow the contaminants to flow through. Similarly, TMAE-or S-fractional columns may be used in place of the Cellucine sulfate column. The two columns can also be combined into a sequential purification step. The S protein is eluted from the column to obtain a purified protein solution. The solution can then be concentrated by tangential flow ultrafiltration and diafiltration using a membrane with the desired molecular weight cut-off.

Specifically, in one method of purifying S protein, virus harvest concentrate is centrifuged at 28,000 Xg for 30 minutes at 4 ℃. The supernatant was discarded and the pellet resuspended in extraction buffer containing 10mM Tris-HCl (pH 7.0), 150mM NaCl, 2% (w/v) Triton X-100 to the original harvest concentrate volume. Pefabloc was added to a final concentration of 5 mM. The suspension was stirred at room temperature for 30 minutes. The supernatant containing soluble S protein was clarified by centrifugation at 28,000 Xg for 30 minutes at 4 ℃. The TMAE-Fractogel column was equilibrated with 10mM Tris-HCl (pH 7.0), 150mM NaCl containing 0.02% Triton X-100. TritonX-100 supernatant containing soluble S protein was applied directly to a TRAE-Fractogel column. The total sample volume and 2 column volumes of 10mM Tris-HCl, pH 7.0, 150mM NaCl (containing 0.02% Triton X-100) were collected. TMAE-Fractogel effluent containing S protein was diluted 3-fold with 10mM Tris-HCl, pH 7.0 (containing 0.02% Triton X-100).

The hydroxyapatite column was equilibrated with 10mM Tris-HCl, pH 7.0, 50mM NaCl, 0.02% Triton X-100. TMAE effluent was added and the column was washed with 2 column volumes of 10mM Tris-HCl, pH 7.0, 50mM NaCl, 0.02% Triton X-100 and 4 column volumes of 5mM sodium phosphate, pH 7.0, 1M NaCl, 0.02% Triton X-100. The protein was eluted with 4 column volumes of 20mM sodium phosphate, pH 7.0, 1M NaCl, 0.02% TritonX-100. The fractions were collected based on A280 and protein content to determine antigen concentration. The purified S protein was ultrafiltered by tangential flow ultrafiltration using 300kDa NMWL membrane.

2. Recombinant production of SARS subunit vaccine

As described above, SARS virus protein can be produced by recombinant expression. Suitable host cells for recombinant expression include bacteria, mammals, insects, yeast, and the like. Recombinant expression can be used to make full-length SARS proteins, fragments thereof, or fusion products thereof.

The fusion peptide can be used to facilitate expression and purification of recombinant SARS protein. For example, adding a marker protein to the SARS antigen to be expressed as a fusion protein containing the marker protein and the SARS antigen to perform expression facilitates recombinant production of SARS polypeptide. Such marker proteins facilitate the purification, detection and stability of the expressed protein. Marker proteins suitable for use in the present invention include poly-arginine-tag (Arg-tag), poly-histidine-tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin binding peptide, cellulose binding domain, SBP-tag, chitin binding domain, glutathione S-transferase tag (GST), lactose binding protein, transcription termination anti-termination factor (NusA), E.coli thioredoxin (TrxA), and protein disulfide isomerase I (DsbA). Preferred marker proteins include His-tag and GST. For a detailed discussion of the marker proteins see Terpe et al, "review of marker protein fusions: from molecular and biochemical bases to commercial systems "(Overview of tag protein fusions: from molecular and biochemical standards to commercial systems), applied Microbiol Biotechnol (2003) 60: 523-533.

After purification, the marker protein may optionally be removed from the expressed fusion protein, i.e., by treatment with specific splicing enzymes as is known in the art. Commonly used proteins include enterokinase, Tobacco Etch Virus (TEV), thrombin, and factor Xa.

Accordingly, the invention also includes SARS virus subunit vaccines comprising the fusion proteins. Preferably, the fusion protein comprises a first amino acid sequence encoded by a SARS virus polynucleotide sequence. The SARS virus polynucleotide sequence encoding the first amino acid sequence includes one or more of the SARS virus polynucleotide sequences identified in the specification and fragments thereof.

The fusion protein may comprise the amino acid sequence of a SARS virus protein or a fragment thereof. The SARS virus protein is selected from one or more of the following SARS virus proteins: p28, P65, Nsp1, Nsp2(3CL protease), Nsp3, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8s Nsp9(RNA polymerase), Nsp10 (helicase), Nsp11, Nsp12, Nsp13, spike, Orf3, Orf4, envelope, matrix, Orf7, Orf8, Orf9, Orf10, Orf11, nucleocapsid, and Orf 13.

In one embodiment, the fusion protein comprises a first amino acid sequence comprising a SARS virus antigen or a fragment thereof. The SARS virus amino acid sequence can contain one or more of the T-epitope sequences identified above.

Preferably, the fusion protein comprises the amino acid sequence of the spike protein of SARS virus or a fragment thereof. Spike protein-specific fragments useful in fusion proteins include the S1 domain and the S2 domain. Other spike protein fragments that may be used in the fusion protein include various regions of the S1 and S2 domains, including the receptor binding region of the S1 domain, the oligomerization region of the S2 domain, the leucine zipper region of the S2 domain, the membrane anchoring region of the S2 domain, the hydrophobic domain region of the S2 domain, the cysteine-rich region of the S2 domain, and the cytoplasmic tail region of the S2 domain (see fig. 19). The amino acid sequences of spike proteins corresponding to these regions can be identified by those skilled in the art, including, for example, using functional predictions (predicted transmembrane helices, predicted N-terminal signal regions, predicted coiled coil regions, etc.) as set forth earlier in this application and homology comparisons with other known baculovirus sequences (see fig. 4F and 5).

The fusion protein may further comprise a second amino acid sequence. The second amino acid sequence may comprise a polypeptide sequence which facilitates expression or purification of the S protein, preferably one of such marker sequences has been discussed above. Alternatively, the second amino acid sequence can comprise a second amino acid sequence of SARS virus. Alternatively, the second amino acid sequence may comprise the amino acid sequence of other viruses or bacteria, including one or more of the viruses or bacteria identified in section I below.

The second amino acid sequence may comprise an amino acid sequence of another respiratory virus. The second amino acid sequence may chelate to an amino acid sequence of a virus selected from the group consisting of: coronavirus, influenza virus, rhinovirus, parainfluenza virus (PIV), Respiratory Syncytial Virus (RSV), adenovirus and metapneumovirus.

In one embodiment, the second amino acid sequence may comprise the amino acid sequence of an adjuvant comprising one or more of the adjuvants identified in section I below.

In one embodiment, the invention includes fusion proteins comprising the amino acid sequence of the spike protein of SARS virus, or a fragment thereof. The fusion protein may further comprise a second amino acid sequence comprising an amino acid sequence selected from the group consisting of a second SARS viral protein, a non-SARS viral protein, a bacterial protein, and an adjuvant.

(a) Bacterial expression of SARS subunit vaccine

In one embodiment, bacterial host cells are used to recombinantly express SARS viral proteins. Bacterial host cells suitable for use in the present invention include, for example, Escherichia coli (E.coli), Bacillus subtilis (Bacillus subtilis), and Streptococcus.

The SARS virus protein may be modified to facilitate bacterial recombinant expression. In particular, the SARS spike protein can be modified to facilitate transport of the spike protein to the surface of a bacterial host cell.

Applicants have revealed strong structural homology between the spike protein of the SARS virus and the NadA protein of neisseria meningitidis. Both proteins have an N-terminal globular "head" domain (amino acids 24-87), a central alpha-helical region (amino acids 88-350) which is highly prone to form coiled-coil structures, and a C-terminal membrane-anchoring domain (amino acids 351-405 of NadA) formed by 4 amphipathic transmembrane beta-strands. In addition, a leucine zipper motif is present in the coiled coil segment. See fig. 19, which shows the structure of SARS spike protein, comeanducci et al, "NadA-a Novel Vaccine candidate for Neisseria meningitidis" (NadA, a Novel Vaccine candidate of Neisseria meningitidis), j.exp.med.195 (11): 1445-1454(2002). In addition, the leucine zipper motif of NadA is present within the coiled coil segment. NadA proteins also form high molecular weight surface-exposed oligomers (corresponding to three or four monomers) that anchor to the meningococcal outer membrane.

When NadA is expressed in e.coli, the full-length protein assembles or anchors to the oligomer of the e.coli outer membrane, similar to the assembly pathway of proteins found in meningococcus. NadA protein lacking the predicted membrane anchoring domain is then secreted into the culture supernatant. This secreted protein is soluble and still organizes into a trimeric form.

Accordingly, the present invention includes a fusion protein comprising the fusion of the amino acid sequence of the spike protein of SARS virus, or a fragment thereof, and the second amino acid sequence of the bacterial adhesion protein, or a fragment thereof. Preferably the adhesion protein is selected from the group consisting of NadA, YadA (from enteropathogenic yersinia) and UspA2 (from Moraxella catarrhalis). Other NadA-like proteins include the serum resistance protein DsrA of haemophilus ducreyi (haempophilus ducreyi), the immunoglobulin binding proteins EibA, C, D and F of escherichia coli, the outer membrane protein 100 of actinomyces actinomycetemcomitans (Actinobacillus actinomycetcomomatans), the saa gene (STEC) carried by the virulent plasmid of the shiga toxigenic strain of escherichia coli, and various bacterial adhesion proteins described in british patent application No.0315022.4 (filed on 26/2003, the contents of which are incorporated herein by reference).

Preferably, the adhesion protein comprises NadA or a fragment thereof.

Such fusion proteins can be used to facilitate recombinant expression of immunogenic portions of the SARS surface antigen, such as spikes. These fusion constructs allow the SARS S1 and/or S2 domains to adopt the native conformation. These fusion proteins are also capable of oligomerizing to form dimers or trimers, allowing the binding of the S1 and/or S2 domains, adopting the conformation of the native SARS spike protein. In addition, these expression constructs facilitate the exposure of SARS spike protein to surfaces.

The fusion protein of the invention preferably comprises a leader peptide of a NadA-like protein, preferably NadA, a polypeptide of the immunogenic "head" region of the spike protein and a stalk region of the NadA-like protein or spike protein. One or more amino acids of, e.g., a leader peptide or membrane anchoring domain, can be cleaved off or removed during expression and processing of the fusion protein.

The handle region contributes to oligomerization of the expressed protein. Optionally, the fusion protein of the invention may further comprise an anchor region of a NadA-like protein. This anchor region allows the expressed fusion protein to anchor and assemble to the bacterial cell surface.

The fusion protein of the invention contains the following constructs:

(i) the NadA leader peptide (which may optionally also contain the first 6 amino acids of the mature NadA protein to facilitate processing of the leader peptide and proper maturation of the protein) is followed by the spike S1 domain. Preferably, the construct contains amino acids 1-29 of NadA (corresponding to the first 6 amino acids of the NadA leader peptide and mature NadA protein, as shown in FIG. 22 and listed below), followed by amino acids 14-662 of the SARS virus spike protein (corresponding to the S1 domain, see FIG. 19 and SEQ ID NO: 6042, and listed below). Specifically, construct (i) comprises SEQ ID NO: 7302.

(ii) The NadA leader peptide (which may optionally also contain the first 6 amino acids of the mature NadA protein to facilitate processing of the leader peptide and proper maturation of the protein) is followed by the spike S1 domain, followed by the stalk and anchor membrane domain of NadA. Preferably, the construct contains amino acids 1-29 of NadA (corresponding to the first 6 amino acids of the NadA leader peptide and mature NadA protein, as shown in FIG. 22 and listed below), followed by amino acids 14-662 of the SARS virus spike protein (corresponding to the S1 domain, see FIG. 19 and SEQ ID NO: 6042, and listed below), followed by amino acids 88-405 of NadA (corresponding to the stalk and anchor membrane domains). Specifically, construct (ii) comprises SEQ ID NO: 7303.

(iii) the NadA leader peptide (which may optionally also contain the first 6 amino acids of the mature NadA protein) is followed by the SARS virus spike protein S1 domain, followed by the NadA stalk domain. Preferably, the construct contains amino acids 1-29 of NadA, followed by amino acids 14-662 of the SARS virus spike protein (corresponding to the S1 domain), followed by amino acids 88-350 of NadA (corresponding to the stalk and anchor membrane domains). Specifically, construct (iii) comprises SEQ id no: 7304.

(iv) the NadA leader peptide (which may optionally also contain the first 6 amino acids of the mature NadA protein) is followed by the SARS virus spike S1 and S2 domains (excluding the putative transmembrane region), followed by the anchor domain of NadA. Preferably, the construct contains amino acids 1-29 of NadA, followed by amino acids 14-1195 of the SARS virus spike protein (corresponding to S1 and S2, excluding the putative transmembrane region), followed by amino acids 351-405 of NadA (corresponding to the NadA anchor domain). Specifically, construct (iii) comprises SEQ ID NO: 7305. alternatively, the NadA anchor domain may comprise amino acids 332 and 405 of NadA.

(v) The NadA leader peptide (which may optionally also contain the first 6 amino acids of the mature NadA protein) is followed by the SARS virus spike S1 and S2 domains (excluding the putative transmembrane region). Preferably, the construct contains amino acids 1-29 of NadA followed by amino acids 14-1195 of the SARS virus spike protein. Specifically, construct (v) comprises seq id NO: 7306.

in each of constructs (i) - (v), the first 23 amino acids were of the NadA leader peptide and the GS dipeptide at residues 679-680 was inserted from the restriction endonuclease site.

In constructs (i), (ii) and (iii), the NadA "head" was replaced by the spike S1 domain, and the fusion protein was anchored to the e.coli outer membrane or secreted into the culture supernatant, respectively. In constructs (iv) and (v), the "head" and "handle" domains of NadA were replaced by the S1 and S2 spike domains; at this time, the two fusion proteins were anchored to the E.coli outer membrane or secreted into the culture supernatant, respectively.

Accordingly, the invention also includes fusion proteins comprising the amino acid sequence of the spike protein of SARS virus, or a fragment thereof, and the second amino acid sequence of the bacterial adhesion protein, or a fragment thereof. Preferably, the amino acid corresponding to the "head" of the adhesion protein is replaced by an amino acid corresponding to the SARS virus spike S1 domain. Alternatively, the amino acids corresponding to the "head" and "handle" domains of bacterial adhesion proteins are replaced with amino acids corresponding to the SARS virus spike protein S1 and S2 domains.

As described above and shown in fig. 19, the S1 domain of spike protein was identified as the globular receptor binding "head" region. The S1 domain of the spike protein preferably comprises SEQ ID NO: 6042 amino acids from about position 14-662. The S1 domain may comprise a shorter amino acid sequence with some amino acids removed from the N-terminal or C-terminal regions. Preferably 3, 5, 7, 9, 13, 15, 20 or 25 amino acids are removed from the N-terminal or C-terminal region. The S1 domain further comprises a sequence identical to SEQ ID NO: the S1 region of 6042 has an amino acid sequence with sequence identity. An example of the S1 domain is SEQ ID NO: 7307.

as described above and shown in FIG. 19, the S2 domain of spike protein was identified as the "stalk" region. The "handle" region comprises an oligomerization domain region, a leucine zipper domain region, a membrane anchoring region, a hydrophobic domain region, a cysteine-rich domain region, and a cytoplasmic tail region. The S2 domain of the spike protein preferably does not comprise a transmembrane region and comprises SEQ ID NO: 6042 amino acid at position 663-1195. The S2 domain may comprise a shorter amino acid sequence with some amino acids removed from the N-terminal or C-terminal regions. Preferably 3, 5, 7, 9, 13, 15, 20 or 25 amino acids are removed from the N-terminal or C-terminal region. The S2 domain further comprises a sequence identical to SEQ ID NO: the S2 region of 6042 has an amino acid sequence with sequence identity. An example of the S2 domain (excluding the transmembrane region) is SEQ ID NO: 7308.

An example of the NadA protein described above is SEQ ID NO: 7309. as mentioned above, the leader sequence of the NadA used in the fusion protein preferably comprises the first 29 amino acids of NadA (including the leader sequence and about 6 amino acids of the NadA noggin). Examples of such leader sequences are set forth below in SEQ ID NOS: 7310 and 7311. Fusion proteins may use shorter amino acid sequences that contain some amino acids removed from the N-terminal or C-terminal regions. Preferably, 1, 2, 3, 4 or 5 amino acids are removed from the N-terminal or C-terminal region of the sequence. The leader sequence for the fusion protein may further comprise a sequence identical to SEQ ID NO: 7310 or SEQ ID NO: 7311 has an amino acid sequence with sequence identity. Preferably, the leader sequence comprises SEQ ID NO: 7311.

optionally, the fusion peptide comprises about the first 6 amino acids of the mature NadA protein to facilitate processing of the leader peptide and proper maturation of the protein. An example of the first 6 amino acids of the mature NadA protein is SEQ ID NO: 7312.

as mentioned above, the stalk and anchor sequence of the NadA used in the fusion protein preferably comprises amino acids from about positions 88-405 of NadA. An example of an amino acid sequence comprising a NadA handle and an anchor region is SEQ ID NO: 7313. an example of an amino acid sequence comprising a NadA stalk region (without an anchor region) is SEQ ID NO: 7314. an example of an amino acid sequence comprising a NadA anchor region is SEQ ID NO: 7315. fusion proteins using stalk (and/or anchor) sequences comprise shorter amino acid sequences with some amino acids removed from the N-terminal or C-terminal regions. Preferably, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids are removed from the N-terminal or C-terminal region of the sequence. The leader sequence for the fusion protein may further comprise an amino acid sequence having sequence identity to 7313.

The fusion proteins of the present invention (including those described above) can be prepared, for example, according to the following methods. Individual fragments (such as the regions described above) can be amplified by PCR using the oligonucleotide primers listed in the following table. (S1L denotes the spike protein fused to the leader peptide of NadA; S2 denotes the handle region of the spike protein with or without a stop codon). Oligonucleotides were designed based on the DNA sequence of neisseria meningitidis (n.meningitidis) B2996 strain NadA and the DNA sequence of SARS virus isolate FRA1 spike. Each oligonucleotide contained a restriction site as a tail to direct cloning into the expression vector pET21 b.

SEQ ID NO: restriction sites

S1L For 7316 NdeI

S1L Rev 7317 BamHI

S2 For 7318 BamHI

S2 Rev 7319 HindIII

S2-end-Point Rev 7320 XhoI

NadA₈₈ For 7321 BamHI

NadA₃₅₀ Rev 7322 XhoI

NadA₃₃₂ For 7323 HindIII

NadA₄₀₅ Rev 7324 XhoI

The individual fragments were subsequently cloned into the pET21b vector and the protein expressed under the control of the inducible T7 promoter. Use of primer S1_L-For and S1_LRev obtained by PCR the S1 domain of the spike protein fused to the NadA leader (S1)_L). The forward oligonucleotide primer contains the NdeI restriction sequence and the sequence encoding the NadA leader peptide as well as the first 6 amino acids of the mature protein. The PCR fragment was cloned as an NdeI/BamHI fragment in pET21b vector opened with the same restriction enzyme. This clone (pET-S1) was then used _L) To clone in sequence the other different domains, such as BamHI/XhoI, BamHI/HindIII or HindIII/XhoI fragments. The BamHI and HindIII restriction sites allow the introduction of the amino acids GS and KL, respectively.

The PCR amplification protocol was as follows: using 200ng of genomic DNA from N.meningitidis 2996 or 10ng of a preparation of plasmid DNA (plasmid pCMVnew, containing the entire gene encoding the spike protein) as template, 40. mu.M oligonucleotide primer in the amplification reaction was used, using 400. mu.M dNTPs solution, 1 XPCR buffer (containing 1.5mM MgCl. sub.₂) 2.5 units TaqIDNA polymerase (using Perkin-Elmer AmpliTaQ or Invitrogen Platinum PfxDNA polymerase).

After pre-incubation of the entire mixture at 95 ℃ for 3 minutes, each sample was subjected to two-step amplification: the first 5 rounds were performed with a hybridization temperature (Tm1) excluding the primer restriction enzyme tail. Then, 30 rounds were performed based on the calculated hybridization temperature (Tm2) for the full-length oligonucleotide. The time of extension at 68 ℃ or 72 ℃ varies depending on the length of the fragment to be amplified. The cycle is completed in a 10 minute extension step at 68 ℃ or 72 ℃.

The amplified DNA was loaded directly onto an agarose gel and Qiagen was used as described by the manufacturer^TMThe GelExtraction Kit purified the DNA fragment corresponding to the correct size band from the gel.

The purified DNA and plasmid vector corresponding to the amplified fragment were digested with the appropriate restriction enzymes, purified using QIAquick PCR purification kit (according to manufacturer's instructions) and subjected to ligation reaction.

The ligation products were transformed into competent E.coli DH5 α and recombinant clones were screened by growing randomly selected clones and extracting plasmid DNA with Qiagen QIAprep Spin Miniprep kit according to the manufacturer's instructions.

The recombinant plasmid was introduced into E.coli BL21(DE3) which was used as an expression host. Single recombinant colonies were inoculated into LB + ampicillin and incubated at 37 ℃ for 14-16 hours. The bacteria can be recovered directly by centrifugation (non-induced conditions), or diluted into fresh medium and incubated at 37 ℃ until OD₆₀₀Between 0.4 and 0.80. Protein expression was induced for 3 hours by the addition of 1mM isopropylthio-. beta. -D-galactoside (IPTG) (induction conditions).

Bacteria were resuspended in SDS-sample buffer 1X and boiled for 5-10 minutes to obtain whole cell lysates. Equal amounts of protein were separated using NuPAGE (Invitrogen) or BIORAD gel systems according to the manufacturer's instructions. Proteins were visualized by coomassie blue staining or transferred to nitrocellulose membranes for western blot analysis. Anti-purified NadA for western blot _351-405Rabbit polyclonal antiserum (1: 3000 dilution) and peroxidase-conjugated secondary antibody (DAKO).

FIGS. 38 and 39 show S1_L、S1_LNadA and S1_L-NadA_{Delta anchor}Results of expression in E.coli. FIG. 37 shows a fusion construct.

Bacterially expressed SARS viral antigens can also be used to prepare compositions containing outer membrane vesicles, wherein the outer membrane vesicles comprise one or more SARS viral antigens.

Outer membrane vesicles ("OMVs"), also known as blebs, refer to vesicles formed from or derived from outer membrane fragments of gram-negative bacteria. OMVs typically comprise Outer Membrane Proteins (OMPs), lipids, phospholipids, periplasmic material and Lipopolysaccharides (LPS). In a process known as vesiculation, gram-negative bacteria often form OMVs when they are heavily infected. OMVs can also be obtained from gram-negative bacteria by some chemical denaturing process, such as detergent extraction. The SARS virus antigen of the invention can also be used to prepare synthetic OMVs or liposomes having a lipid bilayer encapsulating an aqueous core.

OMVs of the invention are preferably lipid vesicles comprising an aqueous core surrounded by a lipid bilayer. The lipid vesicles typically have a monolayer structure (i.e., an aqueous core surrounded by a lipid bilayer), although multilamellar lipid vesicles may also be used in the compositions of the invention. The size of the OMVs is typically in the nanomolar to micromolar range, e.g., from about 1nM to 100. mu.M, more typically from about 10nM to 10. mu.M, preferably from 30nM to 1. mu.M.

The OMVs of the invention are preferably prepared from gram-negative bacteria. Gram-negative bacteria are those which are not resistant to the decolorization treatment in the well-known gram staining method. Gram-negative bacteria are characterized by a complex, polygonal cell wall and typically have an outer polysaccharide capsule. Gram-negative bacteria suitable for use in the manufacture of OMVs include, for example, Neisseria, Moraxella, Chrysomyiame, Acinetobacter, Brucella, Bordetella, Chlamydia, fungi, Actinobacillus, Borrelia (Borelia), Serratia, Campylobacter, helicobacter, Haemophilus, Escherichia, Legionella, Salmonella, Pseudomonas and Yersinia.

The OMVs of the invention preferably contain one or more SARS virus antigens or fragments thereof. The SARS virus antigen can be recombinantly expressed in gram-negative bacterial host cells and then harvested with OMVs.

Antigenic components, such as recombinantly expressed SARS virus antigens, can be located within any or all of the three major compartments of the lipid vesicle, including compartments attached to the inner or outer surface of the lipid vesicle by, for example, a membrane anchoring domain; a compartment inserted into a lipid bilayer, for example wherein the antigenic component itself is a hydrophobic or lipid-based entity; or a compartment located in the aqueous center or core of the lipid vesicle.

Synthetically prepared OMVs or liposomes may be used in the present invention. Such liposomes comprise a number of different lipids or fatty acids. Lipids suitable for inclusion in liposomes of the invention include, but are not limited to, phosphatidylinositol- (4, 5) -diphosphate, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cholesterol, β -oleoyl- γ -palmitoyl, lipopolysaccharide, and galactocerebroside (galactocerbroside).

Suitable methods for extracting OMVs from bacterial material include deoxycholate extraction, Tris/HCl/EDTA extraction and lithium acetate extraction. Preferably the extraction process comprises physically and/or chemically disrupting the bacterial cell outer membrane to allow sufficient release of OMVs for purification and isolation. See, for example, WO 03/051379.

The OMVs of the invention may be enriched and/or have antigenic components added thereto, such as SARS virus antigens, by methods known in the art, including, for example, direct in vitro mixing, where high energy mixing may optionally be performed to facilitate integration of the antigenic components into one compartment of the liposomes. High energy mixing methods suitable for use in the present invention include homogenization, ultrarapid disruption, extrusion, and combinations thereof.

Preferably, the antigenic component, such as a SARS virus antigen, is recombinantly produced using host cells from which the OMV is obtained. In one embodiment, such OMVs are made by introducing into a recombinant host cell a nucleic acid sequence encoding a SARS virus antigen. Preferably, the nucleic acid sequence encoding the SARS virus antigen is under the control of a strong promoter sequence. Preferably, the nucleic acid sequence encoding the SARS virus antigen further comprises an outer membrane targeting signal. For example, a nucleic acid sequence encoding a SARS virus antigen can be fused to a sequence encoding an outer membrane protein naturally produced by a bacterial host. Preferably, the nucleic acid sequence encoding the SARS virus antigen is fused to a signal peptide sequence of an outer membrane protein naturally produced by the bacterial host.

Methods for making optimal OMVs for vaccines are described, for example, in Filip et al, j.bact. (1973) 115: 717-722; davies et al, j.immunol.method (1990) 143: 215-; and WO 01/09350.

In one embodiment, a bacterial host cell, such as E.coli, is transformed to express the SARS spike protein. As described above, the spike protein may be modified to facilitate bacterial expression and transport of the spike protein to the surface of the host cell. The various spike/NadA fusion constructs described above can be used to prepare OMVs of the invention. Preferably, a construct containing the knob domain of spike S1 fused to the handle region of NadA was used to generate OMVs. Such constructs may optionally contain a NadA leader peptide as well as a NadA anchor peptide. A schematic of the structure of these preferred OMV constructs is shown in FIG. 49.

Example 6 describes a method for preparing OMVs of the invention.

(b) Mammalian expression of SARS subunit vaccine

As described above, mammalian host cells are used to recombinantly express SARS virus protein. Mammalian host cells suitable for use in the present invention include, for example, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, and others. Mammalian sources of cells include, but are not limited to, human or non-human primate cells (e.g., MRC-5 (ATCCCL-171), WI-38(ATCC CCL-75), human embryonic kidney cells (293 cells, typically transformed with sheared adenovirus type 5 DNA), monkey kidney Vero cells (including COS7 cells), horses, cattle (e.g., MDBK cells), sheep, dogs (e.g., dog kidney MDCK cells, ATCC CCL34MDCK (NBL2) or MDCK 33016 (deposited under the accession number DSM ACC 2219 as described in WO 97/37001), cats, and mice (e.g., hamster cells, such as BHK21-F, HKCC cells or Chinese hamster ovary cells (CHO cells)) and can be obtained from various developmental stages, including, for example, adult, neonatal, fetal, or embryonic.

The polynucleotide encoding the SARS virus protein may be modified to facilitate or enhance expression. For example, commercially available leader sequences, such as tPA or IgK or interleukin-2, are known in the art for use in recombinant constructs. However, it is preferred to use the native SARS leader sequence. The use of a native leader sequence can ensure that the protein will enter the human cell in the same way as a normal viral infection, which is advantageous for e.g. DNA vaccines expressing the antigen in situ.

As described above, marker sequences may be used in expression constructs to facilitate purification, detection, and stabilization of the expressed protein. Marker proteins suitable for use in the present invention include poly-arginine-tag (Arg-tag), poly-histidine-tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin binding peptide, cellulose binding domain, SBP-tag, chitin binding domain, glutathione S-transferase tag (GST), maltose binding protein, transcription termination anti-termination agent (NusA), E.coli thioredoxin (TrxA), and protein disulfide isomerase I (DsbA). Preferred marker proteins include His-tag and GST. For a detailed discussion of the marker proteins see Terpe et al, "review of marker protein fusions: from molecular and biochemical basis to commercial systems ", Appl Microbiol Biotechnol (2003) 60: 523-533.

After purification, the marker protein may optionally be removed from the expressed fusion protein, i.e., by treatment with specific splicing enzymes as is known in the art. Commonly used proteins include enterokinase, Tobacco Etch Virus (TEV), thrombin and X_aA factor.

One or more amino acid sequences or amino acid domains of the spike protein may be removed to facilitate recombinant expression in a mammal. For example, the entire S2 domain or the spike transmembrane region can be removed. Representative examples of some expression constructs suitable for mammalian expression of full-length or truncated spike glycoproteins are shown in FIG. 40. The polynucleotide sequence representing each construct is shown in SEQ ID NOS 6578-6583. The meaning of each symbol is as follows:

clone name description expression constructs

nShA native leader sequence SEQ ID NO: 6578

Full length spikes

Histidine tag

nS native leader sequence SEQ ID NO: 6579

Full length spikes

nSh Δ TC native leader sequence SEQ ID NO: 6580

Spikes without transmembrane sequence

Histidine tag

nSh Δ TC native leader sequence SEQ ID NO: 6581

Spikes without transmembrane sequence

nS1h native leader sequence SEQ ID NO: 6582

S1 Domain

Histidine tag

nS1 native leader sequence SEQ ID NO: 6583

S1 Domain

The cloned cDNA fragment containing the full-length spike coding sequence and the transmembrane and cytoplasmic domain deleted spike construct (TM-Cy-deleted spike) was inserted into the expression vector, pCMVIII, to form secreted nSh and nSh Δ TC, respectively. Both spike proteins contained a six histidine tag at the C-terminus to aid in the initial qualitative expression of the spike protein. Similar sequences encoding full-length spikes or spikes deleted of the transmembrane and cytoplasmic domains, but not containing a histidine "tag" are readily substituted by those skilled in the art.

The expressed protein was separated into an Aqueous Fraction (AF) and a Detergent Fraction (DF) using the method shown in FIG. 48 to assess the possible localization of the expressed spike construct, and the results of the western blot analysis are shown in FIG. 43. The expression of the above-described vector constructs was evaluated after transfection into COS7 cells. Constructs expressing the full-length spike protein remained in the cell membrane, while constructs expressing the truncated spike protein were either localized in the cytosol (FIG. 43) or secreted into the cell culture medium (FIG. 44). As shown in fig. 43, the full-length spike protein was found to be present in aggregated form in DF (membrane), while the truncated protein was present in monomeric form in AF (cytosol). As shown in fig. 44, the deleted protein (Sh Δ TC) was secreted and a small fraction of the full-length spike protein was detected in the culture medium with rabbit serum.

The recombinantly expressed spike protein may be oligomerized. When the spike proteins are used in vaccines or to generate antibodies specific for the spike proteins, they are preferably oligomeric. To obtain oligomeric spike proteins, the transmembrane domain is preferably retained in a recombinant expression construct. For example, figure 41 compares the Western blot results of COS7 cell lysates obtained using anti-his tag and rabbit anti-SARS antibodies with expressed nSh and nSh Δ TC. As shown, full-length (nSh) formed an aggregation, whereas the truncated (nShATC) spike protein did not. Antibodies against His-tagged proteins identified full-length and truncated spike proteins in both native and reduced forms. Rabbit antisera identified spike proteins only under non-reducing conditions. Spike aggregates or oligomers appear in large numbers in cell lysates of the expressed nSh constructs. Preferably, the oligomeric spike protein forms a homotrimer, as shown in FIG. 47.

In another experiment, as shown in fig. 42, it was demonstrated that oligomerization of the expressed nSh construct was likely due to non-covalent attachment (rather than, for example, disulfide bonds). Such oligomers dissociate to monomers at elevated temperatures (80-100 ℃) but are stable under reducing conditions if not heated.

The recombinantly expressed spike protein is also preferably glycosylated. Glycosylation can be assessed with tunicamycin and glycosidase. FIG. 45 illustrates that glycosylation of the expressed spike protein is not affected by the removal of the transmembrane domain region. Both the full-length (Sh) and truncated (Sh Δ TC) SARS spike proteins are glycosylated.

Preferably, expression of the constructs of the invention is not toxic to mammalian host cells. FIG. 46 demonstrates that expression of the indicated spike construct is not toxic to COS7 host cells.

Methods for transfection, expression, culture, isolation and purification of recombinant proteins in mammalian cell culture are known in the art. For example, the SARS spike construct of the present invention may be expressed in 293 cells. These cells can be cultured and transfected in a static culture or a monolayer culture. For rapid large-scale production of sufficient SARS protein antigen for in vitro or in vivo evaluation, including immunogenicity studies, 293 (human embryonic kidney) cells can be transiently transfected on a large scale to obtain recombinant antigen on the order of milligrams. Alternatively, 293 cells can be transfected in suspension culture on a large scale. Preferably, the expressed SARS protein is harvested from transfected cells 48-72 hours after transfection, or even 72-96 hours or more after transfection.

When the host cell is transfected with the truncated spike expression construct, the expressed spike protein is secreted by the host cell and collected from the cell culture medium. Following concentration, the spike protein can be purified from the culture medium using, for example, GNA lectin followed by DEAE and ceramic hydroxyapatite column chromatography.

When the host cells are transfected with the full-length spike expression construct, the expressed spike protein remains intracellular and can be purified from cells extracted with triton X-100 detergent. The full-length spike protein can then be captured with GNA lectin and purified by hydroxyapatite and SP chromatography.

Chinese Hamster Ovary (CHO) or other eukaryotic (e.g., mammalian) cells stably expressing the SARS virus antigens of the invention can also be produced (e.g., fig. 73). Preferably the cells are CHO cells and these constructs will contain one or more markers or selection genes to select for the desired CHO cells. In one embodiment, the construct comprises a CMV enhancer/promoter, an ampicillin resistance gene, and fused DHFR and an attenuated neomycin gene for screening purposes. The neomycin selection system can then be used to generate stable cell lines in CHOK-1 cells. The selected clones can then be sequenced to verify the integrity of the identified insert, and then transiently transfected with Trans-LT1 polyamine transfection reagent (PanVeraCorp., Madison, Wis.) to assess expression levels and integrity of the expressed protein by ELISA and western blot analysis.

The method for deriving CHO cells stably expressing SARS virus antigen of the present invention comprises a transfection step and a preliminary screening step using a selective medium. Optionally, these steps may be followed by subcloning to ensure purity of the cell line. Cell culture supernatants can be assayed by antigen capture ELISA to quantitatively select and amplify expression levels at all stages.

For full-length spike expression constructs, immunofluorescence staining with rabbit anti-SARS antibodies was used to screen for intracellular expression of methanol-fixed cells. Continuous measurements were performed during the amplification stage of the T75 flask to ensure stability of the expression levels. The molecular weight and integrity of the expressed protein can be checked by PAGE (native and reductive denaturation conditions) followed by immunodetection.

In one embodiment, the pCMV3 vector expressing the full-length or truncated form of SARS-CoV spike protein is introduced into CHOK-1 cells using Trans-LT-1 reagent. On day 1, 1X 10⁶Cells were seeded on 100mM dishes containing non-selective F12 medium + 10% fetal bovine serum +4mM glutamine. Day 2, with DNA: the LT-1 mixture was used to transfect cells and complete F12 medium was used in place of the original medium. After 24-48 hours, each 100mm plate was divided into 4-6 100mm plates according to cell density. The medium was changed to a fully selective medium containing 500. mu.g/ml geneticin (neomycin). All bovine sera used in these procedures were TSE-free and meet current FDA standards. After 24 hours the medium was changed to a fully selective medium supplemented with 500. mu.g/ml neomycin. After 10-14 days, individual colonies were picked, transferred to 96-well plates and cultured in complete selective medium without G418. When approximately 80% of the wells were filled, the 24-hour supernatant of spike-capture ELISA positive clone selection was transferred to 24-well plates. To initiate expression of the full-length spike protein, the methanol-fixed cells were screened using rabbit anti-SARS antibody as an immunofluorescent stain. There were less than 20-30 cell lines after depletion of the low expressing cell line and expression levels were measured after cell lysis using capture ELISA and western. A portion of each cell line was pelleted, weighed, and 1% triton lysis buffer (containing MOPS, NaCl and MgCl) at the same weight ratio of cells to lysis buffer ₂) And (4) cracking. Supernatants were collected after lysis to determine expression levels. 3-4 clones producing structurally and conformationally correct spike proteins at the highest levels were expanded and adapted to low serum suspension culture conditions in a 3 liter bioreactor for scale-up.

Antigen capture ELISA assays for SARS spike protein can be performed as described in the art. A brief description of this measurement method is as follows. 250ng of purified immunoglobulin obtained from rabbit serum immunized with inactivated SARS virus was plated in each well of a 96-well flat-bottom plate (Corning, Corning, NY). The plates were then washed with buffer containing 16% NaCl and 1% Triton X100. Add 100. mu.L of supernatant or lysate sample (diluted in 100mM NaPO)₄0.1% casein, 1mM EDTA, 1% Triton X100, 0.5M NaCl, and 0.01% thimerosal, in a buffer pH 7.5) and incubated at 37 ℃ for 2 hours. Bound antigen with collected SARS + ve serum or high affinityHuman or murine anti-SARS spike protein monoclonal antibodies were reacted (incubation for 1 hour at 37 ℃) and detected with the appropriate species-specific peroxidase conjugated secondary antibody (incubation for 30 minutes at 37 ℃; TAGO, Burlingame, CA). Plates were developed with TMB (Pierce, Rockford, IL) at room temperature and the reaction was stopped with 4N phosphoric acid. The plates were read at 450nm and the concentration of protein per ml of sample was generated according to a standard curve (OD versus protein concentration) prepared from serial dilutions of known concentrations of recombinant spike protein.

Immunodetection assays can also be performed following standard methods already described in the art. Briefly described as follows. 10-20 μ l samples were analyzed on 4-20% SDS PAGE under mild heating under non-reducing/denaturing conditions. Run at constant pressure of 100V for 1.5-2.0 hours to gel. The proteins were then transferred to nitrocellulose membranes (Millipore, Bedford, MA) for 45 minutes using a semi-dry western transfer system (BioRad, Hercules, CA) according to the manufacturer's instructions. The membrane was reacted with polyclonal rabbit anti-spike serum and then with anti-rabbit Ig (molecular probes, Oregon) conjugated to Alexa 688. The blot was scanned with an infrared imaging system (LI-Cor, inc., Lincoln, Nebraska).

Candidate cell lines were selected for highest spike protein expression and highest stability in small (3 liter) suspension cultures. After amplification, the candidate clones were further evaluated for expression level and integrity of expressed protein, and then tested for expression stability in the absence of selection. Selected clones can also be monitored for their ability to maintain the integrity of the intact SARS spike protein gene DNA sequence. For rapid monitoring of expression levels in flasks (T25 or T75) and 3 liter evaluation cultures, the SARS spike protein can be isolated in a semi-quantitative and qualitative purity of the protein in CHO supernatant using a lectin-based method (Gluvanthus Nivalis lectin). For full-length spike proteins, they can be obtained from cells extracted from triton X-100 detergent. The full-length spike protein was then captured on GNA lectin, followed by hydroxyapatite and SP chromatography. The eluted protein was characterized by the following method: 1) polyacrylamide gel electrophoresis (PAGE) and Coomassie staining, 2) immunodetection with rabbit anti-SARS serum, 3) structural analysis by Size Exclusion Chromatography (SEC) and mass spectrometry by MALDI-TOF.

The route and method of immunization with the vaccine of the present invention will be described in detail in the following sections. Examples 7-9 illustrate protocols for recombinant spike protein immunization.

Vaccine testing

Prior to use in humans, it is often necessary to test vaccines in animal models. Mouse models of SARS coronavirus infection are known (Subbarao et al (2004) J Virol 78: 3572-77), and other animals that can be used as models of infection and/or disease include ferrets and monkeys. Accordingly, the present invention provides a non-human animal infected with SARS coronavirus, wherein the animal is preferably a ferret or primate (e.g., monkey or macaque). The animal may be a sterile animal. Preferably the animal is not a cat (Felis domesticus). The animals may or may not show symptoms of SARS disease, e.g. ferrets (Mustela furo) show significant lung pathology after infection. See: martina et al (2003) Nature 425: 915.

E. polynucleotides encoding SARS antigens of the invention

The present invention includes polynucleotides encoding the SARS antigens of the invention. In addition, the invention encompasses polynucleotides optimized (e.g., by codon optimization) for recombinant production of SARS antigens, including polynucleotides encoding each of the SARS fusion constructs described above.

F. Viral vectors or viral particles for delivery of SARS antigens of the invention

The antigens of the invention may be expressed in vivo or in vitro from polynucleotides encoding the antigens. Expression and delivery of the polynucleotides of the invention is facilitated by viral vectors and/or viral particles.

Heterologous genes, including one or more SRAS genes, can be administered in vitro and in vivo using viral (e.g., alphavirus) produced gene delivery systems. These systems can also be used to produce recombinant proteins derived from SARS virus in cultured cells. The gene-based delivery systems of the invention include viral vectors (e.g., adenoviral vectors, poxviral vectors, alphaviral vectors) and non-viral nucleic acid vectors (e.g., DNA, RNA) encoding one or more SARS viral antigens. Polynucleotides encoding SARS virus antigens are incorporated into the genetic vaccine, either alone or in combination (e.g., as a bicistronic construct).

1. Alpha virus

Alphaviruses are members of the togaviridae family that share structural and replicative properties. Sindbis virus (SIN) is the prototype virus for molecular studies of other alphaviruses, and together with Venezuelan equine encephalitis Virus (VEE) and Semliki Forest Virus (SFV) is the most widely used alphavirus that produces heterologous gene expression vectors (Schlesinger and Dubensky (1999) Curr Opin Biotechnol.10: 434-439; Schlesinger (2001) Expert Opin Biol.Ther.1: 177-91).

Alphaviruses have a relatively small positive polarity single-stranded RNA genome, approximately 12kb in length, which is capped and polyadenylated. The RNA interacts with viral capsid protein monomers to form a nucleocapsid, which is then surrounded by a host cell-derived lipid envelope from which the two viral glycoproteins, E1 and E2, protrude to form a "spike" trimer of heterogeneous dimeric subunits. The polyprotein encoded by the two Open Reading Frames (ORFs) is an enzymatically active nonstructural replicase protein (5 'ORF) and a virion structural protein (3' ORF). The structural polyprotein is translated from a highly abundant subgenomic mRNA transcribed from an internal alphavirus potent promoter (Strauss and Strauss (1994) Microbiol. Rev.58: 491-562). Replication of the genome occurs, like RNA, only in the cytoplasm of the host cell.

The most commonly used alphavirus expression vectors exploit the positive strand properties and modular structure of the RNA genome. These vectors are termed "replicons" due to their property of spontaneous amplification and allow the insertion of heterologous sequences in place of the structural polyprotein gene while retaining the 5 '-and 3' -terminal cis-replication signals, the non-structural replicase genes and the subgenomic binding region promoter (Xiong et al (1989) Science 243: 1188-1191; Liljestorom (1991) Bio/Technology 9: 1356-1361). Chimeric alphavirus vectors (and particles) derived from sequences of the divergent virus family have also been described. (see, e.g., U.S. patent application Ser. No. 09/236,140; see also U.S. Pat. Nos. 5,789,245,5,842,723, 5,789,245,5,842,723 and 6,015,694; and WO95/07994, WO 97/38087 and WO 99/18226). The consensus international publication WO 02/099035, filed 12/2002 and incorporated herein by reference in its entirety, describes chimeric alphavirus molecules and improved alphavirus molecules with improved levels of biosafety.

The absence of the structural protein gene makes the alphavirus replicon vector deficient so that RNA amplification and high levels of expression of the heterologous gene occur within the target cell, but cell-cell transmission of the vector is not possible due to the inability to form progeny viral particles. In recent years, several terms have emerged with the same meaning as used to describe alphavirus replicon particles. These terms include recombinant viral particles, recombinant alphaviral particles, alphaviral replicon particles, and replicon particles. However, these terms are used herein to denote a virosome-like unit containing an alphavirus-derived RNA vector replicon. In addition, these terms may refer to a vector, vector construct, or gene delivery vector.

Replicon RNA can be packaged into particles by introducing the replicon RNA into permissive cells (e.g., RNA or DNA transfection, or particle transfection) containing one or more structural protein expression cassettes or "defect helper" constructs encoding alphavirus structural proteins. These structural proteins encoding the constructs can themselves be introduced into cells by transfection with RNA or DNA and typically retain the native alphavirus subgenomic promoter, as well as the 5 '-and 3' -terminal cis signals for co-amplification with the replicon, but lack any replicase genes and RNA packaging signals (Liljestrom (1991) Bio/Technology 9: 1356-. Immortalized cell lines (e.g., packaging cell lines) stably transformed with constructs expressing alphavirus structural proteins provide a means to avoid transient transfection for production (Polo et al (1999) PNAS 96: 4598-4603).

The present invention includes compositions and methods for making replication-defective viral vector particles (e.g., alphavirus replicon particles) for in vitro and in vivo administration of heterologous genes encoding proteins having therapeutic or prophylactic uses, including genes encoding one or more SARS virus antigens.

In one aspect, the invention includes a method of making a replication-defective viral vector particle (e.g., an alphavirus replicon particle), the method comprising the steps of introducing at least one nucleic acid molecule comprising a viral vector (e.g., an alphavirus replicon RNA) into an immortalized cell of the invention under conditions that are complementary to the viral vector (e.g., an alphavirus replicon RNA) and making the viral vector particle (e.g., an alphavirus replicon particle), and isolating the viral vector particle from the cell or cell culture supernatant. In some embodiments, the immortalized cells are grown in suspension, such as perc.6 cells. In other embodiments, the process is carried out at a larger volume, e.g., a volume in liters or more, as in shake flasks, large flasks, Nunc Cell facilities, Corning Cell Cubes, fermenters, etc.).

In some embodiments, the viral vector is an alphavirus replicon that requires the provision of one or more trans alphavirus structural proteins for supplementation within an immortalized cell. At this point, the method of producing alphavirus replicon particles by complementation includes introducing into the immortalized cells one or more nucleic acids (e.g., RNA, DNA) encoding the alphavirus structural proteins (e.g., capsid and/or envelope glycoproteins), which may be transient or permanent, and may be concurrent with or prior to the introduction of the alphavirus replicon RNA. In some embodiments, the alphavirus replicon RNA is introduced into the cell by transfecting RNA that transcribes the replicon RNA in vitro. In other embodiments, the alphavirus replicon RNA is introduced into the cell by transfection with DNA capable of transcription within the cell (e.g., ELVIS). In yet another embodiment, the alphavirus replicon RNA is introduced into the cell by seed infection with alphavirus replicon particles. In some embodiments, the nucleic acid encoding the viral structural protein is a defective helper RNA or DNA capable of transcribing a defective helper RNA in a cell.

"alphavirus RNA replicon vector", "replicon vector" or "replicon" as used herein refers to an RNA molecule capable of directing its own amplification or self-replication in vivo in a target cell. To direct its own amplification, such an RNA molecule should encode a polymerase necessary to catalyze RNA amplification (e.g., alphavirus nonstructural proteins nsP1, nsP2, nsP3, nsP4) and contain, in combination, the cis RNA sequence required for replication identified and utilized by the encoded polymerase. The alphavirus RNA vector replicon should contain elements in the following order: nonstructural proteins mediate 5 'viral or cellular sequences required for amplification (also referred to as 5' CSE, or 5 'cis replication sequences, or cis 5' viral sequences required for replication, or 5 'sequences capable of initiating alphavirus transcription), sequences encoding biologically active alphavirus nonstructural proteins upon expression (e.g., nsP1, nsP2, nsP3, nsP4), and 3' viral or cellular sequences required for nonstructural protein amplification (also referred to as 3 'CSE, or cis 3' viral sequences required for replication, or alphavirus RNA polymerase identification sequences). Alphavirus RNA vector replicons also contain a component to express one or more heterologous sequences, such as an IRES or a viral (e.g., alphavirus) subgenomic promoter (e.g., a binding region promoter), which in some embodiments can be modified to enhance or reduce viral transcription of subgenomic fragments, or to reduce homology to defective helper or structural protein expression cassettes and one or more heterologous sequences to be expressed. The heterologous sequence preferably comprises a gene encoding a protein, which is the gene within the vector replicon closest to the 3' end. The replicon further comprises a polyadenylation sequence.

"recombinant alphavirus particle", "alphavirus replicon particle" and "replicon particle" herein mean a virus particle-like unit containing an alphavirus RNA vector replicon. Typically, the recombinant alphavirus particles comprise one or more alphavirus structural proteins, a lipid envelope, and an RNA vector replicon. Preferably, the recombinant alphavirus particle comprises a nucleocapsid structure contained within a host cell-derived lipid bilayer, such as the cytoplasmic membrane, in which one or more alphavirus envelope glycoproteins (e.g., E2, E1) are embedded. The particles may also contain other components (e.g., targeting elements such as biotin, other viral structural proteins or portions thereof, hybrid envelopes, or other receptor binding ligands) that can direct tropism of the derived alphavirus particles. In general, the interaction between alphaviral RNA and structural proteins necessary for efficient formation of an alphareplicon particle or nucleocapsid may be an RNA-protein interaction between the capsid proteins and the packaging signal or packaging sequence contained within the RNA.

An "alphavirus packaging cell line" refers to a cell that contains one or more alphavirus structural protein expression cassettes and produces recombinant alphavirus particles following introduction of an alphavirus RNA vector replicon, a eukaryotic layered vector promoter system, or recombinant alphavirus particles. The parental cell may be derived from a mammal or a non-mammal. In a preferred embodiment, the packaging cell line is stably transformed with a structural protein expression cassette.

By "defective helper RNA" is meant an RNA molecule that is capable of being amplified to express one or more alphavirus structural proteins in a eukaryotic cell that also contains functional alphavirus nonstructural "replicase" proteins. The alphavirus nonstructural proteins can be expressed in cells by alphavirus RNA replicon vectors or other means. For amplification and expression of structural proteins (mediated by alphavirus nonstructural proteins), the defective helper RNA molecule should contain both the 5 '-terminal and 3' -terminal RNA sequences required for amplification that can be identified and utilized by the nonstructural proteins, as well as components that express one or more alphavirus structural proteins. Thus, alphavirus-deficient helper RNAs contain elements in the following order: a 5 'viral sequence or cellular sequence required for RNA amplification of an alphavirus nonstructural protein (also referred to as a 5' CSE, or a 5 'cis replication sequence, or a cis 5' viral sequence required for replication, or a 5 'sequence capable of initiating alphavirus transcription), a component that expresses one or more alphavirus structural proteins, a gene sequence that encodes one or more alphavirus structural proteins (e.g., C, E2, E1) upon expression, a 3' viral sequence or cellular sequence required for amplification of an alphavirus nonstructural protein (also referred to as a 3 'CSE, or a cis 3' viral sequence required for replication, or an alphavirus RNA polymerase identifying sequence), and preferably comprises a polyadenylation sequence. Typically, the defective helper RNA does not itself encode or express all four alphavirus nonstructural proteins (nsP1, nsP2, nsP3, nsP4) in their entirety, but may encode or express a subset of these proteins or a portion thereof, or contain sequences from one or more nonstructural protein genes, but these sequences contained within the defective helper do not express nonstructural proteins or portions thereof. As a means of expressing alphavirus structural proteins, the defective helper RNA can contain a viral (e.g., alphavirus) subgenomic promoter, which in some embodiments can be modified to regulate transcription of subgenomic fragments, or to reduce homology to replicon RNAs, or to contain other components (e.g., internal ribosome entry sites, ribosome readthrough elements) that affect alphavirus structural protein expression. Preferably, the alphavirus structural protein gene is the gene closest to the 3' end within the defect helper. Furthermore, it is preferred that the defective helper RNA does not contain sequences that facilitate packaging of the RNA-protein into the nucleocapsid, viral particle-like particle or alphavirus replicon particle by interaction with alphavirus structural proteins. Defective helper RNAs are a particular embodiment of alphavirus structural protein expression cassettes.

The alphavirus used in the present invention may be grown in any of the above cell lines suitable for the SARS virus.

Alphavirus replicon particles can be made according to the methods of the invention by using the above-described cell lines (e.g., immortalized cell lines) and many of the published and accepted alphavirus vector methods. Such methods include, for example, transient packaging procedures such as co-transfection of in vitro transformed replicons and defective auxiliary RNAs (Liljestrom, Bio/Technology 9: 1356-1361, 1991; Bredenbeek et al, J.Virol.67: 6439-6446, 1993; Frolov et al, J.Virol.71: 2819-2829, 1997; Pushko et al, Virology 239: 389-401, 1997; U.S. Pat. Nos. 5,789,245 and 5,842,723), or co-transfection of plasmid DNA replicons and defective auxiliary constructs (Dubensky et al, J.Virol.70: 508-519, 1996), and introduction of alpha virus structural protein expression cassettes (e.g., DNA defective helpers) into the immortalized cell lines of the invention to generate stable Packaging Cell Lines (PCL) (Polo et al, AS 96: 4603, 1999; U.S. Pat. No. 5,789, WO 245, WO 5,842, WO-6757; WO 36694, 00/61772, 366942). The stable packaging cell line can then be used to make alphavirus replicon particles. PCL can be transfected with alphavirus replicon RNA transcribed in vitro, with a plasmid DNA replicon (e.g., ELVIS vector), or with alphavirus replicon particles in liquid, and then incubated under conditions and for a time sufficient to produce progeny alphavirus replicon particles in the culture supernatant. In addition, progeny replicon particles can subsequently be passaged in other native PCL cultures by infection, which will result in further amplification and a commercial scale preparation. Importantly, by using defective helper RNA or stabilized PCL based on a "nicked" structural gene configuration, a replicon particle stock free of detectable contamination with RCV can be generated.

After harvesting, the harvest can be passed through a filter (e.g., pore size 0.2uM, 0.45uM, 0.65uM, 0.8uM) to clarify the crude culture supernatant containing the chimeric alphavirus replicon particles. The crude supernatant may optionally be subjected to low speed centrifugation to remove larger cell debris prior to filtration. In one embodiment, an endonuclease (e.g., Benzonase, Sigma # E8263) is added to the preparation of alphavirus replicon particles to digest the exogenous nucleic acid before or after the chromatographic purification step. In addition, the preparation may be concentrated prior to purification by any of a number of well known methods, such as tangential flow ultrafiltration. Crude or clarified alphavirus replicon particles can be concentrated and purified by chromatographic techniques (e.g., ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography), as described in WO01/92552, which is incorporated herein by reference in its entirety. Two or more such purification methods may be used sequentially.

Examples of alphavirus replicon particles encoding SARS virus spike protein antigen

The present invention includes compositions and methods for making replication-defective viral vector particles (e.g., alphavirus replicon particles) that can be used for in vitro and in vivo administration of heterologous genes encoding proteins having therapeutic or prophylactic use, including genes encoding one or more SARS virus antigens.

The following example illustrates a method for preparing alphavirus replicon particles encoding SARS virus spike antigen.

The SARS virus spike gene can be incorporated into alphavirus replicon particles from a variety of alphaviruses, such as sindbis virus, Semliki forest virus (US 5739026), venezuelan equine encephalitis virus (US 6531135), and replicon particle chimeras from more than one alphavirus (US 6376236, WO 02/99035). In addition, the SARS virus spike gene can be incorporated in its intact form (encoding a full-length spike protein) or in modified forms including, for example, sequence deletions or truncations such that the encoded spike protein is less than full-length (e.g., C-terminal is truncated by one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, etc.) amino acids, deleting the transmembrane region or cytoplasmic tail).

For example, the spike gene can be cloned as a full-length gene into a VCR-chim2.1 vector (WO 02/99035) using commercially available restriction endonucleases from one of the other plasmids described herein by standard RT-PCR conditions or by standard subcloning. For the reverse transcription step in RT-PCR, the Superscript preamplification kit (Invitrogen) was used ^TM) And primers SEQ ID NO: 7325 (sp-RT-R):

the amplification step used the cDNA polymerase advantage kit (Clonetech) and two primers Sp-F-BbvCI (SEQ ID NO: 7326) and Sp-R-NotI (SEQ ID NO: 7327):

the forward primer was designed to contain the sequence ccacc (Kozak, 1991 JBC 19867-70) before the ATG codon to optimize the translation efficiency of the spike gene. Also, the forward primer contains a BbvCI restriction site and the reverse primer contains a NotI restriction site for subsequent cloning of the PCR amplified gene.

The PCR product was purified with QIAquick Nucleotide Removal kit (QIAgen), digested with BbvCI and NotI, Gel purified with QIAquick Gel Extraction kit (QIAgen), and ligated to plasmid VCR-Chim2.1 pre-digested with the same enzymes. Clones containing the SARS spike sequence were verified by sequencing and the new construct was named VCR-Chim 2.1-SARSspike.

To generate VEErep/SINenv-SARSspike replicon particles, the plasmids VCR-Chim2.1-SARSspike, VCR-DH-Scap (WO02/99035) and VCR-DH-Sglydl160(WO02/99035) were linearized with the restriction enzyme PmeI and transcribed in vitro as described above (Polo et al, 1999, PNAS 96: 4598-603; WO 02/99035). And the transcripts were co-transfected into BHK cells as described above (Polo et al, 1999, supra; WO 02/99035). Transfected cells were incubated at 34 ℃ and supernatants collected 20-30 hours after electroporation, clarified by centrifugation, and purified by chromatography, as described above (WO 01/92552).

Infection of BHK cells with purified VEErep/SINenv-SARSspike or VEErep/SINenv-GFP (WO 02/99035) replicon particles overnight verified SARS spike protein expression from the replicon particle vectors. In addition, BHK cells were also transfected in parallel with the in vitro transcribed VCR-Chim2.1-SARSspike replicon RNA. Infected cells were lysed 16 hours post infection and lysate samples were analyzed by western blot using antibodies that recognize the spike protein of SARS virus. The proteins on the gel are stained or transferred to a membrane for Western blot analysis using serum from convalescent patients or mouse or rabbit antisera against SARS virus. VEErep/SINenv-sarspike replicon particles are administered to vaccine recipients (e.g., rodents, non-human primates, humans) as described elsewhere herein.

FIG. 67 shows data from western blot analysis results obtained using SARS virus-specific rabbit polyclonal antisera under non-reducing conditions. The western data demonstrated that the SARS spike protein was not only expressed in cells infected with alphavirus replicon particles or transfected with replicon RNA, but that the predominant form of spike protein was homotrimer (fig. 67A). Homotrimeric ligation of similar spike proteins was observed in western blots of SARS virus particles purified from SARS virus infected VERO cell supernatants, this homotrimer being heat labile, as illustrated by its dissociation into monomeric form at 80 ℃ and 100 ℃ (fig. 67B).

To further characterize the expression of SARS spike protein and processing following expression from alphavirus replicon vectors, BHK-21 cells were infected with alphavirus replicon particles expressing full-length spikes. After 6 hours of infection with 5MOI, the infected cells are treated with L-, [ 2 ], [³⁵S]Methionine/cysteine labeling was performed for 1 hour. [³⁵S]Labeled spike proteins were immunoprecipitated with rabbit anti-SARS serum and digested with Endo-H. Both digested and undigested proteins were analyzed by 4% polyacrylamide-SDS PAGE under reducing conditions. As shown in FIG. 55, a full-length spike protein was synthesized, which is an Endo-H sensitive high mannose glycoprotein (gp170, ER form), modified to an Endo-H resistant glycoprotein with complex oligosaccharides (gp180, Golgi form). The conversion of gp170 to gp180 form is completed within 2 hours.

An alphavirus replicon particle that expresses one or more SARS proteins (e.g., VEErep/SINenv-sarspike replicon particle) is administered to a vaccine recipient to induce a SARS-specific immune response (e.g., rodent, ferret, non-human primate, human), as described elsewhere herein. Immunization can be carried out by a variety of routes including, for example, intramuscular, subcutaneous, intradermal, and intranasal. In addition, the alphavirus replicon particles may be used alone or in combination with other immunization methods of the invention (e.g., "prime-boost") or they may be co-administered with alphavirus replicon particles that express antigens from other respiratory pathogens (e.g., influenza virus, parainfluenza virus, respiratory syncytial virus, human hyperpneumovirus). For example, it was demonstrated in mice that immunization of animals with VEErep/SINenv-SARSspike replicon particles induced anti-spike protein antibodies (FIG. 68). These studies in mice also included additional vaccine groups for comparison, including the inactivated SARS virus and recombinant truncated spike protein vaccines described elsewhere herein, as well as plasmid DNA used as primers, followed by boosting with alphavirus replicon particles. The data clearly show that all vaccine groups had very strong immune responses, including the alphavirus replicon particle group. It should be noted that the antibody levels induced by the inactivated SARS virus vaccine used in these experiments were shown to be protective in the SARS virus animal challenge model.

Similarly, genes encoding other SARS virus antigens (e.g., nucleocapsid proteins, membrane glycoproteins), alone or in combination, are cloned into an alphavirus replicon vector to produce alphavirus replicon particles according to the methods of the invention and using standard molecular biology techniques.

Examples of plasmids expressing SARS virus spike alpha Virus

The invention includes the manufacture of plasmid DNA expressing SARS virus antigen for prophylactic or therapeutic immunization against SARS virus infection. In one embodiment, the SARS virus antigen is the spike (S) protein. In one embodiment, the plasmid DNA is alphavirus-based.

The following example illustrates a method for producing alphavirus-based plasmid DNA expressing SARS virus spike (S).

The SARS spike gene can be delivered using any alphavirus-based plasmid DNA replicon, such as ELVS (Dubensky et al, 1996J Virol.70: 508-19), SINCP (WO 01/81609), or VCP (PCT WO 02/99035).

For example, the SARS spike gene is cloned into SINCP using standard RT-PCR techniques. The reverse transcription step was performed using the oligo Sp-RT-R and Superscript preamplification kit (Invitrogen). The amplification step used the cDNA polymerase advantage kit (Clonetech) and the primers Sp-R-NotI and Sp-F-XhoI (SEQ ID NO: 7328).

Sp-F-XhoI primers were designed to contain the sequence ccacc before the ATG codon (Kozak 1991, supra) to optimize the translation efficiency of the spike gene. Also, the primers contained XhoI restriction sites to allow subsequent cloning of the PCR amplified gene.

The PCR product was purified with QIAquick Nucleotide Removal kit (QIAgen), digested with XhoI and NotI, Gel-purified with QIAquick Gel Extraction kit (QIAgen), and ligated to plasmid SINCP pre-digested with the same enzymes. Clones containing the SARS spike sequence were verified by sequencing and the new construct was called SINCP-SARSspike.

Expression of SARS spike gene was verified by transient transfection of BHK cells with 2. mu.g plasmid DNA SINCP-SARSspike or SINCP pre-incubation for 5 minutes by adding 5. mu.l TransIT polyamine reagent (Mirrus) to low serum medium Optimem (Life technologies). Cells were lysed 48 hours post transfection and cell lysate samples were subjected to 8% SDS-PAGE. The proteins on the gel are stained or transferred to a membrane for Western blot analysis using serum from convalescent patients or mouse or rabbit antisera against SARS virus.

The SINCP-sarspike plasmid replicon is administered to a vaccine recipient (e.g., rodent, non-human primate, human) as a formulated or unformulated plasmid vaccine, either alone or in combination with other vaccines of the invention (e.g., "prime-boost"), as described elsewhere herein.

Similarly, genes encoding other SARS viral antigens (e.g., nucleocapsid protein, membrane glycoprotein) are cloned into an alphavirus plasmid replicon vector.

2. Plasmid expression vector

Examples of plasmid DNA expressing SARS virus spike (S)

The following example illustrates a method for producing plasmid DNA expressing SARS virus spike (S).

The SARS virus spike antigen can also be delivered using other plasmid DNA expression vectors (sometimes referred to as "conventional" DNA vaccines) based on polymerase II promoters, such as the CMV promoter. DNA vaccines for spike antigen genes induce an antibody response in mice (Zhao et al, (2004) Acta Biochim et Biophysica Sinica 36: 37-41) and were found to induce virus neutralization and protective immunity in mice (Yang et al, (2004) Nature 428: 561-.

For example, the SARS spike gene was cloned into pCMVKm2(Zur Megel et al, J.Virol., 74: 2628-. The reverse transcription step used oligo Sp-RT-R and Superscript preamplification kit (Invitrogen). The amplification step used the cDNA polymerase advantage kit (Clonetech) and the primers Sp-F-EcoRI (SEQ ID NO: 7329) and Sp-R-XbaI (SEQ ID NO: 7330).

The forward primer was designed to contain the sequence CCACC (Kozak, 1991, supra) before the ATG codon to optimize the translation efficiency of the spike gene. Meanwhile, the forward primer contained an EcoRI restriction site and the reverse primer contained an XbaI restriction site for subsequent cloning of the PCR amplified gene.

The PCR product was purified with QIAquick Nucleotide Removal kit (QIAGEn), digested with XhoI and NotI, Gel purified with QIAquick Gel Extraction kit (QIAGEn), and ligated to plasmid pCMVKm2 pre-digested with the same enzymes. Particles containing SARS spike sequence were verified by sequencing and the new construct was named pCMVKm 2-SARSspike.

Expression of the SARS spike gene was verified by transient transfection of BHK or 293 cells with 2. mu.g plasmid DNA pCMVKm2-SARSspike or pCMVKm2 by adding 5. mu.l TransIT polyamine reagent (Mirrus) to low serum medium Optimem (Life technologies). Cells were lysed 48 hours post transfection and cell lysate samples were subjected to 8% SDS-PAGE. The proteins on the gel are stained or transferred to a membrane for Western blot analysis using serum from convalescent patients or mouse or rabbit antisera against SARS virus.

The plasmid pCMVKm2-SARSspike is administered to a vaccine recipient (e.g., rodent, non-human primate, human) as a formulated or unformulated plasmid vaccine, as described elsewhere herein.

Similarly, genes encoding other SARS viral antigens (e.g., nucleocapsid protein, membrane glycoprotein) are cloned into plasmid expression vectors.

3. Virus-like particles containing SARS antigen

The SARS viral antigens of the present invention can be formulated as virus-like particles ("VLPs"). Thus, the invention includes a virus-like particle (or VLP) comprising one or more SARS virus antigens. Preferably, the VLP comprises one or more SARS virus antigens selected from the group consisting of: spike (S), nucleocapsid (N), membrane (M) and envelope (E). Preferably the VLP comprises at least M and E.

The VLP of the invention comprises at least one particle-forming polypeptide. The particle-forming polypeptide is preferably selected from coronavirus structural proteins. In one embodiment, the particle-forming polypeptide is selected from one or more SARS virus antigens. In another embodiment, the particle-forming polypeptide is selected from the group consisting of structural proteins of a non-SARS coronavirus (e.g., mouse hepatitis virus).

VLPs may be formed when viral structural proteins are expressed in eukaryotic or prokaryotic expression systems. Upon expression, the structural proteins self-assemble to form particles. Alternatively, the viral structural proteins can be isolated from intact viruses and formulated with phospholipids. Such viral structural proteins are referred to herein as "particle-forming polypeptides". VLPs are non-infectious due to the absence of the viral genome, however, these non-replicating viral capsids mimic the structure of the native viral particle.

Due to their structure, VLPs can display many antigenic sites on their surface (similar to the native virus). VLPs are beneficial for live or attenuated vaccines, and because they are not infectious, they are very safe to produce and use. VLPs are known to induce antibody neutralization and T cell responses, and can be presented via both class I and class II MHC pathways.

Previous work to create coronavirus VLPs has shown that E and M proteins alone are sufficient to form coronavirus VLPs. See Fischer et al, j.virol. (1998) 72: 7885-: 2020-2028.

The invention also includes chimeric VLPs comprising particle-forming polypeptides from non-SARS coronaviruses or portions thereof. Such particle-forming polypeptides may comprise the full-length polypeptide of a non-SARS coronavirus. Alternatively, particle-forming fragments may also be used.

In one embodiment, a fragment of the non-SARS particle-forming polypeptide and a fragment of the SARS virus antigen are fused together. For example, such chimeric polypeptides may contain the intracellular and transmembrane domains of a non-SARS particle-forming polypeptide and the extracellular domain of a SARS virus antigen. In one embodiment, the VLP of the invention comprises a chimeric spike protein comprising the intracellular and transmembrane domains of a Mouse Hepatitis Virus (MHV) spike protein, and said chimeric spike protein further comprises the extracellular domain of a SARS spike protein. This VLP also contains the M and E proteins of coronaviruses. The M and E proteins may be selected from any coronavirus, including Mouse Hepatitis Virus (MHV) or SARS. The sample sequences of MHV S, M and E proteins are included in the figure, as above.

Chimeric spike proteins from the extracellular domain of Feline Infectious Peritonitis Virus (FIPV) spike protein fused to the intracellular and transmembrane domains of MHV spike protein have been disclosed. See WO 98/49195 and WO 02/092827. In these chimeric VLP structures, the M and E proteins of MHV form the capsid structure of the VLP. The chimeric spike protein exposes the extracellular domain of the FIPV spike protein to the surface.

The term "virus-like particle" or "VLP" refers herein to a non-replicating empty viral envelope. VLPs are typically composed of one or more viral proteins, such as, but not limited to, those proteins known as capsid, coat, shell, surface and/or envelope proteins or particle-forming polypeptides of these proteins. VLPs may be formed spontaneously by recombinant expression of these proteins in a suitable expression system. Alternatively, the viral structural proteins may be isolated from intact viruses and formulated with phospholipids. Methods of making particulate VLPs are known in the art and will be described in more detail below. The presence or absence of VLPs in the composition may be detected by conventional techniques known in the art, such as electron microscopy, x-ray crystallography, and the like. See, e.g., Baker et al, biophysis.j. (1991) 60: 1445-1456; hagensee et al, J.Virol. (1994) 68: 4503-4505. For example, an aqueous sample of the test VLP preparation being vitrified may be subjected to cryoelectron microscopy and the image recorded under appropriate exposure conditions.

The phrase "particle-forming polypeptide" includes full-length or nearly full-length viral proteins, as well as fragments thereof, or viral proteins with internal deletions, which are capable of forming VLPs under conditions favorable for VLP formation. Thus, the polypeptides may include full-length sequences, fragments, truncated sequences and partial sequences, as well as analogs and prophetic forms of the mentioned molecules. Thus, the term includes deletions, additions and substitutions to the sequence, as long as the polypeptide is still able to form a VLP. Since coat protein variations often occur between viral isolates, the term includes natural variants of a particular polypeptide. The term also includes deletions, additions and substitutions that do not naturally occur in the mentioned proteins, as long as the proteins are still able to form VLPs.

Preferred substitutions are those that are conservative in nature, i.e., substitutions that occur within a class of amino acids that involve their side chains. Specifically, amino acids can be generally classified into 4 types: (1) acidic amino acids: aspartic acid and glutamic acid; (2) basic amino acids: lysine, arginine and histidine; (3) non-polar amino acids: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to expect that replacement of leucine with isoleucine or valine alone, aspartic acid with glycine, threonine with serine, or conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on biological activity. Proteins having almost the same amino acid sequence as the mentioned molecules but with few amino acid substitutions do not substantially affect the immunogenicity of the protein and are therefore also included in the scope of the mentioned polypeptides.

The VLPs of the invention may be formed from any of the following: a viral protein, a particle-forming polypeptide derived from a viral protein, or a combination of viral proteins or fragments thereof, which are capable of forming a particle under suitable conditions. The requirement for the particle-forming viral protein is that if the particle is formed intracytoplasmically in a host cell, the protein must be sufficiently stable in the host cell in which it is expressed so that a virus-like structure is formed and the polypeptide will spontaneously assemble into a virus-like structure within the cell of the recombinant expression system used. If the protein is secreted into the culture medium, the culture conditions can be adjusted to enable VLP formation. Furthermore, the particle-forming protein should not be cytotoxic in the expression host and cannot replicate in the host in which the VLP will be used.

Preferred particle-forming polypeptides include coronavirus M and E proteins, preferably the M and E proteins of SARS.

Methods and suitable conditions for forming particles from various viral proteins are known in the art. For example, VLPs can be made from proteins from: influenza virus (e.g., HA or NA), hepatitis b virus (e.g., core or capsid protein), hepatitis e virus, measles virus, sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, norwalk virus, human papilloma virus, HIV, RNA phage, Q β phage (e.g., coat protein), GA phage, fr phage, AP205 phage, and Ty (e.g., retrotransposon Ty protein p 1). VLPs have been further described in WO 03/024480, WO 03/024481, and Niikura et al, Virology (2002) 293: 273-280; lenz et al, J.immunology (2001) 5246-5355; pinto et al, J.Infectious diseases (2003)188: 327-338; and Gerber et al, J.virology (2001)75(10)：4752-4760。

As described above, VLPs may form spontaneously when a particle-forming protein of interest is recombinantly expressed in a suitable host cell. Thus, VLPs for use in the present invention may be manufactured using recombinant techniques well known in the art. In this case, the genes mentioned which code for the particle-forming polypeptides can be isolated from DNA libraries or directly from the cells and tissues containing them using known techniques. Genes encoding particle-forming polypeptides can also be made synthetically based on known sequences. Nucleotide sequences containing appropriate codons can be designed to obtain the desired specific amino acid sequence. Typically, the codons preferred by the host used to express the sequence are selected (e.g., human codons for human DNA vaccines). The complete sequence is usually prepared by standard methods of overlapping oligonucleotides assembly, and is assembled into the complete coding sequence. See, e.g., Edge, Nature (1981) 292: 756; nambair et al, Science (1984) 223: 1299; jay et al, J.biol.chem. (1984) 259: 6311.

once the coding sequences for the desired particle-forming polypeptides are isolated or synthesized, they may be cloned into any suitable vector or replicon for expression. Many cloning vectors are known to those skilled in the art, and selection of an appropriate cloning vector is a matter of choice. See generally Sambrook et al. The vector is then used to transform a suitable host cell. Suitable expression systems include, but are not limited to, bacterial, mammalian, baculovirus/insect, vaccinia, Semliki Forest Virus (SFV), yeast and xenopus expression systems well known in the art.

Many cell lines suitable for use as host cells for making the VLPs of the invention are known in the art. Suitable mammalian cell lines include, but are not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, and others. Mammalian sources of cells include, but are not limited to, human or non-human primates (e.g., MRC-5(ATCC CCL-171), WI-38(ATCC CCL-75), human embryonic kidney cells (293 cells, typically transformed with sheared adenovirus type 5 DNA), monkey kidney Vero cells (including COS7 cells), horses, cows (e.g., MDBK cells), sheep, dogs (e.g., dog kidney MDCK cells, ATCC CCL34MDCK (NBL2) or MDCK 33016, deposited as DSM ACC 2219 as described in WO 97/37001), cats, and mice (e.g., hamster cells, such as BHK21-F, HKCC cells or Chinese hamster ovary cells (CHO cells)) and can be obtained from various developmental stages, including, for example, adult, neonatal, fetal, or embryonic.

Bacterial hosts suitable for the production of the VLPs of the invention include escherichia coli (e.coli), bacillus subtilis (bacillus subtilis) and streptococcus. Suitable yeast hosts for making the VLPs of the present invention include Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), Candida maltosa (Candida maltosa), Hansenula polymorpha (Hansenula polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia guiagllermonidii, Pichia pastoris (Pichia pastoris), Schizosaccharomyces pombe (Schizosaccharomyces pombe), and Yarrowia lipolytica (Yarrowia lipolytica). Insect cells suitable for producing the VLPs of the present invention include Aedes aegypti (Aedes aegypti), Autographa californica (Autographa californica), Bombyx mori (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), Spodoptera frugiperda (Spodoptera frugiperda), and Ectropis calis (Trichoplusia ni).

Viral vectors can be used to produce particles in eukaryotic cells, such as those from the poxviridae family, including vaccinia virus and avipoxviruses. In addition, vaccinia based on infection/transfection systems, such as Tomei et al, j.virol (1993) 67: 4017-: 1103-. In this system, cells are first transfected in vitro with a vaccinia virus recombinant encoding the T7 phage RNA polymerase. This polymerase only transcribed the template with the T7 promoter. Cell infection was followed by transfection with the DNA of interest driven by the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the host's translation machinery. This method can produce large amounts of RNA and its translation product in cytoplasm at a high level transiently.

Depending on the expression system and host chosen, VLPs can be made by growing host cells transformed with the expression vector under conditions in which the particle-forming polypeptide is expressed and the VLP is capable of growing. The selection of appropriate growth conditions is known to those skilled in the art. If VLPs are formed within cells, then the cells are disrupted by chemical, physical or mechanical means which will lyse the cells but leave the VLPs substantially intact. Such methods are well known to those skilled in the art and are described, for example, in methods of practicing Protein purification applications (Protein purification applications: A Practical applications), (ed. E.L.V.Harris and S.Angal, 1990).

They are then separated by methods that preserve the integrity of the particles, such as by gradient centrifugation, e.g., strontium chloride (CsCl) gradients and sucrose gradients, and the like (see, e.g., Kirnbauer et al, j.virol. (1993) 67: 6929-6936); ion exchange chromatography (including anion exchange chromatography such as DMAE and TMAE), hydroxyapatite chromatography (see WO00/09671), hydrophobic interaction chromatography, gel filtration chromatography and other filtration methods such as nano (nanometric) filtration and ultrafiltration. Preferably at least one anion exchange step is performed during the purification, more preferably at least two anion exchange steps are used.

The VLP formulations of the invention may also be further treated by methods known in the art using high concentrations of reducing agents to de-assemble into smaller protein-containing fractions, followed by re-assembly of the CLP by removal of the reducing agent or addition of excess oxidizing agent. The resulting reassembled VLP may have improved homogeneity, stability and immunogenicity. In addition, other therapeutic or prophylactic agents can be added to VLPs by reassembly. See McCarthy et al, j.virology (1998)72 (1): 32-41. See also WO 99/13056 and WO 01/42780. Reducing agents suitable for use in de-assembling VLPs include thiol reducing agents (e.g., glutathione, beta mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, hydrogen sulfide, and mixtures thereof), which are preferably contained in a buffer of moderate to low ionic strength. The VLPs need to be exposed to the reducing agent for a sufficient period of time to allow a suitable amount of VLPs to be disassembled.

Adjuvants may be added to the VLPs of the invention to enhance the immunogenicity of the SARS virus antigen. Antigens suitable for use in VLPs include those described above. For example, the VLP of the invention may be adsorbed onto an aluminum adjuvant.

The VLPs of the invention may be formulated to enhance their stability. Other components that may enhance the stability of the VLP formulation include salts, buffers, non-ionic surfactants and other stabilizers, such as polymeric polyanionic stabilizers. See WO 00/45841.

The ionic strength of the solution containing the VLP particles may be maintained by the salt. Almost all salts capable of controlling ionic strength can be used. Preferred salts for modulating ionic strength include physiologically acceptable salts, such as NaCl, KCl, Na₂SO₄、(NH₄)₂SO₄Sodium phosphate and sodium citrate. The concentration of the salt component is preferably about 0.10M to 1M. Very high concentrations are not preferred due to the practical limitations of using high salt concentrations for parenteral injection. In contrast, very moderate salt concentrations, such as more suitable physiological concentrations, for example, from about 0.15M to about 0.5M (with 0.15M-0.32M NaCl), are preferred.

Buffers may also enhance the stability of the VLP formulations of the invention. The buffer preferably optimizes the stability of the VLPs while maintaining a pH range that renders the vaccine formulation non-irritating to recipients. The buffer preferably maintains the pH of the vaccine formulation in the range of pH5.5-7.0, more preferably 6.0-6.5. Buffers suitable for use in vaccine formulations are known in the art and include, for example, histidine and imidazole. The concentration of the buffer preferably ranges from about 2mM to about 100mM, more preferably 5mM-20 mM. When VLPs are adsorbed by or otherwise formulated with an aluminum compound, a buffer containing phosphate is generally not preferred.

Nonionic surfactants may be used to enhance the stability of the VLP formulations of the invention. Surfactants suitable for use in vaccine formulations are known in the art and include, for example, polyoxyethylene sorbitan fatty acid esters (polysorbates), such as polysorbate 80 (e.g., TWEEN 80), polysorbate 20 (e.g., TWEEN 20), polyoxyethylene alkyl ethers (e.g., Brij 35, Brij 58), and in addition nonionic surfactants of the Triton X-100, Triton X-114, NP-40, Span 85, and Pluronic series (e.g., Pluronic 121). The concentration of the surfactant is preferably from about 0.0005% to about 0.5% (weight/volume).

Polymeric polyanionic stabilizers may be used to enhance the stability of the VLP formulations of the invention. Polymeric polyanionic stabilizers suitable for use in the present invention contain a single long chain or multiple cross-linked chains; when in solution, either type of chain bears multiple negative charges. Suitable polyanionic polymers include proteins, polyanions, peptides and polynucleic acids. Specific examples include carboxymethylcellulose, heparin, polyamino acids (such as poly (Glu), poly (Asp) and poly (Glu, Phe)), oxidized glutathione, polynucleotides, RNA, DNA and serum albumin. The concentration of the polymeric polyanionic stabilizer is preferably from about 0.01% to about 0.5%, especially from about 0.05 to 0.1% (weight percent).

G. Passive immunization by antibodies to SARS antigens of the invention

The invention includes antibodies specific for the SARS antigens of the invention and methods of treating or preventing SARS virus-related diseases by administering to a mammalian subject an effective amount of SARS antibodies. Antibodies specific for SARS virus can be made by those skilled in the art. Preferably, the antibody is specific for the spike (S) protein of SARS virus. Efficient neutralization of SARS coronavirus with human monoclonal anti-spike antibodies has been reported (Sui et al, (2004) PNAS USA 101: 2536-. The IgG1 form of the monoclonal antibody showed higher affinity (1.59nM) than the scFv form (32.3 nM).

The antibodies of the invention are specific and selective for the SARS antigen.

In one embodiment, the antibodies of the invention are produced by administering a SARS antigen to an animal. The method further comprises isolating the antibody from the animal.

The antibody of the invention may be a polyclonal or monoclonal antibody preparation, a monospecific antiserum, a human antibody, or may be a hybrid or chimeric antibody, such as a humanized antibody, a variant antibody (Fab')₂A fragment, a f (ab) fragment, an Fv fragment, a single domain antibody, a dimeric or trimeric antibody fragment or construct, a microbody, or a functional fragment thereof that binds to the antigen in question.

Antibodies are made using techniques well known to those skilled in the art, described, for example, in U.S. patent nos.4,011,308; 4,722,890, respectively; 4,016,043; 3,876,504, respectively; 3,770,380 and 4,372,745. For example, polyclonal antibodies are generated by immunizing a suitable animal (e.g., mouse, rat, rabbit, sheep, or goat) with an antigen of interest. To enhance immunogenicity, the antigen may be bound to a carrier prior to immunization. Such vectors are well known to those of ordinary skill in the art. Immunization is typically carried out by: the antigen is mixed or emulsified with saline, preferably with an adjuvant such as Freund's complete adjuvant, and the mixture or emulsion is injected parenterally, usually subcutaneously or intramuscularly. Animals are injected 2-6 weeks later with antigen in saline (preferably Freund's incomplete adjuvant) one or more booster immunizations. Antibodies can also be generated by in vitro immunization using methods known in the art. Polyclonal antisera are then obtained from the immunized animals.

By the method of Kohler and Milstein [ (1975) Nature256：495-497]Or the improved method to prepare monoclonal antibody. Typically, mice or rats are immunized as described above. Rabbits may also be used. However, rather than taking blood from the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dispersed into single cells. If desired, spleen cells can be screened by adding a cell suspension (after removal of non-specifically adhered cells) to the protein antigen-coated plate or well. Antigen-specific membrane-bound immunoglobulin-expressing B cells bind to the plate and are not washed away as are other substances in the suspension. The resulting B cells or all isolated splenocytes are then induced to fuse with myeloma cells to form hybridomas, which are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are inoculated by limiting dilution and assayed for antibodies that specifically bind to the immunizing antigen (and not to unrelated antigens) And (4) generating. The selected monoclonal antibody-secreting hybridomas are then cultured in vitro (e.g., in tissue culture flasks or hollow fiber reactors) or in vivo (e.g., in mouse ascites).

Humanized antibodies and chimeric antibodies are also useful in the present invention. Hybrid (chimeric) antibody molecules are generally described in Winter et al, (1991) Nature349: 293-. Humanized antibody molecules are generally described in Riechmann et al, (1988) Nature332: 323-327; verhoeyan et al, (1988) Science239: 1534 — 1536; and british patent application No. gb 2,276,169 (published on 21/9 of 1994). One method of engineering humanized antibodies involves cloning a recombinant DNA containing a promoter, leader region, and mouse antibody gene variable region sequences and human antibody gene constant region exons to produce a mouse-human chimera, the humanized antibody. See generally Kuby, Immunology, third edition, W.H.Freeman and Company, New York (1998), page 136.

Antibody fragments capable of identifying the SARS antigen are also included within the scope of the invention. Many antibody fragments containing an antigen binding site capable of exhibiting the immunological binding characteristics of an intact antibody molecule are known in the art. For example, functional antibody fragments such as F (ab') ₂And (3) fragment. These fragments will contain two antigen binding sites but lack part of the constant region of each heavy chain. Similarly, if desired, Fab fragments containing a single antigen binding site can be made, for example, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments comprising only the variable regions of the heavy and light chains can also be made using standard techniques, such as recombinant methods or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are designated F_v. See, e.g., Inbar et al, (1972) Proc. Nat. Acad. Sci USA69: 2659-2662; hochman et al, (1976) Biochem15: 2706-2710; and Ehrlich et al, (1980) Biochem19：4091-4096。

A single chain Fv ("sFv" or scFv ") polypeptide is a covalently linked V_H-V_LHeterodimers, which are V linked by linker comprising peptide coding_H-and V_LExpressed as a fusion gene encoding a gene. Huston et al, (1988) Proc. nat. Acad. Sci. USA85: 5879-5883. A number of methods have been described for identifying and establishing chemical structures (linkers) that are used to convert light and heavy chain polypeptides of the antibody V regions that naturally aggregate but can be chemically separated into sFy molecules, and sFv molecules that fold into three-dimensional structures substantially similar to those of the antigen binding site. See, for example, U.S. patent nos.5,091,513; 5,132, 405; and 4,946,778. sFv molecules can be made using methods already described in the art. See, e.g., Huston et al, (1988) Proc. Nat. Acad. Sci USA 85: 5879-5338; U.S. patent nos.5,091,513; 5,132,405 and 4,946,778. Design criteria include determining the approximate length of the distance between the C-terminus of one strand and the N-terminus of the other strand, where the linker is typically formed of small hydrophilic amino acid residues that do not coil or form secondary structures. Such methods have been described in the art. See, for example, U.S. patent nos.5,091,513; 5,132,405 and 4,946,778. Suitable linkers typically include polypeptide chains in which glycine and serine can be substituted for each other, and may include insertions of glutamic acid and lysine residues to enhance solubility. Anti-spike scFv antibodies have been reported (Sui et al, (2004) PNAS USA 101: 2536-.

"Mini-antibody" or "microbody" (Minibody) may also be used in the present invention. Microbodies are sFv polypeptide chains that contain an oligomerization region at their C-terminus separated from the sFv by a hinge region. Pack et al (1992) Biochem31: 1579-1584. The oligomerization region comprises a self-associating alpha-helix, such as a leucine zipper, which may be stabilized by other disulfide bonds. The oligomerization region is designed to coincide with the directional folding across the membrane, a process that is thought to favor in vivo folding of the polypeptide into a functional binding protein. Typically, microbodies are made using recombinant methods well known in the art. See, e.g., Pack et al, (1992)Biochem 31: 1579-1584; cumber et al (1992) J.immunology149B：120-126。

Antibodies of the invention can also be produced and characterized by non-conventional methods. For example, a phage display library can be screened for antibodies that bind to the SARS antigen of the invention. See generally, Siegel, "Recombinant Monoclonal Antibody Technology" (Recombinant Monoclonal Antibody Technology), transfus. Clin. biol. (2002)9(1): 15-22; sidhu, "Phage Display for pharmaceutical Biotechnology" (phase Display in pharmaceutical Biotechnology), curr. opin. biotechnol. (2000)11(6): 610-616; sharon et al, "Recombinant Polyclonal antibody libraries" (Recombinant Polyclonal antibodies libraries), comb. chem. high through high Screen (2000)3(3): 185-196; and Schmitz et al, "phage display: molecular tools for antibody production-Review "(phase Display: A Molecular tools for the Generation of Antibodies-Review), planta, (2000)21 SupplA：S106-12。

The antibodies of the invention can be made by administering to an animal a polynucleotide sequence encoding a SARS antigen. The SARS antigen is then expressed in vivo and antibodies specific for the SARS antigen are produced in vivo. The method of using polynucleotides to deliver SARS antigens of the invention is discussed in section 4 below.

The antibodies of the invention are preferably specific for SARS virus.

H. Combinations of one or more of the foregoing vaccines

The compositions of the present invention also comprise combinations of one or more of the above compositions. For example, the invention includes compositions comprising an attenuated SARS virus and a subunit SARS virus antigen.

Combination of SARS antigen and other respiratory viral antigens

The invention also relates to vaccine formulations comprising one or more SARS virus antigens and one or more other respiratory virus antigens. Other respiratory virus antigens suitable for use in the present invention include antigens from the following viruses: influenza virus, Human Rhinovirus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus (RSV), adenovirus, hyperpneumovirus and rhinovirus. Other respiratory viral antigens may be from coronaviruses other than SARS coronavirus, such as NL63 human coronavirus (van der Hoek et al (2004) Nature Medicine 10: 368-. Preferably the other respiratory virus antigen is an influenza virus antigen.

The invention also includes one or more bacterial or viral antigens in combination with a SARS viral antigen. The antigens may be used alone or in any combination. (see, e.g., WO 02/00249, which describes the use of combinations of bacterial antigens). Such compositions may include multiple antigens from the same pathogen, multiple antigens from different pathogens, or multiple antigens from the same and different pathogens. Thus, bacteria, viruses, and/or other antigens may be contained in the same composition, or may be administered separately to the same subject. It is generally desirable to use a combination of antigens to elicit an immune response.

Non-limiting examples of bacterial pathogens that may be used in the present invention include: diphtheria (see, for example, Chapter 3 of the vaccine, 1998, Plutkin and Mortimer eds. (ISBN 0-7216-; 1946-0), Staphylococcus (see, for example, Kuroda et al [ (2001) Lancet 357: 1225-; 1240) Staphylococcus aureus (Staphyloccocusuerueus), cholera, tuberculosis, Clostridium tetani (C.tetani), also known as tetanus (see, for example, vaccine 4, 1998, Plutkin and Mortimer eds. (ISBN 0-7216-; 1946-0), group A and group B streptococci (including Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus agalactiae (Streptococcus agalactiae), and Streptococcus pyogenes (Streptococcus pygeis), as described, for example, in, for example, Nitroson et al, (2000) vaccine J187. J. 19. 331. 35; American 35; Pentium 92; Klebsiella 57; Klotu. 35; Pepti et al.; 35; Pepti 51; Klotu. 35; Pepti 51; Pepti., (1999) infect Dis Clin North Am 13: 227-; ferretti et al, (2001) PNASAS 98: 4658-: 349-355; rappuoli et al, (1991) TIBTECH 9: 232-238), meningitis, Moraxella catarrhalis (see, e.g., McMichael (2000) Vaccine19 suppl.1: s101-107) and other disease states including, but not limited to, Neisseria meningitidis (a, B, C, Y), Neisseria gonorrhoeae (see, e.g., WO 99/24578; WO 99/36544; and WO 99/57280), helicobacter pylori (helicobacter pylori) (e.g., CagA, VacA, NAP, HopX, HopY and/or urease, as described, for example, in WO 93/18150; WO 99/53310; WO 98/04702) and Haemophilus influenzae (Haemophilus influenza). Haemophilus influenzae type B (HIB) (see, e.g., Costantino et al, (1999) Vaccine 17: 1251-1263), Porphyromonas gingivalis (Ross et al, (2001) Vaccine 19: 4135-4132), and combinations thereof.

