WO2005027963A2 - METHODS AND COMPOSITIONS FOR THE GENERATION OF A PROTECTIVE IMMUNE RESPONSE AGAINTS SARS-CoV - Google Patents

METHODS AND COMPOSITIONS FOR THE GENERATION OF A PROTECTIVE IMMUNE RESPONSE AGAINTS SARS-CoV Download PDF

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WO2005027963A2
WO2005027963A2 PCT/US2004/029999 US2004029999W WO2005027963A2 WO 2005027963 A2 WO2005027963 A2 WO 2005027963A2 US 2004029999 W US2004029999 W US 2004029999W WO 2005027963 A2 WO2005027963 A2 WO 2005027963A2
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sars
cov
protein
antibody
cells
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WO2005027963A3 (en
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Gary J. Nabel
Zhi-Yong Yang
Yue Huang
Wing-Pui Kong
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The United States Of America As Represented By Thesecretary Of Health And Human Services, Nih
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to novel methods and compositions for the generation of a protective immune response in humans and other animals against the coronavirus responsible for Severe Acute Respiratory Syndrome (SARS-CoV).
  • SARS-CoV Severe Acute Respiratory Syndrome
  • SARS severe acute respiratory syndrome
  • SARS-CoV a novel coronavirus
  • Coronavimses a genus of the Coronaviridae family, are enveloped, positive-strand, R ⁇ A viruses.
  • coronavimses cause respiratory and enteric diseases in humans and domestic animals (Holmes, 2001, Coronavimses, p. 1187-1203, In Knipe et al. (eds.), Fields Virology, (Lippincott Williams & Wilkins, Philadelphia); Lai and Holmes, 2001, Coronaviridae: The viruses and their replication, p. 1163-1185, In Knipe et al. (eds.), Fields Virology, (Lippincott Williams & Wilkins, Philadelphia)).
  • SARS-CoV is a highly pathogenic vims that causes severe acute respiratory disease, especially in the elderly (World Health Organization. Consensus document on the epidemiology of severe acute respiratory sydrome (SARS). 10-17-2003).
  • Coronavimses can be divided into three serologically distinct groups (Holmes, 2001, Coronavimses, p. 1187-1203, In Knipe et al. (eds.), Fields Virology, (Lippincott Williams & Wilkins, Philadelphia). Phylogenetically, SARS-CoV is not closely related to any of the three groups (Marra et al., 2003, Science 300:1399-1404), although it is most similar to the group II coronavimses (Rest and Mindell, 2003, Infect. Genet. Evol. 3:219-225; Snijder et al., 2003, J. Mol. Biol. 331:991-1004).
  • SARS-CoV genome Although the organization of the SARS-CoV genome is related to animal coronavimses, its genetic sequence is unique. Its genome encodes four structural proteins and two regulatory proteins. Based on a comparison to animal coronavimses, three structural gene products are predicted to be present on the viral envelope, the S, M, and E proteins (Lai and Holmes, 2001, supra).
  • the structure of the SARS-CoV envelope differs in some respects from other budding, enveloped vimses, such as retrovimses and lentivimses, which contain one viral envelope protein.
  • the present invention relates to novel compositions and methods for the inducement of an immunoprotective immune response against SARS-CoV.
  • the invention relates to a vaccine composition comprising a nucleic acid molecule that comprises an S polynucleotide that encodes an extracellular domain portion of a SARS-CoV S protein.
  • the invention in another aspect, relates to a method of protecting a subject from infection by SARS-CoV comprising administering a nucleic acid molecule to the subject, wherein the nucleic acid molecule comprises an S polynucleotide that encodes an extracellular domain portion of a SARS-CoV S protein.
  • the invention in another aspect, relates to a method of treating a, subject for a SARS-CoV infection comprising administering to the subject a SARS-CoV neutralizing antibody.
  • the invention in another aspect, relates to a method of screening for inhibitors of SARS-CoV infection comprising: (a) incubating a cell susceptible to SARS-CoV infection with a SARS-CoV S pseudotyped viral vector in the presence or absence of a test inl ibitory agent; (b) assaying entry of said pseudotyped viral vector into said cell; (c) identifying the test inhibitory agent as an inhibitor of SARS-CoV infection if entry of said pseudotyped viral vector into said cell is reduced in the presence of the test inhibitory agent.
  • the invention in another aspect, relates to a method of measuring the presence or concentration of anti-SARS-CoV neutralizing antibodies in a biological sample of a mammal, wherein said method comprises the steps of: (a) incubating a cell susceptible to SARS-CoV infection with a SARS-CoV S pseudotyped viral vector in the presence or absence of the biological sample; (b) assaying entry of said pseudotyped viral vector into said cell; (c) identifying the biological sample as a positive for the presence of SARS-CoV neutralizing antibodies if entry of said pseudotyped viral vector into said cell is reduced in the presence of biological sample.
  • the invention relates to an immunoassay that measures the presence or concentration of an anti-SARS-CoV S protein antibody in a biological sample of a mammal, wherein said immunoassay comprises the steps of: (a) contacting said biological sample with mammalian expressed S protein antigen, said contacting being under conditions sufficient to permit anti-S-protein antibody if present in said sample to bind to said antigen and form an antigen-anti-S-protein antibody complex; (b) contacting said formed antigen-anti-S-protein antibody complex with an anti-S-protein antibody binding molecule under conditions sufficient to permit said anti-S-protein antibody binding molecule to bind to anti-S- protein antibody of said formed antigen-anti-S-protein antibody complex and form an extended complex; and (c) determining the presence or concentration of said anti-S-protein antibody in said biological sample by determining the presence or concentration of said formed extended complex.
  • the invention in another aspect, relates to a method of protecting a subject from infection by SARS-CoV comprising exposing the subject to isolated S protein.
  • the invention in another aspect, relates to a vaccine composition comprising at least a portion of the extracellular domain of isolated S protein.
  • the invention relates to a vaccme composition
  • a nucleic acid molecule that comprises an S polynucleotide that encodes an extracellular domain portion of a SARS-CoV S protein, wherein the vaccine composition does not exhibit antibody-dependent vims enhancing activity.
  • the invention relates to a vaccme composition comprising at least a portion of the extracellular domain of isolated S protein, wherein the vaccine composition does not exhibit antibody-dependent vims enhancing activity.
  • the invention in another aspect, relates to a method of assaying a vaccine composition for antibody-dependent vims enhancing activity comprising the steps of: (a) generating an immune response to a vaccine composition in an animal; (b) isolating antibodies or antisera from the animal; (c) assaying the ability of the antibodies or antisera to enhance cellular entry of a SARS-CoV vims strain or a corresponding pseudovims; (d) identifying the vaccine composition as having an antibody-dependent vims enhancing activity if the antibodies or antisera enhance cellular entry of the SARS-CoV vims strain or the corresponding pseudotype.
  • Figure 1 shows the infection of Vero (African green monkey kidney epithelial) cells retroviral and lentiviral vectors that have been pseudotyped with the SARS-CoV S protein.
  • Panel A shows the infection of the Vero cell line with the S-pseudotyped lentiviral or retroviral vector expressing luciferase (Wood et al., 1984, Biochem. Biophys. Res. Commun. 124:592-596).
  • Panel B shows that the S glycoprotein, but not the M or E glycoprotein, mediates viral entry by the lentiviral vector S pseudotype
  • Panel C shows the requirement for the cytoplasmic domain of S for infection, as analyzed by the generation of pseudotyped vims using full-length (S) or carboxy-terminal deleted S proteins.
  • Panel C shows a western blot analysis of the expression of the carboxy terminal deleted S variants.
  • Figure 2 shows an analysis of the pH-dependent entry of the SARS-CoV pseudotyped lentiviral vectors. The pseudolentivimses are incubated in the presence of increasing amounts of ammonium chloride (Panel A) or bafilomycin (Sigma, St. Louis, MO) (Panel B). The experiment is performed in triplicate. Data is presented as percentage of activity at the indicated dose compared to no dmg treatment.
  • Figure 3 shows an analysis of the SARS-CoV S pseudotyped lentiviral vector tropism for human and animal cells and a correlation with SARS-CoV infectability.
  • Panel A shows an analysis of the SARS-CoV S pseudo-lentivims tropism for different human cell types. All infections are performed in triplicate. Data is presented as the average ⁇ standard deviation. The results of one of two independent experiments are shown.
  • Panel B shows an analysis of the SARS-CoV S pseudo-lentivims infectivity of renal and lung cells from different animal species. All infections are performed in triplicate. Data is presented as the average ⁇ standard deviation. The results of one of two independent experiments are shown.
  • Panel C shows an analysis of infectivity of SARS-CoV (Urbani) on selected susceptible and resistant cells from Panels A and B above.
  • SARS-CoV (Urbani) is titered on Vero cells as previously described in Subarrao et al., 2004, J. Virol. 78(7):3572-7.
  • a dotted line indicates the detection limit of infectivity of SARS-CoV.
  • Figure 4 shows an analysis of DC-SIGN receptor dependent uptake of
  • Panel A shows the binding of purified SARS-CoV S glycoprotein to cell lines.
  • Cell cultures containing lxlO 6 cells of African green monkey kidney cells (Vero), human T leukemia cells (A3R5 and MT2), or THP-1 myelomonocytic leukemia cells expressing wild type or mutant forms of DC-SIGN (THP-DC-SIGN and THP-DC-SIGN ⁇ 35, respectively) are incubated with purified S protein, which is tagged with a myc and His epitope at amino acid 1190, for 20 minutes on ice.
  • Binding of spike protein to the cells is detected using an FITC labeled anti-His (carboxy-terminal) antibody (Invitrogen, Carlsbad, CA).
  • An FITC labeled IgG isotype is used as a control.
  • Data is analyzed by flow cytometry.
  • Panel B shows an analysis of direct viral entry of SARS-CoV S pseudotyped lentiviral vector (left panel) and cell mediated transfer of the pseudotyped lentiviral vector from THP-1, THP-DC-SIGN and THP-DC-SIGN ⁇ 35 cells (right panel).
  • THP-1, THP-DC-SIGN and THP-DC- SIGN ⁇ 35 Susceptibility of Vero, A3R5, MT2, THP-1, THP-DC-SIGN and THP-DC- SIGN ⁇ 35 to SARS-CoV S pseudotyped lentiviral vector infection is measured after transduction using the luciferase reporter (left panel).