Non-limiting examples of viral pathogens that can be used in the present invention include: meningitis, rhinoviruses, influenza (Kawaoka et al, Virology (1990) 179: 759-767; Webster et al, "Antigenic variation of influenza A viruses" (Antigenic variation of influenza A viruses), p.127-168, compiled in P.Palese and D.W.Kingsbury (ed.), "influenza Genetics" (Genetics of influenza viruses. Springer-Verlag, N.Y.), Respiratory Syncytial Virus (RSV), parainfluenza virus (PIV), rotaviruses (e.g., VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 or NSP5, and other rotavirus antigens, as described in WO 00/26380), etc. other antigens of the invention may also be derived from Amiaviruses, e.g., Amiavirus derived from Ammonia family Fanaturaceae, such as described in et al, e.g., protein 126, Spanish.g., American protein, 2000: 287, et al, (see, e.g., Ammonia., Amano, Spanish., see, et al., Spanish., para, et al., para, entitled, "Antigenic variation of protein, Spanish., para, et al., Spanish., Sp; caliciviridae family; togaviridae (e.g., rubella virus, etc.); flaviviridae, including common flaviviruses (e.g., yellow fever virus, japanese encephalitis virus, serotypes of dengue virus, tick-borne encephalitis virus, west nile virus, st louis encephalitis virus); pestiviruses (e.g., classical swine fever virus, bovine viral diarrhea virus, border disease virus); and hepatitis C virus (e.g., hepatitis A, B and C as described in U.S. Pat. Nos.4,702,909; 5,011,915; 5,698,390; 6,027,729 and 6,297,048); parvoviruses (e.g., parvovirus B19); (ii) the family coronaviridae; reoviridae; bimaviridae; rhabdoviridae (e.g., rabies virus, etc., described in Dressen et al (1997) Vaccine 15 Suppl: s 2-6; MMWR Morb Mortal Wkly Rep.1998Jan 16: 47 (1): 12, 19); filoviridae; paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc., as described in "vaccine" (1998, Plotkin and Mortimer eds. (ISBN 0-7216 1946-0)), (Orthomyxoviridae (e.g., A, B and influenza C virus, etc., as described in "vaccine" (1998, Plotkin and Mortimer eds. (ISBN 0-7216 1946-0)), (Orthomyxoviridae), arenaviridae, Retroviridae (e.g., HTLV-1; HTLV-11; HIV-1 (also known as HTLV-III, LAV, ARV, HTI, R, etc.)), including but not limited to the antigens of HIVIllb, HIVSF2, HIVLAV, HIVI-AL, I-VMIIN, SF162 isolates); HIV-I CM235, HIV-I US 4; HIV-2; simian Immunodeficiency Virus (SIV). In addition, antigens may also be derived from Human Papilloma Virus (HPV) and tick-borne encephalitis virus. For a description of these viruses, and others, see, e.g., Virology (Virology), third edition (w.k. joklik eds., 1988); basic Virology (Fundamental Virology), second edition (ed.b.n. fields and d.m. knit, 1991).

The proteins may be derived from the herpesviridae family, including proteins derived from herpes simplex virus types 1 and 2 (HSV), such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens derived from herpes zoster virus (VZV), EB virus (EBV) and Cytomegalovirus (CMV), including CMV gB and gH (see U.S. Pat. No.4,689,225 and PCT publication W089/07143); and antigens derived from other human herpesviruses, such as HHV6 and HHV 7. (see, e.g., Chee et al (Cytomegaloviruses) (J.K.McDougall eds., Springer-Verlag1990), pp.125-169-]To understand the protein coding content of cytomegalovirus; McGeoch et al [ J.Gen.Virol. (1988)69：1531-1574]To understand the various HSV-1 encoded proteins; U.S. Pat. No.5,171,568 for the understanding of HSV-1 and HSV-2gB and gD proteins and the genes encoding them; baer et al [ Nature (1984)310：207-211]To identify protein coding sequences of the EBV genome; and Davison and Scott [ j.gen.virol. (1986)67：1759-1816]To understand VZV). Herpes Simplex Virus (HSV) rgD2 is a recombinant protein produced by genetically engineered chinese hamster ovary cells. The normal anchor region of this protein is truncated, resulting in secretion of the glycosylated protein into the tissue culture medium. gD2 in CHO media can be purified to greater than 90% purity. Human Immunodeficiency Virus (HIV) env-2-3 is a recombinant form of HIV envelope protein produced by genetically engineered Saccharomyces cerevisiae. This protein has the entire protein region of HIV gp120, but the non-glycosylated protein has been denatured when purified from yeast. HIV gpl20 is a fully glycosylated secreted form of gp120, produced in CHO cells in a similar manner to gD2 described above. Other HSV antigens suitable for use in immunogenic compositions are described in PCT publications WO 85/04587 and WO 88/02634, which are incorporated herein by reference in their entirety. Mixtures of gB and gD antigens (truncated surface antigens lacking the anchor region) are particularly preferred.

Antigens from the hepaciviridae family including Hepatitis A Virus (HAV) (see, e.g., Bell et al, (2000) Peditar infection Dis. J.19: 1187-1188; Iward (1995) APMIS 103: 321-326), Hepatitis B Virus (HBV) (see, e.g., Gerlich et al, (1990) Vaccine 8 Suppl: S63-68-326) may also be conveniently used in the techniques described herein&79-80), Hepatitis C Virus (HCV) (see, e.g., PCT/US88/04125, published european application No. 318216), Hepatitis D Virus (HDV), Hepatitis E Virus (HEV), and Hepatitis G Virus (HGV). For example, the viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, for example, international publications WO 89/04669, WO 90/11089, and WO 90/14436. The invention also includes molecular variants of such polypeptides, for example PCT/US 99/31245; PCT/US99/31273 andas described in PCT/US 99/31272. The HCV genome, which encodes several viral proteins, includes E1 (also known as E) and E2 (also known as E2/NSI) as well as the N-terminal nucleocapsid protein (known as the "core") (see Houghton et al [ Hepatology (1991))14：381-388]To understand the description of HCV proteins, including E1 and E2). Similarly, the sequence of HDV delta-antigen is also known (see, e.g., U.S. patent No.5,378,814), and such antigens may be conveniently used in the compositions and methods of the invention. In addition, antigens from HBV such as core antigen, surface antigen, SAg, and pre-surface sequences pre-S1 and pre-S2 (previously referred to as pre-S) as well as combinations of the above antigens such as SAg/pre-S1, SAg/pre-S2, SAg/pre-S1/pre-S2, and pre-S1/pre-S2 may also be used. See, for example, "HBV vaccine-from laboratory to licensed: case analysis "(HBV Vaccines-from the laboratory to licenses: a case study), selected from Mackett, M. and Williamson, J.D., (Human Vaccines and immunization), pages 159 and 176, to understand HBV structure; and U.S. patent nos.4,722,840,5,098,704, 5,324,513, which are incorporated herein by reference in their entirety; beames et al, J.Virol (1995) 69: 6833 6838, Birnbaum et al, J.Virol (1990)64: 3319-3330; and Zhou et al, J.Virol (1991)65: 5457-5464. These proteins, as well as antigenic fragments thereof, are useful in the compositions and methods of the invention.

Influenza virus is another example of a virus for which the present invention would be particularly effective. In particular, the envelope glycoproteins HA and NA of influenza a are of particular interest for the generation of immune responses. A number of HA subtypes of influenza A have also been identified (Kawaoka et al, Virology (1990)179: 759 and 767; webster et al, "Antigenic variation of influenza A virus" (Antigenic variation of influenza A viruses), pp.127- "168", selected from P.Pase and D.W.Kingsbury (eds.), "influenza Genetics" (Genetics of influenza viruses. Springer-Verlag, N.Y.). Thus, proteins derived from any of these isolates can be used in the compositions and methods described herein.

Non-limiting examples of parasite antigens include those of organisms that cause malaria and lyme disease.

The methods of the invention comprise administering to an animal an immunogenic composition comprising a SARS virus antigen (including one or more inactivated SARS virus, attenuated SARS virus, a SARS split virus preparation, or a recombinant or purified subunit preparation of one or more SARS virus antigens). The immunogenic compositions for use in the present invention may comprise an immunologically effective amount of a SARS virus antigen. An "immunologically effective amount" refers to an amount sufficient to generate an immune response in an animal against SARS antigen.

The immune response preferably comprises the production of antibodies specific for the SARS antigen. The amount of antibody produced will vary depending on the animal used, whether adjuvant is present, and the like.

The immunogenic compositions of the invention also contain one or more adjuvants.

The immunogenic compositions of the invention may be administered mucosally. Suitable mucosal routes of administration include oral, intranasal, intragastric, pulmonary, enteral, rectal, ocular and vaginal routes. The immunogenic composition can be formulated for mucosal administration. For example, when the composition is administered orally, it can be formulated as tablets or capsules (optionally enteric coated), liquids, transgenic plants, and the like. When the composition is to be administered intranasally, it may be in the form of a nasal spray, nasal drops, gel or powder.

The immunogenic compositions of the invention may be administered parenterally. Suitable parenteral routes include Intramuscular (IM), subcutaneous, intravenous, intraperitoneal, intradermal, transdermal and transdermal (see, e.g., WO 98/20734) routes, as well as delivery to the interstitial space. The immunogenic compositions can be prepared in a form suitable for parenteral administration, for example, in sterile or pyrogen-free injectable form.

The vaccines of the present invention may be administered in combination with other immunomodulators. In particular, the composition will generally comprise an adjuvant. Other preferred adjuvants include, but are not limited to, one or more of the following:

A. Mineral-containing composition

Mineral-containing compositions suitable for use as adjuvants in the present invention include mineral salts, such as aluminum and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates etc. (see for example Vaccine design: subunit and adjuvant research, chapters 8 and 9, (1995) Powell and Newman ISBN0-306 and 44867-X.), or mixtures of different minerals and the compounds, which may take any suitable form (e.g. colloidal, crystalline, amorphous etc.), preferably adsorbed. The mineral-containing composition can be made into metal salt particles. See WO 00/23105.

B. Oily emulsion

Oily emulsion compositions suitable for use as adjuvants in the present invention include squalene-water emulsions such as MF59 (5% squalene, 0.5% Tween 80 and 0.5% Span 85, made into submicron particles using a microfluidizer). See WO 90/14837. See also Frey et al, "Comparison of safety, tolerance and immunogenicity of MF59-adjuvanted and unadjuvanted influenza vaccines in non-elderly adults" (Comparison of the safety, tolerability, and understanding of a MF59-adjuvanted influenza Vaccine and a non-adjuvanted influenza Vaccine in non-elderly ads ", Vaccine (2003) 21：4234-4237。

Particularly preferred adjuvants for use in the compositions are submicron oil-in-water emulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as submicron oil-in-water emulsions containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80^TM(Polyoxyethylene sorbitan monooleate) and/or 0.25-1.0% Span 85^TM(sorbitan trioleate) and optionally N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphonoxy) -ethylolAmines (MTP-PE), such as submicron oil-in-water emulsions known as "MF 59" (WO 90/14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, which are incorporated herein by reference in their entirety; and Ott et al, "MF 59-Design and Evaluation of a Safe and effective Human vaccine adjuvants" (MF 59-Design and Evaluation of a Safe and PotentAdjuvant for Human Vaccines) selected from the group consisting of "vaccine Design: subunit and adjuvant research" (Powell, M.F. and Newman, eds. M.J.) Plenum Press, New York, 1995, 277-296). MF59 contains 4-5% w/v squalene (e.g. 4.3%), 0.25-0.5% w/v Tween 80^TMAnd 0.5% w/v Span 85 ^TMAnd optionally with varying amounts of MTP-PE, are formulated into submicron particles using a microfluidizer such as a 110Y microfluidizer (Microfluidics, Newton, MA). For example, the MTP-PE is present in an amount of about 0 to 500. mu.g/dose, more preferably 0 to 250. mu.g/dose, and most preferably 0 to 100. mu.g/dose. The term "MF 59-0" here denotes the above mentioned submicron oil-in-water emulsions without MTP-PE, whereas the term MF59-MTP denotes formulations containing MTP-PE. For example, "MF 59-100" contains 100. mu.g of MTP-PE per dose, and so on. Another submicron oil-in-water emulsion MF69 used herein contains 4.3% w/v squalene, 0.25% w/v Tween 80^TMAnd 0.75% w/v Span 85^TMAnd optionally MTP-PE. Another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 0.4% Tween 80^TM5% Pluronic block polymer L121 and thr-MDP were also microfluidized into submicron emulsions. MF75-MTP denotes preparations containing MTP, for example 100. mu.g MTP-PE/dose.

Submicron oil-in-water emulsions, methods of making such emulsions, and immunostimulants (e.g., muramyl peptides) useful in the compositions of the invention are described in detail in WO 90114837 and U.S. Pat. Nos.6,299,884 and 6,451,325, which are incorporated herein by reference in their entirety.

Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may also be used as adjuvants in the present invention.

C. Saponin preparation

Saponin formulations may also be used as adjuvants in the present invention. Saponins are a heterogeneous group of sterol glycosides and triterpene glycosides found in the bark, leaves, stems, roots and even flowers of many plant species. Saponins from the bark of the Quillaia saponaria Molina tree have been extensively studied as adjuvants. Saponins are also commercially available from smilaxornata (sarsaprilla), gypsophila paniculata (bridges veil) and saponaria officinalis (soapstock). Saponin adjuvant formulations include purified formulations, such as QS21, as well as liquid formulations, such as ISCOMs.

The saponin composition can be purified by high performance thin layer chromatography (HP-LC) and reverse phase high performance liquid chromatography (RP-HPLC). Specific purified fractions obtained using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B, and QH-C. Preferably the saponin is QS 21. One method of making QS21 is disclosed in U.S. patent No.5,057,540. Saponins may also contain sterols, such as cholesterol (see WO 96/33739).

The combination of saponin and cholesterol can be used to form unique particles known as Immune Stimulating Complexes (ISCOMs). ISCOMs typically also contain a phospholipid, such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOMs include one or more of Quil A, QHA and QHC. ISCOMs are also described in EP 0109942, WO 96/11711 and WO 96/33739. Optionally, the ISCOM may be free of other detergents. See WO 00/07621.

For the manufacture of saponin-base-solubilizing adjuvants see Barr et al, "ISCOMs and other saponin-base adjuvants" (ISCOMs and other saponin-base adjuvants ", Advanced Drug Delivery Reviews (1998)32: 247-271. See also Sjolander et al, "ingestion of oral saponins and ISCOM vaccines and adjuvant Activity" (Uptake and adjuvant Activity of organic Delivery vaccines and ISCOMvacines ", Advanced Drug Delivery Reviews (1998)32：321-338。

D. Bacterial or microbial derivatives

Adjuvants suitable for use in the present invention include bacterial or microbial derivatives such as:

(1) non-toxic derivatives of intestinal bacterial Lipopolysaccharide (LPS)

Such derivatives include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3 dMPL). 3dMPL is a mixture of 3-O-deacylated monophosphoryl lipid A and 4, 5 or 6 acylated chains. Preferred "small particle" forms of 3-O-deacylated monophosphoryl lipid A are disclosed in EP 0689454. This "small particle" of 3dMPL is small enough to pass through a 0.22 micron sterile filtration membrane (see EP 0689454). Other non-toxic LPS derivatives include monophosphoryl lipid a mimetics, such as aminoalkyl glucosaminide phosphate derivatives, e.g. RC-529. See Johnson et al (1999) bioorgMedChem Lett 9: 2273-2278.

(2) Lipid A derivatives

Lipid A derivatives include lipid A derivatives from E.coli, such as OM-174. OM-174 is described, for example, in Meraldi et al, "A novel adjuvant OM-174, which is potentially useful in humans, Induces a Protective Response when administered with the C-Terminal Fragment 242-310 of the Synthetic Plasmodium pernici circumsporozoite protein" (OM-174, a New Adjuvant with a Potential for Human Use, an inductor a Protective Response with an added adjuvant with the Synthetic C-Terminal Fragment 242-310 from the Synthetic C-Terminal Fragment of Plasmodium sporozoite protein of Vaccine (2003)21: 2485 and 2491; and Pajak et al, "OM-174 adjuvant induces migration and maturation of murine dendritic cells in vivo" (The AdjuvantOM-174 antigens bath The migration and formation of muscle dendritic cell vivo ", Vaccine (2003)21：836-842。

(3) Immunostimulatory oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing unmethylated cytosine followed by guanine and linked by a phosphate linkage). Bacterial double stranded RNAs or oligonucleotides containing palindromic or poly (dG) sequences have been shown to have immunostimulatory activity.

Cpgs may include nucleoside modificationsDecorative/analog, such as phosphorothioate modifications, and may be double-stranded or single-stranded. Optionally, guanosine may be replaced by its analogs such as 2' -deoxy-7-deazaguanosine. See, for example, Kandimalla et al [ "divergent synthetic nucleotide motif recognition patterns: design and establishment of potent immunomodulatory oligodeoxyribonucleic acid preparations with different cytokine-inducing properties (designing and describing of cell affinity immunomodulatory oligonucleotide preparations with discrete cytokine induction profiles), nucleic acids Research (2003)31(9)：2393-2400](ii) a Examples of possible analogues are described in WO 02/26757 and WO 99/62923. Adjuvant efficacy of CpG oligonucleotides is also described in Krieg, "CpG motif: active ingredients in bacterial extracts? "(CpG motifs: the active ingredient in bacterial extracts: 831-835; McCluskit et al, "strategy for boosting immunization with hepatitis B surface antigen and CpG DNA in mice by Parenteral and mucosal administration" (parental and mucosal prime-boost immunization protocols in mice with hepatitis B surface antigens and pG DNA), FEMS Immunology and Medical Microbiology (2002) 32: 179-185; WO 98/40100; U.S. Pat. Nos. 6,207,646; U.S. Pat. No.6,239,116 and U.S. Pat. No.6,429,199.

CpG sequences may target TLR9, such as GTCGTT or TTCGTT motifs. See, kandiimalla et al, "Toll-like receptor 9: identification and cytokine Induction by novel synthetic CpG DNA Regulation (Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpGDNAs), Biochemical Society Transactions (2003)31(part 3): 654-658. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such as a CpG-B ODN. CpG-A and CpG-B ODN are described in Blackwell et al, "CpG-A induced monocyte IFN-gamma-Inducible Protein is regulated by plasmacytoid dendritic cell-derived IFN-alpha" (CpG-A-induced monoclonal IFN-gamma-induced Protein-10 Production is) Regulated byPlasmacytoid Dendritic Cell Derived IFN-alpha)，J.Immunol.(2003) 170(8)：4061-4068；Krieg，“CpG A-Z”(From A to Z on CpG)，TRENDS in Immunology(2002)23(2): 64-65 and WO 01/95935. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed such that the 5' end is recognized by the receptor. Optionally, two CpG oligonucleotide sequences may be linked at their 3' ends to form an "immunomer". See, e.g., Kandimalla et al, "Secondary Structure of CpG oligonucleotides affecting immunostimulatory Activity" (Secondaryon structures in CpG oligonucleotides affect immunological activity), BBRC (2003) 306: 948-; kandimilla et al, "Toll-like receptor 9: biochemical Society Transactions (2003) by novel synthetic CpG DNA regulatory identification and cytokine induction31(part 3): 664-; bhagat et al, "CpG penta-and hexa-deoxyribo-nucleic acids are potent immunomodulators" (CpGpenta-and hexa-deoxyribonucleotides as potential immunomodulating agents) BBRC (2003)300: 853-.

(4) ADP ribosylating toxins and detoxified derivatives thereof

Bacterial ADP ribosylating toxins and detoxified derivatives thereof are useful as adjuvants in the present invention. Preferably, the protein is derived from escherichia coli (i.e., escherichia coli heat-labile enterotoxin "LT), cholera (" CT "), or pertussis (" PT "). WO 95/17211 describes the use of detoxified ADP ribosylating toxins as mucosal adjuvants and WO 98/42375 describes them as parenteral adjuvants. Preferred adjuvants are detoxified LT mutants, such as LT-K63, LT-R72 and LTRL 92G. The use of ADP ribosylating toxins and their detoxified derivatives, particularly LT-K63 and LT-R72, as adjuvants may be found in the following references, the contents of which are incorporated herein by reference: beignon et al, "enhancing The ability of peptide antigens to stimulate CD4+ T cells and secrete gamma interferon after co-administration of E.coli Heat-Labile enterotoxin LTR72 Mutant to Bare skin" (The LTR72 Mutant of Heat-laboratory Enterotoxin of Escherichia coli Enterprise of Peptide antibiotic to Elicit CD4+ Tand Secrete Gamma interference after application to Bare Skin, Infection and Immunity (2002)70 (6): 3012-3019; pizza et al, "mucosal vaccine: nontoxic derivatives of LT and CT as Mucosal adjuvants "(Mucosal vaccines of non-toxin derivitives of LT and CT asmucosal adjuvants), Vaccine (2001)19: 2534, 2541; pizza et al, "LTK 63and LTR72, two mucosal adjuvants to be tested clinically" (LTK63and LTR72, two mucosally adjuvants for clinical trials) (int. J. Med. Microbiol (2000)290(4-5): 455-461; Scharton-Kersten et al, "transdermal Immunization with Bacterial ADP ribosylating Exotoxins, Subunits and Unrelated Adjuvants" (transdermal Immunization with Bacterial ADP-ribosylating Exotoxins, Subunits and unalated Adovants), Infection and Immunity (2000)68(9): 5306-5313; ryan et al, "mutants of E.coli heat-labile toxins as effective mucosal adjuvants for nasal delivery of acellular pertussis vaccines: the different Effects of the Nontoxic AB Complex and the enzymatic activities on Th1and Th2 Cells "(variants of Escherichia coli Heat-laboratory Toxin as an Effective mucosal Adjuvants for Nasal Delivery of an Enzyme Pertus Vaccine: variants of the Nontoxic AB Complex and Enzyme Activity Th1and Th2 Cells) Infection and Immunity (1999) 67(12): 6270-6280; partidos et al, "Escherichia coli Heat-labile enterotoxin and site-directed mutant thereof LTK63 enhanced proliferative and cytotoxic T cell responses to intranasal co-immune synthetic peptides" (Heat-laboratory of Escherichia coli and site-directed mutant LTK63 enhanced the proliferative and cytotoxic T-cell responses to intranasal co-immune synthetic peptides), Immunol.Lett. (1999)67(3): 209-216; pepporoni et al, "E.coli heat-labile enterotoxin Mutants are safe and effective adjuvants for intranasal delivery of vaccines" (variants of the Escherichia coli heat-labile enterotoxin as safe and Strong)adjuvants for indoor delivery of vitamins), vitamins (2003)2 (2): 285-; and Pine et al, (2002) "Intranasal immunization with influenza vaccine and detoxified mutants of E.coli heat-labile enterotoxin (LTK 63)" (Intra immunological with influenza and a degraded mutant of heat resistant enterotoxin (LTK63)) J. control Release (2002)85 (1-3): 263-270. Amino acid substitutions are preferably based on sequence alignments of subunits of ADP ribosylating toxins A and B, see Domenighini et al, mol. Microbiol (1995) 15(6): 1165-1167, which is incorporated by reference in its entirety.

E. Immunomodulator

Suitable human immunomodulators for use as adjuvants in the present invention include various cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon- γ), macrophage colony stimulating factor and tumor necrosis factor.

F. Bioadhesive and mucoadhesive agents

Bioadhesives and mucoadhesives may also be used as adjuvants in the present invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al, (2001) J.Cont.Rele.70: 267-276) or mucoadhesives such as poly (acrylic acid), polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides and crosslinked derivatives of carboxymethylcellulose. Chitosan and its derivatives may also be used as adjuvants in the present invention. For example WO 99/27960.

G. Microparticles

Microparticles may also be used as adjuvants in the present invention. Microparticles formed from biodegradable non-toxic materials (i.e., particles having a diameter of about 100nm to 150 μm, more preferably about 200nm to 30 μm, and most preferably about 500nm to 10 μm) with poly (lactide-co-glycolide) such as poly (alpha-hydroxy acids), polyhydroxybutyric acid, orthopolyesters, polyanhydrides, polycaprolactone, and the like are preferred, and may optionally be treated to impart a negative charge (e.g., treatment with SDS) or a positive charge (e.g., treatment with a cationic detergent such as CTAB) to their surface.

H. Liposomes

Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No.6,090,406, U.S. Pat. No.5,916,588 and EP 0626169.

I. Polyoxyethylene ethers and polyoxyethylene ester formulations

Adjuvants suitable for use in the present invention include polyoxyethylene ethers and esters. WO 99/52549. Such formulations also include a polyoxyethylene sorbitan ester surfactant in admixture with the octoxynol (WO01/21207), and a polyoxyethylene alkyl ether or ester surfactant in admixture with at least one other non-ionic surfactant such as octoxynol (WO 01/21152).

Preferred polyoxyethylene ethers are selected from the group consisting of: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.

J. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al, "Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazenes" (Preparation of hydrogel microspheres by coacceration of aqueous solutions), Biomaterials (1998)19(1-3): 109-115; and Payne et al, "Release of proteins from Polyphosphazene Matrices" (Protein Release from Polyphosphazene Matrices), adv. drug. delivery Review (1998) 31(3)：185-196。

K. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in the present invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-desmoplamyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetyl-muramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphonoxy) -ethylamine (MTP-PE).

L, imidazoquinolone compounds

Examples of imidazoquinolone compounds suitable for use as adjuvants in the present invention include Imiquimod and its homologs, as further described in Stanley, "mechanism of action and therapeutic potential of Imiquimod and imidazoquinolones" (mechanism of action and therapeutic potential) Clin Exp Dermatol (2002)27(7): 571-577; and Jones, "Resiquimod 3M" (Resiquimod 3M), Curr Opin Investig Drugs (2003)4(2)：214-218。

Virosomes and virus-like particles (VLPs)

Virosomes and virus-like particles (VLPs) may also be used as adjuvants in the present invention. These structures typically contain one or more viral proteins, and are optionally combined or formulated with phospholipids. They are generally nonpathogenic and non-replicating, and do not generally contain any native viral genome. Such viral proteins may be produced recombinantly or isolated from intact viruses. These viral proteins suitable for use in virosomes or VLPs include proteins from the following sources: influenza virus (e.g., HA or NA), hepatitis b virus (e.g., core or capsid protein), hepatitis e virus, measles virus, sindbis virus, rotavirus, foot and mouth disease virus, retrovirus, norwalk virus, human papilloma virus, HIV, RNA phage, Q β phage (e.g., coat protein), GA phage, fr phage, AP205 phage, and Ty (e.g., retrotransposon Ty protein p 1). VLPs are also described in the following documents: WO 03/024480, WO 03/024481, and Niikura et al, "Chimeric Recombinant Hepatitis E Virus-Like Particles as Oral vaccine vectors present Foreign Epitopes" (nucleic Recombinant Hepatitis E viruses-Like Particles as an organic vaccine Presenting against Epitopes), Virology (2002) 293: 273-280; lenz et al, "papillomavirus-Like particles induce Dendritic cell polar Activation" (Papilomargivurs-Like particulate induced Activation of Dendritic Cells), Journal of Immunology (2001) 5246-5355; pinto et al, "Virus-like particles with recombinant HPV-16L1Cellular Immune response of Immunized Healthy Volunteers to Human Papillomavirus (HPV) -16L1 "(Cellular Immunity Responses to Human Papillomavirus (HPV) -16L1health Volumers Immunized with Recombinants HPV-16L1 Virus-Like Particles), Journal of Infectious Diseases (2003)188: 327-338; and Gerber et al, "Human papillomavirus virus-Like Particles Are effective Oral Immunogens when co-administered with E.coli Heat-Labile enterotoxin Mutant R192G or CpG" (Human Papilomavirus-Like Particles Are effective Oral Immunogens by animal 9 organic Immunogens, i.e., platelet antigen peptide fat-sample enzyme Mutant R192G or CpG), Journal of virology (2001)75(10): 4752-4760. Virosomes are also described, for example, in Gluck et al, "New Technology Platforms for Future Development of vaccines" (New Technology Platforms in the Development of vaccines for the Future), Vaccine (2002) 20：B10-B16。

The invention also includes combinations of one or more of the adjuvant types described above. For example, the following adjuvant compositions may be used in the present invention:

(1) saponins and oil-in-water emulsions (WO 99/11241);

(2) saponins (e.g. QS21) + non-toxic LPS derivatives (e.g. 3dMPL) (see WO 94/00153);

(3) saponins (e.g. QS21) + non-toxic LPS derivatives (e.g. 3dMPL) + cholesterol;

(4) saponins (e.g. QS21) +3dMPL + IL-12 (optionally + sterol) (WO 98/57659);

(5)3dMPL in combination with, for example, QS21 and/or an oil-in-water emulsion (see european patent applications 0835318, 0735898 and 0761231);

(6) SAF, containing 10% squalane, 0.4 % Tween 80, 5% Pluronic block polymer L121 and thr-MDP, is made into submicron emulsion by microfluidization or stirred vigorously to form larger particle emulsion.

(7)Ribi^TMAdjuvant System (RAS) (Ribi Immunochem), 2% inSqualene, 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid a (MPL), Trehalose Dimycolate (TDM), and Cell Wall Skeleton (CWS), preferably MPL + CWS (Detox)^TM) (ii) a And

(8) one or more mineral salts (e.g. aluminium salts) + non-toxic derivatives of LPS (e.g. 3 dPML).

Aluminium salts and MF59 are preferred adjuvants for parenteral immunisation. Bacterial toxin mutants are preferred mucosal adjuvants.

As mentioned above, adjuvants suitable for use in the present invention may comprise one or more of:

-escherichia coli heat-labile enterotoxin ("LT"), or a detoxified mutant thereof, such as K63 or R72 mutant;

-cholera toxin ("CT"), or a detoxified mutant thereof;

microparticles (i.e. particles having a diameter of about 100nm to 150 μm, more preferably about 200nm to 30 μm, most preferably about 500nm to 10 μm) formed from biodegradable non-toxic materials (e.g. poly (alpha-hydroxy acids), polyhydroxybutyric acid, orthopolyesters, polyanhydrides, polycaprolactone, etc.);

polyoxyethylene or polyoxyethylene ethers (see international patent application WO 99/52549);

-a polyoxyethylene sorbitan ester surfactant in admixture with an octoxynol (WO01/21207), or a polyoxyethylene alkyl ether or ester surfactant in admixture with at least one other non-ionic surfactant such as an octoxynol (WO 01/21152);

chitosan (for example, International patent application WO 99/27960)

Immunostimulatory oligonucleotides (e.g.CpG oligonucleotides) and saponins (see International patent application WO00/62800)

-immunostimulatory double-stranded RNA.

Aluminum compounds (e.g. aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, aluminum oxychloride, aluminum orthophosphate, aluminum sulfate, etc. (see for example the "vaccine design: subunit and adjuvant research" (edited by Powell and Newman, plenum press, 1995(ISBN 0-306-44867-X)) (hereafter "vaccine design") (chapters 8 and 9), or mixtures of different aluminum compounds, which can take any suitable form (e.g. gel, crystal, amorphous, etc.), and are preferably adsorbed;

MF59 (5% squalene, 0.5% Tween 80 and 0.5% Span 85, formulated as submicron particles with microfluidizer) (see Chapter 10 of vaccine design; see also International patent application WO 90/14837);

liposomes (see "vaccine design" chapters 13 and 14);

ISCOM (see chapter 23 of vaccine design);

SAF, containing 10% squalane, 0.4 % Tween 80, 5% pluronic block polymer L121 and thr-MDP, microfluidized into submicron emulsions or vigorously stirred to form emulsions of larger particle size (see chapter 12 of vaccine design);

ribi Adjuvant System (RAS) (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), and cell wall matrix (CWS), preferably MPL + CWS (Detox)^TM)；

Saponin adjuvants, such as QuilA or QS21 (see "vaccine design" chapter 22), also called Stimulon^TM；

-ISCOMs, possibly free of other detergents (WO 00/07621);

-Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA);

cytokines, such as from the group of interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 etc.), interferons (e.g. interferon- γ), macrophage colony stimulating factor, tumor necrosis factor etc. (see "vaccine design" chapters 27 and 28);

Monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see Chapter 21 vaccine design);

-3dMPL in combination with, for example, QS21 and/or an oil-in-water emulsion (european patent applications 0835318, 0735898 and 0761231);

oligonucleotides containing CpG motifs (see Krieg (2000) Vaccine, 19: 618-622; Krieg (2001) curr. Opin. mol. Ther., 2001, 3: 15-24; WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581, etc.), i.e.containing at least one CG dinucleotide,

polyoxyethylene ethers or esters (International patent application WO 99/52549);

-a polyoxyethylene sorbitan ester surfactant in admixture with an octoxynol (WO01/21207), or a polyoxyethylene alkyl ether or ester surfactant in admixture with at least one other non-ionic surfactant such as an octoxynol (WO 01/21152);

immunostimulatory oligonucleotides (e.g. CpG oligonucleotides) and saponins (WO 00/62800);

immunostimulants and metal salt particles (international patent application WO 00/23105);

-saponins and oil-in-water emulsions (WO 99/11241);

-saponins (e.g. QS21) +3dMPL + IL-12 (optionally + sterol) (WO 98/57659).

Other suitable mucosal or parenteral applications are also available (see, for example, vaccine design: subunit and adjuvant research (edited by Powell and Newman, Plenum Press, 1995(ISBN 0-306-.

Mutants of LT are preferred mucosal adjuvants, in particular the "K63" and "R72" mutants (see for example international patent application WO 98/18928), which will enhance the immune response.

Microparticles are also preferred mucosal adjuvants. They are preferably derived from poly (alpha-hydroxy acids), especially from poly (lactide) ("PLA"), copolymers of D, L-lactide and glycolide or glycolic acid, such as poly (D, L-lactide-co-glycolide) ("PLG" or "PLGA"), or copolymers of D, L-lactide and caprolactone. Microparticles can be derived from different polymeric materials having various molecular weights and, when copolymers such as PLG, which have different lactide to glycolide ratios, the choice of which will be a tremendous choice depending in part on the antigens to be administered simultaneously.

The SARS virus (inactivated or attenuated), viral antigen, antibody or adjuvant of the present invention may be embedded in a microparticle or adsorbed thereto. Embedding within PLG microparticles is preferred. PLG microparticles are described in detail in Morris et al, (1994), Vaccine, 12: 5-11; chapter 13 of mucosal vaccine (edited by Kiyono et al, Academic Press 1996(ISBN012410587)), and vaccine design: subunit and adjuvant studies (edited by Powell and Newman, Plenum Press 1995(ISBN 0-306-.

The LT mutant is advantageously used with the antigen embedded in the microparticle, which will significantly enhance the immune response.

Aluminum compounds and MF59 are preferred adjuvants for parenteral administration.

The composition may comprise an antibiotic.

The immunogenic compositions of the invention may be administered in a single dose, or as part of a regimen. The regimen may include a primary dose and a booster dose, which may be administered mucosally, parenterally, or various combinations thereof.

The methods of the invention also include administering to the animal a composition comprising an effective amount of an antibody of the invention to treat or prevent a SARS virus-associated disease. An "effective amount" of an antibody of the invention is an amount sufficient to provide passive immunoprotection or treatment of an animal. Preferably, the antibodies of the invention are specific for SARS virus antigen.

Methods of treatment may combine immunogenic and antibody compositions. Accordingly, the invention includes a method of treating or preventing a SARS virus-associated disease comprising administering an immunogenic composition comprising an immunologically effective amount of a SARS virus antigen and administering an effective amount of an antibody specific for the SARS virus antigen. The immunogenic composition and the antibody may be administered simultaneously or separately. The invention also includes a composition comprising an immunogenic composition comprising an immunologically effective amount of a SARS virus antigen, and further comprising an effective amount of an antibody specific for a SARS virus antigen

The SARS virus antigens and antibodies of the present invention may be administered in the form of polynucleotides. The SARS virus antigen and/or antibody protein is then expressed in vivo.

The SARS virus antigens and antibodies of the present invention can be delivered using one or more genetic vectors, administered by nucleic acid immunization using standard genetic delivery methods. Methods of delivering genes are known in the art. See, for example, U.S. patent nos.5,399,346, 5,580,859, 5,589,466. The constructs can be delivered (e.g., injected) subcutaneously, epicutaneously, intradermally, intramuscularly, intravenously, mucosally (e.g., nasal, rectal, and vaginal), intraperitoneally, orally, or a combination thereof. Yang et al [ (2004) Nature 428: 561-564] mice were injected intramuscularly with 25. mu.g of plasmid DNA encoding the spike antigen (formulated with 200. mu.l PBS pH 7.4) at weeks 0, 3 and 6 as described.

One example of a replication-defective gene delivery vector that can be used in the practice of the present invention is any alphavirus vector described in, for example, U.S. patent nos.6,342,372; 6,329,201 and WO 01/92552.

Many virus-based systems have been developed to deliver genes to mammalian cells. For example, retroviruses are a convenient platform for gene delivery systems. The selected sequences can be inserted into a vector and packaged into a retrovirus using techniques known in the art. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. A number of retroviral systems have been described (U.S. Pat. No.5,219,740; Miller & Rosman, BioTechniques (1989) 7: 980-; 990; Miller, A.D., Human Gene Therapy (1990) 1: 5-14; Scarpa et al, Virology (1991) 180: 849-; Burns et al, Proc. Natl. Acad. Sci. USA (1993) 90: 8033-; 8037; and Boris-Lawrie & Temin, Cur. Opin. Genet. Devel. (1993) 3: 102-; 109.

A number of adenoviral vectors have also been described. Unlike retroviruses, which are integrated into the host genome, adenoviruses remain extrachromosomal and thus minimize the risk associated with insertional mutagenesis (Haj-Ahmad and Graham, J.Virol. (1986) 57: 267-5: 717 729; seth et al, J.Virol. (1994) 68: 933 940; barr et al, Gene Therapy (1994) 1: 51-58; berkner, k.l. biotechniques (1988) 6: 616-629; and Rich et al, Human Gene Therapy (1993) 4: 461-476). The codon-optimized pattern of adenovirus delivery of genes encoding the SARS coronavirus structural antigen spike S1, membrane proteins and nucleocapsid proteins has been studied in macaques and found to elicit a strong neutralizing antibody response (Gao et al, (2003) Lancet 362 (9399): 1895-.

In addition, various adeno-associated virus (AAV) vector systems can also be used for gene delivery. AAV vectors are readily constructed using techniques well known in the art. See, e.g., U.S. patent nos.5,173,414 and 5,139,941; WO92/01070 (published on 23.1.1992) and WO 93/03769 (published on 4.3.1993); lebkowski et al, molec cell biol. (1988) 8: 3988-; vincent et al, vitamins 90(1990) (Cold spring Harbor Laboratory Press); carter, b.j. current Opinion in Biotechnology (1992) 3: 533-; muzycka, n.current Topics in microbiol.and Immunol. (1992) 158: 97 to 129; kotin, R.M.human Gene Therapy (1994) 5: 793-801; shelling and Smith, Gene Therapy (1994) 1: 165-169; and Zhou et al, j.exp.med. (1994) 179: 1867-1875.

Other vector systems for delivery of polynucleotides by mucosal or other routes are recombinant poxvirus vaccines for enteral administration, described in Small Small, P.A. et al (U.S. Pat. No.5,676,950, published 10/14/1997, incorporated herein by reference), as well as vaccinia viruses and fowlpox viruses. For example, vaccinia virus recombinants expressing the genes can be constructed by the following method. The DNA or antibody coding sequence encoding the SARS antigen or antibody is first inserted into a suitable vector adjacent to the vaccinia promoter and flanking a vaccinia DNA sequence, such as a sequence encoding Thymidine Kinase (TK). The vector was then used to transfect cells that were simultaneously infected with vaccinia. The vaccinia promoter and the gene encoding the sequence of interest can be inserted into the viral genome by homologous recombination. The resulting TK-recombinants may be selected by culturing the cells in the presence of 5-bromodeoxyguanosine and selecting resistant viral plaques therein.

Alternatively, avipoxviruses (e.g., fowlpox and canarypox viruses) may be used to deliver genes encoding SARS virus antigens or antibodies of the present invention. It is known that protective immunity can be provided by administering recombinant fowlpox virus expressing a mammalian pathogen immunogen to a non-avian species. The use of fowlpox vectors is particularly desirable in humans and other mammals, since fowlpox replicates only fruitfully in susceptible birds and therefore does not infect mammalian cells. Methods for producing recombinant fowlpox viruses are known in the art and can be produced using genetic recombinants, according to the methods described above for producing vaccinia viruses. See, for example, WO 91/12882; WO 89/03429; and WO 92/03545. Picornavirus-derived vectors may also be used. (see, e.g., U.S. Pat. Nos.5,614,413 and 6,063,384).

Molecularly coupled carriers, such as Michael et al [ j.biol.chem. (1993) 268: 6866-: 6099-6103] the adenoviral chimeric vector can also be used for gene delivery.

Conveniently, a vaccinia infection/transfection system is employed to provide inducible transient expression of the coding sequence of interest (e.g., a SARS virus antigen or antibody expression cassette) in the host cell. In this system, cells were first infected in vitro with a vaccinia virus recombinant encoding the T7 phage RNA polymerase. This polymerase exhibits exquisite specificity, i.e., it transcribes only the template with the T7 promoter. Following infection, the polynucleotide of interest transfects the cells under the drive of the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the translation machinery of the host cell. This method can produce large amounts of RNA and its translation product in cytoplasm at a high level transiently. See, e.g., Elroy-Stein and Moss, proc.natl.acad.sci.usa (1990) 87: 6743-6747; fuerst et al, Proc.Natl.Acad.Sci.USA (1986) 83: 8122-8126.

As an alternative to infection with recombinant vaccinia or fowlpox viruses, or to gene delivery using other viral vectors, amplification systems can be used which, when introduced into a host cell, will give high levels of expression. In particular, the T7RNA polymerase promoter may be engineered to precede the T7RNA polymerase coding region. Translation of RNA from such a template will result in T7RNA polymerase, which transcribes more template. Simultaneously, a cDNA whose expression is controlled by the T7 promoter is produced. Thus, some of the T7RNA polymerase produced by translation of the amplified template RNA will result in transcription of the desired gene. Since some T7RNA polymerase is necessary to initiate this amplification, T7RNA polymerase can be introduced into the cell along with the template to initiate the transcription reaction. The polymerase may be introduced as a protein or on a plasmid encoding an RNA polymerase. For T7 systems and their use in transformed cells see, for example, WO 94/26911; studier and Moffatt, j.mol.biol. (1986) 189: 113-130; deng and Wolff, Gene (1994) 143: 245-249; gao et al, biochem. biophysis. res. commun. (1994) 200: 1201-1206; gao and Huang, nuc. acids Res, (1993) 21: 2867 2872; chen et al, nuc. acids Res, (1994) 22: 2114-2120; and U.S. patent No.5,135,855.

The immunogenic compositions of the invention may also contain diluents such as water, saline, glycerol, alcohol, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be included in the immunogenic composition.

The immunogenic compositions for use in the present invention may be administered to an animal. Animals suitable for the methods of the invention include humans and other primates, including non-human primates, such as chimpanzees, as well as other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic animals such as dogs and cats; test animals, including rodents, such as mice, rats and guinea pigs; birds, including domestic, wild and game birds, such as chickens, turkeys, and quail birds, ducks, geese, and the like. Animals suitable for use in the present invention may be of any age, including adult and neonatal animals. Transgenic animals may also be used in the present invention.

The immunogenic compositions of the invention can be used to treat or prevent SARS virus-associated disease.

The compositions of the present invention are preferably pharmaceutically or pharmacologically acceptable. In particular, it is desirable that the composition is not biologically or otherwise undesirable, i.e., the substance may be administered to an individual in the form of a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with the components of the composition it contains.

Pharmaceutically acceptable salts can be used in the compositions of the invention, for example, mineral salts (e.g., hydrochloride, hydrobromide, sulfate or sulfate salts) and salts of organic acids (e.g., acetate, propionate, malonate or benzoate salts). Particularly useful proteinaceous substances are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin and other proteins well known to those skilled in the art. The compositions of the present invention may also contain liquids or excipients such as water, saline, glycerol, dextrose, alcohols, and the like, alone or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes can also be used as carriers for the compositions of the invention.

The vaccines of the present invention can be made and tested using SARS-specific reagents or analytical assays. Such analytical assays include, for example: 1) viral titration and plaque assay to quantify infectious viral particles, 2) neutralization assay with constant virus and variable serum dilutions, 3) two-step RT-PCR system (Light Cycler-Roche) to detect minus-strand viral RNA, with target sequence located within the N gene, providing the highest sensitivity possible, and 4) ELISA and western blot analysis to detect and purify viral proteins.

In addition, can generateRabbit polyclonal antiserum to obtain antibody reagents against SARS-CoV (and demonstrate induction of neutralizing antibodies). The sampling protocol for generating this reagent is as follows. The virus is first cultured in suitable cultured cells (e.g., Vero cells) and precipitated by a 20% sucrose (w/v) pad. The precipitate was then treated with a glycerol-potassium tartrate gradient for further purification. The virus-containing fractions were then diluted and pelleted by ultracentrifugation. The precipitate was then dissolved in PBS and washed with C₃H₄O₂(beta-propiolactone, BPL) inactivates the virus. 1X 10 mixed with adjuvant IFA on days 0, 14 and 28⁹Inactivated virions were used to immunize two rabbits Subcutaneously (SC). Rabbit blood was collected on days 0 (pre-vaccination), 13, 28 and 35 (1 week after the third immunization). Sera obtained from this protocol were tested for reactivity against SARS-CoV protein by western blotting and were found to react with the major structural proteins spike (S), membrane (M) and nucleocapsid (N) proteins.

J. Emerging coronavirus vaccines

The SARS epidemic leads to an increased understanding of the viral infection caused by coronaviruses. The vaccines of the present invention may be used to prevent or treat emerging strains of coronavirus, including emerging SARS virus.

The present invention provides a vaccine comprising an inactivated (or killed) human coronavirus, an attenuated human coronavirus, a split human coronavirus preparation, or a recombinant or purified subunit preparation of one or more human coronavirus antigens, wherein the human coronavirus is not a SARS coronavirus. Optionally, the human coronavirus is not a 229E coronavirus. Optionally, the human coronavirus is OC43 coronavirus. Optionally, the human coronavirus is not an NL63 coronavirus. Accordingly, the present invention provides a vaccine as defined above, wherein said human coronavirus is not a SARS coronavirus, not a 229E coronavirus, not an OC43 coronavirus, and not an NL63 coronavirus. Such vaccines are useful for the prevention and/or treatment of human coronavirus infections that are about to occur.

The invention also provides a vaccine comprising: (a) inactivated (or killed) human coronavirus, attenuated human coronavirus, split human coronavirus preparation, or recombinant or purified subunit of one or more human coronavirus antigens, wherein said human coronavirus is not a SARS coronavirus, as defined above; and (b) an inactivated (or killed) human coronavirus, an attenuated human coronavirus, a split human coronavirus preparation, or a subunit preparation of one or more recombinant or purified human coronavirus antigens, wherein the human coronavirus is a SARS coronavirus. The vaccine can be used for preventing and/or treating SARS and other human coronavirus.

In addition to providing a vaccine containing antigens from more than one coronavirus type, the invention also provides a vaccine containing antigens from multiple strains of the same coronavirus, such as antigens from different strains of the SARS coronavirus or different strains of a coronavirus other than the SARS coronavirus. In one embodiment, the vaccine comprises antigens from at least two coronavirus strains, or at least three coronavirus strains. In one embodiment, the vaccine comprises antigens from at least two coronavirus types. In one embodiment, the vaccine comprises from about each known coronavirus type (type I, type II, and type III) to at least one antigen. This vaccine has the current mode of influenza vaccine.

Selection of a coronavirus and/or coronavirus for use in a vaccine of the invention can be based on various criteria. For example, selection can be based on viruses and/or strains that have been detected in the geographic region where the vaccine will be administered (e.g., northern hemisphere or southern hemisphere, specific country, etc.). Selection can be based on animal monitoring (see, e.g., viruses detected in hospitalized patients due to respiratory infections) results. The selection may be performed annually, for example before winter. Vaccines can also be vaccinated annually, also in the influenza vaccine model.

Preferred vaccines should be sufficiently immunogenic to provide a neutralizing immune response, more preferably a protective and/or therapeutic immune response. A particularly preferred vaccine should meet the potency requirements specified by the WHO at that time.

Preferred subunit antigens comprised by the vaccine of the invention are purified spike proteins, more preferably in oligomeric (e.g. trimeric) form. The spike protein may be cleaved into its S1 and S2 products, or not.

The techniques described above for selecting viruses and/or strains for vaccine manufacture can also be used to select HR1 and HR2 sequences of suitable viruses and/or strains available to provide the therapeutic peptides described above.

The diagnostic compositions and methods of the invention

The present invention provides a method for detecting SARS coronavirus. Testing patient samples can be used to detect and diagnose viral infections. Testing donated blood can be used to prevent inadvertent viral transmission during transfusion. The detection method mainly comprises three aspects: detecting SARS virus nucleic acid; detecting SARS virus protein; and detecting an anti-SARS virus immune response. The present invention provides all three of these methods.

When reference is made herein to nucleotide sequence specific oligonucleotide probes and primers, "similar" sequences include those sequences that are at least 90% identical to a known SARSV genomic sequence, and include sequences that are at least 95%, at least 99% identical, and 100% identical to a SARSV genomic sequence over the length of the probe or primer.

The term "target nucleic acid region" or "target nucleic acid" as used herein refers to a nucleic acid molecule having a "target sequence" to be amplified. The target nucleic acid may be single-stranded or double-stranded, and may contain other sequences than the target sequence that are not amplified. The term "target sequence" denotes a specific nucleotide sequence of a target nucleic acid to be amplified. The target sequence may include a region within the target molecule to which the probe hybridizes, under appropriate conditions to which the probe will stably hybridize. "target sequence" may also include composite sequences that bind to oligonucleotide primers and are extended with the target sequence as a template. The target nucleic acid may be single-stranded per se, and the term "target sequence" also refers to a sequence complementary to a "target sequence" present in the target nucleic acid. The term "target sequence" refers to both plus (+) and minus (-) strands if the "target nucleic acid" is originally double-stranded.

The term "primer" or "oligonucleotide primer" as used herein refers to an oligonucleotide that initiates synthesis of a complementary DNA strand when placed under conditions that induce synthesis of a primer extension product, i.e., in the presence of nucleotides and a polymerization inducing agent (e.g., DNA or RNA polymerase) and having a suitable temperature, pH, metal concentration, and salt concentration. The primer is preferably single-stranded for most efficient amplification, but may also be double-stranded. If double stranded, the primer is first treated to separate its two strands and then used to prepare an extension product. The denaturation step is usually accomplished by heating, but it is also possible to treat with alkali and then neutralize. Thus, a "primer" is complementary to the template and complexed with the template by hydrogen bonding or hybridization to give a primer/template complex, thereby initiating synthesis by the action of a polymerase, which primer is extended during DNA synthesis by the addition of a base complementary to the template covalently attached to the 3' end of the primer.

The term "probe" or "oligonucleotide probe" refers to a structure comprised of a polynucleotide as described above that contains a nucleic acid sequence that is complementary to a nucleic acid sequence present in a target nucleic acid analyte. The polynucleotide region of the probe may be comprised of DNA, and/or RNA, and/or synthetic nucleoside analogs. When "oligonucleotide probes" are used for 5' nuclease assays, e.g., TaqMan ^TMThe probe will contain at least one fluorescer and at least one quencher digested by the 5' endonuclease activity used in the reaction to detect any amplified target oligonucleotide sequence. As used herein, the oligonucleotide probe will contain a sufficient number of phosphodiester tendons adjacent to its 5 ' terminus such that the 5 ' to 3 ' nuclease activity employed is effective to degrade the bound probe to separate the fluorescer and quencher. When the oligonucleotide probe is used in the TRA technique, it will be appropriately labelled as described below.

The hybridizing sequences need not have good complementarity to provide stable hybridization. In most cases, stable hybrids are formed when less than about 10% of the mismatched bases are omitted from loops formed by four or more nucleotides. Thus, the term "complementary" as used herein means that the oligonucleotides that form stable duplexes with their "complements" under the conditions of the assay are generally about 90% or more homologous.

The terms "hybridize" and "hybridization formation" refer to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex by Watson-Crick base pairing. When a primer "hybridizes" to a target (template), such a complex (or hybrid) is sufficiently stable to function as a primer, e.g., required by a DNA polymerase, which initiates DNA synthesis.

Stringent hybridization conditions typically include the following requirements: salt concentrations below about 1M, more usually below about 500mM, preferably below about 200 mM; the hybridization temperature can be as low as 5 ℃ but is generally above 22 ℃, more generally above about 30 ℃, and preferably above about 37 ℃. Longer fragments may require higher hybridization temperatures for specific hybridization. Other factors that affect hybridization stringency include the base composition and length of the complementary strands, the presence or absence of organic solvents, and the degree of base mismatching, the combination of parameters used being more important than the absolute value of any single parameter. Other hybridization conditions that may be controlled include buffer type and concentration, solution pH, presence or absence of blocking reagents or blocking protein solutions that reduce background binding (e.g., repetitive sequences) and their concentrations, detergent species and concentrations, polymer-like molecules that increase the relative concentration of polynucleotides, metal ions and their concentrations, chelating agents and their concentrations, and other conditions known in the art. Hybridization conditions of lower stringency and/or more physiological requirements can be used when the labeled polynucleotide amplification cycle switch substrate is bound to a complementary probe polynucleotide during real-time assays for PCR amplification monitoring, such as molecular beacon assay. Such less stringent hybridization conditions also include solution conditions effective for other aspects of the method, such as reverse transcription or PCR.

"biological sample" as used herein refers to a tissue, cell, or bodily fluid sample isolated from a subject, which typically includes antibodies made by the subject. Typical samples include, but are not limited to, blood, plasma, serum, feces, urine, bone marrow, bile, spinal fluid, lymph fluid, skin samples, skin, respiratory, intestinal and genitourinary tracts, tears, saliva, sputum, mucous membranes, milk, blood cells, organs, tissues, living tissue (e.g., lung, liver, kidney), and in vitro cell culture samples including, but not limited to, conditioned media, e.g., recombinant cells and cell components, resulting from the growth of cells and tissues in culture. Other samples may also be used for diagnosis, including fecal samples and nasopharyngeal aspirates.

The term "antibody" includes polyclonal and monoclonal antibody preparations, as well as hybrid, variant, chimeric and humanized antibodies, and hybrid (chimeric) antibody molecules (see, e.g., Winter et al (1991) Nature349: 293-299; and U.S. Pat. No. 4,816,567); f (ab')₂And F (ab) a fragment; fv molecules (non-covalent heterodimers, see, e.g., Inbar et al, (1972) Proc Natl Acad Sci USA 69: 2659-2662; and Ehrlich et al, (1980) Biochem19: 4091-; single chain Fv molecules (sFv) (see, e.g., Huston et al, (1988) Proc Natl Acad Sci USA85: 5879-5883); an oligomer; dimeric and trimeric antibody fragment constructs; microbodies (see, e.g., Pack et al (1992) Biochem31: 1579-1584; cumber et al (1992) J Immunology149B: 120-126); humanized antibody molecules (see, e.g., Riechmann et al (1988) Nature332: 323-327; verhoeyan et al, (1988) Science239: 1534 — 1536; and british patent publication No. gb 2,276,169, published on 9/21 of 1994); and any functional fragment obtained from such a molecule, wherein said fragment retains the specific binding properties of the parent antibody molecule.

The term "monoclonal antibody" as used herein refers to an antibody composition comprising a population of homogeneous antibodies. The term is not limited by the type or source of the antibody and by the manner in which it is prepared. The term includes all immunoglobulins.

Methods for preparing polyclonal and monoclonal antibodies are known in the art. Polyclonal antibodies are generated by immunizing a suitable animal (e.g., mouse, rat, rabbit, sheep, or goat) with an antigen of interest. To enhance immunogenicity, the antibodies may be linked to a carrier prior to immunization. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid coacervates (such as oil droplets or liposomes) and inactivated virus particles. Such vectors are well known to those skilled in the art. In addition, antigens can be conjugated to bacterial toxoids, such as diphtheria, tetanus, cholera toxins, and the like, to enhance their immunogenicity.

When large volumes of serum are required, it is preferred to use rabbits, sheep and goats to prepare polyclonal antisera. Also, because labeled anti-rabbit, anti-sheep and anti-goat antibodies are available, these animals are a good design choice. Immunization is typically carried out by: the antigen is mixed or emulsified in saline, preferably in an adjuvant such as Freund's complete adjuvant ("FCA"), and the mixture or emulsion is injected parenterally, usually subcutaneously or intramuscularly. The immunization is boosted 2-6 weeks later by one or more injections with antigen in saline, preferably Freund's incomplete adjuvant ("FIA"). Antibodies can additionally be generated by in vitro immunization using methods known in the art. Polyclonal antisera are then obtained from the immunized animals.

As mentioned above, the method according to Kohler and Milstein [ (1975) Nature 256: 495-497] or a modified method thereof.

Nucleic acid detection method

Many methods for amplifying target sequences are known, such as Polymerase Chain Reaction (PCR), reverse transcription PCR (RT-PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA), Transcription Mediated Amplification (TMA). These methods are generally described in the following references: (PCR) U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159; (RT-PCR) U.S. Pat. Nos.5,310,652, 5,322,770; (LCR) EP application No. 320,308 (published on 14/6 1989); (SDA) U.S. Pat. Nos.5,270,184 and 5,455,166, and G.T. Walker, "practical application of Strand Displacement amplification" (Empir) And (3) and (1) of chemical Applications of Strand Displacement amplification) selected from PCR Methods and Applications (PCR Methods and Applications): 1-6(1993), Cold Spring Harbor Laboratory Press; (TMA) U.S. Pat. Nos. 5,399,491; l. Malek et al "Nucleic Acid Sequence Based Amplification (NASBA)^TM) ", selected from Methods in Molecular Biology (Methods in Molecular Biology), volume 28, chapter 36: method for analyzing nucleic acids by non-radioactive probes, compiled by 1994 p.g. isaac, Humana Press, inc. PCR methods can include some variation to quantify the target sequence, such as by real-time PCR analysis (as described, inter alia, in U.S. Pat. nos. 5,210,015, 5,487,972, 5,994,056, 6,171,785). (the above references are incorporated herein by reference in their entirety).

One embodiment of the present invention for detecting the presence of SARS virus in a sample comprises the steps of: providing a sample suspected of containing a SARS virus nucleic acid target; amplifying the template sequence contained in said SARS virus nucleic acid target using any known nucleic acid amplification technique, including any of the techniques mentioned herein, using the oligonucleotide primers described herein, particularly those comprising the kits described herein; and detecting the amplified template sequence, wherein the presence of the amplified template sequence indicates the presence of SARS virus in the sample.

Amplification techniques typically involve the use of two primers. When the target sequence is single-stranded, the technique typically includes a preliminary step of making complementary strands to obtain a double-stranded target. The two primers are hybridized to different strands of the double-stranded target and then re-extended. The extension product can serve as a target for further hybridization/extension. The net effect is to amplify the template sequence within the target, the 5 'and 3' ends of the template being determined by the position of the two primers in the target. Alternatively, if one or both of the primers contains a promoter sequence, RNA polymerase can be used to amplify (by transcription) the target (as with TMA).

The present invention provides methods and kits for amplifying and/or detecting template or target sequences in SARSV viral nucleic acids. The invention provides a kit comprising primers for amplifying a template sequence contained in a SARSV nucleic acid target, the kit comprising a first primer and a second primer, wherein the first primer comprises a sequence substantially complementary to a portion of the template sequence and the second primer comprises a sequence substantially complementary to a portion of the complement of the template sequence, wherein the substantially complementary sequences within the primers define the two ends of the template sequence to be amplified. The kit of the present invention further comprises a probe that is substantially complementary to and hybridizes to the template sequence and/or its complement. Such probes can be used in hybridization techniques to detect amplified template, or to isolate (i.e., capture) amplified template or initial target nucleic acid.

The kits of the invention also contain primers and/or probes for generating and detecting internal standards to aid in the quantification of the measurement (e.g., Fille et al, 1997 Biotechniques 23: 34-36).

The kit of the invention also contains a DNA polymerase, which is typically a thermostable DNA polymerase, in which case a non-isothermal amplification procedure will be used. The kit may further comprise a dNTP, a magnesium salt (e.g., MgCl)₂) Buffers, and the like.

The kits of the invention also include more than one pair of primers (e.g., for nested amplification), when one primer is compared to more than one primer pair. The kit may also contain a plurality of probes.

Oligomeric probes and primers

In combination with the nucleic acid detection method of the present invention, oligomers having sequence similarity or complementarity to the SARSV genome can be used. The SARSV genomic sequences referred to herein are used to generate probes and primers for use in assays for detecting nucleic acids in test samples. Probes can be designed based on conserved nucleotide regions of a polynucleotide of interest or non-conserved nucleotide regions of a polynucleotide of interest. Methods for designing probes that optimize the assay are known to those skilled in the art. In general, nucleic acid probes can be generated based on non-conserved regions or unique regions where the highest specificity is desired, or conserved regions when the detection is performed, for example, on different members of a multigene family or on the most closely related nucleotide regions in related animals such as mice and humans.

Oligomers of about 8 or more nucleotides can be prepared as shown herein based on the SARSV genome and/or preferably conserved regions of the SARSV genome, and/or particularly the primer and probe sequences described herein, which can hybridize to the positive strand of the SARSV RNA or its complement, as well as to the SARSV cDNA. These oligomers can serve as probes for detecting (including isolating and/or labeling) polynucleotides comprising SARSV nucleotide sequences, and/or as primers for transcribing and/or replicating target SARSV sequences. The oligomer comprises a targeting polynucleotide sequence consisting of nucleotides complementary to a target SARSV nucleotide sequence; this sequence is sufficiently long and complementary to the SARSV sequence to form a sufficiently stable duplex required for this purpose. For example, if the objective is to isolate an analyte containing a target SARSV sequence by immobilization, the oligomer may contain a polynucleotide region sufficiently long and complementary to the target SARSV sequence to achieve sufficient stability of the duplex capable of immobilizing the analyte on a solid surface by binding of the analyte to the oligomer under the conditions of isolation. Also, for example, if the oligomer is to act as a primer to transcribe and/or replicate a target SARSV sequence in an analyte polynucleotide, the oligomer will contain a polynucleotide region sufficiently long to be sufficiently complementary to the target SARSV sequence to allow, under polymerization conditions, the polymerizing agent to replicate continuously from the primer in the form of a stable duplex containing the target sequence. Also for example, if the oligomer is used as a labeled probe or is to be bound to a multimer, the targeted polynucleotide region will be long enough and sufficiently complementary to form a stable hybridization duplex structure with the labeled probe and/or multimer to enable detection of the duplex. The oligomer contains a minimum of about 4 contiguous nucleotides complementary to the target SARSV sequence; typically, the oligomer contains at least about 8 contiguous nucleotides complementary to the target SARSV sequence, preferably at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides complementary to the target SARSV sequence, up to about 50, 75, 100, 200 contiguous nucleotides or more.

Typically, in amplification-based methods (e.g., PCR, RT-PCR, TMA), oligomers will be used as a set of primers, one member of which has sequence similarity or complementarity to a more conserved portion of the SARSV genome (in coronaviruses) and the other member of which has sequence similarity or complementarity to a less well-conserved portion. The primer set can be used to amplify the target region in a manner well known in the art. In general, the 5 'untranslated region (5' UTR) and the 3 'untranslated region (3' UTR) are the most conserved regions. FIG. 8 shows a comparison of the 5' UTR of several coronaviruses. FIG. 10 shows a comparison of the 3' UTR of several coronaviruses. FIGS. 9 and 11 show the sequences of preferred primers used to amplify the 5 'UTR and 3' UTR, respectively. Other primers and probes are readily designed based on the sequence alignments provided herein.

However, the oligomer need not consist only of sequences complementary to the target SARSV sequence. It furthermore contains nucleotide sequences (e.g.promoters) or other parts which are suitable for the intended use of the oligomer. For example, if the oligomers are used as primers for amplification of SARSV sequences by, for example, PCR, they may contain sequences that, when in duplex form, form a restriction site that facilitates particle amplification. As another example, if the oligomers are to be used as "capture probes" for hybridization assays, they will also comprise a binding partner coupled to an oligomer comprising an oligonucleotide sequence complementary to a target SARSV sequence. The oligomer may comprise or be coupled to other useful types of molecules or sequences known in the art and suitable for various purposes, including labeling nucleotide probes.

Table 4(SEQ ID NOS: 1021-.

For diagnosis and screening, preferred primers and probes for detecting SARS nucleic acid are SEQ ID NOS: 7332-7336 (forward primer), SEQ ID NOS: 7337-7341 (reverse primer) and SEQ ID NOS: 7342 and 7352 (probes). These primers and probes are effective for detecting 3' UTR sequences.

Any of the forward primers described above can be used in combination with any of the reverse primers described above to amplify a SARSV nucleic acid. The amplification product can be detected (or captured) using any of the probes described above. Particularly preferred combinations of forward and reverse primers and probes for detecting amplification products are: forward SEQ ID NO: 7332, reverse SEQ ID NO: 7337. 7338, 7339, or 7341, probe SEQ ID NO: 7342; forward SEQ ID NO: 7333 or 7334, reverse SEQ ID NO: 7340 and SEQ ID NO: 7343 any of 7351 as a probe; forward SEQ ID NO: 7335, reverse SEQ ID NO: 7340 or 7341, the probe may be SEQ ID NO: 7342 and 7352. Other combinations of forward and reverse primers and suitable probes can be readily determined by one skilled in the art from the above information.

Other preferred primers and probes for detecting SARS nucleic acid for diagnosis and screening are SEQ ID NOS: 7353-7362 (forward primer), SEQ ID NOS: 7363-7373 (reverse primer) and SEQ ID NOS: 7374 and 7385 (probes). These primers and probes are effective for detecting sequences in the 5' UTR.

The above primers can be used in combination to amplify a SARSV nucleic acid, in the following combinations: the forward primer is SEQ ID NO: 7353 and 7356, the reverse primer is SEQ ID NO: 7363 and 7368, the amplification product is amplified using the probe SEQ ID NO: 7374 detection (or capture); the forward primer is SEQ ID NO: 7357 and 7362, the reverse primer is SEQ ID NO: 7367. 7369 and 7373, the amplification product is amplified using the probe SEQ ID NO: 7375 and 7385 detection (or capture). Particularly preferred combinations of forward and reverse primers and probes are: the forward primer is SEQ id no: 7353 and 7356, the reverse primer is SEQ ID NO: 7363 and 7366, the probe is SEQ ID NO: 7374; the forward primer is SEQ ID NO: 7357 along with 7358, the reverse primer is SEQ ID NO: 7367. 7369 the probe is SEQ ID NO: 7375 or 7376; the forward primer is SEQ ID NO: 7357 along with 7359, the reverse primer is SEQ ID NO: 7367. 7369 or 7370, the probe is SEQ ID NO: 7375 or 7376. A more preferred combination is SEQ ID NO: 7353 or 7354 with SEQ ID NO: 7363 or 7364, the probe is SEQ ID NO: 7374. other combinations of forward and reverse primers and suitable probes can be readily determined by one skilled in the art from the above information. An eight nucleotide sequence (SEQ ID NO: 7386) that is particularly conserved in the 3 'UTR (about 70-80 bases from the 3' end) of SARS and some other coronaviruses is particularly useful in the identification of SARSV. Primers containing this region are preferably combined with the reverse primer of the sequence region more specific for SARS.

In addition to the above, Intergenic Sequences (IS) characteristic of coronaviruses have been identified in SARSV (see above). IS comprises at least the sequence ACGAAC (SEQ ID NO: 7293) which IS present upstream of each Open Reading Frame (ORF) of the viral genome. The 5 'UTR comprising IS IS spliced at the 5' end of the respective viral mRNA or adjacent to IS. Thus, primers containing IS or its complement can be used to amplify viral nucleic acids, including cDNA made from viral RNA. Thus, the invention includes a set of primers, wherein one primer comprises ACGAAC (SEQ ID NO: 7293) or its complement (SEQ ID NO: 7387) and one primer comprises any suitable sequence or complement from the SARS genome. Useful probes for detecting and/or capturing viral RNA or cDNA made from viral RNA may comprise an IS sequence, or the complement thereof, as described above.

One set of primers used to amplify SARS sequences, particularly by RT-PCR, employs SEQ ID NOs 6562, 6563, 6564 and 6565. Of course, 6562 is 6564 is a sense primer and 6563 and 6565 are antisense primers. Primers SEQID NOS: 6562 and 6565 can be used in a first amplification, and a second nested amplification with primers SEQ id nos: 6563 and 6564. In some embodiments of the invention, all four primers are excluded.

A kit for amplifying and detecting SARS sequences, particularly by RT-PCR, employs SEQ ID NOs6567 and 6568 as primers and SEQ ID NO 6566 as probe (particularly labeled with, e.g., TAMRA and/or FAM) to amplify the sequences. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying and detecting SARS sequences, particularly by RT-PCR, employs SEQ ID NOs7395 and 6568 as primers and SEQ ID NO 6566 as probe (particularly labeled with, e.g., TAMRA and/or FAM) to amplify the sequences. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying SARS sequences, particularly nucleocapsid genes, uses SEQ ID NOs 6560 and 6561 as primers. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying SARS sequence uses SEQ ID NOs 6496, 6497, 6562, 6563, 6564 and 6565 as primers. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying SARS sequence uses SEQ ID NOs 6562, 6563, 6564 and 6565 as primers. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying SARS sequence uses SEQ ID NOs 6500, 6501, 6502 and 6503 as primers. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying SARS sequence uses SEQ ID NOs 6496, 6497, 6500, 6501, 6502, 6503, 6562, 6563, 6564 and 6565 as primers. In some embodiments of the invention, these primers and probes are excluded.

A method for amplifying and detecting SARS sequences (especially by real-time PCR, such as TaqMan^TM) The kit of (a) employs SEQ ID NOs 6567 and 6568 as primers and SEQ ID NO 6566 as probe (especially labelled with, for example, TAMRA and/or FAM) to amplify the sequence. In some embodiments of the invention, these primers and probes are excluded.

A method for amplifying and detecting SARS sequences (especially by real-time PCR, such as TaqMan^TM) The kit of (A) uses SEQ ID NOs 7395 and 6568 as primers and SEQ ID NO 6566 as probe (especially labelled with, for example, TAMRA and/or FAM)And increasing the sequence. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying and detecting SARS sequences employs SEQ ID NOs 6562, 6565 and 6568 as primers and SEQ ID NO 7396 and 7397 as probes (especially labelled with, for example, TAMRA and/or FAM) to amplify the sequences. In some embodiments of the invention, these primers and probes are excluded.

A kit for amplifying and detecting SARS sequence comprises the nucleic acid sequence of SEQ ID NO: 9780 as a forward primer, the primer comprising the nucleotide sequence of SEQ ID NO: 9781 as a reverse primer and a primer comprising the nucleotide sequence of SEQ ID NO: 9782 as a probe.

Preferred sequences for RT-PCR and LightCycler analysis include SEQ ID NOs 6562, 6568, 6565, 7396 and 7397. In some embodiments of the invention, these primers and probes are excluded.

Such oligomers are prepared by methods known in the art, including, for example, excision, transcription, or chemical synthesis. The target sequence and/or region of the genome to which the targeting polynucleotide of the oligomer is complementary is selected according to the purpose. For example, if the objective is to screen biological samples (e.g., blood, respiratory material, liver, lung) for the presence of SARSV, oligomers are preferably used as probes and/or primers and hybridized to conserved regions of the SARSV genome. Some conserved regions of the SARSV genome to which the oligomers can bind have been described herein, such as the 5 'UTR and the 3' UTR.

In basic nucleic acid hybridization assays, single-stranded analyte nucleic acid (DNA or RNA) is hybridized to a nucleic acid probe, and the resulting duplex is detected. The length of the SARSV polynucleotide (native or extended) probe is such that it is capable of detecting unique viral sequences by hybridization. While 6-8 nucleotides are of workable length, sequences containing 10-12 nucleotides are preferred, with about 13, 14, 15, 16, 17, 18, 19, 20, or 21 or more nucleotides appearing to be optimal. Preferably, these sequences will be from regions lacking heterogeneity. These probes can be prepared by conventional methods, including automated oligonucleotide synthesis. Useful probes may be, for example, those derived from regions of the SARSV genome that are not well conserved. Regions of the SARS genome that are not well conserved can be readily determined by sequence alignment as provided herein, as well as other well-known techniques. Sequences complementary to any unique portion of the SARSV genome are also satisfactory. For use as a probe, perfect complementarity is preferred, but this is not necessary as the length of the fragment increases.

To use such probes to detect the presence of a SARSV polynucleotide (e.g., screening contaminated blood or diagnosing an infected individual), a biological sample to be analyzed (including but not limited to blood, serum, lung, liver, mucosa, kidney, saliva, or sputum) can be processed, if necessary, to extract the nucleic acids contained therein. The obtained sample nucleic acid can be subjected to gel electrophoresis or other separation technologies according to molecular weight; alternatively, the nucleic acids can be dot blotted without size separation. To form a hybrid duplex with the targeting sequence of the probe, the target region of the nucleic acid analyte must be in single stranded form. Denaturation will not be required when the sequence is naturally present in single stranded form. However, when the sequence is present in a double-stranded form, it is necessary to denature the sequence. Denaturation can be carried out using various techniques known in the art. After denaturation, the nucleic acid analyte and probe are incubated under conditions that promote stable hybrid formation of the target sequence in the probe with the putative target sequence in the analyte, and the resulting probe-containing duplex is detected.

Detection of the resulting duplex (if present) is typically accomplished using a labeled probe; alternatively, the probe may be unlabeled, but may be detected directly or indirectly by specific binding to a labeled ligand. Suitable labels and methods of labeling probes and ligands are known in the art and include, for example, radiolabels (which may be incorporated by known methods such as nick translation or kinase), biotin, fluorophores, chemiluminescent moieties (such as dioxetanes, especially triggered dioxetanes), enzymes, antibodies and the like.

The probe region used to bind the analyte can be made completely complementary to the SARSV genome. Therefore, high stringency conditions are often required to prevent false positives. However, the use of high stringency conditions is only required when the probe is complementary to a region of the viral genome lacking heterogeneity. The stringency of hybridization is determined by a variety of factors during the hybridization process and washing process, including temperature, ionic strength, length of time, and formamide concentration. These factors are listed in ManiatisT (1982).

Variations of this basic method may also be used and are known in the art, including those that facilitate separation of the duplex to be detected from foreign material and/or amplification of the signal from the labeled moiety. For an understanding of these changes, reference may be made, for example: matthews and Kricka (1988), Analytical Biochemistry 169: 1; landegren et al, (1988), Science 242: 229; and Mittlin (1989), Clinical chem.35: 1819. these documents and the following publications describe assay formats and are incorporated herein by reference. Probes suitable for detection of sarvs in these assays include sequences that hybridize to a target sarvs polynucleotide sequence to form a duplex with an analyte strand, wherein the duplex is sufficiently stable in the detection system to be detected.

One suitable variation is described, for example, in U.S. Pat. No.4,868,105 (filed 9/1989) and EPO publication No.225,807 (published 16/6/1987). These publications describe a solution-phase nucleic acid hybridization assay in which a nucleic acid analyte is hybridized to a set of labeling probes and a set of capture probes. The probe-analyte complexes are coupled by hybridization to a solid-supported capture probe (complementary to the capture probe set). This allows the nucleic acid analyte to be removed from the solution as a solid phase complex. The analyte is in the form of a solid phase complex to facilitate the subsequent separation step in the assay. The set of labeled probes is complementary to the labeled probes bound by hybridization to the solid phase/analyte complex.

Polymerase Chain Reaction (PCR) is a technique for amplifying a desired nucleic acid sequence (target) contained in a nucleic acid or a mixture thereof. In PCR, a pair of excess primers is used to hybridize to the complementary strand of the target nucleic acid. The primers will be extended individually with the target nucleic acid as template by the polymerase. The extension product itself becomes the target sequence and then dissociates from the original target strand. The new primer is then hybridized and extended by the polymerase, and the cycle is repeated to geometrically increase the number of target molecules. PCR is disclosed in U.S. Pat. Nos.4,683,195 and 4,683,202, which are incorporated herein by reference.

Ligase Chain Reaction (LCR) is another method of nucleic acid amplification. In LCR, a probe pair is used that contains two primary (first and second) probes and two secondary (third and fourth) probes, all of which are used in molar excess of the target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous, such that the primary probes are contiguous with each other in a 5' phosphate-3 hydroxyl relationship, such that the two probes are covalently fused or ligated by a ligase to form a fusion product. In addition, a third (secondary) probe may hybridize to a portion of the first probe, and a fourth (secondary) probe may hybridize in similar juxtaposition to a portion of the second probe. Of course, if the target is initially double-stranded, the secondary probe will also hybridize to the complementary strand of the target in the first instance. Once the ligated primary probe strand is separated from the target strand, it will hybridize to the third and fourth probes, allowing them to ligate to form a complementary secondary ligation product. It is important to recognize that the ligation product is functionally equivalent to the target or its complement. Amplification of the target sequence can be achieved through multiple rounds of repeated hybridization and ligation cycles. This technique is described in more detail in EP-A-320308 (published 16.6.1989) by K.Backman et al, EP-A-0439182 (published 31.7.1991) which are incorporated herein by reference.

For the amplification of mRNA, it is within the scope of the invention to reverse transcribe mRNA into cDNA, which is then amplified by polymerase chain reaction (RT-PCR); or a single enzyme in both steps, as described in U.S. Pat. No.5,322,770, which is incorporated herein by reference; or reverse transcription of mRNA into cDNA followed by asymmetric gap ligase chain reaction (RT-AGLCR), as described by r.l. marshall et al [ PCR Methods and Applications 4: 80-84(1994), also incorporated herein by reference.

TMA is described in detail, for example, in U.S. Pat. No.5,399,491, the contents of which are incorporated herein by reference in their entirety. In one example of a routine assay, an isolated nucleic acid sample suspected of containing a SARSV target sequence is mixed with a buffer concentrate containing buffer, salt, lintel, nucleotide triphosphates, primers, dithiothreitol, and spermidine. The reaction is optionally incubated at about 100 ℃ for about 2 minutes to denature all secondary structures. After cooling to room temperature, reverse transcriptase, RNA polymerase and RNase H were added and the mixture was incubated at 37 ℃ for 2-4 hours. The reaction can then be determined by: the product was denatured, probe solution was added, incubated at 60 ℃ for 20 minutes, solution was added to selectively hydrolyze unhybridized probe, the reaction was incubated at 60 ℃ for 6 minutes and the remaining chemiluminescence was measured in a luminometer.

Typically, TMAs comprise the steps of: (a) isolating nucleic acids, including RNA, from a biological sample of interest suspected of being infected with SARSV; and (b) mixing to form a reaction mixture: (i) an isolated nucleic acid, (ii) first and second oligonucleotide primers, the first primer having a complex sequence sufficiently complementary to the 3 ' terminal portion of the RNA target sequence complexed therewith (e.g., the (+) strand, if present), and the second primer having a complex sequence sufficiently complementary to the 3 ' terminal portion of the target sequence of the complement complexed therewith (e.g., the (-) strand), wherein the first oligonucleotide further comprises a sequence from 5 ' to the complex sequence comprising a promoter, (iii) a reverse transcriptase or an RNA and DNA-dependent DNA polymerase, (iv) an enzymatic activity that selectively degrades the RNA strand of an RNA-DNA complex (e.g., rnase H), and (v) an RNA polymerase that recognizes the promoter.

The components of the reaction mixture may be mixed stepwise or in one portion. The reaction mixture is incubated under conditions to form oligonucleotide/target sequences, including conditions for priming of DNA and synthesis of nucleic acids, including ribonucleoside triphosphates and deoxyribonucleoside triphosphates, for a time sufficient to provide multiple copies of the target sequence. The reaction is preferably carried out under conditions suitable to maintain the stability of reaction components such as enzyme components, and the reaction conditions need not be altered or controlled during the amplification reaction. Thus, the reaction may take place under substantially isothermal conditions with substantially constant ionic strength and pH. This reaction generally does not require a denaturation step to isolate the RNA-DNA complex produced by the first DNA extension reaction.

Suitable DNA polymerases include reverse transcriptases, such as Avian Myeloblastosis Virus (AMV) reverse transcriptase (obtained, for example, from Seikaaku America, Inc.) and Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (obtained, for example, from Bethesda Research Laboratories).

Promoters or promoter sequences suitable for incorporation into primers are nucleic acid sequences (which may be naturally occurring, synthetically produced, or products of restriction digestion) that are specifically recognized by RNA polymerases, which recognize and bind to the sequences and initiate the transcription process, thereby producing RNA transcripts. The sequence may optionally contain nucleotide bases that extend beyond the actual recognition site of the RNA polymerase, which may increase stability or sensitivity to degradation processes or increase transcription efficiency. Examples of useful promoters include those recognized by certain phage polymerases, such as the phage T3, T7, or SP6 promoters or E.coli promoters. These RNA polymerases are available from commercial sources, such as New England Biolabs and Epicentre.

Some reverse transcriptases suitable for use in the methods described herein have rnase H activity, such as AMV reverse transcriptase. However, it is preferred to add exogenous RNase H, such as E.coli RNase H, even when AMV reverse transcriptase is used. RNase H can be purchased from, for example, Bethesda Research Laboratories.

RNA transcripts made by these methods can be used as templates to make additional copies of the target sequence by the mechanisms described above. The system is autocatalytic and amplification occurs through autocatalysis without the need for repeated modification or changes in reaction conditions such as temperature, pH, ionic strength, and the like.

Detection can be accomplished by a variety of methods including direct sequencing, hybridization to sequence-specific oligomers, gel electrophoresis, and mass spectrometry. These methods may employ heterogeneous or homogeneous modes, isotopic or non-isotopic labeling, or no labeling at all.

Suitable labels for binding to the primers and/or probes used in the methods of the invention include, but are not limited to: 5-FAM (also known as 5-carboxyfluorescein; also known as spiro (isobenzofuran-1 (3H), 9 ' - (9H) xanthene) -5-carboxylic acid, 3 ', 6 ' -dihydroxy-3-oxo-6-carboxyfluorescein); 5-hexachloro-fluorescein ([4, 7, 2 ', 4', 5 ', 7' -hexachloro- (3 ', 6' -bispecific valeryl fluorescein) -6-carboxylic acid]) (ii) a 6-hexachloro-fluorescein ([4, 7, 2 ', 4', 5 ', 7' -hexachloro- (3 ', 6' -bispecific valeryl fluorescein) -5-carboxylic acid]) (ii) a 5-tetrachloro-fluorescein ([4, 7, 2 ', 7' -tetrachloro- (3 ', 6' -bispentanoyl fluorescein) -5-carboxylic acid ]) (ii) a 6-tetrachloro-fluorescein ([4, 7, 2 ', 7' -tetrachloro- (3 ', 6' -bispentanoyl fluorescein) -6-carboxylic acid]) (ii) a Tetramethylrhodamine (TAMRA), including (i)5-TAMRA (5-carboxytetramethylrhodamine; xanthylium, 9- (2, 4-dicarboxyphenyl) -3, 6-bis (dimethylamino) and (ii)6-TAMRA (6-carboxytetramethylrhodamine; xanthylium, 9- (2, 5-dicarboxyphenyl) -3, 6-bis (dimethylamino); EDANS (5- ((2-aminoethyl) amino) naphthalene-1-sulfonic acid); 1, 5-IAEDANS (5- ((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid); DABCYL (4- ((4- (dimethylamino) phenyl) azo) benzoic acid); cy5 (iododicarbocyanine-5); cy3 (iododicarbocyanine-3); and BODIPY.^TMFL (4, 4-difluoro-5, 7-dimethyl-4-boro-3 a, 4 a-diaza-s-indacene-3-propanoic acid). Preferably, the probe is labeled with both FAM (e.g., labeled at 5 ') and TAMRA (e.g., labeled at 3').

The nucleic acids of the invention may be used in solution or bound to a solid matrix or support, for example in the form of a DNA array.

It is apparent that many variations and many modes of design of the assay methods described herein are possible, as is known in the art. The above description is intended as a guide and one skilled in the art can readily modify the process using techniques well known in the art.

A302 nt amplicon of SARS virus is called "BNI-1" (SEQ ID NO: 9927). It has been sequenced in the Bernhard Nocht Institute in Hamburg, Germany. In 4 months 2003, BNI-1 sequence was published and published in WHO website (http:// www.who.int/csr/sars/documents/en /) and Dorsten et al, "identify a novel coronavirus in Patients with Severe Acute Respiratory Syndrome" (Identification of a novel Coronavir in Patients with Severe Acuter Respiratory Syndrome), New England journal of Medicine, published on-line in http: // www.nejm.org. Both of these reference treatments are incorporated herein by reference in their entirety. Some embodiments of the invention do not include a polypeptide comprising SEQ ID NO: 9927. Some other embodiments of the invention do not include a polypeptide comprising SEQ ID NO: 9927. Some embodiments of the invention do not include a polypeptide comprising SEQ ID NO^s: 9928 and 9959. Some other embodiments of the invention do not include a polypeptide comprising SEQ ID NO^s: 9928 and 9959. Some embodiments of the invention do not include these exclusionary parts.

Immunoassay method

The present invention uses various immunoassay techniques to identify individuals exposed to SARSV, and/or biological samples containing SARSV or antibodies to SARSV.

Immunoassay mode

In fact, the SARSV antigen can be used in any assay format that employs known antigens to detect antibodies. A common feature of all of these assays is that the antigen is contacted with a biological sample suspected of containing antibodies to SARSV under conditions that allow for the presence of any such antibodies in the antigen binding composition. Such conditions are typically physiological temperature, pH and ionic strength, and an excess of antigen is used. The antigen is incubated with the sample and the immune complex composed of the antigen is then detected. Alternatively, anti-SARSV antibodies can be used to detect SARSV antigens present in a biological sample. Antigen/antibody detection may also be combined; for example, HCV detection as described in us patent 6,630,298.

The design of immunoassay formats varies widely and there are many formats, as is known in the art. For example, the protocol may use a solid support or immunoassay. Many assays use labeled antibodies or polypeptides; the label may be, for example, an enzyme label, a fluorescent label, a chemiluminescent label, a radioactive label, or a dye molecule. Assays for amplifying immune complex signals are also known, for example assays using biotin and avidin as well as enzyme-labeled and mediated immunoassays, such as ELISA assays.

Immunoassays can be, but are not limited to, heterogeneous or homogeneous formats, and can be of the standard type or competitive type. In the heterogeneous mode, the polypeptides are typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptides after incubation. Examples of solid supports that can be used include nitrocellulose (e.g., immobilized on a membrane or in a microtiter well format), polyvinyl chloride (e.g., in a sheet or microtiter well), polystyrene latex (e.g., in a microbead or microtiter plate), polyvinylidene fluoride (polyvinylidene fluoride), diazotized paper, nylon membranes, microchips, high or low density biochips, Recombinant Immunoassays (RIBA), microfluidization devices, microbeads, activated beads, and protein A beads. For example, Dynatech Immunlon or Immunlon 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous mode. Separation of the solid support containing the antigenic polypeptide from the test sample typically requires washing before detection of the bound antibody. Both standard and competitive modes are known in the art.

In the homogeneous mode, the test sample is incubated with the antigen mixture in solution. For example, incubation may be conducted under conditions that will precipitate any antigen-antibody complexes formed. Standard and competitive formats for these assays are known in the art.

In the labeling mode, the amount of SARSV antibody in the antibody-antigen complex will be monitored directly. This can be accomplished by determining whether a labeled anti-allogeneic (e.g., anti-human) antibody recognizing an epitope of the anti-SARSV antibody binds as a result of complex formation. In the competitive mode, the amount of SARSV antibody in the sample can be deduced by monitoring the competitive effect of a known amount of labeled antibody (or other competing ligand) in the complex.

The formation of complexes containing anti-SARSV antibodies (in a competitive assay, the amount of competing antibody is detected) can be detected by any of a number of known techniques, depending on the mode employed. For example, conjugates of anti-xenogenic Ig complexed with a label (e.g., an enzyme label) can be used to detect unlabeled SARSV antibodies in the complex.

In immunoprecipitation or agglutination assay formats, SARSV antigens react with antibodies to form a network that precipitates from solution or suspension, forming a visible precipitate layer or film. If no anti-SARSV antibody is present in the test sample, no visible precipitate will form.

Particle Agglutination (PA) assays are of at least three specific types. These assays are used to detect antibodies to various antigens coated on a support. One type of such an assay is the hemagglutination assay, which uses Red Blood Cells (RBCs) sensitized by passive adsorption of antigen (or antibody) to the RBCs. RBCs coated with purified antigen aggregate if antibodies to other specific antigens are present in the body composition.

To eliminate the non-specific reaction that may be present in the hemagglutination assay, RBCs can be replaced in PA with two artificial vectors. Most commonly referred to as latex particles. However, gelatin granules may also be used. Assays using any of these particles can be based on passive agglutination of particles coated with purified antigen.

SARSV antigens are typically packaged in the form of kits for use in these immunoassays. These kits typically contain the native SARSV antigen, a control antibody preparation (positive and/or negative), a labeled antibody as required for the assay format, and a signal generator (e.g., an enzyme substrate) when the label does not directly generate a signal, in separate containers. The native SARSV antigen may have been bound to a solid matrix or isolated with a matrix-binding agent. The kit will also typically include instructions for performing the assay (e.g., paper, tape, CD-ROM, etc.).

Immunoassays using native SARSV antigens have also been used to screen blood to prepare blood supplies that are free of potentially infectious SARSV. The method of preparing a blood supply comprises the following steps. The body composition of the donor, preferably blood or a blood component, is contacted with the native SARSV antigen to generate an immune response between the SARSV antibody (if present) and the SARSV antigen. Detecting whether the reaction forms an anti-SARSV antibody-SARSV antigen complex. Donor blood that does not display antibodies to the native SARSV antigen forms the blood supply.

Antibody preparation

As described above, the assay may use various antibodies bound to a solid support to detect antigen or antigen/antibody complexes formed when SARSV infection is present in a sample. These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be hybrid or chimeric antibodies, such as humanized antibodies, variant antibodies, F (ab')₂Fragments, F (ab) fragments, Fv fragments, single domain antibodies, dimeric or trimeric antibody fragment constructs, microbodies, or functional fragments thereof that bind to the antigen to be detected.

Antibodies are made using techniques well known to those skilled in the art, for example, U.S. patent nos.4,011,308; 4,722,890, respectively; 4,016,043; 3,876,504, respectively; 3,770,380, respectively; and 4,372,745. For example, polyclonal antibodies are generated by immunizing a suitable animal (e.g., mouse, rat, rabbit, sheep, or goat) with an antigen of interest. To enhance immunogenicity, the antigen may be bound to a carrier prior to immunization. Such vectors are well known to those of ordinary skill in the art. Immunization is typically carried out by: the antigen is mixed or emulsified with saline, preferably with an adjuvant such as Freund's complete adjuvant, and the mixture or emulsion is injected parenterally, usually subcutaneously or intramuscularly. The immunization is boosted 2-6 weeks later by one or more injections with protein in saline, preferably Freund's incomplete adjuvant. Antibodies can additionally be generated by in vitro immunization using methods known in the art. Polyclonal antisera are then obtained from the immunized animals.

As described above, monoclonal antibodies can generally be generated by the method of Kohler and Milstein [ (1975) Nature256：495-497]Or an improved method thereof.

As described above, antibody fragments capable of recognizing SARS antigen are also included in the scope of the present invention. Many antibody fragments containing an antigen binding site capable of exhibiting the immunological binding characteristics of an intact antibody molecule are known in the art. For example, F (ab') can be generated by cleaving the constant region of an antibody molecule not responsible for antigen binding with, for example, pepsin₂Fragments to make functional antibody fragments. These fragments will contain two antigen binding sites but lack part of the constant region of each heavy chain. Similarly, if desired, Fab fragments containing a single antigen binding site can be made, for example, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments comprising only the variable regions of the heavy and light chains can also be made using standard techniques, such as recombinant methods or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are designated F_v. See, e.g., Inbar et al, (1972) Proc. Nat. Acad. Sci USA69: 2659-2662; hochman et al, (1976) Biochem15: 2706-2710; and Ehrlich et al, (1980) Biochem 19：4091-4096。

A single chain Fv ("sFv" or scFv ") polypeptide is a covalently linked V_H-V_LHeterodimers consisting of V linked by peptide-encoding linkers_H-and V_LExpressed as a fusion gene encoding a gene. Huston et al, (1988) Proc. nat. Acad. Sci. USA85: 5879-5883. A number of methods have been described to resolve and establish chemical structures (linkers) that are used to convert the light and heavy polypeptide chains of the antibody V regions that naturally aggregate but can be separated by chemical means into sFv molecules that fold into three-dimensional structures substantially similar to those of the antigen binding site. See, for example, U.S. patent nos.5,091,513; 5,132, 405; and 4,946,778. sFv molecules can be made using methods already described in the art. Referring to the description of the preferred embodiment,for example, Huston et al, (1988) Proc. Nat. Acad. Sci USA85: 5879-5338; U.S. patent nos.5,091,513; 5,132,405 and 4,946,778. Design criteria include determining the approximate length of the distance between the C-terminus of one strand and the N-terminus of the other strand, where the linker is typically formed of small hydrophilic amino acid residues that do not coil or form secondary structures. Such methods have been described in the art. See, for example, U.S. patent nos.5,091,513; 5,132,405 and 4,946,778. Suitable linkers typically comprise polypeptide chains of alternating glycine and serine, and may include intervening glutamic acid and lysine residues to enhance solubility.

"miniantibodies" or "microbodies" may also be used in the present invention. Microbodies are sFv polypeptide chains that contain an oligomerization region at their C-terminus separated from the sFv by a hinge region. Pack et al (1992) Biochem31: 1579-1584. The oligomerization region comprises a self-associating alpha-helix, such as a leucine zipper, which may be stabilized by other disulfide bonds. The oligomerization region is designed to match the directional folding across the membrane, a process that is thought to favor in vivo folding of the polypeptide into a functional binding protein. Typically, microbodies are prepared using recombinant methods well known in the art. See, e.g., Pack et al (1992) Biochem31: 1579-1584; cumber et al (1992) J.immunology149B：120-126。

Production of SARS antigen

The SARSV antigens for use in the present invention are typically produced by recombinant methods. Accordingly, polynucleotides encoding SARSV antigens for use in the present invention can be made using standard molecular biology techniques. For example, polynucleotide sequences encoding the above-described molecules can be obtained recombinantly, e.g., by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from vectors known to contain the gene. In addition, the desired gene can be isolated directly from the viral nucleic acid molecule using techniques described in the art, for example, the technique described for HCV in U.S. Pat. No.5,350,671 to Houghton et al. In addition to cloning, genes encoding antigens of interest can also be made synthetically. The molecule can be designed with the appropriate codons for the particular sequence (optimal codons for preferred expression in the chosen host). The complete sequence is usually prepared by standard methods of overlapping oligonucleotides assembly, and is assembled into the complete coding sequence. See, e.g., Edge, Nature (1981) 292: 756; nambair et al, Science (1984) 223: 1299; jay et al, J.biol.chem. (1984) 259: 6311.

Thus, a particular nucleotide sequence may be obtained from a vector containing the desired sequence or sequences synthesized in whole or in part by a variety of oligonucleotide synthesis techniques known in the art, with site-directed mutagenesis and Polymerase Chain Reaction (PCR) techniques being more appropriate. See, e.g., Sambrook, supra. Specifically, one method of obtaining a nucleotide sequence encoding a desired sequence is to anneal a complement set of synthetic overlapping oligonucleotides made in a conventional automated polynucleotide synthesizer, then ligate with a suitable DNA ligase and amplify the ligated nucleotide sequence by PCR. See, e.g., Jayaraman et al, (1991) proc.natl.acad.sci.usa 88: 4084-4088. Furthermore, oligonucleotide-directed synthesis (Jones et al, (1986) Nature 54: 75-82), oligonucleotide-directed mutagenesis of existing oligonucleotide regions (Riechmann et al, (1988) Nature 332: 323-327, and Verhoeyen et al, (1988) Science 239: 1534-1536), and enzymatic filling of gapped oligonucleotides with T4DNA polymerase (Queen et al, (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033) can also be employed in the present invention to provide molecules with altered or enhanced antigen binding capacity and/or reduced immunogenicity.

Once a coding sequence is prepared or isolated, the sequence can be cloned into any suitable vector or replicon. Many cloning vectors are known to those skilled in the art and selection of an appropriate cloning vector is a matter of choice. Suitable vectors include, but are not limited to, plasmids, phages, transposons, cosmids, chromosomes (including artificial chromosomes such as BAC or YAC), or viruses that are capable of replication when combined with appropriate control elements.

The coding sequence is then placed under the control of appropriate control elements, depending on the system used for expression. Thus, the coding sequence may be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operon, so that the DNA sequence of interest is transcribed into RNA by a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence, which may then be removed by post-translational processing by the host. See, for example, U.S. patent nos.4,431,739; 4,425,437, respectively; 4,338,397.

In addition to control sequences, regulatory sequences may be added to control expression of the sequences according to the growth of the host cell, and are known to those skilled in the art, examples of which include those sequences which switch gene expression on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector. For example, enhancer elements may be used herein to increase the expression level of the construct. Examples include the SV40 early gene enhancer (Dijkema et al, (1985) EMBO J.4: 761), the enhancer/promoter from Rous sarcoma virus Long Terminal Repeat (LTR) (Gorman et al, (1982) Proc. Natl. Acad. Sci. USA 79: 6777) and elements from human CMV (Boshart et al, (1985) Cell 41: 521), such as those contained in the CMV intron A sequence (U.S. Pat. No.5,688,688). One or more selectable markers for a replication initiation sequence that replicates spontaneously in a suitable host cell, one or more restriction sites, high copy number potential and a strong promoter may also be included in the expression cassette.

An expression vector is constructed such that a particular coding sequence is located in the vector with the appropriate control sequences, relative to the control sequences, in a position and orientation such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase transcribes the coding sequence in conjunction with the control sequences of the DNA molecule). Modifications to the sequence encoding the molecule of interest may be required to achieve the end purpose. For example, in some cases the sequence must be modified to allow it to bind the control sequence in the proper orientation; i.e. in order to maintain the reading frame. Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence may be cloned directly into an expression vector which already contains the control sequences and appropriate restriction sites.

As mentioned above, it may be desirable to make mutants or analogues of the antigen of interest. The methods are described, for example, in Dasmahapatra et al, U.S. Pat. No.5,843,752 and Zhang et al, U.S. Pat. No.5,990,276. SARSV mutants or analogs for use in such assays can be made by deleting one or more nucleotides within a partial sequence, or an insertion, and/or substitution sequence encoding the polypeptide of interest. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis and the like, are well known to those skilled in the art. See, e.g., Sambrook et al, supra; kunkel, T.A, (1985) proc.natl.acad.sci.usa 82: 448; geisselsoder et al, (1987) BioTechniques 5: 786; zoller and Smith (1983) Methods enzymol.100: 468; Dalbie-McFarland et al, (1982) Proc. Natl. Acad. Sci USA 79: 6409.

Molecules can be expressed in a number of systems, including insect, mammalian, bacterial, viral, and yeast expression systems, which are well known in the art.

For example, insect cell expression systems, such as the baculovirus system, are known to those skilled in the art and are described, for example, in Summers and Smith, the Current agricultural test Bulletin of Texas (Texas agricultural Experiment Bulletin) No.1555 (1987). Materials and methods for baculovirus/insect cell expression systems are available as kits from Invitrogen, San DiegoCalif ("MaxBac" kit). Similarly, bacterial and mammalian cell expression systems are well known in the art and are described, for example, in Sambrook et al, supra. Yeast expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al, 1989) butterworks, London.

Many suitable host cells for use in the above system are also known. For example, mammalian cell lines are known in the art and include immortalized cell lines obtained from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human embryonic kidney cells, human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, and others. Similarly, bacterial hosts such as E.coli, Bacillus subtilis, and Streptococcus may also be used in the expression constructs of the invention. Yeast hosts useful in the present invention include Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), Candida maltosa (Candida maltosa), Hansenula polymorpha (Hansenula polymorpha), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia guilliermondii, Pichia pastoris (Pichia pastoris), Schizosaccharomyces pombe (Schizosaccharomyces pombe), and Yarrowia lipolytica (Yarrowia polylithica). Insect cells for baculovirus expression vectors include Aedes aegypti (Aedes aegypti), Autographa californica (Autographa californica), Bombyx mori (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), Spodoptera frugiperda (Sodoptera frugiperda), and Ectropis calis (Trichoplusia ni).

Nucleic acid molecules containing a nucleotide sequence of interest can be stably integrated into the host cell genome or maintained as stable episomal elements in a suitable host cell using a variety of gene delivery techniques well known in the art. See, for example, U.S. Pat. No.5,399,346.

Depending on the expression system and host chosen, the molecule can be made by growing the host cell transformed with the expression vector described above under conditions in which the protein is expressed. The expressed protein is then isolated from the host cell and purified. If the expression system secretes the protein into the growth medium, the product can be purified directly from the medium. If non-secreted, the product may be isolated from the cell lysate. The selection of suitable growth conditions and recovery methods is known to those skilled in the art.

Examples

To efficiently express the SARSV antigen in Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris), insect cells, and mammalian cells, the domains listed in the following table were cloned into expression vectors. The number of nt sequences is from SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.

-RNA polymerase 1 a: SARS nt 250-13398

-RNA polymerase 1 b: SARS nt 13399-21470

Envelope (homologous to ns2, hemagglutinin-esterase envelope glycoprotein and spike glycoprotein): SARS nt21477-25244

-a membrane: SARS nt 27849-

-a nucleocapsid: SARS nt 28105-29373

The combination of PCR and synthetic oligo was used to generate the domains described above, linked via restriction sites into the following expression vectors:

two-end vector promoter expression host of restriction site

HindIII/SaiI pBS24.1 ADH2/GAPDH AD 3/Saccharomyces cerevisiae

EcoRI/Sal | \ pBS24.1 ADH2/GAPDH/SOD fusion AD 3/Saccharomyces cerevisiae

XbaI/SalI pAO815 AOXI GS 115/Pichia pastoris

EcoRI/BamHI pCMVkm2 CMVp/enhancer/intron A HVK-293/transient transfection

EcoRI/XmaI pCMVIII CMVp/enhancer/intron A CHO Stable cell line

The cell line used by Chiron was selected,

NheI/SalI pBluBac4.5 polyhedrin proteins include: sf9, Sf21, Tn5

Treatment of SARS infection with RNAi

RNA interference or "RNAi" is a term originally created by Fire and co-workers and used to describe the phenomenon of blocking gene expression when double-stranded RNA (dsRNA) is introduced into worms (Fire et al, Nature 391, 806-811 (1998)). RNAi is most likely involved in the degradation of mRNA, leading to sequence-specific post-transcriptional gene silencing in multiple organisms. RNAi is a post-transcriptional process triggered by the introduction of double-stranded RNA that results in gene silencing in a sequence-specific manner. RNAi has been reported to occur naturally in a variety of species including nematodes, trypanosomes, plants and fungi. It is likely to protect organisms from viral infection, regulate transposon activity and eliminate aberrant transcripts.

The first evidence that dsRNA could achieve efficient gene silencing via RNAi came from the study of C.elegans (Caenorhabditis elegans) (Fire et al (1998) Nature, 391: 806-. Subsequent studies in Drosophila melanogaster (Drosophila melanogaster) demonstrated that RNAi is a two-step mechanism (Elbashir et al, (2001) Genes Dev., 15 (2): 188-. First, a long dsRNA is cleaved into a 21-23 nucleotide (nt) fragment called small interfering RNA (siRNA) by an enzyme called dicer. The siRNA then binds to the ribonuclease complex (called RISC, an RNA-induced silencing complex) targeting the complex to a complementary mRNA. RISC then cleaves complementary siRNA opposite the target mRNA, making the mRNA susceptible to other RNA degradation pathways.

RNAi is a phenomenon in which dsRNA corresponding to a target DNA or RNA sequence inhibits or silences gene expression. Even though dsRNA can mediate gene-specific interference in mammalian cells under certain circumstances (Wianny and Zernicka-Goetz (2000) Nature Cell biol.2: 70-75; Svoboda et al (2000) Development 17: 4147-4156), the use of RNAi in mammalian cells is generally limited because dsRNA triggers ds RNA-dependent Protein Kinase (PKR) and thus inactivates the translation factor eIF2a resulting in global inhibition of protein synthesis and often Apoptosis (Gil and Esteban (2000) Apoptosis 5: 107-114).

Gene-specific inhibition in this year using siRNAs of about 21 or 22 base pairs in length corresponding to the target RNA or DNA sequences has been shown to interrupt expression of these target sequences in mammalian cells (Elbashir, S.M., et al, Nature 411: 494-498 (2001)). However, it is not clear whether all RNA or DNA sequences of the genome of a mammalian cell are sensitive to siRNA. It was also not determined whether each mammalian cell type has the necessary mechanism for causing gene-specific inhibition with siRNA. In addition, there are at least two reasons for limiting the use of siRNA: this transient nature of the inhibitory effect is observed in cells administered with siRNA; and in some cases it may be necessary to chemically synthesize the siRNA prior to use (Tuschl T., Nature Biotechnol., 20: 446- & 448 (2002)). Also, the instability of these short synthetic RNAs makes the use of these sirnas as long-term drugs problematic.

To overcome these limitations, the present invention provides a modified siRNA that has improved stability against nuclease degradation while still retaining its ability to inhibit viral replication through RNA interference. This modification of siRNA ribonucleotides is by the addition of a chemical group either by chemical synthesis or in vitro transcription, or one of these methods can be used to prepare longer modified RNAs and cleave them into siRNAs with dicers.

While other gene-specific inhibition methods use chemically modified nucleic acids, such as antisense and ribozyme techniques, such modifications disrupt the critical enzymatic activity necessary to allow these techniques to function. In the case of antisense technology, modifications to ribonucleotides disrupt rnase H activity, and such modifications would abolish the catalytic activity of ribozymes.

The present invention provides a modified double-stranded RNA (dsRNA) molecule that is resistant to nuclease degradation, has a length of about 10-30 nucleotides, and is capable of inactivating a virus in a mammalian cell. The invention also provides a method of inactivating viruses by administering modified small interfering RNAs (siRNAs) that have been modified to make them ribozyme or RNase resistant and retain biological activity that inhibits viral replication by targeting viral RNA sequences.

The invention also relates to a method of making a modified siRNA targeted to a viral RNA sequence, the method comprising preparing a modified double stranded RNA (dsrna) containing at least one modified ribonucleotide in at least one strand spanning the viral genome; and cutting the modified dsRNA segment by using recombinant human cutter to obtain more than one modified siRNA.

The present invention provides modified dsRNA molecules containing about 10-30 nucleotides that mediate target RNA interference in hepatocytes or SRAS infected cells.

RNA interference or RNAi refers herein to the expression of a sequence-specific, or gene-specific, heterogeneous gene (protein synthesis) without causing global inhibition of protein synthesis in cells containing the siRNA. The present invention is not limited by a particular theory of the mechanism of action of RNAi. For example, RNAi may be involved in the degradation of messenger RNA (mRNA) in RNA-induced silencing complex (RISC), preventing transcribed mRNA translation, or it may be involved in methylation of genomic DNA, altering gene transcription. The lack of gene expression by RNAi may be transient, lasting for a short time, or may be stable, permanent, lasting for an indefinite period of time.

The term RNA has art-recognized meanings. In addition, RNA is also used herein to denote double-stranded RNA (dsRNA) or single-stranded RNA (ssrna) or dsRNA containing single-stranded overhangs. dsRNA is used in the present invention to denote short interfering RNA (siRNA), microRNA (miRNA) and small hairpin RNA (shRNA), and RNA is also used to denote messenger RNA (mRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA).

The present invention relates to small interfering RNA (siRNA) that have been chemically modified to provide enhanced stability against nuclease degradation, but which are still capable of binding target RNA that may be present in a cell. When the target RNA is virus-specific RNA, the modified siRNA is capable of binding to the virus-specific RNA and inactivating the virus. The modified siRNA of the present invention contains modified ribonucleotides wherein the siRNA is resistant to enzymatic degradation, such as rnase degradation, and still retains the ability to inhibit viral replication. More specifically, the modified siRNA is modified at the ribose 2' position of the siRNA. The modification occurs at the 2' position of at least one ribonucleotide of the siRNA. The binding of the receptor binding ligand to the siRNA molecule can be used to target the siRNA to a desired cell type. For example, cholesterol-based siRNA, which results from the binding of cholesterol to the 5 '-end or 3' -end of the siRNA molecule, enhances targeting of the siRNA to hepatocytes. Ligands for receptor-mediated siRNA targeting the liver include HBV surface antigen, LDL, etc.

More specifically, the siRNA is modified on at least one pyrimidine, at least one purine, or a combination thereof. However, generally siRNA is modified for all pyrimidines or all purines or a combination of all pyrimidines and all purines. More preferably, the pyrimidines are modified, the pyrimidines being cytosines, cytosine derivatives, uracils, uracil derivatives, or combinations thereof. It is also possible to modify selected ribonucleotides in at least one strand of the siRNA, or the ribonucleotides in both strands of the siRNA can be modified.

Nucleotides containing the pyrimidine bases of RNA (cytosine and uracil) can be chemically modified by adding a molecule at the 2' position of the ribose molecule that inhibits degradation or decomposition of the RNA. 2' -modified pyrimidine nucleotides can be formed in a variety of different ways. This 2' modification increases the stability of the siRNA by rendering the siRNA unaffected by or resistant to nuclease activity. Thus, the 2' modified siRNA has a longer serum half-life and is resistant to degradation compared to unmodified siRNA. The siRNA may also be fully or partially modified.

When the siRNA is chemically modified, it is preferable to add a halide chemical group to the ribonucleotide of the siRNA. Among halides, fluorine is the preferred molecule, but other chemical molecules than fluorine-such as methyl-, methoxyethyl-, and propyl-modifications can also be used. Preferred modifications are however fluoro-modifications, such as 2 ' -fluoro-modifications or 2 ', 2 ' -fluoro-modifications. Thus, in a preferred embodiment of the invention, the siRNA is modified by the addition of a fluorine molecule to the 2' carbon of the pyrimidine ribonucleotide. The siRNA may be fully or partially fluorinated. For example, only cytosine nucleotides need to be fluorinated. Alternatively, only uracil nucleotides need be fluorinated, but both uracil and brittle pyrimidines may be fluorinated. In addition, only one strand (sense or antisense strand) of the siRNA can be fluorinated. Even 2' fluorination of the siRNA moiety is resistant to nucleic acid degradation. Furthermore, it is noted that 2' fluorinated sirnas are not toxic to cells, an unexpected result given that fluorine chemistries are generally toxic to living organisms.

The siRNA of the present invention is designed to interact with a target nucleotide sequence. Most preferably, such target nucleotide sequence is a sequence of a pathogenic agent or pathogen whose gene expression is desired to be inhibited. More preferably, the target nucleotide sequence is a sequence in a viral genome, further such viral genome is from an RNA virus or a DNA virus selected from the group consisting of: hepatitis C Virus (HCV), hepatitis a virus, hepatitis b virus, hepatitis d virus, hepatitis e virus, ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, Human Papilloma Virus (HPV), hyperpneumovirus and coronavirus. The most preferred virus is the SARS virus.

Modified sirnas can be prepared by a number of methods, such as by chemical synthesis, transcription with T7 polymerase, or modification with dicer of modified long double-stranded rna (dsrna) prepared by either of the two methods described above. Dicers can be used to cleave dsRNA containing approximately 500-1000 base pairs, resulting in a mixed population of dsRNA of approximately 21-23 base pairs in length. Furthermore, an unexpected result of using the dicer approach is that the dicer will cleave modified dsRNA strands, such as 2' fluorinated modified dsRNA. Prior to the establishment of this method, dicer was thought to be unable to cleave modified siRNA. The enzymatic cleavage method can be performed using Dicer siRNA Generation Kit available from Gene Therapy Systems (San Diego, Calif.).

Small interfering RNA (siRNA) is defined herein as double-or single-stranded RNA of about 10 to 30 nucleotides in length, more preferably 12 to 28 nucleotides in length, more preferably 15 to 25 nucleotides in length, even more preferably 19 to 23 nucleotides in length, and most preferably 21 to 23 nucleotides in length. The length of the siRNA used herein is determined by the length of one strand of the RNA. For example, an siRNA of 21 nucleotides in length (21-mer) may contain two opposing RNA strands that anneal together to give 19 contiguous base pairs. The two nucleotides remaining at one end of the molecule do not anneal with the opposite strand, thereby forming an "overhang". The overhang may be at the 5 'or 3' end of the dsRNA. Preferably the overhang is at the 3' end of the RNA. Double-stranded RNA with two opposite strands of different lengths will be represented by the longer of the two strands. For example, a dsRNA formed from one strand 21 nucleotides in length annealed to an opposite strand 20 nucleotides in length is considered herein to be a 21-mer (21-mer).

Preferably, the siRNA of the present invention will contain a 3' overhang of about 2-4 bases. More preferably, the 3' overhang is 2 nucleotides in length. More preferably, the 2 nucleotides contained in the 3' overhang are uracils (U).

In one embodiment, the RNA molecule provided herein comprises a nucleotide sequence that is at least 80% identical to the nucleotide sequence of a target pathogenic agent or virus. Preferably, the RNA molecule of the invention is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of the target agent or virus.

In practice, a known computer program, such as the Bestfit program (Wisconsin sequence analysis software package, Unix version 8, genetics computer group, University Research Park, 575Science Drive, Madison, wis.53711), is used to determine whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of the target agent or virus. Bestfit found the best segment of homology between the two sequences using the local homology algorithm of Smith and Waterman (Advances in Applied Mathemitics 2: 482- & 489 (1981)). When using Bestfit or any other sequence comparison program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the invention, parameters need to be set, of course, the percentage of identity is calculated over the full length of the reference nucleotide sequence, in which case gaps of homology of up to 5% of the total number of nucleotides in the reference sequence are permissible.

The present invention provides a method of inactivating a target pathogenic agent, preferably a virus, in a patient, the method comprising administering to the patient an amount of modified siRNA effective to inactivate the target pathogenic agent or virus. RNA interference of a target DNA segment in a cell can be achieved by administering to the cell a dsRNA molecule or siRNA, wherein the nucleotide sequence of the dsRNA molecule corresponds to the nucleotide sequence of the target DNA segment. Preferably, the RNA molecule used to induce the target RNAi is siRNA.

Gene suppression, target suppression, sequence specific suppression, target RNAi, or sequence specific RNAi are used interchangeably herein. Furthermore, sequence-specific inhibition is determined herein by measuring the level of inhibited protein in cells containing siRNA (test cells) and cells not containing the same siRNA (control cells), respectively, and comparing the two measurements. Furthermore, the test cells and the control cells must be from the same source and the same animal. For example, the control cells and test cells may be, but are not limited to, human hepatocytes as well as cell cultures in vitro, or they may be derived from hepatocellular carcinoma. In addition, the control cells and test cells used to determine the level or amount of gene inhibition must be determined under similar (if not identical) conditions.

The phrase "target DNA segment" is used herein to refer to a DNA sequence, including introns or exons, encoding all or part of an mRNA of a target protein for which suppression is desired. A DNA segment may also refer to a DNA sequence that normally regulates the expression of a target protein, including but not limited to a promoter for the target protein. In addition, the DNA segment may or may not be part of the genome of the cell, or may be extrachromosomal DNA, such as plasmid DNA.

The invention also relates to a method of inactivating a virus in a patient comprising administering to the patient an effective amount of a modified siRNA to inactivate the virus. The siRNA is preferably about 10-30 nucleotides in length, more preferably 12-28 nucleotides in length, more preferably 15-25 nucleotides in length, even more preferably 19-23 nucleotides in length, and most preferably 21-23 nucleotides in length. The method preferably uses a 2 'modified siRNA modified at the 2' position of at least one ribonucleotide of said siRNA. The method uses siRNA modified with a chemical group selected from fluoro-, methyl-, methoxyethyl-, and propyl-modifications. Fluoro-modifications are preferred, 2 ' -fluoro-modifications or 2 ', 2 ' -fluoro-modifications are also useful herein and are also preferred.

The modification may be a modification on an siRNA pyrimidine, purine or combination thereof. More preferably, the pyrimidine is modified, such as cytosine, a cytosine derivative, uracil, a uracil derivative, or a combination thereof. In one embodiment, at least one strand of the siRNA contains at least one modified nucleotide, and in another embodiment, both strands of the siRNA contain at least one modified nucleotide.

The pathogenic agent or pathogen, more particularly a virus, for which the method is used may be an RNA virus or a DNA virus, which may be selected from the group consisting of: hepatitis C Virus (HCV), hepatitis a virus, hepatitis b virus, hepatitis d virus, hepatitis e virus, ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, Human Papilloma Virus (HPV), hyperpneumovirus and coronavirus. More preferably, the target virus is SARS virus. The method utilizes siRNA prepared by the following method: (a) identifying a target nucleotide sequence in a viral genome, particularly in SARS virus, to design small interfering rna (sirna); and (b) making the siRNA modified to contain at least one modified nucleotide. More preferably, the siRNA comprises a dsRNA molecule having a first strand with a ribonucleotide sequence that corresponds to a nucleotide sequence that corresponds to a target nucleotide sequence of the virus and a second strand comprising a ribonucleotide sequence that is complementary to the target nucleotide sequence, wherein the first and second strands are separate complementary strands that hybridize to each other to form the dsRNA molecule, and further wherein the first strand ribonucleotide sequence, the second strand ribonucleotide sequence, or the first and second strand ribonucleotide sequences comprise at least one modified nucleotide. In this method, the target nucleotide sequence comprises a conserved nucleotide sequence essential for SARS virus replication selected from the group consisting of SEQ ID NO: 7292. SEQ ID NO: 7293. SEQ ID NO: 7294. SEQ ID NO: 7295. SEQ ID NO: 7296. SEQ ID NO: 7297. SEQ ID NO: 7298. SEQ ID NO: 7299. SEQ ID NO: 7300 and SEQ ID NO: 7301. preferably the nucleotide sequence is selected from SEQ ID NO: 7292 and SEQ ID NO: 7293. more preferably, the nucleotide sequence is SEQ ID NO: 7293.

The sirnas described herein can be made using the modified ribonucleotides described herein. In addition, the modified ribonucleotides of the siRNA used in the method of the invention can be incorporated into the siRNA by chemical synthesis or enzymatic synthesis.

The sirnas described herein may or may not contain a 5' triphosphate group.

The modified siRNA is administered by a method selected from the group consisting of intravenous injection, subcutaneous injection, oral administration, and liposomal delivery. The modified siRNA is aggregated in an organ, tissue or liver of a patient, gastrointestinal tract, respiratory tract, cervix or skin of the patient.

The invention also provides a method for replication of a virus (e.g., SARS virus) in a heterogeneous SARS virus positive cell comprising transfecting the SARS positive cell with a vector capable of directing expression of the modified SARS-specific siRNA. The cells are evaluated to determine whether the marker in the cell has been inhibited by the modified siRNA.

The term patient may here be an animal, preferably a mammal. More preferred subjects are primates, including non-human primates and humans. The terms "subject" and "patient" are used interchangeably.

The treatment methods envisioned by the present invention may be used on subjects that have previously been infected with a virus, or on subjects that have previously been predisposed to infection with the SARS virus. In addition, the methods of the invention can be used to correct or compensate for cellular or physiological abnormalities associated with making a patient susceptible to viral infection, and/or to alleviate symptoms of viral infection in a patient, or as a prophylactic measure in a patient.

Methods of treating a subject with a viral infection include administering the composition to the subject. A composition herein may refer to a pure compound, agent or substance, or a mixture of two or more compounds, agents or substances. The term "agent, substance or compound" as used herein refers to a protein, nucleic acid, carbohydrate, lipid, polymer or small molecule, such as a drug.

In one embodiment of the invention, the composition administered to the subject is a pharmaceutical composition. In addition, the pharmaceutical composition may be administered orally, nasally, parenterally, systemically, intraperitoneally, topically (e.g., by drops or transdermal routes), buccally, or as an oral or nasal spray. The term "parenteral" as used herein means modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable carrier.

"pharmaceutically acceptable carrier" refers to, but is not limited to, a non-toxic solid, semi-solid, or liquid filler, diluent, coating material, or any type of formulation excipient, such as liposomes.

Pharmaceutical compositions of the invention for parenteral injection may contain pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution with injectable sterile solutions or dispersions prior to use. Suitable aqueous or nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. For example, a coating material such as lecithin may be used to maintain the desired particle size in the dispersion, as well as to maintain proper fluidity with surfactants.

The compositions of the present invention may also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Various antibacterial and antifungal agents are added to ensure prevention of the action of microorganisms, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agent such as sucrose, sodium chloride, etc. can also be added. The addition of agents which delay absorption, such as aluminum monostearate and gelatin, can delay the absorption of the injectable pharmaceutical formulation.

In some cases, to prolong the efficacy of the drug, absorption of the drug from subcutaneous or intramuscular injection can be slowed. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of the drug depends on its rate of dissolution, which in turn may depend on the crystallite size and crystalline form. Alternatively, absorption of a parenterally administered drug form can be delayed by dissolving or suspending the drug in an oily vehicle.

Injectable depot forms can be made by forming microcapsule materials of the drug contained within a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particulate polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations can also be made by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by the addition of a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other injectable sterile medium prior to use.

Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be mixed with at least one pharmaceutically acceptable excipient or carrier, for example sodium citrate or dicalcium phosphate, and/or a) fillers or compatibilizers, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retention agents, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as monostearates of acetyl alcohol and glycerol, h) adsorbents, such as kaolin and bentonite, and i) lubricants, such as talc, calcium stearate, magnesium stearate, Solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents.

Similar solid compositions may also be employed as fillers in soft and hard gelatin capsules using lactose and high molecular weight polyethylene glycols and the like as excipients.

Solid dosage forms such as tablets, dragee capsules, pills, and granules may contain coatings or shells such as enteric coatings and other coatings well known in the pharmaceutical art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, over a certain portion of the intestinal tract, optionally in a sustained release manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

The active compounds can also be in the form of microcapsules, if appropriate with one or more of the abovementioned excipients.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and aromatic bases.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Alternatively, the composition may be pressurised or contain a compressed gas, such as nitrogen or a liquefied gas propellant. It is preferred that the propellant medium be liquefied, and that virtually all compositions be such that the active ingredient is not dissolved therein to any substantial extent. The pressurized composition may also contain a surfactant. The surfactant may be a liquid or solid nonionic surfactant, or may be a solid anionic surfactant. Preferably, a solid anionic surfactant in the form of a sodium salt is used.

The compositions of the invention may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid materials, as is known in the art. The liposomes can be formed by dispersing a monolayer or multilayer of hydrated liquid crystals in an aqueous medium. Any physiologically acceptable and metabolizable non-toxic lipid capable of forming liposomes can be used. The composition of the present invention in liposome form may contain, in addition to the compound of the present invention, stabilizers, preservatives, excipients and the like. Preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins). Methods of forming liposomes are known in the art (see, e.g., Prescott, eds., meth.cell biol.14: 33, infra (1976)).

It will be appreciated by those skilled in the art that effective amounts of the agents of the invention may be determined empirically and may be used in purified form or in the form of a pharmaceutically acceptable salt, ester or prodrug, if such is present. The agent may be administered to a subject in need of treatment for a viral infection as a pharmaceutical composition in admixture with one or more pharmaceutically acceptable excipients. It will also be understood that the total daily amount of an agent or composition of the invention when administered to a human patient will be determined by the attending physician within reasonable limits. The specific therapeutically effective amount for any particular patient will depend upon a variety of factors: the type and extent of cellular or physiological response that needs to be achieved; the activity of the particular agent or composition employed; the specific reagents and compositions used; the age, weight, health, sex, and diet of the patient; time of administration, route of administration, and rate of drug excretion; the duration of treatment; drugs for use in compositions or for use with specific agents; and other similar factors well known in the medical arts. For example, it is well known to those skilled in the art to start the administration of an agent at a level lower than that required to achieve the desired therapeutic effect and then gradually increase the dosage until the desired effect is achieved.

Administration can be carried out in a patient-specific manner to provide a blood concentration of the agent that is predetermined using accepted techniques and practices in the art. The patient's dosing regimen may thus be varied to achieve a progressive modulation of blood levels, as measured by HPLC, on the order of 50-1000 ng/ml.

One of ordinary skill in the relevant art will readily appreciate that other suitable modifications and adaptations to the methods and applications described herein may be made without departing from the scope of the invention and any embodiments thereof.

Modified sirnas were prepared by Dharmacon at Lafayette co using conventional chemical synthesis. Each of the C and U in the siRNA duplex (GL2) was replaced with 2 ' -F-U and 2 ' -F-C, except for the 3 ' -end overhang dTdT.

To test the stability of 2' chemically modified siRNA compared to unmodified siRNA (siRNA), the following experiment was performed. 4 ng of siRNA was added to a volume of 20. mu.L of 80% human serum from a healthy donor. The mixture was incubated at 37 ℃ for various periods ranging from 1 minute to 10 days. The same treatment was performed for 2 '-fluoro-modified siRNA (2' -F siRNA). When the incubation process was complete, the mixture was placed on ice and immediately separated by PAGE using a 32P-siRNA control. The 2' modified siRNA is stable compared to unmodified siRNA.

Identification of therapeutic Agents for the treatment of SARS Virus infection

The invention provides methods for treating SARS by administering a therapeutically active agent (e.g., a small molecule compound) to a mammal, as well as methods for identifying a therapeutically active agent (e.g., an effective small molecule) for treating SARS virus infection.

One aspect of the invention provides a method of identifying a therapeutically active agent, the method comprising: (a) contacting the therapeutically active agent with cells infected with SARS virus; (b) the attenuation of SARS-associated enzyme is determined.

In a more specific embodiment, the therapeutically active agent is a small molecule. In other more specific embodiments, the therapeutically active agent is a nucleoside analog (e.g., ribavirin). In other more specific embodiments, the small molecule is a SMIP or peptide immunomodulatory compound. In other more specific embodiments, the therapeutically active agent is a peptoid, an oligopeptide or a polypeptide. In another embodiment, the SARS-associated enzyme is SARS protease. In another embodiment, the SARS-associated enzyme is SARS polymerase. In still other embodiments, the SARS-associated enzyme is a kinase. In still other embodiments, the SARS-associated enzyme is a protease. The furin inhibitor peptidyl chloromethyl ketone prevents cell-cell fusion after MHV infection (de Haan et al, (2004) J Virol), which provides guidance for the treatment of SARS.

The present invention includes a cell-based assay that can be used to screen and identify therapeutically active agents for treating SARS virus infection. Therapeutically active agents of the invention include agents that inhibit, prevent or reduce the replication of the SARS virus. Such agents can be identified by infecting cultured cells (e.g., VERO cells) with SARS virus and evaluating the effect of potential antiviral compounds on SARS virus infection. Assays for determining the effect of a potential antiviral compound on viral replication are well known in the art and may be based on a variety of parameters.

Cell-based assays can be used in high throughput screens to identify therapeutically active compounds from chemical libraries containing potential antiviral compounds. Therapeutically active compounds suitable for use in the present invention may inhibit any SARS virus target necessary for viral replication in whole cells. The efficacy of a therapeutic agent (the ability of a compound to inhibit or inactivate a target virus or cell, resulting in a reduction of the virus in culture) can be measured by assessing the viability and/or proliferation of viable cells in a culture of cells infected with SARS virus.

Many methods are known in the art for measuring cell viability, such as assays that measure enzymes, proteins, nucleotide triphosphates (e.g., ATP), nucleic acids (e.g., mRNA (e.g., GAPDH) or rRNA sequences) or cell metabolites of a cell, such as MTT or MTS. In addition, cell viability and/or proliferation phenomena can be assayed using fluorescent dyes (including, for example, HSV paper) or non-fluorescent dyes (such as propidium diiodide) or labeled DNA.

Alternatively, the effect of a compound or test sample can be determined by directly measuring the amount of virus or virus product in the culture. A method of determining the amount of a virus, viral genome, or viral product comprising: PCR, RT-PCR, TMA, reporter proteins with fluorescent or luminescent properties or enzymatic functions (e.g. luciferase, alkaline phosphatase, GFP), or proteins detectable by antibodies (e.g. EGF) which can be incorporated into the viral genome before the cell culture is infected. In addition, viral products such as viral proteins can be assayed by ELISA or enzyme activity. Methods for identifying viral polynucleotides, viral proteins and antibodies specific for viral proteins have been described above.

Potential antiviral compounds are used in cell-based assays at concentrations of about 10 μ M and the type of compound with therapeutic efficacy is identified by measuring selected parameters such as cell viability/proliferation or viral genome or viral or non-viral derived viral products. Once the compounds are considered active, they can be resynthesized and mimicked. Starting from the identified compounds, many analogs and new compounds were synthesized in successive cycles of optimal synthesis, biological proliferation and modeling to optimize representative structures until in vivo activity was elucidated and optimized.

Suitable cells for use in such assays include those described above as suitable for vaccine production. Preferably the cell is a Vero cell. Human embryonic lung fibroblasts or normal human diploid fibroblasts may also be used in the present invention.

In one embodiment, the invention includes a fluorescence-based cytopathogenic assay to measure the effect of potential antiviral compounds on cell-based assays. An example of such a fluorescence-based cytopathogenic assay is described below.

Microtiter plate (MTP) 1X 10 per well⁴Vero cells were infected with a defined amount of SARS virus, which had the following optimal MOI range: 5-10, 10-25, 25-50, 50-100, 100-500 or 500-1000PFU, a total volume of 200. mu.l of medium (M199 medium supplemented with 5% FCS, 2mM glutamine, 100IU/ml penicillin and 100. mu.g/ml streptomycin), with or without a potential antiviral compound, and 5% CO at 37 ℃₂Incubating for at least 1, 2, 3, 4, 5, 6, or 7 days. Each well of the MTP was washed with PBS (200. mu.l) and then 200. mu.l of PBS containing 10. mu.g/ml fluorescein acetate was added. After incubation at room temperature for 45 minutes, fluorescence was measured at an excitation wavelength of 485nm and an emission wavelength of 538 nm. Determination of IC by plotting non-Linear Profile of antiviral Activity as a function of drug concentration ₅₀The value is obtained.

Other cell-based assays are known in the art, including the GFO assay and the Luc assay. In addition, cell viability can also be measured using a commercially available Promega kit.

In one embodiment, the invention includes a method of measuring the efficacy of a potential antiviral compound by detecting the level of SARS virus RNA in a cell-based assay using RT-PCR. Methods of using RT-PCR are known in the art. An example of such a measurement method is as follows.

Will be 5X 10⁶Vero cells were seeded on tissue cultures. The flasks containing the cells were incubated at 37 ℃ with 5% CO₂The incubation was carried out overnight. Cells were infected with SARS virus (m.o.i.: 1) in the presence and absence of potential antiviral compounds. Optionally, the cells may be treated with the potential compound prior to infection. In any case, appropriate control cell assays were performed.

RNA from infected cells was purified at 2 hours (UL54), 12 hours (UL8) and 16 hours (UL13) post infection and RNA purity was determined quantitatively (Qiagen) (Rneasy kit; 40. mu.l elution) (260nm absorbance). RNA (2. mu.g) was reverse transcribed into cDNA using specific primers (2pmol, using one of the primer pairs described above) according to the SuperScriptII protocol (Invitrogen). Reverse transcription aliquots (2. mu.l) were amplified by PCR. Suitable target SARS gene fragments, i.e.the genes encoding the enzymes, were amplified by PCR (Taq polymerase, Stratagene) for 30 cycles (UL54 and UL 8: heat start at 94 ℃ for 3 minutes, denaturation at 94 ℃ for 1 minute, annealing at 55 ℃ for 1 minute, polymerization at 72 ℃ for 1 minute, UL 13: heat start at 94 ℃ for 3 minutes, denaturation at 94 ℃ for 1 minute, annealing at 60 ℃ for 1 minute, polymerization at 72 ℃ for 1 minute) in a reaction volume of 100. mu.l containing 0.1nmol of each of the above-mentioned suitable oligonucleotides. Aliquots of 8. mu.l from 20-30 PCR cycles (lanes 2-12) were analyzed on 2% agarose gel (Invitrogen) according to the manufacturer's instructions.

The cell-based assays of the invention may optionally employ variants or derivatives of wild-type SARS virus that are attenuated or attenuated in humans and/or animal models (e.g., mice, non-human primates, etc.). The use of such attenuated SARS viruses in screening methods can reduce safety-related problems and vigilance in the pathogenicity of SARS viruses, and can eliminate or reduce the need for high levels of control in assaying and screening compounds.

The present invention includes an enzyme-based assay that can be used to screen for and identify therapeutically active agents for treating SARS virus infection.

One embodiment of the present invention is an assay method comprising the steps of: contacting a known amount of SARS protease in the solution with a peptide comprising a detectable label and a SARS protease cleavage site, wherein SARS protease activity is monitored by measuring the label density of the cleaved product.

In a more specific embodiment, a method for assaying SARS protease is provided, the method comprising contacting a sample solution containing SARS protease with a peptide comprising a fluorescence donor, a fluorescence quencher, and a SARS protein cleavage site, wherein SARS protease activity in the sample is determined by the amount of fluorescence detected by the fluorometer when the peptide is cleaved by the fluorometer.

SRAR therapeutics can be screened using assays based on direct measurement of SARS protease inhibition. Proteins used in such assays, such as 3C-like protease and papain-like protease, can be isolated and purified as described in: seybert et al, j.gen.virol, 78: 71-75, 1997, Ziebuhr et al, adv.exp.med.biol., 440: 115-120, 1998, Sims et al, adv. exp. Med. biol.440: 129-134, 1998, Ziebuhr et al, j.virol, 73: 177-185, 1999, Teng et al, J.Virol., 73: 2658-2666, 1999, Herold et al, J.biol.chem.274: 14918-14925, 1999; and Ziebuhr et al, j.biol.chem.276: 33220-33232, 2001. In addition, example 30 describes a novel method for purifying SARS protease by column chromatography. Example 31 describes a Fluorescence Resonance Energy Transfer (FRET) assay to measure SARS protease activity. Example 31 demonstrates that protease-based assays, such as FRET assays, are suitable for high throughput screening and can be used to screen candidate antiviral compounds. When a SARS protease inhibitor compound is present, the results of the protease assay show a decrease in the amount of fluorescence at a given time as compared to a negative control containing no test compound on a non-inhibitory control compound. Such a method would include the steps of: (a) providing a test solution containing SARS protease; (b) adding a test compound to the test solution; (c) adding a SARS protease substrate to the test solution; and (d) measuring the proteolytic activity of the test solution. In a preferred embodiment, proteolytic activity is measured by fluorescence of the fluorophore product produced by the enzymatic activity of SARS protease.

Attenuated SARS virus variants typically comprise one or more genomic modifications or mutations (e.g., substitutions, deletions, insertions) in the coding region or regions of the protein. Specific examples of the attenuated mutant include, for example, a 5 '-terminal noncoding region, a leader sequence, an intergenic region, a 3' -terminal noncoding region, ORF 1a, ORF 1b, an S gene, an E gene, an M gene, an N gene, or a genetic modification in any nonstructural protein gene other than the ORF 1a/1b region. Preferred attenuating mutations are located in the structural protein (e.g., spike (S)), protease or polymerase domain or non-coding sequence (e.g., 5' -terminal non-coding region, intergenic sequence) of the SARS virus. In addition, cleavage sites can be incorporated or deleted in the spike protein (see, e.g., Gombold et al, J.Virol.67: 4504-4512, 1993; Bos et al, Virology 214: 453-463, 1995), such modifications also being useful for optimizing expression of recombinant spike protein antigens (e.g., for vaccines).

As described herein, various methods are used to obtain attenuated SARS virus variants. These methods include serial passaging of SARS virus in cultured cells (e.g., mammalian cell cultures such as rhesus fetal kidney cells or VERO cells) until the SARS virus is confirmed to be attenuated. Continuous propagation of the virus may be carried out at any temperature at which subculture attenuation can occur, and may be combined with one or more additional mutagenesis steps (e.g., chemical mutagenesis). The attenuated phenotype of a SARS virus mutant obtained after one or more cell culture passages can be readily determined by one skilled in the art. Attenuation is herein taken to mean a reduction in the virulence of the SARS virus in a human subject. Evidence of attenuation is manifested as a reduced level of viral replication or reduced virulence in animal models.

Other methods for making attenuated SARS viruses include passaging the virus in cell culture at sub-optimal temperatures (cold passaging), and introducing the attenuated mutant into the SARS virus genome by random mutagenesis (e.g., chemical mutagenesis with 5-fluorouracil) or by site-directed mutagenesis. The manufacture and production of attenuated RSV vaccines (the methods of which are generally applicable to the SARS virus) is described, for example, in EP 0640128, U.S. patent No.6,284,254, U.S. patent No.5,922,326, U.S. patent No.5,882,651.

The number of passages required to obtain a safe, vaccinable, attenuated virus depends, at least in part, on the conditions employed. Periodic detection of the virulence and immunocompetence of a SARS virus culture in animals (e.g., mice, primates) allows for the convenient determination of parameters for specific tissue culture and temperature combinations.

In another embodiment, the cell-based assay used to screen for antiviral compounds is based on a readout of the expression of a gene product (e.g., a reporter gene product) that is not from the SARS virus. Gene products particularly suitable for the present invention include, but are not limited to, those used in the assays described above.

To obtain such reads, the gene of interest (GOI) encoding the gene reporter gene product must be incorporated into the replicating SARS virus genome or a construct derived from the SARS virus genome (e.g., SARS virus replicon, SARS virus Defective Interfering (DI) RNA). FIG. 13 shows the location of reporter gene incorporation into the SARS virus genome. Preferably, the heterologous reporter gene of interest is inserted between the existing SARS virus genes as shown in FIG. 13. For example, the GOI can be inserted into a position immediately following the stop codon of the SARS virus gene (e.g., ORF 1b, S, E, M, N). The insertion should be positioned to minimize disruption of transcription of the SARS virus gene mRNA. The GOI may also be inserted as an in-frame "fusion" with an existing SARS virus gene, thus maintaining sufficient GOI function for detection. For optimal expression, other SARS virus intergenic sequences (e.g., SEQ ID NO: 7388, with or without other flanking SARS virus sequences) can also be constructed into positions prior to the inserted GOI.

The incorporation of GOI into SARS virus can be accomplished by a variety of techniques by those skilled in the art. For example, one preferred method is targeted RNA recombination, which has the advantage of being able to recombine coronavirus RNA intracellularly (see, e.g., Fischer et al, J.Virol.71: 5148-. Constructs containing the GOI and the desired configuration of flanking SARS virus sequences (e.g., intergenic sequences) (e.g., cDNA for SARS virus defective interfering RNA) are made so that the RNA can be transcribed directly in eukaryotic cells or in vitro and transfected into susceptible cells that are also infected with SARS virus. Recombinant viruses containing a GOI are identified based on expression of a marker encoding the GOI.

Alternatively, the incorporation of the GOI into the SARS virus can be accomplished by one skilled in the art by first assembling a full-length cDNA of the SARS virus, which can be used to make infectious RNA transcripts in vivo (e.g., from the RNA polymerase II promoter) or in vitro (e.g., from a phage promoter). Despite the relatively long genome length, assemblies of such full-length cDNA clones are now readily available to those skilled in the art using standard molecular biology and reverse genetics techniques as well as the genomic sequence of the SARS virus (see, e.g., Thiel et al, J.Gen.Virol., 82: 1273-. The heterologous GOI can be inserted into the full-length SARS virus genomic cDNA using a variety of techniques, e.g., ligation into natural or synthetic restriction sites, PCR (e.g., overlap PCR), and recombination.

Antiviral screening can also be performed with smaller SARS virus recombinants containing the gene of interest, however, further modifications are needed to minimize or eliminate virus-induced cytopathic effects (e.g., CPE). The non-cytopathic derivative of SARS virus can be obtained by various methods by those skilled in the art. For example, a selectable marker (e.g., a drug resistance marker) can be incorporated as a GOI into the SARS virus genome to produce the infectious virus described above (see, e.g., Perri et al, J.Virol., 74: 9802-. Infectious SARS virus or infectious genomic RNA/cDNA containing GOI can then be used to infect/transfect cells (e.g., VERO), with or without prior mutagenesis, and the infected cells then selected using a suitable selection method. Only those cells containing SARS virus that have both a selectable marker and one or more mutations that render the virus non-cell pathogenic will survive and grow in the selection process. The replication of active SARS virus in these cells is readily detected by various detection techniques (e.g., PCR, Northern blot), and such cells can serve as substrates for cell-based screening assays. Mutations that result in the desired non-cytopathic SARS virus phenotype may include nucleotide substitutions, deletions, or additions, which may occur in the coding or non-coding regions of the gene (e.g., 5 'or 3' -end non-coding region, intergenic region, ORF1a, ORF1b, protease domain, polymerase domain). Such mutations are readily identified by sequence exchange with wild-type (e.g., parental) SARS virus and the phenotypic changes exhibited, as well as sequencing of appropriate genomic regions. Similar mutations that reduce or eliminate cytopathic effects can also be used in SARS virus-derived replicon vectors, either by similar direct screening with SARS virus replicons, or by specific construction of replicons based on mutations identified in the infectious SARS virus section above. Furthermore, such mutants may serve as a basis for the attenuated SARS virus derivatives described elsewhere in this application.

Alternatively, in addition to using infectious SARS virus or derivatives thereof for cell-based screening assays, propagation-deficient "replicons" may be constructed and used. Such replicons retain all protein coding sequences and cis-replicating sequences necessary for RNA replication and expression in cells, but delete one or more sequences or genes required for packaging progeny SARS virus (see, e.g., Curtis et al, J.Virol., 76: 1422-1434, 2002). FIG. 14 depicts representative examples of SARS virus replicons of the invention. For example, a SARS virus cDNA construct is generated that lacks one or more (or all) structural protein coding genes, wherein the missing SARS virus genes are replaced by GOI while retaining all transcriptional signals necessary for expression of the GOI. Operably linked to the SARS virus replicon cDNA construct is an RNA polymerase promoter, which can be used to transcribe replicon RNA in vivo (e.g., RNA polymerase II promoter) or in vitro (e.g., phage promoter). SARS replicons can be introduced into susceptible cells as RNA or DNA (depending on the promoter chosen) by transfection, and the transfected cells used to evaluate antiviral compounds. By incorporating one or more mutations such that the replicon has no cytopathic effect on the cell (see above), nucleic acid transfection at each assay is not required.

Alternatively, SARS virus replicons can be packaged into virus-like particles capable of infecting cells without the need to transfect nucleic acid molecules. The requirement for replicon packaging is that substantial SARS virus gene function (e.g., one or more structural proteins) deleted from the replicon be provided in trans in the cell containing the replicon. Replicon RNA can be packaged in a variety of ways by those skilled in the art (see, e.g., Curtis et al, supra: Ortego et al, J.Virol., 76: 11518-11529, 2002). For example, stably transformed cell lines expressing the desired SARS virus gene function, either constitutively or inducibly, can be used. Alternatively, the desired SARS viral gene function can be expressed by a viral vector incorporated into a replicon-containing cell. Alternatively, Defective Interfering (DI) SARS virus-derived RNA containing the desired gene can be incorporated into replicon-containing cells. Such DI constructs used to compensate for lost replicon function are commonly referred to as defective helper RNA or defective helper.

Another configuration useful for the cell-based antiviral screening assay of the present invention utilizes SARS virus-derived DI RNA encoding GOI (see, e.g., Stirrups et al, J.Gen.Virol., 81: 1687-. SARS DI, either as cDNA linked to the RNA polymerase II promoter or as in vitro transcribed RNA, is introduced into susceptible cells also infected with SARS and a readout of the GOI reporter gene product is obtained in the assay.

Replicon-based systems for rapid identification of inhibitors of coronavirus replicase are described in Hertzig et al, (2004) J Gen Virol DOI 10.1099/vir/0/80044-0. Briefly, the system employs a non-cytopathic selectable replicon RNA that can be stably maintained within a eukaryotic cell. The replicon RNA-mediated reporter gene expression can be used as a coronavirus replication marker, and the reporter gene expression can be used for detecting the inhibition effect of a test compound in vitro, so that a replicase inhibitor can be screened at high flux without culturing infectious viruses. Preferred replicon RNAs contain a neomycin resistance gene in the replicase gene, downstream of which a reporter gene (e.g., GFP) can be expressed by replicase-mediated synthesis of a subgenomic mRAN.

Compositions and methods for treating viral infections with SARs

The present invention relates to compositions and methods for treating and/or preventing SARS. The present invention also includes methods for treating and/or preventing SARS by administering a therapeutically effective amount of at least one antiviral compound, such compounds being described in the U.S. patents and published international patent applications listed in tables 1 and 2. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In yet another embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. Lopinavir/ritonavir (Kaletra) protease inhibitors in combination with ribavirin show good clinical efficacy (Chu et al, (2004) Thorax 59: 252-. In another embodiment, the antiviral compound is an RNA-dependent RNA polymerase inhibitor. In another embodiment, the first antiviral compound protease inhibitor is administered with an inhibitor of a second antiviral compound RNA-dependent RNA polymerase. The present invention also provides for the administration of steroidal anti-inflammatory drugs in combination with at least one antiviral compound, such as those described in the references from tables 1 and 2. Combination therapy of steroids with ribavirin has been described in Fujii et al, (2004) J Infect Chem other 10: 1-7. Combination therapy with corticosteroids and interferon alpha-1 has also been reported (Loutfy et al, (2003) JAMA 290: 3222-3228).

The present invention also provides methods for treating and/or preventing SARS by administering by inhalation a therapeutically effective amount of at least one antiviral compound, such compounds being described in the U.S. patents and published international patent applications listed in tables 1 and 2. In another aspect, the antiviral compound can be administered in combination with SMIP, SMIS, or other immunomodulatory compounds (such as those in tables 32 and 33). In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In yet another embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. In another embodiment, the antiviral compound is an RNA-dependent RNA polymerase inhibitor. In another embodiment, the first antiviral compound protease inhibitor is administered with an inhibitor of a second antiviral compound RNA-dependent RNA polymerase. The present invention also provides for the administration of steroidal anti-inflammatory drugs in combination with at least one antiviral compound, such as those described in the references from tables 1 and 2. The steroidal anti-inflammatory drugs may be administered by inhalation to achieve a local effect, or by systemic absorption, for example, by the oral or intravenous route.

The invention also provides a method of treating SARS infection comprising administering a Small Molecule Immunopotentiator (SMIP) compound alone or in combination with an antiviral compound or in combination with a SARS vaccine. In yet another embodiment, the SMIP is a compound described herein or a compound listed in table 32.

The invention also provides a method of treating SARS infection comprising administering an immunosuppressant compound, optionally a Small Molecule Inhibitor (SMIS) compound, either alone or in combination with an antiviral compound. In yet another embodiment, the immunosuppressant compounds are as described herein or listed in table 33.

The invention also provides peptide immunomodulatory compositions, including oligopeptides and polypeptides, capable of affecting an inflammatory response in a patient. In one embodiment, the peptide immunomodulatory compositions are capable of stimulating cytokine production by human cells. In another embodiment, the peptide immunomodulatory compositions are capable of decreasing cytokine levels in humans. Examples of preferred peptide immunomodulatory compositions include those listed in table 35, as well as TGF β 2, TGF β 1, TGF β 3, thymopentin (TP5), β -mercaptopropionyl-arginyl-lysyl-aspartyl-valyl-tyrosyl-cysteinyl amide, colostrum kinin, Lactoferrin (LF), cyclooligopeptide a (CLA), and engulfment peptide (TKPR). The peptide immunomodulatory compositions of the invention may be used alone or in combination with other agents, preferably antiviral compounds, to treat SARS.

The invention also provides a kit for use by a consumer to treat and/or prevent SARS. Such a kit contains: a) a pharmaceutical composition comprising a therapeutically effective amount of at least one antiviral, SMIP, SMIS or other immunomodulatory compound (from those described in the U.S. patents and published international patent applications listed in table 1, table 2, table 34 and table 35) and a pharmaceutically acceptable carrier, vehicle or diluent; b) a container containing the pharmaceutical composition; and optionally, c) instructions describing a method for treating and/or preventing SARS using the pharmaceutical composition. The kit may optionally contain a plurality of compounds for treating SARS, wherein the antiviral compound is selected from the group consisting of a 3C-like protease inhibitor and a papain-like protease inhibitor. In yet another embodiment, the antiviral compound contained in the kit is an inhibitor of RNA-dependent RNA polymerase. When the kit contains more than one antiviral, SMIP, SMIS or other immunomodulatory compound, the compounds contained in the kit may optionally be combined into a pharmaceutical composition.

In another aspect, the invention provides the use of at least one antiviral, SMIP, SMIS or other immunomodulatory compound described in U.S. patents and published international patent applications listed in table 1, table 2, table 34 and table 35 in the manufacture of a medicament for the treatment or prevention of SARS.

Another aspect of the invention provides the use of at least one SMIP compound, or at least one immunosuppressant compound or at least one SMIS compound in the manufacture of a medicament for the treatment or prevention of SARS. Preferred SMIPs, immunosuppressants and SMIS compounds are as described herein.

Unless otherwise indicated, the following terms will be used in section VI of the present application: "compositions and methods for treating viral infections with SARs" means as follows:

"limiting," "treating," and "treatment" are used interchangeably herein and include prophylactic treatment (e.g., prophylactic administration) and palliative treatment, or the effect of providing prophylactic treatment or palliative treatment. The term includes delaying the development of symptoms of SARS and/or reducing the severity of such symptoms that will occur or are expected to occur following a SARS virus infection. The term also includes alleviation of existing symptoms of SARS, prevention of the appearance of other symptoms, alleviation or prevention of metabolic factors underlying the symptoms.

Representative compositions and methods of the invention include: eliminating or reducing the viral load of the SARS virus in a vertebrate (including a human), eliminating or reducing symptoms associated with SARS, and reducing the incidence associated with SARS. In a population of SARS patients, use of the compositions and methods of the invention will result in a reduction in the high mortality associated with SARS.

SARS virus infection and symptoms associated with SARS can be treated by administering the compositions of the invention. The compositions of the invention may be administered systemically. For systemic administration, the compounds described herein can be formulated according to conventional methods for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal, or transdermal) or enteral (e.g., oral or rectal) delivery. Intravenous administration may be by a series of injections or by continuous infusion over a prolonged period of time. Administration by injection or other discrete dispensing route may be carried out at intervals ranging from once a week to once or three times or more per day. Alternatively, the compositions described herein may be administered in a cyclical manner (administration of the composition, then withdrawal, then re-administration of the composition, and so on). Treatment will continue until the desired result is achieved.

The "subject" is a vertebrate, including a human, in need of treatment with the compositions, methods, and kits of the invention. The term "subject" includes males and females unless indicated by sex.

By "co-administering" a combination of antiviral compounds is meant that the components can be administered together as one composition or a portion thereof in a single dosage form. "Co-administration" also includes the administration of multiple antiviral compounds alone, but as part of one treatment procedure or regimen. "Co-administration" also includes administration of a variety of other agents, such as oligopeptides, polypeptides, peptide immunomodulators, nucleic acids, antibodies or vaccines, wherein the compounds or agents are administered separately but as part of a therapeutic procedure or regimen. The components need not be applied simultaneously, although this may be done if desired. "Co-administration" also includes separate administrations at different times, in any order. For example, a patient may use one or more components in the morning and one or more other components in the evening.

By "antiviral compound" is meant herein an antiviral compound as described in the U.S. patents and published international patent applications listed in tables 1 and 2. The U.S. patents and published international patent applications listed in tables 1, 2 and 35 are incorporated herein by reference. In one embodiment, the antiviral compound is an RNA-dependent RNA polymerase. In other preferred embodiments, the antiviral compound is a 3C-like protease inhibitor or a papain-like protease inhibitor. The antiviral compound may be administered in the form of an acid or a soluble alkali or alkaline earth metal salt.

The precise dosage of the antiviral compound will vary depending upon the dosage regimen, and the choice of oral potency of a particular antiviral compound will depend upon the age, weight, sex, and symptoms of the subject, the severity of the condition being treated, and other relevant medical and physical factors. Thus, the precise pharmaceutically effective amount cannot be further specified and can be readily determined by a nurse or clinician.

Generally, the amount of antiviral compound can be selected to reduce the SARS viral load of the subject, and/or to reduce symptoms associated with SARS. For humans, an effective oral dosage of the antiviral compound is generally about 1.5-6000 μ g per kg body weight per day, preferably about 10-2000 μ g per kg body weight per day.

One of ordinary skill in the art will appreciate that certain antiviral, SMIP, SMIS, and immunomodulatory compounds of the invention comprising a 3C-like protease inhibitor, a papain-like protease inhibitor, and an RNA-dependent RNA polymerase inhibitor will contain one or more atoms in a particular stereochemical, tautomeric, or geometric configuration, which yields stereoisomers, tautomers, and configurational isomers. All such isomers and mixtures thereof are included in the present invention when active. Crystalline and amorphous forms of the antiviral compounds of the present invention are also included, as are hydrates, solvates, and isomorphous forms of the antiviral compounds of the present invention.

SMIP compounds of the present invention include those described in published U.S. Pat. Nos.4,547,511 and 4,738,971, having the following general formula (a):

heterocyclic radical

For treating diseases responsive to agents that enhance cell-mediated immunity.

Immunostimulatory oligonucleotides and polynucleotides are described in PCT WO 98/55495 and PCT WO 98/16247. The adjuvants described in U.S. patent application No.2002/0164341 include unmethylated CpG dinucleotides (CpG ODN) and non-nucleic acid adjuvants. U.S. patent application No.2002/0197269 describes compositions containing an antigen, an antigenic CpG-ODN, and a polycationic polymer.

Further, published U.S. patent nos.4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, 5,525,612, WO99/29693 and u.s.ser.no.09/361,544 disclose compounds of general formula (b):

are used as "immune response modifiers".

Other compounds having SMIP and antiviral activity are described in U.S. patent application entitled "thiosemicarbazone as an antiviral and immunopotentiator" (thiosemicarbazone as Anti-Virals and rammunolotters) filed on 29.12.2003, having docket number PP19814.004US, and having the following structure:

a compound of formula c:

wherein: e is absent or selected from alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl or substituted heteroaryl;

l is absent or selected from oxygen, amino, alkenyl, substituted alkenyl, alkoxy, alkylamino, aminoalkyl, heterocyclyl, carbocyclyl, or carbonyl;

w is absent or selected from cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl or substituted heteroaryl;

X is absent or selected from oxygen, amino, alkenyl, substituted alkenyl, alkoxy, alkylamino, aminoalkyl, heterocyclyl, carbocyclyl or carbonyl;

y is selected from cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl or substituted heteroaryl;

y' is absent or selected from F, Cl, Br, I, nitro, alkyl, substituted alkyl or optionally substituted heterocyclyl, amino, alkylamino, dialkylamino;

y' is absent or selected from F, Cl, Br, I, nitro, alkyl, substituted alkyl or optionally substituted heterocyclyl, amino, alkylamino, dialkylamino;

r' is H, alkyl or substituted alkyl;

r' is H, or

R 'and R' together form a heterocyclic ring;

z and Z' are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, alkoxy, substituted alkoxy, aminocarbonyl, alkoxycarbonyl, carboxysulfonyl, methanesulfonyl, substituted or unsubstituted alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, lower alkylcarbonyl, lower alkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, substituted arylalkyl, heteroarylalkyl, heteroaryl, substituted heteroaryl, aminocarbonyl, substituted alkylcarbonyl, alkoxycarbonyl, heteroarylcarbonyl, substituted alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, substituted or unsubstituted alkylcarbonyl, Arylaminocarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, cyclic amidino, cycloalkyl, cyclic imino, arylsulfonyl or arylsulfonamido; or

Z and Z' together form a heterocyclic ring which may optionally be substituted,

and tautomers and pharmaceutically acceptable salts, esters, or prodrugs thereof.

Other SMIP Compounds are described in U.S. patent application 1O/762873 entitled "tryptamine Compounds for Immune Potention" filed hereafter and on 21.1.2004, in which a general embodiment of a compound represented by formula (d) is disclosed:

wherein,

A. b, C, D, E, F, G and H are independently selected from carbon and nitrogen, or A and B and/or C and D may together represent nitrogen or sulphur;

R₁、R₂、R₃、R₄、R₈and R₁₀Independently selected from the group consisting of hydrogen, halogen, lower alkyl, substituted alkyl, cycloalkyl, heterocyclyl, alkylheterocyclyl, substituted heterocyclyl, substituted alkenyl, amino, (substituted alkyl) (alkyl) amino, imino, halogenated lower alkyl, hydroxy, alkoxy, substituted alkoxy, hydroxyalkylthio, nitro, alkylsulfonyl, N-alkylsulfonamide, arylalkyl, arylalkylaryl, arylaryl, aryloxy, arylamino, acylamino, acyloxyamino, alkylaminoacylamino, alkylaminosulfonylamino, alkylamino, alkenylamino, dialkylamino, alkoxyalkylamino, alkoxyalkylheterocyclyl, mercaptoalkoxyalkyl, cyano, formyl, -COOR ₁₁(wherein R is₁₁Is hydrogen, lower alkyl, aryl, heterocyclic, mono-or disaccharide) or-CONR₁₂R₁₃(wherein R is₁₂And R₁₃Independently selected from hydrogen, lower alkyl, aryl, heterocyclic, saccharide, peptide or amino acid residue); or R₂And R₃Together form a six-membered aromatic ring;

R₇and R₉Independently selected from hydrogen, halogen, lower alkyl, halo-lower alkyl, cycloalkyl, heterocyclyl, substituted heterocyclyl or heterocyclylalkyl; and

R₁、R₂、R₃、R₄、R₇、R₈、R₉and R₁₀Absent when the ring atom to which they are attached is sulfur or a doubly-bound nitrogen; or a pharmaceutically acceptable salt, ester, or prodrug thereof,

provided that when A, B, C, D, E, F and H are carbon, R is₁、R₂、R₃、R₄、R₇、R₈、R₉And R₁₀Not all are hydrogen.

In one embodiment, the compounds of formula (I) have a backbone structure, wherein D is nitrogen and A-C and E-H are carbon.

In one embodiment, when D is carbon, at least R₁-R₄And R₇-R₁₀One or both of which are not hydrogen.

In one embodiment, R₁-R₄And R₈And R₁₀At least two independently selected from the group consisting of: hydrogen, halogen, lower alkyl, cycloalkyl, heterocyclic, substituted heterocyclic, alkylheterocyclic, amino, imino, halooligoureaAlkyl, alkoxy, nitro, alkylsulfonyl, arylalkyl, arylalkylaryl, arylaryl, aryloxy, arylamino, acylamino, acyloxyamino, alkylaminoacylamino, alkylaminosulfonylamino, alkylamino, alkenylamino, dialkylamino, alkoxyalkylamino, alkoxyalkylheterocyclyl, mercaptoalkoxyalkyl, cyano, formyl, -COOR ₁₁(wherein R is₁₁Is hydrogen, lower alkyl, aryl, heterocyclic, mono-or disaccharide) and-CONR₁₂R₁₃(wherein R is₁₂And R₁₃Independently selected from hydrogen, lower alkyl, aryl, heterocyclic, saccharide, peptide or amino acid residue); and R when D is nitrogen₄Is absent.

In another embodiment, 4A, B, C, D, E, F, G and H are independently selected from carbon and nitrogen;

R₁、R₂、R₃、R₄、R₈and R₁₀Independently selected from hydrogen, halogen, lower alkyl, substituted alkyl, heterocyclyl, substituted alkenyl, (substituted alkyl) (alkyl) amino, halogenated lower alkyl, hydroxy, alkoxy, substituted alkoxy, hydroxyalkylthio, nitro, N-alkylsulfonamide, cyano, -COOR₁₁(wherein R is₁₁Is hydrogen, lower alkyl, aryl, heterocyclic, mono-or disaccharide) or-CONR₁₂R₁₃(wherein R is₁₂And R₁₃Independently selected from hydrogen, lower alkyl, aryl, heterocyclic, saccharide, peptide or amino acid residue).

For the compounds described herein:

the term "lower alkyl" refers to a branched or straight chain acyclic alkyl group containing 1 to 10 carbon atoms and includes, for example, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and the like.

The term "alkyl" refers to an alkyl group that does not contain heteroatoms. Thus, the term includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl Alkyl, dodecyl, and the like. The phrase also includes branched isomers of straight chain alkyl groups, including but not limited to the following examples: -CH (CH)₃)₂、-CH(CH₃)(CH₂CH₃)、-CH(CH₂CH₃)₂、-C(CH₃)₃、-C(CH₂CH₃)₃、-CH₂CH(CH₃)₂、-CH₂CH(CH₃)(CH₂CH₃)、-CH₂CH(CH₂CH₃)₂、-CH₂C(CH₃)₃、-CH₂C(CH₂CH₃)₃、-CH(CH₃)CH(CH₃)(CH₂CH₃)、-CH₂CH₂CH(CH₃)₂、-CH₂CH₂CH(CH₃)(CH₂CH₃)、-CH₂CH₂CH(CH₂CH₃)₂、-CH₂CH₂C(CH₃)₃、-CH₂CH₂C(CH₂CH₃)₃、-CH(CH₃)CH₂CH(CH₃)₂、-CH(CH₃)CH(CH₃)CH(CH₃)₂、-CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃) And so on. The term also includes cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and such rings substituted with the straight and branched chain alkyl groups described above. The term also includes polycyclic alkyl groups such as, but not limited to, adamantyl, norbornyl, bicyclo [2.2.2]Octyl and such rings substituted with the straight and branched chain alkyl groups described above. Thus, the phrase unsubstituted alkyl includes primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. Unsubstituted alkyl groups may be bonded to one or more carbon, oxygen, nitrogen and/or sulfur atoms in the parent compound. Preferred unsubstituted alkyl groups include straight and branched chain alkyl and cycloalkyl groups containing 1 to 20 carbon atoms. More preferred such unsubstituted alkyl groups contain 1 to 10 carbon atoms, and even more preferred such groups contain 1 to 5 carbon atoms. Most preferred unsubstituted alkyl groups include those containing 1 to 3 carbon atomsStraight and branched chain alkyl radicals of the subgroups, including methyl, ethyl, propyl and-CH (CH)₃)₂。

The phrase "substituted alkyl" refers to unsubstituted alkyl groups as defined above wherein one or more of the bonds to carbon or hydrogen are replaced with bonds to non-hydrogen and non-carbon atoms such as, but not limited to, halogen atoms in halides, e.g., F, Cl, Br, and I; phosphorus atoms in groups such as phosphate esters and alkyl dialkyl phosphates; oxygen atoms in the groups of hydroxyl, alkoxy, aryloxy, and ester groups; sulfur atoms in groups such as thiol groups, alkyl sulfides, aryl sulfides, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amino, amide, alkylamine, dialkylamine, arylamine, alkylarylamine, diarylamine, N-oxide, diimide, and enamine; silicon atom in trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, and the like groups; and other heteroatoms in various other groups. Substituted alkyl groups also include those wherein one or more of the bonds to a carbon or hydrogen atom are replaced with a bond to a heteroatom, such as oxygen in carbonyl, carboxyl, and ester groups; nitrogen in the group of imines, oximes, hydrazones, and nitriles. Preferred substituted alkyl groups include alkyl groups in which one or more bonds to a carbon or hydrogen atom are replaced by one or more bonds to a fluorine atom. An example of substituted alkyl groups are trifluoromethyl and other alkyl groups containing a trifluoromethyl group. Other alkyl groups are those in which one or more of the bonds to a carbon or hydrogen atom are replaced by a bond to an oxygen atom, and thus, the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy, or heterocycloxy group. Other alkyl groups include alkyl groups containing amine, alkylamine, dialkylamine, arylamine, (alkyl) (aryl) amine, diarylamine, heterocyclylamine, (alkyl) (heterocyclyl) amine, (aryl) (heterocyclyl) amine, or diheterocyclylamine groups.

The term "alkoxy" refers to RO-, wherein R is, for example, alkyl, lower alkyl as defined above. Representative examples of lower alkylalkoxy include methoxy, ethoxy, t-butoxy and the like.

The phrase "substituted alkoxy" refers to RO-, wherein R is, for example, alkyl substituted with, for example, halogen. RO is, for example, OCF₃。

The term "alkenyl" refers to a branched or straight chain group containing 2 to 20 carbon atoms and also containing one or more carbon-carbon double bonds. Representative alkenyl groups include isoprenyl, 2-propenyl (i.e., allyl), 3-methyl-2-butenyl, 3, 7-dimethyl-2, 6-octadienyl, 4, 8-dimethyl-3, 7-nonadienyl, 3, 7, 11-trimethyl-2, 6, 10-dodecatrienyl, and the like.

The phrase "substituted alkenyl" refers to substituted alkenyl groups, such as diethyl hex-5-enylphosphonate, as well as alkenyl groups substituted with alkyl or substituted alkyl groups such as dialkyl phosphates or esters such as acetates.

The phrase "dialkylamino" refers to an amino group substituted with two alkyl groups, such as C1-20 alkyl.

The phrase "substituted dialkylamino" refers to dialkylamino substituted with, for example, carboxylate, hydroxy, or alkoxy groups.

The term "hydroxyalkylthio" refers to a thio group having a hydroxyalkyl group attached thereto, wherein the alkyl group is, for example, a lower alkyl group. Examples are hydroxyethylthio, -SCH₂CH₂OH。

The term "N-alkylsulfonamide" means-SO₂NH alkyl, wherein alkyl is, for example, octyl.

The term "alkynyl" refers to a branched or straight chain group containing 2 to 20 carbon atoms and containing one or more carbon-carbon triple bonds. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term "aryl" refers to an aryl group that does not contain heteroatoms. Thus, the term includes, but is not limited to, groups such as phenyl, diphenyl, anthracenyl, naphthyl. Although the phrase "unsubstituted aryl" includes groups containing fused rings, such as naphthalene, it does not include aryl groups in which one of the ring members is attached to another group, such as an alkyl or halo group, for example, tolyl is considered herein as the substituted aryl group described below. A preferred unsubstituted aryl group is phenyl. However, an unsubstituted aryl group may incorporate one or more carbon, oxygen, nitrogen, and/or sulfur atoms in the parent compound.

The phrase "substituted aryl" has the same meaning as aryl, while substituted alkyl has the same meaning as alkyl. However, substituted aryl groups also include aryl groups in which an aromatic carbon is bonded to one of the non-carbon or non-hydrogen atoms described above, and also include aryl groups in which one or more aromatic carbons of the aryl group is bonded to a substituted and/or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This includes bond arrangements in which two carbon atoms of an aryl group are combined with two atoms of an alkyl, alkenyl or alkynyl group to form a fused ring system (e.g., dihydronaphthyl or tetrahydronaphthyl). Thus, the phrase "substituted aryl" includes, but is not limited to, tolyl and hydroxyphenyl.

The term "arylalkyl" refers to a lower alkyl group to which an aryl group is attached. Representative arylalkyl groups include benzyl, phenethyl, hydroxybenzyl, fluorobenzyl, fluorophenethyl, and the like.

The phrase "non-fused arylaryl" refers to a group or substituent in which two aryl groups are not fused or linked to each other. Examples of non-fused aryl compounds include, for example, phenylbiphenyl, diphenyldiazene, 4-methylthio-1-phenylbiphenyl, phenoxybenzene, (2-phenylethynyl) benzene, benzophenone, (4-phenylbut-1, 3-dienyl) benzene, phenylbenzylamine, (phenylmethoxy) benzene, and the like. Preferred substituted non-fused arylaryls include: 2- (phenylamino) -N- [4- (2-phenylethynyl) phenyl ] acetamide, 1, 4-diphenylbiphenyl, N- [4- (2-phenylethynyl) phenyl ] -2- [ benzylamino ] acetamide, 2-amino-N- [4- (2-phenylethynyl) phenyl ] propanamide, 2-amino-N- [4- (2-phenylethynyl) phenyl ] acetamide, 2- (cyclopropylamino) -N- [4- (2-phenylethynyl) phenyl ] acetamide, 2- (ethylamino) -N- [4- (2-phenylethynyl) phenyl ] acetamide, 2- [ (2-methylpropyl) amino ] -N- [4- (2-phenylethynyl) phenyl ] ethanamide Amide, 5-phenyl-2H-benzo [ d ]1, 3-dioxine (5-phenyl-2H-benzol [ d ]1, 3-dioxolene), 2-chloro-1-methoxy-4-phenylbiphenyl, 2- [ (imidazolylmethyl) amino ] -N- [4- (2-phenylethynyl) phenyl ] acetamide, 4-phenyl-1-phenoxybenzene, N- (2-aminoethyl) [4- (2-phenylethynyl) phenyl ] carboxamide, 2- { [ (4-fluorophenyl) methyl ] amino } -N- [4- (2-phenylethynyl) phenyl ] acetamide, 2- { [ (4-methylphenyl) methyl ] amino } -N- [4- (2-phenylethynyl) phenyl ] acetamide, 4-phenyl-1- (trifluoromethyl) benzene, 1-butyl-4-phenylbiphenyl, 2- (cyclohexylamino) -N- [4- (2-phenylethynyl) phenyl ] acetamide, 2- (ethylmethylamino) -N- [4- (2-phenylethynyl) phenyl ] acetamide, 2- (butylamino) -N- [4- (2-phenylethynyl) phenyl ] acetamide, N- [4- (2-phenylethynyl) phenyl ] -2- (4-pyridinylamino) acetamide, N- [4- (2-phenylethynyl) phenyl ] -2- (quinin-3-ylamino) acetamide, N- [4- (2-phenylethynyl) phenyl ] pyrrolidin-2-ylcarboxylate Amide, 2-amino-3-methyl-N- [4- (2-phenylethynyl) phenyl ] butanamide, 4- (4-phenylbut-1, 3-dienyl) phenylamine, 2- (dimethylamino) -N- [4- (4-phenylbut-1, 3-dienyl) phenyl ] acetamide, 2- (ethylamino) -N- [4- (4-phenylbut-1, 3-dienyl) phenyl ] acetamide, 4-ethyl-1-phenylbiphenyl, 1- [4- (2-phenylethynyl) phenyl ] ethyl-1-one, N- (1-carbamoyl-2-hydroxypropyl) [4- (4-phenylbut-1, 3-dienyl) phenyl ] carboxamide, N- [4- (2-phenylethynyl) phenyl ] propionamide, 4-methoxybenzyl-biphenyl-one, phenyl-N-benzamide, (tert-butoxy) -N- [ (4-phenylbiphenyl) methyl ] carboxamide, 2- (3-phenylbiphenoxy) ethyl hydroxamic acid, 3-phenylbiphenylpyruvate, 1- (4-ethoxyphenyl) -4-methoxybenzene and [4- (2-phenylethynyl) phenylpyrrole.

The phrase "non-fused heteroarylaryl" refers to non-fused arylarylarylaryls wherein one aryl is heteroaryl. Examples of heteroarylaryl include, for example, 2-phenylpyridine, phenylpyrrole, 3- (2-phenylethynyl) pyridine, phenylpyrazole, 5- (2-phenylethynyl) -1, 3-dihydropyrimidine-2, 4-dione, 4-phenyl-1, 2, 3-thiadiazole, 2- (2-phenylethynyl) pyrazine, 2-phenylthiophene, phenylimidazole, 3- (2-piperazinylphenyl) furan, 3- (2, 4-dichlorophenyl) -4-methylpyrrole, and the like. Preferred substituted non-fused heteroaryl aryl groups include: 5- (2-phenylethynyl) pyrimidin-2-ylamine, 1-methoxy-4- (2-thienyl) benzene, 1-methoxy-3- (2-thienyl) benzene, 5-methyl-2-phenylpyridine, 5-methyl-3-phenylisoxazole, 2- [3- (trifluoromethyl) phenyl ] furan, 3-fluoro-5- (2-furyl) -2-methoxy-1-prop-2-enylbenzene, (hydroxyimino) (5-phenyl (2-thienyl)) methane, 5- [ (4-methylpiperazinyl) methyl ] -2-phenylthiophene, 2- (4-ethylphenyl) thiophene, methyl-2-phenylthiophene, 4-methylthio-1- (2-thienyl) benzene, 2- (3-nitrophenyl) thiophene, (tert-butoxy) -N- [ (5-phenyl (3-pyridyl)) methyl ] carboxamide, hydroxy-N- [ (5-phenyl (3-pyridyl)) methyl ] amide, 2- (phenylmethylthio) pyridine, and benzylimidazole.

The phrase "non-fused heteroarylheteroaryl" refers to a non-fused arylaryl group in which both aryl groups are heteroaryl. Examples of heteroarylheteroaryl groups include, for example, 3-pyridylimidazole, 2-imidazolylpyrazine and the like. Preferred substituted non-fused heteroaryl groups include: 2- (4-piperazinyl-3-pyridyl) furan, diethyl (3-pyrazin-2-yl (4-pyridyl)) amine and dimethyl {2- [2- (5-methylpyrazin-2-yl) ethynyl ] (4-pyridyl) } amine.

The phrase "fused arylaryl" refers to an aryl group as defined above that is fused to an aryl group and fully conjugated. Representative fused arylaryls include diphenyl, 4- (1-naphthyl) phenyl, 4- (2-naphthyl) phenyl, and the like.

The phrase "fused heteroarylaryl" refers to an aryl group as defined above fused to and fully conjugated with a heteroaryl group. Representative fused heteroarylaryl groups include quinoline, quinazoline, and the like.

The phrase "fused heteroarylheteroaryl" refers to a heteroaryl as defined above fused to and fully conjugated with a heteroaryl. Representative fused heteroarylheteroarylheteroarylheteroaryls include pyrazolopyrimidine (pyrazalopyrimidine), imidazoquinoline, and the like.

The term "aryloxy" refers to RO-, wherein R is aryl. Representative arylalkoxy groups include benzyloxy, phenylethoxy, and the like.

The term "arylalkoxy" refers to a lower alkoxy group having an aryl group attached thereto. Representative arylalkoxy groups include benzyloxy, phenylethoxy, and the like.

The term "aryloxyaryl" refers to an aryl group having an aryloxy group attached thereto. Representative aryloxyaryl groups include 4-phenoxyphenyl, 3-phenoxyphenyl, 4-phenoxy-1-naphthyl, 3-phenoxy-1-naphthyl, and the like.

The term "aryloxyarylalkyl" refers to an arylalkyl group having an aryloxy group attached. Representative aryloxyarylalkyl groups include 4-phenoxyphenylmethyl, 3-phenoxyphenylmethyl, 4-phenoxyphenylethyl, 3-phenoxy-phenylethyl, and the like.

The term "arylalkoxyaryl" refers to an aryl group having an arylalkoxy group attached thereto. Representative arylalkoxyaryl groups include 4-benzyloxyphenyl, 3-benzyloxyphenyl and the like.

The term "arylalkoxyarylalkyl" refers to an arylalkyl group having an arylalkoxy group attached. Representative arylalkoxyarylalkyl groups include 4-benzyloxybenzyl, 3-benzyloxybenzyl, and the like.

The term "cycloalkyl" refers to an alicyclic group containing 3 to 7 carbon atoms and includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term "cycloalkylalkyl" refers to a lower alkyl group to which a cycloalkyl group is attached. Representative examples of cycloalkylalkyl groups include cyclopropylmethyl, cyclohexylmethyl, 2- (cyclopropyl) ethyl, and the like.

The term "halogen" means iodine, bromine, chlorine or fluorine; "halo" refers to iodo, bromo, chloro, or fluoro.

The term "haloalkyl" refers to a lower alkyl group as defined above bearing at least one halogen substituent, e.g., chloromethyl, fluoroethyl, or trifluoromethyl, and the like.

The term "heterocyclyl" (or heterocyclic, heterocyclic) means aromatic and non-aromatic cyclic compounds including monocyclic, bicyclic, and polycyclic cyclic compounds, such as, but not limited to, quinuclidinyl, containing 3 or more ring atoms, one or more of which is a heteroatom, such as, but not limited to N, O and S. Although the phrase "unsubstituted heterocyclyl" includes fused heterocyclic groups, such as benzimidazolyl, it does not include heterocyclic groups in which one of the ring atoms is attached to another group, such as alkyl or halo, such as 2-methylbenzimidazolyl or substituted heterocyclic groups. Examples of heterocyclyl groups include, but are not limited to: unsaturated 3-to 8-membered rings having 1 to 4 nitrogen atoms, such as, but not limited to, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1, 2, 4-triazolyl, 1H-1, 2, 3-triazolyl, 2H-1, 2, 3-triazolyl, etc.), tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.); saturated 3-8 membered rings containing 1-4 nitrogen atoms such as, but not limited to, pyrrolidinyl, imidazolinyl, piperidinyl, piperazinyl; fused unsaturated heterocycles containing 1 to 4 nitrogen atoms, such as, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolinyl, isoquinolinyl, indazolyl, benzotriazolyl; unsaturated 3-to 8-membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1, 2, 4-oxadiazolyl, 1, 3, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, etc.); saturated 3-8 membered rings containing 1-2 oxygen atoms and 1-3 nitrogen atoms, such as, but not limited to, morpholinyl; unsaturated fused heterocyclic rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl, benzooxadiazolyl, benzoxazinyl (e.g., 2H-1, 4-benzoxazinyl, etc.); unsaturated 3-to 8-membered rings containing 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms, such as, but not limited to, thiazolyl, isothiazolyl, thiadiazolyl (e.g., 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 3, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, etc.); saturated 3-8 membered rings containing 1-2 sulfur atoms and 1-3 nitrogen atoms, such as, but not limited to, thiazolidinyl; saturated and unsaturated 3-8 membered rings containing 1-2 sulfur atoms, such as, but not limited to, thienyl, dihydrodithiino, dihydrodithiolyl, tetrahydrothiophene, tetrahydrothiopyran; unsaturated fused heterocyclic ring containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as, but not limited to, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g., 2H-1, 4-benzothiazinyl, etc.), dihydrobenzothiazine (e.g., 2H-3, 4-dihydrobenzothiazine, etc.); unsaturated 3-8 membered rings containing oxygen atoms such as, but not limited to, furyl; unsaturated condensed heterocyclic ring containing 1 to 2 oxygen atoms, such as benzodioxolyl (e.g., 1, 3-benzodioxolyl, etc.); unsaturated 3-to 8-membered rings containing one oxygen atom and 1-2 sulfur atoms, such as, but not limited to, dihydrooxathiahexadienyl; saturated 3-to 8-membered rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms, such as 1, 4-oxathiane; unsaturated fused rings containing 1 to 2 sulfur atoms, such as benzothienyl, benzodiazadienyl; and unsaturated fused heterocycles containing one oxygen atom and 1-2 oxygen atoms, such as benzoxathienylene. Heterocyclyl also includes the above groups in which one or more S atoms in the ring is double bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include tetrahydrothiophene, tetrahydrothiophene oxide, and tetrahydrothiophene 1, 1-dioxide. Preferred heterocyclyl groups contain 5 or 6 ring atoms. More preferred heterocyclic groups include morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1, 2, 3-triazole, 1, 2, 4-triazole, tetrazole, thiomorpholine (wherein the sulfur atom of the thiomorpholine is bonded to one or more oxygen atoms), pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan and tetrahydrofuran.

The phrase "substituted heterocyclyl" refers to a heterocyclyl group as defined above wherein one ring member is bonded to a non-hydrogen atom as described above in substituted alkyl and substituted aryl groups. Examples include, but are not limited to, 2-methylbenzimidazolyl, 5-chlorobenzothiazolyl, 1-methylpiperazinyl, and 2-chloropyridyl. "aminosulfonyl" refers to the group-S (O)₂-NH₂. "substituted aminosulfonyl" refers to the group-S (O)₂-NRR’，Wherein R is lower alkyl and R' is hydrogen or lower alkyl. The term "aralkylaminosulfonylaryl" refers to the group-aryl-S (O)₂-NH-aralkyl, wherein the aralkyl is lower aralkyl.

"carbonyl" means a divalent group-C (O) -.

"carbonyloxy" generally refers to the group-C (O) -O-. Such groups include esters-C (O) -O-R, where R is lower alkyl, cycloalkyl, aryl or lower aralkyl. The term "carbonyloxycycloalkyl" generally refers to "carbonyloxycarbocycloalkyl" and "carbonyloxyheterocycloalkyl", i.e., wherein R is independently carbocycloalkyl or heterocycloalkyl. The term "arylcarbonyloxy" refers to the group-C (O) -O-aryl, wherein aryl is a monocyclic or polycyclic carbocyclic aryl or heterocyclic aryl. The term "aralkylcarbonyloxy" refers to the group-C (O) -O-aralkyl, where aralkyl is loweraralkyl.

The term "sulfonyl" refers to the group-SO₂-. "alkylsulfonyl" means having the structure-SO₂R-wherein R is alkyl. Alkylsulfonyl groups employed in compounds of the present invention are typically lower alkylsulfonyl groups containing from 1 to 6 carbon atoms in the backbone. Thus, alkylsulfonyl groups employed in compounds of the present invention typically include, for example, methylsulfonyl (i.e., R is methyl), ethylsulfonyl (i.e., R is ethyl), propylsulfonyl (i.e., R is propyl), and the like. The term "arylsulfonyl" refers to the group-SO₂-an aryl group. The term "aralkylsulfonyl" refers to the group-SO₂-aralkyl, wherein aralkyl is loweraralkyl. The term "sulfonamido" refers to-SO₂NH₂。

The term "carbonylamino" refers to the divalent group-NH-C (O) -in which the hydrogen atom of the amide nitrogen of the carbonylamino group may be substituted with a lower alkyl, aryl or lower aralkyl group. Such groups include both carbamate (-NH-C (O) -O-R) and amide (-NH-C (O) -O-R), wherein R is a straight or branched lower alkyl, cycloalkyl or aryl group or a lower aralkyl group. The term "lower alkylcarbonylamino" refers to alkylcarbonylamino wherein R is lower alkyl having 1-6 carbon atoms in the backbone structure. The term "arylcarbonylamino" refers to the group-NH-C (O) -R, where R is aryl. Similarly, the term "aralkylcarbonylamino" refers to a carbonylamino group wherein R is a lower aralkyl group.

The term "guanidino" refers to compounds derived from guanidine H2N-C (═ NH) -NH₂Part (c) of (a). Such moieties include moieties that are bound to a nitrogen atom with a formal double bond (the "2" -position of a guanidine, e.g., diaminomethyleneamino (H2N)₂C ═ NH —) and moieties bound to one of the two nitrogen atoms bearing the formal single bond (the "1-" and/or "3" -position of guanidine, for example, H₂N-C (═ NH) -NH-). The hydrogen atom on one of the nitrogen atoms may be substituted with a suitable substituent such as lower alkyl, aryl or lower aralkyl.

Representative cyclic imino and heterocyclic imino groups include, for example, those shown below. These cyclic and heterocyclic imino groups may also be substituted and may be incorporated at various positions as will be apparent to those skilled in the art of smart organic and pharmaceutical chemistry in conjunction with the description herein.

And

representative substituted amidino groups and heterocyclic amidino groups include, for example, those shown below. These amidino and heterocyclic amidino groups may also be substituted, as will be apparent to those skilled in the art of smart organic and pharmaceutical chemistry, in conjunction with the description herein.

And

representative substituted alkylcarbonylamino, alkoxycarbonylamino, aminoalkoxycarbonylamino and arylcarbonylamino groups include, for example, those shown below. These groups may also be substituted as will be apparent to those skilled in the art of fine organic and pharmaceutical chemistry in view of the description herein.

And

representative substituted aminocarbonyl groups include, for example, those shown below. These heterocyclic groups may also be substituted as will be apparent to those skilled in the art of fine organic and pharmaceutical chemistry in view of the description herein.

And

representative substituted alkoxycarbonyl groups include, for example, those shown below. These alkoxycarbonyl groups may also be substituted as will be apparent to those skilled in the art of smart organic and pharmaceutical chemistry in view of the description herein.

And

"substituted" means the explicit replacement of hydrogen with one or more monovalent or divalent groups. Suitable substituents include those described herein for the particular group, as well as hydroxy, nitro, amino, imino, cyano, halo, thio, thioamino, amidino, imino, oxo, oxamido, imino, guanidino, sulfonamido, carboxy, formyl, alkyl, substituted alkyl, halo-lower alkyl, lower alkoxy, halo-lower alkoxy, lower alkoxy-alkyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio, aminoalkyl, cyanoalkyl, benzyl, pyridyl, pyrazolyl, pyrrole, thiophene, imidazolyl and the like.

The term "linking moiety" refers to a covalent bond or a non-cyclized divalent group, e.g., -CO-, -O-, -S-, -CH₂-, -NH-, and substituted or unsubstituted alkyl, alkenyl, alkynyl, carbonyl, alkoxycarbonyl, as defined herein.