  • Cell mediated pseudoviral transfer by THP-1, THP-DC-SIGN or THP-DC-SIGN ⁇ 35 cells is assessed by incubating the particular THP cell type (3xl0 4 cells) with pseudotyped lentiviral vector for two hours at 37°C. After incubation, cells are washed three times before they are added to a cell culture containing 3xl0 4 target cells. Cells are collected 72 hours after coincubation for luciferase assays.
  • Panel C shows the inhibition of direct infection and cell-mediated transfer of SARS-CoV S pseudo lentivirus by mouse anti-SARS-CoV S antisemm.
  • SARS-CoV S pseudotyped lentiviral vector is exposed to mouse control or anti-S specific antisemm at mdicated dilutions for sixty minutes at 37°C before adding to Vero cells (left panel).
  • the indicated THP-DC-SIGN cells are incubated with pseudovimses as described for Panel B above, followed by incubation with Vero cells in the presence or absence of mouse anti-SARS-CoV S specific antisemm for 48 hours. After the 48-hour incubation period, cells are collected for luciferase assays.
  • Figure 5 shows the uptake and transfer of GFP-Vpr labeled SARS-CoV S pseudotyped lentiviral vector and SARS-CoV by mature and human myeloid dendritic cells (mDC).
  • Panel A comprises confocal microscopic images of cells showing the uptake of GFP-Vpr labeled SARS-CoV S pseudotyped lentiviral vector by mDC, and the subsequent transfer of the pseudotyped lentiviral vector from mDC to renal epithelial cells.
  • the mDC cells are infected with the GFP-Vpr labeled SARS-CoV S pseudotyped lentivims for 30 minutes at 37°C.
  • the mDCs are then added onto human renal epithelial cells (786-0; 3xl0 4 cells per well, plated a day before) in eight well coverslip slides (Nalge Nunc, Naperville, IL) at a ratio of 1 : 1. Uptake, polarization and transfer are assessed by confocal microscopy with representative cells. The arrow indicates the transfer of labeled vims from mDCs to 786-0 cells.
  • Panel B shows that human mature mDCs are not directly infected by SARS-CoV (Urbani strain) but instead promote cell-mediated infection of susceptible target cells.
  • Vero cells and immature and mature dendritic cells are incubated with SARS-CoV (Urbani) for one hour in 96 well dishes (2xl0 4 cells/well). After incubation, cells are washed and maintained in culture medium for 72 hours before collection of culture supernatants for analysis of viral titer (left panel).
  • SARS-CoV Urbani
  • Mature dendritic cells are also infected for one hour with SARS-CoV, washed, detached with trypsin, and replated onto 96-well-dishes with Vero cells (2xl0 4 cells/well; 1:1 ratio) in the presence or absence of mouse anti-SARS-CoV S antisemm (1 : 100 dilution) Cells are maintained in culture for 72 hours before supernatants are collected for analysis of viral titer (right panel). Viral titer is measured as described in Subarrao et al. ( 2004, J. Virol. 78(7):3572-7). Vims yield is expressed as TCID 50 per ml. Dotted lines indicate the detection limit of SARS-CoV.
  • Figure 6 shows the inhibition of viral pseudotyping using two types of inhibitors.
  • Panel A shows the inhibitory activity of a peptide aptamer (Peptide 1 (HR2)) of the HR2 coiled-coil region of SARS-CoV S protein (residues 1154 to 1190 of SEQ ID NO:3) vs an unrelated peptide (Control).
  • the Peptide 1 (HR2) sequence is LGDISGINASWNIQKEIDRL (SEQ ID NO:l) and the control peptide sequence is MFIFLLFLTLTSGSDLDR (SEQ ID NO:2). Both peptides are purified by high-pressure liquid chromatography.
  • Panel B shows the inhibitory activity of human convalescent antisera from a patient infected with SARS (SARS) vs. non-immune control sera (Control).
  • Figure 7 shows a schematic representation of SARS-CoV S glycoprotein cDNAs and the expression of the encoded recombinant proteins.
  • Panel A shows the stmcture of the cDNAs used.
  • Panel B shows a western blot analysis of the expression of the recombinant SARS-CoV S proteins after transfection of the indicated plasmid expression vectors in 293T cells. Arrows indicate specific S ⁇ CD (upper) and S ⁇ TM (lower) bands.
  • Figure 8 shows immune responses to SARS-CoV DNA vaccination in BALB/c mice.
  • Panel B shows antibody responses induced by plasmid DNA vaccination against the SARS-CoV S protein.
  • End-point dilution enzyme linked immunosorbent assay titres of SARS-CoV S- specific antibodies (left panel) in semm of vaccinated animals collected 10 days after the final boost are determined by optical density as described in the Materials and Methods of Example 2 herein.
  • Figure 9 shows that DNA vaccinated BALB/c mice are protected against pulmonary SARS-CoV replication after challenge. Immunization and challenge are performed in mice as described in Subbarao et al., 2004, J. Virol. 78, 3572-
  • FIG. 10 shows the results of a determination of the immune mechanism of protection, using the techniques of T-cell depletion, adoptive transfer and antibody passive transfer.
  • Panel B shows a lack of protection against SARS-CoV replication in the lungs after adoptive T-cell transfer from vaccinated mice.
  • the recipient mice are challenged 24 hours after adoptive T-cell transfer.
  • Panel C shows that protection against SARS-CoV replication in the lungs is provided by the passive transfer of immune IgG from vaccinated mice.
  • Figure 11 shows the confirmation of T cell depletion in lung tissues.
  • Flow cytometry with monoclonal antibodies to mouse CD4 (left column) or CD8 (right column) is performed on lymphocytes derived from the lungs of BALB/c mice depleted with rat anti-mouse monoclonal antibodies to CD4, CD8, and/or CD90 antibodies, individually or in the indicated combinations as described in the Materials and Methods section of Example 2 herein.
  • Single-cell suspensions are prepared from lungs harvested at 48 hours after injection.
  • Lung tissues are minced with scissors, resuspended in 5 ml of an enzyme mixture (RPMI 1640, 12 mg collagenase type I, and 100 ⁇ g DNAase) at 37°C for 30 min, followed by mincing and passage through a 40 ⁇ m nylon cell strainer. Cells are washed once with PBS, and resuspended in PBS.
  • an enzyme mixture RPMI 1640, 12 mg collagenase type I, and 100 ⁇ g DNAase
  • 2x 10 6 are used for staining on ice for 30 min using rat anti-mouse CD3-PE(clone 17A2) (BD-Pharmingen, San Diego, CA), rat anti- mouse CD4-PerCP(clone RM4-5) (BD-Pharmingen, San Diego, CA), rat anti- mouse CD8-APC(clone 53-6.7) (BD-Pharmingen, San Diego, CA), followed by washing with 1 ml ice cold PBS. The cells are finally resuspended in PBS+1% formaldehyde for fluorescence activated cell sorting analysis. 200,000 cells are collected and analyzed using Flowjo software. Figure 12 shows the confirmation of T cell depletion in spleen tissues.
  • Flow cytometry with monoclonal antibodies to mouse CD4 (left column) or CD8 (right column) is performed on lymphocytes derived from the spleens of BALB/c mice depleted with rat anti-mouse monoclonal antibodies to CD4, CD8, and/or CD90 antibodies, individually or in the indicated combinations as described in the Materials and Methods section of Example 2 herein.
  • Single-cell suspensions are prepared from spleens harvested at 48 hours after injection. Spleen tissues are homogenized, resuspended in PBS, and passed through a 40 ⁇ m nylon cell strainer (BD Falcon, Bedford, MA). Cells are washed once with PBS, and resuspended in PB S .
  • 2 x 10 6 are used for staining on ice for 30 min using rat anti-mouse CD3 - PE(clone 17A2) (BD-Pharmingen, San Diego, CA), rat anti-mouse CD4- PerCP(clone RM4-5) (BD-Pharmingen, San Diego, CA), rat anti- mouse CD8- APC(clone 53-6.7) (BD-Pharmingen, San Diego, CA), followed by washing with 1 ml ice cold PBS. The cells are finally resuspended in PBS+1% formaldehyde for fluorescence activated cell sorting analysis. 200,000 cells are collected and analyzed using Flowjo software.
  • Figure 13 shows an ELISA analysis of semm samples from SARS patients. Eleven semm samples collected from five patients are analyzed for the presence of antibodies against SARS-CoV proteins using mammalian expressed S protein based ELISA. ELISA analyses of patient sera (1:133 dilution) for IgM, IgG and IgA against SARS-CoV spike (S) protein antigen (upper panel) or SARS-CoV nucleocapsid (N) protein antigen (lower panel) are shown at different time points after the onset of symptoms. Samples from each patient are represented by a different symbol. Solid black symbols represent samples that tested positive for SARS-CoV antibody as measured using the traditional vims-infected cell lysate based ELISA and immunofluorescence assays (IF A), whereas open symbols represent samples that tested negative using these methods.
  • S SARS-CoV spike
  • N SARS-CoV nucleocapsid
  • Figure 14 shows an analysis of SARS-CoV neutralizing antibody levels in patient sera.
  • Neutralizing antibody levels are measured using a SARS-CoV S pseudotyped lentiviral vector encoding the luciferase reporter as the neutralization target.
  • Each patient sample is represented by a different symbol.
  • Solid black symbols represent samples that tested positive for SARS-CoV antibody as measured using the traditional vims infected cell lysate based ELISA and IFA methods, whereas open symbols represent samples that tested negative using these methods.
  • Lower luminescence values indicate a lower level of pseudotyped lentivims infection due to higher viral neutralization activity in the semm samples. Results at a semm dilution of 1 : 50 are shown.
  • Figure 15 shows results demonstrating the incorporation of diverse S proteins into lentiviral vectors with comparable efficiency. Plasmids encoding the indicated S proteins are cotransfected with lentiviral expression vectors into 293T cells. Forty-eight hours after transfection, supernatants are harvested and mn on sedimentation gradients as described in Example 3 herein. A representative Western blot analysis of gradient fractions for S(GD03T0013) is shown in Panel A. Panel B shows quantification of sedimentation gradient fractions for the indicated strains.
  • Figure 16 shows an analysis of the sensitivity to antibody neutralization of lentivims vectors pseudotyped with alternative S proteins and an analysis of antibody dependent enhancement of vims entry using pseudovimses constructed with the S protein from two palm civet virases.