The term "SMIP compound" refers to a small molecule immune enhancing compound, including small molecule compounds having a molecular weight of less than about MW 1000g/mol, preferably MW 800g/mol, capable of stimulating or modulating a pro-inflammatory response in a patient. In one embodiment, the SMIP compound is capable of stimulating cytokine production by human peripheral blood mononuclear cells. Preferred SMIP compounds and derivatives thereof include, for example, amino azavinyl compounds, indolizine compounds, acyl piperazine compounds, indole dione compounds, Tetrahydroisoquinoline (THIQ) compounds, anthraquinone compounds, indandione compounds, phthalimide (pthalimide) compounds, benzocyclodione compounds, aminobenzimidazole quinolinone (ABIQ) compounds, hydroxyphthalimide (hydrapthalimide) compounds, pyrazolopyrimidine compounds, quinazolinone compounds, quinoxaline compounds, triazine compounds, tetrahydropyrrolidinoquinoxaline compounds, pyrrole compounds, biphenylone compounds, sterol compounds, and isoxazole compounds.

The term "SMIS compound" refers to small molecule immunosuppressant compounds, including small molecule compounds having a molecular weight of less than about MW 1000g/mol, preferably MW 800g/mol, which are capable of inhibiting or modulating a pro-inflammatory response in a patient.

The acyl piperazine compounds described herein include compounds of formula (III), as follows:

wherein,

R₉selected from the group consisting of substituted or unsubstituted aryl, heteroaryl, arylalkyl, arylalkenyl, heteroarylalkyl, and heteroarylalkenyl;

R₁₀is a substituted or unsubstituted alkyl group;

n is an integer of 0 to 2; and

if D is₁Is carbon, then D₂Is oxygen, D₃Is absent, and D₄Selected from the group consisting of substituted or unsubstituted aryl, heteroaryl, carbocyclyl, alkoxyaryl, fused arylaryl, fused arylheteroaryl and fused heteroarylaryl; or,

if D is₁Is nitrogen, then D₂Is nitrogen, D₄Is absent, and D₃Selected from the group consisting of substituted or unsubstituted aryl, heteroaryl, carbocyclyl, alkoxyaryl, fused arylaryl, fused arylheteroaryl and fused heteroarylaryl.

The indole dione compounds described herein include compounds of formula (IV), as described below:

wherein,

R₁₁and R₁₂Independently selected from the group consisting of H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid (carbocyclic acid), and substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl and carbocyclyl; and

R₁₃Selected from the group consisting of substituted or unsubstituted aryl, heteroaryl, arylalkyl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl and alkylbenzyl.

Tetrahydroisoquinoline (THIQ) compounds described herein include compounds of formula (V), as described below:

wherein,

l is a covalent bond or is selected from-CH₂-。-CO-、-O-、-S-、CHF、-NH-、-NR₂₀-, wherein R₂₀Is a lower alkyl group;

R₁₄selected from hydrogen, halogen or substituted or unsubstituted alkyl;

R₁₅selected from the group consisting of substituted or unsubstituted carbocyclyl, aryl, arylalkyl, alkoxyaryl, heteroaryl, heterocyclyl;

R₁₆selected from hydrogen, halogen or substituted or unsubstituted alkyl;

R₁₇selected from hydrogen, halogen or substituted or unsubstituted alkyl; and

R₁₈and R₁₉Is independently selected from_HHydroxyl, halogen, alkoxy, amino, unsubstituted alkyl, substituted alkyl, and alkylamino.

The benzocyclodione compounds described herein include compounds of formula (VI), as described below:

wherein,

e is selected from NR₂₅Or CR₂₆R₂₇；

R₂₁、R₂₃And R₂₄Independently selected from the group consisting of H, hydroxy, halo, alkoxy, amino, unsubstituted alkyl, substituted alkyl, and alkylamino;

R₂₂selected from the group consisting of H, hydroxy, halogen, alkoxy, amino and unsubstituted or substituted alkyl and alkylamino, arylalkyl, heteroarylalkyl, aryl, heteroaryl, arylcarbonyl, heterocyclyl, heterocyclylalkyl and heteroarylcarbonyl;

R₂₅Selected from the group consisting of substituted or unsubstituted aryl, heteroaryl, heterocyclyl, carbocyclyl, arylalkyl, heteroarylalkyl, and heterocycloalkyl;

R₂₆selected from the group consisting of H, halogen, hydroxy, amino, and substituted or unsubstituted alkyl, carbonylalkyl, and alkylcarbonylalkyl; and

R₂₇selected from the group consisting of aryl, arylalkyl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, carbocyclyl, arylcarbonylalkyl, and arylalkylcarbonyl.

The amino nitrogen heterovinyl compounds described herein include compounds of formula (VII), as follows:

wherein,

g is S or NH;

R₂₈selected from the group consisting of H, and substituted or unsubstituted alkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl groups;

q is selected from the group consisting of hydrogen, substituted alkyl, unsubstituted alkyl and aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, fused or non-fused arylaryl, substituted arylaryl, arylheteroaryl, substituted arylheteroaryl, heteroarylheteroaryl, and substituted heteroarylheteroaryl;

V₁selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, alkoxy, substituted alkoxy, aminocarbonyl, alkoxycarbonyl, carboxysulfonyl, methanesulfonyl and substituted or unsubstituted alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, lower alkylcarbonyl, lower alkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, substituted arylaminocarbonylamino, heteroarylamino, substituted aryl, arylalkyl, substituted arylalkyl, heteroarylcarbonyl, methanesulfonyl, arylcarbonyl, aralkylcarbonyl, and substituted or unsubstituted alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, alkylcarbonyloxy, alkylcarbonylamino, alkylaminocarbonylamino, Aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, cycloamidino, cycloalkyl, cycloimino, arylsulfonyl or arylsulfonamido; and

V₂Selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, alkoxy, substituted alkoxy, aminocarbonyl, alkoxycarbonylCarboxysulfonyl, methanesulfonyl and substituted or unsubstituted alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, lower alkylcarbonyl, lower alkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, cycloamidino, cycloalkyl, cycloimino, arylsulfonyl and arylsulfonamido.

Lactam compounds described herein include compounds of formula (VIII), as described below:

Wherein,

W₁selected from-OH, -OR₃₆、-NR₃₇R₃₈；

W₂Selected from O, S, NR₃₉

R₂₉And R₃₀Together form a substituted or unsubstituted 5-6 membered ring, all of which are carbon atoms or contain at least one O, N or S atom;

R₃₅and R₃₉May be the same or different and is selected from H, -OH, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, -C (═ O) H, -C (═ O) -alkyl, or-C (═ O) -aryl;

R₃₁、R₃₂、R₃₃and R₃₄Can be the same or different and are independently selected from H, Cl, Br, F, I, -NO₂、-CN、-OH、-OR₄₀、-NR₄₁R₄₂、-C(＝O)R₄₃-SH, substituted and unsubstitutedSubstituted amidino, substituted and unsubstituted guanidino, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkynyl, substituted and unsubstituted heterocyclic, substituted and unsubstituted alkylaminoalkyl, substituted and unsubstituted dialkylaminoalkyl, substituted and unsubstituted arylaminoalkyl, substituted and unsubstituted diarylaminoalkyl, substituted and unsubstituted (alkyl) (aryl) aminoalkyl, substituted and unsubstituted heterocyclylalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted heterocyclylaminoalkyl, substituted and unsubstituted diheteroylaminoalkyl, substituted and unsubstituted (alkyl) (heterocyclic) aminoalkyl, substituted and unsubstituted (aryl) (heterocyclic) aminoalkyl, substituted and unsubstituted hydroxyalkyl, substituted and unsubstituted alkoxyalkyl, substituted and unsubstituted alkoxyalkylalkyl, substituted and unsubstituted alkoxylalkyl, substituted and unsubstituted alkoxylalkylamino, substituted and unsubstituted cycloalkylalkyl, substituted and unsubstituted heterocyclylalkylamino, substituted and unsubstituted cycloalkylalkyl, Substituted and unsubstituted aryloxyalkyl and substituted or unsubstituted heterocyclyloxyalkyl;

R₃₆Selected from the group consisting of substituted and unsubstituted alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heterocyclyl, substituted and unsubstituted heterocyclylalkyl, -C (═ O) H, -C (═ O) -alkyl, -C (═ O) -aryl, -C (═ O) O-alkyl, -C (═ O) O-aryl, -C (═ O) NH-alkyl₂C (═ O) NH (alkyl), — C (═ O) NH (aryl), — C (═ O) N (alkyl)₂-C (═ O) N (aryl)₂-C (═ O) N (alkyl) (aryl), -NH₂NH (alkyl), -NH (aryl), -N (alkyl)₂N (alkyl) (aryl), -N (aryl)₂-C (═ O) NH (heterocyclyl), -C (═ O) N (heterocyclyl)₂-C (═ O) N (alkyl) (heterocyclyl) or-C (═ O) N (aryl) (heterocyclyl);

R₃₇selected from H, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, or substituted and unsubstituted heterocyclyl;

R₃₈selected from the group consisting of H, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heterocyclyl, -OH, alkoxy, aryloxy, -NH₂Substituted and unsubstituted heterocyclylalkyl groups, substituted and unsubstituted aminoalkyl groups, and substituted and unsubstituted alkylamino groupsArylalkyl, substituted and unsubstituted dialkylaminoalkyl, substituted and unsubstituted arylaminoalkyl, substituted and unsubstituted diarylaminoalkyl, substituted and unsubstituted (alkyl) (aryl) aminoalkyl, substituted and unsubstituted alkylamino, substituted and unsubstituted arylamino, substituted and unsubstituted dialkylamino, substituted and unsubstituted diarylamino, substituted and unsubstituted (alkyl) (aryl) amino, -C (═ O) H, -C (═ O) -alkyl, -C (═ O) -aryl, -C (═ O) O-alkyl, -C (═ O) O-aryl, -C (═ O) NH-alkyl ₂C (═ O) NH (alkyl), — C (═ O) NH (aryl), — C (═ O) N (alkyl)₂-C (═ O) N (aryl)₂-C (═ O) N (alkyl) (aryl), -C (═ O) -heterocyclyl, -C (═ O) -O-heterocyclyl, -C (═ O) NH (heterocyclyl), -C (═ O) -N (heterocyclyl)₂C (═ O) -N (alkyl) (heterocyclyl), -C (═ O) -N (aryl) (heterocyclyl, substituted and unsubstituted heterocyclyl aminoalkyl, substituted and unsubstituted diheterocyclylaminoalkyl, substituted and unsubstituted (alkyl) (heterocyclyl) aminoalkyl, substituted and unsubstituted (aryl) (heterocyclyl) aminoalkyl, substituted and unsubstituted hydroxyalkyl, substituted and unsubstituted alkoxyalkyl, substituted and unsubstituted aryloxyalkyl, or substituted and unsubstituted heterocyclyloxyalkyl;

R₄₁selected from H, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, or substituted and unsubstituted heterocyclyl;

R₄₂selected from the group consisting of H, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heterocyclyl, -C (═ O) H, -C (═ O) -alkyl, -C (═ O) -aryl, -C (═ O) NH₂C (═ O) NH (alkyl), — C (═ O) NH (aryl), — C (═ O) N (alkyl)₂-C (═ O) N (aryl)₂a-C (═ O) N (alkyl) (aryl), -C (═ O) O-alkyl, -C (═ O) O-aryl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkylaminoalkyl, substituted and unsubstituted dialkylaminoalkyl, substituted and unsubstituted arylaminoalkyl, substituted and unsubstituted diarylaminoalkyl, substituted and unsubstituted (alkyl) (aryl) aminoalkyl, substituted and unsubstituted heterocyclylalkyl -C (═ O) -heterocyclyl, -C (═ O) -O-heterocyclyl, -C (═ O) NH (heterocyclyl), -C (═ O) -N (heterocyclyl)₂-C (═ O) -N (alkyl) (heterocyclyl), -C (═ O) -N (aryl) (heterocyclyl), substituted and unsubstituted heterocyclylaminoalkyl, substituted and unsubstituted diheteroylaminoalkyl, substituted and unsubstituted (heterocyclyl) (alkyl) aminoalkyl, substituted and unsubstituted (heterocyclyl) (aryl) aminoalkyl, substituted and unsubstituted hydroxyalkyl, substituted and unsubstituted alkoxyalkyl, substituted and unsubstituted aryloxyalkyl, or substituted and unsubstituted heterocyclyloxyalkyl; and

R₄₃selected from H, -NH₂NH (alkyl), -NH (aryl), -N (alkyl)₂-N (aryl)₂N (alkyl) (aryl), -NH (heterocyclyl), -N (heterocyclyl) (alkyl), -N (heterocyclyl) (aryl), -N (heterocyclyl)₂Substituted and unsubstituted alkyl, substituted and unsubstituted aryl, -OH, substituted and unsubstituted alkoxy, substituted and unsubstituted heterocyclyl, substituted and unsubstituted aryloxy, heterocyclyloxy, -NHOH, -N (alkyl) OH, -N (aryl) OH, -N (alkyl) O-alkyl, -N (aryl) O-alkyl, -N (alkyl) O-aryl or-N (aryl) O-aryl.

Preferably R₂₉And R₃₀Together form a substituted or unsubstituted benzene ring.

The hydroxyphthalimide (hydrophthalamide) compounds described herein include compounds of formula (IX), as follows:

wherein,

R₄₄selected from substituted or unsubstituted aryl, heteroaryl, arylalkyl, heteroarylalkyl, fused arylaryl, non-fused arylaryl, fused heteroarylaryl, non-fused heteroarylaryl, fused arylheteroaryl or non-fused arylheteroaryl;

R₄₅、R₄₇、R₄₉and R₅₁May be the same or different and is independently selected from the group consisting of H, nitro, halogen, amino, hydroxy, cyano, carbocyclic acid, and substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl or carbocyclyl; and

R₄₆、R₄₈、R₅₀and R₅₂May be the same or different and is independently selected from H, halogen, and substituted or unsubstituted alkyl.

The biphenylketone compounds described herein include compounds of formula (X), as described below:

wherein,

R₅₃independently selected from H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, carbocyclyl;

R₅₄Independently selected from H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, carbocyclyl; and

o and p are integers from 0 to 4.

Isoxazole compounds described herein include compounds of formula (XI) as follows:

wherein,

R₅₅selected from the group consisting of substituted or unsubstituted aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl groups;

R₅₆selected from the group consisting of substituted or unsubstituted aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl groups; and

R₅₇selected from the group consisting of H, halogen, hydroxy, or substituted or unsubstituted alkyl, aryl, heteroaryl, heterocyclyl, and carbonyl.

The steroid compounds described in this application include compounds of formula (XII), as follows:

wherein,

R₅₈selected from the group consisting of substituted or unsubstituted aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl groups.

Preferably R₅₈Is a pyrone substituent.

The quinazolinone compounds described herein include compounds of formula (XIII), as follows:

wherein,

R₅₉selected from the group consisting of H, halogen, hydroxy, and substituted or unsubstituted alkyl, aminoalkyl, alkylaminoalkyl, alkoxy, dialkylaminoalkyl, hydroxyalkyl, alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl groups;

R₆₀selected from substituted or unsubstituted aryl, heteroaryl, arylalkyl, heteroarylalkyl, and heterocyclylalkyl groups; and

R₆₁、R₆₂、R₆₃and R₆₄May be the same or different and is independently selected from H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkyl, and carbocyclyl.

The pyrrole compounds described herein include compounds of formula (XIV), as follows:

wherein,

R₆₅selected from the group consisting of H, hydroxyl, and substituted or unsubstituted alkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, heteroarylaminoalkyl, arylaminoalkyl, heteroaryloxyalkyl, and aryloxyalkyl groups;

R₆₆、R₆₇、R₆₈And R₆₉May be the same or different and is independently selected from H, nitro, halogen, amino, hydroxy, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, carboxy, nitro, cyano, carbocyclic acid, cycloalkyl,arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, and carbocyclyl.

Other preferred azole compounds include those of the formula (XV):

wherein:

K₁is nitrogen, oxygen or optionally substituted carbon;

w is absent or selected from-O-, -S (O) -, -SO₂-、-NH-、-NH-CO-、-NR’CO-、-NHSO₂-、-NR’SO₂-、-CO-、-CO₂-、-CH₂-、-CF₂-, CHF, -CONH-, -CONR ' -, or-NR ' -, wherein R ' is alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl, heterocycle;

ar is an optionally substituted aryl, heteroaryl or protecting group;

R₇₀and R₇₀' is independently selected from hydrogen and methyl;

R₇₁、R₇₂、R₇₃and R₇₄Independently selected from hydrogen, hydroxy, and optionally substituted lower alkyl, cyclolower alkyl, cycloaminoalkyl, alkylaminoalkyl, lower alkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl, or heteroaryl;

R₇₅And R₇₈Independently selected from the group consisting of hydrogen, halo, and optionally substituted lower alkyl, cycloalkyl, alkoxy, amino, aminoalkoxy, carbonyloxy, aminocarbonyloxy, alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cycloimino, heterocycloiminoamino, amidino, cycloamidino, heterocycloiamidino, guanidino, aryl, heteroaryl, heterocycloikyl, heterocydoamino, aminoxy, cycloamidino, heterocycloimidamino, arylcarbonylamino, arylcarbonylaCyclocarbonyloxy, heteroarylcarbonyloxy and arylsulfonamido;

R₇₆selected from the group consisting of hydrogen, aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl;

R₇₇selected from the group consisting of hydrogen, hydroxy, halo, carboxy, nitro, amino, amido, amidino, imino, cyano, sulfonyl, methanesulfonyl, or substituted or unsubstituted alkyl, alkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, lower alkylcarbonyl, lower alkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, cyclosulfonamido, cyclothioamino, Cyclic amidino, heterocyclic amidino, cycloalkyl, cyclic imino, heterocyclic imino, guanidino, aryl, heteroaryl, heterocyclic, heterocycloalkyl, arylsulfonyl and arylsulfonamido groups;

The anthraquinone compound of the present invention includes, for example, a compound of the formula (XVI):

wherein,

R₇₉、R₈₀、R₈₁and R₈₂Can be the same or different and is independently selected from the group consisting of H, nitro, halogen, amino, hydroxy, cyano, carbocyclic acids, and substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, sulfonyl, aminosulfonyl, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylArylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, and carbocyclyl; and

R₈₃and R₈₄Together form a substituted or unsubstituted 5-6 membered ring which is all carbon or contains 1-2 heteroatoms selected from O, S or N.

The quinoxaline compounds described herein include partially non-conjugated tricyclic compounds, optionally substituted with nitrogen heteroatoms, and preferred embodiments of the quinoxaline are shown in (XVII):

wherein,

J₁is a group of C or N, or a group of N,

J₁' is selected from the group consisting of H, substituted aryl, unsubstituted aryl, substituted heteroaryl, and unsubstituted heteroaryl;

J₂is a group of C or N, or a group of N,

J₂' is selected from the group consisting of H, substituted aryl, unsubstituted aryl, substituted heteroaryl, and unsubstituted heteroaryl;

J₃Selected from-CO-, -NH-, or-N ═ N;

if J is₄is-O-, then J₄' absent; or,

if J is₄Is ═ C-, then J₄' is selected from H and substituted or unsubstituted alkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, arylalkyl, aminoalkyl, alkylamino and alkylthio; and

R₈₅、R₈₆、R₈₇、R₈₈and R₈₉May be the same or different and is independently selected from H, nitro, halogen, amino, hydroxy, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted,Alkoxy, alkylcarbonyl, alkylcarbonylamino, sulfonyl, aminosulfonyl, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl and carbocyclyl.

Triazine compounds refer to substituted 6-membered heterocyclic rings, the entire ring being substituted with 3 nitrogen atoms. Preferred embodiments of the present invention include those shown by structural formulas (XVIII), (XIX) and (XX):

wherein,

R₉₀selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, arylalkyl, and arylalkenyl;

R₉₁And R₉₃Independently selected from H and unsubstituted alkyl;

R₉₁is an aryl group; preferably a phenyl group,

wherein,

R₉₄selected from the group consisting of H, amino, alkyl, aminoalkyl and halogen;

R₉₅selected from the group consisting of substituted or unsubstituted aryl, arylamino, arylalkylamino, heteroaryl, heteroarylamino and heteroalkylamino;

R₉₆and R₉₇Independently selected from H, halogen or alkyl, preferably methyl; or,

R₉₆can form a double bond directly with the nitrogen atom, as indicated by the dashed line in the above formula; and

wherein,

R₉₈selected from H, substituted alkyl or unsubstituted alkyl; preferably a methyl group, and more preferably a methyl group,

R₉₉selected from H, substituted alkyl or unsubstituted alkyl; preferably an ethyl group is used as the solvent,

R₁₀₀selected from substituted or unsubstituted aryl, heteroaryl, alkoxyaryl, arylalkyl or heteroarylalkyl.

The indolizine compounds described herein include those described by the following formula (XXI):

wherein,

a is selected from-O-, -S-, -NH-or-NR₈-；

W is selected from-CH₂-, -O-, -S-, -NH-or-NR-₈-；

R₇Selected from the group consisting of carbocyclyl, non-fused carbocyclyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted fused arylheteroaryl, unsubstituted fused arylheteroaryl, substituted non-fused arylaryl, or unsubstituted non-fused arylaryl;

R₆Selected from substituted or unsubstituted aryl and heteroaryl; and

R₈independently a substituted or unsubstituted alkyl group.

The pyrazolopyrimidine compound described herein includes a compound represented by the following formula (XXII):

wherein,

R₁₀₁selected from H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, sulfonyl, aminosulfonyl, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, and carbocyclyl;

R₁₀₂selected from H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid, or substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, and carbocyclyl;

R₁₀₃selected from the group consisting of H, nitro, halogen, amino, hydroxyl, cyano, carbocyclic acid, trifluoromethyl, and substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, and carbocyclyl;

R₁₀₄Selected from the group consisting of H and substituted or unsubstituted aryl, heteroaryl, arylalkoxy, heteroarylalkoxy, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, carbocyclylalkyl, carbocyclyl;

R₁₀₅selected from H or substituted or unsubstitutedAryl, heteroaryl, arylalkoxy, heteroarylalkoxy, arylalkylamino, arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy, heterocyclylalkyl, carbocyclylalkyl, carbocyclyl of (a);

wherein R is₁₀₄And R₁₀₅At least one of which is not H.

SMIP compounds identified by in vitro (cellular or non-cellular assays) or in vivo methods are described in methods 1 and 2 below.

Pharmaceutical compositions containing the compounds of the present invention may be in any form suitable for the intended method of administration, including, for example, solutions, suspensions or emulsions. Solutions, suspensions and emulsions are generally prepared using liquid carriers. Liquid carriers useful in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvents, pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier can contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity modifiers, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols such as ethanol, and polyhydric alcohols such as glycerol. Suitable oils include, for example, soybean oil, peanut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the vehicle may also contain oleate esters, such as ethyl oleate, isopropyl myristate, and the like. The compositions of the present invention may also be in the form of microparticles, microcapsules, liposome capsules, and the like, as well as mixtures of two or more thereof.

Other additives include immunostimulants known in the art. Immunostimulatory oligonucleotides and polynucleotides are described in PCT WO 98/55495 and PCT WO 98/16247. U.S. patent application No.2002/0164341 describes adjuvants including unmethylated CpG dinucleotides (CpG ODN) and non-nucleic acid adjuvants. U.S. patent application No.2002/0197269 describes compositions containing an antigen, an antigenic CpG-ODN, and a polycationic polymer. Other immunostimulatory additives that have been described in the art may be used, for example, as described in U.S. patent nos. 5,026,546; U.S. patent nos. 4,806,352; and U.S. patent No.5,026,543.

Controlled release delivery systems may be used, such as controlled diffusion matrix systems or erodible systems, for example as described in: lee's "diffusion-Controlled matrix system" (pp. 155-198) and Ron and Langer's "erodible system" (pp. 199-224) published in the Controlled drug delivery discussion (Treatise on Controlled drug delivery), A.Kydonieus, Marcel Dekker, Inc., New York 1992. The matrix may be, for example, a biodegradable material that can spontaneously degrade in situ or in vivo, for example, by hydrolysis or enzymatic cleavage (e.g., protease cleavage). The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Examples of polymers having cleavable linkages include polyesters, polyanhydrides, polysaccharides, poly (phosphate esters), polyamides, polyurethanes, poly (iminocarbonates), and poly (phosphazenes).

The compounds of the present invention may be administered enterally, orally, parenterally, sublingually, by inhalation spray, rectally, or topically, in the form of unit dose formulations containing conventional non-toxic carriers, adjuvants, and vehicles which are pharmaceutically acceptable. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subcutaneous, rectal, and the like. Topical administration also includes transdermal administration, such as transdermal patches or iontophoretic devices. The term parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

Injectable preparations, for example sterile injectable solutions or oily suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-propanediol. Acceptable carriers and solvents that may be employed include water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with suitable non-irritating excipients such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and therefore melt in the rectum to release the drug.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose or starch. Typically, such dosage forms may also contain other substances in addition to inert diluents, for example, lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may contain buffering agents. In addition, tablets or pills may have enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. The composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

When referring to the mode of administration, it should be emphasized that it is a combination of therapeutic agents that provides a synergistic effect, whether the first and second agents are administered together or separately. Thus, the two agents may be administered together in a single dose or separately at different times and intervals.

An effective amount of a compound of the invention generally includes any dose sufficient to detectably treat a viral infection.

Successful treatment of a subject as described herein can result in the reduction or alleviation of symptoms of a subject afflicted with a drug or biological disorder, e.g., to prevent further progression of the disease or to prevent the disease.

The amount of active ingredient that may be combined with the carrier materials to form a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug composition and the severity of the particular disease undergoing therapy. The therapeutically effective amount in a given situation is readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

The compounds of the invention may be administered in the form of liposomes. Liposomes are typically prepared with phospholipids or other lipid substances, as is known in the art. Liposomes are formed by dispersing a monolayer or multilayer of hydrated liquid crystals in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The composition of the present invention in the form of liposome contains, in addition to the compound of the present invention, a stabilizer, a preservative, an excipient and the like. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods of forming liposomes are known in the art. See, e.g., Prescott, eds., "methods in Cell Biology" (Mthods in Cell Biology), volume XIV, Academic Press, New York, N.W., p.33, see (1976) below.

Although the SMIP compounds of the present invention may be administered as the sole active agent, they may also be used in combination with one or more other agents for the treatment of SARS. Other representative agents for use in combination with the compounds of the present invention to treat viral infections include, for example, interferon, ribavirin, gancylovir, and the like.

When other active agents are used in combination with the compounds of the present invention, the other active agents are generally used in therapeutic amounts as indicated in the physician's docket reference (PHYSICIANS' DESK REFERENCE, PDR, 53 rd edition, 1999), which is incorporated herein by reference, or in therapeutically effective amounts up to the level of ordinary skill in the art.

The compounds of the invention and other therapeutically active agents may be administered at the recommended maximum clinical dose or at lower doses. The dosage level of the active compound in the compositions of the invention may be varied depending upon the route of administration, the severity of the disease and the response of the patient to achieve the desired therapeutic response. The combined administration may be as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions administered at the same time or at different times, or the therapeutic agents can be administered as a single composition.

The compounds of the invention can be readily synthesized using the methods described herein or other methods well known in the art.

The compounds may be used in the form of salts of inorganic or organic acids. These salts include, but are not limited to: acetate, oxalate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, salicylate, pectate (pectate), persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate tartrate, thiocyanate, p-toluenesulfonate and undecanoate. The basic nitrogen-containing groups may additionally be quaternized with such agents as lower alkyl halides, for example methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfides such as dimethyl, diethyl, dibutyl and diamyl sulfides; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides, and the like. Thus, water-soluble or oil-soluble or water-or oil-dispersible products can be obtained.

Examples of acids which may be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulphuric acid and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Base addition salts can be prepared in situ in the final isolation and purification steps of the compounds of formula (I) or by reacting the carboxylic acid moiety with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with an ammonium or organic primary, secondary or tertiary amine, respectively. Pharmaceutically acceptable salts include, but are not limited to: cations of alkali or alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to: ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for forming base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

Various compounds and their synthesis are described in international patent application publication No. WO02/18327 (benzamide and pyridyl amide based compounds); WO0222598 and WO02/18383 (ABIQ-based compounds); and WO 02/81443 (phthalamide (pthalamide) -based compounds), all of which are within the context of the present invention, are useful for enhancing immunity. The entire disclosures of these U.S. patents and international publications are incorporated herein by reference. Other compounds or intermediates of interest to the present invention are purchased from commercial sources and used in the following processes: the chemical structures of interest were mapped into the ACD-SC database (from MDL Information Systems). Search the following companies/research institutes for identified compound supplier and purchase information: ASDI, ASINEX, BIONET, CHEMBRIDGE, CHEMDIV, CHEMPEX, CHEMSAR, COMGENEX, CSC, INTERBIOSCREEN, LABOTEST, MAYBRIDGE, MICROROSOURCE/GENESIS, OLIVIA, ORION, PEAKDALE, RYAN SCIENTIFIC, SPECS, TIMTEC, U OF FLORIDA and ZELINSKY.

Azalene compounds

Scheme 1

The compounds of the present invention containing a benzimidazole core may be prepared by a number of methods familiar to those skilled in the art. In one approach, appropriately functionalized diamines may be coupled with various thioisocyanates to form thiourea intermediates. Cyclization to form the benzimidazole moiety may be effected under known conditions, such as treatment with a carbodiimide or an alkyl halide. Alternatively, the diamine can be reacted sequentially with carbonyldiimidazole and phosphorus oxychloride and then coupled with the appropriate amine.

The compound having an oxazole structure can be prepared according to the above-mentioned method or other known conventional methods. Haviv et al (j.med.chem.1988, 31, 1719) describe a method of assembling oxazole cores wherein hydroxyaniline is treated with potassium ethyl xanthate. The resulting sulfonylbenzoxazole may then be chlorinated and coupled with an amine.

Compounds containing a benzothiazole core may also be prepared by known methods. The ortho-halothioisothiocyanate may be reacted with an amine to form a thiourea. Then reduced with NaH to form a thiazole ring.

Benzothiazoles are typically substituted by the methods of the present invention, for example, by the following synthetic routes:

synthesis of 4- [ (2- { [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } -

1H-benzimidazolyl-6-yl) oxy ] -N-methylpyridine-2-carboxamide

The following compound 4- [ (2- { [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } -1H-benzimidazol-6-yl) oxy ] -N-methylpyridine-2-carboxamide (159322) was synthesized:

step 1 Synthesis of 4- [ (4-amino-3-nitrophenyl) oxy]-N-methylpyridine-2-carboxamide: the mixture containing 4-amino-3-nitrophenol (1eq) and potassium bis (trimethylsilyl) amide (2eq) was stirred in dimethylformamide at room temperature for 2 hours. To the mixture were added (4-chloro (2-pyridyl)) -N-methylcarboxamide (1eq) and potassium carbonate (1.2eq) and stirred at 90 ℃ for 3 days. The reaction mixture was then concentrated and partitioned between ethyl acetate and water. The organic layer was separated, washed with brine, dried, filtered and concentrated in vacuo to give a brown solid. Purification on silica gel (2% triethylamine/50% ethyl acetate in hexane) afforded 4- [ (4-amino-3-nitrophenyl) oxy) as an orange solid]-N-methylpyridine-2-carboxamide. The product had satisfactory NMR. HPLC, 3.39 min; MS: MH⁺=289。

Step 2. Synthesis of 4- [ (3, 4-diaminophenyl) oxy]-N-methylpyridine-2-carboxamide: will contain [4- (3-amino-4-nitrophenoxy) (2-pyridyl)]The mixture of-N-is dissolved in methanol and hydrogenated with a catalytic amount of 10% Pd/C until the yellow color disappears to give the product amine. HPLC, 2.5 mins; MS: MH ⁺＝259。

Step 3 Synthesis of 4- [ (2- { [ 4-chloro-3- (trifluoromethyl) phenyl ] amino } -1H-benzimidazol-6-yl) oxy ] -N-methylpyridine-2-carboxamide:

dissolving in tetrahydrofuran and containing 4- [ (3, 4-diaminophenyl) oxy]A mixture of (E) -N-methylpyridine-2-carboxamide (1eq) and 4-chloro-3- (trifluoromethyl) benzeneisothiocyanate (1eq) was stirred at room temperature for 16 hours to give the corresponding thiourea. To the resulting mixture was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (2eq) and the mixture was stirred for another 10 hours. The mixture was concentrated and partitioned between ethyl acetate and water. The organic layer was washed with brine and dried. Purification by HPLCThe reaction product was converted to 4- [ (2- { [ 4-chloro-3- (trifluoromethyl) phenyl group]Amino } -1H-benzimidazol-6-yl) oxy]-N-methylpyridine-2-carboxamide. MS: MH⁺＝462

Synthesis of 4- ({2- [ (4-bromophenyl) amino ] -1-methyl-

1H-benzimidazolyl-5-yl } oxy) -N-methylpyridine-2-carboxamide

The following compound 4- ({2- [ (4-bromophenyl) amino ] -1-methyl-1H-benzimidazol-5-yl } oxy) -N-methylpyridine-2-carboxamide (161651) was synthesized:

step 1 Synthesis of 4- { [ 3-amino-4- (methylamino) phenyl group]Oxygen } -N-methylpyridine-2-carboxamide: 4- [ (4-amino-3-nitrophenyl) oxy ]A solution of (E) -N-methylpyridine-2-carboxamide (1eq) in methylene chloride was treated with trifluoroacetic anhydride (1eq) and stirred at 0 ℃ for 10 minutes. The mixture was washed with saturated NaHCO₃The solution was quenched. The organic layer was separated and washed with water, brine, dried and evaporated. MS: MH⁺＝385.2

Benzyltrimethylammonium chloride (1eq) dimethyl sulfate (1.2eq) was added to a solution of trifluoroacetamide (1eq) in a mixture of toluene, acetonitrile and sodium hydroxide (50%). The biphasic mixture was stirred at room temperature overnight and evaporated. The mixture was added to ethyl acetate, washed with water, brine, dried and evaporated. The crude product was purified by column chromatography eluting with 1: 1 hexane and ethyl acetate, then 2% triethylamine (1: 1 hexane and ethyl acetate) to give N-methyl-4- { [4- (methylamino) -3-nitrophenyl) as an orange-red solid]Oxy } pyridine-2-carboxamide. MS: MH⁺＝303.1。

A solution of nitromethylaniline in methanol was treated with 5% palladium on carbon and stirred at room temperature under hydrogen for 15 minutes (until the yellow colour disappeared). The mixture was filtered and the filtrate was concentrated to give 0.36 g of diamine 4- { [ 3-amino-4- (methyl) amineAmino) phenyl]Oxy } -N-methylpyridine-2-carboxamide. MS: MH ⁺＝273.3。

Step 2 Synthesis of 4- ((2- [ (4-bromophenyl) amino)]-1-methyl-1H-benzimidazolyl-5-yl } oxy) -N-methylpyridine-2-carboxamide: diamine 4- { [ 3-amino-4- (methylamino) phenyl]A solution of oxygen } -N-methylpyridine-2-carboxamide (1eq) in methanol was treated with 4-bromophenyl isothiocyanate (1eq) and stirred at 60-65 ℃ for 2 hours. The reaction mixture was cooled to room temperature, methyl iodide (1eq) was added and stirred at 60 ℃ overnight. The reaction was cooled to room temperature, evaporated, poured into ethyl acetate, washed with water and brine, dried and evaporated under reduced pressure. Column chromatography using a gradient solvent system (hexane and ethyl acetate, and 1: 1 methylene chloride in methylene chloride and acetone or 5% methanol) gave the product as a semi-white powder. MS: MH⁺＝452.3

Aminobenzimidazolyl quinolinones

Compounds of structure I are synthesized from simple starting molecules shown in schemes 1-4 and are exemplified in the examples. Compounds of structure I are generally prepared from aromatic compounds substituted with amine and carboxylic acid groups as shown in scheme 1.

Scheme 2

As shown in scheme 2, a substituted aromatic compound such as a substituted or unsubstituted 2-aminobenzoic acid can be reacted with an acid halide such as methyl 2- (chlorocarbonyl) acetate to produce an amide, which will react with a substituted or unsubstituted 1, 2-diaminobenzene. The resulting product is a 4-hydroxy-substituted compound of structure I. One skilled in the art will recognize that the methods outlined in scheme 1 can be modified to produce a variety of compounds.

A method for preparing 4-amino substituted compounds of structure I is shown in scheme 3. As shown in scheme 3, the 4-amino substituted compounds of structure I can be synthesized using amine and nitrile substituted aromatic compounds. Compounds such as ethyl 2-cyanoacetate may be reacted with ethanol to produce ethyl 3-ethoxy-3-iminopyruvate hydrochloride. Followed by reaction with substituted or unsubstituted 1, 2-phenylenediamine to produce substituted or unsubstituted ethyl 2-benzimidazolyl-2-ylacetate. Substituted or unsubstituted ethyl 2-benzimidazolyl-2-ylacetate is reacted with an aromatic compound containing amine and nitrile groups, such as substituted or unsubstituted 2-aminobenzonitrile, and a base, such as lithium bis (trimethylsilyl) amide or a lewis acid, such as tin tetrachloride, to give a substituted or unsubstituted 4-amino-substituted compound of structure I.

Scheme 3

Scheme 4 shows a general synthetic route to the synthesis of 4-dialkylamino and 4-alkylamino compounds of structure I. Scheme 3 shows that the 4-hydroxy substituted compounds of structure I can be converted to 4-chloro derivatives by reaction with phosphorus oxychloride or thionyl chloride. The 4-chloro derivative may then be reacted with an alkylamine or dialkylamine to produce the corresponding 4-alkylamino or 4-dialkylamino derivative. Deprotection gives the final 4-alkylamino or 4-dialkylamino compound of structure I. Other groups that can also be reacted in this manner with the 4-chloro derivative include, but are not limited to, ROH, RSH, and CuCN.

Scheme 4

As shown in scheme 5, substituted or unsubstituted 2-benzimidazolyl-2-yl acetates prepared according to schemes 3 and 4 can be used to synthesize compounds of structure I having H, an alkyl group, an aryl group, or a heterocyclic group at the 4-position.

Thiosemicarbazone (thiosemarbanone)

General Process for the preparation of thiosemicarbazones

Scheme 6

A solution of aldehyde (1.0 equiv.) and thiosemicarbazone (1.05 equiv.) in acetic acid was stirred overnight. Removal of excess acetic acid gives a residue, which is washed with ethanol or purified by preparative HPLC to give the thiosemicarbazone.

Scheme 7

A solution of the aldehyde (1.0 eq), thiosemicarbazone (1.05 eq) and acetic acid (0.1 eq) in methanol was stirred overnight. Methanol is removed to give a residue, which can be used in scheme 6.

Scheme 8

Arylamine (2.1 equivalents) was added to a solution of { [ (1E) -1-aza-2- (4-fluoro-3-nitrophenyl) vinyl ] amino } -aminomethane-1-thione in ethanol. The solution was stirred at room temperature until the initial fluoride disappeared. The solution was purified to the product.

Scheme 9

A mixture of 4- (diethylamino) -2-hydroxybenzaldehyde (1 eq), benzyl bromide (1.2 eq) and powdered potassium carbonate in ethanol was stirred at room temperature for 2 days. The ethanol was removed and the residue was dissolved in ethyl acetate and water. NaHCO for organic layer ₃Washed with aqueous solution and brine, and Na₂SO₄Dried and concentrated. The residue was purified on silica gel eluting with ethyl acetate/hexane to give 4- (diethylamino) -2-benzoyloxy-benzaldehyde.

The aldehyde is converted to the thiosemicarbazone according to scheme 7.

Scheme 10

A solution of 3, 4-difluorobenzonitrile (1 equivalent), amine (1.5 equivalents) and DIEA (2 equivalents) in NMP was heated in a Smith microwave oven (Personal Chemistry) for 30 minutes. The reaction mixture was purified over silica gel to give 4-substituted 3-fluorobenzonitrile.

DIBAL-H (1M in toluene, 1.5 equiv.) is added to a toluene solution of nitrile at-78 ℃. The reaction mixture was allowed to warm to room temperature and stirred for 16 h, quenched with methanol/ethyl acetate/brine (1: 4). After stirring at room temperature for 30 min, the solution was extracted with ethyl acetate (3 ×). The combined organic layers were washed with NaHCO₃Aqueous, brine, and concentrated. The aldehyde is purified on silica gel or directly converted to the thiosemicarbazone (scheme 7).

Scheme 11

A solution of 2, 4, 5-trifluorobenzonitrile (1 equivalent) and 4-arylpiperazine (1.2 equivalents) and DIEA (1.2 equivalents) in THF was heated at 80 ℃ for 2 hours. The mixture was purified on silica gel to give 4-substituted 2, 5-difluorobenzonitrile.

Scheme 12

To the alcohol (1.0 eq) was added a solution of potassium tert-butoxide in THF (1M, 1.1 eq). After 5 minutes, this solution was added to a solution of 4-N-substituted-2, 5-difluorobenzonitrile (1 eq) in THF. The reaction mixture was stirred at room temperature overnight and quenched with aqueous ammonium chloride. The aqueous layer was extracted with ethyl acetate (3 ×). The combined organic layers were washed with brine and concentrated to give a residue which was purified to give 4-N-substituted-2-O-substituted-5-fluorobenzonitrile.

Following the procedure of scheme 10, DIBAL-H is used to reduce 4-N-substituted-2-O-substituted-5-fluorobenzonitrile to give 4-N-substituted-2-O-substituted-5-fluorobenzaldehyde.

The aldehyde is converted to the corresponding thiosemicarbazone using scheme 7.

Scheme 13

A solution of 4-N-substituted-2, 5-difluorobenzonitrile (1 equivalent), amine (1.5 equivalents) and DIEA (2 equivalents) in NMP was heated in a Smith microwave oven (Personal Chemistry) for 30 minutes. The reaction mixture was purified by silica gel to give 4-N-substituted-2-N-substituted-5-fluorobenzonitrile.

DIBAL-H reduction of 4-N-substituted-2-N-substituted-5-fluorobenzonitrile with DIBAL-H affords 4-N-substituted-2-N-substituted-5-fluorobenzaldehyde according to the procedure of scheme 10.

Preparation of amino {3- [5- (3-chlorophenyl) (2-furyl) ] (2-pyrazolinyl) } methane-1-thione

MeMgBr (3.0 equiv.) in ether was added to a solution of 5- (3-chlorophenyl) furan-2-carbaldehyde (5- (3-chlorophenyl) furan-2-carbaldehyde) (1.0 equiv.) in THF at 0 deg.C and stirred for 45 minutes. The reaction was quenched with water, diluted with ether and filtered through Celite (Celite). The organic layer was separated and washed with brine, over MgSO₄Drying and concentrating to obtain 1- [5- (3-chlorophenyl) -2-furyl]Ethan-1-ol.

CH at secondary alcohol (1.0 equivalent) ₂Cl₂Adding MnO into the solution₂(10 equivalents). The reaction was stirred overnight, filtered through Celite, and concentrated to give 1- [5- (3-chlorophenyl) -2-furyl]Ethan-1-one.

Concentrated hydrochloric acid (cat.) was added to a mixture of ketone (1.0 eq.), paraformaldehyde (2.0 eq.), dimethylamine hydrochloride (2.0 eq.), and molecular sieve in ethanol. The reaction was refluxed overnight under nitrogen and concentrated. A few drops of HCl were added and the mixture was made up with DCM and water. The organic layer was discarded. The aqueous layer was made basic and extracted with DCM (3 ×). The organic layer was washed with brine, over MgSO₄Drying and concentrating to obtain 3- (dimethylamino) -1- [5- (3-chlorophenyl) (2-furyl)]Propan-1-one.

Is prepared from sulfurSemicarbazone (1.0 eq) was dissolved in MeOH while heated under nitrogen. To the reaction was added aqueous sodium hydroxide (6M, 9.0 equiv). Adding 3- (dimethylamino) -1- [5- (3-chlorophenyl) (2-furyl) dropwise to the reaction mixture]Propan-1-one (1.0 equiv) in methanol. The solvent was removed, the residue dissolved in DCM and washed with water, brine, over MgSO₄Dried and concentrated. The final compound was purified by preparative HPLC to give amino {3- [5- (3-chlorophenyl) (2-furyl)](2-pyrazolinyl) } methane-1-thione; LC/MS m/z306.2(MH +); rt 3.06 min.

Scheme 14

CHCl in 4-pyridylmethylamine (1.0 equiv.) and triethylamine (2.0 equiv.)₃Adding CS into the solution₂(1.0 eq)) and stirred overnight. The reaction was cooled to 0 ℃ and ethyl chloroformate (1.0 equiv) was added dropwise. The reaction was stirred at 0 ℃ for 15 minutes and then at room temperature for 2 hours, followed by addition of (tert-butyl) oxycarbohydrazide (1.2 equivalents). After stirring for another 1 h, the mixture was stirred with aqueous citric acid (5%) and saturated NaHCO₃And brine, over MgSO₄Dried and concentrated. The desired Boc-protected thiosemicarbazone was purified by column chromatography.

To a solution of Boc-protected thiosemicarbazone (1.0 eq) in DCM was added a solution of HCl in dioxane (2M, 8.3 eq) and stirred for 15 min. MeOH was then added to dissolve the precipitate, and additional furfural and a small amount of acetic acid (0.5mL) were added. The mixture was stirred overnight and the solvent was removed to give a residue which was purified by preparative-HPLC to give the thiosemicarbazone.

Synthesis of 4- [4- (4-methylpiperazin-1-yl) phenoxymethyl ] benzaldehyde

In the process of coolingTo 0 ℃ of 4-piperazin-1-ylphenol (1 equivalent) in CHCl₃To the solution was added dropwise di-tert-butyl carbonate (1 equivalent) of CHCl₃And (3) solution. The solution was stirred at 0 ℃ for 1 hour, then removed from the cold bath and stirred at room temperature for 18 hours. NaHCO for organic solution ₃The aqueous solution and brine were washed over MgSO₄Dried and concentrated and the crude material was used without purification.

At room temperature under nitrogen, the resulting 4- (1-BOC-piperazin-4-yl) phenol (1 equivalent) in anhydrous CH₃Adding anhydrous CH slowly dropwise into CN solution₃Slurry of NaH (1 eq) in CN. The slurry was stirred at room temperature for 2 hours, then the solid was filtered and Et₂And O washing.

Sodium 4- (1-BOC-piperazin-4-yl) phenolate (1 eq) was mixed with methyl 4-bromomethylbenzoate (1 eq) in anhydrous acetone and heated at 60 ℃ under reflux for 18 hours. The slurry was filtered and the filtrate was concentrated to give crude methyl 4- [4- (1-BOC-piperazin-4-yl) phenoxymethyl ] benzoate, which was used without purification.

LiAlH in anhydrous THF cooled to 0 ℃ under nitrogen₄(4 equiv.) of the slurry 4- [4- (1-BOC-piperazin-4-yl) phenoxymethyl was added slowly dropwise]Methyl benzoate (1 eq) in anhydrous THF. Once completely added, the slurry was heated to reflux at 80 ℃ for 1 hour. The slurry was then cooled to 0 ℃ and treated with water, 10% aqueous NaOH, and then water. The resulting solid was filtered, the filtrate diluted with chloroform, washed with brine, and then passed over MgSO₄Drying and concentrating to obtain crude 4- [4- (4-methylpiperazin-1-yl) phenoxymethyl ]Benzyl alcohol, used without purification.

Oxalyl chloride (1.1 equiv) dissolved in DCM was added dropwise to a solution of DMSO (2.6 equiv) in anhydrous DCM cooled to-78 ℃ under nitrogen. The solution was stirred at-78 ℃ for 5 minutes, and then 4- [4- (4-methylpiperazin-1-yl) phenoxymethyl was added dropwise]A solution of benzyl alcohol (1eq) in DCM and stirred for a further 30 minutes at-78 ℃. Triethylamine (2.5 equivalents) was added slowly and the solution was allowed to reach room temperature. Solution NaHCO₃The aqueous solution and brine were washed over MgSO₄Drying and concentrating to obtain crude 4- [4- (4-methylpiperazin-1-yl) phenoxymethyl]Benzaldehyde, which is converted to the thiosemicarbazone according to scheme 7.

Azole compounds

Scheme 15

Synthesis of azoles

Preparation of tert-butyl (2E) -3- (2, 4-dichlorophenyl) prop-2-enoate (2).

At room temperature under argon, with thorough stirring, cinnamate (1eq), tert-butanol (4eq), DMAP (1.4eq), and CH₂Cl₂To the solution was added pure DIC (1.4 eq). (Note-the cinnamate must be completely dissolved in solution and therefore may require slight heating-allow the solution to cool to room temperature and then add DIC₂Cl₂DIC was carefully diluted and added and an ice bath was prepared. ) After stirring for 8 hours, a white precipitate formed in the reaction. The reaction was checked by TLC, eluting with 25% EtOAc in hexanes (R of the product) _f0.9). Transfer all reaction to separatory funnel (with CH)₂Cl₂Washing). The organic mixture was treated with citrate, saturated NaHCO₃Aqueous solution, water and brine. The organic layer was dried (Na)₂SO₄) Filtered and concentrated to dryness to give a crude product as an oil. Will be coarseThe resulting oil was mixed with hexane and stirred for 30 minutes. The precipitate formed was filtered through celite and the filtrate was evaporated. The hexane mixture was transferred to a silica plug core and eluted with EtOAc/hexane (97: 2 v/v). The first eluted UV active was collected and evaporated to give 2 with a purity > 99% (75-80% yield).

Preparation of tert-butyl 4- (2, 4-dichlorophenyl) pyrrole-3-carboxylate (3).

Anhydrous ether was added to NaH (1.5eq, oily dispersed phase) under argon. The ether was removed by decantation through syringe and NaH was suspended in fresh ether under argon. A solution of TOSMIC (1.1eq) and 2(1eq) in a mixture of ether and DMSO was added dropwise to the stirred NaH suspension over a period of 20-30 minutes at 0 ℃. The addition was slightly exothermic and gas was formed. After addition, the reaction was allowed to return to room temperature. The reaction was then subjected to TLC (25% EtOAc/hexanes, R of the UV-active product)_f0.4) and LCMS until the reaction is complete (about 2-3 hours). After the reaction is finished, saturated NH is used ₄Aqueous Cl carefully quench the reaction (slowly added to avoid strong gas evolution and exotherm) and dilute with ether. The layers were separated and the organic phase was washed with saturated NaHCO₃Aqueous solution, water and brine. The crude black solid can be purified by recrystallization. Best results were obtained by direct recrystallization from a hot EtOAc/hexane (1: 3v/v) mixture or by dissolving the crude product in slightly hot EtOAc and then adding hexane (the volume of hexane was about twice the volume of EtOAc). The hot solution was then allowed to cool to room temperature and aged overnight. The crystals were first filtered and then washed with hexane to give the product 99% pure in 60-70% yield.

Preparation of tert-butyl 4- (2, 4-dichlorophenyl) -1- [3- (1, 3-dioxabenzo [ c ] oxazolin-2-yl) propyl ] pyrrole-3-carboxylate (4).

Solid NaH (1.5eq, oily dispersed phase) was added to a small portion of a solution of pyrrole 3(1eq) and 3-bromopropylphthalimide (1.2eq) in DMF under stirring at room temperature under argon. Note-some gas formation, but the temperature does not appear to rise above 40-50 ℃. The reaction was stirred at room temperature under argon for 1.5 hours. Then TLC (CH)₂Cl₂Acetonitrile (95: 5v/v), R of UV-active product_f0.5) and LCMS. After the reaction is finished, saturated NH is used ₄Aqueous Cl carefully quenched the reaction (slowly added to avoid intense gas evolution and exotherm). Then saturated NaHCO was added₃Aqueous solution to avoid emulsification and extraction of the basic organic mixture with ether. The combined ether layers were washed with saturated NaHCO₃Washing with aqueous solution, water, brine, and passing over Na₂SO₄Dried, filtered and concentrated to dryness to give the crude product. The crude product was purified by elution through silica with EtOAc/hexanes (1: 4 v/v). The purified product contains some residual 3-bromopropylphthalimide, which does not interfere with the subsequent synthetic steps. The material was removed and used without further purification. Assuming that the yield is constant.

Preparation of tert-butyl 1- (3-aminopropyl) -4- (2, 4-dichlorophenyl) pyrrole-3-carboxylate (5).

Phthalimidopyrrole (pthalimido pyrrolo) 4(1eq) was dissolved in ethanol and hydrazine (3eq) at room temperature under nitrogen. After heating and refluxing, white precipitate was formed. The reaction was subjected to TLC (CH)₂Cl₂Acetonitrile (95: 5v/v), R of UV-active product_f0.2) and LCMS stirred at reflux until reaction was complete (about 2 hours). After the reaction was complete, the reaction was cooled to room temperature and the medium was filtered off in vacuo through a fine sintered glass filter. The filtrate was concentrated under reduced pressure to a viscous solid. The crude product was taken up in ethanol/EtOAc (1: 1v/v), stirred and the precipitate was filtered off in the same manner as described above. Concentrating the filtrate under reduced pressure And then dried under vacuum for 10-15 minutes. The process of adding ethanol/EtOAc, filtering and concentrating was repeated one or more times, or as needed, to remove most of the white precipitate and residual hydrazine. The product was then dried under vacuum overnight. The resulting material was used without further purification. Once dried, the reaction yielded a glassy product (yield of about 87% for 2 steps).

Preparation of tert-butyl 1- {3- [ (6-amino-5-nitro (2-pyridyl)) amino ] propyl } -4- (2, 4-dichlorophenyl) pyrrole-3-carboxylate (7).

To a premixed anhydrous reagent of pyrrole 5(1eq) and 6-chloro-3-nitro-2-pyridylamine (6) (1.1eq) in powder form was added DMA followed by Hunig's base (2eq) and subsequent stirring at room temperature. The reaction was then heated to 80 ℃ overnight. The reaction was then subjected to TLC (EtOAc/hexanes (1: 1v/v), R of the UV-active yellow product_f0.25), HPLC and LCMS. After the reaction was judged complete by HPLC, the reaction was cooled to 70 ℃. Ethylenediamine (anhydrous) was then added to the reaction to destroy any residual unreacted chloropyridine 6. After stirring at 70 ℃ for 15 minutes, the reaction was cooled and saturated NaHCO was added₃The aqueous solution was quenched. The aqueous mixture was extracted with EtOAc and the combined organic layers were extracted with saturated NaHCO ₃The aqueous solution, water, brine were washed, dried, filtered, and concentrated to dryness to give the crude product as a tan solid. The crude product was purified by flash chromatography eluting with EtOAc/hexane (4: 6 v/v). The purified SnAr adduct 7 was isolated in 58% yield as a yellow solid.

Preparation of 1- {3- [ (6-amino-5-nitro (2-pyridyl)) amino ] propyl } -4- (2, 4-dichlorophenyl) pyrrole-3-carboxylic acid (8).

Pyrrole tert-butyl ester 7(1eq), water (. 1%) and CH in a test tube at room temperature₂Cl₂To the mixture was added TFA (catalytic amount) with stirring. The tube was stirred at room temperature until the reaction was complete (about 12 hours). The reaction was then concentrated at room temperature under reduced pressure and dried under vacuum. Redissolving the crude residue in CH₂Cl₂And concentrated under reduced pressure at room temperature. The resulting material was used as a TFA salt in the final coupling step without further purification.

Preparation of N- ((1S) -2-hydroxy-isopropyl) (1- {3- [ (6-amino-5-nitro (2-pyridyl)) amino ] propyl } -4- (2, 4-dichlorophenyl) pyrrol-3-yl) carboxamide (9,).

(2S) - (+) -2-aminopropan-1-ol (1.5eq) was added under argon at room temperature to a mixture of acid (8) (1eq), HBTU (1.5eq), Hunig' S base (2eq) and DMF (premixed in tube in this order). The reaction was stirred for 3-4 hours until the end of the reaction was indicated by LCMS and HPLC. The reaction mixture was then diluted with EtOAc and with NaHCO ₃Washed and concentrated to give a powder in 70% yield.

The compounds in the examples were named using ACD Name software (version 5.07, 2001/11/14) available from Advanced Chemistry Development, Inc. Some compounds and starting materials are named using standard IUPAC nomenclature.

The compounds in table 34 were synthesized using the synthetic methods described in the above examples and schemes and screened according to methods 1 and 2 below. The precursors used are readily known to those skilled in the art and are available from Aldrich (Milwaukee, Wis.) or Acros Organics (Pittsburgh, Pa.).

Method for screening SMIP/SMIS compound

Method 1

Candidate small molecule immunopotentiators can be identified in vitro. Screening is performed in vitro based on the ability of the compound to activate immune cells. One marker of such activity is the induction of cytokine production (e.g., the production of TNF-. alpha.). Small molecules that induce apoptosis were identified as having this activity. These small molecule immunopotentiators have potential uses as adjuvants and immunotherapies.

In the assay procedure for Small Molecule Immunopotentiators (SMIPs) (high throughput screening (HTS)), human Peripheral Blood Mononuclear Cells (PBMCs) (500,000/mL in RPMI 1640 medium supplemented with 10% FCS) were added to 96-well plates (100,000 per well) that already contained 5 μ M compound formulated in DMSO. PBMC were incubated at 37 ℃ with 5% CO ₂The cells were incubated for 18 hours. They were tested for their ability to produce cytokines in response to small molecule compounds using a modified sandwich ELISA.

Briefly, secreted TNF was measured in the supernatant of PBMC cultures using plates conjugated with a primary antibody for capture, followed by sandwich formation with a biotinylated anti-TNF secondary antibody. The biotinylated secondary antibody was then detected with avidin-europium and the amount of europium bound was determined by time-resolved fluorescence. In this experiment, the TNF-inducing activity of SMIP compounds was demonstrated as an increase in Europim counts over cells incubated in RPMI medium alone. "hits" were selected on the basis of their TNF-inducing activity at an optimal dose relative to LPS (1. mu.g/ml), a potent inducer of TNF. The robustness of the assay and low background allowed routine selection of hits with about 10% LPS activity, typically 5-10 times background (cells only). It was then necessary to confirm whether the selected hit compounds induced cytokine production by multiple blood donors at low concentrations. Those compounds with constant activity at concentrations of 5 μ M or less are considered to be compatible with the purpose of this assay. The assay can be readily modified to screen compounds that are effective at higher or lower concentrations.

Method 2

Each of the compounds in Table 34 induces TNF- α production by human peripheral blood mononuclear cells. Many of these compounds show TNF- α -inducing activity below 20 μ M. Many compounds show TNF- α -inducing activity below 5 μ M. Many compounds show activity inducing TNF- α production below 1.5. mu.M.

For this reason, each R group of any of the compounds listed in table 34 is preferred. Furthermore, each of these compounds is preferred due to each compound's excellent activity, and preferably as a member of the group including any or all other compounds, and each compound preferred in methods of modulating immune enhancement and methods of treating biological conditions related thereto, for example, may be used as a vaccine adjuvant. It is also preferred to use each compound in the manufacture of a medicament for use in vaccination, immune enhancement, reduction of tumor growth and treatment of biological conditions resulting therefrom. In addition to the above methods, methods for measuring other cytokines (e.g., IL 1-beta, IL-12, IL-6, IFN-gamma, IL-10, etc.) are well known in the art and can be used to find active SMIP compounds of the present invention.

Compounds that cause TNF- α production at higher concentrations, such as 100. mu.M, 200. mu.M or 300. mu.M, in the above assays can be used. For example, Roxorubine (Loxoribine) can lead to efficient TNF- α production at 300 μ M (see Pope et al, Immunostimulatory Compound 7-Allyl-8-oxoguanosine (Roxoribine) Induces a significant Subset of Murine Cellular immunocytokines (Immunostimulation Compound 7-Allyl-8-oxoguanosine (Loxoribine) industries a Distingt Subset of Murine Cytokines Cellular Immunology 162: 333-.

The present invention also includes isotopically-labeled antiviral compounds, which are identical in structure to those recited above, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the antiviral compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³¹P、³²P、³⁵S、¹⁸F and³⁶and (4) Cl. Containing isotopes and/or other atoms as described aboveOther isotopes of the antiviral compounds of the present invention, derivatives thereof, and pharmaceutically acceptable salts of said compounds and said derivatives are included within the scope of the present invention. Certain isotopically-labelled antiviral compounds of the present invention, for example, into which a radioactive isotope such as³H and¹⁴c, are useful in drug and/or substrate tissue distribution assays. Due to tritium (i.e. tritium)³H) And carbon-14 (i.e.¹⁴C) Isotopes are particularly preferred because they are easy to prepare and detect. In addition, heavier isotopes such as deuterium (i.e. deuterium) are used²H) Substitution may provide certain therapeutic benefits due to higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and is therefore preferred in some circumstances. Isotopically-labeled antiviral compounds and derivatives thereof of the present invention can generally be prepared by known procedures or by procedures described in the references, and readily available isotopically-labeled reagents can be substituted for non-isotopically-labeled reagents.

According to the present invention, methods of administering an effective amount of a SMIP compound as an adjuvant are provided. Immunogenic compositions comprising the SMIP compound, an antigen and optionally other adjuvants are also provided.

As an adjuvant, SMIP compounds can be combined with antigens and delivery systems to form the final immunogenic composition or vaccine product.

As an immunotherapy, SMIP compounds may be used alone or in combination with other therapies for treating SARS.

One of ordinary skill in the art will appreciate that the physiologically active antiviral compound SMIP or SMIS having accessible hydroxyl groups is typically administered in the form of a pharmaceutically acceptable ester. The antiviral compounds of the present invention may be effectively administered as esters formed from their hydroxyl groups, as would be envisioned by those skilled in medicinal chemistry. It has long been known in the art of pharmaceutical chemistry that the rate and duration of action of antiviral compounds can be adjusted by selecting the appropriate ester group.

Otherification useful in combination with the therapeutic agents described hereinThe compound comprises Pentoxifylline (PTX), methylprednisolone, trimetrexin (Neutrexin), hydrastin (thymosin alpha-1), optionally substituted 5-carbamimidoyl (aminomethiminino) -3-methyl-4-isoxazole carboxylic acid phenylamide, cyclosporine A (CsA), 6-oxo-1, 4, 5-thiazine [2, 3-b ] ]Quinazoline, 3-amino-2 (1H) -thiooxy-4 (3H) -quinazolinone, gangciclovir, glycyrrhizin, tetracyclines, aminoglycosides, quinolone, bicyclam (1, 4-bis (1, 4, 8, 11-tetraazacyclotetradecan-1-ylmethyl) benzeneoctanedioic acid dihydrate), rapamycin, wortmannin, enrazepril, roquine/rimantane, inactivin, DNCB, AG7088, 9-aminocamptothecin (CPT-11), loxobine, bromoprimine, onanase (Onoscapine), and related compounds^_(ranpirnase), statins (such as lovastatin-Mevacor)^_pravastatin-Pravachol^_simvastatin-Zocor^_fluvastatin-Lescol^_atorvastatin-Lipitor^_And rosuvastatin-Crestor^_)。

The term "effective amount" herein denotes the amount of antiviral compound in the compositions, kits and methods of the invention that is capable of treating the disease in question. The particular dose of the compounds of the present invention administered will, of course, be determined on a case by case basis, including, for example, the compound administered, the route of administration, the condition of the patient, and the severity of the condition being treated.

The dosage of the antiviral compound of the present invention administered to a subject may vary and is dependent upon the judgment of the clinician. It should be noted that it is necessary to adjust the dosage of the compound when administered in the form of a salt (e.g., laurate), as salts can provide some molecular weight variation.

The following dosages, as well as others mentioned elsewhere in this specification, are on average for a human weighing about 65-70 kg. A skilled physician will be able to determine the dosage required for a subject weighing outside the range of 65-70kg based on the medical history of the subject and whether the subject has certain illnesses, such as diabetes. Calculation of the dosage of the free base form (e.g., salt or hydrate) of the other forms of the drug can be accomplished by simply scaling the molecular weights of the drug species involved.

Typically, the pharmaceutical composition will include at least one antiviral compound in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water, borate buffered saline containing trace metals, and the like. The formulations may also include one or more excipients, preservatives, solubilizers, buffers, lubricants, fillers, stabilizers, and the like. Methods of preparation are well known in The art and are described, for example, in Remington's pharmaceutical Sciences, Mack pub. Co., New Jersey (1991) or in Remington's pharmaceutical Sciences and practices, 20 th edition, Lippincott Williams & Wilkins, Baltimore, Maryland (2000), incorporated herein by reference.

The pharmaceutical compositions used in the present invention may be in the form of sterile pyrogen-free liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches, or other forms known in the art.

Many active ingredient antiviral compounds are known to be absorbed from the digestive tract, and therefore oral administration of the compounds is generally preferred for reasons of simplicity. However, administration of the compounds intravenously, subcutaneously, transdermally or as a rectally or vaginally absorbed suppository is equally effective if desired. All conventional types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, buccal tablets, suppositories, and suspensions. The compositions are formulated to contain a daily dose or a convenient portion of a daily dose in dosage unit form, which may be in the form of a tablet or capsule, or a convenient volume of liquid.

Capsules are prepared by mixing one or more compounds with a suitable diluent and encapsulating the appropriate amount of the mixture. Commonly used diluents include inert powdered materials such as many different types of starch, powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), cereal flour and similar edible powders.

Tablets are made by direct compression, by wet granulation or dry granulation. The formulation typically includes diluents, binders, lubricants, and disintegrants, as well as one or more compounds. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (e.g., sodium chloride) and powdered sugar. Cellulose derivative powders may also be used. Typical tablet binders are starch, gelatin and sugars (e.g., lactose, fructose, glucose, etc.). Natural and synthetic gums may also be used, including acacia, alginate, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethyl cellulose and waxes are also commonly used as binders.

Lubricants are often required in tablet formulations to prevent the tablet and punch from sticking to the die. The lubricant is selected from smooth solids such as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

Tablet disintegrants are substances that swell when wet to break the tablet and release one or more compounds. They include starches, clays, celluloses, algins and gums, more specifically such as corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, natural sponge flour, cation exchange resins, alginic acid, guar gum, citrus pulp and carboxymethyl cellulose, sodium lauryl sulfate also being used.

Tablets are also often sugar coated with flavors and blocking agents, or coated with film forming protective agents to modify the dissolution characteristics of the tablet. The compounds can also be formulated as chewable tablets, which requires the use of relatively large amounts of a pleasant tasting substance, such as mannitol, in the formulation, as is now well established in the art.

Typical bases may be used when it is desired to administer the compound as a suppository. Cocoa butter is a traditional suppository base that may be modified by the addition of waxes to raise its melting point slightly. Suppository bases which are miscible with water are widely used and include, inter alia, polyethylene glycols of various molecular weights.

The effect of the compound can be delayed or prolonged by suitable formulation. For example, pellets of the compound that dissolve slowly can be prepared and incorporated into tablets or capsules. This technique can also be improved by making several pellets with different dissolution rates and filling a mixture of these pellets in a capsule. The tablets or capsules may be coated with a film that resists dissolution within a predetermined time. Even parenteral preparations can be prepared in long-acting form by dissolving or suspending one or more compounds in an oily or emulsifying vehicle which slowly disperses in serum.

The pharmaceutical combination of the present invention may be administered in a controlled release formulation (e.g. a slow release or a fast release formulation). Such controlled release formulations of the pharmaceutical combinations of the present invention may be prepared by methods well known to those skilled in the art. The method of administration will be determined by a clinician or other person skilled in the art after evaluation based on the condition and needs of the patient.

The term "prodrug" refers to a compound that is converted in vivo (e.g., by hydrolysis in blood) to form the antiviral compounds of the present invention. The transformation may occur by various mechanisms, such as by hydrolysis in blood. Detailed descriptions of prodrugs are provided in t.higuchi and w.stella, "prodrugs as novel delivery Systems" (Pro-drugs as novelderlivery Systems), a.c.s. symposium series, volume 14, and "biologically reversible Carriers in Drug Design" (biological in Drug Design), ed by Edward b.roche, american pharmaceutical Association Pergamon Press, 1987. The term "prodrug" also includes combinations of one or more antiviral compounds that are prodrugs of each other into a single molecule that can be converted to produce the various antiviral compounds of the present invention.

For example, if the antiviral compounds of the present invention contain a carboxylic acid functional group, the prodrug may comprise an ester formed by replacing the hydrogen atom of the carboxylic acid group with a group such as (C)₁-C₈) Alkyl, (C)₂-C₁₂) Alkanoyloxymethyl, 1- (alkanoyloxy) ethyl having 4 to 9 carbon atoms, 1-methyl-1- (alkanoyloxy) -ethyl having 5 to 10 carbon atoms, 3 to 6 alkanoyloxymethyl groupsAlkoxycarbonyloxymethyl of carbon atom, 1- (alkoxycarbonyloxy) ethyl of 4 to 7 carbon atoms, 1-methyl-1- (alkoxycarbonyloxy) ethyl of 5 to 8 carbon atoms, N- (alkoxycarbonyl) aminomethyl of 3 to 9 carbon atoms, 1- (N- (alkoxycarbonyl) amino) ethyl of 4 to 10 carbon atoms, 3-phenylprop [ c ] ethyl]Furanonyl, 4-crotonolactonyl, gamma-butyryl-4-yl, di-N, N- (C)₁-C₂) Alkylamino radical (C)₂-C₃) Alkyl (e.g. beta-dimethylaminoethyl), carbamoyl- (C)₁-C₂) Alkyl, N-di (C)₁-C₂) Alkylcarbamoyl- (C)₁-C₂) Alkyl and pyrido-, pyrrolo-or morpholino (C)₂-C₃) An alkyl group.

Similarly, if the antiviral compounds of the present invention contain an alcohol functional group, prodrugs can be formed by replacing the hydrogen atom of the alcohol group with a group such as (C) ₁-C₆) Alkanoyloxymethyl, 1- ((C)₁-C₆) Alkanoyloxy) ethyl, 1-methyl-1- ((C)₁-C₆) Alkanoyloxy) ethyl group, (C)₁-C₆) Alkoxycarbonyloxymethyl, N- (C)₁-C₆) Alkoxycarbonylaminomethyl, succinyl, (C)₁-C₆) Enol group, alpha-amino group (C)₁-C₄) An enol group, an arylacyl group and an alpha-aminoacyl or alpha-aminoacyl-alpha-aminoacyl group, wherein each alpha-aminoacyl group is independently selected from a naturally occurring L-amino acid, P (O) (OH)₂、-P(O)(O(C₁-C₆) Alkyl radical)₂Or a glycosyl group (residue resulting from removal of the hydroxyl group in the hemiacetal form of the carbohydrate).

If the antiviral compounds of the present invention contain an amine functional group, prodrugs can be formed by replacing the hydrogen atom of the amine group with a group such as R^X-carbonyl, R^XO-carbonyl, NR^XR^X1-carbonyl group, wherein R^XAnd R^X1Are each independently ((C)₁-C₁₀) Alkyl, (C)₃-C₇) Cycloalkyl, benzyl, or R^X-the carbonyl group isNatural alpha-aminoacyl or natural alpha-aminoacyl-natural alpha-aminoacyl, -C (OH) C (O) OY^X(wherein Y is^XIs H, (C)₁-C₆) Alkyl or benzyl), -C (OY)^X0)Y^X1Wherein Y is^X0Is (C)₁-C₄) Alkyl radical, Y^X1Is ((C)₁-C₆) Alkyl, carboxyl (C)₁-C₆) Alkyl, amino (C)₁-C₄) Alkyl or mono-N-or di-N, N- (C)₁-C₆) Alkylaminoalkyl, -C (Y)^X2)Y^X3Wherein Y is^X2Is H or methyl, Y^X3Is mono-N-or di-N, N- (C)₁-C₆) Alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.

The compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Antiviral, SMIP, SMIS or other immunomodulatory compounds are prepared or obtained as described herein and as described in the U.S. patents and published international patent applications set forth in Table 1, Table 2, Table 34 and Table 38. The antiviral compound may be formulated in a pharmaceutically acceptable composition suitable for delivery to the lung. Specific formulations include dry powders, liquid solutions or suspensions suitable for use as sprays and sprays for use in metered dose inhalers. The preparation of such formulations is well known to those skilled in the art and is described in U.S. Pat. Nos. 5,814,607 and 5,654,007, and in the U.S. patents and published International patent applications set forth in Table 3, which are incorporated herein by reference.

Dry powder formulations will contain the antiviral compound in dry (optionally lyophilized form) form, preferably having a particle size within the range of deposition in the lung. The particle size range for deposition in the lung is typically between 1-5 μm. When systemic delivery of antiviral compounds by absorption from the lungs into the bloodstream is desired, the particle size of the antiviral compound formulation is typically between 0.1 and 2 μm. The preferred particle size range may be produced by methods such as jet milling, spray drying and solvent precipitation. Dry powder devices typically require a powder mass of between about 1mg and 100mg to produce an aerosolizable dose. Thus, the antiviral compound will typically be mixed with a pharmaceutically acceptable bulk dry powder. Preferred bulk dry powders include sucrose, lactose, trehalose, Human Serum Albumin (HSA), phospholipids and glycine, as well as those described in the literature listed in table 3. The dry powder can be administered to a subject using a conventional dry powder inhaler. For liquid formulations, the antiviral compound may be dissolved in any physiologically acceptable carrier known for delivery of nebulizable formulations. Such carriers include aqueous solutions of buffered and unbuffered water-soluble compounds, as well as physiological solutions, including saline (preferably 0.2-2N NaCl). Other liquid carriers, such as ethanol, propylene glycol, and ethanol-propylene compositions, may also be used if the solubility of the antiviral compound is limited. The antiviral compound may also be administered as a solid suspension.

For administration by inhalation, the compositions for use in accordance with the present invention are typically delivered in the form of an aerosol spray presentation via pressurized packs or a nebulizer, with the use of a propellant, such as air, dichlorodifluoromethane, dichlorotetrafluoroethane, or other suitable gas. Preferably, the antiviral compound formulations of the present invention are incorporated into aerosol propellants to produce inhalable particles as described above for the dry powder formulations. The particles are suspended in a propellant and may optionally be coated with a surfactant to enhance their dispersion. When a compressed aerosol is used, a valve may be provided to deliver a pre-metered amount to define the dosage unit.

Commercially available jet nebulizers are available and can be used to deliver nebulized antiviral compounds to a subject. Such spray atomizers include, but are not limited to: a product supplied by AeroTech 11(CIS-US, Bedford, Mass.). In addition, to deliver aerosolized antiviral compounds to the lungs of the subject, a source of oxygen may be added to the nebulizer to provide a flow rate of, for example, 10L/min. Generally, during spontaneous breathing, the interval between inhalations through the mask is preferably 5 to 40 minutes. The present invention provides novel compositions comprising a suitable carrier and an aerosolized antiviral compound in an amount sufficient to reduce or alleviate the viral load and symptoms of SARS in a SARS patient. Such a dose may be lower than the corresponding systemic dose used to reduce or alleviate the viral load and symptoms of SARS in SARS patients.

The antiviral, SMIP, SMIS and immunomodulatory compositions of the invention can be administered with sterol anti-inflammatory drugs to treat SARS and SARS symptoms. Examples of the sterol anti-inflammatory agents of the present invention include hydrocortisone, prednisolone dexamethasone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, betamethasone, and the like.

The antiviral compounds of the compositions of the present invention are largely aerosolized into particles that deliver the drug to the terminal bronchioles and respiratory bronchioles. For effective delivery of antiviral compounds in aerosol form to the lung bronchial space of the respiratory tract, the mean average diameter of the aerosol particles formed must be predominantly between 1 and 5 μm. The formulation must also not adversely affect the function of the respiratory tract. Thus, the formulation must contain sufficient drug formulated under these conditions to deliver the drug effectively while avoiding adverse reactions.

For liquid solutions and suspensions, a nebulizer can be selected from commercially available nebulizers. Jet atomizers available from medical and PariLCS, LC Plus under the name sidessteam 0 and from Pari respiratory equipment, Richmond, Virginia under the name eFlow are typical examples of atomizers suitable for practicing the invention. Ultrasonic nebulizers can produce particles of about 1-5 μm in size, for example, both Aerosonic from DeVilbiss and UltraAire from Omron are suitable.

Advantageously, the advantages of the present invention also provide the consumer with a kit for treating and/or preventing SARS. Such a kit comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of at least one compound as set forth in tables 34 and 35 or in the U.S. patents and published international patent applications set forth in tables 1, 2, and 35 and a pharmaceutically acceptable carrier, vehicle, or diluent; (b) a container for containing the pharmaceutical composition; and, optionally; (c) instructions for using the pharmaceutical composition to treat and/or prevent SARS are described. The kit may optionally comprise a plurality of antiviral compounds for treating SARS, wherein the antiviral compounds are selected from the group consisting of 3C-like protease inhibitors and papain-like protease inhibitors. In another embodiment, the kit comprises an antiviral compound that is an RNA-dependent RNA polymerase inhibitor. When the kit contains multiple antiviral compounds, the antiviral compounds in the kit can optionally be administered in combination with the same pharmaceutical composition.

As used herein, a "kit" includes containers containing different compositions, such as separate bottles or separate foil packs. The container may be of any conventional shape or form known in the art, made of pharmaceutically acceptable materials, such as a carton or carton, a glass or plastic bottle or can, a resealable bag (e.g., for containing "refill" tablets in different containers), or a blister pack (containing a packaging location where individual doses are extruded according to a treatment regimen). The container used may depend on the precise dosage form involved, e.g. a conventional cardboard box is not used to contain a liquid suspension. It is feasible to use multiple containers together in a package that sells unit dosage forms. For example, the tablets may be contained in bottles, and the bottles may be contained in boxes.

An example of such a kit is called a blister pack. Blister packs are well known in the packaging industry and are widely used for packaging unit pharmaceutical dosage forms (tablets, capsules, etc.). Blister packs are generally composed of a relatively rigid sheet material covered by a foil, preferably of transparent plastics material. During packaging, depressions will be formed in the plastic foil. The depressions may be of the size and shape of a single tablet or capsule to be packaged, or may be of a size and shape corresponding to a plurality of tablets and/or capsules to be packaged. The tablets or capsules are then placed in the respective depressions and the plastic foil is sealed with a relatively stiff sheet material at the surface of the plastic foil opposite to the direction in which the depressions are formed. As a result, the tablets or capsules are sealed individually or together as desired in the depressions between the plastic foil and the sheet. Preferably the strength of the sheet is such that an opening can be formed in the sheet in the depression by manually applying pressure to the depression so that the tablet or capsule can be removed from the blister pack. The tablet or capsule can then be removed from the opening.

It may be desirable to provide a written memory aid containing information and/or instructions for a doctor, pharmacist or subject, such as in the form of numbers against a tablet or capsule corresponding to the number of days a tablet or capsule should be taken, or in the form of a card containing the same type of information. Other examples of such memory aids are calendars printed on cards, such as "first week, Monday, Tuesday", …, "second week, Monday, Tuesday, …", and so forth. Other variations of memory assistance are readily apparent. The "daily dose" may be a tablet or capsule or several tablets or capsules to be taken on a given day. Also, a daily dose of one or more components of the kit may consist of a single tablet or capsule, while a daily dose of one or more other components of the kit may consist of several tablets or capsules.

Other particular embodiments of the kit are dispensers designed to dispense daily doses at a time in the order in which they are used. Preferably the dispenser is equipped with a memory aid to facilitate compliance with the treatment regimen. An example of such a memory aid is a mechanical counter which can indicate the number of daily doses that have been dispensed. Another example of such a memory aid is a battery-driven microchip memory equipped with a liquid crystal display or audible prompting signals, for example, to indicate the date the last daily dose was taken and/or to prompt the time the next dose was taken.

Examples

EXAMPLE 1 examples of SARS virus isolates

A SARS virus (FRA) was isolated from a clinical specimen from a patient of Frankfurt, Germany. Isolates were grown in Vero cells. The SARS virus RNA was extracted and amplified by RT-PCR. The nucleotide sequence of the viral genome was determined by direct sequencing of the PCR products. Computer analysis is used to predict genomic features, compare the genome to previously known coronavirus and the sequence of different SARS virus isolates.

Isolation and sequencing are more specifically performed as follows. After passage 3 of SARS virus in Vero cells, the cells were centrifuged by ultracentrifugation from 3X 10 ⁷Viral particles were purified from the cell supernatant. Viral RNA was extracted by Triazol method (Gibco-BRL). Viral RNA (200ng) was transcribed into cDNA using avian RNaseH-thermostable reverse transcriptase following the manufacturer's instructions (Thermoscript RT System, Invitrogen). Briefly, 50pmol oligo (dT) was used₂₀(SEQ ID NO: 7389) or 25ng of random hexamer initiated the RT reaction in a final volume of 20. mu.l. Amplification and sequencing of the SARS genome is accomplished by direct sequencing of the PCR product, obtained using: i) specific primers from conserved regions of homologues found by multiple comparisons of known coronaviruses; ii) oligonucleotides designed around the short sequence of the SARS isolate, the sequence being available on the web via the WHO network laboratory; iii) degenerate primers for amplification of the cDNA mixture, the primers having multiple overlapping fragments with the final product. Gap closure was recognized by long-range PCR with High Fidelity Taq (Roche) using primers designed on the selected fragment. Sequences were collected by primer walking using BigDye terminator chemistry (Applied Biosystems) and an automated DNA sequencer (3700 capillary mode, Applied Biosystems). After the first pass through the entire genome was obtained, a set of forward and reverse primers was used to amplify and sequence the genome de novo, using an average 2kb template DNA segment. AutoAssembler (applied biosystems) reads from overlapping fragments were automatically pooled and manually edited for contiguous 29,740 bp.

The computer analysis sequence was performed as follows. The GCG Wisconsin software package (version 10.0) was used for in silico analysis of gene and protein sequences. The PSORT program (http:// PSORT. nib. ac. jp /) is used for local prediction. For secondary structure analysis, the PHD software is used, which can obtain http at the following website: // cubic. bioc. columbia. edu/predictprotein/. The PSI-BLAST algorithm is used for homology searching (http:// www.ncbi.nlm.nih.gov/BLAST) using a non-redundant protein database. ClustalW is used to obtain multiple alignments of gene and protein sequences. The LearnCoil-VMF program was used to predict the coiled-coil region in the spike protein (http:// Learncoil-VMF. lcs. mit. edu/cgi-bin/VMF). Leucine zipper was predicted with program 2ZIP, which can be found in http: v/2 zip. molgen. mpg.de.

Phylogenetic analyses were performed using the proximity join algorithm performed by the program NEIGHBOR in the phylogenetic inference software package (PhyLIP) (Felsenstein J1993, a program distributed by the authors). Bootstrap analysis is usually performed with 100 replicates using the program Seqboot. The dendrogram is processed and displayed in TreeView. The program HMMER was used to generate sequence distributions from multiple sequence alignments of the S1 domain of the spike protein. Subsequently, the HMMPFAM program was used to compare the SARS spike S1 domain to these distributions.

The genome of the SARS virus isolate is 29,740 bases in length and the overall structure of the genome is similar to that of 3 known coronavirus groups. Starting from the 5' end of the leader sequence, the untranslated region (UTR) and the 2 overlapping open reading frames encoding a polyprotein containing the enzymes necessary for replication can be identified. These are followed by spikes (S), envelope (E), matrix (M), nucleocapsid (N) structural proteins and another 8 ORFs specific for SARS virus. The UTR of poly (A) was isolated at the 3' -end of the genome. The overall homology to coronavirus groups 1, 2 and 3 is low, so the SARS virus belongs to a new coronavirus group (group 4). More detailed spike protein amino acid sequence analysis showed that SARS virus isolates were more closely related to coronavirus group 2.

The complete genome length of the SARS virus isolate is 29,740 bp. This sequence is available on Genbank and has a GC content of 40.8%, comparable to that of known viruses of the same family. The genome structure is similar to other coronavirus structures. There are 14 open reading frames predicted. The major structures of the genome and gene products are shown in fig. 17 and table 10. A comparison between the SARS genome and coronavirus groups 1, 2 and 3 is reported in figure 18.

Nucleotides 1-73 comprise a predicted RNA leader followed by a 197 nucleotide untranslated region (UTR). The UTR is followed by 2 overlapping open reading frames (ORF1a, ORF1b) that cover two thirds of the genome (nucleotides 265. sup. -. sup. 21485). They encode a large polyprotein that is predicted to be processed by viral proteases to produce replicase complexes. The 3' part of the genome contains genes encoding 4 structural proteins (S, spike, E, envelope, M, matrix glycoprotein and N, nucleocapsid) and 8 predicted ORFs with unknown function (fig. 17). Finally, at the 3' end of the genome, we found the 2 nd UTR with 340 bases followed by a poly (A) stretch. We have identified a putative Intergenic (IG) sequence, also known as Transcription Associated Sequence (TAS), which is a typical feature of coronaviruses. The IG sequence is characterized by 6-18 nucleotides at the 3' end of the leader sequence and can be found in front of each gene. IG sequences play a key role in RNA transcription and replication thereof. The SARS virus IG sequence is characterized by the sequence SEQ ID NO: 7293 and is present 9 times in the genome (FIG. 17). The leader sequence and IG sequence are specific for each coronavirus and represent the specific signals of the virus.

Replicase regions

The replicase gene, ORF1ab (SEQ ID NO: 7232), consisting of 2 overlapping ORFs, ORF1a and ORF1b, was translated into a single polyprotein by frame-shifting the ribosome at position 13,393 within the polymerase coding region. See Brierley et al, Embo J1987: 6(12): 3779-3785. As expected, a stem-loop sequence was present 10 base pairs downstream of this position (SEQ ID NO: 7390; 5' -CGGTGTAAGTGCAGCCCGT CTTACACCG-3'). This polyprotein is co-or post-translationally cleaved into multiple proteins by its own encoded proteases. Using the cleavage consensus sequence and mimicking with other coronaviruses, we mapped the possible cleavage sites of this polyprotein and identified 14 products, including the leader protein p28, MHV p65 protein homolog and the other 12 proteins named nsp13 from nsp1 (nsp, nonstructural proteins) (fig. 17 and table 10). Amino acid sequence analysis suggested that there were some functional motifs within the putative ORF1ab protein. We specified 2 potential proteases (nsp1 and nsp2), a 1-class growth factor motif within ORF1a, while in ORF1b we identified RNA polymerase (nsp9) and predicted helicase (nsp 10). Other predicted cleavage products (nsp3, nsp) 4. nsp5, nsp6, nsp11, nsp12, and nsp13) are proteins whose functions are unknown. Many of these proteins are presumed to be present in the RNA replication complex, which is associated with membrane structures in infected cells. nsp3 and nsp4 specifically comprise hydrophobic domains. As shown in fig. 18, the SARS replicase region has a similar composition to that of coronavirus groups 1, 2 and 3; however, overall amino acid conservation was low (table 11). Most conserved proteins are polymerases and helicases.

Nsp1 is a papain-like cysteine protease (PLP) that cleaves the first 2 protein products (leader protein p28 and p65 homologs). Within nsp1 of MHV, 2 domains with papain-like activity were mapped (Kanjaahaluethai et al (2000) J.Virol 74 (17): 7911-21), which are also conserved in bovine infectious gastroenteritis virus (TGV) and human 229E coronavirus. However, by sequence alignment with SARS nsp1, we identified the only PLP domain containing catalytic residues Cys833 and His 994.

Nsp2 is a chymotrypsin-picornavirus 3C protease (3CLp) responsible for post-translational processing of the other 12 proteins, most of which cleave at Q/A or Q/S sites (Ziebuhr et al (1999) J.virol73 (1): 177-85). Nsp2 also has autoproteolytic activity. The major catalytic residues are better conserved in other coronaviruses and are located at positions His41 and Cys 145. Furthermore, even the conserved amino acids Tyr161 and His163 are found in the SARS 3CLp sequence and are thought to be involved in substrate recognition and essential for proteolytic activity (Hegyi et al (2002) J. Gen Virol 83(Pt 3): 581-593).

The invention includes SEQ ID NO: 9960 and the 1ab sequence of SEQ ID NO: 9961, including fragments, variants, homologs, and the like.

Structural region

Analysis of the nucleotide sequence of the 3' portion of the SARS genome identified 12 predicted open reading frames. They encode within 8.2kb and include 4 structural proteins S, E, M and N, common to all coronaviruses and 8 predicted ORFs, which are specific for this virus. The SARS-specific IG sequence upstream of most ORFs (FIGS. 17&18) suggests that most genes may be transcribed independently. Interestingly, the same sequences as in group 2IG were also present at the end of the RNA leader and in front of the matrix-encoding genes and ORFs.

Spikes are type I glycoproteins that form large spikes on virion surfaces and are responsible for receptor binding and membrane fusion (Gallagher (2001) Adv Exp Med Biol 494: 183-92). The protein is 1255 residues long and has 17 predicted N-glycosylation sites. It has a 13aa leader peptide and a 17aa C-terminal membrane anchor sequence (1202-1218). Some (MHV, HCoV-OC43, AIBV and BCoV) but not all (TGV, FIPV, HCoV-229E) coronavirus spike proteins were proteolytically cleaved in 2 subunits S1 and S2. It is assumed that S1 forms a globular head that remains non-covalently linked to the C-terminal membrane anchor. Cleavage is mediated by a basic amino acid sequence that resembles the consensus sequence of the furin cleavage site (Garten et al, Biochimie 1994; 76 (3-4): 217-225). However, in the case of this SARS virus isolate, we failed to identify this sequence, indicating that the S protein of this SARS virus isolate is unlikely to be cleaved during maturation. Secondary structure prediction indicates that the globular structure of the spike protein is conserved in all known coronaviruses. The S1 domain is formed primarily by beta sheets and may adopt globular folds, whereas in the S2 domain, a broad alpha helical region is predicted. In addition, the LearnCoil-VMF program specifically designed to identify similar regions of the coiled-coil in the viral membrane fusion protein predicts 2 coiled-coils within S2 spanning amino acids 900-1005 and 1151-1185, respectively (FIG. 19). All 2 coiled coil regions contain a leucine zipper motif, which is also present in the spikes of all coronaviruses. Leucine zipper is known to promote protein oligomerization; since the spike proteins of TGV and MHV form heterotrimers (Delmas et al, J Virol 1990; 64 (11): 5367-) -5375) (Godeke et al, J Virology 2000; 74 (3): 1566-) -1571, it is conceivable that it plays a role in promoting and/or stabilizing similar quaternary structures in the SARS leucine zipper. The spike protein plays a major role in coronavirus biology, as the S1 domain comprises a receptor binding domain and a virus neutralizing epitope, while the S2 domain is involved in the membrane fusion process essential for virus infectivity. As expected, multiple alignments of different spike proteins showed a large degree of variability within the S1 domain, while S2 was more conserved.

The envelope protein E is a very short polypeptide of 76 amino acids and is involved in morphogenesis of the virion envelope (Godet et al, Virology 1992; 188 (2): 666-. Computer analysis predicted there were long transmembrane domains near the N-terminus and 2N-glycosylation sites. The amino acid similarity with other coronaviruses is low, and the greatest homology is with the small envelope protein of infectious gastroenteritis virus (TGV).

The matrix glycoprotein (M) is a 221-residue polypeptide with a predicted molecular weight of 25 kDa. The topology predicted by computer analysis consists of a short amino-terminal ectodomain, 3 transmembrane segments, and a carboxy-terminus located inside the viral envelope. The SARS M glycoprotein was N-glycosylated at the N-terminus, mimicked by the matrix glycoprotein of TGV, avian infectious gastroenteritis virus (AIBV) and hypervirulent MHV-2 strains. The SARS M glycoprotein showed the highest similarity to the group 2 virus (table 11).

Finally, nucleocapsid protein N is a phosphoprotein of 397 residues in length that interacts with viral genomic RNA to form the nucleocapsid. The level of conservation with other coronaviruses was low, ranging from 26.9% identity with HCoV-229E to 37.4% identity with bovine coronavirus (BcoV) (Table 11). Epitope analysis of nucleocapsid proteins has been accomplished (Li et al (2003) Geno Prot & Bioinfo 1: 198-206), where the epitope position at the C-terminus of the protein is located in the amino acid sequence of SEQ ID NO: 7394 (amino acid 371-407 of SEQ ID NO: 6052).

In addition to the above basic proteins, many viruses express a group of other peptides that are generally not essential for viability but which affect the infection potential of the virus (de Haan et al, Virology 2002; 296 (1): 177-189). These proteins are generally conserved among members of the same serogroup, but differ significantly between groups. For this reason, they are generally called histone proteins (FIG. 11). The members of group 1, represented here by HcoV-229E, have 2 group-specific genes, located between the S and E genes and sometimes 1 or 2 ORFs downstream of the N gene, preceding the 3' UTR region of the genome. The group 2 virus, with MHV as the prototype, has 2 group-specific genes between ORF1b and S (2a and HE) and 2 other group-specific genes between the S and E genes. Finally, group 3 viruses are represented by the prototype AIBV, with 2 group-specific genes between the S and E genes and 2 other group-specific genes between the M and N genes.

All other group-specific ORFs encode proteins whose role has not been determined, except for the hemagglutinin esterase HE, which has demonstrated both hemagglutinating and acetyl esterase enzymatic activities.

Interestingly, the specific gene arrangement unique in the SARS genome and the predicted ORFs did not show any significant homology to the ORFs present in other coronaviruses, nor to any other known proteins from different organisms. Like the viruses of groups 1 and 3, SARS lacks HE hemagglutinin and does not contain ORFs between ORF1b and the S gene. Furthermore, 2 predicted ORFs (ORF3 and ORF4) are encoded in the region between S and E, with the overlap acting over most of their length. ORF3 has an IG sequence 2bp upstream of the ATG start codon. In contrast to the other groups, SARS contains 5 predicted ORFs in the region between the M and N genes. ORF7 is located 10 bases downstream of the M gene stop codon and has an IG sequence 155 nucleotides upstream of the ATG start codon. Similarly, ORF8 and ORF10 present IG just upstream of the ATG start codon. On the other hand, the 5' ends of ORF9 and ORF11 are briefly stacked with flanking genes, for which reason they do not require IG to activate transcription. ORF12 is completely superimposed with the N gene and has very low homology with the 22kDa protein of the MHV virus, which is encoded in the corresponding region.

Although there is no indication of possible localization and function derived from sequence similarity, ORFs 3, 7, and 8 contain hydrophobic segments, suggesting association with membrane structure. In addition, the longest ORF3 in the SARS-specific gene is the only ORF that encodes a peptide containing a large number of predicted O-glycosylation sites (Table 11). Predicted N-glycosylation sites were identified in ORF3, ORF11 and ORF 12.

The 2 shorter ORFs in the nonstructural region are SEQ ID NOS: 9965 and 9966. The invention includes polypeptides having these sequences, as well as fragments, variants, and the like.

Phylogenetic analysis

The serological grouping was confirmed in the past using the frequency of substitutions within 922 conserved bases of the 11 coronavirus pol genes from 3 different serogroups to show that the variability among the members of each serogroup is much less than that between the different serogroups (Stephensen et al, Virus Res 1999; 60 (2): 181-9). We used the 922bp region of the SARS pol gene and compared it to the same fragments from the other 12 coronaviruses. The resulting dendrogram showed that the SARS virus was different from the other 3 coronavirus groups (fig. 20). Similar results were obtained using the full-length amino acid sequences of pol, 3 CL-protease and helicase from the replicase regions and the full-length amino acid sequences of spikes and matrix glycoproteins from the structural regions (data not shown). These data confirm the complete genome of the SARS virus cluster in a new coronavirus group (group 4).

To gain a clearer understanding of the possible evolutionary relationships, we performed analyses using consensus sequences for protein prediction domains. We specifically generated a consensus sequence of the S1 domains of the spike proteins from group 1 and group 2, and then we compared them to the S1 domain of SARS spikes. No consensus could be generated from group 3, as only the spike protein of AIBV is known. Interestingly, the tree constructed by comparing SARS 1 with the consensus generated from group 2 spike proteins differs from the tree in fig. 20, showing that the relationship between SARS and coronavirus group 2 is much more intimate (fig. 21A). Further analysis showed that 19 of the 20 cysteines present in the SARS S1 domain were spatially conserved with the group 2 consensus sequence, while only 5 maintained within the group 1 and group 3 sequences (fig. 21B). Given the fundamental role that cysteine plays in protein folding, it is likely that the SARS 1 domain shares a similar spatial structure with coronavirus group 2.

Sequence variability between SARS coronaviruses

We compared the FRA sequence to the 4 complete SARS genomes available on the web. A total of 30 mutations were detected. 9 of these mutations were silent, while 21 resulted in amino acid substitutions (Table 12). Within ORF1a, 3 silent and 7 productive mutations (productive mutations) were detected. In ORF1b, there were 5 silent and 3 productive mutations. One productive mutation is caused by 2 nucleotide substitutions, the 2 nucleotide substitutions resulting in a single amino acid change. 5 changes were localized in the spike protein, 4 of which were productive and 1 silent. There were 2 productive mutations in ORF3 and matrix glycoprotein M. There were 1 productive mutations in ORF10 and nucleocapsid protein N.

The total difference between FRA and TOR2 was 9 nucleotides, resulting in 2 silent mutations and 7 amino acid changes. The total difference between FRA and Urbani is 12 nucleotides, resulting in 5 silent mutations and 7 amino acid changes. For CUHK, there are 16 nucleotides in difference, 5 of which are silent mutations. For FRA and HKU 14, the nucleotide changes produced 4 silent and 9 productive mutations.

Example 2 production, inactivation and purification of intact SARS Virus, purification Using MCS chromatography resin, followed by Density gradient ultracentrifugation

SARS virus isolate FRA1 (EMBL: AY310120) was passaged on VERO cells cultured in DMEM (Gibco: Cat. No. 21969-035, batch No. 3078864), penicillin/streptomycin (Gibco: Cat. No. 15070-063, batch No. 1120042) and 3% FCS (Gibco: Cat. No. 10270-106, batch No. 40F6130K) at 37 ℃ with 5% CO₂. Trypsin (Gibco: catalog No. 25300-054, batch No. 3078729) was used to detach the cells.

For virus production, passage 3 was used to inoculate VERO cells with moi of-0.1. Cells were incubated with virus at 37 ℃ for 1 hour in infectious medium (DMEM without PS, FCS); after 1 hour, cells were washed 2 times and incubated for an additional 48 hours at 37 ℃ in the presence of 3% FCS and antibiotics. Supernatants were collected 48 hours post infection (p.i.) and pre-cleared by centrifugation at 3000rpm4 ℃ for 10 minutes.

The SARS virus was inactivated by treatment with beta-propiolactone (BPL) at 4 deg.C (1: 2000) for 18 hours followed by 37 deg.C for 3 hours. To test whether the virus was successfully inactivated, VERO cells were incubated with 10ml of BPL-treated supernatant for 4 days at 37 ℃; subsequently, the supernatant was transferred to fresh VERO cell culture and incubated for an additional 4 days. Cells were examined for cytopathic effect (CPE).

200ml of BPL-inactivated SARS virus collection was then clarified with a 0.65 μm-pore size filter (47mm diameter) to allow passage of virus particles and to retain cell debris. The filter unit was connected to a Masterflex pump which achieved a constant flow rate of 40 ml/min.

MCS chromatography purification step

The filtered viral suspension was then subjected to MCS chromatography. The MCS column was prepared as follows. 27ml of slurry produced 14ml of sedimented resin, which was packed with a G _ tec Superperformance column (diameter 1.0cm, height 15.7cm, volume 12.33 ml). 1% column volume of 1% acetone solution was injected into the column and the column ran at 100 cm/hour flow rate. Subsequently, HETP, N and A_sValues were calculated as HETP: 0.056cm, N/m: 1790 and A_s＝1.20。

The amount of protein in the purified solution after the MCS chromatography step was evaluated using the dioctyl cocate (BCA) method ((Interchim) (see, e.g., http: www.piercenet.com/files/BCA. pdf) and electrophoresis.

SDS-PAGE according to Laemmli, Nature (1970) 227: 680 and 685. Samples of SDS-PAGE were diluted to a protein concentration of 77. mu.g/ml. Different protein concentrations were added depending on the type of gel used (10/12/15 wells, Novex/Invitrogen):

number of holes	Protein dilution concentration	Sample application	Protein/pore
				10 holes	77μg/ml	20μl	1μg
12 holes	77μg/ml	15-20μl	0.75-1μg
				15 holes	77μg/ml	10μl	0.5μg

Samples for reducing SDS-PAGE were prepared as follows:

	26 μ l sample or diluted sample
		+ 10. mu.l NuPage sample buffer (4X) SDS NP0003
	+ 4. mu.l of TCEP Bondbreaker solution 77720 (1: 2 in MilliQ water)
		Final volume:	40μl

the sample was heated at 70 ℃ for 10 minutes or left at room temperature for 1 hour (the sample was left at room temperature to prevent coagulation/complex formation of coronavirus M protein), followed by centrifugation at 14,000 for about 1 minute in a bench top centrifuge.

Labeling for gels was prepared as follows. Bands of gels containing less than 1. mu.g of protein were readily visualized by silver staining procedure using silver staining kit protein plus a staining procedure (Pharmacia Biotech).

Western blotting was performed as follows. Semi-dry blotting techniques were used to transfer proteins from SDS gels to nitrocellulose membranes. 0.8mA/cm for transfer²The current of (2) was carried out for 1 hour. Rabbit polyclonal antibodies against SARS virus were used for immunodetection using Western Breeze, Novex chromogenic Western blot immunoassay kit (Novex/Invitrogen).

The chromatogram of the inactivated SARS MCS capture step is shown in FIG. 27. To assess purity, the MCS chromatography fraction was analyzed by silver staining on NuPage 10% or 4-12% Bis-Tris-gel (Novex) with heating at 70 ℃ for 10 min under reducing conditions (FIG. 28). These fractions were also analyzed by western blot analysis (fig. 29) under the same conditions to assess purity, using PAK 11/03 SARS Cov 270603, neutralizing titer 1: 512 (this antibody was used for this and subsequent western blots). The purity was estimated as follows:

sample (I)	Volume/ml	[ protein ]]/μg/ml	Total protein/mg	Step protein recovery/%)
					Coronavirus collectionArticle (A)	100	2547.6	254.76	100
After filtration, the sample is added	100	2440.3	244.03	95.8
					Flows through	85	2321.4	197.32	77.5
Washing machine	49.32	468.5	23.11	9.1
					Peak 1	12.12	252.7	3.062	1.2
Total recovery	-	-	464.4	86.5

B. Density gradient ultracentrifugation step

The eluted SARS virus fraction was then subjected to density gradient ultracentrifugation using a bucket rotor for further purification of the inactivated virus. The peak of 3ml MCS was loaded on a linear gradient (15-60% sucrose; 17ml 15% and 17ml 60% sucrose in a gradient mixer). The separation was performed with a Beckman SW 28 rotor at 20,000rpm for 2 hours.

The sucrose and protein content in the linear gradient ultracentrifugation section is shown in the following table, the graph in fig. 30, and the purity estimate in fig. 31:

In part	Fraction size/ml	[ sucrose ]]/％	[ protein ]]/μg/ml
				1	2	61	96.12
2	2	59.4	98.62
				3	2	57.5	87.63
4	2	54.5	86.91
				5	2	50.5	79.9
6	2	47.2	74.3
				7	2	43.7	68.05
8	2	40.2	60.43
				9	2	37.2	57.38
10	2	34	53.12
				11	2	30	50.63
12	2	25.7	35.02
				13	2	22.4	35.33
14	2	19.5	39.25
				15	2	15.5	69.79
16	2	8.5	169.03
				17	2	8.5	128.96

The protein concentration of fraction 11 was again measured against a standard curve (FIG. 31 SDS-gel) prepared in 30% sucrose and giving a protein concentration of 3.67. mu.g/ml (0.05. mu.g on gel). The M protein appears to be absent from this preparation, which may be caused by the sample handling process (heating the sample).

The protein concentration measurements of table 2 may differ due to sucrose interference of this assay.

Example 3 production, inactivation and purification of intact SARS Virus, purification Using MCS chromatography resin, followed by Density gradient ultracentrifugation

Inactivated SARS virus was prepared as described in the examples above.

MCS chromatography purification step

In this example, 200ml of inactivated SARS virusThe collection was subjected to MCS chromatography. The chromatogram of the inactivated SARS virus purification capture step using MCS is shown in figure 32, protein recovery in the table below and purity estimation in figure 33.

Sample (I)	Volume/ml	[ protein ]]/μg/ml	Total protein/mg	Step protein recovery/%)
					Coronavirus collection	200	2239.2	447.83	100
After filtration, the sample is added	200	2245.1	449.02	100.3
					Flows through	185	2126.3	393.37	87.8
Washing machine	49.32	450.1	22.2	5.0
					Peak 1	4.43	1245.6	5.52	1.2
Total recovery	-		421.08	93.7

B. Density gradient ultracentrifugation step

The 3.5ml MCS peak fractions were then loaded onto a linear gradient (15-40% sucrose; 16ml 15% and 16ml 40% sucrose in a gradient mixer). The separation was performed with a Beckman SW 28 rotor at 20,000rpm for 2 hours.

The sucrose and protein content in the linear gradient ultracentrifugation section is shown in the following table and in the graph in fig. 34:

pipe	Fraction size/ml	[ sucrose ]]/％	[ eggWhite colour (Bai)]/μg/ml
				1	2	40	45.86
2	2	39	45.68
				3	2	37.5	44.14
4	2	35.5	37.82
				5	2	33.5	34.48
6	2	31.5	31.76
				7	2	30.5	29.49
8	2	28	30.87
				9	2	25.5	31.7
10	2	23.5	26.74
				11	2	21.75	23.58
12	2	20	35.33
				13	2	18	96.38
14	2	14.5	523.79
				15	2	8	941.97
16	2	8	696.7

Protein recovery is shown in the table below and purity estimates are shown in figure 35. Electron micrographs of density gradient portions 8, 9 and 10 are shown in fig. 36:

step (ii) of	Volume/ml	Protein/ug/ml	Total protein/mg	Step protein%
					Sample application	3.5ml	1245.6	4359.6	100
Bulk protein fraction	3.5ml	720.8	4324.9	99.2
					Viral bee parts	8ml	29.7	237.6	5.5
Total recovery			4562.5	104.7

EXAMPLE 4 immunization of mice with inactivated SARS Virus

Mice were immunized subcutaneously with 5 μ g of BPL-inactivated SARS-CoV particles (BPL-SARS-CoV) on days 0, 14 and 28, either alone or with Alum or MF59 as adjuvants. Sera were collected at 0 (pre-immunization), 13 (post-1 st immunization), 28 (post-2 nd) and 35 days (1 week post-3 rd immunization). Evaluation of neutralizing antibodies blocked SARS-CoV infection of Vero cells in vitro. After 3 immunizations, the neutralizing titer ranged from 1: 100 to 1: 1000, at levels similar to those present in the serum of patients in the SARS convalescent period. As shown in the table below, the adjuvant-free vaccine induced neutralizing antibodies after the 3 rd immunization, which were significantly improved by inclusion of the adjuvant, which appeared after the 2 nd immunization and the overall titer increased after the subsequent 3 rd immunization.

Immunogens	Neutralizing potency
					Before one	After 1 st	After 2 nd time	After 3 rd time
	BPL-SARS-CoV+MF59(5μg)	＜1∶20	＜1∶20	1∶158					1∶630
BPL-SARS-CoV+Alum(5μg)					＜1∶20	＜1∶20	1∶67	1∶612
	BPL-SARS-CoV(5μg)	＜1∶20	＜1∶20	＜1∶20					1∶71
PBS					＜1∶20	＜1∶20	＜1∶20	＜1∶20

EXAMPLE 5 immunization of Balb/c mice with inactivated SARS Virus

A Balb/c mouse model for SARS infection was developed (Subbarao et al (2004), J.Virol., 78: 3572-77). In this model, 10 was used in Balb/c mice⁴TCID₅₀Intranasal inoculation of the virus. TCID was detected in infected mice lungs 48 hours after inoculation₅₀Viral titers increased 2-log. Although viral replication was easily detected, mice did not show any symptoms of SARS disease and spontaneously cleared the virus 1 week after vaccination. The reduction in viral titer in previously immunized animals compared to control animals demonstrates the protective effect of the vaccine evaluated.

In this example, 4 Balb/c mice per group were immunized 3 times ( days 0, 14, 28) with 5. mu.g of BPL-inactivated SARS-CoV particles, alone or in combination with MF59, and 10 on day 43⁴TCID₅₀SARS-CoV challenge. 2 days after virus challenge, mice were euthanized, turbinates (NT) and lung SARS-CoV were quantified and the mean virus titer was measured for each mouse. Control groups received PBS alone, or influenza virus (FLU), with or without MF59 adjuvant. The data is as follows (see also FIG. 51), where 4 mice are tested per group and the virus titers are expressed as log ₁₀TCD₅₀Per gram of tissue:

immunogens	Viral replication in the lungs of challenged mice		Virus replication in the turbinate of challenged mice
					Infection/# assay	Mean (. + -. SE) Virus Titers	Infection/# assay	Mean (. + -. SE) Virus Titers
	PBS
		4/4	6.3±0.3	3/4	2.8±0.35
	MF-59 alone					4/4	6.1±0.13	3/4	3.0±0.38
FLU vaccine (5 ug)		4/4	6.3±0.07	3/4	2.9±0.36
	FLU vaccine (5 μ g) + MF-59					4/4	6.0±0.19	4/4	3.0±0.11
BPL-SARS-CoV(5μg)		_	1.6±0.13*	0/4	Not detected**
	BPL-SARS-CoV(5μg)+MF-59					0/4	Not detected*	0/4	Not detected**

In comparison to PBS immunized mice, the two-tailed student's t-test showed: p < 0.00001 or P ═ 0.025

As shown, no virus could be detected in BPL-SARS-CoV immunized mice. The lower limit of infectious virus detection in 10% w/v lung homogenate suspension was 1.5log₁₀TCID₅₀Pergm, 5% w/v turbinate suspension the limit was 1.8log₁₀TCID₅₀And/gm. Thus, the virus titer in the immunized mammal is lower than these initial values.

Thus, the inactivated SARS-CoV vaccine was effective in preventing viral infection, with only 1 of 8 mice vaccinated, with or without MF59 adjuvant. No similar protection was observed in the control group, which was either PBS diluent, MF59 adjuvant or influenza vaccine, with or without MF59 adjuvant.

Neutralization titers of sera taken from animals in challenge studies were evaluated 2 weeks after the 1 st immunization, 1 week after the 2 nd immunization, and 1 week after the 3 rd immunization. Mice immunized with the vaccine adjuvanted with MF59 had developed a neutralization titer of 1: 71 after the 2 nd immunization, increasing to 1: 588 after the 3 rd immunization, while mice receiving the vaccine without adjuvant did not have any neutralizing activity after the 2 nd immunization and the neutralization titer after the 3 rd immunization was 1: 64. The sera of the mice of each control group did not show any neutralizing activity. These data not only clearly demonstrate the ability of the inactivated SARS-CoV vaccine to induce protective SARS neutralizing antibodies, but also the beneficial effect of making the vaccine with adjuvant on increasing neutralizing titer.

Example 6 preparation of OMV containing SARS Virus antigen

Coli (e.coli) was transfected with the plasmid of interest (encoding the SARS virus antigen). A single colony with the plasmid of interest was grown overnight at 37 ℃ in 20ml LB/Amp (100. mu.g/ml) liquid culture. Bacteria were diluted 1: 30 in 1.0L fresh medium and grown at 30 ℃ or 37 ℃ until OD₅₅₀To 0.6-0.8. The recombinant protein was expressed induced with IPTG at a final concentration of 1.0 mM. After 3 hours of incubation, the bacteria were harvested by centrifugation at 8000 Xg 4 ℃ for 15 minutes and resuspended in 20ml of 20mM Tris-HCl (pH 7.5) and intact protease inhibitor (Boehringer-Mannheim)^TM). All subsequent processes were performed at 4 ℃ or on ice.

Cells were disrupted by sonication, using a Branson Sonifier 450, and centrifuged at 5000 Xg for 20 minutes to pellet unbroken cells and inclusion bodies. The supernatant containing membrane and cell debris was centrifuged at 50000g (Beckman Ti50, 29000rpm) for 75 minutes, washed with 20mM Bis-tris propane (pH 6.5), 1.0M NaCl, 10% (v/v) glycerol and pelleted again at 50000g for 75 minutes. The pellet was resuspended in 20mM Tris-HCl (pH 7.5), 2.0% (v/v) sarcosyl, intact protease inhibitor (1.0mM EDTA, final concentration) and incubated for 20 minutes to dissolve the intima. Cell debris was pelleted by centrifugation at 5000g for 10 min and the supernatant was centrifuged at 75000g for 75 min (Beckman Ti50, 33000 rpm). The outer membrane was washed with 20mM Tris-HCl (pH 7.5) and centrifuged at 75000g for 75 minutes or overnight. The OMVs were finally resuspended in 500. mu.l of 20mM Tris-HCl (pH 7.5), 10% v/v glycerol. The protein concentration was estimated by a standard Bradford assay (Bio-Rad), while the protein concentration of the inner membrane fraction was determined by a DC protein assay (Bio-Rad). The different fractions from the separation process were determined by SDS-PAGE.

Example 7 immunogenicity, dose and route Placement of recombinant spike proteins in mice

The immunogenicity, route and dose of the inventive recombinant spike proteins in mice were evaluated using the following detailed procedures. The administered antigen preferably elicits neutralizing antibodies at least in the range of 1/100-1/1000. Increased doses of antigen can be tested, ranging from 5 to 20 μ g of recombinant spike antigen, alone or in combination with equal volumes of MF 59-citrate, and SC or IM administered to anesthetized mice in 100 μ l of inoculum. Groups of BALB/c mice were sensitized on day 0 and boosted on days 14 and 28, 6 per treatment.

Group of	Treatment of	Dose/route	Sampling interval 1	Number of mice
					1-3	Rec-spike protein	20，10，5μg/SC	7, 21, 35, 42 days	6 per dose level
4-6	Rec-spike protein	20，10，5μg/SC	7	6 per dose level
					7-9	Rec-spike protein	20，10，5μg/IM	7, 21, 35, 42 days	6 per dose level
10-12	Rec-spike protein	20，10，5μg/IM	7	6 per dose level
					13-15	Rec-spike-MF 59	20，10，5μg/SC	7, 21, 35, 42 days	6 per dose level
16-18	Rec-spike-MF 59	20，10，5μg/SC	7	6 per dose level
					19-21	Rec-spike-MF 59	20，10，5μg/IM	7, 21, 35, 42 days	6 per dose level
22-24	Rec-spike-MF 59	20，10，5μg/IM	7	6 per dose level
					25	MF59	NA/SC	7, 21, 35, 42 days	6+6 (killed on days 7 and 42)
27	MF59	NA/IM	7, 21, 35, 42 days	6+6 (killed on days 7 and 42)
					29	Salt water	NA/SC	7, 21, 35, 42 days	6+6 (killed on days 7 and 42)
31	Salt water	NA/IM	7, 21, 35, 42 days	6+6 (killed on days 7 and 42)

This procedure can also be used to assess the Th1/Th2 profile of the specific immune response elicited by the recombinant spike protein. Neutralizing and spike-specific antibody titers were assessed on days 7, 21 and 35; determining the IgG2avs IgG1 isotype of the spike specific antibody at days 21 and 35; determining lymph node and spleen T cell anti-crevicular proliferation in vitro at days 7 and 42, respectively; spleen T cells were evaluated at day 42 for IFN- γ and IL-4 production against recombinant spike protein from SARS-CoV. Peripheral blood was collected on days 7, 21 and 35; lymph node cells were collected on day 7 and spleen cells were collected on day 42. The neutralizing and spike specific antibody titers and isotypes were determined by SARS-CoV infection of Vero cells and ELISA, respectively. Lymph node and spleen T cell proliferation by³[H]Thymidine uptake determination. The frequency of spleen IFN-. gamma.and IL-4 production by T lymphocytes was determined by ELISPOT and FACS.

Example 8 immunogenicity, dose and route Placement of spike proteins in rabbits

The recombinant spike proteins of the invention were evaluated for immunogenicity, route and dose in rabbits using the following detailed procedures. Increased doses of antigen can be tested, ranging from 5 to 40 μ g of recombinant spike antigen, alone or in combination with equal volumes of MF 59-citrate, and SC or IM administered to anesthetized animals in 200 μ l of inoculum. The new zealand white female rabbit group was immunized as shown in the table below, 10 animals per treatment. Animals were sensitized on day 0 and boosted on days 14 and 28. Peripheral blood was collected on days 7, 21 and 35. Neutralizing and spike specific antibody titers were determined by inhibition of SARS-CoV infection of Vero cells and ELISA, respectively.

Group of	Treatment of	Dose/route	Sampling interval	Number of rabbits
					1-4	Full length spike proteins	40，20，10，5μg/SC	7, 21, 35 days	10 per dose level
5-8	Full length spike proteins	40，20，10，5μg/IM	7, 21, 35 days	10 per dose level
					9-12	Truncated spike proteins	40，20，10，5μg/SC	7, 21, 35 days	10 per dose level
13-16	Truncated spike proteins	40，20，10，5μg/IM	7, 21, 35 days	10 per dose level
					17-20	Full-length spike protein-MF 59	40，20，10，5μg/SC	7, 21, 35 days	10 per dose level
21-24	Full-length spike protein-MF 59	40，20，10，5μg/IM	7, 21, 35 days	10 per dose level
					25-28	Truncated spike protein-MF 59	40，20，10，5μg/SC	7, 21, 35 days	10 per dose level
29-32	Truncated spike protein-MF 59	40，20，10，5μg/IM	7, 21, 35 days	10 per dose level
					33	MF59	NA/SC	7, 21, 35 days	10
34	MF59	NA/IM	7, 21, 35 days	10
					35	Saline	NA/SC	7, 21, 35 days	10
36	Saline	NA/IM	7, 21, 35 days	10

Example 9 immunogenicity and dosing of recombinant spike proteins in ferrets

The recombinant spike proteins of the invention were evaluated for immunogenicity and dosage in ferrets using the following detailed procedures. 3 groups of ferrets, 6 were used for treatment, which were immunized with recombinant SARS-CoV spike protein from a CHO cell line, either alone or mixed with equal volume of MF 59-citrate, and SC was administered to anesthetized animals in 200. mu.l of inoculum. The recombinant spike protein vaccine was tested at the time of eliciting the highest neutralizing antibody titers in mice, 35 days after the 2 nd boost. Animals were sensitized on day 0 and boosted on days 14 and 28. Peripheral blood was collected on days 7, 21 and 35. Neutralizing and spike specific antibody titers were determined by inhibition of SARS-CoV infection of Vero cells and ELISA, respectively.

Group of	Treatment of	Dose/route	Sampling interval	Number of ferrets
					1&2	Rec-spike protein	Y mug or 2Y mug/ SC	7, 21, 35 days	6
3&4	Rec-spike protein + MF59	Y mug or 2Y mug/ SC	7, 21, 35 days	6
					5	Salt water	NA/ SC	7, 21, 35 days	6

The 3 ferrets, 6 groups, used in the immunogenicity study above can then be used to assess the efficacy of the recombinant spike protein in protecting the immunized animals from infection and/or disease. After 2 weeks of the last intratracheal augmentation, use 10⁶Intermediate tissue culture infectious dose units (TCID)₅₀) The SARS-CoV Utah strain of (1) attacks the anesthetized animals. SARS-CoV infection was assessed by removing nasal, pharyngeal (faringeal) and rectal swabs from animals 20 days after challenge, as described above (12). The presence of SARS-CoV in the sample material was assessed by RT-PCR and Vero cell infection assay. Animals were monitored for clinical signs of SARS by assessing sleep time, temperature, respiratory symptoms, diarrhea, body weight and survival. Protection was determined by the amount and duration of viral release and the severity of disease symptoms and percentage of surviving animals.

Example 10 expression of spike proteins for immunization

SARS-CoV spike glycoprotein is expressed in full-length and truncated forms, constructed using nSh and nSh Δ TC pCMVIII as described above, and 2 both with a hexahistidine tag. Expression of the vector constructs was assessed 48 hours after transfection into 293 and COS7 cells. Full-length spike protein was detected only in cell lysates by western blotting (nSh), but not in the media (fig. 52).

Most SARS-CoV full-length spike protein was expressed as a high-molecular glycoprotein in transiently transfected COS7 cells running at 540kDa on a non-reducing gel (FIG. 53). gp540 is thermally unstable, indicated by complete dissociation into monomeric forms upon boiling (gp170 & gp180), but it is resistant to DTT treatment. These data suggest that the recombinant spike protein associates non-covalently to homotrimers (gp 540). The presence of spike proteins in homotrimeric associations was also confirmed in the inactivation and purification of SARS-CoV virions. Western blot analysis of virion proteins gave almost identical results, using the same conditions as for the determination of spike protein characteristics (figure 54).

Example 11: spike protein processing

To characterize spike protein processing, BHK-21 cells were infected with an alphavirus replicon particle expressing the full-length spike of SARS-CoV. After 6 hours of infection with MOI 5, the infected cells are treated with L-, [ 2 ], [³⁵S]Methionine/cysteine labeling was done for 1 hour and followed to 4 hours. [³⁵S]Labeled spike protein immunization by anti-SARS rabbit serumThe pellet was pelleted and digested with Endo-H. Both digested and undigested proteins were analyzed by SDS-PAGE (4% polyacrylamide). As shown in FIG. 55, the full-length spike protein was synthesized as an Endo-H sensitive high mannose glycoprotein (gp170, ER form) that underwent modification to an Endo-H resistant glycoprotein with intact oligosaccharides (gp180, Golgi form). The conversion of gp170 to gp180 form occurred within 2 hours (fig. 56).

Example 12: high level of protein expression

To develop a system for rapid expression of protein antigens, 293 (human embryonic kidney) cells were transfected with DNA to obtain milligram quantities of recombinant antigen. The most common method of culturing and transfecting 293 cells is in static or monolayer culture. These processes were modified for the production of secreted or intracellular proteins by large scale transfection of 293 cells in suspension and expansion of transfected cells in suspension culture. Some initial experiments were performed on 100ml scale cultures to determine optimal conditions such as cell number, transfection reagent type (FuGENE 6, Lipitoid or RO-1538) and DNA to transfection reagent ratio. FuGENE 6 was the best transfection reagent on a pre-experimental basis.

Comparing the kinetics of gene expression with those of other viral envelope glycoproteins, the data suggest that stable protein expression reaches a maximum approximately 72 to 96 hours after transfection, followed by a significant decrease, with the peak reaching being dependent on the gene of interest. Thus, using optimal conditions, the transfection method scales from 100ml to 4 liters. 4L cultures can be used to rapidly produce 2-10 mg protein antigen. To facilitate antigen purification and maximize yield and recovery of purified protein, transfection conditions were optimized by using serum-free media.

Bulk transfection methods were used to express both truncated and full-length spike proteins. The kinetics of expression of the truncated form of the spike protein is shown in FIG. 56A. The expression of the truncated form of the spike protein reached a maximum at about 48 hours and stabilized up to 72 hours, so cultures were harvested 72 hours after transfection.

The collected medium was concentrated 20X and used to purify the truncated spike protein by a very simple purification strategy in which the spike truncated form was captured on GNA lectin, followed by DEAE and ceramic hydroxyapatite column chromatography. The purified proteins were analyzed by silver staining on SDS-PAGE (FIG. 56B) and also by Western blotting (FIG. 56C). Early efforts were able to purify the truncated form of the spike protein at > 95% purity and recover approximately 50%. The molecular weight of the truncated form of the spike protein is approximately 170-180 kDa.

Full-length spike proteins were expressed in 293 cells using a bulk transfection strategy. Expression data suggest that like the truncated form, expression is highest at about 48 hours post transfection and remains stable up to 72 hours. However, in contrast to the truncated form and what was expected, the full-length protein was not secreted but remained intracellular as indicated by the absence of any signal in the western blot of cell culture supernatants. The full-length form of the protein was purified from cells extracted with Triton X-100 detergent. The full-length spike protein was subsequently captured on GNA lectin, followed by hydroxyapatite and SP chromatography. The calculated molecular weight of the full-length spike protein is approximately 600kDa, which is close to the theoretical mass of the trimer.

Example 13: SARS virus seed culture

The SARS-CoV reference seed Virus was propagated only in Vero cells tested, this Virus was used to produce the Master Virus (Master) and the Working Virus Seeds (Working Virus Seeds) under GMP. Clinical samples from SARS-CoV infected patients respiratory tract were inoculated into documented Vero cells using assay media. Virus-containing medium was collected 4 days after infection and designated passage 1 (P1). Round 2 virus propagation was again performed on qualified VERO cells, inoculated with 1ml of 100-fold diluted P1 virus per T-75 flask with assay medium. Culture supernatants were collected 3 days post infection and stored at-80 ℃ as P2 reference stock virus without plaque purification.

The Vero cell bank for further generation of SARS-CoV was prepared from a specific cell subset that had not been used since the emergence of transmissible spongiform encephalopathies (e.g. since 1980). A study cell bank of these cells was prepared with the indicated new zealand derived fetal bovine serum. For this study cell bank, a Master Cell Bank (MCB) was made under GMP conditions and only well-defined and well-controlled media and additives were used. The cell banks were tested for the absence of foreign agents according To applicable US, EU and international guidelines (see Points To Consider "cell line characteristics for production of biologicals", FDA/CBER 7/1993; ICH Q5D Draft 6 "cell substrate", 1996, 10/23; CPMP/ICH/294/95 "biotech product quality guide notes: obtaining cell substrates and characterization for production of biotech/biologicals" ( step 4, 97, 7, 16 days); WHO Final Draft "requirements for use of animal cells as in vitro substrates for production of biologicals" 1997, 3, 7 days). This cell bank also needs to be tested for tumorigenicity and identity.

The reference virus was plaque purified and amplified in qualified Vero cells without FCS to produce master and working seeds. Another option that helps ensure the purity of the master seed and facilitates safety assessment is to pellet SARS-CoV and resuspend it in PBS. Viral suspensions were made up to 60% (w/w) sucrose with crystalline sucrose, and the suspensions were transferred to centrifuge tubes and covered with 50, 40, 30 and 20% (w/w) sucrose solutions in PBS. Gradient centrifugation for 72 hours followed by fractionation. The virus-containing fractions were diluted and the virions were reprecipitated by ultracentrifugation. RNA from the viral pellet is isolated and transfected into qualified Vero cells, where "infectious" positive strand RNA results in the production of infectious virus that can be plaque purified and amplified to produce additional primary and working seeds from the purified viral RNA.

Viral seeds are tested for the absence of adventitious agents (see, e.g., 21CFR, no. § 630.35, safety test, revised 4/1, 1994) and for identity, using high-potency neutralizing antisera prepared from a separate source. Virus seed safety tests for vaccine purposes are routinely performed by service laboratories. Broad spectrum PCR tests may be added to the test and/or alternatively.

Example 14: scaling up virus production and inactivation

The generation, inactivation and purification procedures of inactivated SARS-CoV with sufficient structural integrity to elicit a protective neutralizing antibody response in animal models include: vero cells infected with virus, m.o.i. 0.01, no FCS and antibiotics; collecting the culture medium, centrifuging and removing, inactivating BPL, and then testing whether complete inactivation is achieved through verification; the inactivated material was filtered, subjected to MCS-column purification, and further purified by sucrose gradient centrifugation.

When this basic operation is used for larger scale commercial use, some modifications and improvements may be developed. First, cell culture and infection methods can be adapted to roller bottles as an intermediate step to rapidly produce in existing BSL 3+ equipment for preliminary testing. Full commercial production typically uses fermentation processes in closed systems, but roller bottle systems can be completed more quickly. Roller bottles provide a true suspension culture system for Vero cells, which has various technical and safety advantages over microcarrier culture. Suspension cultures can be grown to any desired fermentation scale without interfering with the closed system between cell generations because trypsinization is not required.

To scale up the infection process in roller bottles to 30-50 liters per batch, the optimal m.o.i. and harvest fractions for the selected medium and culture conditions should first be determined. For larger scale, methods of safely collecting and handling larger volumes of highly infectious material should be used, and therefore cell separation by centrifugation can be replaced by other methods, such as filtration through disposable cartridges.

The above described MCS chromatography and gradient purification steps are easily scaled up to batch volumes of up to 50 liters. However, for larger scales, to improve purity, ultrafiltration and sterile filtration steps are used. Nuclease treatment to remove host cell DNA is also included.

Example 15: large scale analytical methods

Analytical methods for SARS coronavirus include viral titration methods, immunological and physicochemical methods (ELISA, PAGE, western blot with specific antisera against purified whole virus, etc.) for quantitative purification of antigen and characterization. Other analytical tests include: fast yield test, through asymmetric domain flow separation and laser particle detection and counting; western blot using specific antisera against individual viral proteins; for testing of remaining host cell DNA.

The remaining DNA tests are generally done by hybridization, such as the use limit test. This test was performed according to established and other cell line validated methods. In addition, Threshold can be used^TMA method.

To generate specific antibodies, all ORFs from the structural and non-structural gene regions of SARS-CoV are expressed using recombinant proteins. ORFs can be cloned and expressed in E.coli, if desired, in eukaryotic vectors such as baculovirus. This provides a sufficient amount of purified stable protein for immunization of mice and rabbits to generate polyclonal and monoclonal antibodies against SARS proteins and to establish specific ELISA assays. Different expression vectors can be tested to maximize the yield of the recombinant protein in soluble form, e.g., different vectors, 1 containing a sequence encoding 6N-terminal histidine residues and the other 1 containing the glutathione-S-transferase protein fused to the C-terminus of the SARS protein. Recombinant proteins can be purified by single step column chromatography on nickel-chelating agarose or glutathione-sepharose 4B resins. These processes are very rapid and generally produce proteins of 60-90% purity, suitable for culturing specific antisera (Pizza et al (2000) Science 287: 1816-20). 5 mice and 2 rabbits for each recombinant protein were immunized with 20 and 50 μ g of recombinant protein SC, respectively, IFA as adjuvant, on days 0, 14 and 28. Sera were collected on days 7, 21 and 35 to assess specific titers before euthanasia of animals, which were euthanased for blood collection and spleen removal.

To detect impurities (e.g. proteins obtained from Vero cells) in vaccine preparations, rabbit serum reacted with Vero-obtained proteins may be used. This antiserum was obtained by immunizing rabbits with at least 10. mu.g of Vero cell lysate, CFA/IFA. The reactivity of sera with Vero-derived proteins can be tested in Western blots. For more specific antisera directed against specifically relevant cells that tend to co-purify with the virus-derived proteins, mock-infected cell cultures that were subjected to the purification process can be prepared for collection and use in immunizing rabbits.

Methods can be developed to determine serum neutralization titers from immunized animals and humans without the limitation of using infectious SARS-CoV in BSL-3+ laboratories. One such strategy is to develop an ELISA assay for measuring antibodies against a target protein using a recombinant antigen, specifically a spike protein or an epitope obtained from a spike. Appropriate antigen epitopes enable correlation to be established between ELISA values and virus neutralization measurements. This method compares specific and protective antibody titers more rapidly and more efficiently (higher yields). This ELISA test is also an ideal tool for monitoring specific antibodies in safety assays where hundreds of animal sera must be tested.

Another strategy is to combine structural elements from 2 pathogenic SARS-CoV with non-pathogenic coronavirus Mouse Hepatitis Virus (MHV) to construct chimeric virus-like particles (VLPs) that can be labeled. The assay is based on the fusion of octadecylrhodamine (R18) -labeled VLPs with cells (Hoekstra et al (1984) Biochemistry 23: 5675-81). The method relies on the reduction in self-quenching of fluorescence of R18 incorporated into VLPs upon fusion with the cell membrane. Coronavirus VLPs appear to mimic natural virions, relating to their appearance in Electron Microscopy (EM) and their biological activity. However, since they do not contain viral RNA, they are subsequently unable to cause productive infection (Vennema et al (1996) EMBO J15: 2020-. The VLP system can be used with Mouse Hepatitis Virus (MHV) strain A59(MHV-A59) (Godeke et al (2000) J Virol 74: 1566-15), which contains the chimeric S protein. Protein chimeras, which consist of the extracellular domain of SARS-CoV and the transmembrane and intracellular domains (64C-terminal amino acid residues) from the MHV spike protein, are capable of co-expression with MHV M (membrane) and E (envelope) proteins in OST-7 cells (Godeke et al.). VLPs secreted in the supernatant were collected, purified and labeled with octadecylrhodamine (R18) (Hoekstra et al). A constant amount of VLPs were incubated with serial serum dilutions for 1 hour at 37 ℃ in 96-well plates. Subsequently, angiotensin converting enzyme 2(ACE2) was added to cells expressing the SARS-CoV receptor and the degree of fusion was measured using a fluorescence spectrophotometer.

The final strategy was to monitor the ability of serum to inhibit cell-cell fusion interactions between cells expressing the SARS-CoV S protein and a human cell line expressing angiotensin converting enzyme 2(ACE2), which is a functional receptor for SARS-CoV (Li et al). This reporter-based assay utilizes the fluorescence shift (green to blue) of the fluorogenic substrate CCF2/AM (AM-acetoxymethyl) upon cleavage by beta-lactamase (Bla) as a cell-cell fusion readout (ZLokarnik et al (1998) Science 279: 84-88). For this assay, cell lines derived from BHK were generated which stably expressed Bla and SARS-CoV proteins. In addition, a human cell line expressing ACE2 on its surface was used. BHK cells expressing the S protein on their surface and expressing Bla in the cytoplasm were incubated with serial dilutions of the sera to be tested at 37 ℃ for 1 hour. ACE2 expressing cell lines were loaded with 1 μ M CCF2/AM for 1 hour at 22 deg.C, washed 2 times with PBS, and co-cultured with BHK cells. In the case of cell-cell fusion, Bla cleaves the substrate, producing a green-blue transition, excited at 409 nm. Thus, inhibition of fusion by serum provides a detectable change.

Example 16: stable inactivation of SARS-CoV

Although the purified inactivated SARS-CoV vaccine can induce an effective neutralizing antibody response in animals, it is relatively unstable and can benefit from formulations to improve stability over an acceptable period of time. Suitable formulation variations include the use of different buffer systems, pH ranges, stabilizing excipients (e.g., sugars and sugar alcohols, amino acids, etc.), and the like. The stability test can be performed in real time at normal storage temperature or in an accelerated manner by increasing the temperature. Thus, vaccine stability performance has increased to about 1 year or more. Lyophilized vaccine preparations can also be used to extend the life of the vaccine itself, allowing for increased stability with more additives during lyophilization.

Example 17: optimized dosage and schedule for inactivating viruses

Animal models of SARS-CoV infection have been reported, including mice, ferrets, and macaques. As shown in example 4 above, mice immunized with the BPL-SARS-CoV vaccine achieved neutralizing antibody titers ranging from 1: 100 to 1: 1000, similar to the levels found in convalescent patients, and 100% were protected from challenge virus infection. While mouse challenge models are limited to infection rather than disease, ferrets and macaques are useful models of human SARS disease. 2 to 4 days after inoculation with SARS-CoV, it was found that both ferrets and macaques shed infectious SARS-CoV particles from the throat, nose and pharynx, as evidenced by RT-PCR and/or virus isolation of Vero cells. At about the same time, the infected animals become drowsy, manifest respiratory distress and eventually die. Histologically, SARS-CoV infection in these animals was associated with lung lesions of varying severity, similar to that found in biopsied lung tissue and autopsy material from SARS patients. As these models become available, latency studies using vaccines can be first performed in mice for immune readings, and the efficacy of optimal doses and schedules can be assessed in ferret and macaque models.

Initial studies in mice were used to determine the optimal dose and schedule required to elicit the highest neutralizing antibody levels, with titers ranging at least from 1/100-1/1000. In parallel with the assessment of neutralizing activity, additional features of the humoral and cellular immune responses can be studied. Sera from immunized mice can be specifically assessed for spike-specific antibody response isotype (IgG1 vs. Similarly, the frequency of IFN- γ and IL-4 production by splenic CD4+ T cells in response to BPL-SARS-CoV particles can be assessed by ELISPOT and ELISA. These experiments help to gain insight into the quality of T cell responses that assist in eliciting protective antibody responses.

Increased vaccine doses (e.g., from 5 to 20 μ g BPL-SARS-CoV, alone or mixed with equal volume of MF 59-citrate) can be tested and SC administered to anesthetized mice in 100 μ l of inoculum. Groups of BALB/c mice were sensitized on day 0 and boosted on days 14 and 28 with 10 mice per treatment. Secondary endpoints (Secondary endpoints) compare the kinetics of neutralizing the relative spike-specific antibody titers and evaluate the Th1/Th2 distribution of the specific immune response, thus neutralizing and spike-specific antibody titers were evaluated 7, 21 and 35 days after sensitization and after 2, 3, 4 and 5 months. IgG2a and IgG1 titers of spike-specific antibodies were determined at 21, 35 days and 2, 3, 4 and 5 months after sensitization. Splenic T cells were evaluated at day 42 and at the end of 5 months for proliferation of anti-recombinant spike protein from SARS-CoV and for IFN-. gamma.and IL-4 production. Peripheral blood was collected after days 7, 21 and 35 and 2, 3, 4 and 5 months. Splenocytes were harvested at day 42 and at the end of 5 months. Neutralization and spike-specific antibody titers and isotypes were determined by inhibiting infection of Vero cells and ELISA, respectively. The splenocytes proliferate through³[H]Thymidine uptake determination. CD4⁺The frequency of spleen IFN-. gamma.and IL-4 production by T lymphocytes was determined by ELISPOT and FACS analysis.

Based on mouse results, the BPL-SARS-CoV vaccine was able to test induced protective neutralizing antibody titers in ferrets. Ferrets were immunized according to a similar schedule as mice and with doses that elicited the highest neutralizing antibody titers at day 35 after the 2 nd boost in mice. Groups 3 ferrets were immunized with BPL-SARS-CoV, either alone or mixed with equal volume of MF 59-citrate, and SC was administered to anesthetized animals in 200. mu.l of inoculum, 6 per treatment. Animals were sensitized on day 0 and boosted on days 14 and 28. Peripheral blood was collected on days 7, 21 and 35. Neutralizing and spike specific antibody titers were determined by inhibition of SARS-CoV infection of Vero cells and ELISA, respectively. Groups of ferrets were used to evaluate the efficacy of BPL-SARS-CoV to protect immunized animals from infection and/or disease. 2 weeks after the last fortification, 10 weeks⁶Intermediate tissue culture infectious dose units (TCID)₅₀) The SARS-CoV CDC strain of (A) attacks anesthetized animals intratracheally. SARS-CoV infection was assessed by removing nasal, pharyngeal and rectal swabs from animals 20 days after challenge (Martina et al, supra). The presence of SARS-CoV in the sample material was assessed by RT-PCR and Vero cell infection assay. Animals were monitored for clinical signs of SARS disease by assessing sleep time, temperature, respiratory symptoms, diarrhea, body weight and survival. Protection was determined by the amount and duration of viral release, duration and severity of disease symptoms, and percentage of surviving animals. Subsequently, the preparation giving the highest neutralizing antibody titer on day 35 can be tested against 2-fold higher doses of BPL-SARS-CoV given in the same preparation and with the same protocol.

Additional studies can assess the immunogenicity and efficacy of candidate vaccines in non-human primates. 3 component older rhesus macaques were immunized with BPL-SARS-CoV, either alone or mixed with equal volume of MF 59-citrate, and SC was administered to anesthetized animals in 500. mu.l inoculum, 4 per treatment. The BPL-SARS-CoV vaccine can be tested after the 2 nd boost in ferretsDose testing resulting in the highest neutralizing antibody titer on day 35. Animals were sensitized on day 0 and boosted at 3 and 6 weeks. Peripheral blood was collected at weeks 1, 4 and 7. Secondary endpoints assessed the Th1/Th2 distribution of specific immune responses. Thus, neutralization and spike-specific antibody titers and the frequency of IFN-. gamma.and IL-4 production by peripheral blood CD4+ T cells in response to recombinant SARS-CoV spike protein were evaluated at weeks 1, 4, and 7. Neutralizing and spike specific antibody titers were determined by inhibition of SARS-CoV infection of Vero cells and ELISA, respectively. Intracellular cytokine staining and FACS analysis CD4 for quantification of IFN-. gamma.and IL-4 production⁺T cells. Macaques can also be used to assess the efficacy of BPL-SARS-CoV in protecting immunized animals from infection and/or disease. 2 weeks after the last fortification, 10 weeks⁶Intermediate tissue culture infectious dose units (TCID)₅₀) The anaesthetized animals were challenged with the SARS-CoV CDC strain of (5 ml volume). A few drops of virus could also be administered in the conjunctiva, 0.5ml in the nose and the rest in the trachea. SARS-CoV infection was assessed by removing nasal, pharyngeal and rectal swabs from animals 20 days after challenge (Fouchier et al (200300 Nature 423: 240.) the presence of SARS-CoV in the sample material was assessed by RT-PCR and Vero cell infection assays.

Mouse

Group of	Treatment of	Dose/route	Sampling interval	Number of mice
					1-3	BPL-SARS- CoV	20，10，5μg/ SC	7, 21, 35 days; 2, 3, 4, 5 months;	10 per dose level
4-6	BPL-SARS- CoV	20，10，5μg/SC	42 days	10 per dose level
					7-9	BPL-SARS- CoV MF59	20，10，5μg/ SC	7, 21, 35 days; 2, 3, 4, 5 months;	10 per dose level
10-12	BPL-SARS- CoV MF59	20，10，5μg/SC	42 days	10 per dose level
					13	MF59	NA/ SC	7，21，35 days; 2, 3, 4, 5;	10+10 (kill at the end of day 42 and 5 months)
14	Salt water	NA/ SC	7, 21, 35 days; 2, 3, 4, 5 months;	10+10 (kill at the end of day 42 and 5 months)

Ferret

Group of	Treatment of	Route of the way	Sampling interval	Number of ferrets
					1	BPL-SARS- CoV	SC				7, 21, 35 days	6
2	BPL-SARS-CoV- MF59	SC												7, 21, 35 days	6
					3	Salt water	SC				7, 21, 35 days	6

Macaque

Group of	Treatment of	Pathway(s)	Sampling interval	Number of macaques
					1	BPL-SARS- CoV	SC				1, 4, 7 weeks	4
2	BPL-SARS-CoV- MF59	SC												1, 4, 7 weeks	4
					3	Salt water	SC				1, 4, 7 weeks	4

Example 18: human T cell response

As a precursor to the start of human clinical studies, the reactivity of peripheral blood T lymphocytes from healthy donors with different HLA haplotypes can be assessed using in vitro priming techniques (Abrignani et al (1990) Proc Natl Acad Sci USA 87: 6136-40). The purpose of this study was to first indicate immunodominant T cell epitopes in SARS-CoV proteins. Briefly, PBMCs from 20 healthy donors with different HLA haplotypes were cultured in media containing 5% autologous serum in the presence of different concentrations of SARS-BPL-CoV particles ranging from 0.5 to 20. mu.g/ml. Expression of activation markers was assessed after 24 and 48 hours. After 12 hours and 15 days of culture, the frequency of IFN-. gamma.and IL-4 producing T lymphocytes was evaluated, and 100U/ml recombinant human IL-2 was present. Activated cytokines that produced CD4T lymphocytes were sorted out and finally cloned as single cells using FACS techniques. CD4+ T cell repertoire from human subjects with different HLA was assessed by proliferation assay of clones of CD4+ T cell line and anti-self EBV-transformed cell line loaded with a 15-mer overlay peptide derived from the most relevant structural and non-structural proteins of SARS-CoV.

When turning to the actual human trial, safety and immune response in healthy adults were assessed following intramuscular immunization using increasing doses of BPL-inactivated SARS-CoV vaccine, including MF59 adjuvant or omitted, depending on clinical data. In cohort 1, 3/4 immunizations were given at 0, 1, 6 months, and in cohorts 2 and 3, immunizations were given at 0, 1, 2, 6 months and 0, 2, 6 weeks, respectively. The test was performed with blind observation and placebo as control. Subjects were randomized to dose waterAnd (7) flattening. Parameters of the immune response to be measured include serum neutralizing antibodies, ELISA antibodies and IFN-. gamma.producing peripheral blood CD4+ T cells, stained by intracellular cytokines.

Group of	Antigen dose (μ g)	Administration schedule	Number of subjects treated	Number of subjects with placebo	Sampling interval
						A1
	10	0, 1, 6 months	18	6	0, 1, 2, 6, 7 months
						A2
	20	0, 1, 6 months	18	6	0, 1, 2, 6, 7 months
						B1
	10	0, 1, 2, 6 months	18	12	0, 1, 2, 6, 7 months
						B2
	20	0, 1, 2, 6 months	18	12	0, 1, 2, 6, 7 months
						C1
	10	0, 2, 6 weeks	18	12	0, 2, 6, 10, 30 weeks
						C2
	20	0, 2, 6 weeks	18	12	0, 2, 6, 10, 30 weeks

Example 19: selection of CHO cell lines for spike protein expression

Methods for obtaining Chinese Hamster Ovary (CHO) cell lines stably expressing viral envelope glycoproteins with intact protein conformation, proper glycosylation and effective binding of neutralizing antibodies are well established for HIV and HCV (Srivastava et al (2002) J Virol 76: 2835-47; Srivastava et al (2003) J Virol 77: 11244-. The same technique can be applied to SARS-CoV for the generation of 2 different stable CHOK-1 cell lines, producing either full-length or truncated SARS spike protein. The spike protein can be expressed using the constructs described herein, but without the 6-His tag. The ability of these proteins to produce neutralizing antibodies in immunized animals and their expression levels in CHOK-1 cells can be compared.

A spike-expressing pCMV3 vector can be used to obtain stable CHOK-1 cell lines containing the CMV enhancer/promoter, ampicillin resistance, fused DHFR and attenuated neomycin gene for selection purposes. Stable cell lines can be generated in CHOK-1 cells using a neomycin-based selection system. Clones can be sequenced to confirm the integrity of the insert, transient transfection with Trans-LT1 polyamine transfection reagent (PanVera Corp., Madison, Wis.) can be used to assess expression levels and also expression protein integrity by ELISA and Western blot analysis.

Initial CHO cells were selected to be free of TSE/BSE contamination and risk was based on relevant regulatory criteria. To construct cell lines, the procedure involves transfection, primary selection with selective media, followed by subcloning to ensure cell line purity. Cell supernatants can be assayed by antigen capture ELISA to quantify expression levels for all selection and amplification stages. For full-length spike expression, internal expression of methanol-fixed cells was screened by immunofluorescence staining using rabbit anti-SARS antibody. Continuous measurements during the T-75 flask amplification stage can be used to ensure expression levels. The molecular weight and integrity of the expressed protein can be checked by PAGE under native and reducing and denaturing conditions, followed by immunodetection.

The pCMV3 vector expressing the full-length or truncated form of SARS-CoV spike protein can be introduced into CHOK-1 cells using Trans-LT-1 reagent and non-selective medium. 24-48 hours after transfection, cells divide at a 1: 5 ratio and the medium can be changed to a selective medium containing 500. mu.g/ml neomycin, depending on cell density. Any bovine serum used in these procedures was from a TSE-free source and met regulatory standards. After 10 to 14 days, individual colonies were picked and transferred to 96-well plates and cultured in complete non-selective medium. When approximately 80% of the wells were full, the 24 hour supernatant could be screened by spike capture ELISA. To initiate expression of the full-length spike protein, cells can be fixed with methanol and screened by immunofluorescence staining using rabbit anti-SARS antibody. After removal of low expressing cell lines, less than 20-30 cell lines, capture ELISA and western blot can then be used to determine expression levels after cell lysis. A portion of each cell line can be pelleted, weighed and lysed in 1% Triton lysis buffer for determination of expression levels. The 3 to 4 clones producing the highest level of structurally and conformationally correct spike protein can be scaled up to a 3 liter bioreactor and adapted to low serum suspension culture conditions for expansion.

Antigen capture ELISA assays for SARS spike protein can be performed with 96-well flat-bottom plates coated with 250ng of purified immunoglobulin per well, obtained from rabbit serum immunized with inactivated SARS virus. A sample of either supernatant or lysate was added and incubated at 37 ℃ for 2 hours. SARS binding to relative pool of antigens^+veSerum or high affinity monoclonal antibody, the monoclonal antibody is human or mouse SARS spike protein, and is detected with proper specific peroxidase conjugated secondary antibody. The plates were developed with TMB substrate (Pierce, Rockford, IL), read at 450nm wavelength, and the protein concentration per ml sample was obtained from a standard curve (OD vs. protein concentration) based on serial dilutions of a known concentration of recombinant spike protein.

Immunodetection assays were also performed, following standard methods described in Srivastava et al (2002) supra. Briefly, 10-20. mu.l samples were analyzed on 4-20% SDS PAGE under non-reducing/mildly heated denaturing conditions. Subsequently, the protein was transferred to nitrocellulose membrane and reacted with polyclonal anti-spiked rabbit serum, followed by Alexa688(Molecular Probes, Oregon) conjugated anti-rabbit Ig. The spot is scanned with an infrared imaging system.

The highest expressing candidate cell lines were screened for spike protein expression and stability in small scale (3 liter) perfusion bioreactors. Candidate clones were further evaluated for expression level and expressed protein integrity, and then tested for expression stability without selection. Selected clones were tested for maintaining the integrity of the integrated SARS spike protein gene DNA sequence. For rapid monitoring of expression levels in vials and 3 liter evaluation cultures, a lectin-based method (Gluvanthus Nivalis lectin) was developed for isolating SARS spike protein to a level of purity that allows for semi-quantification of protein in CHO supernatant and characterization. Full-length spikes were obtained from cells extracted with Triton X-100 detergent, subsequently captured on GNA lectin, followed by hydroxyapatite and SP chromatography. The eluted protein is then characterized by: (1) polyacrylamide gel electrophoresis (PAGE) and Coomassie staining, (2) immunodetection with anti-SARS rabbit serum, (3) structural characterization by Size Exclusion Chromatography (SEC) and mass spectrometry by MALDI-TOF.

The productivity of a CHO cell line expressing SARS spike protein should be at least 2mg/L and 3mg/100gm cells for full length spike protein, the cells being at steady state cell density. The yield from a 45 day, 2.5 liter bioreactor was 1000mg crude protein.

Example 20: purification of spike proteins for human vaccines

For the purpose of purifying the SARS spike protein for the production of GMP-grade material for human use, the following basic procedure is used, all steps being performed at 2-8 ℃: the starting material was concentrated CHO cell culture supernatant (20-30X), thawed and filtered through a 0.45 μm membrane; this material is a heavily contaminating protein from the culture as well as DNA; the 1 st purification step is affinity chromatography using Gluvanthus Nivalis (GNA), a lectin that preferentially recognizes terminal mannose containing carbohydrates; capture of glycosylated proteins, including SARS spike protein, non-glycosylated proteins and DNA that do not bind to the column; the GNA column is then subjected to 2 chromatography steps operating in flow-through mode; the anion exchanger DEAE and ceramic hydroxyapatite (cHAP); DEAE binds some contaminating supernatant proteins and DNA, while cHAP binds any contaminating serum proteins; the full-length spike protein was purified from the cell pellet; cells were lysed with Triton X-100, followed by capture of full-length spike protein on GNA lectin, followed by hydroxyapatite and SP chromatography.

The purified SARS spike can be further processed to remove foreign viruses: inactivating the virus at pH 3.51 hours; the sample was then concentrated and diafiltered into pH 4 buffer, finally the purified protein was captured with SP resin; the spike protein binds to this resin and many viruses flow through.

The spike protein was eluted, concentrated and diafiltered into buffer. The bulk product produced was then filtered through a DV50 virus removal membrane followed by 0.2 μm membrane filtration. The bulk of the material produced is filled into suitable containers, such as 3.0ml vials, 100 grade laminar flow purge hoods.

The assay for each purification step included protein concentration, endotoxin (LAL), bioburden and recovery.

Prior to human administration, efficacy testing can assess the specific ability of a vaccine to affect a particular response in vitro or in vivo assays. In vivo immunogenicity was determined by a quantitative group of 10 mice using different doses of protein antigen. Serum was analyzed for the presence of IgG antibodies by ELISA. The passing criteria are based on the number of seropositive vaccine-treated animals compared to reference criteria. Other tests include overall safety, sterility, purity, vaccine identity (using ELISA specific for spike protein), quantity & protein concentration (UV spectrophotometric absorption process, based on molar absorbance of aromatic amino acids).

Stability tests were performed on the bulk drug substance and the final container product. The bulk products were evaluated at-60 ℃ (recommended storage conditions), 25 ± 2 ℃ and 40 ± 2 ℃ temperatures, protected from light, at time points of 0, 3, 6, 9 and 12 months. The final container product was tested at-60 ℃ in reverse at 5 + -3 ℃, 25 + -2 ℃ and 40 + -2 ℃ at time points of 0, 3, 6, 9, 12 months. Stability indicating assays may include appearance, pH, protein content, SDS-PAGE, size exclusion HPLC, vessel/closure integrity, performed on single samples in bulk and triple final vessel material assays.

The proteins purified by this method can be evaluated in mice, rabbits and ferrets, as described above, and based on the results of examples 4, 5, 8 and 9 above.

Initial experiments were performed in mice to determine the optimal dose and schedule of GMP spike protein required to elicit the highest neutralizing antibody levels, with titers at least in the range of 1/100-1/1000. Spike proteins were tested in the range of 5 to 40 μ g, and mice were anesthetized in 100 μ l inoculum, either alone or mixed with an equal volume of MF 59-citrate. Groups of BALB/c mice were immunized, 10 mice per treatment. Animals were sensitized on day 0 and boosted on days 14 and 28. Secondary endpoint comparisons neutralize the kinetics of the relative spike-specific antibody titers and are used to assess the Th1/Th2 distribution of specific immune responses. Assessment of neutralization and spike specific antibody titers at 7, 21 and 35 days post-sensitization and after 2, 3, 4 and 5 months; IgG2a and IgG1 titers of spike-specific antibodies were determined at 21 and 35 days and 2, 3, 4 and 5 months after sensitization; evaluating spleen T cell proliferation and IFN-gamma and IL-4 production against recombinant spike protein from SARS-CoV at day 42 and at the end of 5 months; peripheral blood was collected after days 7, 21 and 35 and 2, 3, 4 and 5 months; splenocytes were harvested at day 42 and at the end of 5 months; neutralizing and spike specific antibody titers and isotypes were determined by inhibition of SARS-CoV infection of Vero cells and ELISA, respectively. The splenocytes proliferate through ³[H]Thymidine uptake determination. CD4⁺The frequency of spleen IFN-. gamma.and IL-4 production by T lymphocytes was determined by ELISPOT and FACS analysis.

Second, the optimal dose and schedule of recombinant spike protein in ferrets was determined. Based on the mouse results, the spike vaccine that gave the highest antibody neutralization titers was tested against 2-fold higher doses of recombinant spike protein given in the same formulation. 3 groups of ferrets were SC immunized under anesthesia with 200. mu.l inoculum, 6 per treatment. Animals were sensitized on day 0 and boosted on days 14 and 28. Peripheral blood was collected on days 7, 21 and 35. Neutralizing and spike specific antibody titers were determined by inhibition of SARS-CoV infection of Vero cells and ELISA, respectively. Similar to the previous ferret study, groups of ferrets were used to evaluate the efficacy of vaccines to protect immunized animals from infection and/or disease.

The immunogenicity and efficacy of the candidate vaccine can also be assessed in non-human primates. 3 component older rhesus macaques were immunized with recombinant SARS-CoV spike protein, either alone or mixed with equal volume of MF 59-citrate, and SC was administered to anesthetized animals in 500. mu.l inoculum, 4 per treatment. Spike protein vaccines were tested at the dose that elicited the highest neutralizing antibody titer in ferrets on day 35. Animals were sensitized on day 0 and boosted at 3 and 6 weeks. Peripheral blood was collected at weeks 1, 4 and 7. The secondary endpoint assessed the Th1/Th2 profile of the specific immune response as described above (neutralization and spike-specific antibody titers, frequency of IFN-. gamma.and IL-4 production by peripheral blood CD4+ T cells in response to recombinant spike protein, assessed at weeks 1, 4 and 7).

Finally, IM recombinant SARS vaccine with MF59 adjuvant was subjected to human phase, placebo control, dose escalation, safety/immunogenicity trials. The test evaluates safety and immune response in healthy adults following immunization with increasing doses of SARS recombinant vaccine with MF59 adjuvant, administered intramuscularly. 3/4 immunizations were given at 0, 1, 6 months. The trial was observer blind and placebo controlled. Subjects were randomized to individual dose levels. The immunoreactive parameters to be measured included serum neutralizing antibodies, ELISA antibodies and IFN- γ producing peripheral blood CD4+ T cells, stained by intracellular cytokines:

group of	Vaccine antigen dose (μ g)	Administration schedule	Number of subjects treated	Subjects with placebo (MF59)Number of	Sampling interval
						A1	50	0, 1, 6 months	18	6	0, 1, 2, 6, 7 months
A2
		100	0, 1, 6 months	18	6	0, 1, 2, 6, 7 months

Example 21: comparison of inactivated Virus with purified spike protein

The immunogenicity and efficacy of inactivated virus and purified spike protein can be compared in non-human primates. 3 component old cynomolgus monkeys were immunized with recombinant SARS-CoV spike protein from CHO cell line or with BPL-SARS-COV, 4 per treatment, the doses and formulations used resulted in the highest neutralizing antibody titers in previous immunogenicity challenge experiments, and SC was administered to anesthetized animals in 500. mu.l inoculum. Animals were sensitized on day 0 and boosted at 3 and 6 weeks. Peripheral blood was collected at weeks 1, 4 and 7. The secondary endpoints assessed the Th1/Th2 distribution of the specific immune response, as described above.

Group of	Treatment of	Dose/route	Sampling interval	Number of macaques
					1	Rec-spike protein + or-MF 59	Yμg/ SC	1, 4, 7 weeks	4
2	BPL-SARS-CoV + or-MF 59	Yμg/ SC	1, 4, 7 weeks	4
					3	Salt water	NA/ SC	1, 4, 7 weeks	4

Example 22: expression in Yeast

Yeast is a useful and inexpensive eukaryotic expression system. The protein expressed by yeast is used for recombining hepatitis B virus vaccine, and recombining SARS antigen can also be expressed in total yeast for vaccine application. Yeast expression is also convenient for antigen production, for use in the preparation of monoclonal and polyclonal antibodies, or for use in serological assays.

Cloning of nucleocapsid protein (N) and 2 different forms of spike glycoprotein (S) from SARS coronavirus FRA strain (AY310120) for expression in saccharomyces cerevisiae (S. cerevisiae):

SARS N: amino acids 1-422 (coordinates 28120 of AY310120 strain 29388) -FIG. 65

SARS spike: amino acids 14-1195 (transmembrane domain and cytoplasmic tail deleted) -FIG. 66

SARS spike: amino acids 14-662(S1 Domain)

To generate the S1 construct, an approximately 3733bp XhoI-NotI fragment was used as the starting point, which encodes the full-length spike glycoprotein. PCR was used to amplify the full-length gene in 2 segments: 2440bp of XbaI-BlnI and 1306bp of BlnI-SalI. These fragments were separately subcloned into commercial vectors (Novagen): pT7Blue2XbaI-BlnI (5 'end of spike glycoprotein) and pT7Blue2BlnI-SalI (3' end of spike glycoprotein; FIG. 58). The following primers were used for the subsequent PCR reaction: spk-1 (5') SEQ ID NO: 9785 (b); spk-2 (5') SEQ ID NO: 9786; spk-3 (5') SEQ ID NO: 9787; spk-4 (5') SEQ ID NO: 9788.

Coli HB101 competent cells were transformed with PCR ligation products and plated on Luria agar plates containing 100. mu.g/ml ampicillin. Desired clones were identified by micro-screen DNA analysis. After sequence confirmation and plasmid amplification of the desired subclones, the internal SalI site present in the XbaI-BlnI portion of the spike sequence needs to be removed to facilitate future cloning into the yeast expression vector (BamHI-SalI). Therefore, we prepared CelII-MfeI vector from pT7Blue2XbaI-BlnI (5' terminal spike) to remove the 143bp sequence containing SalI site. The kinase-modified oligonucleotide DS1-6(SEQ ID NOS: 9789-9794) was subsequently ligated into the CelII-MfeI vector to replace the 143bp removed for SalI site mutation (no amino acid change), yielding pT7Blue2. XbaI-BlnI. DELTA. sal.

5 'XbaI-BlnI (from pT7Blue2. XbaI-BlnI. DELTA. Sal) and 3' BlnI-SalI (from pT7Blue2BlnI-SalI) spike glycoproteins were inserted and purified by gel, and they were ligated into p893-1XbaI-SalI vector (vector obtained from pLitmus 38(New England Biolabs), and the alpha factor leader sequence was cloned into the BamHI-Sal I site of MCS). The resulting full-length SARS Spike coding sequence was designated p893-1.SARS Spike 1255#9 (FIG. 58).

Coli HB101 competent cells were transformed with oligonucleotides in place of the ligation products and plated on Luria agar plates containing 100. mu.g/ml ampicillin. Desired clones were identified by micro-screen DNA analysis. After confirming the positive clone sequence, pT7Blue2 Xba-Bln. DELTA.Sal was selected as a template for PCR reaction to amplify the spike S11967 bpXba-Sal fragment. The fragment was subsequently subcloned into the p893-1Xba-Sal vector, the sequence was confirmed, and was designated p893-1.Spike S1#11 (FIG. 59).

For cloning into the Saccharomyces cerevisiae expression vector pBS24.1, the 5 'end of the S1 sequence had to be modified from XbaI to HindIII to allow ligation with the 3' HindIII end of the ADH2/GAPDH BamHI-HindIII promoter fragment. The AgeI-SalI 1943bp fragment was gel purified from pT7Blue2Xba-Bln Δ Sal (supra). This fragment was ligated into the pSP72HindIII-SalI commercial subcloning vector (designated pSP72.SARS Spike S1# 2; FIG. 59) together with an oligonucleotide (S1-1+ S1-2, creating the necessary 5' HindIII site) which acted upon the HindIII-AgeI 30bp kinase. S1-1 has the sequence of SEQ ID NO: 9795 and S1-2 have SEQ ID NO: 9796.

after confirming the sequence of the positive clone from the micro-screening DNA analysis, the HindIII-SalI fragment was gel purified. The 1365bp BamHI-HindIII ADH2/GAPDH promoter fragment was ligated into the pBS24.1 BamHI-SalI vector along with the 1973bp HindIII-SalI S1 fragment to create a genetically engineered pd. SARS spike S1#2 expression plasmid (FIG. 60).

S. cerevisiae strain AD3 transformed pd. sars spike S1#2, and individual transformants were examined for expression after depletion of glucose in the culture medium. The recombinant protein was expressed at high levels in yeast as detected by coomassie blue staining. Yeast cell-specific transformation of SARS S1 expression plasmid Using Invitrogen S.c. EasyComp ^TMAnd (4) a transformation kit. The expression is shown in FIG. 57.

Spike 1195 protein does not contain the Transmembrane (TM) region or cytoplasmic tail present in the full-length SARS construct, and to express this protein, the following series of genetic manipulations were performed:

the BlnI-DraI 1056bp fragment was gel purified from pT7Blue2BlnI-SalI #11 (supra). This fragment was ligated with a 68bp oligonucleotide (DRS1+ 2; SEQ ID NOS: 9797 & 9798) synthetic pair acting as DraI-SalI kinase into pT7Blue2BlnI-SalI vector (FIG. 61). Coli HB101 competent cells were transformed with oligonucleotides in place of the ligation products and plated on Luria agar plates containing 100. mu.g/ml ampicillin. Desired clones were identified by micro-screen DNA analysis. After the sequence was confirmed, the clone was named pT7Blue2BlnI-Sal spike 1195# 7. The 1126bp BlnI-SalI fragment encoding the end of spike 11953' was gel purified.

To generate the XbaI-SalI spike 1195 fragment, a 3109bp XbaI-PciI fragment was isolated from p893-1.SARS spike 1255#9 (supra) and a 457bp PciI-SalI fragment from pT7Blue2.SARS spike 1195#7 (supra). The 2 fragments were cloned into p893-1XbaI-SalI vector, resulting in p893-1.SARS spike 1195#34 plasmid (FIG. 62).

To clone SARS spike 1195 into the pBS24.1 s.cerevisiae expression vector, the SARS spike 11955' end must be modified from XbaI to HindIII as done for the spike S1 expression clone described above. First, a 2416bp AgeI-BlnI fragment was isolated from p893-1.SARS spike 1195# 34. This fragment was ligated into pT7Blue2HindIII-BlnI vector together with a synthetic HindIII-AgeI 30bp oligonucleotide (S1 protein was generated as described above for expression in Saccharomyces cerevisiae). Coli HB101 competent cells were transformed with oligonucleotides in place of the ligation products and plated on Luria agar plates containing 100. mu.g/ml ampicillin. Desired clones were identified by micro-screen DNA analysis. After confirmation of positive clone sequences and plasmid amplification of pT7Blue2.SARS 11955' HindIII-BlnI #10 (FIG. 63), we isolated a 402bp HindIII-NcoI fragment and a 2044bp NcoI-BlnI fragment (FIG. 63). HindIII-BlnI isolation was performed in 2 steps to avoid cloning events involving an internal HindIII site located at nucleotide number 1319 of the spike 1195 protein.

To assemble the BamHI-SalI expression cassette of spike 1195 into the pBS24.1 vector, E.coli HB101 competent cells were transformed into the pBS24.1BamHI-SalI vector with BamHI-HindIII (ADH2/GAPDH promoter), HindIII-NcoI 402bp fragment, NcoI-BlnI 2044bp and BlnI-SalI 1126bp fragments. Samples were plated on Luria agar plates containing 100. mu.g/ml ampicillin. The desired clone was identified by micro-screen DNA analysis, thus producing a genetically engineered pd.sars spike 1195#10 (fig. 64).

S. cerevisiae strain AD3 transformed pd. sars spike 1195#10 and individual transformants were examined for expression following glucose depletion in the culture medium. Recombinant proteins were detected by coomassie blue staining. Yeast cells were specifically transformed with the SARS S1 expression plasmid using the Invitrogen s.c. easycomptm transformation kit.

Example 23: expression in mammalian cell lines

A cDNA fragment containing the S protein ORF, 1255 amino acids in S protein ORF, was amplified by RT-PCR from SARS virus RNA (Frankfurt isolate) grown in Vero cells. The amplified PCR fragment was cloned into pBlueScript vector, sequenced, and the consensus spike sequence was aggregated to generate the full-length SARS spike clone pBSnSh. pBSnSh is transcribed in vitro and then translated in rabbit reticulocyte lysates, producing a single polypeptide with an estimated molecular weight of 140 kDa.

This plasmid insert was recloned via XhoI and Not I into the mammalian expression vector pCMVIII (Srivastava et al (2003) J.Virol.77: 11244-11259) to yield construct nSh (FIG. 74A). The PCR fragment contained a spike protein of 1195 amino acids, which deleted the Transmembrane (TM) domain and the cysteine rich cytoplasmic tail (Cy), which was amplified and the pCMVIII vector was cloned to generate the construct nSh Δ TC (fig. 74B). Both 2 constructs were C-terminally labeled with 6 histidine residues to aid in characterization. The XhoI/NotI fragment without histidine tag was subcloned into the alphavirus replicon vector backbone pvcrchim2.1 for generation of alphavirus replicon particle chimeras expressing the S protein. Generation and characterization of replication-defective alphavirus vector particles was essentially performed as described previously (Perri et al (2003) J.Virol.77: 10394. 10403; Polo et al (1999) PNAS USA.96: 4598. 4603). The resulting alphavirus vector particles were designated VEE/SIN.

COS7 cells and BHK-21 cells were maintained in Dulbecco's modified Eagle's Medium supplemented with 10% fetal bovine serum, at 37 ℃ and 5% CO in air₂. COS7 cells were transfected with an expression plasmid (nSh, nSh. delta. TC) using a transfection kit (Transit-COS, Mirus) following the manufacturer's instructions. Cells were washed 1 time with ice PBS and lysed with 1 Xlysis buffer (20mM MOPS, 10mM NaCl, 1.5mM MgCl) ₂And 1% Triton X-100) in buffer containing the intact protease inhibitor (Roche). After incubation on ice for 30 minutes, the debris was removed by centrifugation. Lysates were clarified or purified or used directly for western blotting.

To purify the secreted spike protein, the medium was collected from the transfected cells and centrifuged at 12,000rpm for 10 minutes to remove cell debris. The clarified medium was applied to a ConA-Sepharose column (Vector Lab). The column was washed well with 20mM sodium phosphate buffer, followed by elution of bound protein with 1M methyl α -D-mannopyranoside (MMP), 1M NaCl in 20mM sodium phosphate buffer. The column section containing the SARS-CoV spike protein was applied to the MagneHis protein purification System (Promega) following the manufacturer's recommended protocol.

For Western blot analysis, proteins were separated by 4-20% SDS-PAGE and subsequently electrophoretically transferred to nitrocellulose membrane (Invitrogen). The membrane was blocked in blocking buffer (5% skim milk in PBS and 0.1% tween 20), incubated with the indicator antibody for 1 hour at room temperature, washed and probed with horseradish peroxidase (HRP) conjugated secondary antibody (Biosource), followed by chemiluminescence (ECL system, Amersham) and exposure to X-ray film. The antibody used was a mouse monoclonal anti-histidine antibody (anti-His-tagged monoclonal antibody, Novagen), a rabbit polyclonal anti-peptide antibody against SARS-CoV spike protein (SmPab, Abgent) or rabbit anti-SARS serum (2BE) obtained by immunizing rabbits with purified SARS-CoV virions. The latter cell culture neutralization titer was 1/2,500. Unless otherwise indicated, antibodies were used at 1/1,000 for anti-histidine antibodies and SmPab, and 1/10,000 for anti-SARS rabbit serum.

Some spike proteins were treated with peptide-N glycosidase F (PNGase F). Cell lysates were diluted in 0.5% SDS and 1% β -mercaptoethanol and denatured at 100 ℃ for 10 min. After 2-fold dilution with 1% NP-40 in 50mM sodium phosphate (pH 7.5), the sample was treated with PNGase F (NEB) at 37 ℃ for 1 hour. The enzyme treated samples were analyzed by 4-12% SDS-PAGE under reducing conditions. For partial digestion with PNGase, the cell lysate was diluted with 50mM sodium phosphate (pH 6.0) containing 0.75% Triton-X and treated with PNGase F (Calbiochem) for 3 hours at 37 ℃. The enzyme treated samples were analyzed by 4-20% SDS-PAGE under non-reducing conditions.

Western blots of cells 48 hours post transfection are shown in figure 75. When the lysates were boiled and analyzed under reducing SDS-PAGE conditions, S protein doublets were detected in the cell lysates, estimated to have molecular weights of 170-180 kDa (FIG. 75A, lane 3). This doublet appears to be due to differential glycosylation of 1 polypeptide product after pretreatment of cell lysates with PNGase F, which reduces the doublet to a single species of-140 kDa (fig. 75A, lane 4). This is the expected size predicted from the amino acid sequence of the full-length, intact polypeptide product. This experiment shows that full-length SARS-CoV is expressed as a single, uncleaved polypeptide in mammalian cells, but has 2 different glycosylation forms, gp170 and gp180, respectively. Unlike the full-length sequence encoding 2S glycoforms that were not secreted, a single species S.DELTA.protein product of 160kDa was detected in both cell lysates (FIG. 75A, lane 5) and cell culture medium (FIG. 75A, lane 3).

To further characterize the intracellular processing of the S protein, BHK21 cells were infected with defective alphavirus particles expressing full-length S, as described above. After 6 hours of infection with MOI 5, the infected cells are treated with L-, [ 2 ], [³⁵S]The methionine/cysteine pulse was labeled for 1 hour and followed for 2 or 4 hours. Immunoprecipitation [ alpha ], [³⁵S]Labeled S protein, rabbit antiserum raised against inactivated, purified virus, followed by Endo H digestion. Endo H treatment consisted of dilution with sample buffer (50mM sodium phosphate, 0.1% SDS, 50mM DTT, pH 6.0) and boiling for 5 minutes. After denaturation, it was further diluted with 0.75% Triton-X100 and treated with endoglycosidase H (endo H) at 37 ℃ for 3 hours following the manufacturer's instructions (Calbiochem). The enzyme-treated samples were added to a loading buffer containing 0.1% SDS and DTT and analyzed by 8% SDS-PAGE.

Both digested and undigested proteins were boiled in SDS and analyzed by reducing SDS-PAGE (FIG. 55). After 1 hour pulse, the S protein appeared as gp170 single component, sensitive to Endo H (lanes 1 and 2). After a 2 hour pulse, the new species (gp180) appeared together with gp170 in approximately equal proportions (lane 3). After 4 hours of pulsing, the gp180 species was the major S protein component (lane 5), now resistant to Endo H (lanes 5 and 6). This data is consistent with gp170 being an ER-resident glycoprotein with a high mannose chain and gp180 corresponding to a Golgi processing glycoprotein with Endo H resistant complex oligosaccharides.

The Endo H sensitivity of C-terminal deleted S.DELTA.proteins purified from cell culture media was also tested. As shown in fig. 76, S Δ observed in cell lysates was found to be sensitive to Endo H (lanes 1 and 2), while S Δ was secreted in cell culture medium against Endo H (lanes 3 and 4). This result is consistent with the glycoprotein being synthesized in the ER in an immature form before transfer to the Golgi apparatus, which adds complex carbohydrates and subsequently secretes proteins.

As described above, the S protein expressed in COS7 cells was detected in Western blot analysis of cell lysates, which were in the form of gp170/gp180 doublets, and the cell lysates were fully denatured by boiling in the presence of DTT. However, when the same cell lysate was not heat denatured prior to Western blot analysis by SDS-PAGE, most of the S proteins detected were high molecular glycoproteins in the range of 440-669kDa (FIG. 77, lane 1). The 500kDa species was treated against 10mM DTT (lane 3) and did not dissociate into the monomeric form unless the lysate was first heat denatured at 100 deg.C (lane 4). In contrast, the oligomeric form of the test protein (thyroglobulin), whose quaternary structure is maintained by disulfide bonds, is converted to the subunit form by 10mM DTT treatment. These data suggest that the-500 kDa oligomeric form of the S protein is not disulfide-linked and is heat labile. To determine the heat sensitivity of the S protein-500 kDa species, the heat denaturation experiment was repeated, but without DTT. As shown in FIG. 78, heat denaturation of the-500 kDa protein alone at 100 ℃ was sufficient to convert it to the gp170/180 unimer form (lane 4). Using a 80 ℃ heat denaturation step, a similar ratio of 500kDa and monomeric forms was detected.

To further investigate whether this-500 kDa species represents oligomers of the S protein in native conformation, this protein was derived from Vero cell cultures using comparative analysis of the S glycoprotein obtained from virions. After SDS PAGE, purified virions were dissolved in 1% SDS and subjected to Western blot analysis. The presence of the-500 kDa spike oligomers was confirmed in the virion particles (FIG. 79, lane 1). In addition, the oligomer to homopolymer transition produced by heat denatured soluble virions was the same as that observed with full length recombinant S (lanes 2, 3). The oligomeric nature of virions S was further analyzed in cross-linking experiments. Aliquots of inactivated virus from sucrose gradient fractions were treated with 10% SDS at 1% final concentration and diluted 2-fold with 0.2M triethanolamine-HCl (pH 8, Sigma); dimethine (DMS; Pierce Chemical Co.) from a freshly prepared solution (10mg/ml in 0.2M triethanolamine-HCl) was then added to a final concentration of 3.3 mg/ml. After 2 hours at room temperature, the samples were concentrated with Centricon-30, electrophoresed on a 4% polyacrylamide gel and analyzed by silver staining. Both untreated and DMS-crosslinked virion proteins were heat denatured and the effect of heat on maintaining oligomeric structure was analyzed by SDS-PAGE and silver staining (FIG. 80). Without crosslinking, thermal denaturation results in the substitution of monomeric species for the 500kD spike protein species. In contrast, in cross-linked proteins, the-500 kD and monomeric species do not change significantly upon heating. These data support that the 500kD protein is an oligomer of non-covalently bound S-monomeric protein. After cross-linking and boiling, the-500 kD species move, diffusing somewhat slower than the untreated form. This mobility shift may be due to structural changes caused by boiling. In addition, a small 300kD protein species could be found, which could represent a non-dissociating S dimer.

To more accurately estimate the size of recombinant-500 kDS species expressed in COS7 cells, COS7 cell lysates containing S protein oligomers were fractionated by size exclusion column chromatography. The major portion of the 500kD oligomer is co-eluted with the 572kD marker protein. Together, these experiments suggest that the-500 kD S species observed in COS7 cell lysates may be homotrimers of S protein monomers.

The oligomeric state of S.DELTA.spike protein was also examined after expression in COS7 cells. As shown in FIG. 81, the presence of recombinant S.DELTA.protein in the cell lysate was also detected in the high molecular weight form in the range of-500 kDa when the lysate was not heated prior to SDS-PAGE and Western blot analysis (lane 1). However, the oligomerization efficiency of intracellular S.DELTA.protein appeared to be much lower (< 10%) than the full-length S protein under the same Western blot analysis conditions. Heat sensitivity testing of this 500kDa protein showed that the S.DELTA.oligomers were more heat labile than the full-length S oligomers, as evidenced by > 90% conversion of all 500kDa species to the monomeric Sd form at 80 ℃ (lane 2). Also (FIG. 82), most of the secreted S.DELTA.proteins were found in the form of a homopolymer, while the-500 kDa species were barely detectable (only when the protein was overdosed for Western blot analysis) (lane 1). At temperatures above 80 ℃, all secreted S Δ proteins detected were unimers (lanes 2, 3).

Glycosylation of 500kDa protein, study of the effect of deglycosylation on antibody binding. Recombinant COS7 lysate was treated with PNGase F under non-denaturing conditions (as described above) and subjected to western blot analysis. As shown in fig. 83, deglycosylation did not affect the anti-histidine Mab antibody binding to the treated S oligomers (lanes 2, 3). However, it affected the reactivity with rabbit antiserum raised against the purified virus (lane 6). DTT alone when omitted from SDS-PAGE samples, this antiserum bound S obtained from the virion in western blot analysis, indicating that it recognizes predominantly discrete conformational epitopes. This antiserum also showed high virus neutralizing antibody titers. It does not bind deglycosylated, recombinant S, suggesting that carbohydrates actively act on the more ordered, naturally-structured S polypeptide oligomers.

The difference between recombinant S and S.DELTA.proteins is the presence or absence of TM-and Cys-rich domains at the C-terminus. This difference predicts that upon lysis of the transfected cells, full length S is associated with the membrane fraction and Sd is in the soluble fraction. Thus, nSh or nSh Δ TC transfected cells were lysed under hypotonic conditions and the soluble cytosolic fraction was separated from the insoluble membrane fraction by centrifugation (fig. 48). As shown in FIG. 84, S proteins of the-500 kDa and 180/170kDa species were found in the membrane fraction (DF) (lane 4), but could not be detected in the soluble cytosolic fraction (AF) (lane 3). However, truncated S.DELTA.proteins of the monomeric species (gp170) were found in all 2 fractions (lanes 5, 6). This suggests that the C-terminal TM-and Cys-rich domains are required for the S protein to anchor to the cell membrane.

Cell localization of S and S Δ proteins in COS7 cells was analyzed by indirect immunofluorescence microscopy. 48 hours after transfection, cells were either fixed directly with 2% paraformaldehyde, without detergent for cell surface staining, or treated with detergent followed by Cytofix/Cytoperm solution for intracellular staining. The fixed cells were then stained with rabbit anti-SARS serum (2BE) and FITC-conjugated antibody. nSh transfected cells showed S protein foci indicating Golgi localization (FIG. 85A), while nSh Δ TC transfected cells showed uniform distribution of S Δ protein throughout the cytoplasm indicating ER localization (FIG. 85B). Although intact S protein on the surface of transfected cells was also observed in non-fixed cells (fig. 85D), no S Δ could be detected on the cell surface (fig. 85E). These results indicate the role of TM-and Cys-rich domains in anchoring S proteins to the plasma membrane. Although the TM region alone may act as a membrane anchor, the potential role of the Cys-rich region remains to be determined.

Thus, the SARS recombinant full-length S protein is an N-linked glycoprotein with an estimated molecular weight of 170-180,000 kDa. Deglycosylation with PNGase F produced a polypeptide of the expected size for use in the non-cleaved, encoded polypeptide (140 kDa). Expression of the full-length SARS-CoV S gene transiently and stably in a variety of mammalian cells including COS7, 293, BHK21, and Huh7 cell lines consistently produced the S protein doublet (gp170/180) as detected by Western blot analysis. Pulse-chase analysis of transfected cells demonstrated that SARS-CoV S protein was initially synthesized as an Endo H-sensitive gp170 species, followed by a gradual evolution of the Endo H-resistant gp180 form, probably due to the addition of complex carbohydrates and Golgi.

Recombinant S protein is not secreted into the cell culture medium unless the C-terminal 60 amino acids containing the TM region and the Cys-rich tail are deleted.

The quaternary structure of the full-length recombinant S protein was studied using cross-linking treatment, heat denaturation and size fractionation analysis. The results are consistent with the presence of recombinant S protein as a-500 kDa homotrimer. Similar analysis of S obtained from virions also yielded the same results. Other enveloped RNA viruses have reported this trimeric structure: hemagglutinin HA of influenza virus, E1-E2 heterodimer of alphavirus, and G protein of vesicular stomatitis virus. Incubation under reducing conditions showed that the SARS-CoV S trimer structures are non-covalently associated and very stable. The S oligomers present in the cell lysates showed resistance to 10mM DTT reduction, 1% SDS detergent treatment and heat denaturation up to 60 ℃. Incubation at temperatures > 80 ℃ resulted in dissociation of the trimeric complex, as evidenced by a decrease in trimer with a concomitant increase in the monomeric band. Temperature-induced appearance of highly mannosylated gp170(ER monomeric form) and complex glycosylated gp180 (golgi monomeric form), suggesting that trimerization may occur before transport of the monomeric spike protein to the intermediate golgi. This is consistent with other reports on TGEV, influenza HA, vesicular stomatitis G protein. For these proteins, trimerization is reported to occur before the complex oligosaccharide is added to the golgi.

The C-terminal truncated forms of S in oligomeric and monomeric form were found in cell lysates with frequencies of 10% and 90%, respectively. The truncated proteins secreted into the culture medium were found to be well glycosylated and almost all in the form of a unimer. We conclude that the C-terminal 60 amino acids of the S glycoprotein contain a membrane anchoring region that affects trimerisation efficiency. In protein S trimerization, the C-terminal region may be required to initiate a series of events and the three-stranded coiled-coil structure in the S2 stem domain provides more stabilizing power as observed in influenza virus HA oligomers.

Example 24: CHO cell for spike protein expression

A CHO cell line stably expressing the full-length or truncated SARS-CoV spike protein was prepared. Several stably transfected CHO cell lines were obtained and fig. 73 shows western blot data from a representative set of clones.

Example 25: expression in E.coli

All SARS-CoV ORFs (FIG. 17, Table 10) were cloned into pET vector and expressed as C-terminal His-tagged fusion proteins in E.coli. Proteins less than 16kD were also expressed as N-terminal GST (glutathione S-transferase) fusion proteins using pET vectors.

Nsp1 and Nsp2 are 2 SARS-CoV proteins with proteolytic activity, which are not expressed as full-length proteins due to toxicity in E.coli. The genes were cloned in different ratios to separate the catalytic residues in the resulting recombinant protein (Cys 833/His994 for Nsp 1; His41/Cys145 for Nsp 2) which was: nucleotide 2719-5214 from AY 310120; nsp1B from nucleotide 5218-7371; nsp1C from nucleotides 7372-; nsp2A from nucleotides 9985-10416; nsp2B from nucleotides 10476-.

Nsp9(SEQ ID NO: 9775) was divided into 2 parts: nsp9A from nucleotides 13371-14756; nsp9B from nucleotide 14757-16166.

Matrix (M), ORF3 and ORF7 contained 3, 2 and 1 transmembrane domains, respectively. These proteins were expressed as deletion proteins, excluding the first 100 amino acids (M and ORF3) or the first 18 amino acids including the hydrophobic region (ORF 7).

The cloning sequences are shown in Table 26.

The 2-step strategy was used to amplify the cloned sequences. In step 1, a DNA fragment containing more than 1 gene or a single gene is amplified, using the sequenced cDNA as a template. Amplification of 11 cDNA sequences: (1) a fragment, designated amplC1, comprising the gene encoding protein E, protein M, orf 7-8-9-10; (2) a fragment, designated amplC2, comprising the gene encoding orf 3-4; (3) a fragment, designated amplC5, comprising the genes encoding the proteins Nsp12 and Nsp 13; (4) an Nsp11 gene; (5) p28 and P65 genes; (6) nsp1B and Nsp1C gene portions; (7) a fragment, designated amplC9, comprising the genes encoding the proteins Nsp2 and Nsp 3; (8) a fragment, designated amplNsp4-7, comprising the genes encoding the proteins Nsp4, Nsp5, Nsp6, Nsp7 and for amplifying the Nsp9A gene part; (9) an Nsp9B gene portion and an Nsp10 gene; (10) a fragment, designated amploc, comprising the genes encoding the proteins Orf11, nucleocapsid protein (N) and Orf 12; (11) the Nsp1A gene portion. The primers used in step 1 are given in table 27:

In step 2, a single gene is amplified, using the DNA fragment from step 1 of amplification as a template. The primers are shown in Table 28.