  • lentivims vectors that are pseudotyped with the indicated SARS-CoV S glycoproteins from human isolates are incubated with purified IgG from mice vaccinated with a DNA expression vector encoding SfJrbani) or with a negative control sera from non- immune mice that was subtracted from immune sera. Percent inhibition, assessed by luciferase reporter gene expression, is calculated as the reduction in luciferase activity relative to values achieved in the absence of sera.
  • Panel B shows an analysis of antibody-dependent enhancement of pseudovims entry with psuedovirases constructed with S-protein from two palm civet SARS-CoV isolates.
  • Panel C shows an analysis of antibody neutralization of pseudovimses constructed with the S protein from the Urbani strain (SfJJrbani)), the resistant human strain (S (GD03T0013)), and two palm civet strains (S(SZ3) and S(SZ16)) using human neutralizing mabs (S3.1, S127, and SI 11) derived from EBV-transformed B lymphocytes.
  • Figure 17 shows the differential sensitivity to ACE-2 inhibition of pseudovimses constructed with the S protein from the Urbani strain or the GD03T0013 strain and the relative resistance of S(GD03T0013) and S(SZ3) pseudotyped vimses to homologous neutralization.
  • Panel A shows the results obtained when the indicated pseudovimses are incubated with increasing amounts of soluble recombinant hACE-2. Entry is assessed using the luciferase reporter gene.
  • Panel B shows that immunization with homologous S protein (i.e S protein from GD03T001) does not result in sera with increased neutralization activity against pseudovims constructed with the S(GD03T0013).
  • Panel C shows that sera from mice immunized with full length S(SZ3) neutralizes SfJJrbani) pseudotyped viras but does not neutralize S(SZ3) pseudotyped vims.
  • Figure 18 shows the results of experiments to define the genetic determinants of S glycoprotein sensitivity to antibody neutralilation or antibody enhancement.
  • Panel A shows a schematic diagram of a neutralization sensitive S protein ( SfJJrbani)), a neutralization resistant S protein (palm civet S(SZ3)), and chimeric S proteins.
  • Panel B shows an analysis of luciferase gene transfer efficiency into 786-0 cells of pseudovimses constructed with the indicated wild type and chimeric S proteins (left panel). The dashed line indicates the background levels of gene transfer in the absence of S.
  • An analysis of the inhibition or enhancement of the indicated S pseudotypes using a neutralizing monoclonal antibody to S shows the dependence on the hACE-2 binding domain (right panel).
  • Panel C shows an analysis of anti-S antisera with SfJJrbani) protein or S(SZ3) protein.
  • a western blot analysis compares the amount of S protein in transfected cell supernatants (left panel, Input) with the amount of protein immunoprecipitated with S-antisera (right panel, IP).
  • Figure 19 shows the results of experiments to identify immunogens and mabs that circumvent antibody-dependent enhancement of viras entry and the results of a biochemical analysis of the mechanism of enhancement.
  • Panel A shows a neutralization profile of purified IgG from mice vaccinated with a DNA expression vector encoding a secreted form of S( Urbani) (S(l 153) - terminated at aa 1153) against the indicated human or civet pseudovimses.
  • Panel B shows a Western blot analysis of the biochemical interaction of purified IgG from S(l 153) immunized mice with the following S proteins and chimeric S proteins: S(Urbani), SU, US, and S(SZ3).
  • the present invention relates to novel compositions and methods related to the development of a vaccine composition for providing immunity to SARS-CoV.
  • SARS-CoV refers to the causal agent of SARS, and to evolutionarily derived variants of such coronavims that cause respiratory disease in humans and other animals.
  • the methods and compositions of the present invention may be used in any species affected by SARS-CoV, including humans and non-human animals (e.g., non-human mammals and birds).
  • SARS-CoV S protein SARS-CoV spike protein
  • the invention further reflects the recognition that viral transmission can occur through cell-mediated transfer by dendritic cells.
  • the invention further reflects the recognition that exposure of a subject to isolated SARS-CoV S protein via DNA vaccination induces a robust immune response.
  • An immune response will typically comprise a humoral immune response, and will preferably also provide a cytotoxic cell response, and may involve B cells, T cells, and other types of lymphoid cells. Cytotoxic immunity complements the humoral system by eliminating the infected cells.
  • neutralizing antibodies refers to antibodies that bind to SARS-CoV viras particles and interfere with the ability of the vims particles to infect cells.
  • an "immunogenic SARS-CoV compound” is a proteinaceous molecule (i.e., a SARS-CoV protein, or a polypeptide derivative of a SARS-CoV protein) that: (A) possesses an epitope that binds to antibody that is immunologically reactive with a protein of the SARS-CoV; or (B) induces a recipient host to produce antibody that is immunologically reactive with the SARS- CoV.
  • Such epitope may be a contiguous region of the protein, or may be discontinuous and formed through the three-dimensional conformation of the molecule.
  • the immunogenic SARS-CoV compound will be a SARS-CoV S compound that will: (A) possess an epitope that binds to antibody that is immunologically reactive with SARS-CoV S protein; or (B) induce a recipient host to produce antibody that is immunologically reactive with SARS- CoV S protein.
  • polypeptide derivative is intended to refer to a polypeptide of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more amino acid residues which has a sequence that is identical to a sequence of a SARS-CoV protein, or which contains insertions, deletions or substitutions from the sequence of a SARS-CoV protein that nevertheless permit such polypeptide derivative to function as an immunogenic SARS-CoV compound when administered into a recipient.
  • some amino acid sequences of the polypeptides described herein can be varied without negatively affecting the immunogenic nature of the polypeptides and nucleic acids of the invention.
  • Acceptable modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • the same type of modification may be present in the same or varying degrees at several sites in a given compound, and a given compound may contain many types of modifications.
  • Compounds may have more than one type of modification.
  • the immunogenic SARS-CoV compounds of the present invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods.
  • Immunogenic SARS-CoV protein compounds can be prepared in any suitable manner (e.g., isolated from naturally occurring polypeptides, produced via recombinant techniques, produced synthetically or chemically, or produced by a combination of such methods.
  • SEQ ID NO:3 (GenBank AY278741) provides the amino acid sequence of the SARS-CoV S protein of the SARS-CoV (Urbani strain). (SEQ ID NO:3)
  • Prefe ⁇ ed immunogenic SARS-CoV compounds of the invention comprise at least a portion of the extracellular domain of the S protein, which is composed of residues 14 to 1190 of SEQ ID NO:3.
  • such portion will comprise 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 300, 500, 1000 or more amino acid residues whose sequence is identical to a sequence of the extracellular domain of the S protein, or which contains one or more insertions, deletions or substitutions (e.g., >95% identical, >90% identical, >80% identical, >70% identical) from the sequence of the extracellular domain of the S protein, that nevertheless permit such immunogenic SARS-CoV compounds to function as an immunogenic SARS-CoV compound when administered into a recipient.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Bmtlag et al., 1990, Comp. App. Biosci.6:231 -245.
  • a sequence alignment the query and subject sequences are both amino acid sequences.
  • the result of said global sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total residues of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%).
  • a 90 residue subject sequence is compared with a 100 residue query sequence.
  • deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual co ⁇ ections are to be made for the purposes of the present invention.
  • SARS-CoV S protein of SEQ ID NO:3 is encoded by residues 21,492 - 25,259 (SEQ ID NO:4) of the SARS-CoV genome (GenBank AY278741):
  • Preferred nucleic acid molecules ofthe invention will also comprise a polynucleotide encoding a transmembrane polypeptide, refe ⁇ ed to herein as a transmembrane polynucleotide.
  • the polynucleotide may encode a transmembrane portion ofa naturally occurring S protein.
  • the transmembrane portion ofthe S protein ofSEQ ID NO:3 comprises residues 1191 to 1216.
  • the transmembrane polypeptide may comprise this polypeptide sequence or a variant or fragment thereof, or it may be an unrelated transmembrane polypeptide that serves the purpose of anchoring the extracellular domain portion in the cell membrane.
  • Nucleic acid molecules of the invention may also comprise a polynucleotide encoding a cytoplasmic domain. However, it is contemplated as part of this invention that a cytoplasmic domain is not necessary and that the inclusion of a cytoplasmic domain may be undesirable.
  • Preferred nucleic acid molecules of the invention will also comprise a polynucleotide encoding a signal polypeptide, referred to herein as a signal polynucleotide, which directs the secretion of the polypeptide encoded by the polynucleotide or polynucleotide construct of the present invention.
  • a signal polynucleotide which directs the secretion of the polypeptide encoded by the polynucleotide or polynucleotide construct of the present invention.
  • a naturally occurring leader sequence or a functional derivative thereof, is employed.
  • a completely synthetic (i.e., an amino acid sequence not occurring in nature) amino acid coding sequence that functions as a leader sequence can be constructed by those skilled in the art utilizing recombinant DNA techniques, and may be employed.
  • the signal polynucleotide may be that which is ordinarily associated with the SARS-CoV S protein in nature or it may be a heterologous sequence derived from another secreted protein.
  • nucleic acid molecules of the invention encode immunogenic compounds using human preferred codons. Human preferred codons are well known in the art and are described for example, in Haas et al., 1996, Curr. Biol. 6:315-324.
  • the nucleic acid molecules of the invention will be optionally operatively linked to one or more regulatory elements (promoters, translation initiation sites, etc.) so as to permit the expression of the encoded polypeptides in a recipient cell.
  • the nucleic acid molecules will not contain such regulatory elements, and will require cellular processes (such as recombination, integration into nuclear or mitochondrial DNA, etc.) in order to produce the encoded polypeptides.
  • silent substitutions, additions and deletions which do not substantially alter the three dimensional stracture of the extracellular domain of the S protein or that do not alter the ability of the nucleic acid constract encoding the S protein, or the S protein, to induce SARS-CoV neutralizing antibodies.
  • conservative substitutions For example, aromatic amino acids that can be conservatively substituted for one another include phenylalanine, tryptophan, and tyrosine.
  • Hydrophobic amino acids that can be conservatively substituted for one another include leucine, isoleucine, and valine.
  • Polar amino acids that can be conservatively substituted for one another include glutamine and asparagine.
  • Basic amino acids that can be conservatively substituted for one another include arginine, lysine, and histidine.
  • Acidic amino acids that can be conservatively substituted for one another include aspartic acid and glutamic acid.
  • Small amino acids that can be conservatively substituted for one another include alanine, serine, threonine, methionine, and glycine.
  • immunogenic SARS-CoV compounds comprise polynucleotides encoding at least two SARS CoV proteins, or portions thereof, capable of forming synthetic nucleocapsids upon co- transfection.
  • one aspect of the invention relates to the recognition that co-transfection of polynucleotides encoding the following combinations of SARS CoV proteins results in the formation of nucleocapsids: N, M and S proteins; N, M and E proteins; N and M proteins; and N, M, S and E proteins.
  • the invention relates to the following: co-transfection of polynucleotides encoding the N, M and S proteins, or portions of these proteins; co-transfection of polyncleotides encoding the N, M and E proteins, or portions of these proteins; co- transfection of polyncleotides encoding the M and N proteins, or portions of these proteins; and co-transfection of polyncleotides encoding the N, M, S and E proteins, or portions of these proteins.
  • the invention also relates to the recognition that co-transfection of polynucleotides encoding the N, M, and S proteins, or portions of these proteins, results in the formation of budding virus or corona-like structures.
  • co-transfected polynucleotides comprise polynucleotides that encode the N, M and S proteins, or portions of these proteins, wherein co- transfection results in the formation of budding vims or the formation of corona- like stractures.
  • Preferred immunogenic SARS-CoV compounds of the invention will comprise polynucleotides encoding 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 300, 500, 1000 or more amino acid residues of the SARS CoV S, M, N or E whose sequence is identical to a sequence of the S, M, N or E protein, or which contains one or more insertions, deletions or substitutions (e.g., >95% identical, >90%> identical, >80% identical, >70% identical) from the sequence of the S, M, N or E proteins, that nevertheless permit such immunogenic SARS-CoV compounds to function form nucleocapsids, preferably corona-like stractures, when administered into a recipient.
  • the protein sequences of S, M, N, and E proteins, and the nucleic acid sequences encoding those proteins may be found at Genbank Accession No. AY278741.
  • the protein sequences of the M, N or E SARS-CoV proteins may be readily ascertained by one of skill in the art.
  • the protein sequences for SARS CoV (Urbani strain) are as follows:
  • E protein (Genbank Accession No. AAP13443.1) (SEQ ID NO: 5) YSFVSEETG TLIVNSVLLF LAFWFLLVT LAILTALRLC AYCCNIV VS LVKPTVYVYS RVKNLNSSEG VPDLLV
  • MADNGTITVE ELKQLL ⁇ Q N LVIGFLFLA IMLLQFAYSN RNRFLYIIKL VFLWLLWPVT LACFVLAAVY RI WVTGGIA IA ACIVGLM LSYFVASFR LFARTRSMWS FNPETNILLN
  • co-tranfection may occur via serial transfection (i.e. administration to the subject) of the polynucleotides, wherein one or more polynucleotides encoding at least one nucleocapsid forming protein (M, N, S or E), or portions thereof, are administered initially, with a second administration of at least one polynucleotide encoding at least one additional nucleocapsid forming protein.
  • serial transfection i.e. administration to the subject
  • one or more polynucleotides encoding at least one nucleocapsid forming protein M, N, S or E
  • a "SARS-CoV vaccine composition” is a pharmaceutically acceptable composition that, when administered to a subject, exposes the subject to an immunogenic SARS-CoV compound, and induces the formation of an immune response against SARS-CoV.
  • the SARS-CoV vaccine composition will comprise the immunogenic SARS-CoV compound.
  • the SARS-CoV vaccine composition will comprise one or more nucleic acid molecules that upon administration to the subject directs the synthesis or expression of an immunogenic SARS-CoV compound.
  • the vaccine compositions of the present invention will comprise one or more nucleic acid molecules that encode at least a portion of the S protein, and most preferably at least a portion of the extracellular domain of the S protein.
  • the nucleic acid molecules of such vaccme compositions may be single stranded or double-stranded, and may be circular or linear. Such molecules may be DNA or RNA, or composed of both DNA and RNA.
  • the nucleic acid molecules of such vaccine compositions may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • nucleic acid molecules can be present in a single construct, e.g., on a single plasmid, or in separate constructs, e.g., on separate plasmids.
  • nucleic acid molecules will preferably additionally comprise a regulatory element such as a promoter, a transcription terminator, etc. or a secretory or other signal peptide or domain.
  • Vaccination via the introduction of a nucleic acid molecule encoding the S protein reduces SARS-CoV replication by six orders of magnitude in the lungs of vaccinated subjects.
  • the administration of such vaccine compositions will mediate the induction of neutralizing antibodies for SARS-CoV in a human or non- human animal (e.g., canine, feline, porcine, ovine, avine, equine, bovine, simian, etc.).
  • the immune response induced is preferably, but need not be, clinically immunoprotective.
  • an "immunoprotective" immune response is an immune response that reduces the growth, infectivity, pathogenicity, or viability of an infectious agent in a human or a non-human animal.
  • a clinically immunoprotective immune response is an immunoprotective response that significantly attenuates, and more preferably prevents, the growth, infectivity, pathogenicity, or viability of an infectious agent in a human or a non-human animal.
  • the methods and compositions of the present invention will serve to protect an uninfected subject so as to substantially prevent or attenuate symptoms or SARS-CoV infection.
  • symptoms include, but are not limited to high fever (>38°C), dry cough, shortness of breath or breathing difficulties, pneumonia, headache, muscular stiffness, loss of appetite, malaise, confusion, rash and diarrhea.
  • the methods and compositions of the invention will serve to prevent an individual from becoming infected with SARS-CoV.
  • the methods and compositions of the invention will serve to reduce the time frame for persistence of the viras in infected individuals (referred to herein as "persistence time").
  • the persistence time of the viras in infected individuals can be assayed in a number of different ways.
  • persistence time of the vims can be detected via the use of samples obtained from mouth, nose or throat swabs or from nasal washings. Samples may be analyzed for the presence of infectious particles using, for example, techniques that are described in the Examples herein.
  • vaccine compositions of the invention are effective in reducing the persistence time of the vims, as detected by any selected sample procedure and assay procedures, by about 15 %, 30%, 45%, 60%, 75% or 90% in vaccinated subjects as compared to unvaccinated control subjects.
  • an "antibody-dependent vims enhancing activity" with respect to a vaccine composition or an immunogenic compound refers to the ability of a particular immunogenic compound or vaccine composition to elicit an antibody response that enhances cellular entry of any strain of the SARS-CoV viras, particularly strains of SARS-CoV that are isolated from animals, preferably non-human mammals, such as, for example, the Palm Civet (Paguma larvata), the raccoon dog, or the Chinese ferret badger.
  • antibody-dependent virus enhancing activity can be assessed, for examples, using the techniques described in Example 5 herein.
  • vaccine compositions or immunogenic compounds that that do not possess an antibody depedent viras enhancing activity will comprise a DNA molecule that encodes the SARS-CoV S protein that is truncated at or near the juncture of the extracellular domain and the transmembrane domain (i.e. within 10 amino acids, preferably within 5 amino acids, upstream or downstream, of amino acid 1191 of SEQ ID NO:3), to result in cellular secretion of the extracellular domain portion, or a portion thereof, of the SARS-CoV S protein.
  • the vaccme composition will comprise an RNA molecule that encodes an immunogenic SARS-CoV compound.
  • the RNA is in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • Methods for introducing RNA sequences into vertebrate cells are described in U.S. Patent No. 5,580,859.
  • the RNA may be in the form of an RNA vims genome.
  • an RNA virus genome of the present invention is noninfectious, (i.e., does not result in the production of infectious viras particles in vertebrate cells).
  • RNA viras genomes include, but are not limited to, alphaviras genomes, picornaviras genomes, and retroviras genomes.
  • Methods for the in vivo introduction of non-infectious viral genomes to vertebrate tissues are well known to those of ordinary skill in the art and are described, e.g., in Altman-Hamamdzic et al. , 1997, EXPRESSION OF BETA-GALACTOSIDASE IN MOUSE BRAIN: UTILIZATION ' OF A NOVEL NONREPLICAT ⁇ V ⁇ SINDBIS VIRUS VECTOR As A NEURONAL GENE DELIVERY SYSTEM, Gene ⁇ ierapy 4:815-822, in U.S.
  • RNA viras genomes packaged in a viral coat e.g., a picornaviras coat or an alphaviras coat
  • Viral replicons i.e., non-infectious RNA viras genomes packaged in a viral coat, e.g., a picornaviras coat or an alphaviras coat, are also useful for efficient administration of RNA. See, e.g., U.S. Patents Nos. 5,766,602; 5,614,413, and PCT Publication No. WO 95/07994.
  • the vaccine composition will comprise a DNA molecule that encodes an immunogenic SARS-CoV compound.
  • a DNA molecule is typically a component of an expression vector.
  • a typical expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing.
  • Suitable DNA viras genomes include he ⁇ esviras genomes, adenoviras genomes, adeno-associated vims genomes, and poxvims genomes.
  • cellular elements can also be used (e.g., the human actin promoter, metallothionem promoter). In humans, CMV IEP is preferred.
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109), VR1051, VR1055, and pcDNA3 (Invitrogen, San Diego, Calif).
  • vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109), VR1051, VR1055, and pcDNA3 (Invitrogen, San Diego, Calif).
  • CMV cytomegalovirus
  • transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • Promoters may be a cell-specific promoter and direct substantial transcription of the DNA only in predetermined cells or the promoter may not have cell type specificity.
  • Other transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the S polynucleotide or polynucleotide construct to direct cell-specific transcription.
  • a nucleic acid molecule of a SARS-CoV vaccine composition of the present invention is part of a circular or linearized plasmid, which is preferably non-infectious (i.e., does not result in the production of infectious vims particles in vertebrate cells), and noninitegrating (i.e., does not integrate into the genome of vertebrate cells).
  • a linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease.
  • non-infectious means that the vector is not capable of eliciting the production of infectious vims particles upon its entry into a recipient cell.
  • a non-infectious vector containing a nucleic acid molecule of a SARS-CoV vaccine composition of the present invention can contain functional sequences from non-mammalian (e.g., viral or bacterial) species, but does not contain at least one viral sequence necessary for the production of infectious virion particles.
  • non-integrating is intended to denote that the nucleic acid molecule of the SARS-CoV vaccine composition does not functionally integrate into the genome of recipient cells.