Among the proteins whose expression can be observed, the proteins are either in the inclusion body (insoluble) or in a soluble form. Purification is carried out on suitable materials. Table 29 shows the molecular weight of the expression fragments of SARS-CoV ORFs, whether they were cloned (+ or-), whether expression of the cloned fragments (+ or-), and the protein form selected for purification.

When the protein is a soluble His-tagged product, a single colony is streaked and grown overnight at 37 ℃ on LB/Amp (100. mu.g/ml) agar plates. The isolated colonies from this plate were inoculated into 20ml LB/Amp (100. mu.g/ml) liquid medium and grown overnight at 37 ℃ with shaking. The overnight cultures were diluted 1: 30 into 1.0L LB/Amp (100. mu.g/ml) liquid medium and grown at the optimum temperature (30 or 37 ℃) until OD550nm reached 0.6-0.8. Recombinant protein expression was induced by the addition of IPTG (final concentration 1.0mM) and the culture was incubated for a further 3 hours. The bacteria were collected by centrifugation at 8000 Xg for 15 minutes at 4 ℃. The bacterial pellet was resuspended in 10ml of cold buffer A (300mM NaCl, 50mM phosphate buffer, 10mM imidazole, pH 8.0). Cells were disrupted by sonication (French press) on ice, using a Branson sonicator 450, 40W for 30 seconds 4 times, and centrifuged at 13000 Xg for 30 minutes at 4 ℃. Supernatant was mixed with 150. mu. lNi ²⁺Resin (previously equilibrated with buffer a) and incubated at room temperature, gently stirred for 30 min. The resin was high Flow Chelating Sepharose Fast Flow (Pharmacia) and was prepared according to the manufacturer's instructions. The preparation was centrifuged in portions at 700 Xg 4 ℃ for 5 minutes and the supernatant discarded. The resin was washed 2 times (batch) with 10ml buffer A for 10 minutes, resuspended in 1.0ml buffer A and loaded onto a disposable column. The resin was washed successively with buffer A at 4 ℃ until the OD280nm reached 0.02-0.01. The resin was further washed with cold buffer B (300mM NaCl, 50mM phosphate buffer, 20mM imidazole, pH 8.0) until the flow-through OD280nm reached 0.02-0.01. His fusion proteins were eluted by adding 700. mu.l of cold elution buffer C (300mM NaCl, 50mM phosphate buffer, 250mM imidazole, pH 8.0), and fractions were collected until OD_280nmIndicating that all recombinant proteins were obtained. 20 μ l aliquots of each elutionThe fractions were analyzed by SDS-PAGE. Protein concentration was estimated using the Bradford assay.

When the protein is an insoluble product, the inclusion bodies are purified as follows: cells were homogenized in 25ml of 0.1M Tris HCl pH 7, 1mM EDTA at 4 deg.C (5g wet weight) using an ultraturrax (10000 rpm); adding 1.5mg lysozyme per gram of cells; mix briefly with ultraturrax and incubate for 30 min at 4 ℃; disrupting the cells using sonication or high pressure homogenization; to digest the DNA, MgCl was added ₂To a final concentration of 3mM and DNase to a final concentration of 10ug/ml, incubation at 25 ℃ for 30 minutes, adding 0.5 volume of 60mM EDTA, 6% Triton x-100, 1.5M NaClpH 7.0 to the solution, and incubation at 4 ℃ for 30 minutes; 31000g 4 ℃ centrifugation for 10 min; the pellet was resuspended in 40ml of 0.1M Tris HClpH 7.0, 20mM EDTA using ultraturrax; 31000g 4 ℃ centrifugation for 10 min; IB precipitate was stored at-20 ℃.

The expression results are shown in FIGS. 86 to 105. Examples of purity and yield are given in table 30.

Example 26: maintenance of key epitopes on truncated spike antigens

The reactivity of the human monoclonal antibodies with neutralizing activity with the purified truncated spike protein was tested in an ELISA assay. Briefly, ELISA plates were coated with truncated forms of spike protein at a concentration of 1. mu.g/ml (100. mu.l/well) and the plates were incubated overnight at 4 ℃. The plates were washed, non-specific binding sites blocked, then different dilutions of antibody were added and the plates incubated for 1 hour at room temperature. At the end of the incubation, the plates were washed and bound antibody was detected using horseradish peroxidase (HRP) conjugated anti-human IgG and appropriate substrate. The optimal density for each well was recorded at 405nm using an ELISA reader. The data are shown in fig. 69, clearly demonstrating that the neutralizing epitope recognized by the mAb is preserved and exposed on the recombinant truncated spike protein.

Example 27: different spike vaccines

The purified truncated spike protein was used to immunize mice and the level of antibody binding induced by the anti-truncated spike protein was determined by ELISA assay. Briefly, 1 group of 10 mice was immunized with 3 μ g of truncated spike protein, MF59 as adjuvant, at 0, 4 and 8 week intervals. Serum samples were collected from these animals and antibodies induced by the truncated spike protein were determined in an ELISA assay. An additional group of 8 mice were immunized with 75 μ g of DNA encoding the truncated form of the spike protein on the PLG particles, immunized at 0, 4 and 13 week intervals, sera collected and analyzed for anti-spike antibodies as above.

The distribution of induced bound antibody in each group was plotted as Geometric Mean Titer (GMT). The purified truncated spike protein apparently induced a strong antibody response more efficiently than plasmid DNA vaccines expressing the truncated spike antigen and delivered with PLG microparticle preparations. Further comparison of the antibody responses induced by inactivated BPL-SARS-CoV (which has been shown to be protective) in the same mouse strain showed that the purified truncated spike protein was in the same range as the magnitude of the antibody response induced by the inactivated virus vaccine (FIG. 70).

The neutralizing potential of antibodies induced by recombinant truncated spike proteins or DNA expressing the same spike antigen was also assessed. The GMT values obtained for group 2 are shown in FIG. 71. Based on these data, it appears that the purified protein is more significantly effective in inducing a neutralizing antibody response against the SARS-CoV spike. Furthermore, the neutralization titer usually induced by the purified truncated spike protein is comparable to that induced by the inactivated SARS-CoV vaccine.

Figure 72 shows a comparison of antibody binding levels (ELISA, X-axis) with neutralizing titers (Y-axis). In general, there is a good correlation between binding and neutralizing antibodies. The bottom left panel shows the ratio 2 weeks after 3 rd immunization with DNA vaccine; the top right panel shows the ratio 2 weeks after the 2 nd immunization with the protein vaccine. All 2 vaccine forms showed constant association.

In further experiments, the ability of DNA vaccines to elicit an immune response in mice was investigated. Mice were immunized with the pCMV-nSdTC plasmid, without or with PLG particles. Serum from mice was then used as a staining antibody against cultured 293 cells transfected with full-length or truncated spikes. Cells were centrifuged and the pellet lysed prior to testing. Test antibodies, anti-culture supernatants and anti-cell lysates. As shown in figure 112, mouse sera were able to detect spike proteins in cell lysates expressing full-length spikes and in cell supernatants expressing truncated spike proteins. Results were comparable to the staining observed with rabbit serum, which was obtained after immunization with whole death vaccine. Anti-spike antibodies can be induced using DNA vaccination.

Example 28: expression cassette in pCMV

The sequence of plasmid pCMVKm2 is represented by SEQ ID NO: 9923. The gene encoding the full-length form (pCMVKm2SARS spike nS; SEQ ID NO: 9921) or the Δ TC form (pCMVKm2SARS spike nS Δ TC; SEQ ID NO: 9922) spike protein was inserted into this basic vector.

Mice were immunized with these vectors and similar vectors encoding N, M or E proteins. Vectors encoding the same protein but with optimal codon usage were also prepared. Codons were optimized for efficient human expression starting from the FRA sequence (GenBank: AY 310120). The optimal sequence is: n (SEQ ID NO: 9924); m (SEQ ID NO: 9925); e (SEQ ID NO: 9926).

After administration, protein expression can be detected by immunofluorescence in all cases. For example, figure 106 shows immunofluorescence (with anti-SARS rabbit serum) results after administration of vectors encoding the optimal N antigen, showing high levels of expression. Mice receiving the control vehicle alone showed no fluorescence.

FIG. 107 compares the immunofluorescence (with Abgent anti-M antibody) of native M sequence (107A) or codon optimized M sequence (107B). Similarly, fig. 108 compares the immunofluorescence (with Abgent anti-E antibody) of the native E sequence (108A)) or codon optimized E sequence (108B).

4 groups of mice (8 per group) were immunized with: (1) SARS nS spikes, nSdTC truncated spikes and N proteins; (2) pCMV-SARS-nSdTC: DNA + DNA-PLG at 0, 4 and 13 weeks; (3) CMV-nS: DNA + DNA-PLG + VEE/SINRep at 0, 4 and 9 weeks; (4) VEE/SIN Rep-SARS-nS, 3 times, at 0, 4 and 13 weeks. Sera from all groups recognized the SARS nS and nSdTC proteins and also showed viral binding and neutralizing activity.

Example 29: spike protein cleavage

To investigate the effect of protease cleavage on the SARS-CoV spike protein, the protein was expressed in E.coli in a variety of forms, including: (1) full length S1-S2; (2) alone S1; (3) seven HRs 1 and (4) seven HRs 2. The expressed protein was used to culture immune rabbit serum, which was then used to present western blots of Vero cells, with or without SARS-CoV infection of the cells.

FIG. 109 shows a Western blot cultured with 1: 10000 diluted antibody, antibody anti-S1 domain or uncleaved S1-S2 domain. FIG. 110 shows a Western blot incubated with antibodies raised against 4 proteins each, diluted 1: 10000. The difference in antigen reactivity is significant.

Fig. 111 shows similar data. Each serum was tested for 4 lanes, which 4 lanes are, from left to right: (a) 1: 500 diluted serum, SARS-CoV-infected cells; (b) serum at 1: 500 dilution, non-infected cells; (c) serum, SARS-CoV-infected cells, diluted 1: 2500; (d) serum at 1: 2500 dilution, non-infected cells. Again, the difference in antigen reactivity is significant.

FIG. 109-111 shows the presence of multiple spike protein forms in infected Vero cells, approximately 75kDa, 90kDa, 180kDa and > 250kDa in size. Cleaving (only a small fraction of) the spike protein, either intracellularly or after particle release.

If the enzymatic cleavage of the mouse hepatitis coronavirus spike protein is inhibited, cell-cell fusion (syncytia form) is also inhibited, but virus-cell is not (de Haan et al (2004) J Virol). Syncytia were observed in vivo in the lungs of SARS-infected patients, but were not found in Vero cell cultures of SARS-CoV. Thus, inhibition of spike protein cleavage can be used to prevent syncytial formation and associated pathology, even though viral infectivity may not be blocked.

Example 30: purification of SRAS protease

Cells were grown to mid-log phase at 37 ℃ and induced with 0.2% L-arabinose. Cells were harvested by centrifugation and resuspended in Lysis Buffer (LB) containing 20mM Tris pH 7.5, 500mM NaCl, 5% glycerol V/V, 0.05% Triton X-100, 5mM ME, 5mM imidazole and the complete protease inhibitor (-) EDTA. Benzonase was added to the lysate at a final concentration of 50U/ml. Subsequently, the cells were lysed by 2 passes through a pre-chilled microfluidizer. Lysates were clarified by high speed centrifugation at 44,000 Xg. The clarified lysate was applied to a prepared Pharmacia chelating FF column, charged with nickel sulfate. After applying the lysate, the column was washed with 5 column volumes of LB followed by 5 column volumes of LB with the addition of 45mM imidazole. The column was then eluted with LB supplemented with 250mM imidazole. The purity of the isolated SARS protease was 50%. The protease containing fractions were collected, adjusted to 5mM EDTA and applied to a Superdex200 gel filtration column equilibrated in 20mM Tris pH 7.5, 150mM NaCl, 5% V/V glycerol, 0.05% Triton X-100 and 5mM DTT. The purity of the isolated SARS protease was 70%. Again, the protease containing fractions were collected and subsequently stored at-80 ℃ until use. Activity assay, mass spectrometry and western blot analysis were used to positively identify proteins (figure 133). All steps were done in pre-chilled buffer, keeping as much of the preparation at 4 ℃ as possible.

Western blot of SARS protease purification fractions

The operation is as follows: briefly, protein concentration was based on absorbance at 280nm and purity was assessed by coomassie staining of the gel. Proteins were run on a 4-20% gradient gel and transferred to nitrocellulose. Subsequently, the spots were blocked with 3% BSA, probed with mouse IgG anti-7 His, and then with HRP-conjugated mouse IgG secondary antibody. Spots were presented using the ECL kit (pharmacia Biotech). The results are shown in FIG. 133, where A is the fractionated column pool loaded with 50, 100 and 200ng of target protein and B is the immobilized metal affinity column pool loaded with 50, 100 and 200ng of target protein.

Example 31: continuous fluorescence resonance energy transfer (FRER) enzyme assay

Peptides containing EDANS, fluorescence donors, DABCYL, fluorescence quenchers (DABCYL-VNSTLQ SGLRK-EDANS) were synthesized by syn. The peptide contains a cleavage site Gln-Ser in the middle. Meyers et al, A.C. Academic Press, London, 1998, 726-. The proteolytic activity of SARS protease was kinetically followed by measuring the level of formation of cleavage products containing the fluorescent donor SGLRK-EDANS using a Hitachi fluorometer (F-4500FL Spec.) set at 340nm excitation and 490nm emission wavelengths. 5L of 5mM peptide stock in DMSO solution was added to the reaction mixture containing 295L of standard buffer (75mM Tris-HCl, 25mM NaOAc, 25mM Bis-Tris, 25mM glycine, 5mM EDTA and 1mM EDTA, pH 7.4) and 100ul of buffer or 100ul of 3.6uM protease stock. The kinetic curve was followed for 6 minutes (reaction linearity, R2 value 0.998 (fig. 134)). Fluorescence formation (proteolytic reaction) may be enzyme dependent, with the fluorescence formed in the 6 minute time frame tripling when the enzyme concentration is tripling.

It is to be understood that the present invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention.

TABLE 1 U.S. Patents and published International patent applications

Publication number	Title	Pub. Date:
			US-3927216	1, 2, 4-triazole E-3-Carboxamides (1, 2, 4-Triazol E-3-Carboxamides For Inhibiting viral infections)	12/16/1975
US-4010269	Antiviral Quinazoline Compositions And Methods Of Use thereof	3/1/1977
			US-4065570	Antiviral 5- (Substituted benzylidene) caprolactams (Antiviral 5- (substitated Benzal) Hydantoins)	12/27/1977
US-4089965	Thiazolylphenylguanidines (Thiazolylphenylguanidines As Antirhinovirus Agents) As anti-rhinovirus Agents	5/16/1978
			US-4122191	Anti-rhinovirus Agents (Antirhinovirus Agents)	10/24/1978
US-4192895	Anti-rhinovirus Agents (Antirhinovirus Agents)	3/11/1980
			US-4254144	Substituted benzonitrile (Substituted benzathines Having biological Activity) with Antiviral Activity	3/3/1981
US-4264617	Antiviral 5- (Substituted benzylidene) caprolactams (Antiviral 5- (substitated Benzal) Hydantoins)	4/28/1981
			US-4287188	Purine Derivatives (Purine Derivatives)	9/1/1981
US-4327088	Phosphonoxy-or glycosylglycosylglycosyloxy-Substituted allylbenzenes, Compositions And Uses Thereof (phosphor-Orycosyloxy-treated Acrylophenones, Compositions And Uses Thereof)	4/27/1982
			US-4332820	Substituted benzonitrile (Substituted benzathines Having biological Activity) with Antiviral Activity	6/1/1982
US-4349568	Sulfur-Substituted Diphenyl Ethers (sulfor-substitated Diphenyl Ethers Having Antiviral Activity) with Antiviral Activity	9/14/1982
			US-4352792	3-alkoxy Brass Antiviral agent (3-Alkoxyflavanone Antiviral Agents)	10/5/1982
US-4371537	Sulfur-Substituted Phenoxypyridines (Sulfur-Substituted Phenoxypyridines Having antiviral activity) Having antiviral activity	2/1/1983
			US-4423053	2-Amino-5- (O-Sulphamidophenyl) -1, 3, 4-thiadiazole Derivatives As Antiviral Agents And Process For their preparation (Derivatives Of2-Amino-5- (O-Sulphamidophenyl) -1, 3, 4-Thiadiazol As Antiviral Agents And A Process For the preparation Thereof)	12/27/1983
US-4505929	Sulfur-Substituted Diphenyl Ethers (sulfor-substitated Diphenyl Ethers Having Antiviral Activity) with Antiviral Activity	3/19/1985
			US-4526897	The hypertensive agents Isoindolin-2-Yl-aminoimidazoline And Isoindolin-2-Yl-guanidine (hypertentive isoidonolin-2-Yl-aminoimidazoline And hind isoidonolin-2-Yl-Guanidines)	7/2/1985
US-4558134	Some Phenoxy-Pyridine-nitriles (novel Phenoxy-Pyridine-carbonitrile-carboxylic acids) with antiviral activity	12/10/1985
			US-4629729	2-Alkylamino-4, 6-dihalopyrimidines conferring Anti-Viral Activity (Endowed With Anti-Viral Activity2-Alkylamino-4, 6-Dihalo Pyrimidines)	12/16/1986
US-4636492	Inhibition Of Viral protein Activity By Peptide halomethyl ketones (Inhibition Of Viral Protease Activity By Peptide halomethyl ketones)	1/13/1987
			US-4652552	Tetrapeptide Methyl Ketone Inhibitors Of Viral proteins (Tetrapeptide Methyl Ketone Inhibitors Of Viral proteins)	3/24/1987
US-4724233	Therapeutic use Of phosphonomethoxyalkyl adenine (therapeutic Application Of phosphonomethoxylkenyl adenosines)	2/9/1988
			US-4738984	Anti-rhinovirus Agents (Antirhinovirus Agents)	4/19/1988
US-4847246	Antiviral Compositions From Fireflies And methods Of Use thereof	7/11/1989
			US-4855283	Pharmaceutically active N- (2-aminoamido-2-deoxy-hexosyl) -amides, -aminoFormic ester And application thereof (NovelPharmaceutically Active N- (2-Aminoacylamido-2-Deoxy-Hexosyl) -Amides, -carbanates And-Ureas)	8/8/1989
US-4885285	Phosphorus Compounds, method For the production thereof And Use thereof (Phosphorus Compounds, Processes For the Manufacture of the same, And Use thereof)	12/5/1989
			US-4956351	Antiviral Pharmaceutical composition (Antiviral Pharmaceutical Compositions Containing Cyclodextrins)	9/11/1990
US-5001125	Anti-Virally Active pyridazine pyridazines (Anti-viral Active pyridazines)	3/19/1991
			US-5036072	Antiviral Agent (Antiviral Agent)	7/30/1991
US-5070090	heterocyclic-Substituted morpholinyl alkylphenol Ethers against picornaviruses (immobilized morpholino alkylphenols Ethers)	12/3/1991
			US-5100893	Anti-picomavirus pyridazines (antipicornaviral pyridazines)	3/31/1992
US-5112825	Anti-rhinovirus hetero-amine-Substituted Pyridazines (Antirhinoviral Heteroamines-Substituted Pyridazines)	5/12/1992
			US-5157035	Antiviral Active pyridazines (Anti-viral Active pyridazines)	10/20/1992
US-5240694	Combined Antiviral And anti-mediator Treatment Of the common cold (Combined Antiviral And anti-timer Treatment Of common Cold)	8/31/1993
			US-5242924	Tetrazolyl- (Phenoxy and phenoxyalkyl) -piperidylpyridazines (Tetrazolyl- (Phenoxy and phenoxyyalkyl) -piperidylpyridazines As Antiviral Agents) As Antiviral Agents	9/7/1993
US-5278184	Buffered Derivatives Of Pyrrole And Pyrrolidine suitable for the treatment Of rhinovirus infections (Synthetic Derivatives Of Pyrrole And Pyrrolidine)	1/11/1994

	Suitable For The Therapy Of Infections Caused By Rhinoviruses)
			US-5364865	Phenoxy-And Phenoxyalkyl-Piperidines (Phenoxy-And phenoxyakyl-Piperidines As antiviral Agents) As antiviral agents	11/15/1994
US-5453433	Thiadiazole-based substance And anti-picornaviral composition	9/26/1995
			US-5492689	Combined viral inhibitory anti-media (COVAM) therapy of the Common cold (Combined viral inhibitor (COVAM) treatment of Common Colds)	2/20/1996
US-5514679	Therapeutic phenoxyalkylpyridazines And Intermediates thereof (Therapeutic phenoxy pyridines And intermedia therapeutics)	5/7/1996
			US-5514692	Substituted Quinoline Derivatives (Substituted Quinoline Derivatives Useful As Antipiconavir Agents) for use As anti-picornaviral agents	5/7/1996
US-5523312	Anti-small RNA virus Agents (Antipicornaviral Agents)	6/4/1996
			US-5545653	Anti-Viral Compounds (Anti-Viral Compounds)	8/13/1996
US-5552420	Therapeutic phenoxyalkyl pyrroles And phenoxyalkyl azines (Therapeutic phenoxy thioazoles And phenoxy alkylazines)	9/3/1996
			US-5567719	Thiadiazoles And Their Use As anti-small RNA virus Agents (Thiadiazoles And Their Use As Antipicornaviral Agents)	10/22/1995
US-5580897	1, 2-dithiines (1, 2-Dithiins Having Antifungal Activity) with Antifungal Activity	12/3/1996
			US-5618821	Therapeutic phenoxyalkylheterocycles (therapeutic phenoxy phenylalkylheterocycles)	4/8/1997
US-5618849	Orally Active Antiviral Compounds (Orally Active Antiviral Compounds)	4/8/1997
			US-5648354	1, 2-dithiines (1, 2-Dithiins Having Antifungal Activity) with Antifungal Activity	7/15/1997
US-5650419	Thiadiazoles And Their Use As anti-small RNA virus Agents (Thiadiazoles And Their Use As Antipicornaviral Agents)	7/22/1997
			US-5693661	Anti-Viral Compounds (Anti-Viral Compounds)	12/2/1997
US-5721261	Therapeutic phenoxyalkyl pyrroles And phenoxyalkyl azines (Therapeutic phenoxy phenylsulkylazoles And phenoxy ethylene glycols)kylazines)	2/24/1998
			US-5725859	Plant-Based Therapeutic Agent With viral inhibiting and antiviral effects (Plant-Based Therapeutic Agent With viral and antiviral effects)	3/10/1998
US-5750527	Thiadiazoles And Their Use As anti-small RNA virus Agents (Thiadiazoles And Their Use As Antipicornaviral Agents)	5/12/1998
			US-5750551	Treatment of virological Diseases (Treatment For Viral Diseases)	5/12/1998
US-5762940	Methods And Compositions For Inhibiting Or destroying Viruses Or Retroviruses (Methods And Compositions For Inhibiting Or destroying Viruses Or Retroviruses)	6/9/1998
			US-5763461	Therapeutic phenoxyalkyl heterocycles (Therapeutic phenyl Phenoxyalkylheterocycles)	6/9/1998
US-5821242	Anti-Viral Compounds (Anti-Viral Compounds)	10/13/1998
			US-5821257	Thiadiazoles And Their use As anti-picornaviral Agents	10/13/1998
US-5821331	Anti-small RNA virus Agents (Anti-Picornaviral Agents)	10/13/1998
			US-5846986	Therapeutic phenoxyalkyl pyrroles And phenoxyalkyl azines (Therapeutic phenoxy thioazoles And phenoxy alkylazines)	12/8/1998
US-5856530	Anti-picornaviral Compounds And Methods For their use And Preparation (anti-picornaviral Compounds And Methods For the use And Preparation)	1/5/1999
			US-5891874	Anti-Viral compounds (Anti-Viral Compound)	4/6/1999
US-5962487	Anti-picornaviral Compounds And Methods For their use And Preparation (anti-picornaviral Compounds And Methods For the use And Preparation)	10/5/1999
			US-6004933	Cysteine Protease Inhibitors (Cysteine Protease Inhibitors)	12/21/1999
US-6020371	Anti-picornaviral compounds, compositions Containing Them, And Methods of Their Use And preparation (anti-picornaviral compositions Containing the same And Methods For the same Use)	2/1/2000
			US-6087374	Anti-Viral Compounds (Anti-Viral Compounds)	7/11/2000
US-6114327	Anti-Viral Compounds (Anti-Viral Compounds)	9/5/2000
			US-6117844	Methods And compositions For Antiviral Therapy (Method And Composition For Antiviral Therapy)	9/12/2000
US-6194447	Bis (benzimidazole) Derivatives (bis (benzamidazole) Derivatives Serving As Potasassages) As Potassium blockers	2/27/2001
			US-6214799	Anti-picornaviral Compounds And Methods For their use And Preparation (anti-picornaviral Compounds And Methods For the use And Preparation)	4/10/2001
US-6277891	Nitrogen monoxide Inhibits Rhinovirus Infection (nitrile Oxide inhibitors Rhinovirus Infection)	8/21/2001
			US-6294186	Antimicrobial Compositions containing benzoic acid analogs And Metal salts (Antimicrobial Compositions Comprising A benzoic acid Analog And A Metal Salt)	9/25/2001
US-6331554	Anti-picornaviral Compounds, Compositions Containing Them, And Methods of Their Use And preparation (antibiotic Compositions, Compositions Containing Them, And Methods For Their Use)	12/18/2001
			US-6358971	Anti-Viral Compounds (Anti-Viral Compounds)	3/19/2002
US-6362166	Anti-picornaviral Compounds And Methods For Their use And preparation (anti-picornaviral Compounds And Methods For Their)	3/26/2002

	Use And Preparation)
			US-6414004	3-Substituted 5-Aryl-4-Isoxazolecarbonitriles (3-substistuted 5-Aryl-4-isoxazo carboxylic acids HavingOptiral Activity) with antiviral Activity	7/2/2002
US-6420591	Carbamates And compositions thereof, Methods of using them to treat Cancer, Inflammation Or Viral Infection (carbonates And complexes of the disease, And Methods For the Use of the Treating Cancer, Inflammation, Or Viral Infection)	7/16/2002
			US-6469018	Compound (Compounds)	10/22/2002
US-6498178	IMPDH Enzyme inhibitor (Inhibitors Of IMPDH Enzyme)	12/24/2002
			US-6514997	Anti-picornaviral compounds And Compositions, Their Pharmaceutical Uses, And Their synthetic substances (anti-picornaviral compounds And compounds, the therapeutic Uses, And the Materials For the Synthesis)	2/4/2003
US-6525043	Use Of Ion Channel modulators (Use Of Ion Channel Modulating Agents)	2/25/2003
			US-6531452	Anti-picornaviral compounds And Compositions, Their Pharmaceutical Uses, And Their synthetic substances (anti-picornaviral compounds And compounds, the therapeutic Uses, And the Materials For the Synthesis)	3/11/2003
US-6534489	Organophosphorus Compounds And Use Thereof (organophosphorous Compounds And Use Thereof The of)	3/18/2003
			WO00/06529	Diketoacid Derivatives As polymerase Inhibitors (Diketoacid-Derivatives As Inhibitors Of microorganisms)	2/10/2000
WO00/25791	Pyridin-4-Yl Or Pyrimidin-4-Yl Substituted Pyrazines (Pyridin-4-Yl Or Pyrimidin-4-Yl Substituted Pyrazines)	5/11/2000
			WO00/27423	Methods And Compositions For Treating Common Cold Symptoms (Methods And Compositions For Treating Common Cold Symptoms)	5/18/2000
WO00/34308	Protein Transduction System And method Of Use Thereof (Protein Transduction System And Methods Of Use Thereof)	6/15/2000
			WO00/39348	Methods And Compositions For Identifying Protease Modulators (Methods And Compositions For Identifying Protease Modulators)	7/6/2000
WO00/40243	New Compounds (Novel Compounds)	7/13/2000
			WO00/50037	Nitrosylated And Nitrosylated Proton pump inhibitors, Compositions And Methods Of Use thereof (Nitrosated And Nitrosylated Proton Pump inhibitors, Compositions And Methods Of Use)	8/31/2000
WO00/56331	Impdh Enzyme inhibitor (Inhibitors Of Impdh Enzyme)	9/28/2000
			WO00/56757	Immunomodulator Steroids, In Particular the hemihydrate Of 16.alpha. -Bromoepiandrosterone (immunomodulating Steroids, In particulate the hemihydrate Of 16.alpha. -Bromoepiandosterone)	9/28/2000
WO00/66096	New application And medicine Of Antiepileptic (New Indication For Use Of anti-epileptic Agents And medicinal)	11/9/2000
			WO00/78746	Antiviral Agents (Antiviral Agents)	12/28/2000
WO01/00199	Compounds with antiviral activity (Compound infected From Salvia specificities Having antiviral Activity) Obtained From the genus Salvia	1/4/2001
			WO01/00585	Pyrazolidinol Compounds (Pyrazolidinol Compounds)	1/4/2001
WO01/02551	Virus-Like particles, their preparation and their use, preferably in drug screening and functional genomics (Virus Like)Particles，Their Preparation And Their Use Preferably In Pharmaceutical Screening And Functional Genomics)	1/11/2001
			WO01/03681	Use Of flavonoids, Coumarins And related compounds for the treatment Of Infections (Use Of Flavones, Coumarins And related Compounds To Treat wounds)	1/18/2001
WO01/05396	Use Of Cobalt Chelates For Treating Or Preventing viral infections (Use Of Cobalt complexes For Treating Or Preventing viral Infection)	1/25/2001
			WO01/10894	Anti-picornaviral compounds And Compositions, Their Pharmaceutical Uses, And Their synthetic substances (anti-picornaviral compounds And compounds, the therapeutic Uses, And the Materials For the Synthesis)	2/15/2001
WO01/19322	Csaids is used for Rhinovirus Infection (Use Of Csaids In Rhinovirus Infection)	3/22/2001
			WO01/19822	Antiviral Agents (Antiviral Agents)	3/22/2001
W001/22920	Colon And Colon Cancer-Associated Polynucleotides And polypeptides (Colon And Colon Cancer Associated Polynucleotides And polypeptides)	4/5/2001
			WO01/25188	Novel Carbamates And Ureas (Novel Carbamates And Ureas)	4/12/2001
WO01/31016	Processed Human cytokines Phc-1 And Phc-2(Processed Human cytokines Phc-1 And Phc-2)	5/3/2001
			WO01/37837	3, 4-Dihydro- (1h) -Quinazolin-2-Ones And Their Use As Csbp/P38 Kinase Inhibitors (3, 4-Dihydro- (1h) -Quinazolin-2-Ones And Their Use As Csbp/P38 Kinase Inhibitors)	5/31/2001
WO01/38312	3, 4-Dihydro- (1h) Quinazolin-2-one compounds As Csbp/P38 Kinase Inhibitors (3, 4-Dihydro- (1h) Quinazolin-2-onecomdots As Csbp/P38 Kinase Inhibitors)	5/31/2001
			WO01/38313	3, 4-Dihydro- (1h) Quinazolin-2-one compounds As Csbp/P38 Kinase Inhibitors (3, 4-Dihydro- (1h) Quinazolin-2-onecomdots As Csbp/P38 Kinase Inhibitors)	5/31/2001
WO01/38314	3, 4-Dihydro- (1h) Quinazolin-2-one compounds As Csbp/P38 Kinase Inhibitors (3, 4-Dihydro- (1h) Quinazolin-2-onecomdots As Csbp/P38 Kinase Inhibitors)	5/31/2001
			WO01/40189	Anti-picornaviral compounds And Compositions, Their Pharmaceutical Uses, And Their synthetic substances (anti-picornaviral compounds And compounds, the therapeutic Uses, And the Materials For the Synthesis)	6/7/2001
WO01/49303	Multivalent electronically Active Compositions And methods of making And using the same (Multivalent Electron Active Compositions And)	7/12/2001

	Methods Of Making And Using Same)
			WO01/60393	Selective Destruction Of cell infection With human immunodeficiency Virus (Selective Destruction Of Cells induced With human immunodeficiency Virus)	8/23/2001
WO01/62726	2-Oxo-1-Pyrrolidine Derivatives, process for producing the same And use thereof (2-Oxo-1-Pyrrolidine Derivatives, process for preparing the same And use thereof)	8/30/2001
			WO01/79167	Anti-picornaviral compounds And Compositions, Their Pharmaceutical Uses, And Their synthetic substances (anti-picornaviral compounds And compounds, the therapeutic Uses, And the Materials For the Synthesis)	10/25/2001
WO01/90047	Novel Mmp-2/Mmp-9 Inhibitors (Novel Mmp-2/Mmp-9 Inhibitors)	11/29/2001
			WO01/90129	Prevention And Treatment of infections And Other Diseases With Mono-And polysaccharide-Based Compounds (Therapeutic And Therapeutic Treatment of infectious And Other Diseases With Mono-And polysaccharide-Based Compounds)	11/29/2001
WO01/92499	Nucleic Acid Molecules Encoding protein A interact With Ser/Thr Kinase Akt (Nucleic Acid Molecules Encoding A protein interacting With Ser/Thr Kinase Akt)	12/6/2001
			WO01/93883	Therapeutic agent-Iii (Therapeutic Agents-Iii)	12/13/2001
WO01/93884	Therapeutic agent-I (Therapeutic Agents-I)	12/13/2001
			WO01/93885	Therapeutic agent-Ii (Therapeutic Agents-Ii)	12/13/2001
WO01/96297	Anti-picornaviral compounds And Compositions, Their Pharmaceutical Uses, And Their synthetic substances (anti-picornaviral compounds And compounds, the therapeutic Uses, And the Materials For the Synthesis)	12/20/2001
			WO02/04413	Chiral Integrin Modulators And Methods Of Use Thereof (Chiral Integrin Modulators And Methods Of Use Thereof)	1/17/2002
WO02/11743	Treatment Of Prostate Cancer (Treatment Of Prostate Cancer)	2/14/2002
			WO02/12477	Enhanced Replication Of Hcv Rna (Enhanced Replication Of Hcv Rna)	2/14/2002
WO02/14343	Immunosuppressive, anti-inflammatory And analgesic compounds (immunological compounds)	2/21/2002
			WO02/24145	Antiviral Substances From Plant cuticle And epithelium materials (Antiviral substructures From Plant Current Material)	3/28/2002
WO02/28351	Recombinant Mucin-Binding protein From Streptococcus pneumoniae (Recombinant Mucin Binding Proteins From Steptococcus sp. moniae)	4/11/2002
			WO02/30442	Methods For Treating Cytokine-Mediated liver Injury (Method For Treating Cytokine Mediated liver Injury)	4/18/2002
WO02/34771	Nucleic Acids And Proteins From streptococcal Groups A And B (Nucleic Acids And Proteins From Streptococcus Groups A) &B)	5/2/2002
			WO02/44737	Diagnostic And therapeutic Compositions And Methods relating To the G Protein-Coupled Receptor (Gpcr) anaphylatoxin C3a Receptor (Diagnostic And therapeutic Compositions And Methods of use) (Gpcr) Anaphyloxin C3a Receptor	6/6/2002
WO02/50045	Antiviral Agents (Antiviral Agents)	6/27/2002
			WO02/51413	Macrocyclic Anti-Viral compounds (Macrocyclic Anti-Viral Compounds)	7/4/2002
WO02/53138	Treatment of tumor Lesions (Treatment For Inhibiting neutring Neoplastic stresses)	7/11/2002
			WO02/57425	Nucleoside Derivatives As RNA-Dependent RNA Viral Polymerase Inhibitors (nucleotide Derivatives As Inhibitors of RNA-Dependent RNA Viral Polymerase)	7/25/2002
WO02/59083	New Compounds (Novel Compounds)	8/1/2002
			WO02/60875	Nicotinamide diaryl Derivatives As Pde4 isomerase inhibitors (Nicotinamide bisaryl Derivatives Useful As inhibitors of inhibition of Pde4 Isozymes)	8/8/2002
WO02/60898	Used as Pde4 isomerase inhibitorThiazolyl-, Oxazolyl-, Pyrrolyl-And imidazolate-Acid Amide Derivatives Of (Thiazolyl-, Oxazolyl-, Pyrrolyl-, And Imidazolyl-Acid Amide Derivatives Useful Usul As Inhibitors Of Pde4 Isozymes)	8/8/2002
			WO02/69903	Nucleosides, their Preparation And Use as inhibitors Of RNA Viral polymerase (nucleotides, Preparation Of And Of Use Of Rna Viral polymerase)	9/12/2002
WO02/72022	Substituted Tetracycline Compounds (Substituted tetracyclic Compounds As Antifungal Agents) As Antifungal Agents	9/19/2002
			WO02/72031	Substituted Tetracycline Compounds (Substituted Tetracycline Compounds As synergistic antibiotic Agents) As synergistic antifungal Agents	9/19/2002
WO02/76939	Cysteine Protease Inhibitors (Cysteine Protease Inhibitors)	10/3/2002
			W002/77021	Proteins And Nucleic Acids of Streptococcus Pneumoniae (Streptococcus Pneumoniae Proteins And Nucleic Acids)	10/3/2002
WO02/79401	Novel Rgs9 Protein Binding Interactions And Methods Of use thereof (Novel Rgs9 Protein Binding Interactions And Methods Of use thereof)	10/10/2002
			WO02/82041	Production And Use Of novel peptide-Based reagents For Use With bispecific receptors (Production And Use Of Novelpeptide-Based reagents For Use With Bi-Specific Antibodies)	10/17/2002
WO02/87465	Dual Targeting Compositions And Methods for viral infection And cancer cells (Compositions And Methods Of Double-targeted Virus	11/7/2002

	Infections And Cancer Cells)
			WO02/87500	Viral Enzyme Activated Protoxophores and their Use To Treat Viral Infections (Viral Enzyme Activated Protoxophores and Use Of Same To Treat Viral Infections)	11/7/2002
WO02/88091	Inhibiting Human Rhinovirus 2a Cysteine Protease (Inhibitors Of Human Rhinovirus 2a Cysteine Protease)	11/7/2002
			WO02/89832	Pharmaceutical composition For Preventing Or Treating Th1 And Th2 Cell-Related Diseases By regulating Th1/Th2Ratio (pharmaceutical compositions For correcting Or Treating Th1 And Th2 Cell Related Diseases By modulation The Th1/Th2Ratio.)	11/14/2002
WO02/92779	Method For Enriching tissue with Long-Chain polyunsaturated fatty Acids (Method For Enriching Tissues In Long Chain dye and fatty Acids)	11/21/2002
			WO02/94185	Conjugates And Compositions For Delivery of cells (Conjugates And Compositions For Cellular Delivery)	11/28/2002
WO02/94868	Staphylococcus Aureus Proteins And Nucleic Acids (Staphylococcus Aureus Proteins And Nucleic Acids)	11/28/2002
			WO02/96867	Protein Kinase Inhibitors (Inhibitors Of Protein Kinase For The Treatment Of diseases)	12/5/2002
WO02/98424	Novel Anti-infective agent (Novel Anti-infections)	12/12/2002
			WO03/04489	Compositions And Methods For Inhibiting Prenyltransferases (Compositions And Methods For Inhibiting Prenyltransferases)	1/16/2003
WO03/08628	Enzyme Nucleic Acid Peptide Conjugates (enzymic Nucleic Acid Peptide Conjugates)	1/30/2003
			WO03/15744	Chitin Microparticles And Their Medical Uses (Chitin Microparticles And Their Medical Uses)	2/27/2003
WO03/20222	Dioxolane And oxathiolane derivatives As RNA Dependent RNA Viral Polymerase Inhibitors (Dioxolane And OxathiolaneDerivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase)	3/13/2003
			WO03/20270	Oxadiazolyl-phenoxyalkylisoxazoles, compositions thereof, and methods of using the sameMethods For Their Use as anti-small RNA viral Agents (Oxadiazolyl-phenoxyisoxazoles, Compositions of the theory of added Methods For the theory Use of Asanti-Picornaviral Agents)	3/13/2003
WO03/20271	Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof, And Methods of Their Use as anti-small RNA viral Agents (Oxadiazolyl-phenoxyylisoxazoles, Compositions of Methods For using same For the same Use of anti-Picornaviral Agents)	3/13/2003
			WO03/20712	Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof, And Methods of Their Use as anti-small RNA viral Agents (Oxadiazolyl-phenoxyylisoxazoles, Compositions of Methods For using same For the same Use of anti-Picornaviral Agents)	3/13/2003
WO86/03412	Improvements in therapy for The Control And prevention of Rhinovirus Infections (Improvements Relating To The Treatment Control And preservation of Rhinovirus Infections)	6/19/1986
			WO86/03971	Antiviral Agents (Antiviral Agents)	7/17/1986
WO88/09669	Non-toxic microorganism And application thereof (Avirule microorganisms And Uses Therefor)	12/15/1988
			WO92/03475	Enterovirus Peptides (Enterovirus Peptides)	3/5/1992
WO92/22520	Orally Active Antiviral Compounds (Orally Active Antiviral Compounds)	12/23/1992
			WO92/22570	Small RNA virus protease Inhibitors (Inhibitors Of Picornavirus Proteases)	12/23/1992
WO94/00012	Nucleic Acids And Methods For their Use in the control Of viral pathogens (Nucleic Acids And Methods Of Use Of the same For controlling viral pathogen Pathologens)	1/6/1994
			WO95/03821	Prosaposin And Cytokine Derived Peptides (Prosaposin And Cytokine dependent Peptides Astherpeutic Agents) as therapeutic Agents	2/9/1995
WO95/09175	Ring-Expanded Nucleosides And Nucleotides (Ring-Expanded Nucleotides And Nucleotides)	4/6/1995
			WO95/11992	Antiviral Compounds (Antiviral Compounds)	5/4/1995
WO95/31198	Thiadiazoles And Their Use As anti-small RNA virus Agents (Thiadiazoles And Their Use As Antipicornaviral Agents)	11/23/1995
			WO95/31438	Therapeutic phenoxyalkyl heterocycles (Therapeutic phenyl oxalkylheters)ocycles)	11/23/1995
WO95/31439	Therapeutic Phenoxyalkylpyridazines And Intermediates thereof (Therapeutic phenoxy pyridines And intermedia therapeutics)	11/23/1995
			WO95/31452	Therapeutic phenoxyalkyl pyrroles And phenoxyalkyl azines (Therapeutic phenoxy thioazoles And phenoxy alkylazines)	11/23/1995
WO95/34595	Antiviral Dendrimers	12/21/1995
			WO95/35103	Pharmaceutical compositions And methods Of Treatment For preventing And/Or treating Viral Infections And Optionally inflammation (A Pharmaceutical Composition For The Prevention And/Or Treatment Of Viral Infections And Option inflammation And inflammation Of Viral Infections And inflammation Of inflammation	12/28/1995
WO96/05836	Method for Treating Cold Symptoms with Pentoxifylline (Methods Of Treating Cold Symptoms Using Pentoxylline)	2/29/1996
			WO96/05854	Preparation of a composition containing cyclosporin A Or Fk506 Or Rapamycin And Xanthine derivatives (Combination Preparation, continuous Cyclosporin A Or Fk506 Or Rapamycin And A Xanthine Derivative)	2/29/1996
WO96/09822	Anti-small RNA virus Agents (Antipicornaviral Agents)	4/4/1996
			WO96/11211	Selective Inhibition begins with internal RNA Translation (Selective Inhibition Of Internally Initiated Rna Translation)	4/18/1996

WO96/22689	Multi-Component RNA catalyst And application Thereof (Multiple Component Rna Catalysts And Uses of the same)	8/1/1996
			WO96/40641	Sulfonamide Derivatives As adjuvants (Sulfonamide Derivatives As Cell addition Modulators)	12/19/1996
WO97/08553	Targeting Proteins Of Gram-Positive bacterial Cell Wall (Targeting Of Proteins To The Cell Wall Of Gram-Positive Bacteria)	3/6/1997
			WO97/34566	Electrophilic Ketones For treating herpes virus Infections (electrophophilie Ketones For The Treatment Of herpes viruses Infections)	9/25/1997
WO97/41137	Of anthocyanidins and anthocyanidin derivativesApplications (Use Of Anthocynidinin And Anthocynidinin Derivatives)	11/6/1997
			WO97/43305	Picornavirus 3C protease Inhibitors And methods Of Their Use And Preparation (Inhibitors Of Picomavir 3C proteins And methods For the Use Of same And Preparation)	11/20/1997
WO97/47270	Novel antiviral Compounds (Novel Anti-Viral Compounds)	12/18/1997
			WO98/03572	Linear Antiviral Polymers (Antiviral Linear Polymers)	1/29/1998
WO98/07745	Compositions And Methods For Treating infections with Indolicidin analogs (Compositions And Methods For Treating infectious diseases using analogs Of Indolicidin)	2/26/1998
			WO98/11778	Antimicrobial Treatment of Herpes Simplex virus and Other Infectious Diseases (Antimicrobial Treatment For viruses Simplex Virus and Other Infectious Diseases)	3/26/1998
WO98/22495	Anti-kinin Compounds And their use (Antikinin Compounds And Uses Thereof)	5/28/1998
			WO98/31363	Anti-Viral Compounds (Anti-Viral Compounds)	7/23/1998
WO98/31374	Methods Of Treating rhinovirus Infections (Method Of Treating rhinovirus Infections)	7/23/1998
			WO98/32427	Treatment and Prevention Of infection With Bioactive substances Encapsulated in a biodegradable-Biocompatible Polymeric Matrix (Therapeutic treatment and Prevention Of infection With A Bioactive Material Encapsulated With Bioactive biodegradable-Biocompatible Polymeric Matrix)	7/30/1998
WO98/34601	Method For Inhibiting Intracellular virus Replication (Method For Inhibiting Intracellular Viral Replication)	8/13/1998
			WO98/42188	Antimicrobial Prevention And treatment of Human immunodeficiency viruses And Other Infectious Diseases (Antimicrobial preservation And treatment of Human immunodeficiency Virus And Other Infectious Diseases)	10/1/1998
WO98/43950	Anti-picornaviral compounds, Compositions Containing Them, And Methods of Their Use (antibodies Compositions, Compositions Containing Them, And Methods For Their Use)	10/8/1998
			WO98/49190	Substituted Oxadiazole Cysteine Protease Inhibitors (Substituted Oxadiazole Cysteine Protease Inhibitors)	11/5/1998
WO98/55120	Anti-Viral Compounds (Anti-Viral Compounds)	12/10/1998
			WO99/30699	Cysteine protease modulators (Anti-Viral Compounds)	6/24/1999
WO99/31122	Anti-picornaviral Compounds And Methods For their use And Preparation (anti-picornaviral Compounds And Methods For the use And Preparation)	6/24/1999
			WO99/54317	Cysteine Protease Inhibitors (Cysteine Protease Inhibitors)	10/28/1999
WO99/55663	Impdh Enzyme inhibitor (Inhibitors Of Impdh Enzyme)	11/4/1999
			WO99/57135	Anti-picornaviral compounds, their preparation and use (Antipico)rnaviral Compounds，Their Preparation And Use)	11/11/1999
WO99/59587	Anti-Viral Compounds (Anti-Viral Compounds)	11/25/1999
			WO99/61437	Novel 2-Alkyl Substituted Imidazole Compounds (Novel 2-Alkyl Substituted Imidazole Compounds)	12/2/1999

TABLE 2 U.S. Patents and published International patent applications

Publication number	Title	Pub. Date:
			WO02/69903	nucleosides, their Preparation And Use as inhibitors Of RNA Viral polymerase (nucleotides, Preparation Of And Of Use Of Rna Viral polymerase)	9/12/2002
WO02/48116	Hepatitis C Virus Ns3 Protease inhibitor (Inhibitors Of Hepatitis C Virus Ns3 Protease)	6/20/2002
			WO02/48157	Imidazolinones And related derivatives As Hepatitis C Virus Ns3 Protease Inhibitors (Imidazolidiones And Their relative Inhibitors As Hepatitis C viruses Ns3 Protease Inhibitors)	6/20/2002
WO02/61048	In Vitro Replication System For RNA-Dependent RNA Polymerase (Rdrp) Viruses (In Vitro System For Replication of Rna-Dependent Rna Polymerase (Rdrp) Viruses)	8/8/2002
			WO03/02518	Novel 2, 4-Difluorobenzamide Derivatives (Novel 2, 4-Difluorobenzamide Derivatives Asantibral Agents) as antiviral Agents	1/9/2003
WO02/79187	Methoxy-1, 3, 5-Triazine Derivatives (Methoxy-1, 3, 5-Triazine Derivatives As Antiviral Agents) As Antiviral Agents	10/10/2002
			WO01/78648	6-Methylnicotinamide Derivatives (6-Methylnicotinamide Derivatives As Antiviral Agents) As Antiviral Agents	10/25/2001
WO01/12214	MYCOPHENOLATE MOFETIL conjugated with PEG-IFN-Alpha (MYCOPHENOLATE MOFETIL IN ASSOCIATION WITHPEG-IFN-. Alpha.)	2/22/2001
			WO02/100415	4 '-Substituted Nucleosides (4' -subsampled Nucleosides)	12/19/2002
WO02/18404	Nucleoside Derivatives (nucleotide Derivatives)	3/7/2002

WO02/94289	Antiviral Nucleoside Derivatives (Antiviral Nucleoside Derivatives)	11/28/2002
			WO96/39500	Hepatitis C Virus-Specific Oligonucleotides (Oligonucleotides specificic For Hepatitis C Virus)	12/12/1996
WO03/00713	Nucleoside compound of HCV (nucleotide Compounds In Hcv)	1/3/2003
			WO01/60381	Nucleoside analogues With formamidine-Modified bicyclic base (nucleotide Analogs With Carboxamidine-Modified BicyclicBase)	8/23/2001
WO02/03997	Pyrido [2, 3-D]Pyrimidines and pyrimido [4, 5-D]Pyrimidine nucleosides (Pyrido [2, 3-D)]Pyrimidine AndPyrimido[4，5-D]Pyrimidine Nucleosides)	1/17/2002
			WO97/26883	Modulation Of Th1/Th2 Cytokine Expression By Ribavirin3 And Ribavirin3 Analogs In activated T-thymus dependent cells (Modulation Of Th1/Th2 Cytokine Expression By y Ribavirin3 And Ribavirin3 antigens In activated-Lymphocytes)	7/31/1997
WO03/26589	Methods And Compositions For Treating hepatitis C Virus with 4 '-Modified Nucleosides (Methods And Compositions For Treating hepatitis C Virus Using 4' -Modified Nucleosides)	4/3/2003
			WO03/26675	Methods And Compositions For treating flaviviruses And Pestiviruses with 4 '-Modified nucleosides (Methods And Compositions For treating flaviviruses And Pestiviruses Using 4' -Modified nucleosides)	4/3/2003
WO97/30067	Sugar-Modified Gapped Oligonucleotides (Sugar-Modified Gapped Oligonucleotides)	8/21/1997
			WO01/47883	Fused-Ring Compounds And their Use As pharmaceuticals (Fused-Ring Compounds And Use of As Drugs)	7/5/2001
WO03/00254	Fused ring Compounds And their Use as pharmaceuticals (Fused Cyclic Compounds And Medicinal Use therof)	1/3/2003
			WO02/100354	Pyrrolo [2, 3-D]Pyrimidine nucleoside analogues (Pyrrolo [2, 3-D)]Pyrimidine Nucleoside Analogs)	12/19/2002
WO01/55111	Diaryl Compounds, Their Preparation And therapeutic Use (Biaryl Compounds, Heat preference And Use In Therapy)	8/2/2001
			WO01/16149	2-Azapurine Compounds And uses thereof (2-Azapurine Compounds And Cold Their Use)	3/8/2001
WO01/85770	Whistle Virus Ii (Sentinel Virus Ii)	11/15/2001
			WO02/12263	Pyrazolo [3, 4-D ] containing purine-2, 6-diamines]Pyrimidine analogue Nucleic Acid binding compounds and uses thereof (Nucleic Acid binding Compounds binding Pyrazolo [3, 4-D)]Pyrimidine Analogues Of Purin-2，6-Diamine And Their Uses)	2/14/2002
JP 2001-247550 A2	Fused Ring compounds And their medical Use (Condensed Ring Compound And Its Medicinal Use)	9/11/2001
			6210675	PT-NANB Hepatitis polypeptide (PT-NANB Hepatitis Polypeptides)	4/3/2001
6451991	Sugar-Modified Gapped Oligonucleotides (Sugar-Modified Gapped Oligonucleotides)	9/17/2002
			5830455	Methods Of Treatment with therapeutic compositions Of Interferon-alpha And Radical-Free Scavengers (Method Of Treatment Using ATherapeutic Combination Of alpha-Interferon And Free Radical Scavengers)	11/3/1998
5908621	Polyethylene Glycol Modified Interferon Therapy (Polyethylene Glycol Modified Interferon Therapy)	6/1/1999
			5990276	Synthetic Inhibitors Of Hepatitis C Virus NS3 protease (Synthetic Inhibitors Of Hepatitis C Virus NS3 Prptase)	11/23/1999
6172046	Combination Therapy For eliminating detectable HCV-RNA In Patients with Chronic Hepatitis C Infection (Combination Therapy For Eradisiting detectable HCV-RNA In Patients Having viral Hepatitis C Infection)	1/9/2001
			6177074	Polyethylene Glycol Modified Interferon Therapy (Polyethylene Glycol Modified Interferon Therapy)	1/23/2001
6326137	Hepatitis C Virus Protease-Dependent Chimeric Pestivirus (Hepatitis C Virus Protease-Dependent Chimeric Pestivirus)	12/4/2001
			6434489	Hepatitis C Virus NS5B Polymerase conjugate and method Of crystallization thereof (Compositions Of Hepatitis C viruses NS5B Polymerase and methods For Crystallizing Same)	8/13/2002
6461605	Continuous Low Dose Cytokine Infusion Therapy (Continuous Low-Dose Cytokine Infusion Therapy)	10/8/2002
			6472373	Combination Therapy For eliminating detectable HCV-RNA In Patients with Chronic Hepatitis C Infection (Combination Therapy For Eradisiting detectable HCV-RNA In Antiviral Therapy regional Hepatitis C Infection)	10/29/2002
6524570	Polyethylene Glycol Modified Interferon Therapy (Polyethylene Glycol Modified Interferon Therapy)	2/25/2003
			WO00/37097	Ribavirin-Interferon alpha induced HCV combination therapy (Ribavirin-Interferon Alfa Induction Hcv Comb)ination Therapy)	6/29/2000
WO00/37110	Ribavirin-Pegylated Interferon alpha-induced HCV combination Therapy (Ribavirin-Pegylated Interferon infection HCV combination Therapy)	6/29/2000
			WO00/62799	HCV Combination Therapy comprising Ribavirin (Hcv Combination Therapy, Containing Ribavirin Inarsenic With antibiotics) in Combination With Antioxidants	10/26/2000
WO01/58929	Azapeptides (Azapeptides Useful In The Treatment Of Hepatitis Of The Hepatitis C)	8/16/2001
			WO02/32414	Ribavirin-Pegylated Interferon alpha-induced HCV combination therapy (Ribavirin-Pegylated Interferon Alfa HCV combination therapy)	4/25/2002
WO03/24461	HCV Combination Therapy (Hcv Combination Therapy)	3/27/2003
			WO93/20835	Treatment Of Hepatitis With Gm-Csf (Treatment Of Hepatitis With Gm-Csf)	10/28/1993
WO96/36702	Soluble Active Hepatitis C Virus Protease (Soluble, Active Hepatitis C Virus Protease)	11/21/1996
			WO97/16204	Continuous Low Dose Cytokine Infusion Therapy (Continuous Low-Dose Cytokine Infusion Therapy)	5/9/1997
WO97/43310	Hepatitis C Virus Ns3 Protease synthesis inhibitor (Synthetic Inhibitors Of Hepatitis C Virus Ns3 Protease)	11/20/1997

WO98/48840	Polyethylene Glycol-Interferon Alpha Conjugates (Polyethylene Glycol-Interferon Alpha Conjugates For therapeutics Infection) For treating Infection	11/5/1998
			WO99/15194	Combination Therapy For eliminating detectable HCV-RNA In Patients with Chronic Hepatitis C Infection (Combination Therapy For Eradiciting detectable Hcv-Rna In Patients Having viral Hepatitis C Infection)	4/1/1999
WO99/59621	Ribavirin And Interferon Alpha Combination therapy for Antiviral therapy of Patients with primary chronic hepatitis C Infection (Combination therapy viral Ribavirin And Interferon Alpha In Antiviral Treatment nasal Patients Having G Chronic hepatitis C Infection)	11/25/1999
			WO02/100846	Treating or preventing yellow diseaseCompounds And Methods of viral infection (Compounds And Methods For The Treatment Or prevention of Flavivirus Infections)	12/19/2002
WO02/100851	Compounds And Methods For treating Or preventing Flavivirus Infections (Compounds And Methods For The Treatment of pollution of flaviviruses Infections)	12/19/2002
			5241053	Fusion protein containing Glycoprotein Gd And LTB Of HSV-1 (Fused Proteins Comprising Gd Of HSV-1And LTB)	8/31/1993
5556946	Interleukin-2/Viral Antigen Protein chimeras (hterleukin-2/Viral Antigen Protein chimeras)	9/17/1996
			6087484	Enhancement Of Ribozyme Catalytic activity by 2 '-O-Substituted Facilitator oligonucleotides (Enhancement Of Ribozyme Catalytic activity A2' -O-immobilized protease Oligonucleotide)	7/11/2000
5830905	Compounds, Compositions And Methods For treating hepatitis C (Compounds, Compounds And Methods For treating hepatitis C)	11/3/1998
			6316492	Methods For Treating Or Preventing Viral Infections and related Diseases (Methods For Treating Or Preventing Viral Infections and associated Diseases)	11/13/2001
6440985	Methods For Treating Viral Infections (Methods For Treating Viral Infections)	8/27/2002
			WO00/10573	Compounds, Compositions And methods for Treating Or Preventing Viral Infections And related Diseases (Compounds, Compositions And methods for Treating Or Preventing Viral Infections And Associated Diseases)	3/2/2000
WO00/13708	Treating or preventing virusesMethods For Treating Infections and related Diseases (Methods For Treating Or presenting Viral Infections and infected Diseases)	3/16/2000
			WO00/18231	Methods For Treating Or Preventing Viral Infections and related Diseases (Methods For Treating Or Preventing Viral Infections and associated Diseases)	4/6/2000
WO99/51781	Hepatitis C Virus Ns5b Compositions And Methods Of use thereof (Hepatitis C viruses Ns5b Compositions And Methods Of use thereof)	10/14/1999
			6323180	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	11/27/2001
6143715	Peptide analogue of Hepatitis C Inhibitor (Hepatitis C Inhibitor Peptide Analogues)	11/7/2000
			6329379	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	12/11/2001
6329417	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	12/11/2001
			6410531	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	6/25/2002
6420380	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	7/16/2002
			6448281	Viral Polymerase Inhibitors (Viral Polymerase Inhibitors)	9/10/2002
6479508	Viral Polymerase inhibitors (Viral Polymerase In)hibitors)	11/12/2002
			6534523	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	3/18/2003
WO00/09543	Hepatitis C Inhibitor tripeptide (hepatis C Inhibitor Tri-Peptides)	2/24/2000
			WO00/09558	Hepatitis C Inhibitor peptide (Hepatitis C Inhibitor Peptides)	2/24/2000
WO00/59929	Macrocyclic peptide Activity Against Hepatitis C Virus (Macrocyclic Peptides Active Against The Hepatitis C Virus)	10/12/2000
			WO02/04425	Viral Polymerase Inhibitors (Viral Polymerase Inhibitors)	1/17/2002
WO02/70739	HCV Polymerase Inhibitor Assay (Hcv Polymerase Inhibitor Assay)	9/12/2002
			WO03/07945	Viral Polymerase Inhibitors (Viral Polymerase Inhibitors)	1/30/2003
WO03/10140	Viral Polymerase Inhibitors (Viral Polymerase Inhibitors)	2/6/2003
			WO03/10141	Viral Polymerase Inhibitors (Viral Polymerase Inhibitors)	2/6/2003
WO99/07734	Peptide analogue of Hepatitis C Inhibitor (Hepatitis C Inhibitor Peptide Analogues)	2/18/1999
			WO01/16379	Hepatitis C Virus Replication Inhibitors (Hepatitis C Virus Replication Inhibitors)	3/8/2001
WO02/07761	Inhibiting the production And Replication of viral viruses (inhibition of Replication C Virus Processing And Replication)	1/31/2002
			WO02/57287	Nucleoside Derivatives As RNA-Dependent RNA Viral Polymerase Inhibitors (nucleotide Derivatives As Inhibitors of RNA-Dependent RNA Viral Polymerase)	7/25/2002
WO02/57425	Nucleoside Derivatives As RNA-Dependent RNA Viral Polymerase Inhibitors (nucleotide Derivatives As Inhibitors of RNA-Dependent RNA Viral Polymerase)	7/25/2002

WO02/70651	Virus Reporter protein Particles (Viral Reporter Particles)	9/12/2002
			WO03/20222PCT/US2003/041493	Dioxolane And oxathiolane derivatives As Inhibitors Of RNA-Dependent RNA Viral Polymerase (Dioxolane And Oxathiolanelanderivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase) Thiosemicarbazones As antiviral And immunopotentiators (Thiosemicazoles As Anti-vitamins And antigens)	3/13/200301/10/2003

Table 3: U.S. patents and published International patent applications relating to inhalation techniques for delivery of the antiviral compounds of the present invention

Publication number	Title	Pub. Date:
			5740794	apparatus and method for dispensing dry powder medicaments (Apparatus and methods for dispensing dry powder medicaments)	4/21/1998
5775320	Method and apparatus for delivering aerosolized medicaments	7/7/1998
			5785049	Method and apparatus for dispensing dry powder medicaments	7/28/1998
5814607	Pulmonary delivery of parathyroid hormone active fragments (Pulmonary delivery of active fragments of parathyroid hormone)	9/29/1998
			5826633	Powder filling system, device and method(Powder filling systems，apparatus and methods)	10/27/1998
5458135	Method and apparatus for delivering aerosolized medicaments	10/17/1995
			5607915	Pulmonary delivery of parathyroid hormone active fragments (Pulmonary delivery of active fragments of parathyroid hormone)	3/4/1997
5654007	Method and system for processing dispersible fine powders	8/5/1997
			5922354	Method and system for processing dispersible fine powders	7/13/1999
5928469	Method for storing substances (Process for storage of materials)	7/27/1999
			5976574	Process for spray drying organic solvent suspensions of hydrophobic drugs (Processes for spraying drying hydrophilic drugs in organic solvents subspensions)	11/2/1999
5985248	Process for spray drying hydrophobic drugs and compositions thereof	11/16/1999
			5994314	Compositions and methods for delivering nucleic acids to the lung (Compositions and methods for nucleic acid delivery to the lung)	11/30/1999
5997848	Methods and compositions for pulmonary delivery of insulin (Methods and compositions for pulmonary delivery of insulin)	12/7/1999
			6001336	Process for spray drying aqueous suspensions of hydrophobic drugs and compositions thereof (Processes for spraying aqueous suspensions of hydrophilic drugs and compositions of the same)	12/14/1999
6019968	Dispersible antibody compositions and methods for making and using same	2/1/2000
			6051256	Dispersible macromolecular compositions and methods for their preparation and use (Dispersible macromolecular compositions and methods for their preparation and use)	4/18/2000
6071428	Stable compositions (Stable compositions)	6/6/2000
			6077543	System and method for spray drying hydrophobic drugs containing hydrophilic excipients (Systems and)processes for spray dryinghydrophobic drugs with hydrophilic extipients)	6/20/2000
6080721	Pulmonary delivery of parathyroid hormone active fragments (Pulmonary delivery of active fragments of parathyroid hormone)	6/27/2000
			6089228	Apparatus and method for dispensing dry powder medicaments (Apparatus and methods for dispensing dry powder medicaments)	7/18/2000
6103270	Method and system for processing dispersible fine powders	8/15/2000
			6123936	Methods and compositions for dry powder formulations of interferons (Methods and compositions for the dry powder formulations of interferons)	9/26/2000
6136346	Powdered pharmaceutical preparation with improved dispersibility	10/24/2000
			6138668	Method and apparatus for delivering aerosolized medicaments	10/31/2000
6165463	Dispersible antibody compositions and methods of making and using same	12/26/2000
			6182712	Powder filling apparatus and method of use	2/6/2001
6187344	Powdered pharmaceutical preparation with improved dispersibility	2/13/2001
			6207135	Gas particles for ultrasonic diagnosis and method for producing the same (gases for ultrasonic diagnosis and process for the same)	3/27/2001
6231851	Methods and compositions for dry powder formulations of interferons (Methods and compositions for the dry powder formulations of interferons)	5/15/2001

6257233	Dry powder dispersion device and method of use thereof (Dry powder dispersing and methods for the use)	7/10/2001
			6258341	Stable glass statePowder preparation (Stable glass state powder formulations)	7/10/2001
6267155	Powder filling systems, devices and methods	7/31/2001
			6294204	Method for producing microcapsules having uniform morphology and microcapsules produced by the Method	9/25/2001
6303582	Compositions and methods for delivering nucleic acids to the lung (Compositions and methods for nucleic acid delivery to the lung)	10/16/2001
			6309623	Stable articles for metered dose inhalers (Stabilized formulations for use in metered dose inhalers)	10/30/2001
6309671	Stable glassy state powder formulations (Stable glass state powder formulations)	10/30/2001
			6358530	Powdered pharmaceutical preparation with improved dispersibility	3/19/2002
6365190	System and method for spray drying hydrophobic drugs with hydrophilic excipients	4/2/2002
			6372258	Methods for spray drying drugs and hydrophobic amino acids (Methods of spraying-drying a drug and a hydrophosphonic amino acid)	4/16/2002
6423344	Dispersible macromolecular compositions and methods of making and using the same	7/23/2002
			6426210	Material Storage (Storage of materials)	7/30/2002
6433040	Stable bioactive preparations and methods of use thereof (Stabilized bioactive preparations and methods of use)	8/13/2002
			6440337	Method and apparatus for producing granules (Method and apparatus for the formation of granules)	8/27/2002
RE37872	Material Storage (Storage of materials)	10/8/2002
			6479049	Methods and compositions for dry powder formulations of interferons (Methods and compositions for the dry powder formulations of interferons)	11/12/2002
6503411	Stable compositions (Stable compositions)	1/7/2003
			6509006	Devices, compositions and methods for hydrophobic nebulization of drugs to the lung (Devices compositions and methods for the delivery of airborne agents)	1/21/2003
6514496	Dispersible antibody compositions and methods of making and using same	2/4/2003
			6518239	Dry powder compositions (dry powder compositions) having improved dispersibility	2/11/2003
6543448	Device for dispersing dry powder medicineAnd methods (appatatus and methods for dispersing dry powder documents)	4/8/2003
			6546929	Dry powder dispensing apparatus and method of use	4/15/2003
WO 00/15262	Pulmonary delivery of dry powder active agents (dry powder active agent pulmonary delivery)	3/23/2000
			WO 93/00951	Method and apparatus for delivering aerosolized medicaments	1/21/1993
WO 94/07514	Pulmonary delivery of parathyroid hormone active fragments (pulmonary delivery of active fragments of parathyroid hormone)	4/14/1994
			WO 95/24183	Methods and compositions for pulmonary delivery of insulin (methods and compositions for pulmonary delivery of insulin)	9/14/1995
WO 95/31479	Methods and compositions for dry powder formulations of interferons (methods and compositions for the delivery of interferons)	11/23/1995
			WO 96/09085	Apparatus and method for dispensing dry powder medicaments (appatatus and methods for dispensing dry powder containers)	3/28/1996
W0 96/32096	Powdered pharmaceutical preparation with improved dispersibility	10/17/1996
			WO 96/32116	Compositions and methods for delivering nucleic acids to the lung	10/17/1996
WO 96/32149	Pulmonary delivery of aerosolized medicaments (pulmonary delivery of airborne agents)	10/17/1996
			WO 96/32152	Dry powder alpha 1-antitrypsin pulmonary delivery (pulmony administration of dry powder alpha 1-antiprysin)	10/17/1996
WO 96/40068	Method and system for processing dispersible fine powders	12/19/1996
			WO 97/41031	Powder filling systems, devices and methods	11/6/1997
WO 97/41833	Dispersible macromolecular compositions and methods of making and using the samer preparation and use)	11/13/1997
			WO 98/16205	Stable glassy state powder formulations	4/23/1998
WO 98/29096	Aerosolized hydrophobic drug (aerosolized hydrophobic drug)	7/9/1998
			WO 98/29098	Process for spray drying an aqueous suspension of a hydrophobic drug containing a hydrophilic excipient and composition produced by this process (process for spray drying an aqueous suspension of a hydrophilic drug with a hydrophilic excipient and a prepared by process	7/9/1998
WO 98/29140	Process and composition for spray drying an organic solvent suspension of a hydrophobic drug containing a hydrophilic excipient (processes and compositions for spray drying hydrophilic drugs in organic solvents of hydrophilic excipients)	7/9/1998
			WO 98/29141	Process for spray drying hydrophobic pharmaceutical solutions containing hydrophilic excipients and compositions produced by this process	7/9/1998

	drying solutions of hydrophobic drugs with hydrophilic excipients and compositions prepared by suchprocesses)
			WO 99/19215	Powder filling apparatus and method	4/22/1999
WO 99/42124	Liquid crystal forms of cyclosporin (liquid crystal systems of cyclosporins)	8/26/1999
			WO 99/47196	Delivery of aerosolized active agent (delivery)	9/23/1999
WO 99/62495	Dry powder dispensing apparatus and method of use	12/9/1999
			WO 00/21594	Delivery of atomized active agent with flow resistance modulation (flow resistance modulated atomized active agent delivery)	4/20/2000
WO OO/61178	Pulmonary delivery of dry powder formulations for infertility treatment	10/19/2000
			WO 00/72904	Device and method for metering and dispensing aerosolized medicamentsispensing metered amount of aerosolizedmedication)	12/7/2000
WO 01/00263	Systems and methods for aerosolizing pharmaceutical formulations	1/4/2001
			WO 01/00312	Spray drying process for preparing dry powders	1/4/2001
WO 01/32144	Dry powder compositions (dry powder compositions) having improved dispersibility	5/10/2001
			WO 01/43529	Easy to remove powder container (refills to failure of the extraction of powders)	6/21/2001
WO 01/43530	System and method for removing powder from containers	6/21/2001
			WO 01/43802	System and method for handling packaging powders (systems and methods for handling packaging packaged powders)	6/21/2001
WO 01/44764	Non-destructive quality sensing system and method (systems and methods for non-destructive mass sensing)	6/21/2001
			WO 01/87393	System, device and method for opening a powder to be liquefied (systems, devices and methods for exposing a powder to be liquefied)	11/22/2001
WO 01/93932	Locking mechanism for aerosol drug delivery devices (lockout mechanism for aerosol drug delivery devices)	12/13/2001
			WO 02/09669	Apparatus and method for producing particles having a narrow particle size distribution and particles produced thereby	2/7/2002
WO 02/11695	Inhalable spray-dried 4-spiral-wrapped protein powder (attenuated aggregation) with minimal agglomeration	2/14/2002
			WO 02/49619	Induced phase transformation method (induced phase transformation method for the production of microparticles containing a hydrophilic active agent)	6/27/2002
WO 02/49620	Induced phase transformation method (induced phase transformation method for the production of fine particles containing a hydrophobic active agent)	6/27/2002
			WO 02/54868	Pulmonary delivery of polyene antifungal agents (pulmonary delivery of polyene antifungal agents)	7/18/2002
WO 02/87542	Novel methods and compositions for delivery of macromolecules to or through the respiratory tract	11/7/2002
			WO 02/100548	Centrifugal rotating drum for treating sticky powder	12/19/2002
WO 03/00326	Powder atomization device and method (powder atomization apparatus and method)	1/3/2003
			WO 03/00329	Flow rate regulators and methods for aerosol drug delivery devices	1/3/2003

Table 4: forward and reverse primers for SARSV nucleic acid amplification

Logarithm of formation	Forward primer SEQIDNO	Forward primer origin	Forward primer end point	Forward primer Tm	Forward primer% GC	Reverse primer SEQIDNO	Reverse primer origin	Reverse primer end point	Reverse primer Tm	Reverse primer% GC	Primer TmDiff	Length of product	Product Tm	Product% GC	Annealing score	Optimum annealing temperature
																	1	1021	12726	12746	51.3	47.6	3521	12996	12977	50.2	40	1	271	75	42.8	26	52.6
2	1022	12236	12256	51.2	42.9	3522	12993	12975	51.4	47.4	0.2	758	76.4	42.5	26	54
																	3	1023	12373	12391	50.8	47.4	3523	12993	12975	51.4	47.4	0.6	621	76.4	43	26	53.8
4	1024	12236	12256	51.2	42.9	3524	12996	12977	50.2	40	0.9	761	76.4	42.3	26	53.6

Table 5: primer and method for producing the same

Forward primer SEQ ID NO and common ordinate		Reverse primer SEQ ID NO and common ordinate		TM(Forward and reverse) (. degree. C.)		Product Length (bp)
							6076	1-19	6171	199-183	50.1	50.3	199
6077	149-169	6172	334-315	51.5	52.4	186
							6078	292-310	6173	560-541	50.8	51.1	269
6079	598-619	6174	749-731	52.6	50.6	152
							6080	721-742	6175	930-912	50.4	50.3	210
6081	888-912	6176	1077-1058	52.8	51.2	190
							6082	984-1003	6177	1149-1131	51.1	51.1	166
6083	1157-1175	6178	1479-1460	50.9	51.6	323
							6084	1420-1441	6179	1700-1680	51.2	50.7	281
6085	1685-1707	6180	1834-1811	53.8	53.7	150
							6086	1740-1764	6181	1987-1963	53.4	52.2	248
6087	2007-2025	6182	2251-2232	50.3	50.1	245
							6088	2226-2245	6183	2385-2366	50.4	50.1	160
6089	2428-2446	6184	2749-2728	50.1	50.3	322
							6090	2742-2763	6185	2893-2875	50.6	51.4	152
6091	2823-2844	6186	3082-3058	50.4	52.3	260
							6092	3007-3031	6187	3185-3164	51.9	51.0	179
6093	3234-3254	6188	3497-3478	51.1	51.3	264
							6094	3453-3476	6189	3647-3627	51.8	52.1	195
6095	3601-3622	6190	3877-3853	52.5	53.6	277
							6096	4007-4027	6191	4158-4135	51.1	51.4	152
6097	4141-4165	6192	4316-4295	51.3	50.8	176
							6098	4366-4387	6193	4567-4544	54.6	55.4	202
6099	4488-4508	6194	4708-4690	50.7	50.3	221
							6100	4658-4677	6195	4994-4974	50.5	51.2	337
6101	4902-4922	6196	5115-5092	50.5	51.4	214
							6102	5239-5260	6197	5450-5430	50.8	50.9	212
6103	5366-5389	6198	5560-5542	50.5	51.8	195
							6104	5593-5612	6199	5860-5836	50.8	51.6	268
6105	6042-6062	6200	6291-6271	50.4	51.1	250
							6106	6271-6291	6201	6483-6463	51.1	50.2	213
6107	7017-7040	6202	7171-7153	52.4	52.8	155
							6108	7253-7272	6203	7504-7486	50.3	50.3	252
6109	7415-7434	6204	7677-7654	54.5	53.6	263
							6110	7615-7635	6205	7821-7798	51.1	52.8	207
6111	7728-7746	6206	7936-7915	51.7	50.1	209
							6112	7845-7867	6207	7994-7970	52.7	53.4	150
6113	8011-8029	6208	8189-8170	51.4	50.6	179
							6114	8143-8166	6209	8300-8281	52.2	50.8	158
6115	8221-8239	6210	8388-8369	51.0	51.1	158
							6116	8553-8575	6211	8931-8915	51.8	50.3	379
6117	8867-8886	6212	9254-9236	50.7	50.6	388
							6118	9244-9267	6213	9597-9573	51.9	53.4	354
6119	9620-9640	6214	9990-9969	51.3	51.3	371
							6120	10009-10027	6215	10188-10171	50.2	50.2	180
6121	10093-10113	6216	10244-10223	52.4	50.6	152
							6122	10242-10265	6217	10608-10589	51.2	51.0	367
6123	10549-10571	6218	10783-10763	53.7	55.2	235

6124	10766-10785	6219	10930-10912	52.0	51.1	165
							6125	11065-11085	6220	11305-11287	50.7	50.0	241
6126	11265-11287	6221	11429-11405	54.5	53.5	165
							6127	11552-11571	6222	11730-11709	52.0	50.4	179
6128	11705-11726	6223	11869-11848	50.1	50.2	165
							6129	11801-11824	6224	11984-11967	51.5	50.4	184
6130	12040-12058	6225	12254-12235	52.3	51.9	215
							6131	12235-12253	6226	12406-12388	50.1	50.1	172
6132	12366-12384	6227	12730-12712	51.7	52.2	365
							6133	12727-12748	6228	12994-12976	50.8	50.3	268
6134	12948-12966	6229	13224-13201	50.7	51.7	277
							6135	13175-13196	6230	13324-13300	54.3	55.1	150
6136	13237-13258	6231	13545-13526	52.9	52.9	309
							6137	13790-13810	6232	13963-13945	50.9	50.7	174
6138	14080-14098	6233	14280-14257	51.5	51.0	201
							6139	14405-14427	6234	14561-14540	50.2	50.9	157
6140	14882-14906	6235	15046-15024	50.9	51.5	165
							6141	14951-14976	6236	15145-15124	53.1	52.9	195
6142	15113-15134	6237	15275-15257	51.6	50.8	163
							6143	15211-15230	6238	15383-15363	50.2	50.1	173
6144	15364-15387	6239	15528-15506	54.0	52.1	165
							6145	15456-15477	6240	15605-15585	52.0	53.2	150
6146	15513-15532	6241	15897-15876	51.2	50.4	385
							6147	15837-15856	6242	15999-15978	52.3	50.8	163
6148	16073-16096	6243	16301-16277	51.7	52.8	229
							6149	16245-16266	6244	16404-16380	50.3	52.0	160
6150	16366-16385	6245	16515-16492	52.9	53.8	150
							6151	16553-16571	6246	16777-16758	53.4	51.5	225
6152	16832-16852	6247	17026-17004	51.0	51.6	195
							6153	16982-17001	6248	17359-17340	51.2	50.2	378
6154	17354-17372	6249	17511-17490	51.3	50.4	158
							6155	17422-17443	6250	17573-17552	50.2	51.1	152
6156	17603-17623	6251	17769-17748	50.7	51.5	167
							6157	17728-17746	6252	17883-17862	50.9	51.2	156
6158	18011-18030	6253	18163-18140	52.9	51.9	153
							6159	18076-18098	6254	18225-18205	54.4	55.0	150
6160	18270-18292	6255	18432-18413	51.9	51.4	163
							6161	18352-18373	6256	18648-18629	51.3	50.8	297
6162	18550-18571	6257	18702-18684	50.4	51.9	153
							6163	18720-18738	6258	19004-18983	50.6	51.0	285
6164	18960-18981	6259	19109-19085	54.7	54.3	150
							6165	19065-19089	6260	19217-19195	52.8	51.7	153
6166	19310-19329	6261	19476-19454	50.2	52.1	167
							6167	19569-19589	6262	19719-19701	50.5	51.8	151
6168	19707-19731	6263	19856-19833	55.7	55.9	150
							6169	19771-19792	6264	19921-19901	50.1	50.2	151
6170	19833-19851	6265	19986-19966	50.9	50.7	154

Table 6: primer and method for producing the same

Forward primer SEQ ID NO and common ordinate		Reverse primer SEQ ID NO and common ordinate		TM(Forward and reverse) (. degree. C.)		Product Length (bp)
							6266	20110-20132	6305	20425-20404	51.9	50.9	316

6267	20468-20492	6306	20617-20596	53.2	53.5	150
							6268	20557-20578	6307	20891-20871	50.4	50.6	335
6269	20838-20856	6308	21037-21015	52.5	52.0	200
							6270	21096-21116	6309	21295-21272	50.1	51.7	200
6271	22173-22194	6310	22414-22395	52.4	51.0	242
							6272	22320-22342	6311	22501-22479	54.8	54.3	182
6273	22532-22552	6312	22695-22675	50.6	50.0	164
							6274	22712-22736	6313	22873-22852	56.7	55.5	162
6275	22842-22861	6314	23086-23067	51.0	52.8	245
							6276	23151-23170	6315	23395-23376	51.4	50.3	245
6277	23307-23326	6316	23524-23501	51.1	51.1	218
							6278	23615-23635	6317	23776-23758	50.7	50.2	162
6279	23838-23857	6318	23996-23977	50.4	50.6	159
							6280	24030-24051	6319	24407-24386	57.6	55.7	378
6281	24388-24407	6320	24581-24563	50.4	50.1	194
							6282	24559-24579	6321	24938-24921	52.0	50.4	380
6283	24922-24941	6322	25184-25166	50.1	51.2	263
							6284	25201-25220	6323	25400-25382	51.1	51.4	200
6285	25363-25381	6324	25646-25627	51.1	50.5	284
							6286	25656-25681	6325	25839-25814	54.5	56.4	184
6287	25761-25782	6326	25982-25961	54.6	54.3	222
							6288	26039-26058	6327	26189-26166	54.0	53.0	151
6289	26184-26205	6328	26333-26310	50.9	51.8	150
							6290	26422-26442	6329	26660-26641	51.3	50.2	239
6291	26571-26589	6330	26739-26715	51.7	53.2	169
							6292	26733-26752	6331	26960-26941	51.1	52.2	228
6293	26866-26885	6332	27139-27117	50.7	51.9	274
							6294	27300-27321	6333	27458-27439	51.2	50.2	159
6295	27361-27380	6334	27579-27558	52.4	51.1	219
							6296	27718-27740	6335	27917-27901	50.7	50.0	200
6297	28041-28059	6336	28207-28189	50.8	50.8	167
							6298	28166-28189	6337	28411-28393	52.2	52.9	246
6299	28395-28414	6338	28671-28653	51.5	50.2	277
							6300	28654-28672	6339	28821-28800	50.6	52.3	168
6301	28867-28885	6340	29184-29166	51.5	51.6	318
							6302	29183-29204	6341	29360-29342	50.4	50.4	178
6303	29262-29279	6342	29626-29606	50.1	50.2	365
							6304	29538-29559	6343	29690-29670	50.0	50.4	153

Table 7: primer and method for producing the same

Name (R)	SEQ ID NO：	Common ordinate		Name (R)	SEQ ID NO：	Common ordinate
							AB4f	6344	19869-19888		CB1r	6367	28011-28030
AB5f	6345	20238-20257		CB2r	6368	27671-27690
							BC1f	6346	20581-20600		CB3r	6369	27301-27320
BC2f	6347	20950-20969		CB4r	6370	26931-26950
							BC3f	6348	21339-21358		CB5r	6371	26575-26594
BC4f	6349	21708-21727		CB6r	6372	26191-26210
							BC5f	6350	22041-22060		CB7r	6373	25841-25860
BC6f	6351	22410-22429		CB8r	6374	25476-25495
							BC7f	6352	22759-22778		CB9r	6375	25126-25145
BC8f	6353	23131-23150		CB10r	6376	24791-24810
							BC9f	6354	23500-23519		CB11r	6377	24422-24441

BC10f	6355	23841-23860		CB12r	6378	24031-24050
							BC11f	6356	24210-24229		CB13r	6379	23673-23692
BC12f	6357	24560-24579		CB14r	6380	23298-23317
							BC13f	6358	24941-24960		CB15r	6381	22928-22947
BC14f	6359	25310-25329		CB16r	6382	22567-22586
							BC15f	6360	25675-25694		CB17r	6383	22196-22215
BC16f	6361	26044-26063		CB18r	6384	21831-21850
							BC17f	6362	26413-26432		CB19r	6385	21431-21450
BC18f	6363	26763-26782		CB20r	6386	21073-21092
							BC19f	6364	27132-27151		CB21r	6387	20715-20734
BC20f	6365	27491-27510		BA1r	6388	20345-20364
							BC21f	6366	27845-27864		BA2r	6389	19969-19988
				BA3r	6390	19599-19618
											BA4r	6391	19228-19247
				BA5r	6392	18852-18871

Table 8: primer and method for producing the same

Name (R)	SEQ ID NO	Common ordinate	Name (R)	SEQ ID NO	Common ordinate
						F1F2F3F4F5F6F7F8F9F10F11F12F13F14F15F16F17F18F19F20F21F22F23F24F25F26F27F28F29F30F31F32F33F34	6393639463956396639763986399540064016402640364046405640664076408640964106411641264136414641564166417641864196420642164226423642464256426	1-19292-310721-742984-10031420-14411740-17642226-22452742-27633007-30313453-34764007-40274366-43874658-46775239-52605593-56126271-62917253-72727615-76357845-78678143-81668553-85759244-926710009-1002710242-1026510766-1078511265-1128711705-1172612040-1205812366-1238412948-1296613237-1325814080-1409814882-1490615113-15134	R1R2R3R4R5R6R7R8R9R10R11R12R13R14R15R16R17R18R19R20R21R22R23R24R25R26R27R28R29R30R31R32R33R34	6441644264436444644564466447644864496450645164526453645464556456645764586459646064616462646364646465646664676468646964706471647264736474	334-315749-7311077-10581479-14601834-18112251-22322749-27283082-30583497-34783877-38534316-42954708-46905115-50925560-55426291-62717171-71537677-76547936-79158189-81708388-83699254-92369990-996910244-1022310783-1076311305-1128711730-1170911984-1196712406-1238812994-1297613324-1330013963-1394514561-1454015145-1512415383-15363

F35F36F37F38F39F40F41F42F43F44F45F46F47F48

64276428642964306431643264336434643564366437643864396440

15364-1538715513-1553216073-1609616366-1638516832-1685217354-1737217603-1762318011-1803018270-1829218550-1857118960-1898119310-1932919707-1973119833-19851

R35R36R37R38R39R40R41R42R43R44R45R46R47

6475647664776478647964806481648264836484648564866487

15605-1558515999-1597816404-1638016777-1675817359-1734017573-1755217883-1786218225-1820518648-1862919004-1898319217-1919519719-1970119921-19901

Table 9: primer and method for producing the same

	Name (R)	SEQ ID NO：
			1	CB12R	6488
2	R0010	6489
			3	R0011	6490
4	R0012	6491
			5	BNI-ED	6492
6	BNI-EU	6493
			7	SAR1S-U	6494
8	SAR1As-D	6495
			9	SAR1S	6496
10	SAR1As	6497
			11	IN2-U	6498
12	IN4-D	6499
			13	IN-2	6500
14	IN-4	6501
			15	IN-6	6502
16	IN-7	6503
			17	COR1-U	6504
18	COR2-D	6505
			19	COR-1	6506
20	COR-2	6507
			21	HKUF-U	6508
22	HKUR-D	6509
			23	HKU-F	6510
24	HKU-R	6511
			25	1451-D	6512
26	1451-U	6513
			27	690-D	6514
28	690-U	6515
			29	690-D2	6516
30	690-U2	6517
			31	EMC7-D	6518
32	EMC7-U	6519
			33	EMC7-D2	6520
34	EMC7-U2	6521
			35	EMC8-D	6522
36	EMC8-U	6523

	Name (R)	SEQ ID NO：
			37	EMC8-D2	6524
38	EMC8-U2	6525
			39	EMC11-D	6526
40	EMC11-U	6527
			41	ORF1B-D	6528
42	ORF1B-U	6529
			43	ORFS-D	6530
44	ORFS-U	6531
			45	E7-717F	6532
46	E8-85R	6533
			47	E8-307F	6534
48	E11-771F	6535
			49	E11-96R	6536
50	CON1-F	6537
			51	CON1-U	6538
52	CON2-F	6539
			53	CON2-R	6540
54	CON3-F	6541
			55	CON3-R	6542
56	15-F	6543
			57	15-R	6544
58	15-F2	6545
			59	15-R2	6546
60	13-F	6547
			61	13-R	6548
62	13-F2	6549
			63	13-R2	6550
64	CONTIG-F	6551
			65	QT3-R	6552
66	QT3-F	6553
			67	QIN-R	6554
68	QIN-F	6555
			69	AB1-F	6556
70	AB2-F	6557
			71	AB3-F	6558
72	AB1-R	6559

Table 10: characterization of the predicted protein and open reading frame for SARS virus

	SARS ORF(SEQ ID NO)	Length (aa)	Function of	Cleavage site	Feature(s)	Consd*
							ORF1a	P28(9766)	179	Leader protein	179(G/G)#		+
P65(9767)	639	MHV p65 cleavage product homologs	818(G/A)		+
						Nsp1(9768)		2422##	Papain-like protease, cleaving the first two proteins	3240(Q/S)	Sulfatase domain, zinc binding domain	+
Nsp2(9769)	306	3C-like protease, cleavage protein nsp1-nsp12	3546(Q/G)		+
						Nsp3(9770)		290	？	3836(Q/S)	5 TMDs	+
Nsp4(9771)	83	？	3919(Q/A)	1 TMD	+
						Nsp5(9772)		198	？	4117(Q/N)		+
Nsp6(9773)	113	？	4230(Q/A)		+
						Nsp7(9774)		139	？	4369(Q/S)	Hypothetical growth factor-like motifs	+
ORF1b	Nsp9(9775)	932	RNA polymerase	5298(Q/A)									+
						Nsp10(9776)	601	The putative helicase Tanner et al, (2003) J Biol Chem 278: 39578-82	5899(Q/A)	Metal binding domains, ATP/GTP binding domains	+
	Nsp11(9777)	527	？	6426(Q/S)								+
						Nsp12(9778)	346	？	6772(Q/A)		+
	Nsp13(9779)	298	？	-								+
						Structural region	Spike (S) (6042)		Major antigenic determinant comprising a receptor binding domain		Leader peptide, 1 TMD, 17N-glycosylation site		+
Orf3(6043)	274	？		2 TMD, 1N-glycosylation site, 10O-glycosylation site	-
							Orf4(6044)	154	？			-
Envelope (E) (6045)	76	Associated with viral envelopes		1 TMD, 2N-glycosylation site	+
							Substrate (M) (6046)	221	Transmembrane proteins associated with the viral envelope		3 TMD, 1N-glycosylation site	+
Orf7(6047)	63	？		1 TMD	-
							Orf8(6048)	122	？		1 TMD	-
Orf9(6049)	44	？		Surface association	-
							Orf10	39	？		Surface association	-
Orf11(6050)	84	？		1N-glycosylation site	-
							Nucleocapsid (N) (6052)	422	Andviral genomic RNA association		Phosphoproteins	+
Orf13	98	？		1O-glycosylation site	-

TMD: a predicted transmembrane domain.

Cons^d*: + denotes the presence of the corresponding protein in at least one other coronavirus

#: or cleavage after Gly-Gly (i.e., at G/A) to give a 180mer product

# #: the 2422mer product can also be cleaved after residue 1922 (Gly-2740 of SEQ ID NO: 6039) to yield a peptide containing a Zn binding motif

The 1922mer product Plpr of (SEQ ID NO: 7254) and a 500mer product.

Table 11: protein homology between SARS and other coronaviruses

The numbers indicate the ratio of amino acid identity between the SARS protein and the corresponding gene product of the other coronavirus. More conservative pairs are shown in bold;

the more variable pairs are underlined.

	Group 1			Group 2		Group 3
							Protein	229E	TGV	PEDV	MHV	BCoV	AIBV
Replicase regions
							Leader protein p28p65 homolog nsp1(PLP protease) nsp2(3CL protease) nsp3nsp4nsp5nsp6nsp7	＜20＜2025.540.43038.648.245.153.8	＜202325.843.82742.242.938.954.5	＜202325.444.629.439.843.945.156.1	27＜20295034.247.546.845.156.2	＜20203048.435.546.147.346.955.4	＜20＜20254128.537.338.739.858.3
nsp9 (polymerase) nsp10 (helicase) nsp11nsp12nsp13	59.860.752.343.156.4	59.66253.74354.4	6062.352.345.455.3	67.367.257.645.963	66.968.657.64565	62.458.9 52 40.2 53.4
							Structural region
Spike (S) envelope (E) matrix glycoprotein (M) nucleocapsid (N)	28.833*30.6 26.9	31.6*27.932.530.1	30.32034.829.5	31.12340.837.3	3126.541.937.4	32.7*23.232.531.5

These three alignments were obtained on only one fragment of the whole protein.