  • the construct can, for example, be a non-replicating polynucleotide, or a specific replicating polynucleotide genetically engineered to lack the ability to mediate integration into the genome of a recipient cell.
  • a non-integrating nucleic acid molecule lacks a functional sequence required for the integration of a polynucleotide into the genome of a recipient cell.
  • nucleic acid molecule form depends in part on the desired kinetics and duration of expression.
  • DNA is preferred, especially DNA plasmids.
  • RNA is preferred, especially messenger RNA, since RNA is rapidly translated into polypeptide, but is degraded more quickly than DNA.
  • An "operable association" is achieved when a nucleic acid molecule encoding a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the molecule under the influence or control of the regulatory sequence(s).
  • Two DNA molecules (such as an immunogenic SARS- CoV compound-encoding molecule and a promoter associated with the 5' end of such molecule) are "operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA molecules does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or (3) interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with an immunogenic SARS-CoV compound- encoding molecule if the promoter is capable of effecting transcription of that molecule.
  • nucleic Acid Vaccine Compositions of the Present Invention Numerous methods for delivering nucleic acid molecules are known in the art. The most convenient way to delivery the polynucleotide constructs is in a plasmid (DNA) vector. Alternatively, a viral vector can be used. A number of viral based systems have been developed for transfecting mammalian cells. For example, nucleic acid molecules of the invention can be inserted into a vector and packaged as retroviral particles using techniques known in the art. A number of retroviral systems have been described (U.S. Pat. No.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al.
  • Additional viral vectors which will find use for delivering the recombinant nucleic acid molecules of the present invention, include, but are not limited to, those derived from the pox family of vimses, including vaccinia viras and avian poxvirus.
  • DNA vaccines and methods of their manufacture and delivery that may be used in accordance with the present invention are disclosed in US Patents Nos. 5,589,466; 5,620,896; 5,641,665; 5,703,055; 5,707,812; 5,846,946; 5,861,397; 5,891,718; 6,022,874; 6,147,055; 6,214,804; 6,228,844; 6,399,588; 6,413,942; 6,451,769, European Patent Documents EP 1165140A2; EP1006796A1 and EP0929536A1; and PCT Patent Publications WO00/57917; WO00/73263; WO01/09303; WO03/028632; W094/29469; WO95/29703; and W098/14439.
  • Administration may be by needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., "gene guns” or pneumatic "needle less” injectors) Med-E-Jet (Vahlsing, H., et al. (1994) "IMMUNIZATION WITH PLASMID DNA USING A PNEUMATIC GUN,” J. Immunol. Methods 171:11-22), Pigjet (Schrijver, R.S. et al. (1997) “IMMUNIZATION OF CATTLE WITH A BHV1 VECTOR VACCINE OR A DNA VACCINE BOTH CODING FOR THE G PROTEIN OF BRSV," Vaccine 15:1908-1916), Biojector (Davis, H.L.
  • particle accelerators e.g., "gene guns” or pneumatic "needle less” injectors
  • Med-E-Jet Vahlsing, H., et al. (1994) "IMMUNIZATION WITH PLASMID
  • administration is into muscle tissue, i.e., skeletal muscle, smooth muscle, or myocardium.
  • muscle i.e., skeletal muscle, smooth muscle, or myocardium.
  • the muscle is skeletal muscle.
  • the DNA can be operably linked to a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • a polynucleotide construct, or composition comprising a polynucleotide or polynucleotide construct is delivered to any tissue including, but not limited to those disclosed herein, such that the polynucleotide or polynucleotide construct is free from association with liposomal formulations and charged lipids.
  • the polynucleotide, polynucleotide construct, or composition is delivered to a tissue other than brain or nervous system tissue, for example, to muscle, skin, or blood, in any composition as described herein.
  • the vaccine composition is delivered to the interstitial space of a tissue.
  • Interstitial space comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels.
  • the DNA vaccine preferably in the form of plasmid DNA, may be administered (especially by injection) into tissue and voltage pulses applied between electrodes disposed in the tissue, thus applying electric fields to cells of the tissue.
  • the electrically-mediated enhancement covers administration using either iontophoresis or electroporation in vivo. Suitable techniques of electroporation and iontophoresis are provided by Singh, J. et al. (1989) "TRANSDERMAL DELIVERY OF DRUGS BY IONTOPHORESIS: A REVIEW,” Drug Des. Deliv. 4:1-12; Theiss, U. et al. (1991) "IONTOPHORESIS-IS THERE A FUTURE FOR CLINICAL APPLICATION?,” Methods Find. Exp. Clin. Pharmacol.
  • the waveform of the electrical signal provided by the pulse generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train or a bipolar oscillating pulse train.
  • the waveform, electric field strength and pulse duration are dependent upon the type of cells and the DNA that are to enter the cells via electrical-mediated delivery and thus are determined by those skilled in the art in consideration of these criteria.
  • Any number of known devices may be used for delivering the DNA vaccine and generating the desired electric field. Examples of suitable devices include, but are not limited to, a single needle probe, a bipolar probe and a combination needle and plate probe.
  • methods such as continuous-flow electroporation may be employed (See, U.S. Patents Nos. 6,485,961; 6,090,617; 6,074,605; 5,720,921; 5,612,207; and 5,098,843).
  • Nucleic acid molecules of the present invention may be formulated into any of the various compositions and may be used in any of the methods disclosed herein.
  • plasmid DNA e.g., plasmid DNA, ' derivatives of plasmid DNA, mRNA, linear DNA, viral genomes, or polynucleotide fragments contained therein
  • aqueous compositions used in vivo use of sterile pyrogen-free water is preferred.
  • Such formulations will contain an effective amount of a polynucleotide or polynucleotide constract together with a suitable salt and/or auxiliary agent as disclosed herein, in order to prepare pharmaceutically acceptable compositions suitable for optimal administration to a vertebrate.
  • Insoluble polynucleotides or polynucleotide constructs may be solubilized in a weak acid or weak base, and then diluted to the desired volume, for example, with an aqueous solution of the present invention.
  • the pH of the solution may be adjusted as appropriate.
  • a pharmaceutically acceptable additive can be used to provide an appropriate osmolarity.
  • compositions of the present invention may include one or more transfection facilitating materials that facilitate delivery of polynucleotides or polynucleotide constructs to the interior of a cell, and/or to a desired location within a cell.
  • transfection facilitating materials include, but are not limited to lipids, preferably cationic lipids; inorganic materials such as calcium phosphate, and metal (e.g., gold or tungsten) particles (e.g., "powder” type delivery solutions); peptides, including cationic peptides, targeting peptides for selective delivery to certain cells or intracellular organelles such as the nucleus or nucleolus, and amphipathic peptides, i.e.
  • helix forming or pore forming peptides helix forming or pore forming peptides; basic proteins, such as histories; asialoproteins; viral proteins (e.g., Sendai vims coat protein); pore-forming proteins; and polymers, including dendrimers, star- polymers, "homogenous" poly-amino acids (e.g., poly-lysine, poly-arginine), "heterogeneous" poly-amino acids (e.g., mixtures of lysine & glycine), copolymers, polyvinylpyrcolidinone (PVP), and polyethylene glycol (PEG).
  • dendrimers star- polymers
  • homoogenous poly-amino acids e.g., poly-lysine, poly-arginine
  • heterogeneous poly-amino acids e.g., mixtures of lysine & glycine
  • copolymers polyvinylpyrcolidinone (P
  • auxiliary agents of the present invention that facilitate and enhance the entry of a polynucleotide or polynucleotide construct into vertebrate cells in vivo, may also be considered "transfection facilitating materials.”
  • Certain embodiments of the present invention may include lipids as a transfection facilitating material, including cationic lipids (e.g., DMRIE, DOSPA, DC-Choi, GAP-DLRIE), basic lipids (e.g., steryl amine), neutral lipids (e.g., cholesterol), anionic lipids (e.g., phosphatidyl serine), and zwitterionic lipids (e.g., DOPE, DOPC).
  • cationic lipids are 5-carboxyspermylglycine dioctadecylamide
  • Cationic cholesterol derivatives are also useful, including ⁇ 3.beta.-[N- N',N'-dimethylamino)ethane]-carbomoyl ⁇ -cholesterol (DC-Chol).
  • Dimethyldioctdecyl-ammonium bromide (DDAB), N-(3-aminopropyl)-N,N-(bis- (2-tetradecyloxyethyl))-N-methyl-ammonium bromide (PADEMO), N-(3- aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methy- l-ammonium bromide (PADELO), N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)pro- pyl-ammonium bromide (PATELO), and N.sup.l-(3-ammopropyl)((2-dodecyloxy)e- thyl)- N.sup.2-(2-dodecyloxy)ethyl-l-piperazinaminium bromide (GALOE-BP)
  • Non-diether cationic lipids such as DL-l,2-dioleoyl-3-dimethylamin- opropyl-.beta.-hydroxyethylammonium (DORI diester), l-0-oleyl-2-oleoyl-3- dimethylaminopropyl-.beta.-hydroxyethylammonium (DORI ester/ether), and their salts promote in vivo gene delivery.
  • Preferred cationic lipids comprise groups attached via a heteroatom attached to the quaternary ammonium moiety in the head group.
  • a glycyl spacer can connect the linker to the hydroxyl group.
  • Cationic lipids for use in certain embodiments of the present invention include DMRIE (( ⁇ )-N-(2-hydroxyethyl)-N,N-dimethyl-2- ,3-bis(tetradecyloxy)-l- propanaminium bromide), and GAP-DMORIE ((+)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-l-pro- panaminium bromide), as well as ( ⁇ )-N,N-dimethyl-N-[2-(sperminecarboxamido)et- hyl]-2,3-bis(dioleyloxy)-l- propaniminium pentahydrochloride (DOSPA), ( ⁇ )-N-(2-aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanimini- um bromide (.beta.
  • DMRIE-derived cationic lipids that are useful for the present invention are ( ⁇ )-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-l- propanaminium bromide (GAP-DDRIE), ( ⁇ )-N-(3-aminopropyl)-N,- N-dimethyl- 2,3 -(bis-tettadecyloxy)-l -propanaminium bromide (GAP-DMRIE), ( ⁇ )-N-((N"- methyl)-N'-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GMU-DMRIE), ( ⁇ )-N-(2-hydrpxyethyl)-N,N-dimeth- yl-2,3- bis(dodecyloxy)-l -propanaminium bromide
  • a cationic lipid that may be used in concert with the vaccine compositions of the present invention is a "cytofectin.”
  • a "cytofectin” refers to a subset of cationic lipids that incorporate certain structural features including, but not limited to, a quaternary ammonium group and/or a hydrophobic region (usually with two or more alkyl chains), but which do not require amine protonation to develop a positive charge. Examples of cytofectins may be found, for example, in U.S. Patent No. 5,861,397.
  • Cytofectins that may be used in the present invention, include DMRIE (( ⁇ )-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- pr- opanaminium bromide), GAP-DMORIE (( ⁇ )-N-(3-aminopropyl)-N,N- dimethyl-2,- 3 -bis(syn-9-tetradeceneyloxy)-l -propanaminium bromide), and GAP- DLRIE (( ⁇ )-N-(3-aminopro ⁇ yl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-l- propanamini- um bromide).
  • DMRIE (( ⁇ )-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- pr- opanaminium bromide)
  • the cationic lipid may be mixed with one or more co-lipids.
  • co- lipid refers to any hydrophobic material that may be combined with the cationic lipid component and includes amphipathic lipids, such as phospholipids, and neutral lipids, such as cholesterol.
  • amphipathic lipids such as phospholipids
  • neutral lipids such as cholesterol.
  • Cationic lipids and co-lipids may be mixed or combined in a number of ways to produce a variety of non-covalently bonded macroscopic stractures, including, for example, liposomes, multilamellar vesicles, unilamellar vesicles, micelles, and simple films.
  • a prefe ⁇ ed class of co-lipids is the zwitterionic phospholipids, which include the phosphatidylethanolamines and the phosphatidylcholines.
  • the co-lipids are phosphatidylethanolamines, such as, for example, DOPE, DMPE and DPyPE. DOPE and DPyPE are particularly preferred.
  • the most preferred co-lipid is DPyPE, which comprises two phytanoyl substituents inco ⁇ orated into the diacylphosphatidylethano- lamine skeleton.
  • the cationic lipidxo-lipid molar ratio may range from about 9: 1 to about 1 :9, or from about 4: 1 to about 1 :4, or from about 2: 1 to about 1 :2, or about 1:1.
  • a solvent such as chloroform
  • evaporation of the cationic lipid/co-lipid solution under vacuum to dryness as a film on the inner surface of a glass vessel (e.g., a Rotovap round-bottomed flask).
  • a glass vessel e.g., a Rotovap round-bottomed flask
  • the amphipathic lipid component molecules self-assemble into homogenous lipid vesicles.
  • lipid vesicles may subsequently be processed to have a selected mean diameter of uniform size prior to combining with, for example, plasmid DNA according to methods known to those skilled in the art.
  • plasmid DNA DNA sequence
  • the sonication of a lipid solution is described in Feigner, P.L., et al. (1987) "LIPOFECTION: A HIGHLY EFFICIENT, LIPID-MEDIATED DNA-TRANSFECTION PROCEDURE,” Proc Natl. Acad. Sci. USA 84:7413-7417 and in U.S. Patent No. 5,264,618.
  • the polynucleotide or polynucleotide constructs are combined with lipids by mixing, for example, a plasmid DNA solution and a solution of cationic lipid:co-lipid liposomes.
  • concentration of each of the constituent solutions is adjusted prior to mixing such that the desired final plasmid DNA/cationic lipid: co-lipid ratio and the desired plasmid DNA final concentration will be obtained upon mixing the two solutions.
  • the various components of the composition e.g., plasmid DNA, cationic lipid: co-lipid liposomes, and any other desired auxiliary agents, transfection facilitating materials, or additives are each prepared in 2.5 mM sodium phosphate and then simply mixed to afford the desired complex.
  • the desired final solution is to be, e.g., 2.5 mM sodium phosphate
  • certain components of the composition e.g., the auxiliary agent and/or cationic lipid:co-lipid liposomes
  • a volume of water which is less than that of the final volume of the composition
  • certain other components of the composition e.g., the plasmid DNA
  • the final composition is in an aqueous solution of 2.5 mM sodium phosphate.
  • the plasmid DNA could be prepared in 5.0 mM sodium phosphate at one half the final volume
  • the auxiliary agent and/or cationic lipidxo- lipid liposome is prepared in water at one half the final volume, and then these two elements are mixed together to produce the final composition.
  • the cationic lipid: co-lipid liposomes are preferably prepared by hydrating a thin film of the mixed lipid materials in an appropriate volume of aqueous solvent by vortex mixing at ambient temperatures for about 1 minute.
  • the thin films are prepared by admixing chloroform solutions of the individual components to afford a desired molar solute ratio followed by aliquoting the desired volume of the solutions into a suitable container.
  • the solvent is removed by evaporation, first with a stream of dry, inert gas (e.g. argon) followed by high vacuum treatment.
  • inert gas e.g. argon
  • a transfection facilitating material can be used alone or in combination with one or more other transfection facilitating materials.
  • Two or more transfection facilitating materials can be combined by chemical bonding (e.g., covalent and ionic such as in lipidated polylysine, PEGylated polylysine) (Toncheva, V., et al. (1998) "NOVEL VECTORS FOR GENE DELIVERY FORMED BY SELF-ASSEMBLY OF DNA WITH POLY(L-LYSINE) GRAFTED WITH HYDROPHILIC POLYMERS,” Biochim. Biophys.
  • chemical bonding e.g., covalent and ionic such as in lipidated polylysine, PEGylated polylysine
  • hydrophobic and amphiphilic additives such as, for example, sterols, fatty acids, gangliosides, glycolipids, lipopeptides, liposaccharides, neobees, niosomes, prostaglandins and sphingolipids, may also be included in the compositions of the present invention.
  • these additives may be included in an amount between about 0.1 mol % and about 99.9 mol % (relative to total lipid).
  • these additives comprise about 1-50 mol % and, most preferably, about 2-25 mol %.
  • Preferred additives include lipopeptides, liposaccharides and steroids.
  • a vaccme composition may comprise multiple copies of a single polynucleotide constract or several different polynucleotide constructs.
  • the vaccine composition comprises immunogenic polypeptides
  • the composition may comprise multiple copies of a single immunogenic polypeptide, or several different immunogenic polypeptides.
  • the vaccine composition may comprise a combination of polynucleotide constructs and immunogenic polypeptides.
  • vaccine compositions of the invention may be coadministered or sequentially administered, and administered in conjunction with other vaccine compositions.
  • the nucleic acid molecules may be introduced ex vivo into cells, which cells are then introduced into the subject to be vaccinated. In preferred embodiments of ex vivo administration, the cells are derived from the subject to be vaccinated.
  • ex vivo cells are cells into which the polynucleotide construct is introduced, for example, by transfection, lipofection, electroporation, bombardment, or microinjection.
  • the cells contaimng the polynucleotide constract are then administered in vivo into mammalian tissue (see, for example, see Belldegrun, A., et al. (1993) "HUMAN RENAL CARCINOMA LINE TRANSFECTED WITH INTERLEUKIN-2 AND/OR INTERFERON ALPHA GENE(S): IMPLICATIONS FOR LIVE CANCER VACCINES," J. Natl. Cancer Inst. 85: 207-216; Ferrantini, M. et al.
  • a vaccine composition is administered in vivo, such that the vaccine is inco ⁇ orated into the local cells at the site of administration.
  • the local cells subsequently express the immunogenic compound in an amount sufficient to provide an immunoprotective effect against SARS-CoV.
  • the vaccine composition can be administered either within ex vivo cells or free of ex vivo cells or ex vivo cellular material.
  • the polynucleotide construct is administered free of ex vivo cells or ex vivo cellular material.
  • the vaccines of the present invention may thus be administered by inhalation, or interdermally, intracavity (e.g., oral, vaginal, rectal, nasal, peritoneal, ventricular, or intestinal), intradermally, intramuscularly, intranasally, intraocularly, intraperitoneally, intrarectally, intratracheally, intravenously, orally, subcutaneously, transdermally, or transmucosally (i.e., across a mucous membrane) in a dose effective for the production of neutralizing antibody and resulting in protection from infection or disease.
  • intracavity e.g., oral, vaginal, rectal, nasal, peritoneal, ventricular, or intestinal
  • intradermally intramuscularly, intranasally, intraocularly, intraperitoneally, intrarectally, intratracheally, intravenously, orally, subcutaneously, transdermally, or transmucosally (i.e., across a mucous membrane) in a
  • the present vaccine can generally be administered in the form of a spray for intranasal administration, or by nose drops, inhalants, swabs on tonsils, or a capsule, liquid, suspension or elixirs for oral administration.
  • the vaccine may be in the form of single dose preparations or in multi-dose flasks, which can be used for mass vaccination programs.
  • Remington 's Pharmaceutical Sciences Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.
  • Any mode of administration can be used so long as the mode results in the expression of SARS-CoV immunogenic compounds in the desired tissue, in an amount sufficient to be detectable, and/or prophylactically or therapeutically effective.
  • Methods to detect such a response include serological methods to detect the polypeptide in semm, e.g., western blotting, staining tissue sections by immunohistochemical methods, measuring an immune response generated by the mammalian against the polypeptide, and measuring the activity of the polypeptide.
  • the vaccine compositions of the present invention can be lyophilized to produce a vaccine composition in a dried form for ease in transportation and storage.
  • the vaccine compositions of the present invention may be stored in a sealed vial, ampule or the like.
  • the vaccine is dissolved or suspended (e.g., in sterilized distilled water) before administration.
  • An inert carrier such as saline or phosphate buffered saline or any such carrier in which the vaccine composition has suitable solubility, may be used.
  • the vaccine composition may be prepared in the form of a mixed vaccine that contains one or more additional antigens so long as such additional antigens do not interfere with the effectiveness of the SARS-CoV vaccine of the invention, and the side effects and adverse reactions are not increased additively or synergistically.
  • the vaccine compositions of the present invention can be associated with chemical moieties, which may improve the vaccine's solubility, abso ⁇ tion, biological half-life, etc.
  • the moieties may alternatively decrease the toxicity of the vaccine, eliminate or attenuate any undesirable side effect of the vaccine, etc.
  • Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.
  • the immunogenic SARS-CoV vaccine compositions of the invention can be formulated according to known methods for preparing pharmaceutical compositions, whereby the substance to be delivered is combined with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their preparation are described, for example, in Remington's Pharmaceutical Sciences, 16.th Edition, A. Osol, Ed., Mack Publishing Co., Easton, Pa. (1980), and Remington's Pharmaceutical Sciences, 19.sup.th Edition, A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995).