Table 12: differences of five SARS isolates nucleotide and amino acid

		FRA*	TOR2*	Urbani*	CUHK*	HKU*
								Position °	Base/amino acid	Base/amino acid	Base/amino acid	Base/amino acid	Base/amino acid
							ORF1a	2557	A/Thr	G/Ala	G/Ala	G/Ala	G/Ala
2601	T/Val				C
						7746		G/Pro			T
7919	C/Ala		T/Val
						7930		G/Asp				A/Asn
8387	G/Ser				C/Thr
						8416		G/Arg				C/Thr
9404	T/Val			C/Ala
						9479		T/Val			C/Ala
11448	T/Ile	C	C	C	C
ORF1b	13494	GT/Val					AG/Ser
						16622		C/Ala		T
	17564	T/Asp			C/Glu
						17846		C/Arg			T
	18065	G/Lys					A
						18965		A/Ile	T	T	T	T
	19064	A/Glu		G	G
						19084		T/Ile	C/Thr	C/Thr	C/Thr	C/Thr
Spike of the heart						21721		G/Gly			A/Asp
	22222	T/Ile			C/Thr
						23220		T/Ser	G/Ala
	24872	T/Leu		C
						24933		T/Phe	C/Leu	C/Leu	C/Leu	C/Leu
ORF3						25298		G/Gly	A/Arg
	25569	T/Met					A/Lys
Substrate	26600	T/Val	C/Ala	C/Ala	C/Ala
						26857		T/Ser		C/Pro
ORF10						27827		T/Cys			C/Arg
Nucleocapsid						28268		T/Ile	C/Thr	C/Thr	C/Thr	C/Thr

SARS coronavirus FRA (accession number AY310120)

SARS coronavirus TOR2 (accession number AY274119)

SARS coronavirus Urbani (accession number AY278741)

SARS coronavirus CUHK-W1 (accession number AY278554)

HKU-39849 of SARS coronavirus (accession number AY278491)

The position is based on the FRA sequence.

Tables 13 to 25: SEQ ID NOS: t epitope prediction for 6039-

Epitope prediction in http: html, using a minimum score of 0.5 and BIMAS matrix, select the largest of the 20 results. This analysis revealed epitopes for both 9mer and 10 mer.

Table 13: SEQ ID NO: 6039 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1867	SEQ ID NO：7400	8％	450
2	4139	SEQ ID NO：7401	5.55％	312.5
					3	88	SEQ ID NO：7402	4％	225
4	4249	SEQ ID NO：7403	3.55％	200
					5	4059	SEQ ID NO：7404	2.22％	125
6	2027	SEQ ID NO：7405	1.6％	90
					7	3413	SEQ ID NO：7406	1.11％	62.5
8	1823	SEQ ID NO：7407	0.88％	50
					9	2798	SEQ ID NO：7408	0.88％	50
10	220	SEQ ID NO：7409	0.8％	45
					11	3738	SEQ ID NO：7410	0.8％	45
12	4182	SEQ ID NO：7411	0.8％	45
					13	4174	SEQ ID NO：7412	0.66％	37.5
14	1940	SEQ ID NO：7413	0.55％	31.25
					15	38	SEQ ID NO：7414	0.48％	27
16	1231	SEQ ID NO：7415	0.44％	25
					17	1613	SEQ ID NO：7416	0.44％	25
18	3645	SEQ ID NO：7417	0.44％	25
					19	4192	SEQ ID NO：7418	0.44％	25
20	378	SEQ ID NO：7419	0.4％	22.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1867	SEQ ID NO：7420	8％	450
2	1495	SEQ ID NO：7421	4％	225
					3	3921	SEQ ID NO：7422	2.4％	135
4	486	SEQ ID NO：7423	2.22％	125
					5	4139	SEQ ID NO：7424	2.22％	125
6	62	SEQ ID NO：7425	1.6％	90
					7	1190	SEQ ID NO：7426	1.6％	90
8	1284	SEQ ID NO：7427	1.6％	90
					9	3284	SEQ ID NO：7428	1.6％	90
10	2921	SEQ ID NO：7429	1.2％	67.5

11	349	SEQ ID NO：7430	0.8％	45
					12	789	SEQ ID NO：7431	0.8％	45
13	1185	SEQ ID NO：7432	0.8％	45
					14	4184	SEQ ID NO：7433	0.8％	45
15	1313	SEQ ID NO：7434	0.64％	36
					16	3948	SEQ ID NO：7435	0.48％	27
17	149	SEQ ID NO：7436	0.44％	25
					18	941	SEQ ID NO：7437	0.44％	25
19	1390	SEQ ID NO：7438	0.44％	25
					20	1613	SEQ ID NO：7439	0.44％	25

HLA A3-9 mers
					Maximum possible scoring with this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1010	SEQ ID NO：7440	1.48％	180
2	3155	SEQ ID NO：7441	1.48％	180
					3	1229	SEQ ID NO：7442	1.23％	150
4	2405	SEQ ID NO：7443	0.88％	108
					5	2	SEQ ID NO：7444	0.74％	90
6	2304	SEQ ID NO：7445	0.74％	90
					7	2358	SEQ ID NO：7446	0.74％	90
8	3160	SEQ ID NO：7447	0.74％	90
					9	3771	SEQ ID NO：7448	0.74％	90
10	4007	SEQ ID NO：7449	0.74％	90
					11	3079	SEQ ID NO：7450	0.66％	81
12	4045	SEQ ID NO：7451	0.66％	81
					13	1081	SEQ ID NO：7452	0.49％	60
14	3268	SEQ ID NO：7453	0.49％	60
					15	4144	SEQ ID NO：7454	0.49％	60
16	614	SEQ ID NO：7455	0.37％	45
					17	728	SEQ ID NO：7456	0.37％	45
18	1537	SEQ ID NO：7457	0.37％	45
					19	313	SEQ ID NO：7458	0.32％	40
20	1744	SEQ ID NO：7459	0.32％	40

HLA A3-10 mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	62	SEQ ID NO：7460	4.44％	540
2	2151	SEQ ID NO：7461	2.46％	300
					3	633	SEQ ID NO：7462	2.22％	270
4	1158	SEQ ID NO：7463	2.22％	270

5	2565	SEQ ID NO：7464	2.22％	270
					6	2298	SEQ ID NO：7465	1.77％	216
7	3159	SEQ ID NO：7466	1.11％	135
					8	640	SEQ ID NO：7467	0.98％	120
9	2186	SEQ ID NO：7468	0.74％	90
					10	3869	SEQ ID NO：7469	0.74％	90
11	2308	SEQ ID NO：7470	0.66％	81
					12	786	SEQ ID NO：7471	0.55％	67.5
13	749	SEQ ID NO：7472	0.49％	60
					14	1080	SEQ ID NO：7473	0.49％	60
15	2358	SEQ ID NO：7474	0.49％	60
					16	3955	SEQ ID NO：7475	0.49％	60
17	714	SEQ ID NO：7476	0.37％	45
					18	1081	SEQ ID NO：7477	0.37％	45
19	1170	SEQ ID NO：7478	0.37％	45
					20	1228	SEQ ID N0：7479	0.37％	45

HLA A24-9 mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	3797	SEQ ID NO：7480	37.57％	600
2	4202	SEQ ID NO：7481	37.57％	600
					3	3189	SEQ ID NO：7482	25.05％	400
4	1864	SEQ ID NO：7483	23.14％	369.6
					5	1066	SEQ ID NO：7484	22.54％	360
6	2143	SEQ ID NO：7485	22.54％	360
					7	2693	SEQ ID NO：7486	22.54％	360
8	1426	SEQ ID NO：7487	18.78％	300
					9	1238	SEQ ID NO：7488	18.03％	288
10	3768	SEQ ID NO：7489	18.03％	288
					11	797	SEQ ID NO：7490	15.03％	240
12	1882	SEQ ID NO：7491	15.03％	240
					13	1490	SEQ ID NO：7492	13.77％	220
14	2237	SEQ ID NO：7493	13.77％	220
					15	95	SEQ ID NO：7494	12.52％	200
16	1821	SEQ ID NO：7495	12.52％	200
					17	2289	SEQ ID NO：7496	12.52％	200
18	3080	SEQ ID NO：7497	12.52％	200
					19	3660	SEQ ID NO：7498	12.52％	200
20	4354	SEQ ID NO：7499	12.52％	200

HLA A24-10 mers

Maximum possible score Using this molecular type				1596.672
					Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
1	2143	SEQ ID NO：7500	37.87％	604.8
					2	1159	SEQ ID NO：7501	26.30％	420
3	1650	SEQ ID NO：7502	26.30％	420
					4	1150	SEQ ID NO：7503	18.78％	300
5	2763	SEQ ID NO：7504	18.78％	300
					6	3165	SEQ ID NO：7505	18.78％	300
7	3201	SEQ ID NO：7506	15.03％	240
					8	3694	SEQ ID NO：7507	15.03％	240
9	4204	SEQ ID NO：7508	15.03％	240
					10	1692	SEQ ID NO：7509	13.77％	220
11	797	SEQ ID NO：7510	12.52％	200
					12	1610	SEQ ID NO：7511	12.52％	200
13	1789	SEQ ID NO：7512	12.52％	200
					14	1881	SEQ ID NO：7513	12.52％	200
15	3090	SEQ ID NO：7514	12.52％	200
					16	3763	SEQ ID NO：7515	12.52％	200
17	2569	SEQ ID NO：7516	11.27％	180
					18	194	SEQ ID NO：7517	9.39％	150
19	1771	SEQ ID NO：7518	9.39％	150
					20	2488	SEQ ID NO：7519	9.39％	150

HLA A 0201-9 mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	2308	SEQ ID NO：7520	0.20％	8144.13515256
2	3729	SEQ ID NO：7521	0.10％	4047.23088
					3	3574	SEQ ID NO：7522	0.09％	3547.4996634
4	3615	SEQ ID NO：7523	0.06％	2722.682592
					5	3159	SEQ ID NO：7524	0.05％	1999.734264
6	2339	SEQ ID NO：7525	0.03％	1551.92907744
					7	2201	SEQ ID NO：7526	0.03％	1521.53694
8	3559	SEQ ID NO：7527	0.02％	1174.38939504
					9	3085	SEQ ID NO：7528	0.02％	1146.296448
10	4070	SEQ ID NO：7529	0.02％	970.4103696
					11	3708	SEQ ID NO：7530	0.02％	958.92888
12	3098	SEQ ID NO：7531	0.02％	942.678
					13	1362	SEQ ID NO：7532	0.02％	900.6984
14	3563	SEQ ID NO：7533	0.01％	735.86016
					15	3774	SEQ ID NO：7534	0.01％	687.655656
16	4242	SEQ ID NO：7535	0.01％	685.78272

17	2340	SEQ ID NO：7536	0.01％	668.37342936
					18	650	SEQ ID NO：7537	0.01％	640.1983392
19	3862	SEQ ID NO：7538	0.01％	620.57772
					20	2860	SEQ ID NO：7539	0.01％	607.88448

HLA A 0201-10 mers
					Use thisMaximum possible score for molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	2307	SEQ ID NO：7540	0.40％	15915.66281448
2	2201	SEQ ID NO：7541	0.12％	4772.09313
					3	3558	SEQ ID NO：7542	0.05％	2295.04855632
4	1772	SEQ ID NO：7543	0.04％	1759.6656
					5	3087	SEQ ID NO：7544	0.03％	1215.76896
6	2339	SEQ ID NO：7545	0.02％	1116.29986272
					7	2308	SEQ ID NO：7546	0.02％	970.14776112
8	3061	SEQ ID NO：7547	0.02％	836.2525104
					9	2748	SEQ ID NO：7548	0.01％	726.706344
10	3837	SEQ ID NO：7549	0.01％	720.8292
					11	59	SEQ ID NO：7550	0.01％	650.3112
12	2877	SEQ ID NO：7551	0.01％	620.22996
					13	4114	SEQ ID NO：7552	0.01％	559.8936
14	805	SEQ ID NO：7553	0.01％	484.4565072
					15	1655	SEQ ID NO：7554	0.01％	437.48208
16	611	SEQ ID NO：7555	0.00％	319.9392
					17	1961	SEQ ID NO：7556	0.00％	305.94186
18	1223	SEQ ID NO：7557	0.00％	289.08792
					19	852	SEQ ID NO：7558	0.00％	285.67242
20	2139	SEQ ID NO：7559	0.00％	284.845869

HLA A 1101-9 mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	4200	SEQ ID NO：7560	50％	18
2	281	SEQ ID NO：7561	25％	9
					3	3236	SEQ ID NO：7562	25％	9
4	509	SEQ ID NO：7563	16.66％	6
					5	848	SEQ ID NO：7564	16.66％	6
6	2193	SEQ ID NO：7565	16.66％	6
					7	3542	SEQ ID NO：7566	16.66％	6
8	541	SEQ ID NO：7567	15％	5.4
					9	1748	SEQ ID NO：7568	12.5％	4.5
10	829	SEQ ID NO：7569	11.11％	4

11	1149	SEQ ID NO：7570	11.11％	4
					12	2027	SEQ ID NO：7571	11.11％	4
13	2576	SEQ ID NO：7572	11.11％	4
					14	873	SEQ ID NO：7573	8.33％	3
15	2725	SEQ ID NO：7574	8.33％	3
					16	3541	SEQ ID NO：7575	8.33％	3
17	1837	SEQ ID NO：7576	7.5％	2.7
					18	2475	SEQ ID NO：7577	7.5％	2.7
19	2703	SEQ ID NO：7578	7.5％	2.7
					20	1823	SEQ ID NO：7579	6.66％	2.4

HLA A 1101-10 mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	3541	SEQ ID NO：7580	50％	18
2	281	SEQ ID NO：7581	25％	9
					3	1495	SEQ ID NO：7582	25％	9
4	2303	SEQ ID NO：7583	25％	9
					5	2616	SEQ ID NO：7584	25％	9
6	48	SEQ ID NO：7585	16.66％	6
					7	1394	SEQ ID NO：7586	16.66％	6
8	1499	SEQ ID NO：7587	16.66％	6
					9	1862	SEQ ID NO：7588	16.66％	6
10	1163	SEQ ID NO：7589	11.11％	4
					11	4006	SEQ ID NO：7590	11.11％	4
12	4344	SEQ ID NO：7591	11.11％	4
					13	633	SEQ ID NO：7592	10％	3.6
14	119	SEQ ID NO：7593	8.33％	3
					15	1190	SEQ ID NO：7594	8.33％	3
16	1195	SEQ ID NO：7595	8.33％	3
					17	1725	SEQ ID NO：7596	8.33％	3
18	2728	SEQ ID NO：7597	8.33％	3
					19	2895	SEQ ID NO：7598	8.33％	3
20	3033	SEQ ID NO：7599	8.33％	3

HLA B7-9 mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1335	SEQ ID NO：7600	4.44％	240
2	2580	SEQ ID NO：7601	4.44％	240
					3	1703	SEQ ID NO：7602	3.70％	200
4	113	SEQ ID NO：7603	2.22％	120

5	168	SEQ ID NO：7604	2.22％	120
					6	2842	SEQ ID NO：7605	2.22％	120
7	4027	SEQ ID NO：7606	2.22％	120
					8	3680	SEQ ID NO：7607	1.66％	90
9	2085	SEQ ID NO：7608	1.48％	80
					10	2492	SEQ ID NO：7609	1.48％	80
11	2660	SEQ ID NO：7610	1.48％	80
					12	2906	SEQ ID NO：7611	1.48％	80
13	3346	SEQ ID NO：7612	1.48％	80
					14	4038	SEQ ID NO：7613	1.48％	80
15	1163	SEQ ID NO：7614	1.11％	60
					16	1457	SEQ ID NO：7615	1.11％	60
17	2351	SEQ ID NO：7616	1.11％	60
					18	2471	SEQ ID NO：7617	1.11％	60
19	3499	SEQ ID NO：7618	1.11％	60
					20	3635	SEQ ID NO：7619	1.11％	60

HLA B7-10 mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1703	SEQ ID NO：7620	3.70％	200
2	17	SEQ ID NO：7621	2.22％	120
					3	3008	SEQ ID NO：7622	2.22％	120
4	4106	SEQ ID NO：7623	2.22％	120
					5	3450	SEQ ID NO：7624	1.66％	90
6	113	SEQ ID NO：7625	1.48％	80
					7	195	SEQ ID NO：7626	1.48％	80
8	307	SEQ ID NO：7627	1.48％	80
					9	780	SEQ ID NO：7628	1.48％	80
10	1000	SEQ ID NO：7629	1.48％	80
					11	1072	SEQ ID NO：7630	1.48％	80
12	1404	SEQ ID NO：7631	1.48％	80
					13	1980	SEQ ID NO：7632	1.48％	80
14	2262	SEQ ID NO：7633	1.48％	80
					15	2543	SEQ ID NO：7634	1.48％	80
16	2906	SEQ ID NO：7635	1.48％	80
					17	3077	SEQ ID NO：7636	1.48％	80
18	3175	SEQ ID NO：7637	1.48％	80
					19	4195	SEQ ID NO：7638	1.48％	80
20	4251	SEQ ID NO：7639	1.48％	80

Table 14: SEQ ID NO: 6040 epitope

HLA A1-9 mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	20	SEQ ID NO：7640	0.04％	2.25
2	91	SEQ ID NO：7641	0.01％	1
					3	125	SEQ ID NO：7642	0.01％	0.75
4	56	SEQ ID NO：7643	0.00％	0.5
					5	145	SEQ ID NO：7644	0.00％	0.5

HLA A1-10 mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	20	SEQ ID NO：7645	0.01％	0.9
2	56	SEQ ID NO：7646	0.00％	0.5
					3	71	SEQ ID NO：7647	0.00％	0.5
4	144	SEQ ID NO：7648	0.00％	0.5

HLA A3-9 mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	115	SEQ ID NO：7649	0.24％	30
2	87	SEQ ID NO：7650	0.04％	6
					3	80	SEQ ID NO：7651	0.03％	4.05
4	125	SEQ ID NO：7652	0.01％	1.8
					5	39	SEQ ID NO：7653	0.01％	1.5
6	56	SEQ ID NO：7654	0.01％	1.5
					7	135	SEQ ID NO：7655	0.00％	1.2
8	91	SEQ ID NO：7656	0.00％	1
					9	119	SEQ ID NO：7657	0.00％	1
10	141	SEQ ID NO：7658	0.00％	0.9
					11	150	SEQ ID NO：7659	0.00％	0.6
12	137	SEQ ID NO：7660	0.00％	0.54

HLA A3-10 mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	36	SEQ ID NO：7661	0.24％	30
2	144	SEQ ID NO：7662	0.06％	8
					3	101	SEQ ID NO：7663	0.03％	4
4	99	SEQ ID NO：7664	0.02％	3.6
					5	80	SEQ ID NO：7665	0.02％	2.7
6	125	SEQ ID NO：7666	0.01％	1.6875

7	71	SEQ ID NO：7667	0.01％	1.5
					8	118	SEQ ID NO：7668	0.01％	1.5
9	40	SEQ ID NO：7669	0.01％	1.35
					10	5	SEQ ID NO：7670	0.00％	0.9
11	56	SEQ ID NO：7671	0.00％	0.9
					12	107	SEQ ID NO：7672	0.00％	0.6
13	135	SEQ ID NO：7673	0.00％	0.6
					14	141	SEQ ID NO：7674	0.00％	0.6
15	148	SEQ ID NO：7675	0.00％	0.6
					16	116	SEQ ID NO：7676	0.00％	0.5

HLA A24-9 mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	153	SEQ ID NO：7677	1.05％	16.8
2	80	SEQ ID NO：7678	0.75％	12
					3	123	SEQ ID NO：7679	0.50％	8
4	137	SEQ ID NO：7680	0.50％	8
					5	9	SEQ ID NO：7681	0.45％	7.2
6	77	SEQ ID NO：7682	0.45％	7.2
					7	112	SEQ ID NO：7683	0.45％	7.2
8	73	SEQ ID NO：7684	0.41％	6.6
					9	32	SEQ ID NO：7685	0.37％	6
10	110	SEQ ID NO：7686	0.37％	6
					11	140	SEQ ID NO：7687	0.37％	6
12	143	SEQ ID NO：7688	0.37％	6
					13	18	SEQ ID NO：7689	0.30％	4.8
14	54	SEQ ID NO：7690	0.30％	4.8
					15	108	SEQ ID NO：7691	0.30％	4.8
16	141	SEQ ID NO：7692	0.30％	4.8
					17	92	SEQ ID NO：7693	0.27％	4.4
18	33	SEQ ID NO：7694	0.25％	4
					19	49	SEQ ID NO：7695	0.25％	4
20	111	SEQ ID NO：7696	0.25％	4

HLA A24-10 mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	142	SEQ ID NO：7697	12.52％	200
2	110	SEQ ID NO：7698	0.75％	12
					3	99	SEQ ID NO：7699	0.50％	8
4	8	SEQ ID NO：7700	0.45％	7.2

5	140	SEQ ID NO：7701	0.45％	7.2
					6	32	SEQ ID NO：7702	0.37％	6
7	17	SEQ ID NO：7703	0.30％	4.8
					8	53	SEQ ID NO：7704	0.30％	4.8
9	76	SEQ ID NO：7705	0.30％	4.8
					10	107	SEQ ID NO：7706	0.30％	4.8
11	111	SEQ ID NO：7707	0.30％	4.8
					12	72	SEQ ID NO：7708	0.27％	4.4
13	91	SEQ ID NO：7709	0.27％	4.4
					14	31	SEQ ID NO：7710	0.25％	4
15	127	SEQ ID NO：7711	0.25％	4
					16	139	SEQ ID NO：7712	0.25％	4
17	80	SEQ ID NO：7713	0.22％	3.6
					18	38	SEQ ID NO：7714	0.18％	3
19	118	SEQ ID NO：7715	0.18％	3
					20	49	SEQ ID NO：7716	0.12％	2

HLA A 0201-9 mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	Accounting for maximum scorePercentage of	Score recording
					1	80	SEQ ID NO：7717	0.00％	171.96732
2	147	SEQ ID NO：7718	0.00％	51.46848
					3	143	SEQ ID NO：7719	0.00％	11.6146182
4	56	SEQ ID NO：7720	0.00％	11.304684
					5	10	SEQ ID NO：7721	0.00％	10.34586
6	6	SEQ ID NO：7722	0.00％	6.56830734
					7	26	SEQ ID NO：7723	0.00％	6.07614
8	141	SEQ ID NO：7724	0.00％	5.981472
					9	148	SEQ ID NO：7725	0.00％	5.194044
10	9	SEQ ID NO：7726	0.00％	4.299183
					11	137	SEQ ID NO：7727	0.00％	4.299183
12	130	SEQ ID NO：7728	0.00％	4.138344
					13	84	SEQ ID NO：7729	0.00％	3.42792
14	27	SEQ ID NO：7730	0.00％	3.383484
					15	2	SEQ ID NO：7731	0.00％	3.381
16	62	SEQ ID NO：7732	0.00％	3.251556
					17	23	SEQ ID NO：7733	0.00％	2.9542005
18	99	SEQ ID NO：7734	0.00％	1982232
					19	33	SEQ ID NO：7735	0.00％	1.86921
20	111	SEQ ID NO：7736	0.00％	1.76402985

HLA A 0201-10 mers

Maximum possible score Using this molecular type				3925227.1
					Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
1	5	SEQ ID NO：7737	0.00％	159.9696
					2	25	SEQ ID NO：7738	0.00％	69.552
3	80	SEQ ID NO：7739	0.00％	36.5148
					4	107	SEQ ID NO：7740	0.00％	21.3624
5	148	SEQ ID NO：7741	0.00％	17.73576
					6	61	SEQ ID NO：7742	0.00％	13.9104
7	147	SEQ ID NO：7743	0.00％	11.304684
					8	53	SEQ ID NO：7744	0.00％	8.230458
9	17	SEQ ID NO：7745	0.00％	7.3086111
					10	110	SEQ ID NO：7746	0.00％	6.174104475
11	9	SEQ ID NO：7747	0.00％	6.0858
					12	99	SEQ ID NO：7748	0.00％	5.6823984
13	2	SEQ ID NO：7749	0.00％	3.188283
					14	41	SEQ ID NO：7750	0.00％	2.206413
15	135	SEQ ID NO：7751	0.00％	2.076624
					16	76	SEQ ID NO：7752	0.00％	2.005692
17	23	SEQ ID NO：7753	0.00％	1.798209
					18	40	SEQ ID NO：7754	0.00％	1.68996456
19	39	SEQ ID NO：7755	0.00％	1.516482
					20	118	SEQ ID NO：7756	0.00％	1.2683304

HLA A 1101-9 mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	91	SEQ ID NO：7757	2.77％	1

HLA A 1101-10 mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Note the bookIs divided into
					1	101	SEQ ID NO：7758	33.33％	12
2	71	SEQ ID NO：7759	2.77％	1
					3	90	SEQ ID NO：7760	1.66％	0.6

HLA B7-9 mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	49	SEQ ID NO：7761	2.22％	120
2	9	SEQ ID NO：7762	1.11％	60
					3	73	SEQ ID NO：7763	0.66％	36
4	33	SEQ ID NO：7764	0.37％	20

5	137	SEQ ID NO：7765	0.37％	20
					6	141	SEQ ID NO：7766	0.37％	20
7	77	SEQ ID NO：7767	0.22％	12
					8	112	SEQ ID NO：7768	0.22％	12
9	143	SEQ ID NO：7769	0.22％	12
					10	81	SEQ ID NO：7770	0.14％	8
11	13	SEQ ID NO：7771	0.09％	5
					12	69	SEQ ID NO：7772	0.09％	5
13	18	SEQ ID NO：7773	0.07％	4
					14	32	SEQ ID NO：7774	0.07％	4
15	54	SEQ ID NO：7775	0.07％	4
					16	80	SEQ ID NO：7776	0.07％	4
17	92	SEQ ID NO：7777	0.07％	4
					18	108	SEQ ID NO：7778	0.07％	4
19	111	SEQ ID NO：7779	0.07％	4
					20	123	SEQ ID NO：7780	0.07％	4

HLA B7-10 mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	99	SEQ ID NO：7781	0.74％	40
2	17	SEQ ID NO：7782	0.37％	20
					3	8	SEQ ID NO：7783	0.22％	12
4	72	SEQ ID NO：7784	0.22％	12
					5	91	SEQ ID NO：7785	0.22％	12
6	127	SEQ ID NO：7786	0.11％	6
					7	31	SEQ ID NO：7787	0.07％	4
8	32	SEQ ID NO：7788	0.07％	4
					9	53	SEQ ID NO：7789	0.07％	4
10	76	SEQ ID NO：7790	0.07％	4
					11	107	SEQ ID NO：7791	0.07％	4
12	110	SEQ ID NO：7792	0.07％	4
					13	111	SEQ ID NO：7793	0.07％	4
14	140	SEQ ID NO：7794	0.07％	4
					15	9	SEQ ID NO：7795	0.05％	3
16	19	SEQ ID NO：7796	0.05％	3
					17	33	SEQ ID NO：7797	0.03％	2
18	93	SEQ ID NO：7798	0.03％	2
					19	102	SEQ ID NO：7799	0.03％	2
20	129	SEQ ID NO：7800	0.02％	1.5

Table 15: SEQ ID NO: 6041 epitope

HLA A1-9 mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1818	SEQ ID NO：7801	1.6％	90
2	373	SEQ ID NO：7802	1.33％	75
					3	681	SEQ ID NO：7803	1.33％	75
4	74	SEQ ID NO：7804	0.88％	50
					5	786	SEQ ID NO：7805	0.88％	50
6	1495	SEQ ID NO：7806	0.88％	50
					7	88	SEQ ID NO：7807	0.8％	45
8	357	SEQ ID NO：7808	0.8％	45
					9	1271	SEQ ID NO：7809	0.8％	45
10	1799	SEQ ID NO：7810	0.8％	45
					11	1393	SEQ ID NO：7811	0.48％	27
12	386	SEQ ID NO：7812	0.44％	25
					13	2304	SEQ ID NO：7813	0.44％	25
14	198	SEQ ID NO：7814	0.4％	22.5
					15	840	SEQ ID NO：7815	0.4％	22.5
16	2359	SEQ ID NO：7816	0.4％	22.5
					17	1194	SEQ ID NO：7817	0.32％	18
18	1546	SEQ ID NO：7818	0.32％	18
					19	2200	SEQ ID NO：7819	0.22％	12.5
20	996	SEQ ID NO：7820	0.2％	11.25

HLA A1-10 mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	995	SEQ ID NO：7821	10％	562.5
2	1303	SEQ ID NO：7822	2.22％	125
					3	1582	SEQ ID NO：7823	2％	112.5
4	1456	SEQ ID NO：7824	1.6％	90
					5	772	SEQ ID NO：7825	1.11％	62.5
6	181	SEQ ID NO：7826	0.88％	50
					7	632	SEQ ID NO：7827	0.88％	50
8	2281	SEQ ID NO：7828	0.88％	50
					9	1586	SEQ ID NO：7829	0.8％	45
10	2109	SEQ ID NO：7830	0.8％	45
					11	745	SEQ ID NO：7831	0.55％	31.25
12	1916	SEQ ID NO：7832	0.53％	30
					13	966	SEQ ID NO：7833	0.44％	25
14	1387	SEQ ID NO：7834	0.44％	25
					15	2263	SEQ ID NO：7835	0.44％	25

16	2457	SEQ ID NO：7836	0.26％	15
					17	1057	SEQ ID NO：7837	0.22％	12.5
18	2562	SEQ ID NO：7838	0.22％	12.5
					19	74	SEQ ID NO：7839	0.17％	10
20	298	SEQ ID NO：7840	0.17％	10

HLA A3-9 mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	536	SEQ ID NO：7841	3.33％	405
2	986	SEQ ID NO：7842	2.46％	300
					3	805	SEQ ID NO：7843	1.64％	200
4	2345	SEQ ID NO：7844	1.48％	180
					5	2481	SEQ ID NO：7845	0.55％	67.5
6	204	SEQ ID NO：7846	0.49％	60
					7	895	SEQ ID NO：7847	0.44％	54
8	1512	SEQ ID NO：7848	0.44％	54
					9	2491	SEQ ID NO：7849	0.37％	45
10	436	SEQ ID NO：7850	0.32％	40
					11	917	SEQ ID NO：7851	0.32％	40
12	1176	SEQ ID NO：7852	0.32％	40
					13	1517	SEQ ID NO：7853	0.29％	36
14	466	SEQ ID NO：7854	0.24％	30
					15	1784	SEQ ID NO：7855	0.24％	30
16	2039	SEQ ID NO：7856	0.24％	30
					17	2124	SEQ ID NO：7857	0.24％	30
18	1049	SEQ ID NO：7858	0.22％	27
					19	2200	SEQ ID NO：7859	0.22％	27
20	2598	SEQ ID NO：7860	0.22％	27

HLA A3-10 mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	392	SEQ ID NO：7861	2.46％	300
2	2230	SEQ ID NO：7862	1.48％	180
					3	590	SEQ ID NO：7863	1.11％	135
4	697	SEQ ID NO：7864	1.11％	135
					5	919	SEQ ID NO：7865	0.74％	90
6	1354	SEQ ID NO：7866	0.74％	90
					7	1430	SEQ ID NO：7867	0.74％	90
8	2534	SEQ ID NO：7868	0.74％	90
					9	202	SEQ ID NO：7869	0.49％	60

10	488	SEQ ID NO：7870	0.49％	60
					11	922	SEQ ID NO：7871	0.49％	60
12	1735	SEQ ID NO：7872	0.49％	60
					13	2281	SEQ ID NO：7873	0.49％	60
14	1894	SEQ ID NO：7874	0.44％	54
					15	2552	SEQ ID NO：7875	0.44％	54
16	555	SEQ ID NO：7876	0.37％	45
					17	1134	SEQ ID NO：7877	0.37％	45
18	1149	SEQ ID NO：7878	0.29％	36
					19	283	SEQ ID NO：7879	0.24％	30
20	917	SEQ ID NO：7880	0.24％	30

HLA A24-9 mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	2375	SEQ ID NO：7881	36.07％	576
2	1751	SEQ ID NO：7882	28.93％	462
					3	195	SEQ ID NO：7883	25.05％	400
4	2306	SEQ ID NO：7884	21.04％	336
					5	806	SEQ ID NO：7885	20.66％	330
6	1252	SEQ ID NO：7886	18.78％	300
					7	160	SEQ ID NO：7887	15.03％	240
8	517	SEQ ID NO：7888	15.03％	240
					9	375	SEQ ID NO：7889	12.52％	200
10	1275	SEQ ID NO：7890	12.52％	200
					11	2175	SEQ ID NO：7891	12.52％	200
12	2207	SEQ ID NO：7892	12.52％	200
					13	2343	SEQ ID NO：7893	12.52％	200
14	443	SEQ ID NO：7894	11.27％	180
					15	668	SEQ ID NO：7895	7.51％	120
16	1825	SEQ ID NO：7896	6.88％	110
					17	1690	SEQ ID NO：7897	4.69％	75
18	159	SEQ ID NO：7898	3.75％	60
					19	2550	SEQ ID NO：7899	3.75％	60
20	1949	SEQ ID NO：7900	3.38％	54

HLA A24-10 mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	641	SEQ ID NO：7901	45.09％	720
2	809	SEQ ID NO：7902	24.80％	396
					3	1209	SEQ ID NO：7903	22.54％	360

4	216	SEQ ID NO：7904	18.03％	288
					5	159	SEQ ID NO：7905	15.03％	240
6	528	SEQ ID NO：7906	15.03％	240
					7	799	SEQ ID NO：7907	15.03％	240
8	1436	SEQ ID NO：7908	15.03％	240
					9	2219	SEQ ID NO：7909	15.03％	240
10	1065	SEQ ID NO：7910	13.77％	220
					11	1953	SEQ ID NO：7911	13.15％	210
12	1966	SEQ ID NO：7912	12.52％	200
					13	2600	SEQ ID NO：7913	12.52％	200
14	71	SEQ ID NO：7914	9.39％	150
					15	380	SEQ ID NO：7915	9.39％	150
16	1989	SEQ ID NO：7916	9.39％	150
					17	342	SEQ ID NO：7917	8.76％	140
18	1071	SEQ ID NO：7918	8.76％	140
					19	2570	SEQ ID NO：7919	6.88％	110
20	2550	SEQ ID NO：7920	6.26％	100

HLA A 0201-9 mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1632	SEQ ID NO：7921	0.09％	3607.31448
2	1640	SEQ ID NO：7922	0.04％	1748.2560912
					3	1776	SEQ ID NO：7923	0.03％	1492.58592
4	2512	SEQ ID NO：7924	0.03％	1434.16845
					5	1073	SEQ ID NO：7925	0.03％	1338.876
6	230	SEQ ID NO：7926	0.01％	685.78272
					7	1001	SEQ ID NO：7927	0.01％	559.8936
8	716	SEQ ID NO：7928	0.01％	558.27486
					9	2280	SEQ ID NO：7929	0.01％	511.19781048
10	590	SEQ ID NO：7930	0.01％	469.6692
					11	664	SEQ ID NO：7931	0.01％	442.076389524
12	1094	SEQ ID NO：7932	0.00％	382.536
					13	1735	SEQ ID NO：7933	0.00％	382.536
14	1625	SEQ ID NO：7934	0.00％	342.4606344
					15	1974	SEQ ID NO：7935	0.00％	336.885048
16	2382	SEQ ID NO：7936	0.00％	319.9392
					17	2417	SEQ ID NO：7937	0.00％	319.9392
18	744	SEQ ID NO：7938	0.00％	256.416670125
					19	108	SEQ ID NO：7939	0.00％	232.52724
20	390	SEQ ID NO：7940	0.00％	228.0411084

HLA A 0201-10 mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	2511	SEQ ID NO：7941	0.38％	15126.90795
2	1608	SEQ ID NO：7942	0.05％	2049.4656
					3	2572	SEQ ID NO：7943	0.04％	1879.5921264
4	255	SEQ ID NO：7944	0.03％	1566.6522795
					5	895	SEQ ID NO：7945	0.03％	1338.876
6	1171	SEQ ID NO：7946	0.02％	1107.960876
					7	1691	SEQ ID NO：7947	0.01％	782.95521024
8	20	SEQ ID NO：7948	0.01％	549.9372312
					9	1632	SEQ ID NO：7949	0.01％	479.041993296
10	2280	SEQ ID NO：7950	0.01％	472.418344576987
					11	1963	SEQ ID NO：7951	0.00％	358.73928
12	1955	SEQ ID NO：7952	0.00％	331.093464
					13	741	SEQ ID NO：7953	0.00％	318.652488
14	523	SEQ ID NO：7954	0.00％	278.7876
					15	1073	SEQ ID NO：7955	0.00％	266.6988828
16	2489	SEQ ID NO：7956	0.00％	243.432
					17	777	SEQ ID NO：7957	0.00％	218.5730664
18	1737	SEQ ID NO：7958	0.00％	218.0785572
					19	589	SEQ ID NO：7959	0.00％	210.538251
20	229	SEQ ID NO：7960	0.00％	205.230564

HLA A 1101-9 mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	2337	SEQ ID NO：7961	33.33％	12
2	2156	SEQ ID NO：7962	25％	9
					3	492	SEQ ID NO：7963	20％	7.2
4	18	SEQ ID NO：7964	16.66％	6
					5	332	SEQ ID NO：7965	16.66％	6
6	415	SEQ ID NO：7966	16.66％	6
					7	2479	SEQ ID NO：7967	16.66％	6
8	1495	SEQ ID NO：7968	11.11％	4
					9	2035	SEQ ID NO：7969	11.11％	4
10	1349	SEQ ID NO：7970	10％	3.6
					11	1194	SEQ ID NO：7971	8.33％	3
12	1648	SEQ ID NO：7972	8.33％	3
					13	96	SEQ ID NO：7973	6.66％	2.4
14	764	SEQ ID NO：7974	6.66％	2.4
					15	986	SEQ ID NO：7975	6.66％	2.4

16	2345	SEQ ID NO：7976	6.66％	2.4
					17	698	SEQ ID NO：7977	5.55％	2
18	1355	SEQ ID NO：7978	5.55％	2
					19	1987	SEQ ID NO：7979	5.55％	2
20	2085	SEQ ID NO：7980	5.55％	2

HLA A 1101-10 mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	2083	SEQ ID NO：7981	33.33％	12
2	2123	SEQ ID NO：7982	25％	9
					3	2147	SEQ ID NO：7983	16.66％	6
4	331	SEQ ID NO：7984	12.5％	4.5
					5	1035	SEQ ID NO：7985	11.11％	4
6	1064	SEQ ID NO：7986	11.11％	4
					7	2154	SEQ ID NO：7987	11.11％	4
8	1048	SEQ ID NO：7988	7.5％	2.7
					9	202	SEQ ID NO：7989	6.66％	2.4
10	721	SEQ ID NO：7990	6.66％	2.4
					11	2109	SEQ ID NO：7991	6.66％	2.4
12	2230	SEQ ID NO：7992	6.66％	2.4
					13	1306	SEQ ID NO：7993	5.55％	2
14	1622	SEQ ID NO：7994	5.55％	2
					15	1772	SEQ ID NO：7995	5.55％	2
16	1796	SEQ ID NO：7996	5.55％	2
					17	186	SEQ ID NO：7997	5％	1.8
18	414	SEQ ID NO：7998	5％	1.8
					19	697	SEQ ID NO：7999	5％	1.8
20	1175	SEQ ID NO：8000	5％	1.8

HLA B7-9 mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1447	SEQ ID NO：8001	14.81％	800
2	642	SEQ ID NO：8002	3.70％	200
					3	34	SEQ ID NO：8003	2.22％	120
4	186	SEQ ID NO：8004	1.48％	80
					5	244	SEQ ID NO：8005	1.48％	80
6	459	SEQ ID NO：8006	1.48％	80
					7	1475	SEQ ID NO：8007	1.48％	80
8	1867	SEQ ID NO：8008	1.48％	80
					9	2032	SEQ ID NO：8009	1.48％	80

10	2047	SEQ ID NO：8010	1.48％	80
					11	2335	SEQ ID NO：8011	1.48％	80
12	622	SEQ ID NO：8012	1.11％	60
					13	1375	SEQ ID NO：8013	1.11％	60
14	1617	SEQ ID NO：8014	0.92％	50
					15	1023	SEQ ID NO：8015	0.83％	45
16	286	SEQ ID NO：8016	0.74％	40
					17	490	SEQ ID NO：8017	0.74％	40
18	810	SEQ ID NO：8018	0.74％	40
					19	1420	SEQ ID NO：8019	0.74％	40
20	1854	SEQ ID NO：8020	0.74％	40

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1617	SEQ ID NO：8021	3.70％	200
2	752	SEQ ID NO：8022	2.22％	120
					3	1552	SEQ ID NO：8023	2.22％	120
4	154	SEQ ID NO：8024	1.48％	80
					5	165	SEQ ID NO：8025	1.48％	80
6	383	SEQ ID NO：8026	1.48％	80
					7	1501	SEQ ID NO：8027	1.48％	80
8	2093	SEQ ID NO：8028	1.48％	80
					9	2564	SEQ ID NO：8029	1.48％	80
10	622	SEQ ID NO：8030	1.11％	60
					11	1086	SEQ ID NO：8031	1.11％	60
12	1262	SEQ ID NO：8032	1.11％	60
					13	1556	SEQ ID NO：8033	1.11％	60
14	845	SEQ ID NO：8034	1％	54
					15	286	SEQ ID NO：8035	0.74％	40
16	490	SEQ ID NO：8036	0.74％	40
					17	552	SEQ ID NO：8037	0.74％	40
18	1858	SEQ ID NO：8038	0.74％	40
					19	2107	SEQ ID NO：8039	0.74％	40
20	2582	SEQ ID NO：8040	0.74％	40

Table 16: SEQ ID NO: 6042 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	846	SEQ ID NO：8041	2.22％	125

2	798	SEQ ID NO：8042	1.6％	90
					3	787	SEQ ID NO：8043	0.88％	50
4	1178	SEQ ID NO：8044	0.88％	50
					5	637	SEQ ID NO：8045	0.8％	45
6	557	SEQ ID NO：8046	0.44％	25
					7	1020	SEQ ID NO：8047	0.44％	25
8	282	SEQ ID NO：8048	0.32％	18
					9	1241	SEQ ID NO：8049	0.24％	13.5
10	466	SEQ ID NO：8050	0.22％	12.5
					11	727	SEQ ID NO：8051	0.2％	11.25
12	706	SEQ ID NO：8052	0.17％	10
					13	324	SEQ ID NO：8053	0.16％	9
14	752	SEQ ID NO：8054	0.16％	9
					15	54	SEQ ID NO：8055	0.13％	7.5
16	554	SEQ ID NO：8056	0.13％	7.5
					17	590	SEQ ID NO：8057	0.12％	6.75
18	569	SEQ ID NO：8058	0.08％	5
					19	613	SEQ ID NO：8059	0.08％	5
20	90	SEQ ID NO：8060	0.08％	4.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1241	SEQ ID NO：8061	4.8％	270
2	967	SEQ ID NO：8062	0.8％	45
					3	1010	SEQ ID NO：8063	0.48％	27
4	426	SEQ ID NO：8064	0.44％	25
					5	809	SEQ ID NO：8065	0.44％	25
6	1178	SEQ ID NO：8066	0.44％	25
					7	787	SEQ ID NO：8067	0.22％	12.5
8	958	SEQ ID NO：8068	0.22％	12.5
					9	727	SEQ ID NO：8069	0.2％	11.25
10	610	SEQ ID NO：8070	0.17％	10
					11	12	SEQ ID NO：8071	0.13％	7.5
12	1181	SEQ ID NO：8072	0.12％	6.75
					13	373	SEQ ID NO：8073	0.11％	6.25
14	602	SEQ ID NO：8074	0.11％	6.25
					15	20	SEQ ID NO：8075	0.04％	2.5
16	32	SEQ ID NO：8076	0.04％	2.5
					17	53	SEQ ID NO：8077	0.04％	2.5
18	400	SEQ ID NO：8078	0.04％	2.5
					19	557	SEQ ID NO：8079	0.04％	2.5

20

667

SEQ ID NO：8080

0.04％

2.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	768	SEQ ID NO：8081	0.82％	100
2	808	SEQ ID NO：8082	0.49％	60
					3	85	SEQ ID NO：8083	0.24％	30
4	663	SEQ ID NO：8084	0.24％	30
					5	1245	SEQ ID NO：8085	0.14％	18
6	288	SEQ ID NO：8086	0.09％	12
					7	50	SEQ ID NO：8087	0.08％	10
8	320	SEQ ID NO：8088	0.07％	9
					9	402	SEQ ID NO：8089	0.07％	9
10	798	SEQ ID NO：8090	0.07％	9
					11	902	SEQ ID NO：8091	0.06％	8.1
12	364	SEQ ID NO：8092	0.05％	6.75
					13	297	SEQ ID NO：8093	0.04％	6
14	992	SEQ ID NO：8094	0.04％	6
					15	38	SEQ ID NO：8095	0.03％	4.5
16	249	SEQ ID NO：8096	0.03％	4.5
					17	706	SEQ ID NO：8097	0.03％	4.05
18	1204	SEQ ID NO：8098	0.03％	4.05
					19	1178	SEQ ID NO：8099	0.03％	4
20	343	SEQ ID NO：8100	0.02％	3.6

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	255	SEQ ID NO：8101	1.48％	180
2	180	SEQ ID NO：8102	0.55％	67.5
					3	768	SEQ ID NO：8103	0.49％	60
4	1177	SEQ ID NO：8104	0.49％	60
					5	380	SEQ ID NO：8105	0.24％	30
6	100	SEQ ID NO：8106	0.18％	22.5
					7	786	SEQ ID NO：8107	0.16％	20
8	1217	SEQ ID NO：8108	0.16％	20
					9	207	SEQ ID NO：8109	0.14％	18
10	1183	SEQ ID NO：8110	0.14％	18
					11	38	SEQ ID NO：8111	0.09％	12
12	52	SEQ ID NO：8112	0.09％	12
					13	8	SEQ ID NO：8113	0.06％	8

14	679	SEQ ID NO：8114	0.06％	8
					15	73	SEQ ID NO：8115	0.05％	6.75
16	1204	SEQ ID NO：8116	0.05％	6.075
					17	50	SEQ ID NO：8117	0.04％	6
18	774	SEQ ID NO：8118	0.04％	6
					19	845	SEQ ID NO：8119	0.04％	6
20	214	SEQ ID NO：8120	0.04％	5.4

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1118	SEQ ID NO：8121	19.84％	316.8
2	51	SEQ ID NO：8122	18.78％	300
					3	161	SEQ ID NO：8123	18.78％	300
4	434	SEQ ID NO：8124	18.78％	300
					5	365	SEQ ID NO：8125	13.77％	220
6	736	SEQ ID NO：8126	12.52％	200
					7	620	SEQ ID NO：8127	7.51％	120
8	1068	SEQ ID NO：8128	7.51％	120
					9	817	SEQ ID NO：8129	3.75％	60
10	336	SEQ ID NO：8130	3.44％	55
					11	687	SEQ ID NO：8131	3.13％	50
12	254	SEQ ID NO：8132	2.34％	37.5
					13	627	SEQ ID NO：8133	1.87％	30
14	950	SEQ ID NO：8134	1.75％	28
					15	28	SEQ ID NO：8135	1.56％	25
16	408	SEQ ID NO：8136	1.56％	25
					17	159	SEQ ID NO：8137	1.31％	21
18	1166	SEQ ID NO：8138	1.26％	20.16
					19	45	SEQ ID NO：8139	1.25％	20
20	185	SEQ ID NO：8140	1.25％	20

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	438	SEQ ID NO：8141	27.55％	440
2	489	SEQ ID NO：8142	22.54％	360
					3	254	SEQ ID NO：8143	18.78％	300
4	354	SEQ ID NO：8144	11.27％	180
					5	406	SEQ ID NO：8145	11.27％	180
6	1047	SEQ ID NO：8146	11.27％	180
					7	473	SEQ ID NO：8147	7.51％	120

8	350	SEQ ID NO：8148	6.26％	100
					9	769	SEQ ID NO：8149	6.26％	100
10	193	SEQ ID NO：8150	5.63％	90
					11	479	SEQ ID NO：8151	3.13％	50
12	0	SEQ ID NO：8152	2.70％	43.2
					13	813	SEQ ID NO：8153	1.87％	30
14	739	SEQ ID NO：8154	1.50％	24
					15	782	SEQ ID NO：8155	1.50％	24
16	1186	SEQ ID NO：8156	1.31％	21
					17	910	SEQ ID NO：8157	1.05％	16.8
18	128	SEQ ID NO：8158	0.93％	15
					19	183	SEQ ID NO：8159	0.93％	15
20	1069	SEQ ID NO：8160	0.93％	15

HLA A0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	1041	SEQ ID NO：8161	0.01％	484.2379773
2	981	SEQ ID NO：8162	0.00％	382.536
					3	957	SEQ ID NO：8163	0.00％	342.4606344
4	896	SEQ ID NO：8164	0.00％	232.6931712
					5	1173	SEQ ID NO：8165	0.00％	201.447432
6	733	SEQ ID NO：8166	0.00％	171.86796
					7	410	SEQ ID NO：8167	0.00％	135.45252
8	786	SEQ ID NO：8168	0.00％	119.463012
					9	150	SEQ ID NO：8169	0.00％	102.17550222
10	1	SEQ ID NO：8170	0.00％	94.98737754
					11	595	SEQ ID NO：8171	0.00％	93.239424
12	1095	SEQ ID NO：8172	0.00％	89.41779
					13	1166	SEQ ID NO：8173	0.00％	87.58584
14	845	SEQ ID NO：8174	0.00％	79.642008
					15	734	SEQ ID NO：8175	0.00％	73.47672
16	802	SEQ ID NO：8176	0.00％	71.872056
					17	1213	SEQ ID NO：8177	0.00％	71.872056
18	105	SEQ ID NO：8178	0.00％	50.232
					19	939	SEQ ID NO：8179	0.00％	49.13352
20	130	SEQ ID NO：8180	0.00％	48.732354

HLA A0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	372	SEQ ID NO：8181	0.04％	1896.33528

2	410	SEQ ID NO：8182	0.02％	1134.00849744
					3	162	SEQ ID NO：8183	0.01％	685.3897512
4	1076	SEQ ID NO：8184	0.01％	640.90320525
					5	1196	SEQ ID NO：8185	0.01％	623.742666372
6	353	SEQ ID NO：8186	0.01％	446.7384576
					7	50	SEQ ID NO：8187	0.00％	375.97824
8	733	SEQ ID NO：8188	0.00％	271.863864
					9	130	SEQ ID NO：8189	0.00％	235.6873848
10	415	SEQ ID NO：8190	0.00％	185.679
					11	297	SEQ ID NO：8191	0.00％	177.496704
12	1	SEQ ID NO：8192	0.00％	152.42160582
					13	56	SEQ ID NO：8193	0.00％	110.013876
14	732	SEQ ID NO：8194	0.00％	101.0988
					15	6	SEQ ID NO：8195	0.00％	98.26704
16	261	SEQ ID NO：8196	0.00％	91.60164
					17	1040	SEQ ID NO：8197	0.00％	76.98537
18	928	SEQ ID NO：8198	0.00％	71.2908
					19	1188	SEQ ID NO：8199	0.00％	69.81282
20	1094	SEQ ID NO：8200	0.00％	52.5987

HLA A1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	402	SEQ ID NO：8201	25％	9
2	902	SEQ ID NO：8202	22.5％	8.1
					3	288	SEQ ID NO：8203	11.11％	4
4	85	SEQ ID NO：8204	6.66％	2.4
					5	706	SEQ ID NO：8205	6.66％	2.4
6	456	SEQ ID NO：8206	5.55％	2
					7	920	SEQ ID NO：8207	5.55％	2
8	535	SEQ ID NO：8208	5％	1.8
					9	364	SEQ ID NO：8209	3.33％	1.2
10	438	SEQ ID NO：8210	3.33％	1.2
					11	798	SEQ ID NO：8211	3.33％	1.2
12	808	SEQ ID NO：8212	3.33％	1.2
					13	937	SEQ ID NO：8213	3.33％	1.2
14	956	SEQ ID NO：8214	3.33％	1.2
					15	557	SEQ ID NO：8215	2.77％	1
16	1218	SEQ ID NO：8216	2.77％	1
					17	784	SEQ ID NO：8217	2.5％	0.9
18	249	SEQ ID NO：8218	2.22％	0.8
					19	768	SEQ ID NO：8219	2.22％	0.8

20

1178

SEQ ID NO：8220

2.22％

0.8

HLA A1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	38	SEQ ID NO：8221	13.33％	4.8
2	807	SEQ ID NO：8222	12.5％	4.5
					3	100	SEQ ID NO：8223	11.11％	4
4	380	SEQ ID NO：8224	11.11％	4
					5	767	SEQ ID NO：8225	10％	3.6
6	533	SEQ ID NO：8226	8.33％	3
					7	967	SEQ ID NO：8227	6.66％	2.4
8	919	SEQ ID NO：8228	5.55％	2
					9	305	SEQ ID NO：8229	5％	1.8
10	211	SEQ ID NO：8230	3.33％	1.2
					11	511	SEQ ID NO：8231	3.33％	1.2
12	1177	SEQ ID NO：8232	3.33％	1.2
					13	429	SEQ ID NO：8233	2.77％	1
14	758	SEQ ID NO：8234	2.77％	1
					15	797	SEQ ID NO：8235	2.5％	0.9
16	255	SEQ ID NO：8236	2.22％	0.8
					17	986	SEQ ID NO：8237	2.22％	0.8
18	1157	SEQ ID NO：8238	2.22％	0.8
					19	170	SEQ ID NO：8239	1.66％	0.6
20	893	SEQ ID NO：8240	1.66％	0.6

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	200	SEQ ID NO：8241	1.48％	80
2	1243	SEQ ID NO：8242	1.48％	80
					3	123	SEQ ID NO：8243	0.74％	40
4	248	SEQ ID NO：8244	0.66％	36
					5	1036	SEQ ID NO：8245	0.66％	36
6	494	SEQ ID NO：8246	0.37％	20
					7	495	SEQ ID NO：8247	0.37％	20
8	523	SEQ ID NO：8248	0.37％	20
					9	842	SEQ ID NO：8249	0.37％	20
10	932	SEQ ID NO：8250	0.37％	20
					11	274	SEQ ID NO：8251	0.33％	18
12	588	SEQ ID NO：8252	0.22％	12
					13	656	SEQ ID NO：8253	0.22％	12

14	657	SEQ ID NO：8254	0.22％	12
					15	767	SEQ ID NO：8255	0.22％	12
16	911	SEQ ID NO：8256	0.22％	12
					17	939	SEQ ID NO：8257	0.22％	12
18	1007	SEQ ID NO：8258	0.22％	12
					19	1170	SEQ ID NO：8259	0.22％	12
20	1206	SEQ ID NO：8260	0.22％	12

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	505	SEQ ID NO：8261	4.44％	240
2	312	SEQ ID NO：8262	3.70％	200
					3	141	SEQ ID NO：8263	1.11％	60
4	1006	SEQ ID NO：8264	0.66％	36
					5	411	SEQ ID NO：8265	0.44％	24
6	122	SEQ ID NO：8266	0.37％	20
					7	134	SEQ ID NO：8267	0.37％	20
8	184	SEQ ID NO：8268	0.37％	20
					9	367	SEQ ID NO：8269	0.37％	20
10	402	SEQ ID NO：8270	0.37％	20
					11	494	SEQ ID NO：8271	0.37％	20
12	560	SEQ ID NO：8272	0.37％	20
					13	626	SEQ ID NO：8273	0.37％	20
14	931	SEQ ID NO：8274	0.37％	20
					15	956	SEQ ID NO：8275	0.37％	20
16	1117	SEQ ID NO：8276	0.37％	20
					17	1169	SEQ ID NO：8277	0.37％	20
18	1196	SEQ ID NO：8278	0.37％	20
					19	247	SEQ ID NO：8279	0.22％	12
20	273	SEQ ID NO：8280	0.22％	12

Table 17: SEQ ID NO: 6043 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	168	SEQ ID NO：8281	0.2％	11.25
2	212	SEQ ID NO：8282	0.08％	4.5
					3	223	SEQ ID NO：8283	0.08％	4.5
4	104	SEQ ID NO：8284	0.04％	2.5
					5	170	SEQ ID NO：8285	0.04％	2.5

6	99	SEQ ID NO：8286	0.04％	2.25
					7	188	SEQ ID NO：8287	0.02％	1.35
8	180	SEQ ID NO：8288	0.02％	1.25
					9	219	SEQ ID NO：8289	0.02％	1.25
10	18	SEQ ID NO：8290	0.01％	1
					11	226	SEQ ID NO：8291	0.01％	1
12	98	SEQ ID NO：8292	0.01％	0.625
					13	151	SEQ ID NO：8293	0.01％	0.625
14	10	SEQ ID NO：8294	0.01％	0.6
					15	13	SEQ ID NO：8295	0.00％	0.5
16	32	SEQ ID NO：8296	0.00％	0.5
					17	70	SEQ ID NO：8297	0.00％	0.5
18	78	SEQ ID NO：8298	0.00％	0.5
					19	82	SEQ ID NO：8299	0.00％	0.5
20	145	SEQ ID NO：8300	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	99	SEQ ID NO：8301	0.8％	45
2	223	SEQ ID NO：8302	0.8％	45
					3	188	SEQ ID NO：8303	0.48％	27
4	206	SEQ ID NO：8304	0.2％	11.25
					5	253	SEQ ID NO：8305	0.17％	10
6	174	SEQ ID NO：8306	0.13％	7.5
					7	97	SEQ ID NO：8307	0.04％	2.5
8	257	SEQ ID NO：8308	0.04％	2.5
					9	179	SEQ ID NO：8309	0.04％	2.25
10	162	SEQ ID NO：8310	0.02％	1.25
					11	196	SEQ ID NO：8311	0.02％	1.25
12	219	SEQ ID NO：8312	0.02％	1.25
					13	18	SEQ ID NO：8313	0.01％	1
14	246	SEQ ID NO：8314	0.01％	1
					15	38	SEQ ID NO：8315	0.01％	0.75
16	33	SEQ ID NO：8316	0.00％	0.5
					17	69	SEQ ID NO：8317	0.00％	0.5
18	81	SEQ ID NO：8318	0.00％	0.5
					19	104	SEQ ID NO：8319	0.00％	0.5
20	116	SEQ ID NO：8320	0.00％	0.5

HLA A3-9mers
		Maximum possible score Using this molecular type	12150

Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
						1	104	SEQ ID NO：8321	0.98％	120
2	123	SEQ ID NO：8322	0.74％	90
					3	82	SEQ ID NO：8323	0.44％	54
4	106	SEQ ID NO：8324	0.11％	13.5
					5	99	SEQ ID NO：8325	0.08％	10.8
6	127	SEQ ID NO：8326	0.08％	10
					7	71	SEQ ID NO：8327	0.07％	9
8	1	SEQ ID NO：8328	0.06％	8.1
					9	113	SEQ ID NO：8329	0.04％	6
10	84	SEQ ID NO：8330	0.03％	4.5
					11	109	SEQ ID NO：8331	0.03％	4.05
12	58	SEQ ID NO：8332	0.02％	3
					13	138	SEQ ID NO：8333	0.02％	3
14	44	SEQ ID NO：8334	0.02％	2.7
					15	81	SEQ ID NO：8335	0.02％	2.7
16	226	SEQ ID NO：8336	0.02％	2.7
					17	184	SEQ ID NO：8337	0.01％	1.8
18	102	SEQ ID NO：8338	0.01％	1.215
					19	39	SEQ ID NO：8339	0.00％	1.2
20	234	SEQ ID NO：8340	0.00％	0.9

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	99	SEQ ID NO：8341	1.33％	162
2	81	SEQ ID NO：8342	0.44％	54
					3	104	SEQ ID NO：8343	0.24％	30
4	51	SEQ ID NO：8344	0.16％	20
					5	122	SEQ ID NO：8345	0.11％	13.5
6	71	SEQ ID NO：8346	0.07％	9
					7	69	SEQ ID NO：8347	0.04％	6
8	223	SEQ ID NO：8348	0.04％	5.4
					9	84	SEQ ID NO：8349	0.03％	4.5
10	63	SEQ ID NO：8350	0.02％	3.6
					11	138	SEQ ID NO：8351	0.02％	3
12	201	SEQ ID NO：8352	0.01％	1.8
					13	44	SEQ ID NO：8353	0.01％	1.35
14	83	SEQ ID NO：8354	0.01％	1.35
					15	116	SEQ ID NO：8355	0.00％	1.2
16	46	SEQ ID NO：8356	0.00％	0.9
					17	183	SEQ ID NO：8357	0.00％	0.81

18	57	SEQ ID NO：8358	0.00％	0.6
					19	93	SEQ ID NO：8359	0.00％	0.6
20	113	SEQ ID NO：8360	0.00％	0.6

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	198	SEQ ID NO：8361	13.15％	210
2	105	SEQ ID NO：8362	9.39％	150
					3	210	SEQ ID NO：8363	4.69％	75
4	75	SEQ ID NO：8364	3.15％	50.4
					5	85	SEQ ID NO：8365	2.63％	42
6	205	SEQ ID NO：8366	2.10％	33.6
					7	77	SEQ ID NO：8367	1.87％	30
8	158	SEQ ID NO：8368	0.65％	10.5
					9	103	SEQ ID NO：8369	0.56％	9
10	227	SEQ ID NO：8370	0.55％	8.8704
					11	32	SEQ ID NO：8371	0.54％	8.64
12	74	SEQ ID NO：8372	0.50％	8
					13	131	SEQ ID NO：8373	0.50％	8
14	54	SEQ ID NO：8374	0.46％	7.5
					15	99	SEQ ID NO：8375	0.45％	7.2
16	44	SEQ ID NO：8376	0.37％	6
					17	62	SEQ ID NO：8377	0.37％	6
18	87	SEQ ID NO：8378	0.37％	6
					19	89	SEQ ID NO：8379	0.37％	6
20	154	SEQ ID NO：8380	0.37％	6

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	105	SEQ ID NO：8381	22.54％	360
2	204	SEQ ID NO：8382	17.53％	280
					3	209	SEQ ID NO：8383	3.13％	50
4	75	SEQ ID NO：8384	1.87％	30
					5	85	SEQ ID NO：8385	1.87％	30
6	77	SEQ ID NO：8386	1.12％	18
					7	74	SEQ ID NO：8387	0.84％	13.44
8	210	SEQ ID NO：8388	0.56％	9
					9	226	SEQ ID NO：8389	0.55％	8.8704
10	98	SEQ ID NO：8390	0.54％	8.64
					11	198	SEQ ID NO：8391	0.46％	7.5

12	67	SEQ ID NO：8392	0.45％	7.2
					13	152	SEQ ID NO：8393	0.43％	7
14	43	SEQ ID NO：8394	0.37％	6
					15	63	SEQ ID NO：8395	0.37％	6
16	72	SEQ ID NO：8396	0.37％	6
					17	89	SEQ ID NO：8397	0.37％	6
18	101	SEQ ID NO：8398	0.37％	6
					19	107	SEQ ID NO：8399	0.37％	6
20	111	SEQ ID NO：8400	0.37％	6

HLA A0201-9mers
					Using such moleculesMaximum possible score of type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	138	SEQ ID NO：8401	0.21％	8532.082944
2	106	SEQ ID NO：8402	0.10％	3977.8497792
					3	44	SEQ ID NO：8403	0.03％	1243.078056
4	71	SEQ ID NO：8404	0.00％	348.872832
					5	234	SEQ ID NO：8405	0.00％	243.432
6	51	SEQ ID NO：8406	0.00％	130.26096
					7	109	SEQ ID NO：8407	0.00％	91.182672
8	81	SEQ ID NO：8408	0.00％	73.342584
					9	88	SEQ ID NO：8409	0.00％	70.386624
10	1	SEQ ID NO：8410	0.00％	65.32728732
					11	38	SEQ ID NO：8411	0.00％	47.876409
12	76	SEQ ID NO：8412	0.00％	36.8637882
					13	46	SEQ ID NO：8413	0.00％	30.889782
14	211	SEQ ID NO：8414	0.00％	21.616753941
					15	201	SEQ ID NO：8415	0.00％	19.657134
16	102	SEQ ID NO：8416	0.00％	18.4318941
					17	199	SEQ ID NO：8417	0.00％	16.496865
18	74	SEQ ID NO：8418	0.00％	15.783256167
					19	62	SEQ ID NO：8419	0.00％	13.9968225
20	99	SEQ ID NO：8420	0.00％	10.31851392

HLA A0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	78	SEQ ID NO：8421	0.01％	556.494246
2	138	SEQ ID NO：8422	0.01％	395.245972224
					3	84	SEQ ID NO：8423	0.00％	201.554244
4	71	SEQ ID NO：8424	0.00％	143.65707264
					5	44	SEQ ID NO：8425	0.00％	132.54624

6	76	SEQ ID NO：8426	0.00％	84.78671286
					7	8	SEQ ID NO：8427	0.00％	69.552
8	211	SEQ ID NO：8428	0.00％	52.7237901
					9	113	SEQ ID NO：8429	0.00％	47.99088
10	61	SEQ ID NO：8430	0.00％	37.4509575
					11	93	SEQ ID NO：8431	0.00％	31.24872
12	137	SEQ ID NO：8432	0.00％	31.1384304
					13	37	SEQ ID NO：8433	0.00％	27.531
14	55	SEQ ID NO：8434	0.00％	22.9153278
					15	98	SEQ ID NO：8435	0.00％	22.1063618985
16	108	SEQ ID NO：8436	0.00％	21.55457052
					17	63	SEQ ID NO：8437	0.00％	21.3624
18	45	SEQ ID NO：8438	0.00％	19.657134
					19	200	SEQ ID NO：8439	0.00％	19.657134
20	104	SEQ ID NO：8440	0.00％	13.87622016

HLA A1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	58	SEQ ID NO：8441	5.55％	2
2	125	SEQ ID NO：8442	1.66％	0.6
					3	226	SEQ ID NO：8443	1.66％	0.6
4	229	SEQ ID NO：8444	1.66％	0.6

HLA A1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	122	SEQ ID NO：8445	2.22％	0.8
2	228	SEQ ID NO：8446	2.22％	0.8

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	97	SEQ ID NO：8447	0.66％	36
2	86	SEQ ID NO：8448	0.37％	20
					3	37	SEQ ID NO：8449	0.33％	18
4	62	SEQ ID NO：8450	0.33％	18
					5	32	SEQ ID NO：8451	0.22％	12
6	102	SEQ ID NO：8452	0.22％	12
					7	227	SEQ ID NO：8453	0.22％	12
8	53	SEQ ID NO：8454	0.11％	6
					9	1	SEQ ID NO：8455	0.07％	4

10	44	SEQ ID NO：8456	0.07％	4
					11	56	SEQ ID NO：8457	0.07％	4
12	64	SEQ ID NO：8458	0.07％	4
					13	74	SEQ ID NO：8459	0.07％	4
14	76	SEQ ID NO：8460	0.07％	4
					15	87	SEQ ID NO：8461	0.07％	4
16	106	SEQ ID NO：8462	0.07％	4
					17	131	SEQ ID NO：8463	0.07％	4
18	23	SEQ ID NO：8464	0.03％	2
					19	157	SEQ ID NO：8465	0.03％	2
20	166	SEQ ID NO：8466	0.03％	2

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	119	SEQ ID NO：8467	3.33％	180
2	264	SEQ ID NO：8468	1.48％	80
					3	98	SEQ ID NO：8469	0.66％	36
4	27	SEQ ID NO：8470	0.37％	20
					5	86	SEQ ID NO：8471	0.37％	20
6	31	SEQ ID NO：8472	0.22％	12
					7	63	SEQ ID NO：8473	0.22％	12
8	96	SEQ ID NO：8474	0.22％	12
					9	101	SEQ ID NO：8475	0.22％	12
10	226	SEQ ID NO：8476	0.22％	12
					11	157	SEQ ID NO：8477	0.14％	8
12	176	SEQ ID NO：8478	0.14％	8
					13	238	SEQ ID NO：8479	0.14％	8
14	36	SEQ ID NO：8480	0.11％	6
					15	53	SEQ ID NO：8481	0.11％	6
16	61	SEQ ID NO：8482	0.11％	6
					17	3	SEQ ID NO：8483	0.07％	4
18	40	SEQ ID NO：8484	0.07％	4
					19	55	SEQ ID NO：8485	0.07％	4
20	74	SEQ ID NO：8486	0.07％	4

Table 18: SEQ ID NO: 6044 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	69	SEQ ID NO：8487	0.04％	2.5

2	89	SEQ ID NO：8488	0.02％	1.5
					3	141	SEQ ID NO：8489	0.01％	1
4	113	SEQ ID NO：8490	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	21	SEQ ID NO：8491	0.02％	1.5
2	88	SEQ ID NO：8492	0.02％	1.5
					3	8	SEQ ID NO：8493	0.02％	1.25
4	31	SEQ ID NO：8494	0.00％	0.5
					5	112	SEQ ID NO：8495	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	60	SEQ ID NO：8496	1.23％	150
2	77	SEQ ID NO：8497	1.11％	135
					3	141	SEQ ID NO：8498	0.49％	60
4	95	SEQ ID NO：8499	0.32％	40
					5	128	SEQ ID NO：8500	0.08％	10
6	113	SEQ ID NO：8501	0.04％	6
					7	69	SEQ ID NO：8502	0.01％	2
8	22	SEQ ID NO：8503	0.01％	1.8
					9	42	SEQ ID NO：8504	0.01％	1.8
10	78	SEQ ID NO：8505	0.00％	1.2
					11	32	SEQ ID NO：8506	0.00％	1
12	54	SEQ ID NO：8507	0.00％	0.9
					13	74	SEQ ID NO：8508	0.00％	0.9
14	28	SEQ ID NO：8509	0.00％	0.6
					15	36	SEQ ID NO：8510	0.00％	0.6
16	48	SEQ ID NO：8511	0.00％	0.6
					17	118	SEQ ID NO：8512	0.00％	0.6
18	4	SEQ ID NO：8513	0.00％	0.5

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	94	SEQ ID NO：8514	0.49％	60
2	48	SEQ ID NO：8515	0.16％	20
					3	128	SEQ ID NO：8516	0.16％	20
4	60	SEQ ID NO：8517	0.12％	15

5	127	SEQ ID NO：8518	0.12％	15
					6	25	SEQ ID NO：8519	0.04％	6
7	95	SEQ ID NO：8520	0.04％	6
					8	141	SEQ ID NO：8521	0.04％	6
9	41	SEQ ID NO：8522	0.04％	5.4
					10	77	SEQ ID NO：8523	0.04％	5.4
11	116	SEQ ID NO：8524	0.04％	5.4
					12	91	SEQ ID NO：8525	0.03％	4
13	4	SEQ ID NO：8526	0.01％	2
					14	112	SEQ ID NO：8527	0.01％	1.8
15	113	SEQ ID NO：8528	0.01％	1.35
					16	12	SEQ ID NO：8529	0.00％	1.2
17	31	SEQ ID NO：8530	0.00％	1
					18	32	SEQ ID NO：8531	0.00％	1
19	15	SEQ ID NO：8532	0.00％	0.9
					20	27	SEQ ID NO：8533	0.00％	0.9

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Note the bookIs divided into
					1	61	SEQ ID NO：8534	14.46％	231
2	16	SEQ ID NO：8535	3.13％	50
					3	120	SEQ ID NO：8536	1.87％	30
4	41	SEQ ID NO：8537	0.60％	9.6
					5	71	SEQ ID NO：8538	0.45％	7.2
6	21	SEQ ID NO：8539	0.37％	6
					7	53	SEQ ID NO：8540	0.37％	6
8	65	SEQ ID NO：8541	0.37％	6
					9	121	SEQ ID NO：8542	0.37％	6
10	74	SEQ ID NO：8543	0.36％	5.76
					11	20	SEQ ID NO：8544	0.35％	5.6
12	79	SEQ ID NO：8545	0.35％	5.6
					13	105	SEQ ID NO：8546	0.33％	5.28
14	48	SEQ ID NO：8547	0.30％	4.8
					15	88	SEQ ID NO：8548	0.30％	4.8
16	106	SEQ ID NO：8549	0.30％	4.8
					17	37	SEQ ID NO：8550	0.27％	4.4
18	70	SEQ ID NO：8551	0.27％	4.4
					19	18	SEQ ID NO：8552	0.25％	4
20	57	SEQ ID NO：8553	0.22％	3.6

HLA A24-10mers

Maximum possible score Using this molecular type				1596.672
					Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
1	120	SEQ ID NO：8554	1.87％	30
					2	73	SEQ ID NO：8555	0.54％	8.64
3	19	SEQ ID NO：8556	0.52％	8.4
					4	78	SEQ ID NO：8557	0.52％	8.4
5	104	SEQ ID NO：8558	0.49％	7.92
					6	61	SEQ ID NO：8559	0.46％	7.5
7	47	SEQ ID NO：8560	0.45％	7.2
					8	36	SEQ ID NO：8561	0.41％	6.6
9	52	SEQ ID NO：8562	0.37％	6
					10	64	SEQ ID NO：8563	0.30％	4.8
11	70	SEQ ID NO：8564	0.30％	4.8
					12	105	SEQ ID NO：8565	0.30％	4.8
13	123	SEQ ID NO：8566	0.30％	4.8
					14	69	SEQ ID NO：8567	0.27％	4.4
15	20	SEQ ID NO：8568	0.25％	4
					16	66	SEQ ID NO：8569	0.25％	4
17	83	SEQ ID NO：8570	0.25％	4
					18	86	SEQ ID NO：8571	0.25％	4
19	101	SEQ ID NO：8572	0.25％	4
					20	119	SEQ ID NO：8573	0.25％	4

HLA A0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	62	SEQ ID NO：8574	0.00％	136.1646
2	85	SEQ ID NO：8575	0.00％	69.6969
					3	47	SEQ ID NO：8576	0.00％	60.153786
4	121	SEQ ID NO：8577	0.00％	52.5182736
					5	74	SEQ ID NO：8578	0.00％	49.13352
6	23	SEQ ID NO：8579	0.00％	21.99582
					7	78	SEQ ID NO：8580	0.00％	19.42488
8	114	SEQ ID NO：8581	0.00％	14.6900655
					9	4	SEQ ID NO：8582	0.00％	11.304684
10	79	SEQ ID NO：8583	0.00％	8.4687081
					11	122	SEQ ID NO：8584	0.00％	6.0996
12	100	SEQ ID NO：8585	0.00％	5.382
					13	105	SEQ ID NO：8586	0.00％	4.981593
14	25	SEQ ID NO：8587	0.00％	4.968
					15	115	SEQ ID NO：8588	0.00％	4.966482
16	24	SEQ ID NO：8589	0.00％	4.4815221585

17	111	SEQ ID NO：8590	0.00％	4.128201
					18	94	SEQ ID NO：8591	0.00％	3.67632
19	34	SEQ ID NO：8592	0.00％	3.47553
					20	12	SEQ ID NO：8593	0.00％	3.30993

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	77	SEQ ID NO：8594	0.00％	147.97188
2	62	SEQ ID NO：8595	0.00％	143.59176
					3	113	SEQ ID NO：8596	0.00％	106.83684
4	78	SEQ ID NO：8597	0.00％	83.526984
					5	86	SEQ ID NO：8598	0.00％	83.526984
6	74	SEQ ID NO：8599	0.00％	69.552
					7	121	SEQ ID NO：8600	0.00％	61.06776
8	12	SEQ ID NO：8601	0.00％	50.232
					9	44	SEQ ID NO：8602	0.00％	26.082
10	4	SEQ ID NO：8603	0.00％	18.3816
					11	0	SEQ ID NO：8604	0.00％	17.38386
12	72	SEQ ID NO：8605	0.00％	17.1396
					13	22	SEQ ID NO：8606	0.00％	16.21914
14	122	SEQ ID NO：8607	0.00％	14.02908
					15	64	SEQ ID NO：8608	0.00％	11.161854
16	46	SEQ ID NO：8609	0.00％	10.34586
					17	54	SEQ ID NO：8610	0.00％	8.846145
18	47	SEQ ID NO：8611	0.00％	7.575080337
					19	131	SEQ ID NO：8612	0.00％	7.452
20	114	SEQ ID NO：8613	0.00％	6.735366

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	69	SEQ ID NO：8614	5.55％	2
2	22	SEQ ID NO：8615	5％	1.8
					3	77	SEQ ID NO：8616	5％	1.8
4	141	SEQ ID NO：8617	3.33％	1.2
					5	60	SEQ ID NO：8618	2.22％	0.8
6	95	SEQ ID NO：8619	2.22％	0.8
					7	36	SEQ ID NO：8620	1.66％	0.6

HLA A 1101-10mers
		Maximum possible score Using this molecular type	36

Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
						1	41	SEQ ID NO：8621	3.33％	1.2
2	68	SEQ ID NO：8622	3.33％	1.2
					3	94	SEQ ID NO：8623	3.33％	1.2
4	31	SEQ ID NO：8624	2.77％	1
					5	127	SEQ ID NO：8625	2.5％	0.9

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	48	SEQ ID NO：8626	0.74％	40
2	20	SEQ ID NO：8627	0.37％	20
					3	121	SEQ ID NO：8628	0.33％	18
4	18	SEQ ID NO：8629	0.07％	4
					5	21	SEQ ID NO：8630	0.07％	4
6	37	SEQ ID NO：8631	0.07％	4
					7	41	SEQ ID NO：8632	0.07％	4
8	53	SEQ ID NO：8633	0.07％	4
					9	65	SEQ ID NO：8634	0.07％	4
10	70	SEQ ID NO：8635	0.07％	4
					11	71	SEQ ID NO：8636	0.07％	4
12	74	SEQ ID NO：8637	0.07％	4
					13	79	SEQ ID NO：8638	0.07％	4
14	88	SEQ ID NO：8639	0.07％	4
					15	105	SEQ ID NO：8640	0.07％	4
16	106	SEQ ID NO：8641	0.07％	4
					17	124	SEQ ID NO：8642	0.07％	4
18	1	SEQ ID NO：8643	0.03％	2
					19	120	SEQ ID NO：8644	0.03％	1.8
20	11	SEQ ID NO：8645	0.02％	1.2

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	66	SEQ ID NO：8646	1.48％	80
2	123	SEQ ID NO：8647	0.74％	40
					3	20	SEQ ID NO：8648	0.37％	20
4	64	SEQ ID NO：8649	0.22％	12
					5	119	SEQ ID NO：8650	0.11％	6
6	54	SEQ ID NO：8651	0.09％	5
					7	19	SEQ ID NO：8652	0.07％	4
8	36	SEQ ID NO：8653	0.07％	4

9	47	SEQ ID NO：8654	0.07％	4
					10	52	SEQ ID NO：8655	0.07％	4
11	69	SEQ ID NO：8656	0.07％	4
					12	70	SEQ ID NO：8657	0.07％	4
13	73	SEQ ID NO：8658	0.07％	4
					14	78	SEQ ID NO：8659	0.07％	4
15	83	SEQ ID NO：8660	0.07％	4
					16	86	SEQ ID NO：8661	0.07％	4
17	101	SEQ ID NO：8662	0.07％	4
					18	104	SEQ ID NO：8663	0.07％	4
19	105	SEQ ID NO：8664	0.07％	4
					20	15	SEQ ID NO：8665	0.03％	2

Table 19: SEQ ID NO: 6045 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	4	SEQ ID NO：8666	0.02％	1.35
2	66	SEQ ID NO：8667	0.02％	1.35
					3	33	SEQ ID NO：8668	0.02％	1.25
4	44	SEQ ID NO：8669	0.01％	1
					5	50	SEQ ID NO：8670	0.01％	1
6	14	SEQ ID NO：8671	0.01％	0.75
					7	48	SEQ ID NO：8672	0.01％	0.75
8	11	SEQ ID NO：8673	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	4	SEQ ID NO：8674	0.12％	6.75
2	66	SEQID NO：8675	0.12％	6.75
					3	10	SEQ ID NO：8676	0.00％	0.5
4	28	SEQ ID NO：8677	0.00％	0.5
					5	32	SEQ ID NO：8678	0.00％	0.5
6	47	SEQ ID NO：8679	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	17	SEQ ID NO：8680	0.24％	30
2	44	SEQ ID NO：8681	0.07％	9

3	19	SEQ ID NO：8682	0.06％	8.1
					4	50	SEQ ID NO：8683	0.04％	5.4
5	29	SEQ ID NO：8684	0.03％	4
					6	52	SEQ ID NO：8685	0.02％	3.24
7	54	SEQ ID NO：8686	0.02％	3
					8	11	SEQ ID NO：8687	0.01％	1.8
9	37	SEQ ID NO：8688	0.01％	1.8
					10	25	SEQ ID NO：8689	0.01％	1.35
11	10	SEQ ID NO：8690	0.00％	0.9
					12	16	SEQ ID NO：8691	0.00％	0.9
13	35	SEQ ID NO：8692	0.00％	0.6

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	49	SEQ ID NO：8693	0.44％	54
2	17	SEQ ID NO：8694	0.22％	27
					3	10	SEQ ID NO：8695	0.14％	18
4	16	SEQ ID NO：8696	0.07％	9
					5	32	SEQ ID NO：8697	0.04％	6
6	19	SEQ ID NO：8698	0.01％	1.8
					7	29	SEQ ID NO：8699	0.00％	1.2
8	23	SEQ ID NO：8700	0.00％	0.9
					9	26	SEQ ID NO：8701	0.00％	0.9

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	18	SEQ ID NO：8702	1.87％	30
2	24	SEQ ID NO：8703	0.65％	10.5
					3	9	SEQ ID NO：8704	0.52％	8.4
4	12	SEQ ID NO：8705	0.52％	8.4
					5	28	SEQ ID NO：8706	0.52％	8.4
6	42	SEQ ID NO：8707	0.52％	8.4
					7	57	SEQ ID NO：8708	0.52％	8.4
8	66	SEQ ID NO：8709	0.52％	8.4
					9	55	SEQ ID NO：8710	0.51％	8.25
10	0	SEQ ID NO：8711	0.48％	7.7
					11	22	SEQ ID NO：8712	0.45％	7.2
12	10	SEQ ID NO：8713	0.37％	6
					13	25	SEQ ID NO：8714	0.37％	6
14	30	SEQ ID NO：8715	0.37％	6

15	19	SEQ ID NO：8716	0.35％	5.6
					16	40	SEQ ID NO：8717	0.31％	5
17	3	SEQ ID NO：8718	0.30％	4.8
					18	65	SEQ ID NO：8719	0.30％	4.8
19	14	SEQ ID NO：8720	0.27％	4.32
					20	56	SEQ ID NO：8721	0.25％	4

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	55	SEQ ID NO：8722	18.78％	300
2	18	SEQ ID NO：8723	2.63％	42
					3	21	SEQ ID NO：8724	2.25％	36
4	2	SEQ ID NO：8725	1.87％	30
					5	24	SEQ ID NO：8726	1.87％	30
6	11	SEQ ID NO：8727	0.52％	8.4
					7	40	SEQ ID NO：8728	0.52％	8.4
8	65	SEQ ID NO：8729	0.42％	6.72
					9	9	SEQ ID NO：8730	0.37％	6
10	8	SEQ ID NO：8731	0.35％	5.6
					11	27	SEQ ID NO：8732	0.35％	5.6
12	41	SEQ ID NO：8733	0.35％	5.6
					13	57	SEQ ID NO：8734	0.31％	5
14	17	SEQ ID NO：8735	0.25％	4
					15	29	SEQ ID NO：8736	0.25％	4
16	64	SEQ ID NO：8737	0.25％	4
					17	16	SEQ ID NO：8738	0.22％	3.6
18	10	SEQ ID NO：8739	0.18％	3
					19	13	SEQ ID NO：8740	0.18％	2.88
20	23	SEQ ID NO：8741	0.08％	1.4

HLA A 0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	19	SEQ ID NO：8742	0.03％	1310.8823136
2	15	SEQ ID NO：8743	0.02％	1082.4143022
					3	16	SEQ ID NO：8744	0.02％	1040.33238624
4	49	SEQ ID NO：8745	0.00％	382.536
					5	25	SEQ ID NO：8746	0.00％	342.863529264
6	56	SEQ ID NO：8747	0.00％	63.28397376
					7	12	SEQ ID NO：8748	0.00％	40.19736105
8	10	SEQ ID NO：8749	0.00％	21.3624

9	22	SEQ ID NO：8750	0.00％	19.7762418
					10	26	SEQ ID NO：8751	0.00％	12.6684
11	20	SEQ ID NO：8752	0.00％	11.544666
					12	37	SEQ ID NO：8753	0.00％	10.4328
13	32	SEQ ID NO：8754	0.00％	8.4456
					14	23	SEQ ID NO：8755	0.00％	6.2888049
15	47	SEQ ID NO：8756	0.00％	6.0858
					16	3	SEQ ID NO：8757	0.00％	4.582929078
17	18	SEQ ID NO：8758	0.00％	4.4855150505
					18	28	SEQ ID NO：8759	0.00％	4.2923589
19	62	SEQ ID NO：8760	0.00％	2.88098391
					20	27	SEQ ID NO：8761	0.00％	1.699677

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	17	SEQ ID NO：8762	0.16％	6459.14167272
2	19	SEQ ID NO：8763	0.01％	607.88448
					3	25	SEQ ID NO：8764	0.00％	126.83304
4	11	SEQ ID NO：8765	0.00％	63.16728165
					5	15	SEQ ID NO：8766	0.00％	53.54651988
6	37	SEQ ID NO：8767	0.00％	28.51632
					7	14	SEQ ID NO：8768	0.00％	21.8247414
8	29	SEQ ID NO：8769	0.00％	21.3624
					9	26	SEQ ID NO：8770	0.00％	19.42488
10	3	SEQ ID NO：8771	0.00％	17.2167282
					11	48	SEQ ID NO：8772	0.00％	15.7068219
12	12	SEQ ID NO：8773	0.00％	9.8581266
					13	27	SEQ ID NO：8774	0.00％	7.3086111
14	39	SEQ ID NO：8775	0.00％	7.10976
					15	23	SEQ ID NO：8776	0.00％	5.7419523
16	22	SEQ ID NO：8777	0.00％	4.599126
					17	45	SEQ ID NO：8778	0.00％	2.5495155
18	31	SEQ ID NO：8779	0.00％	2.52747
					19	52	SEQ ID NO：8780	0.00％	2.383605
20	20	SEQ ID NO：8781	0.00％	2.332847151

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	44	SEQ ID NO：8782	3.33％	1.2

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	3	SEQ ID NO：8783	0.37％	20
2	12	SEQ ID NO：8784	0.37％	20
					3	22	SEQ ID NO：8785	0.37％	20
4	56	SEQ ID NO：8786	0.37％	20
					5	30	SEQ ID NO：8787	0.22％	12
6	9	SEQ ID NO：8788	0.07％	4
					7	10	SEQ ID NO：8789	0.07％	4
8	19	SEQ ID NO：8790	0.07％	4
					9	25	SEQ ID NO：8791	0.07％	4
10	28	SEQ ID NO：8792	0.07％	4
					11	42	SEQ ID NO：8793	0.07％	4
12	65	SEQ ID NO：8794	0.07％	4
					13	35	SEQ ID NO：8795	0.05％	3
14	66	SEQ ID NO：8796	0.02％	1.2
					15	15	SEQ ID NO：8797	0.01％	1
16	47	SEQ ID NO：8798	0.01％	1
					17	20	SEQ ID NO：8799	0.01％	0.6
18	23	SEQ ID NO：8800	0.00％	0.5
					19	27	SEQ ID NO：8801	0.00％	0.5

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	27	SEQ ID NO：8802	0.37％	20
2	8	SEQ ID NO：8803	0.07％	4
					3	9	SEQ ID NO：8804	0.07％	4
4	11	SEQ ID NO：8805	0.07％	4
					5	17	SEQ ID NO：8806	0.07％	4
6	29	SEQ ID NO：8807	0.07％	4
					7	41	SEQ ID NO：8808	0.07％	4
8	52	SEQ ID NO：8809	0.07％	4
					9	64	SEQ ID NO：8810	0.07％	4
10	65	SEQ ID NO：8811	0.07％	4
					11	3	SEQ ID NO：8812	0.03％	2
12	23	SEQ ID NO：8813	0.03％	2

13	21	SEQ ID NO：8814	0.02％	1.2
					14	15	SEQ ID NO：8815	0.01％	1
15	35	SEQ ID NO：8816	0.01％	0.6
					16	39	SEQ ID NO：8817	0.01％	0.6
17	12	SEQ ID NO：8818	0.00％	0.5
					18	22	SEQ ID NO：8819	0.00％	0.5
19	45	SEQ ID NO：8820	0.00％	0.5

Table 20: SEQ ID NO: 6046 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	186	SEQ ID NO：8821	2.22％	125
2	156	SEQ ID NO：8822	0.88％	50
					3	14	SEQ ID NO：8823	0.08％	4.5
4	0	SEQ ID NO：8824	0.04％	2.5
					5	29	SEQ ID NO：8825	0.04％	2.5
6	85	SEQ ID NO：8826	0.04％	2.5
					7	168	SEQ ID NO：8827	0.04％	2.5
8	133	SEQ ID NO：8828	0.02％	1.35
					9	111	SEQ ID NO：8829	0.02％	1.125
10	61	SEQ ID NO：8830	0.01％	1
					11	7	SEQ ID NO：8831	0.01％	0.9
12	131	SEQ ID NO：8832	0.01％	0.9
					13	211	SEQ ID NO：8833	0.01％	0.625
14	4	SEQ ID NO：8834	0.00％	0.5
					15	43	SEQ ID NO：8835	0.00％	0.5
16	95	SEQ ID NO：8836	0.00％	0.5
					17	136	SEQ ID NO：8837	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	133	SEQ ID NO：8838	0.04％	2.7
2	84	SEQ ID NO：8839	0.04％	2.5
					3	167	SEQ ID NO：8840	0.04％	2.5
4	186	SEQ ID NO：8841	0.04％	2.5
					5	131	SEQ ID NO：8842	0.04％	2.25
6	14	SEQ ID NO：8843	0.03％	1.8
					7	205	SEQ ID NO：8844	0.02％	1.25
8	111	SEQ ID NO：8845	0.02％	1.125

9	60	SEQ ID NO：8846	0.01％	1
					10	188	SEQ ID NO：8847	0.01％	0.75
11	211	SEQ ID NO：8848	0.01％	0.625
					12	26	SEQ ID NO：8849	0.00％	0.5
13	94	SEQ ID NO：8850	0.00％	0.5
					14	135	SEQ ID NO：8851	0.00％	0.5
15	168	SEQ ID NO：8852	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	Accounting for maximumPercentage of points	Score recording
					1	43	SEQ ID NO：8853	0.24％	30
2	90	SEQ ID NO：8854	0.14％	18
					3	148	SEQ ID NO：8855	0.09％	12
4	4	SEQ ID NO：8856	0.05％	6.75
					5	24	SEQ ID NO：8857	0.04％	6
6	19	SEQ ID NO：8858	0.04％	5.4
					7	136	SEQ ID NO：8859	0.04％	5.4
8	54	SEQ ID NO：8860	0.03％	4.5
					9	32	SEQ ID NO：8861	0.03％	4
10	14	SEQ ID NO：8862	0.02％	3.6
					11	59	SEQ ID NO：8863	0.02％	3.6
12	88	SEQ ID NO：8864	0.02％	3
					13	87	SEQ ID NO：8865	0.02％	2.7
14	29	SEQ ID NO：8866	0.01％	L 8
					15	48	SEQ ID NO：8867	0.01％	1.8
16	115	SEQ ID NO：8868	0.01％	1.8
					17	186	SEQ ID NO：8869	0.01％	1.8
18	106	SEQ ID NO：8870	0.01％	1.5
					19	53	SEQ ID NO：8871	0.01％	1.35
20	173	SEQ ID NO：8872	0.00％	1.2

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	24	SEQ ID NO：8873	0.22％	27
2	54	SEQ ID NO：8874	0.18％	22.5
					3	135	SEQ ID NO：8875	0.08％	10.8
4	51	SEQ ID NO：8876	0.07％	9
					5	13	SEQ ID NO：8877	0.06％	8.1
6	26	SEQ ID NO：8878	0.04％	6
					7	31	SEQ ID NO：8879	0.04％	6

8	90	SEQ ID NO：8880	0.04％	6
					9	43	SEQ ID NO：8881	0.03％	4.5
10	19	SEQ ID NO：8882	0.03％	4.05
					11	169	SEQ ID NO：8883	0.02％	3
12	87	SEQ ID NO：8884	0.02％	2.7
					13	84	SEQ ID NO：8885	0.01％	1.8
14	88	SEQ ID NO：8886	0.01％	1.8
					15	94	SEQ ID NO：8887	0.01％	1.8
16	64	SEQ ID NO：8888	0.00％	1.2
					17	131	SEQ ID NO：8889	0.00％	1.2
18	99	SEQ ID NO：8890	0.00％	1
					19	53	SEQ ID NO：8891	0.00％	0.9
20	85	SEQ ID NO：8892	0.00％	0.9