  • compositions of the present invention may be in the form of an emulsion, gel, solution, suspension, etc.
  • the pharmaceutical composition can also contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives.
  • Administration of pharmaceutically acceptable salts of the polynucleotides described herein is preferred.
  • Such salts can be prepared from pharmaceutically acceptable non-toxic bases including organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, basic amino acids, and the like.
  • the amount of polypeptides or nucleic acid molecules in a vaccine composition of the present invention depends on many factors, including the age and weight of the subject, the delivery method and route, the type of treatment desired, and the type of nucleic acid molecule being administered.
  • the optimal amount of polypeptides or nucleic acid molecules of the invention that should be administered to a subject to induce a protective immune response or neutralizing antibodies in a subject can be readily determined by one of skill in the art.
  • composition of the present invention containing nucleic acids contains from about 1 ng to about 30 mg of an immunogenic SARS-CoV nucleic acid molecule or polynucleotide constract, more preferably, from about 100 ng to about 10 mg of an immunogenic SARS-CoV nucleic acid molecule or polynucleotide construct.
  • compositions of the present invention may include about 1 ng of an immunogenic SARS-CoV nucleic acid molecule, about 5 ng of an immunogenic SARS-CoV nucleic acid molecule, about 10 ng of an immunogenic SARS-CoV nucleic acid molecule, about 50 ng of an immunogenic SARS-CoV nucleic acid molecule, about 100 ng of an immunogenic SARS-CoV nucleic acid molecule, about 500 ng of an immunogenic SARS-CoV nucleic acid molecule, about 1 ⁇ g of an immunogenic SARS-CoV nucleic acid molecule, about 5 ⁇ g of an immunogenic SARS-CoV nucleic acid molecule, about 10 ⁇ g of an immunogenic SARS-CoV nucleic acid molecule, about 50 ⁇ g of an immunogenic SARS-CoV nucleic acid molecule, about 100 ⁇ g of an immunogenic SARS-CoV nucleic acid molecule, about 150 ⁇ g of an immunogenic SARS-CoV nucleic acid molecule
  • composition of the present invention containing an immunogenic SARS-CoV polypeptide contains from about 1 ng to about 30 mg of an immunogenic SARS-CoV polypeptide, more preferably, from about 100 ng to about 10 mg of an immunogenic SARS-CoV polypeptide.
  • compositions of the present invention may include about 1 ng of an immunogenic SARS-CoV polypeptide, about 5 ng of an immunogenic SARS-CoV polypeptide, about 10 ng of an immunogenic SARS-CoV polypeptide, about 50 ng of an immunogenic SARS-CoV polypeptide, about 100 ng of an immunogenic SARS- CoV polypeptide, about 500 ng of an immunogenic SARS-CoV polypeptide, about 1 ⁇ g of an immunogenic SARS-CoV polypeptide, about 5 ⁇ g of an immunogenic SARS-CoV polypeptide, about 10 ⁇ g of an immunogenic SARS-CoV polypeptide, about 50 ⁇ g of an immunogenic SARS-CoV polypeptide, about 100 ⁇ g of an immunogenic SARS-CoV polypeptide, about 150 ⁇ g of an immunogenic SARS- CoV polypeptide, about 200 ⁇ g of an immunogenic SARS-CoV polypeptide, about 250 ⁇ g of an immunogenic SARS-CoV polypeptide
  • the vaccine compositions of the present invention may be used in concert with adjuvants and other compounds to enhance their immunologic effect.
  • Adjuvants that enhance production of SARS-CoV-specif ⁇ c antibodies include, but are not limited to, various oil formulations such as stearyl tyrosine (ST, see U.S. Patent No. 4,258,029), the dipeptide MDP, saponin, aluminum hydroxide, and lymphatic cytokine.
  • Mucosal adjuvants include cholera toxin B subunit (CTB), a heat labile enterotoxin (LT) from E. coli, and Emulsomes (Pharmos, LTD.,
  • the adjuvant alum (aluminum hydroxide) or ST may be used for administration to humans.
  • aqueous pharmaceutical compositions used in vivo sterile pyrogen-free water is preferred.
  • Such formulations will contain an effective amount of the substance together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions suitable for administration to a human or animal.
  • Insoluble polynucleotides or polynucleotide constructs may be solubilized in a weak acid or weak base, and then diluted to the desired volume, for example, with an aqueous solution of the present invention. The pH of the solution may be adjusted as appropriate.
  • a pharmaceutically acceptable additive can be used to provide an appropriate osmolarity.
  • salt is a substance produced from the reaction between acids and bases, which comprises a metal (cation) and a nonmetal (anion).
  • Salt crystals may be "hydrated” i.e., contain one or more water molecules.
  • Such hydrated salts when dissolved in an aqueous solution at a certain molar concentration, are equivalent to the corresponding anhydrous salt dissolved in an aqueous solution at the same molar concentration.
  • salts that are readily soluble in an aqueous solution are preferred.
  • saline or "normal saline” as used herein refer to an aqueous solution of about 145 mM to about 155 mM sodium chloride, preferably about 154 mM sodium chloride.
  • phosphate buffered saline or PBS refer to an aqueous solution of about 145 mM to about 155 mM sodium chloride, preferably about 154 sodium chloride, and about 10 mM sodium phosphate, at a pH ranging from about 6.0 to 8.0, preferably at a pH ranging from about 6.5 to about 7.5; most preferably at pH 7.2.
  • Certain embodiments of the present invention are drawn to vaccme compositions comprising nucleic acid molecules dissolved in a salt solution which improves entry of the polynucleotide or polynucleotide construct into vertebrate cells in vivo.
  • Preferred salts in which to dissolve a polynucleotide or polynucleotide construct of the present invention include but are not limited to sodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium pyruvate, potassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium pyruvate, disodium DL-.alpha.-glycerol-phosphate, and disodium glucose-6-phosphate.
  • Phosphate salts of sodium or potassium can be either the monobasic form, e.g., NaHP0 4 , or the dibasic form, e.g., Na 2 HP0 4 , but a mixture of the two, resulting in a desired pH, is most preferred.
  • the most prefe ⁇ ed salts are sodium phosphate or potassium phosphate.
  • sodium phosphate or “potassium phosphate,” refer to a mixture of the dibasic and monobasic forms of each salt to present at a given pH.
  • Salts of the present invention are preferably dissolved in aqueous solution at concentrations which enhance entry of nucleic acids of the invention into mammalian cells in vivo, and/or enhance polypeptide expression, relative to saline, PBS, or water.
  • nucleic acid molecules of the invention are dissolved in a salt solution of about 150 mM NaHP0 4 , Na 2 HP0 4 , or NaHC0 3 .
  • auxiliary agent is a substance mcluded in a composition for its ability to enhance, relative to a composition which is identical except for the inclusion of the auxiliary agent, the entry of polynucleotides or polynucleotide constructs into vertebrate cells in vivo, and/or the in vivo expression of polypeptides encoded by such polynucleotides or polynucleotide constructs.
  • Auxiliary agents of the present invention include nonionic, anionic, cationic, or zwitterionic surfactant or detergents, with nonionic, anionic, cationic, or zwitterionic surfactant or detergents, with nonionic surfactant or detergents being preferred, chelators, Dnase inhibitors, agents that aggregate or condense nucleic acids, emulsifying or solubilizing agents, wetting agents, gel- forming agents, and buffers.
  • Suitable auxiliary agents include non-ionic detergents and surfactant such as poloxamers.
  • Poloxamers are a series of non-ionic surfactant that are block copolymers of ethylene oxide and propylene oxide.
  • the poly(oxyethylene) segment is hydrophilic and the poly(oxypropylene) segment is hydrophobic.
  • the physical forms are liquids, pastes or solids.
  • the molecular weight ranges from 1000 to greater than 16000.
  • the basic structure of a poloxamer is HO-- [CH 2 CH 2 0] x -[CH 2 CHO(CH 3 )] y ⁇ [CH 2 CH 2 0] x -H, where x and y represent repeating units of ethylene oxide and propylene oxide respectively.
  • the propylene oxide (PO) segment is sandwiched between two ethylene oxide (EO) segments, (EO--PO--EO).
  • EO ethylene oxide
  • y's distinguishes individual poloxamers.
  • the ethylene oxide segment is sandwiched between two propylene oxide segments, (PO--EO--PO)
  • the resulting stracture is a reverse poloxamer.
  • the basic stracture of a reverse poloxamer is HO ⁇ [CH(CH 3 )CH 2 0)] x ⁇ [CH 2 CH 2 0] y ⁇ [CH 2 C-HO(CH 3 )]x ⁇ H.
  • Poloxamers that may be used in concert with the methods and compositions of the present invention include, but are not limited to commercially available poloxamers such as Pluronic L121 (avg. MW:4400), Pluronic L101 (avg. MW:3800), Pluronic L81 (avg. MW:2750), Pluronic L61 (avg. MW:2000), Pluronic L31 (avg. MW: 1100), Pluronic L122 (avg. MW:5000), Pluronic L92 (avg. MW:3650), Pluronic L72 (avg. MW:2750), Pluronic L62 (avg. MW:2500), Pluronic L42 (avg.
  • Pluronic L63 (avg. MW:2650), Pluronic L43 (avg. MW: 1850), Pluronic L64 (avg. MW:2900), Pluronic L44 (avg. MW:2200), Pluronic L35 (avg. MW:1900), Pluronic P123 (avg. MW:5750), Pluronic P103 (avg. MW:4950), Pluronic P104 (avg. MW:5900), Pluronic P84 (avg.
  • Reverse poloxamers of the present invention include, but are not limited to Pluronic R31R1 (avg. MW:3250), Pluronic R 25R1 (avg. MW:2700), Pluronic R17R1 (avg. MW:1900), Pluronic R31R2 (avg. MW:3300), Pluronic R25R2 (avg. MW:3100), Pluronic Rl 7R2 (avg. MW:2150), Pluronic Rl 2R3 (avg.
  • poloxamers include compounds that are block copolymer of polyethylene and polypropylene glycol such as Synperonic L121, Synperonic L122, Synperonic P104, Synperonic P105, Synperonic P123, Synperonic P85, and Synperonic P94; and compounds that are nonylphenyl polyethylene glycol such as Synperonic NP10, Synperonic NP30, and Synperonic NP5.