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	196	SEQ ID NO：8893	27.55％	440
2	44	SEQ ID NO：8894	18.78％	300
					3	36	SEQ ID NO：8895	12.52％	200
4	92	SEQ ID NO：8896	12.52％	200
					5	109	SEQ ID NO：8897	2.70％	43.2
6	25	SEQ ID NO：8898	1.87％	30
					7	93	SEQ ID NO：8899	1.12％	18
8	12	SEQ ID NO：8900	0.75％	12
					9	123	SEQ ID NO：8901	0.70％	11.2
10	7	SEQ ID NO：8902	0.64％	10.368
					11	17	SEQ ID NO：8903	0.52％	8.4
12	139	SEQ ID NO：8904	0.52％	8.4
					13	193	SEQ ID NO：8905	0.46％	7.5
14	6	SEQ ID NO：8906	0.45％	7.2
					15	19	SEQ ID NO：8907	0.45％	7.2
16	110	SEQ ID NO：8908	0.45％	7.2
					17	114	SEQ ID NO：8909	0.45％	7.2
18	210	SEQ ID NO：8910	0.45％	7.2
					19	46	SEQ ID NO：8911	0.42％	6.72
20	52	SEQ ID NO：8912	0.37％	6

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	92	SEQ ID NO：8913	7.51％	120

2	42	SEQ ID NO：8914	2.63％	42
					3	109	SEQ ID NO：8915	2.25％	36
4	23	SEQ ID NO：8916	1.87％	30
					5	34	SEQ ID NO：8917	0.75％	12
6	6	SEQ ID NO：8918	0.64％	10.368
					7	45	SEQ ID NO：8919	0.63％	10.08
8	196	SEQ ID NO：8920	0.62％	10
					9	44	SEQ ID NO：8921	0.56％	9
10	40	SEQ ID NO：8922	0.55％	8.8
					11	62	SEQ ID NO：8923	0.46％	7.5
12	193	SEQ ID NO：8924	0.46％	7.5
					13	18	SEQ ID NO：8925	0.45％	7.2
14	113	SEQ ID NO：8926	0.45％	7.2
					15	56	SEQ ID NO：8927	0.37％	6
16	176	SEQ ID NO：8928	0.37％	6
					17	16	SEQ ID NO：8929	0.35％	5.6
18	138	SEQ ID NO：8930	0.35％	5.6
					19	127	SEQ ID NO：8931	0.33％	5.28
20	36	SEQ ID NO：8932	0.31％	5

HLA A 0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	Account forPercentage of maximum score	Score recording
					1	13	SEQ ID NO：8933	0.04％	1793.676528
2	87	SEQ ID NO：8934	0.03％	1415.3832
					3	24	SEQ ID NO：8935	0.01％	618.0996816
4	19	SEQ ID NO：8936	0.00％	223.23708
					5	12	SEQ ID NO：8937	0.00％	210.36400875
6	51	SEQ ID NO：8938	0.00％	198.30859992
					7	53	SEQ ID NO：8939	0.00％	194.477328
8	88	SEQ ID NO：8940	0.00％	180.58536756
					9	106	SEQ ID NO：8941	0.00％	169.74828
10	54	SEQ ID NO：8942	0.00％	70.09848
					11	59	SEQ ID NO：8943	0.00％	43.42032
12	94	SEQ ID NO：8944	0.00％	41.792058
					13	20	SEQ ID NO：8945	0.00％	37.46088108
14	63	SEQ ID NO：8946	0.00％	35.73520902
					15	22	SEQ ID NO：8947	0.00％	20.5916435109
16	47	SEQ ID NO：8948	0.00％	12.233222865
					17	66	SEQ ID NO：8949	0.00％	12.2199
18	56	SEQ ID NO：8950	0.00％	11.486706
					19	67	SEQ ID NO：8951	0.00％	6.416172

20

117

SEQ ID NO：8952

0.00％

5.827464

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	43	SEQ ID NO：8953	0.10％	3977.8497792
2	24	SEQ ID NO：8954	0.02％	836.2525104
					3	51	SEQ ID NO：8955	0.02％	815.616432
4	49	SEQ ID NO：8956	0.01％	660.3245145
					5	19	SEQ ID NO：8957	0.00％	251.837856
6	59	SEQ ID NO：8958	0.00％	159.9696
					7	12	SEQ ID NO：8959	0.00％	155.245377
8	45	SEQ ID NO：8960	0.00％	141.1974531
					9	21	SEQ ID NO：8961	0.00％	117.22672269
10	53	SEQ ID NO：8962	0.00％	84.55536
					11	87	SEQ ID NO：8963	0.00％	65.5671672
12	13	SEQ ID NO：8964	0.00％	64.88888616
					13	153	SEQ ID NO：8965	0.00％	49.13352
14	178	SEQ ID NO：8966	0.00％	26.082
					15	18	SEQ ID NO：8967	0.00％	24.802259691
16	116	SEQ ID NO：8968	0.00％	21.5616168
					17	65	SEQ ID NO：8969	0.00％	20.77383
18	86	SEQ ID NO：8970	0.00％	15.7068219
					19	27	SEQ ID NO：8971	0.00％	12.3159135
20	46	SEQ ID NO：8972	0.00％	11.45624789925

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	4	SEQ ID NO：8973	12.5％	4.5
2	136	SEQ ID NO：8974	3.33％	1.2
					3	156	SEQ ID NO：8975	3.33％	1.2
4	140	SEQ ID NO：8976	1.66％	0.6

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	169	SEQ ID NO：8977	5.55％	2
2	94	SEQ ID NO：8978	3.33％	1.2

HLA B7-9mers
		Maximum possible score Using this molecular type	5400

Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
						1	146	SEQ ID NO：8979	0.74％	40
2	154	SEQ ID NO：8980	0.74％	40
					3	80	SEQ ID NO：8981	0.66％	36
4	139	SEQ ID NO：8982	0.33％	18
					5	83	SEQ ID NO：8983	0.22％	12
6	209	SEQ ID NO：8984	0.22％	12
					7	7	SEQ ID NO：8985	0.11％	6
8	3	SEQ ID NO：8986	0.07％	4
					9	6	SEQ ID NO：8987	0.07％	4
10	12	SEQ ID NO：8988	0.07％	4
					11	19	SEQ ID NO：8989	0.07％	4
12	24	SEQ ID NO：8990	0.07％	4
					13	38	SEQ ID NO：8991	0.07％	4
14	46	SEQ ID NO：8992	0.07％	4
					15	56	SEQ ID NO：8993	0.07％	4
16	110	SEQ ID NO：8994	0.07％	4
					17	114	SEQ ID NO：8995	0.07％	4
18	123	SEQ ID NO：8996	0.07％	4
					19	129	SEQ ID NO：8997	0.07％	4
20	166	SEQ ID NO：8998	0.07％	4

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	56	SEQ ID NO：8999	1.48％	80
2	40	SEQ ID NO：9000	0.74％	40
					3	127	SEQ ID NO：9001	0.74％	40
4	170	SEQ ID NO：9002	0.74％	40
					5	140	SEQ ID NO：9003	0.27％	15
6	35	SEQ ID NO：9004	0.22％	12
					7	79	SEQ ID NO：9005	0.22％	12
8	82	SEQ ID NO：9006	0.22％	12
					9	208	SEQ ID NO：9007	0.22％	12
10	209	SEQ ID NO：9008	0.22％	12
					11	80	SEQ ID NO：9009	0.16％	9
12	129	SEQ ID NO：9010	0.14％	8
					13	138	SEQ ID NO：9011	0.11％	6
14	73	SEQ ID NO：9012	0.09％	5
					15	2	SEQ ID NO：9013	0.07％	4
16	5	SEQ ID NO：9014	0.07％	4
					17	6	SEQ ID NO：9015	0.07％	4

18	16	SEQ ID NO：9016	0.07％	4
					19	18	SEQ ID NO：9017	0.07％	4
20	24	SEQ ID NO：9018	0.07％	4

Table 21: SEQ ID NO: 6047 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	53	SEQ ID NO：9019	2％	112.5
2	10	SEQ ID NO：9020	0.08％	4.5
					3	33	SEQ ID NO：9021	0.02％	1.5
4	3	SEQ ID NO：9022	0.00％	0.5
					5	27	SEQ ID NO：9023	0.00％	0.5
6	29	SEQ ID NO：9024	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	10	SEQ ID NO：9025	0.8％	45
2	52	SEQ ID NO：9026	0.2％	11.25
					3	50	SEQ ID NO：9027	0.04％	2.5
4	32	SEQ ID NO：9028	0.02％	1.5
					5	48	SEQ ID NO：9029	0.02％	1.35
6	27	SEQ ID NO：9030	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	38	SEQ ID NO：9031	1.85％	225
2	17	SEQ ID NO：9032	0.02％	3.6
					3	2	SEQ ID NO：9033	0.02％	2.7
4	37	SEQ ID NO：9034	0.01％	1.8
					5	27	SEQ ID NO：9035	0.01％	1.35
6	13	SEQ ID NO：9036	0.00％	0.675
					7	14	SEQ ID NO：9037	0.00％	0.6

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	13	SEQ ID NO：9038	0.04％	6
2	37	SEQ ID NO：9039	0.01％	2.025

32	SEQ ID NO：9040	0.00％	0.9
				419	SEQ ID NO：9041	0.00％	0.675
516	SEQ ID NO：9042	0.00％	0.54

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	20	SEQ ID NO：9043	1.25％	20
2	6	SEQ ID NO：9044	0.52％	8.4
					3	5	SEQ ID NO：9045	0.51％	8.25
4	35	SEQ ID NO：9046	0.36％	5.76
					5	31	SEQ ID NO：9047	0.35％	5.6
6	43	SEQ ID NO：9048	0.27％	4.4
					7	13	SEQ ID NO：9049	0.26％	4.2
8	32	SEQ ID NO：9050	0.21％	3.36
					9	2	SEQ ID NO：9051	0.11％	1.8
10	9	SEQ ID NO：9052	0.10％	1.68
					11	8	SEQ ID NO：9053	0.09％	1.5
12	15	SEQ ID NO：9054	0.09％	1.5
					13	23	SEQ ID NO：9055	0.09％	1.5
14	27	SEQ ID NO：9056	0.08％	1.4
					15	24	SEQ ID NO：9057	0.07％	1.2
16	7	SEQ ID NO：9058	0.06％	1
					17	17	SEQ ID NO：9059	0.06％	1
18	10	SEQ ID NO：9060	0.05％	0.9
					19	39	SEQ ID NO：9061	0.04％	0.792
20	47	SEQ ID NO：9062	0.04％	0.792

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	5	SEQ ID NO：9063	2.63％	42
2	34	SEQ ID NO：9064	0.54％	8.64
					3	30	SEQ ID NO：9065	0.52％	8.4
4	19	SEQ ID NO：9066	0.50％	8
					5	50	SEQ ID NO：9067	0.33％	5.28
6	12	SEQ ID NO：9068	0.26％	4.2
					7	31	SEQ ID NO：9069	0.21％	3.36
8	26	SEQ ID NO：9070	0.15％	2.52
					9	8	SEQ ID NO：9071	0.13％	2.1
10	22	SEQ ID NO：9072	0.12％	2
					11	23	SEQ ID NO：9073	0.11％	1.8

12	6	SEQ ID NO：9074	0.09％	1.5
					13	14	SEQ ID NO：9075	0.09％	1.5
14	16	SEQ ID NO：9076	0.09％	1.5
					15	7	SEQ ID NO：9077	0.06％	1
16	48	SEQ ID NO：9078	0.04％	0.75
					17	0	SEQ ID NO：9079	0.04％	0.72
18	9	SEQ ID NO：9080	0.04％	0.72
					19	47	SEQ ID NO：9081	0.04％	0.66
20	39	SEQ ID NO：9082	0.03％	0.6

HLA A 0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	15	SEQ ID NO：9083	0.00％	14.1442686
2	27	SEQ ID NO：9084	0.00％	9.598176
					3	22	SEQ ID NO：9085	0.00％	9.5634
4	9	SEQ ID NO：9086	0.00％	5.546246013
					5	2	SEQ ID NO：9087	0.00％	5.526462816
6	24	SEQ ID NO：9088	0.00％	4.88163753
					7	17	SEQ ID NO：9089	0.00％	3.699285408
8	31	SEQ ID NO：9090	0.00％	2.29699206
					9	6	SEQ ID NO：9091	0.00％	2.0016040674
10	7	SEQ ID NO：9092	0.00％	0.91287
					11	49	SEQ ID NO：9093	0.00％	0.71805678
12	16	SEQ ID NO：9094	0.00％	0.6694257042
					13	12	SEQ ID NO：9095	0.00％	0.6539828625

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	16	SEQ ID NO：9096	0.00％	34.28765802
2	19	SEQ ID NO：9097	0.00％	18.9368775
					3	14	SEQ ID NO：9098	0.00％	14.1442686
4	27	SEQ ID NO：9099	0.00％	11.406528
					5	26	SEQ ID NO：9100	0.00％	10.9304361558
6	34	SEQ ID NO：9101	0.00％	5.580927
					7	6	SEQ ID NO：9102	0.00％	4.865742
8	9	SEQ ID NO：9103	0.00％	2.64106953
					9	50	SEQ ID NO：9104	0.00％	2.6275752
10	30	SEQ ID NO：9105	0.00％	2.29699206
					11	7	SEQ ID NO：9106	0.00％	0.86083641
12	42	SEQ ID NO：9107	0.00％	0.7049592

13	22	SEQ ID NO：9108	0.00％	0.6628440357
					14	2	SEQ ID NO：9109	0.00％	0.6530644656

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	37	SEQ ID NO：9110	15％	5.4
2	38	SEQ ID NO：9111	2.22％	0.8

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	37	SEQ ID NO：9112	7.5％	2.7

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	35	SEQ ID NO：9113	3.70％	200
2	17	SEQ ID NO：9114	0.11％	6
					3	6	SEQ ID NO：9115	0.07％	4
4	20	SEQ ID NO：9116	0.07％	4
					5	31	SEQ ID NO：9117	0.07％	4
6	43	SEQ ID NO：9118	0.07％	4
					7	7	SEQ ID NO：9119	0.03％	2
8	23	SEQ ID NO：9120	0.02％	1.2
					9	24	SEQ ID NO：9121	0.02％	1.2
10	10	SEQ ID NO：9122	0.01％	0.9

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	35	SEQ ID NO：9123	0.09％	5
2	19	SEQ ID NO：9124	0.07％	4
					3	30	SEQ ID NO：9125	0.07％	4
4	34	SEQ ID NO：9126	0.07％	4
					5	7	SEQ ID NO：9127	0.03％	2
6	16	SEQ ID NO：9128	0.03％	1.8
					7	23	SEQ ID NO：9129	0.02％	1.2
8	50	SEQ ID NO：9130	0.02％	1.2
					9	9	SEQ ID NO：9131	0.01％	1

Table 22: SEQ ID NO: 6048 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	66	SEQ ID NO：9132	0.44％	25
2	80	SEQ ID NO：9133	0.08％	5
					3	93	SEQ ID NO：9134	0.04％	2.7
4	11	SEQ ID NO：9135	0.04％	2.5
					5	89	SEQ ID NO：9136	0.04％	2.25
6	48	SEQ ID NO：9137	0.01％	1
					7	3	SEQ ID NO：9138	0.00％	0.5
8	9	SEQ ID NO：9139	0.00％	0.5
					9	56	SEQ ID NO：9140	0.00％	0.5
10	101	SEQ ID NO：9141	0.00％	0.5
					11	106	SEQ ID NO：9142	0.00％	0.5
12	110	SEQ ID NO：9143	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	30	SEQ ID NO：9144	0.4％	22.5
2	88	SEQ ID NO：9145	0.12％	6.75
					3	48	SEQ ID NO：9146	0.04％	2.5
4	55	SEQ ID NO：9147	0.02％	1.25
					5	13	SEQ ID NO：9148	0.01％	0.9
6	79	SEQ ID NO：9149	0.01％	0.75
					7	93	SEQ ID NO：9150	0.01％	0.675
8	2	SEQ ID NO：9151	0.00％	0.5
					9	8	SEQ ID NO：9152	0.00％	0.5
10	65	SEQ ID NO：9153	0.00％	0.5
					11	66	SEQ ID NO：9154	0.00％	0.5
12	80	SEQ ID NO：9155	0.00％	0.5
					13	105	SEQ ID NO：9156	0.00％	0.5
14	109	SEQ ID NO：9157	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	109	SEQ ID NO：9158	0.74％	90
2	3	SEQ ID N0：9159	0.24％	30
					3	111	SEQ ID NO：9160	0.12％	15
4	106	SEQ ID NO：9161	0.07％	9
					5	95	SEQ ID NO：9162	0.05％	6.075

6	101	SEQ ID NO：9163	0.04％	6
					7	110	SEQ ID NO：9164	0.02％	3.6
8	84	SEQ ID NO：9165	0.02％	3
					9	80	SEQ ID NO：9166	0.02％	2.7
10	37	SEQ ID NO：9167	0.01％	2.25
					11	9	SEQ ID NO：9168	0.01％	2
12	54	SEQ ID NO：9169	0.01％	2
					13	99	SEQ ID NO：9170	0.01％	1.35
14	1	SEQ ID NO：9171	0.01％	1.215
					15	11	SEQ ID NO：9172	0.00％	0.9
16	15	SEQ ID NO：9173	0.00％	0.9
					17	69	SEQ ID NO：9174	0.00％	0.6
18	5	SEQ ID NO：9175	0.00％	0.54
					19	103	SEQ ID NO：9176	0.00％	0.54

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	75	SEQ ID NO：9177	0.49％	60
2	109	SEQ ID NO：9178	0.29％	36
					3	22	SEQ ID NO：9179	0.14％	18
4	15	SEQ ID NO：9180	0.04％	6
					5	110	SEQ ID NO：9181	0.01％	2.25
6	95	SEQ ID NO：9182	0.01％	1.8
					7	101	SEQ ID NO：9183	0.01％	1.35
8	43	SEQ ID NO：9184	0.00％	1
					9	2	SEQ ID NO：9185	0.00％	0.9
10	5	SEQ ID NO：9186	0.00％	0.9
					11	7	SEQ ID NO：9187	0.00％	0.9
12	107	SEQ ID NO：9188	0.00％	0.9
					13	102	SEQ ID NO：9189	0.00％	0.81
14	3	SEQ ID NO：9190	0.00％	0.75
					15	8	SEQ ID NO：9191	0.00％	0.6
16	103	SEQ ID NO：9192	0.00％	0.54

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	88	SEQ ID NO：9193	1.66％	26.6112
2	77	SEQ ID NO：9194	0.77％	12.32
					3	18	SEQ ID NO：9195	0.56％	9
4	108	SEQ ID NO：9196	0.56％	9

5	92	SEQ ID NO：9197	0.54％	8.64
					6	96	SEQ ID NO：9198	0.54％	8.64
7	73	SEQ ID N0：9199	0.46％	7.5
					8	40	SEQ ID NO：9200	0.45％	7.2
9	104	SEQ ID NO：9201	0.42％	6.72
					10	8	SEQ ID NO：9202	0.41％	6.6
11	21	SEQ ID NO：9203	0.37％	6
					12	102	SEQ ID NO：9204	0.37％	6
13	22	SEQ ID NO：9205	0.25％	4
					14	68	SEQ ID NO：9206	0.25％	4
15	106	SEQ ID NO：9207	0.22％	3.6
					16	1	SEQ ID NO：9208	0.18％	3
17	79	SEQ ID NO：9209	0.18％	3
					18	93	SEQ ID NO：9210	0.18％	3
19	101	SEQ ID NO：9211	0.18	3
					20	37	SEQ ID NO：9212	0.15％	2.4

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	100	SEQ ID NO：9213	0.93％	15
2	18	SEQ ID NO：9214	0.78％	12.6
					3	98	SEQ ID NO：9215	0.52％	8.4
4	73	SEQ ID NO：9216	0.46％	7.5
					5	91	SEQ ID NO：9217	0.45％	7.2
6	103	SEQ ID NO：9218	0.42％	6.72
					7	7	SEQ ID NO：9219	0.41％	6.6
8	21	SEQ ID NO：9220	0.37％	6
					9	46	SEQ ID NO：9221	0.37％	6
10	93	SEQ ID NO：9222	0.37％	6
					11	96	SEQ ID NO：9223	0.37％	6
12	101	SEQ ID NO：9224	0.37％	6
					13	77	SEQ ID NO：9225	0.25％	4
14	92	SEQ ID NO：9226	0.22％	3.6
					15	105	SEQ ID NO：9227	0.22％	3.6
16	2	SEQ ID NO：9228	0.18％	3
					17	53	SEQ ID NO：9229	0.18％	3
18	36	SEQ ID NO：9230	0.12％	2
					19	55	SEQ ID NO：9231	0.12％	2
20	102	SEQ ID NO：9232	0.11％	1.8

HLA A 0201-9mers

Maximum possible score Using this molecular type				3925227.1
					Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
1	84	SEQ ID NO：9233	0.01％	441.342216
					2	102	SEQ ID NO：9234	0.00％	63.16728165
3	107	SEQ ID NO：9235	0.00％	51.882640425
					4	1	SEQ ID NO：9236	0.00％	43.8816609
5	95	SEQ ID NO：9237	0.00％	33.40165248
					6	2	SEQ ID NO：9238	0.00％	24.66305226
7	92	SEQ ID NO：9239	0.00％	22.64458905
					8	103	SEQ ID NO：9240	0.00％	20.70206586
9	47	SEQ ID NO：9241	0.00％	11.175953184
					10	94	SEQ ID NO：9242	0.00％	8.452983
11	15	SEQ ID NO：9243	0.00％	8.1793152
					12	8	SEQ ID NO：9244	0.00％	4.993461
13	5	SEQ ID NO：9245	0.00％	4.57284528
					14	99	SEQ ID NO：9246	0.00％	3.999468528
15	105	SEQ ID NO：9247	0.00％	2.231322
					16	20	SEQ ID NO：9248	0.00％	1.3524
17	62	SEQ ID NO：9249	0.00％	0.8631693
					18	6	SEQ ID NO：9250	0.00％	0.824619
19	57	SEQ ID NO：9251	0.00％	0.72105
					20	58	SEQ ID NO：9252	0.00％	0.7147572

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	101	SEQ ID NO：9253	0.03％	1243.078056
2	3	SEQ ID NO：9254	0.01％	592.944462
					3	106	SEQ ID NO：9255	0.00％	94.2678
4	5	SEQ ID NO：9256	0.00％	43.42032
					5	107	SEQ ID NO：9257	0.00％	33.30332334
6	102	SEQ ID NO：9258	0.00％	32.53181778
					7	54	SEQ ID NO：9259	0.00％	27.324
8	7	SEQ ID NO：9260	0.00％	21.3624
					9	1	SEQ ID NO：9261	0.00％	13.723479
10	95	SEQ ID N0：9262	0.00％	13.00344192
					11	94	SEQ ID NO：9263	0.00％	10.01276388
12	99	SEQ ID NO：9264	0.00％	5.6615328
					13	39	SEQ ID NO：9265	0.00％	3.6304212
14	111	SEQ ID NO：9266	0.00％	2.53368
					15	103	SEQ ID NO：9267	0.00％	2.475394803
16	14	SEQ ID NO：9268	0.00％	2.4519012

17	19	SEQ ID NO：9269	0.00％	2.07604992
					18	29	SEQ ID NO：9270	0.00％	1.8179154
19	57	SEQ ID NO：9271	0.00％	1.52076
					20	47	SEQ ID NO：9272	0.00％	1.27712376

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	80	SEQ ID NO：9273	3.33％	1.2
2	69	SEQ ID NO：9274	1.66％	0.6
					3	109	SEQ ID NO：9275	1.66％	0.6

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	22	SEQ ID NO：9276	11.11％	4

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	22	SEQ ID NO：9277	3.70％	200
2	77	SEQ ID NO：9278	2.22％	120
					3	104	SEQ ID NO：9279	0.22％	12
4	40	SEQ ID NO：9280	0.11％	6
					5	8	SEQ ID NO：9281	0.07％	4
6	21	SEQ ID NO：9282	0.07％	4
					7	68	SEQ ID NO：9283	0.07％	4
8	92	SEQ ID NO：9284	0.07％	4
					9	102	SEQ ID NO：9285	0.07％	4
10	46	SEQ ID NO：9286	0.03％	2
					11	98	SEQ ID NO：9287	0.03％	2
12	103	SEQ ID NO：9288	0.03％	2
					13	88	SEQ ID NO：9289	0.02％	1.2
14	105	SEQ ID NO：9290	0.01％	0.9
					15	43	SEQ ID NO：9291	0.01％	0.6
16	79	SEQ ID NO：9292	0.01％	0.6
					17	95	SEQ ID NO：9293	0.01％	0.6
18	107	SEQ ID NO：9294	0.00％	0.5

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording

1	46	SEQ ID NO：9295	1.48％	80
					2	98	SEQ ID NO：9296	1.48％	80
3	91	SEQ ID NO：9297	0.37％	20
					4	103	SEQ ID NO：9298	0.37％	20
5	7	SEQ ID NO：9299	0.07％	4
					6	21	SEQ ID NO：9300	0.07％	4
7	101	SEQ ID NO：9301	0.07％	4
					8	107	SEQ ID NO：9302	0.03％	2
9	67	SEQ ID NO：9303	0.02％	1.2
					10	93	SEQ ID NO：9304	0.02％	1.2
11	69	SEQ ID NO：9305	0.01％	1
					12	39	SEQ ID NO：9306	0.01％	0.6
13	77	SEQ ID NO：9307	0.01％	0.6
					14	22	SEQ ID NO：9308	0.00％	0.5

Table 23: SEQ ID NO: 6049 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	0	SEQ ID NO：9309	0.2％	11.25
2	35	SEQ ID NO：9310	0.01％	0.9
					3	4	SEQ ID NO：9311	0.00％	0.5
4	5	SEQ ID NO：9312	0.00％	0.5
					5	10	SEQ ID NO：9313	0.00％	0.5
6	19	SEQ ID NO：9314	0.00％	0.5
					7	21	SEQ ID NO：9315	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	0	SEQ ID NO：9316	0.2％	11.25
2	5	SEQ ID NO：9317	0.04％	2.5
					3	33	SEQ ID NO：9318	0.02％	1.5
4	3	SEQ ID NO：9319	0.02％	1.25
					5	9	SEQ ID NO：9320	0.00％	0.5
6	18	SEQ ID NO：9321	0.00％	0.5
					7	20	SEQ ID NO：9322	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording

1	4	SEQ ID NO：9323	0.14％	18
					2	16	SEQ ID NO：9324	0.11％	13.5
3	23	SEQ ID NO：9325	0.06％	8.1
					4	18	SEQ ID NO：9326	0.03％	4.05
5	21	SEQ ID NO：9327	0.01％	2.025
					6	9	SEQ ID NO：9328	0.01％	1.8
7	15	SEQ ID NO：9329	0.01％	1.8
					8	25	SEQ ID NO：9330	0.01％	1.8
9	12	SEQ ID NO：9331	0.00％	0.9
					10	19	SEQ ID NO：9332	0.00％	0.9
11	20	SEQ ID NO：9333	0.00％	0.9
					12	2	SEQ ID NO：9334	0.00％	0.81
13	22	SEQ ID NO：9335	0.00％	0.81
					14	10	SEQ ID NO：9336	0.00％	0.6

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	20	SEQ ID NO：9337	0.16％	20.25
2	9	SEQ ID NO：9338	0.09％	12
					3	16	SEQ ID NO：9339	0.07％	9
4	18	SEQ ID NO：9340	0.07％	9
					5	22	SEQ ID NO：9341	0.06％	8.1
6	4	SEQ ID NO：9342	0.03％	4.05
					7	15	SEQ ID NO：9343	0.03％	4.05
8	12	SEQ ID NO：9344	0.02％	3.6
					9	3	SEQ ID NO：9345	0.00％	0.9
10	33	SEQ ID NO：9346	0.00％	0.6
					11	2	SEQ ID NO：9347	0.00％	0.54
12	24	SEQ ID NO：9348	0.00％	0.54

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	8	SEQ ID NO：9349	18.78％	300
2	11	SEQ ID NO：9350	1.87％	30
					3	28	SEQ ID NO：9351	1.50％	24
4	7	SEQ ID NO：9352	0.75％	12
					5	17	SEQ ID NO：9353	0.56％	9
6	14	SEQ ID NO：9354	0.46％	7.5
					7	23	SEQ ID NO：9355	0.37％	6
8	13	SEQ ID NO：9356	0.36％	5.76

9	2	SEQ ID NO：9357	0.35％	5.6
					10	16	SEQ ID NO：9358	0.35％	5.6
11	9	SEQ ID NO：9359	0.30％	4.8
					12	21	SEQ ID NO：9360	0.26％	4.2
13	5	SEQ ID NO：9361	0.25％	4
					14	4	SEQ ID NO：9362	0.22％	3.6
15	0	SEQ ID NO：9363	0.18％	3
					16	19	SEQ ID NO：9364	0.18％	3
17	10	SEQ ID NO：9365	0.15％	2.4
					18	18	SEQ ID NO：9366	0.13％	2.1
19	25	SEQ ID NO：9367	0.06％	1.1
					20	15	SEQ ID NO：9368	0.05％	0.9

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	8	SEQ ID NO：9369	22.54％	360
2	7	SEQ ID NO：9370	1.25％	20
					3	17	SEQ ID NO：9371	0.65％	10.5
4	15	SEQ ID NO：9372	0.52％	8.4
					5	4	SEQ ID NO：9373	0.45％	7.2
6	22	SEQ ID NO：9374	0.37％	6
					7	12	SEQ ID NO：9375	0.36％	5.76
8	27	SEQ ID NO：9376	0.30％	4.8
					9	14	SEQ ID NO：9377	0.28％	4.5
10	20	SEQ ID NO：9378	0.26％	4.2
					11	10	SEQ ID NO：9379	0.25％	4
12	3	SEQ ID NO：9380	0.18％	3
					13	18	SEQ ID NO：9381	0.18％	3
14	9	SEQ ID NO：9382	0.15％	2.4
					15	24	SEQ ID NO：9383	0.10％	1.65
16	16	SEQ ID NO：9384	0.07％	1.2
					17	13	SEQ ID NO：9385	0.06％	1
18	11	SEQ ID NO：9386	0.05％	0.9
					19	1	SEQ ID NO：9387	0.05％	0.84

HLA A 0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	12	SEQ ID NO：9388	0.10％	4267.988928
2	23	SEQ ID NO：9389	0.03％	1360.69088544
					3	9	SEQ ID NO：9390	0.01％	569.948832

4	16	SEQ ID NO：9391	0.00％	309.0498408
					5	15	SEQ ID NO：9392	0.00％	79.73570448
6	2	SEQ ID NO：9393	0.00％	51.109542
					7	18	SEQ ID NO：9394	0.00％	45.25539984
8	25	SEQ ID NO：9395	0.00％	34.28765802
					9	22	SEQ ID NO：9396	0.00％	26.532116325
10	5	SEQ ID NO：9397	0.00％	25.26691266
					11	21	SEQ ID NO：9398	0.00％	4.72873208445
12	11	SEQ ID NO：9399	0.00％	2.638538265
					13	8	SEQ ID NO：9400	0.00％	2.4274552038
14	4	SEQ ID NO：9401	0.00％	1.7415324
					15	20	SEQ ID NO：9402	0.00％	1.6025526
16	13	SEQ ID NO：9403	0.00％	1.453803297
					17	35	SEQ ID NO：9404	0.00％	1.36878336
18	3	SEQ ID NO：9405	0.00％	0.824619
					19	33	SEQ ID NO：9406	0.00％	0.513774

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	22	SEQ ID NO：9407	0.09％	3636.068421648
2	4	SEQ ID NO：9408	0.02％	1107.960876
					3	15	SEQ ID NO：9409	0.02％	836.2525104
4	16	SEQ ID NO：9410	0.00％	150.9313176
					5	12	SEQ ID NO：9411	0.00％	76.55002416
6	1	SEQ ID NO：9412	0.00％	49.0273014
					7	10	SEQ ID NO：9413	0.00％	42.1638414747
8	20	SEQ ID NO：9414	0.00％	9.29480508
					9	24	SEQ ID NO：9415	0.00％	9.2669346
10	13	SEQ ID NO：9416	0.00％	7.96581954
					11	21	SEQ ID NO：9417	0.00％	5.051306761875
12	5	SEQ ID NO：9418	0.00％	2.6941464
					13	11	SEQ ID NO：9419	0.00％	2.3839914
14	34	SEQ ID NO：9420	0.00％	1.465422
					15	2	SEQ ID NO：9421	0.00％	0.70794
16	9	SEQ ID NO：9422	0.00％	0.6513048
					17	19	SEQID NO：9423	0.00％	0.51882640425

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	33	SEQ ID NO：9424	1.66％	0.6

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	13	SEQ ID NO：9425	0.22％	12
2	2	SEQ ID NO：9426	0.07％	4
					3	9	SEQ ID NO：9427	0.07％	4
4	16	SEQ ID NO：9428	0.07％	4
					5	23	SEQ ID NO：9429	0.07％	4
6	5	SEQ ID NO：9430	0.02％	1.2
					7	15	SEQ ID NO：9431	0.01％	1

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	4	SEQ ID NO：9432	0.07％	4
2	10	SEQ ID NO：9433	0.07％	4
					3	12	SEQ ID NO：9434	0.07％	4
4	15	SEQ ID NO：9435	0.07％	4
					5	22	SEQ ID NO：9436	0.07％	4
6	13	SEQ ID NO：9437	0.02％	1.2

Table 24: SEQ ID NO: 6050 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	47	SEQ ID NO：9438	0.01％	0.75
2	21	SEQ ID NO：9439	0.00％	0.5
					3	53	SEQ ID NO：9440	0.00％	0.5

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	16	SEQ ID NO：9441	0.04％	2.5
2	71	SEQ ID NO：9442	0.04％	2.5
					3	47	SEQ ID NO：9443	0.02％	1.5
4	62	SEQ ID NO：9444	0.01％	0.9

5	20	SEQ ID NO：9445	0.00％	0.5
					6	38	SEQ ID NO：9446	0.00％	0.5

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	54	SEQ ID NO：9447	0.02％	2.7
2	17	SEQ ID NO：9448	0.01％	2
					3	3	SEQ ID NO：9449	0.01％	1.8

HLA A3-10mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	22	SEQ ID NO：9450	0.09％	12
2	16	SEQ ID NO：9451	0.01％	2
					3	54	SEQ ID NO：9452	0.00％	0.9

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	70	SEQ ID NO：9453	2.10％	33.6
2	7	SEQ ID NO：9454	1.12％	18
					3	60	SEQ ID NO：9455	0.46％	7.5
4	54	SEQ ID NO：9456	0.37％	6
					5	14	SEQ ID NO：9457	0.31％	5
6	19	SEQ ID NO：9458	0.30％	4.8
					7	47	SEQ ID NO：9459	0.30％	4.8
8	12	SEQ ID NO：9460	0.25％	4
					9	15	SEQ ID NO：9461	0.25％	4
10	67	SEQ ID NO：9462	0.25％	4
					11	21	SEQ ID NO：9463	0.18％	3
12	37	SEQ ID NO：9464	0.06％	1
					13	27	SEQ ID NO：9465	0.03％	0.5

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	14	SEQ ID NO：9466	12.52％	200
2	7	SEQ ID NO：9467	0.93％	15
					3	11	SEQ ID NO：9468	0.75％	12
4	60	SEQ ID NO：9469	0.56％	9
					5	18	SEQ ID NO：9470	0.45％	7.2

6	46	SEQ ID NO：9471	0.45％	7.2
					7	53	SEQ ID NO：9472	0.37％	6
8	69	SEQ ID NO：9473	0.35％	5.6
					9	66	SEQ ID NO：9474	0.25％	4
10	20	SEQ ID NO：9475	0.12％	2
					11	47	SEQ ID NO：9476	0.07％	1.2
12	36	SEQ ID NO：9477	0.06％	1
					13	26	SEQ ID NO：9478	0.04％	0.75
14	70	SEQ ID NO：9479	0.04％	0.72

HLA A 0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	54	SEQ ID NO：9480	0.02％	881.199
2	26	SEQ ID NO：9481	0.00％	95.013
					3	61	SEQ ID NO：9482	0.00％	93.69648
4	19	SEQ ID NO：9483	0.00％	40.2894864
					5	74	SEQ ID NO：9484	0.00％	12.6684
6	35	SEQ ID NO：9485	0.00％	10.34586
					7	69	SEQ ID NO：9486	0.00％	3.3704706
8	13	SEQ ID NO：9487	0.00％	1.656
					9	15	SEQ ID NO：9488	0.00％	1.47537042
10	68	SEQ IDNO：9489	0.00％	0.966
					11	22	SEQ ID NO：9490	0.00％	0.942678
12	12	SEQ ID NO：9491	0.00％	0.7669695
					13	36	SEQ ID NO：9492	0.00％	0.52661835

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	61	SEQ ID NO：9493	0.00％	93.69648
2	25	SEQ ID NO：9494	0.00％	63.33035625
					3	34	SEQ ID NO：9495	0.00％	50.232
4	53	SEQ ID NO：9496	0.00％	45.2838375
					5	26	SEQ ID NO：9497	0.00％	14.35752
6	27	SEQ ID NO：9498	0.00％	2.8557858
					7	17	SEQ ID NO：9499	0.00％	2.3973222
8	36	SEQ ID NO：9500	0.00％	1.798209
					9	69	SEQ ID NO：9501	0.00％	1.03521597
10	67	SEQ ID NO：9502	0.00％	0.966
					11	68	SEQ ID NO：9503	0.00％	0.910938
12	11	SEQ ID NO：9504	0.00％	0.7669695

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	17	SEQ ID NO：9505	2.22％	0.8

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	16	SEQ ID NO：9506	5.55％	2

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	27	SEQ ID NO：9507	0.37％	20
2	54	SEQ ID NO：9508	0.22％	12
					3	70	SEQ ID NO：9509	0.22％	12
4	67	SEQ ID NO：9510	0.11％	6
					5	12	SEQ ID NO：9511	0.07％	4
6	15	SEQ ID NO：9512	0.07％	4
					7	19	SEQ ID NO：9513	0.07％	4
8	49	SEQ ID NO：9514	0.03％	2
					9	69	SEQ ID NO：9515	0.03％	1.8
10	47	SEQ ID NO：9516	0.02％	1.2
					11	5	SEQ ID NO：9517	0.01％	1
12	9	SEQ ID NO：9518	0.01％	1
					13	35	SEQ ID NO：9519	0.01％	1
14	37	SEQ ID NO：9520	0.01％	0.6
					15	68	SEQ ID NO：9521	0.01％	0.6

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	69	SEQ ID NO：9522	0.66％	36
2	53	SEQ ID NO：9523	0.22％	12
					3	5	SEQ ID NO：9524	0.13％	7.5
4	66	SEQ ID NO：9525	0.11％	6
					5	11	SEQ ID NO：9526	0.07％	4
6	27	SEQ ID NO：9527	0.07％	4
					7	46	SEQ ID NO：9528	0.07％	4
8	18	SEQ ID NO：9529	0.02％	1.2
					9	9	SEQ ID NO：9530	0.01％	1
10	26	SEQ ID NO：9531	0.01％	1

11	25	SEQ ID NO：9532	0.01％	0.75
					12	17	SEQ ID NO：9533	0.01％	0.6
13	36	SEQ ID NO：9534	0.01％	0.6
					14	68	SEQ ID NO：9535	0.01％	0.6
15	35	SEQ ID NO：9536	0.00％	0.5
					16	42	SEQ ID NO：9537	0.00％	0.5
17	73	SEQ ID NO：9538	0.00％	0.5

Table 25: SEQ ID NO: 6052 epitope

HLA A1-9mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	365	SEQ ID NO：9539	0.8％	45
2	397	SEQ ID NO：9540	0.44％	25
					3	229	SEQ ID NO：9541	0.32％	18
4	103	SEQ ID NO：9542	0.17％	10
					5	338	SEQ ID NO：9543	0.17％	10
6	251	SEQ ID NO：9544	0.16％	9
					7	79	SEQ ID NO：9545	0.11％	6.25
8	119	SEQ ID NO：9546	0.10％	6
					9	361	SEQ ID NO：9547	0.08％	5
10	60	SEQ ID NO：9548	0.04％	2.25
					11	101	SEQ ID NO：9549	0.04％	2.25
12	278	SEQ ID NO：9550	0.04％	2.25
					13	23	SEQ ID NO：9551	0.02％	1.25
14	164	SEQ ID NO：9552	0.02％	1.25
					15	165	SEQ ID NO：9553	0.02％	1.25
16	295	SEQ ID NO：9554	0.02％	1.25
					17	172	SEQ ID NO：9555	0.01％	0.9
18	0	SEQ ID NO：9556	0.01％	0.75
					19	311	SEQ ID NO：9557	0.01％	0.75
20	78	SEQ ID NO：9558	0.01％	0.625

HLA A1-10mers
					Maximum possible score Using this molecular type				5625
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	114	SEQ ID NO：9559	1.11％	62.5
2	134	SEQ ID NO：9560	0.8％	45
					3	365	SEQ ID NO：9561	0.8％	45
4	77	SEQ ID NO：9562	0.66％	37.5
					5	103	SEQ ID NO：9563	0.44％	25

6	23	SEQ ID NO：9564	0.22％	12.5
					7	338	SEQ ID NO：9565	0.17％	10
8	361	SEQ ID NO：9566	0.17％	10
					9	324	SEQ ID NO：9567	0.11％	6.25
10	375	SEQ ID NO：9568	0.11％	6.25
					11	79	SEQ ID NO：9569	0.04％	2.5
12	295	SEQ ID NO：9570	0.04％	2.5
					13	346	SEQ ID NO：9571	0.04％	2.5
14	378	SEQ ID NO：9572	0.03％	2
					15	251	SEQ ID NO：9573	0.03％	1.8
16	214	SEQ ID NO：9574	0.02％	1.125
					17	160	SEQ ID NO：9575	0.01％	1
18	172	SEQ ID NO：9576	0.01％	0.9
					19	229	SEQ ID NO：9577	0.01％	0.9
20	376	SEQ ID NO：9578	0.01％	0.9

HLA A3-9mers
					Maximum possible score Using this molecular type				12150
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	229	SEQ ID NO：9579	0.49％	60
2	361	SEQ ID NO：9580	0.27％	33.75
					3	330	SEQ ID NO：9581	0.16％	20
4	218	SEQ ID NO：9582	0.09％	12
					5	338	SEQ ID NO：9583	0.04％	6
6	352	SEQ ID NO：9584	0.04％	6
					7	103	SEQ ID NO：9585	0.04％	5.4
8	291	SEQ ID NO：9586	0.01％	2
					9	241	SEQ ID NO：9587	0.01％	1.8
10	290	SEQ ID NO：9588	0.01％	1.8
					11	316	SEQ ID NO：9589	0.01％	1.8
12	222	SEQ ID NO：9590	0.01％	1.35
					13	266	SEQ ID NO：9591	0.01％	1.35
14	53	SEQ ID NO：9592	0.00％	1
					15	100	SEQ ID NO：9593	0.00％	0.9
16	138	SEQ ID NO：9594	0.00％	0.9
					17	240	SEQ ID NO：9595	0.00％	0.9
18	119	SEQ ID NO：9596	0.00％	0.675
					19	44	SEQ ID NO：9597	0.00％	0.6
20	161	SEQ IDNO：9598	0.00％	0.6

HLA A3-10mers
		Maximum possible score Using this molecular type	12150

Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
						1	338	SEQ ID NO：9599	0.49％	60
2	160	SEQ ID NO：9600	0.32％	40
					3	352	SEQ ID NO：9601	0.24％	30
4	361	SEQ ID NO：9602	0.18％	22.5
					5	103	SEQ ID NO：9603	0.13％	16.2
6	290	SEQ ID NO：9604	0.07％	9
					7	351	SEQ ID NO：9605	0.07％	9
8	44	SEQ ID NO：9606	0.04％	6
					9	228	SEQ ID NO：9607	0.03％	4.05
10	394	SEQ ID NO：9608	0.02％	3
					11	240	SEQ ID NO：9609	0.02％	2.7
12	100	SEQ ID NO：9610	0.01％	1.8
					13	114	SEQ ID NO：9611	0.01％	1.8
14	93	SEQ ID NO：9612	0.01％	1.5
					15	134	SEQ ID NO：9613	0.01％	1.5
16	221	SEQ ID NO：9614	0.01％	1.35
					17	330	SEQ ID NO：9615	0.00％	1.2
18	112	SEQ ID NO：9616	0.00％	0.9
					19	218	SEQ ID NO：9617	0.00％	0.9
20	55	SEQID NO：9618	0.00％	0.6

HLA A24-9mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	345	SEQ ID NO：9619	1.50％	24
2	306	SEQ ID NO：9620	0.75％	12
					3	222	SEQ ID NO：9621	0.54％	8.64
4	111	SEQ ID NO：9622	0.51％	8.25
					5	159	SEQ ID NO：9623	0.45％	7.2
6	219	SEQ ID NO：9624	0.45％	7.2
					7	283	SEQ ID NO：9625	0.45％	7.2
8	266	SEQ ID NO：9626	0.42％	6.72
					9	56	SEQ ID NO：9627	0.41％	6.6
10	131	SEQ ID NO：9628	0.37％	6
					11	214	SEQ ID NO：9629	0.37％	6
12	297	SEQ ID NO：9630	0.37％	6
					13	86	SEQ ID NO：9631	0.31％	5
14	122	SEQ ID NO：9632	0.31％	5
					15	48	SEQ ID NO：9633	0.30％	4.8
16	105	SEQ ID NO：9634	0.30％	4.8
					17	213	SEQ ID NO：9635	0.30％	4.8

18	323	SEQ ID NO：9636	0.30％	4.8
					19	338	SEQ ID NO：9637	0.30％	4.8
20	399	SEQ ID NO：9638	0.30％	4.8

HLA A24-10mers
					Maximum possible score Using this molecular type				1596.672
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	65	SEQ ID NO：9639	0.93％	15
2	306	SEQ ID NO：9640	0.75％	12
					3	95	SEQ ID NO：9641	0.66％	10.56
4	36	SEQ ID NO：9642	0.60％	9.6
					5	385	SEQ ID NO：9643	0.50％	8
6	111	SEQ ID NO：9644	0.46％	7.5
					7	104	SEQ ID NO：9645	0.45％	7.2
8	214	SEQ ID NO：9646	0.45％	7.2
					9	221	SEQ ID NO：9647	0.45％	7.2
10	277	SEQ ID NO：9648	0.45％	7.2
					11	150	SEQ ID NO：9649	0.37％	6
12	152	SEQ ID NO：9650	0.37％	6
					13	158	SEQ ID NO：9651	0.37％	6
14	171	SEQ ID NO：9652	0.37％	6
					15	343	SEQ ID NO：9653	0.37％	6
16	110	SEQ ID NO：9654	0.34％	5.5
					17	85	SEQ ID NO：9655	0.31％	5
18	47	SEQ ID NO：9656	0.30％	4.8
					19	213	SEQ ID NO：9657	0.30％	4.8
20	218	SEQ ID NO：9658	0.30％	4.8

HLA A 0201-9mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	222	SEQ ID NO：9659	0.03％	1267.10434728
2	226	SEQ ID NO：9660	0.00％	69.552
					3	316	SEQ ID NO：9661	0.00％	50.232
4	351	SEQ ID NO：9662	0.00％	31.24872
					5	159	SEQ ID NO：9663	0.00％	13.6235739
6	406	SEQ ID NO：9664	0.00％	11.4264
					7	165	SEQ ID NO：9665	O.00％	8.14407
8	238	SEQ ID NO：9666	0.00％	7.0518
					9	138	SEQ ID NO：9667	0.00％	5.112072
10	130	SEQ ID NO：9668	0.00％	3.00547233
					11	303	SEQ ID NO：9669	0.00％	2.59578

12	157	SEQ ID NO：9670	0.00％	2.412585
					13	219	SEQ ID NO：9671	0.00％	2.103255861
14	305	SEQ ID NO：9672	0.00％	1.86369
					15	158	SEQ ID NO：9673	0.00％	1.646892
16	331	SEQ ID NO：9674	0.00％	1.614048
					17	399	SEQ ID NO：9675	0.00％	1.442246832
18	324	SEQ ID NO：9676	0.00％	1.319625
					19	312	SEQ ID NO：9677	0.00％	1.233099
20	262	SEQ ID NO：9678	0.00％	0.966

HLA A 0201-10mers
					Maximum possible score Using this molecular type				3925227.1
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	221	SEQ ID NO：9679	0.00％	309.0498408
2	112	SEQ ID NO：9680	0.00％	98.26704
					3	330	SEQ ID NO：9681	0.00％	98.26704
4	158	SEQ ID NO：9682	0.00％	36.31608
					5	218	SEQ ID NO：9683	0.00％	24.0754248
6	124	SEQ ID NO：9684	0.00％	12.2199
					7	55	SEQ ID NO：9685	0.00％	10.467576
8	315	SEQ ID NO：9686	0.00％	7.7274204
					9	350	SEQ ID NO：9687	0.00％	4.296699
10	405	SEQ ID NO：9688	0.00％	4.286487
					11	388	SEQ ID NO：9689	0.00％	4.054785
12	322	SEQ ID NO：9690	0.00％	3.883803
					13	130	SEQ ID NO：9691	0.00％	3.428691903
14	45	SEQ ID NO：9692	0.00％	3.411230625
					15	132	SEQ ID NO：9693	0.00％	2.99943
16	410	SEQ ID NO：9694	0.00％	2.63718
					17	316	SEQ ID NO：9695	0.00％	2.48686074
18	104	SEQ ID NO：9696	0.00％	2.477311485
					19	164	SEQ ID NO：9697	0.00％	2.2011
20	282	SEQ ID NO：9698	0.00％	2.16591

HLA A 1101-9mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	361	SEQ ID NO：9699	16.66％	6
2	53	SEQ ID NO：9700	2.77％	1
					3	240	SEQ ID NO：9701	1.66％	0.6
4	241	SEQ ID NO：9702	1.66％	0.6

HLA A 1101-10mers
					Maximum possible score Using this molecular type				36
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	361	SEQ ID NO：9703	16.66％	6
2	93	SEQ ID NO：9704	8.33％	3
					3	338	SEQ ID NO：9705	3.33％	1.2
4	134	SEQ ID NO：9706	2.77％	1
					5	228	SEQ ID NO：9707	2.5％	0.9
6	160	SEQ ID NO：9708	2.22％	0.8
					7	239	SEQ ID NO：9709	1.66％	0.6
8	240	SEQ ID NO：9710	1.66％	0.6
					9	257	SEQ ID NO：9711	1.66％	0.6
10	379	SEQ ID NO：9712	1.66％	0.6

HLA B7-9mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	105	SEQ ID NO：9713	14.81％	800
2	66	SEQ ID NO：9714	1.48％	80
					3	93	SEQ ID NO：9715	0.92％	50
4	257	SEQ ID NO：9716	0.55％	30
					5	323	SEQ ID NO：9717	0.37％	20
6	211	SEQ ID NO：9718	0.22％	12
					7	219	SEQ ID NO：9719	0.22％	12
8	403	SEQ ID NO：9720	0.18％	10
					9	343	SEQ ID NO：9721	0.14％	8
10	12	SEQ ID NO：9722	0.11％	6
					11	113	SEQ ID NO：9723	0.11％	6
12	48	SEQ ID NO：9724	0.07％	4
					13	56	SEQ ID NO：9725	0.07％	4
14	150	SEQ ID NO：9726	0.07％	4
					15	153	SEQ ID NO：9727	0.07％	4
16	159	SEQ ID NO：9728	0.07％	4
					17	213	SEQ ID NO：9729	0.07％	4
18	216	SEQ ID NO：9730	0.07％	4
					19	222	SEQ ID NO：9731	0.07％	4
20	283	SEQ ID NO：9732	0.07％	4

HLA B7-10mers
					Maximum possible score Using this molecular type				5400
Sorting	Initiation site	Sequence of	As a percentage of the maximum score	Score recording
					1	36	SEQ ID NO：9733	1.48％	80

2	150	SEQ ID NO：9734	1.48％	80
					3	343	SEQ ID NO：9735	1.48％	80
4	12	SEQ ID NO：9736	1.11％	60
					5	308	SEQ ID NO：9737	1.11％	60
6	130	SEQ ID NO：9738	0.37％	20
					7	55	SEQ ID NO：9739	0.22％	12
8	210	SEQ ID NO：9740	0.22％	12
					9	218	SEQ ID NO：9741	0.22％	12
10	201	SEQ ID NO：9742	0.18％	10
					11	121	SEQ ID NO：9743	0.14％	8
12	391	SEQ ID NO：9744	0.13％	7.5
					13	112	SEQ ID NO：9745	0.11％	6
14	385	SEQ ID NO：9746	0.11％	6
					15	47	SEQ ID NO：9747	0.07％	4
16	66	SEQ ID NO：9748	0.07％	4
					17	95	SEQ ID NO：9749	0.07％	4
18	104	SEQ ID NO：9750	0.07％	4
					19	152	SEQ ID NO：9751	0.07％	4
20	158	SEQ ID NO：9752	0.07％	4

Table 26: cloning sequence for E.coli expression

ORF	DNA length bp	Cloning
				pET	pGEX
		P28	537			NdeI/XhoI
P65	1917			NheI/HindIII
		Nsp1A	2495			NheI/XhoI
Nsp1B	2153			NdeI/XhoI
		Nsp1C	2612			NdeI/XhoI
Nsp2A
		431	NdeI/XhoI	BamHI/XhoI
Nsp2B
		426	NdeI/XhoI	BamHI/XhoI
Nsp3
		870	NdeI/XhoI
Nsp4					249	NdeI/XhoI	BamHI/XhoI
	Nsp5	594	NheI/XhoI
Nsp6					339	NdeI/XhoI	BamHI/XhoI
	Nsp7	417	NdeI/XhoI	BamHI/XhoI
Nsp9A					1385	NheI/XhoI
	Nsp9B	1409	NdeI/XhoI
Nsp10					1803	NheI/XhoI
	Nsp11	1581	NdeI/XhoI
Nsp12					1038	NdeI/HindIII
	Nsp13	897	NdeI/XhoI
Spike (S1)					1946	NdeI/XhoI
	Spike (S2)	1598	NdeI/XhoI
Spike (S1-S2)					3545	NdeI/XhoI
	HR1	287	NdeI/XhoI	BamHI/XhoI
HR2					146	NdeI/XhoI	BamHI/XhoI
	ORF3 100	525	NdeI/XhoI
ORF4					465	NdeI/XhoI
	Envelope (E)	231	NdeI/XhoI	BamHI/XhoI
Substrate (M) 100					366	NdeI/XhoI	BamHI/XhoI
	ORF7 18	137	NdeI/XhoI	BamHI/XhoI
ORF8					369	NdeI/XhoI	BamHI/XhoI
	ORF9	135	NdeIzXhoI	BamHI/XhoI
ORF10					120	NheI/XhoI	BamHI/XhoI
	ORF11	255	NdeI/XhoI	BamHI/XhoI
Nucleocapsid (N)					1269	NdeI/EcoRI
	ORF12	297	NdeI/EcoRI	BamHI/EcoRI

Table 27: primer and method for producing the same

ORF	Forward primer	Reverse primer
			P28	9803	9818
P65	9804	9819
			Nsp1A	9805	9820
Nsp1B	9806	9821
			Nsp1C	9807	9822
Nsp2+Nsp3	9808	9823
			Nsp4-Nsp7	9809	9824
Nsp9A	9810	9825
			Nsp9B	9811	9826
Nsp10	9812	9827
			Nsp11	9813	9828
Nsp12-Nsp13	9814	9829
			ORF3-ORF4	9815	9830
Env-ORF10	9816	9831
			ORF11-ORF12	9817	9832

Table 28: primer and method for producing the same

ORF	Forward primer	Reverse primer
			Nsp2A	SEQ ID NO：9833	SEQ ID NO：9858
Nsp2B	SEQ ID NO：9834	SEQ ID NO：9859
			Nsp3	SEQ ID NO：9835	SEQ ID NO：9860
Nsp4	SEQ ID NO：9836	SEQ ID NO：9861
			Nsp5	SEQ ID NO：9837	SEQ ID NO：9862
Nsp6	SEQ ID NO：9838	SEQ ID NO：9863
			Nsp7	SEQ ID NO：9839	SEQ ID NO：9864
Nsp12	SEQ ID NO：9840	SEQ ID NO：9865
			Nsp13	SEQ ID NO：9841	SEQ ID NO：9866
Spike S1	SEQ ID NO：9842	SEQ ID NO：9867
			Spike S2	SEQ ID NO：9843	SEQ ID NO：9868
Spikes S1-S2	SEQ ID NO：9844	SEQ ID NO：9869
			HR1	SEQ ID NO：9845	SEQ ID NO：9870
HR2	SEQ ID NO：9846	SEQ ID NO：9871
			Orf3Δ100	SEQ ID NO：9847	SEQ ID NO：9872
Orf4	SEQ ID NO：9848	SEQ ID NO：9873
			Env E	SEQ ID NO：9849	SEQ ID NO：9874
Matrix M.DELTA.100	SEQ ID NO：9850	SEQ ID NO：9875
			Orf7Δ18	SEQ ID NO：9851	SEQ ID NO：9876
Orf8	SEQ ID NO：9852	SEQ ID NO：9877
			Orf9	SEQ ID NO：9853	SEQ ID NO：9878
Orf10	SEQ ID NO：9854	SEQ ID NO：9879
			Orf11	SEQ ID NO：9855	SEQ ID NO：9880
Nucleocapsid N	SEQ ID NO：9856	SEQ ID NO：9881
			Orf12	SEQ ID NO：9857	SEQ ID NO：9882

Table 29: cloning, purification and expression in E.coli

SARS CoV ORFs	M.WKd	Cloning	Expression of	Purification of
					P28	19,7	+	+	his sol
P65
		70,3	+	+	his sol
	Nsp1A (N-terminal)					91,6	+	+	his ins
Nsp1B (core)		80,8	+	-
	Nsp1C (C-terminal)					95,3	+	-
Nsp2A (N-terminal)		15,8	+	+	his ins
	Nsp2B (C-terminal)					15,5	+	+	his sol
Nsp3
		31,9	+	-
	Nsp4					9,1	+	+	his sol
Nsp5
		21,8	+	+	his sol
	Nsp6
		12,4	+	+	his sol
Nsp7
		15,3	+	+	his ins
	Nsp9A (N-terminal)					50,8	+	-
Nsp9B (C-terminal)		51,6	+	+	his ins
	Nsp10					66	-
Nsp11		58	-
	Nsp12					38	-
Nsp13		32,7	+	+	his ins
	Spike (S1-his)					71,3	+	+	his ins
Spike (S2-his)		58,6	Cloning	-
	Spike (S1S2-his)					130	+	+	his ins
HR1		11	+	+	his ins
	HR2					5,4	+	+	his sol
ORF3Δ100
1		19,1	+	-
	ORF4					16,9	+	+	his ins (three-body)
Envelope (E)		34,3	+	+	gst ins(IB)
	Matrix (M) Δ 100					13,3	+	+	his ins
ORF7Δ182		31	+	+	gst sol
	ORF8					39,5	+	+	gst ins(IB)
ORF9		30,8	+	+	gst sol
	ORF10					30,3	+	+	gst ins(IB)
ORF11		35,2	+	+	gst ins(IB)
	Nucleocapsid (N)					43,6	+	+	his ins
ORF12		36,7	+	+	his ins

Table 30: coli expression, purification and yield

Protein	Marking	Purity (%)	Yield (mg/l)
				Nsp2A (N-terminal)	His	95	1.7
Nsp2B (C-terminal)	His	95	4.1
				Nsp4	His	95	12.6
Nsp5	His	95	5.88
				Nsp6	His	95	8.1
P28	His	95	1
				P65	His	80	0.553
HR2	His	95	11.9
				HR1	His	80	2.64
Nsp1A	His	95	0.267
				Spikes S1-S2	His	80	0.381
Matrix M	His	85	12.4
				ORF7	GST	85	4.9

Table 31: primer and method for producing the same

SEQ ID NO：	Sorting	Model (model)	Local part	(position)
					10235	F1	1	1	(106)
10236	F2	2	1	(728)
					10237	F3	3	1	(112)
10238	F4	5	2	(1331)
					10239	F5	6	1	(12)
10240	F6	6	1	(346)
					10241	F7	8	1	(904)
10242	F8	9	1	(1016)
					10243	F9	9	1	(1015)
10244	F10	9	1	(719)
					10245	F11	9	1	(720)
10246	F12	10	1	(724)
					10247	R1	2	1	(1283)
10248	R2	4	1	(756)
					10249	R3	4	1	(758)
10250	R4	5	2	(259)
					10251	R5	6	1	(54)
10252	R6	7	1	(648)
					10253	R7	8	1	(948)
10254	R8	8	1	(260)
					10255	R9	9	1	(1282)
10256	R10	9	1	(950)
					10257	R11	9	1	(756)
10258	R12	10	1	(132)

Table 32: primer and method for producing the same

Primer list: (Forward direction)
						Sorting	Score recording		Sequence of	(position)
Model (model)	Local part
		F1F2F3F4F5F6F7F8F9F10F11F12F13F14F15F16F17F18F19F20F21F22F23F24F25	7777799101111121214141617171717202028282929	1111111111111111111111111	SEQ ID NO：10352SEQ ID NO：10353SEQ ID NO：10354SEQ ID NO：10355SEQ ID NO：10356SEQ ID NO：10357SEQ ID NO：10358SEQ ID NO：10359SEQ ID NO：10360SEQ ID NO：10361SEQ ID NO：10362SEQ ID NO：10363SEQ ID NO：10364SEQ ID NO：10365SEQ ID NO：10366SEQ ID NO：10367SEQ ID NO：10368SEQ ID NO：10369SEQ ID NO：10370SEQ ID NO：10371SEQ ID NO：10372SEQ ID NO：10373SEQ ID NO：10374SEQ ID NO：10375SEQ ID NO：10376		(290)(291)(294)(292)(293)(198)(199)(33)(200)(299)(298)(297)(35)(34)(300)(295)(296)(175)(36)(202)(201)(204)(203)(269)(268)
Primer List (reverse)
						Sorting	Model (model)	Local part	Sequence of	(position)
R1R2R3R4R5R6R7R8R9R10R11R12R13R14R15R16R17R18R19R20R21R22R23R24R25	791111121213141415161717171718202021222829323536	1111111111111111111111111	SEQ ID NO：10377SEQ ID NO：10378SEQ ID NO：10379SEQ ID NO：10380SEQ ID NO：10381SEQ ID NO：10382SEQ ID NO：10383SEQ ID NO：10384SEQ ID NO：10385SEQ ID NO：10386SEQ ID NO：10387SEQ ID NO：10388SEQ ID NO：10389SEQ ID NO：10390SEQ ID NO：10391SEQ ID NO：10392SEQ ID NO：10393SEQ ID NO：10394SEQ ID NO：10395SEQ ID NO：10396SEQ ID NO：10397SEQ ID NO：10398SEQ ID NO：10399SEQ ID NO：10400SEQ ID NO：10401	(337)(229)(230)(338)(207)(338)(231)(80)(232)(82)(340)(83)(206)(82)(337)(341)(340)(233)(79)(213)(236)(317)(391)(57)(237)
						Primer list (left): SEQ ID NOs：10402-10433					Primer list (right): SEQ ID NOs：10434-10464
Primer list (forward): SEQ ID NOs：10465-10484					Primer list (reverse): SEQ ID NOs：10485-10504

Table 33: primer and method for producing the same

Primer list (Forward)
					Sorting	Score recording		Sequence of stepsColumn(s) of	(position)
Model (model)	Local part
		F1F2F3F4F5F6F7F8F9F10F11F12F13F14F15F16F17F18F19F20F21F22F23F24F25F26F27F28F29F30F31F32F33F34F35F36F37F38F39F40F41F42F43F44F45F46F47F48F49F50	12234445555666666667777777778888888999999101010101010111111	11111111111111111111111111111111111111111111111111		SEQ ID NO：10580SEQ ID NO：10581SEQ ID NO：10582SEQ ID NO：10583SEQ ID NO：10584SEQ ID NO：10585SEQ ID NO：10586SEQ ID NO：10587SEQ ID NO：10588SEQ ID NO：10589SEQ ID NO：10590SEQ ID NO：10591SEQ ID NO：10592SEQ ID NO：10593SEQ ID NO：10594SEQ ID NO：10595SEQ ID NO：10596SEQ ID NO：10597SEQ ID NO：10598SEQ ID NO：10599SEQ ID NO：10600SEQ ID NO：10601SEQ ID NO：10602SEQ ID NO：10603SEQ ID NO：10604SEQ ID NO：10605SEQ ID NO：10606SEQ ID NO：10607SEQ ID NO：10608SEQ ID NO：10609SEQ ID NO：10610SEQ ID NO：10611SEQ ID NO：10612SEQ ID NO：10613SEQ ID NO：10614SEQ ID NO：10615sEQ ID NO：10616SEQ ID NO：10617SEQ ID NO：10618SEQ ID NO：10619SEQ ID NO：10620SEQ ID NO：10621SEQ ID NO：10622SEQ ID NO：10623SEQ ID NO：10624SEQ ID NO：10625SEQ ID NO：10626SEQ ID NO：10627SEQ ID NO：10628SEQ ID NO：10629	(637)(439)(440)(729)(696)(697)(111)(867)(868)(869)(640)(438)(437)(436)(732)(635)(457)(458)(636)(854)(855)(581)(853)(342)(343)(112)(94)(642)(638)(639)(730)(641)(731)(326)(325)(517)(701)(208)(209)(702)(210)(634)(694)(693)(728)(695)(95)(455)(456)(454)

Primer List (reverse)
						Sorting	Score recording		Sequence of	(position)
Model (model)	Local part
		R1R2R3R4R5R6R7R8R9R10R11R12R13R14R15R16R17R18R19R20R21R22R23R24R25R26R27R28R29R30R31R32R33R34R35R36R37R38R39R40R41R42R43R44R45R46R47R48R49R50	11233444445566666677777777888889999101010101011111111111112121212	11111111111111111111111111111111111111111111111111	SEQ ID NO：10630SEQ ID NO：10631SEQ ID NO：10632SEQ ID NO：10633SEQ ID NO：10634SEQ ID NO：10635SEQ ID NO：10636SEQ ID NO：10637SEQ ID NO：10638SEQ ID NO：10639SEQ ID NO：10640SEQ ID NO：10641SEQ ID NO：10642SEQ ID NO：10643SEQ ID NO：10644SEQ ID NO：10645SEQ ID NO：10646SEQ ID NO：10647SEQ ID NO：10648SEQ ID NO：10649SEQ ID NO：10650SEQ ID NO：10651SEQ ID NO：10652SEQ ID NO：10653SEQ ID NO：10654SEQ ID NO：10655SEQ ID NO：10656SEQ ID NO：10657SEQ ID NO：10658SEQ ID NO：10659SEQ ID NO：10660SEQ ID NO：10661SEQ ID NO：10662SEQ ID NO：10663SEQ ID NO：10664SEQ ID NO：10665SEQ ID NO：10666SEQ ID NO：10667SEQ ID NO：10668SEQ ID NO：10669SEQ ID NO：10670SEQ ID NO：10671SEQ ID NO：10672SEQ ID NO：10673SEQ ID NO：10674SEQ ID NO：10675SEQ ID NO：10676SEQ ID NO：10677SEQ ID NO：10678SEQ ID NO：10679		(367)(666)(464)(669)(750)(720)(465)(370)(668)(135)(901)(667)(609)(464)(665)(486)(356)(758)(366)(368)(136)(675)(366)(608)(884)(120)(355)(671)(756)(751)(666)(242)(543)(724)(482)(121)(662)(750)(719)(242)(484)(375)(728)(373)(998)(486)(881)(882)(244)(1003)
Primer list (left): SEQ ID NOs：10680-10974						Primer list (right): SEQ ID NOs：10975-11282
					Primer list (forward): SEQ ID NOs：11283-11302					Primer list (reverse): SEQ ID NOs：11303-11322

Watch 34

Watch 35

TABLE 35 continuation

TABLE 35 continuation

TABLE 35 continuation

Brief description of the sequences

SEQ ID NO：	Description of the invention
		1	TOR from the genome science center of the great Britain Columbia Canada2Genome assembly draft of isolate sequences. TOR2_ draft _ genome _ assembly _120403 Release I
2	CDC SARS-CoV strain sequence complete nucleotide sequence (Urbani strain)
		3-20	Group-specific coronavirus gene product > Feline Infectious Peritonitis Virus (FIPV)3/4 ═ ORF 3 b; 5/6 ═ ORF 3X; 7/8 ═ ORF 3A > dogCoronavirus 9/10 ═ ORF 7 b; 11/12 ═ ORF 7a > avian infectious bronchitis virus 13/14 ═ ORF 5 b; 15/16 ═ ORF 5 a; 17/18 ═ ORF 3 a; 19/20 ═ ORF 3b
21-520	500 primers in the left part
		521-1020	500 primers in the right part
1021-3520	Forward primers of Table 4
		3521-6020	Reverse primers of Table 4
6021-6026	The primers of FIG. 9
		6027-6033	The primers of FIG. 11
6034-6038	Primers http from the following website: new jm.org/cgi/print/NEJMoa030781v2. pdf
		6039-6051	PEP 1-PEP 13
6052	Extended PEP13
		6053-6056	229E human coronavirus sequences
6057-6060	TGV sequences
		6061-6064	PEDV sequences
6065-6068	Bovine coronavirus sequences
		6069-6071	Murine hepatitis virus sequences
6072-6075	AIBV sequence
		6076-6170	Primer sequence (Forward)
6171-6265	Primer sequences (reverse)
		6266-6304	Primer sequence (Forward)
6305-6343	Primer sequences (reverse)
		6344-6366	Primer sequence (Forward)
6367-6392	Primer sequences (reverse)
		6393-6440	Primer sequence (Forward) F1-F48
6441-6487	Primer sequence (reverse) R1-R47
		6488-6559	Primer sequences
6560-6568	Primer sequences
		6569	Nsp2 protease (3CL-PRO) sequence within SARS coronavirus
6570-72	Nsp2 protease from bovine IBV, MHV and BcoV (3CLp)
		6573	nsp2 protease consensus sequence
6574-6577	IG sequence of FIG. 18
		6578	nSh expression construct in pCMVIII
6579	nS expression constructs in pCMVIII
		6580	nSh Δ TC expression construct in pCMVIII
6581	nS delta TC expression construct in pCMVIII
		6582	nS1h expression construct in pCMVIII
6583	nS1 expression construct in pCMVIII
		6584-6585	cDNA amplification primer
6585-6587	RT-PCR primer
		6588-6809	FIG. 23 shows a constitutional sequence (. gtoreq.4 amino acids)
6810-7179	FIG. 24 shows a constitutional sequence (. gtoreq.4 amino acids)
		7180-7187	SEQ ID NO: n-glycosylation sites within 6039

7188-7189	FIG. 25 composition sequence
		7190	SEQ ID NO: 7188 fragment
7191	Encoding the amino acid sequence of SEQ ID NO: 7190 of the polynucleotides
		7192	SEQ ID NO: amino acids 879-1005 of 6042
7193	SEQ ID NO: amino acid 879-980 of 6042
		7194	SEQ ID NO: amino acids 901-1005 of 6042
7195	SEQ ID NO: amino acid 1144 of 6042-
		7196	SEQ ID NO: amino acids 1144-1196 of 6042
7197-7199	Membrane fusion peptide regions
		7200-7206	NadA-based polypeptides
7207-7223	SEQ ID NO: n-glycosylation sites within 6042
		7224-7231	Sliding area
7232	Orflab polyprotein
		7233-7244	Orflab polyprotein
7245-7247	X of SEQ ID NOS 7233-72442Sequence of
		7248-7253	Orflab polyprotein
7254	Zinc binding domain 2 position
		7255-7271	SEQ ID NOS: n-glycosylation sites in 6040-41, 6043, 6045-46, 6050-51
7272-7291	Polypeptides and polynucleotides
		7292-7293	Intergenic sequence
7294-7301	Nucleotides following intergenic sequence at 5' end of SARSV genome
		7302-7306	NadA constructs
7307-7308	SEQ ID NO: 6042 fragment
		7309	NadA sequence
7310-7311	NadA leader sequence
		7312-7315	Amino acid sequence of NadA
7316-7324	PCR primer
		7325-7330	Primer and method for producing the same
7331	CCACC sequence
		7332-7336	3' UTR forward primer
7337-7341	3' UTR reverse primer
		7342-7352	3' UTR probes
7353-7362	5' UTR forward primer
		7363-7373	5' UTR reverse primer
7374-7385	5' UTR probes
		7386	Conserved 8 nucleotides
7387	SEQ ID NO: 7293 reverse component of
		7388	Intergenic sequence
7389	Poly T
		7390	Stem-loop sequence
7391-7392	Polyglycine linker
		7393	Polyhistidine tag
7394	Nucleocapsid epitope sites
		7395	Antisense primer
7396-7397	Probe needle
		7398-7399	SEQ ID NO: 6042 antigenic fragments
7400-7639	SEQ ID NO: t epitope analysis of 6039
		7640-7800	SEQ ID NO: t epitope analysis of 6040
7801-8040	SEQ ID NO: t epitope analysis of 6041
		8041-8280	SEQ ID NO: t epitope analysis of 6042
8281-8486	SEQ ID NO: t epitope analysis of 6043
		8487-8665	SEQ ID NO: t epitope analysis of 6044
8666-8820	SEQ ID NO: t epitope analysis of 6045

8821-9018	SEQ ID NO: t epitope analysis of 6046
		9019-9131	SEQ ID NO: t epitope analysis of 6047
9132-9308	SEQ ID NO: t epitope analysis of 6048
		9309-9437	SEQ ID NO: t epitope analysis of 6049
9438-9538	SEQ ID NO: t epitope analysis of 6050
		9539-9752	SEQ ID NO: t epitope analysis of 6052
9753-9763	Spike protein amplification primers, particularly for spike fragments
		9764-9765	SEQ ID NO: n-glycosylation site in 6039
9766-9779	ORFlab cleavage products (Table 10)
		9780-9782	Forward primer, reverse primer, probe
9783-9784	Lysine-rich regions
		9785-9798	Oligonucleotides for expression in Saccharomyces cerevisiae
9799-9802	Sequences of FIGS. 65 and 66
		9803-9882	Primers for cloning of Escherichia coli
9883-9885	FIG. 3A, 3B, 3C BCV nucleotide sequences
		9886-9891	BCV amino acids of FIGS. 4A, 4B, 4C, 4D, 4E, 4FSequence of
9892	BCV 5′UTR
		9893	BCV 3′UTR
9894-9896	FIGS. 3A, 3B, 3C MHV nucleotide sequences
		9897-9902	FIGS. 4A, 4B, 4C, 4D, 4E, 4F MHV amino acid sequence
9903-9904	FIG. 3A, 3B AIBV nucleotide sequence
		9905-9909	FIG. 4A, 4B, 4D, 4E, 4F AIBV amino acid sequence
9910	AIBV 5′UTR
		9911	AIBV 3′UTR
9912-9913	FIG. 3B, 3C nucleotide sequences of HOBMPRO, HOBHEGA
		9914-9918	Human CoV amino acid sequences of FIGS. 4A, 4B, 4C, 4E, 4F
9919	HCoV-OC43 5′UTR
		9920	HCoV-OC43 3′UTR
9921-9923	pCMVKm2 vector
		9924-9926	Codon-preferred N, M and E sequences
9927	BNI-1
		9928-9959	From SEQ ID NO: 9927 derived constituent amino acid sequence of 4aa or more
9960	ORFlab variants
		9961	ORFla variants
9962	Spike protein variants
		9963	Membrane protein variants
9964	Nucleocapsid protein variants
		9965-9966	Terminal ORF
9967	Complete genome of FRA

Claims (119)

1. An isolated polypeptide of SARS virus.

2. The polypeptide of claim 1, wherein the polypeptide is a spike (S) polypeptide, an envelope (E) polypeptide, a membrane (M) polypeptide, a hemagglutinin-esterase polypeptide (HE), a nucleocapsid (N) polypeptide, an ORF1a polypeptide, an ORF1ab polypeptide, a proteolytic fragment of an ORF1a polypeptide, or a proteolytic fragment of an ORF1ab polypeptide.

3. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOs: 6039, 7232, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050 or 6052.

4. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having > 75% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 977, 4, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633, and 11636.

5. The polypeptide of claim 1, wherein the polypeptide comprises a fragment of at least 10 contiguous amino acids of an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11552, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633, and 11636.

6. A polypeptide comprising an amino acid sequence having > 80% sequence identity to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11552, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633, and 11636.

7. A polypeptide comprising an amino acid sequence comprising a fragment of at least 10 contiguous amino acids of an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11552, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633, and 11636. 8. A polypeptide comprising a sequence identical to SEQ ID NO: 6042 an amino acid sequence having > 80% sequence identity, and/or comprising a sequence comprising SEQ ID NO: 6042 at least 10 contiguous amino acids, wherein the polypeptide is in trimeric form.

9. A nucleic acid encoding the polypeptide of any one of claims 1-8.

10. The nucleic acid of claim 9, comprising an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOS: 7191, 7273, 7275, 7277, 7279, 7281, 7283, 7285, 7287, 7289, 7291, 7292, 7293, 9968, 10066, 10084, 10299, 10505, 11323, 11563, 11639 and 11640.

11. A polynucleotide comprising a nucleotide sequence having > 80% sequence identity to a nucleic acid according to claim 9 or claim 10.

12. A polynucleotide comprising a fragment of at least 10 contiguous nucleotides of the nucleic acid of claim 9 or claim 10.

13. An antibody recognizing the polypeptide of any one of claims 1 to 8.

14. The antibody of claim 13, wherein the antibody recognizes a polypeptide comprising SEQ ID NO: 6042 or a fragment thereof.

15. The antibody of claim 14, wherein the antibody recognizes a polypeptide comprising SEQ ID NO: 6042, or a fragment thereof.

16. The antibody of claim 13, wherein the antibody is a monoclonal antibody,

17. The antibody of claim 13, wherein the antibody is a human antibody.

18. An immunoassay for detecting SARS virus antigen in a sample comprising the step of contacting the sample with an antibody according to any one of claims 13 to 17.

19. An immunoassay for detecting antibodies against SARS virus antigens in a sample comprising the step of contacting the sample with the polypeptide of any one of claims 1-8.

20. A method of detecting antibodies against SARS virus antigens in a sample comprising contacting the sample with the polypeptide of any one of claims 1-8 under conditions suitable for the polypeptide to bind to the antibodies, if present, and detecting binding of the polypeptide to the antibodies.

21. A method of detecting a SARS virus antigen in a sample comprising contacting the sample with an antibody of any one of claims 13-17 under conditions suitable for the antibody to bind to the antigen, if present, and detecting binding of the antibody to the antigen.

22. A vaccine for treating or preventing Severe Acute Respiratory Syndrome (SARS) comprises inactivated SARS virus, killed SARS virus, attenuated SARS virus, split SARS virus preparation or at least one purified SARS virus antigen.

23. The vaccine of claim 22, comprising the purified polypeptide of any one of claims 1-8.

24. The vaccine of claim 22 or claim 23, wherein the antigen is a purified SARS virus antigen in the form of VLPs.

25. The vaccine of any one of claims 22-24, further comprising an adjuvant.

26. The vaccine of claim 25, wherein the adjuvant is an aluminum salt or is MF59.

27. The vaccine of any one of claims 22-26, comprising more than one SARS virus antigen.

28. The vaccine of claim 27, wherein the antigen is selected from S, E, N and M.

29. The vaccine of claim 22, comprising inactivated SARS virus.

30. The vaccine of claim 29, wherein the virus is inactivated by chemical or physical means.

31. The vaccine of claim 30, wherein the inactivation comprises treating the virus with an effective amount of one or more agents selected from the group consisting of: detergent, formaldehyde, formalin, beta-propiolactone and UV light.

32. The vaccine of claim 30, wherein the inactivation comprises treating the virus with an effective amount of one or more agents selected from the group consisting of: methylene blue, psoralen and carboxyfullerene (C60).

33. The vaccine of claim 30, wherein the inactivation comprises treating the virus with an effective amount of one or more agents selected from the group consisting of: binary ethylamine, acetyl aziridine and gamma irradiation.

34. The vaccine of claim 31, wherein the inactivation comprises treatment with beta-propiolactone.

35. The vaccine of claim 34, wherein the beta-propiolactone is used at a concentration of 0.01-0.5%.

36. The vaccine of claim 34, wherein the beta-propiolactone is used at a concentration of 0.5-0.2%.

37. The vaccine of claim 34, wherein the beta-propiolactone is used at a concentration of 0.025-0.1%.

38. A method of inactivating SARS virus comprising exposing the virus to an inactivating agent at refrigeration temperatures for 12 to 24 hours and then raising the temperature for 3 hours to hydrolyse any remaining inactivating agent.

39. The method of claim 38, wherein the inactivating agent is beta-propiolactone.

40. The method of claim 38, wherein the refrigeration temperature is between 0 ℃ and 8 ℃.

41. The method of claim 38, wherein the elevated temperature is between 33 ℃ and 41 ℃.

42. A method of making an inactivated SARS vaccine, comprising:

a. inoculating SARS virus in mammalian cell culture;

b. culturing the infected cells;

c. harvesting the supernatant containing SARS virus;

d. inactivating SARS virus; and

e. purifying the inactivated SARS virus.

43. The method of claim 42, wherein the mammalian cell culture is derived from one or more cell types selected from the group consisting of: fibroblasts, endothelial cells, hepatocytes, keratinocytes, immune cells, breast cells, smooth muscle cells, melanocytes, nerve cells, prostate cells, kidney cells, skeletal cells, liver cells, retinoblasts, and stromal cells.

44. The method of claim 42, wherein the mammalian cell culture is derived from a cell culture selected from the group consisting of: a human cell, a non-human primate cell, a HeLa cell, a human diploid cell, a rhesus fetal lung cell, a human embryonic kidney cell, a Vero cell, an equine cell, a bovine cell, a ovine cell, a canine cell, a feline cell, or a rodent cell.

45. The method of claim 42, wherein the mammalian cell culture is derived from Vero cells or rhesus fetal kidney cells.

46. The method of claim 42, wherein the mammalian cell is cultured in serum-free media.

47. The method of claim 42, wherein the mammalian cell is cultured in a protein-free medium.

48. The method of claim 42, wherein the seeding step comprises attaching SARS virus to the cell culture for 60-300 minutes.

49. The method of claim 42, wherein the seeding step is performed at 25-40 ℃.

50. The method of claim 42, wherein the purifying step comprises one or more treatments selected from the group consisting of: gradient centrifugation, ultracentrifugation, continuous flow ultracentrifugation, chromatography, polyethylene glycol precipitation, and ammonium sulfate precipitation.

51. The method of claim 42, wherein the purifying step comprises one or more treatments selected from the group consisting of: ultrafiltration and diafiltration.

52. The method of claim 50, wherein the chromatographic process comprises one or more chromatographic processes selected from the group consisting of: ion exchange chromatography, size exclusion chromatography and liquid affinity chromatography.

53. The method of claim 52, wherein the chromatographic treatment comprises the use of one or more chromatography resins selected from the group consisting of: anionic resins and cationic resins.

54. The method of claim 52, wherein the ion exchange chromatography process comprises a first step using a strong anion exchange resin and a second step using a strong cation exchange resin.

55. The method of claim 50, wherein the gradient centrifugation purification step comprises density gradient centrifugation.

56. The method of claim 42, wherein the purifying step comprises a first chromatographic purification and a second gradient centrifugation.

57. The method of claim 56, wherein the first chromatographic purification comprises liquid affinity chromatography.

58. The method of claim 56, wherein the second step gradient centrifugation comprises density gradient centrifugation.

59. A single stranded oligonucleotide comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 21-6020, 6076-.

60. A single stranded oligonucleotide comprising a complementary sequence to the oligonucleotide of claim 59.

61. The oligonucleotide of claim 59 or claim 60, comprising 10-30 nucleotides.

62. The oligonucleotide of claim 61, comprising SEQ ID NO: 7292. SEQ ID NO: 7293, the nucleotide sequence of SEQ ID NO: 7292 or the complement of SEQ ID NO: 7293.

63. A kit comprising primers for amplifying a template sequence contained within a nucleic acid target of SARS virus, the kit comprising a first primer and a second primer, wherein said first primer comprises a sequence substantially complementary to a portion of said template sequence and said second primer comprises a sequence substantially complementary to a portion of the complement of said template sequence, wherein the substantially complementary sequences within said primers define the two ends of the template sequence to be amplified.

64. The kit of claim 63, wherein the template sequence is contained in SEQ ID NO: 1 and/or SEQ ID NO: 2 in (c).

65. The kit of claim 63 or claim 64, wherein the first primer comprises the sequence of SEQ ID NO: 1, and a second primer comprising a fragment of 8 or more nucleotides of SEQ ID NO: 1 of 8 or more nucleotides of the complementary sequence.

66. The kit of claim 63 or claim 64, wherein the first primer comprises the sequence of SEQ ID NO: 2, and a second primer comprising a fragment of 8 or more nucleotides of SEQ ID NO: 2, or a fragment of 8 or more nucleotides of the complementary sequence.

67. The kit of claim 63, wherein the first primer is the oligonucleotide of any one of claims 59-62 and the second primer is the oligonucleotide of any one of claims 59-62.

68. The kit of any one of claims 63-67, further comprising a labeled probe comprising the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2, or the complement of said fragment, which fragment is located within said template sequence.

69. The kit of any one of claims 63-68, wherein the first primer and/or second primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: SEQ ID NOS: 21-6020, 6076-.

70. The kit of any one of claims 63-68, wherein the first primer and/or the second primer comprises a complement of a nucleotide sequence selected from the group consisting of SEQ ID NO: SEQ ID NOS: 21-6020, 6076-.

71. A method for detecting the presence of SARS virus in a sample, the method comprising providing a sample suspected of containing a SARS virus nucleic acid target, amplifying a template sequence contained in the SARS virus nucleic acid target using the kit of any of claims 63-70, and detecting the amplified template sequence, wherein the presence of the amplified template sequence is indicative of the presence of SARS virus in the sample.

72. The method of claim 71, wherein the amplification is accomplished by: polymerase chain reaction, transcription mediated amplification, reverse transcription PCR, ligase chain reaction, strand displacement amplification or nucleic acid sequence based amplification.

73. A double-stranded RNA molecule of about 10 to about 30 nucleotides in length capable of inactivating SARS coronavirus in a mammalian cell.

74. The double stranded RNA of claim 73, wherein the sequence of one strand is at least 90% identical to a target sequence, wherein the target sequence is SEQ ID NO: 1 and/or SEQ ID NO: 2.

75. The double stranded RNA according to claim 73 or claim 74, wherein the target sequence comprises a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7299, 7300 and 7301.

76. The double stranded RNA of any one of claims 73-75, comprising at least one modified nucleotide.

77. A method of treating a patient with SARS comprising administering to the patient a therapeutically effective dose of less than 1000g/mol of the molecule.

78. The method of claim 77, wherein said molecule has an aromatic region and one or more heteroatoms selected from O, S or N.

79. A method of treating a patient with SARS comprising administering to the patient a therapeutically effective amount of a compound selected from the group consisting of: nucleoside analogs, peptoids, oligopeptides, polypeptides, protease inhibitors, 3C-like protease inhibitors, papain-like protease inhibitors, or inhibitors of RNA-dependent RNA polymerase.

80. A method of treating a patient with SARS, the method comprising administering to the patient a steroidal anti-inflammatory drug and at least one antiviral compound.

81. A method of treating a patient with SARS, the method comprising administering to the patient a therapeutically effective amount of a compound selected from the group consisting of: acyclovir, ganciclovir, vidarabidine, foscamet, cidoflorir, amantidine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, an antiviral compound listed in table 1, an antiviral compound listed in table 2, or an interferon.

82. The method of claim 81, wherein the interferon is interferon- α or interferon- β.

83. The method of any one of claims 77-82, wherein the molecule or compound is delivered by inhalation.

84. A method of identifying a therapeutically active agent, the method comprising the steps of: (a) contacting a therapeutically active agent with cells infected with SARS virus; (b) the attenuation of the SARS-associated enzyme is determined.

85. A viral vector or particle for the in vivo delivery of a nucleic acid according to claim 9 or claim 10.

86. The viral vector of claim 85, wherein the vector is an adenoviral vector, a poxviral vector, or an alphaviral vector.

87. An alphavirus replicon particle comprising one or more SARS virus antigens.

88. The replicon particle of claim 87 wherein the SARS virus antigen is a spike protein.

89. The replicon particle of claim 87, wherein the particle comprises a replicon derived from Venezuelan Equine Encephalitis (VEE) virus and further comprises an envelope derived from sindbis virus (SIN) or Semliki Forest Virus (SFV).

90. A vaccine comprises one or more SARS virus antigens and one or more respiratory virus antigens.

91. The vaccine of claim 90, wherein the respiratory virus antigen is selected from the group consisting of: influenza virus, Human Rhinovirus (HRV), parainfluenza virus (PIV), Respiratory Syncytial Virus (RSV), adenovirus, hyperpneumovirus and rhinovirus.

92. The vaccine of claim 91, wherein the respiratory virus antigen is from influenza virus.

93. The vaccine of claim 90, wherein the respiratory viral antigen is from a coronavirus other than the SARS virus.

94. A polypeptide comprising the amino acid sequence of SEQ ID NO: 6042, a surface-exposed immunogenic fragment.

95. The polypeptide of claim 94, wherein said fragment does not comprise the amino acid sequence of SEQ ID NO: 6042 the last 50 amino acids from the C-terminus.

96. The polypeptide of claim 94, wherein said fragment does not comprise the amino acid sequence of SEQ ID NO: 6042 transmembrane domain.

97. The polypeptide of claim 94, wherein said fragment does not comprise the amino acid sequence of SEQ ID NO: the C-terminal cytoplasmic domain of 6042.

98. The polypeptide of claim 94, wherein the fragment does not comprise an N-terminal signal sequence.

99. A polypeptide comprising a sequence selected from SEQ ID NOS: 9968 and 10066.

100. The polynucleotide of claim 99, wherein the polynucleotide comprises a nucleotide sequence identical to a sequence selected from SEQ id nos: 9968 and 10066 have a nucleic acid sequence with > 80% sequence identity.

101. A polypeptide comprising a sequence selected from SEQ ID NOS: 9968 and 10066, wherein said fragment does not comprise the entire nucleic acid sequence of SEQ ID NO: 10033.

102. an isolated polypeptide comprising an amino acid sequence encoded by the sequence of any one of claims 99-101.

103. The polypeptide of claim 102, comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS: 9969-10032, 10067, and 10015.

104. The polypeptide of claim 103, wherein the amino acid sequence is selected from the group consisting of: SEQ ID NOS: 9997, 9998 and 10015.

105. An expression construct for recombinant expression of a spike protein of SARS virus, wherein said construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 6578 and 6583.

106. A mammalian cell line stably expressing SARS virus antigen.

107. The cell line of claim 106, wherein the cell line is a Chinese Hamster Ovary (CHO) cell.

108. The cell line of claim 106, wherein the SARS virus antigen is spike protein or a fragment thereof.

109. The cell line of claim 106, wherein the spike protein is truncated to remove transmembrane sequences.

110. A method of identifying a therapeutically active agent, the method comprising the steps of: (a) contacting a therapeutically active agent with a buffer comprising SARS enzyme; and (b) determining the attenuation of the SARS enzyme.

111. The method of claim 110, wherein the SARS enzyme is SARS protease.

112. The method of claim 111, wherein the buffer further comprises a peptide having a SARS protease cleavage site.

113. The method of claim 110, wherein the assaying is accomplished by a fluorescence assay.

114. The vaccine of any one of claims 22-37 and 90-93, further comprising an adjuvant.

115. The vaccine of claim 114, wherein the adjuvant is SMIP.

116. The vaccine of claim 115, wherein the SMIP compound is selected from the group consisting of: acylpiperazines, tryptanthrin, indolone, tetrahydroisoquinoline, benzocyclodione, amino-azavinyl compounds, thiosemicarbazones, lactams, aminobenzimidazole quinolinone, hydroxyphthalimides, benzophenones, isoxazoles, sterols, quinazolinones, pyrroles (pyroles), anthraquinones, quinoxalines, triazines, indoles, and pyrazolopyrimidines, or pharmaceutically acceptable salts, esters, or prodrugs thereof.

117. A method of vaccinating a subject comprising administering to the subject the vaccine of any one of claims 22-37 and 90-93.

118. The method of claim 117 further comprising administering a SMIP.

119. The method of treating a patient of any one of claims 77-82, further comprising administering at least one SMIP compound.

120. The method of treating a patient of any one of claims 77-82, further comprising administering at least one SMIS compound.

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