  • Suitable auxiliary agents include non-ionic detergents and surfactants such as Pluronic F68, Pluronic F77, Pluronic F108, Pluronic F127, Pluronic P65, Pluronic P85, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic P123, Pluronic L31, Pluronic L43, Pluronic L44, Pluronic L61, Pluronic L62, Pluronic L64, Pluronic L81, Pluronic L92, Pluronic LlOl, Pluronic L121, Pluronic R17R4, Pluronic R25R4, Pluronic R25R2, IGEPAL CA 630, NONIDET NP-40, Nonidet P40, Tween-20, Tween-80, Triton X-100, Triton X-l 14, Thesit; the anionic detergent sodium dodecyl sulfate (SDS); the sugar stachyose; the condensing agent DMSO; and the chel
  • auxiliary agents Nonidet P40, Triton X-100, Pluronic F68, Pluronic F77, Pluronic F108, Pluronic P65, Pluronic P103, Pluronic L31, Pluronic L44, Pluronic L61, Pluronic L64, Pluronic L92, Pluronic R17R4, Pluronic R25R4 and Pluronic R25R2.
  • a most preferred auxiliary agent is Pluronic R25R2.
  • compositions of the present invention comprise about 5 ng to about 30 mg of a suitable polynucleotide or a polynucleotide construct, or an active fragment or variant thereof, and about 0.001% (w/v) to about 2.0% (w/v) of Pluronic R 25R4, preferably about 0.002% (w/v) to about 1.0% (w/v) of Pluronic R 25R4, more preferably about 0.01% (w/v) to about 0.01% (w/v) of Pluronic R 25R4, with about 0.01% (w/v) of Pluronic R 25R4 being the most prefe ⁇ ed; about 0.001% (w/v) to about 2.0% (w/v) of Pluronic R 25R2, preferably about 0.001% (w/v) to about 1.0% (w/
  • the vaccme composition can be solubilized in a buffer prior to administration.
  • Suitable buffers include, for example, phosphate buffered saline (PBS), normal saline, Tris buffer, and sodium phosphate vehicle (100-150 mM preferred).
  • Insoluble polynucleotides can be solubilized in a weak acid or base, and then diluted to the desired volume with a neutral buffer subh as PBS.
  • the pH of the buffer is suitably adjusted, and moreover, a pharmaceutically acceptable additive can be used in the buffer to provide an appropriate osmolarity within the lipid vesicle.
  • Preferred salt solutions and auxiliary agents are disclosed herein.
  • a systemic delivery embodiment is particularly preferred for treating non- localized disease conditions.
  • a local delivery embodiment can be particularly useful for treating disease conditions that might be responsive to high local concentration.
  • systemic and local delivery can be combined.
  • U.S. Patents Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and PCT publication W094/29469 provide methods for delivering compositions comprising naked DNA, or DNA cationic lipid complexes to mammals.
  • compositions used in of the present invention can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 16 th Edition, A. Osol, ed., Mack Publishing Co., Easton, Pa. (1980), and Remington's Pharmaceutical Sciences, 19 th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), both of which are inco ⁇ orated herein by reference in their entireties.
  • the composition is preferably administered as an aqueous solution, it can be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art.
  • the composition may contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives.
  • compositions used in vivo the use of sterile pyrogen-free water is preferred.
  • Such formulations will contain an effective amount of a polynucleotide or polynucleotide constract together with a suitable amount of an aqueous solution in order to prepare pharmaceutically acceptable compositions suitable for administration
  • kits for use in treating SARS-CoV comprising an administration means and a container means containing a vaccine composition of the present invention.
  • the polynucleotide or polynucleotide constract of such composition is in the amount of 1 ng to 30 mg, more preferably in the amount of 100 ng to 20 mg.
  • the container in which the composition is packaged prior to use can comprise a hermetically sealed container enclosing an amount of the lyophilized formulation or a solution containing the formulation suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose.
  • the composition is packaged in a sterile container, and the hermetically sealed container is designed to preserve sterility of the pharmaceutical formulation until use.
  • vaccine compositions of the invention may be used as part of a prime-boost protocol wherein a first vaccine composition that comprises a SARS-CoV antigen, a prime vaccine composition, is administered in conjunction with a boost vaccine composition that comprises a SARS-CoV antigen that differs in form from the SARS-CoV antigen of the prime vaccine composition.
  • a first vaccine composition that comprises a SARS-CoV antigen a prime vaccine composition
  • a boost vaccine composition that comprises a SARS-CoV antigen that differs in form from the SARS-CoV antigen of the prime vaccine composition.
  • boost vaccine composition that comprises a SARS-CoV antigen that differs in form from the SARS-CoV antigen of the prime vaccine composition.
  • boost vaccine composition that comprises a SARS-CoV antigen that differs in form from the SARS-CoV antigen of the prime vaccine composition.
  • both the prime and boost vaccine compositions are vaccine compositions of the invention they expose the subject to the SARS-CoV S protein in different forms.
  • the boost vaccine composition may be administered at the same time as the prime vaccme composition or it may be administered at some time following the initial administration of the prime vaccine composition.
  • the prime and boost vaccine compositions may be administered via the same route or they may be administered via different routes. If the prime and boost vaccine compositions are administered at the same time they may be administered as part of the same formulation or as different formulations. Both the prime vaccine composition and the boost vaccine composition may be administered one or several times. Thus some doses of the prime vaccine may be administered after the administration of a dose of the boost vaccine. It is within the skill of one with ordinary skill in the art to optimize prime boost combinations, including the optimization of routes of vaccine administration and timing for vaccine administrations.
  • Prefe ⁇ ed prime-boost combinations of the invention include, but are not limited to, the following exemplary combinations: (i) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccine composition comprising live attenuated SARS-CoV viras; (ii) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccme composition comprising live viral vectors that expresses SARS-CoV S proteins, wherein prefe ⁇ ed viral vectors include the expression of the SARS-CoV S or N protein; (iii) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccine composition comprising isolated SARS- CoV S protein; (iv) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccine composition comprising isolated SARS- CoV N protein or a plasmid that
  • either of the vaccine compositions may be the prime or the boost vaccine.
  • a vaccine composition comprising a plasmid encoding a SARS-CoV S protein is used as the prime vaccine.
  • viral vectors used to deliver either the prime or the boost vaccine composition are advenovirus vectors and vaccinia vims vectors (e.g. MVA).
  • the present invention relates to methods of reducing the effect or symptoms of SARS-CoV infection in a subject via the administration of SARS-CoV neutralizing antibodies.
  • the subject may be already infected with the viras or may be at risk for potential infection with the virus.
  • the methods and compositions of the present invention are used to elicit antisera and antibodies reactive against SARS-CoV vims, and an effective amount of an antibody reactive with SARS- CoV is administered to such patient.
  • the employed antibodies will preferably not possess an antibody-dependent vims enhancing activity.
  • an "antibody-dependent vims enhancing activity" refers to the ability of the antibody to enhance cellular entry of any strain of SARS-CoV or to enhance cellular entry of a pseudotype vims constructed with a SARS-CoV S protein.
  • the employed antibodies may be derived from human antisera, from non- human mammalian origin, or may be monoclonal, recombinant, single-chain, or humanized. Antigen-binding fragments of such antibodies (e.g., Fab and F(ab) 2 fragments) may alternatively be employed. If desired, such administration can be provided in concert with administration of the vaccine compositions of the invention in order to provoke a long-term immunity to SARS-CoV infection.
  • the dosage administered will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of the above compounds that is in the range of from about 1 pg/kg to 500 mg/kg (body weight of patient), although a lower or higher dosage may be administered.
  • the invention relates to an immunoassay that measures the presence or concentration of an anti-SARS-CoV S protein antibody (refe ⁇ ed to herein as an "anti-S-protein antibody”) in a biological sample of a mammal.
  • the immunoassay comprises the following step: (a) contacting said biological sample with mammalian expressed SARS-CoV S protein antigen (refe ⁇ ed to herein as an "S-protein antigen"), said contacting being under conditions sufficient to permit anti-S-protein antibody if present in said sample to bind to said S-protein antigen and form an S-protein antigen-anti-S-protein antibody complex; (b) contacting said formed S-protein antigen-anti-S-protein antibody complex with an anti-S-protein antibody binding molecule, said contacting being under conditions sufficient to permit said anti-S-protein antibody binding molecule to bind to anti-S-protein antibody of said formed S-protein antigen-anti-S-protein antibody complex and form an extended complex; and (c) determining the presence or concentration of said anti-S-protein antibody in said biological sample by determining the presence or concentration of said formed extended complex.
  • S-protein antigen mammalian expressed SARS-CoV S protein antigen
  • a "mammalian expressed SARS-CoV S-protein antigen” refers to any isolated protein or polypeptide that can specifically react with anti- SARS-CoV-S-protein antibodies and that is expressed in mammalian cells.
  • S- protein antigens may comprise the entire S protein or portions thereof.
  • Prefe ⁇ ed S protein antigens comprise at least a portion of the extracellular domain of the S- protein (i.e. at least a portion of residues 14 to 1190 of SEQ ID NO:3.
  • S-protein antigens may comprise homologs of the S protein, or portions thereof.
  • Prefe ⁇ ed S protein homologs are highly homologous to at least a portion of the extracellular domain of the S protein of a SARS-CoV vims.
  • prefe ⁇ ed S protein homologs will comprise a polypeptide sequence that is at least 20, preferably at least 30 , more preferably at least 40 amino acids long and that is at least 80% identical to a portion of the extracellular domain of the S protein, more preferably at least 90% identical, and more preferably at least 95% identical to at least a portion of the extracellular domain of the S protein.
  • Mammalian expressed S-protein antigens are preferably produced using the techniques of recombinant DNA technology wherein a mammalian expression vector comprising a nucleic acid sequence that encodes an S-protein antigen is introduced into a mammalian cell. The mammalian cell is then cultured under conditions that allow for the production of the S-protein antigen.
  • Various mammalian expression systems are well known in the art and an artisan of ordinary skill in the art could readily design and operate such a system.
  • anti SARS-CoV S protein antibody refers to any of IgM,
  • IgG IgG, or IgA, molecules.
  • a biological sample is assayed for the presences of anti-SARS-CoV S protein antibodies that are IgG molecules.
  • assay formats may be used in accordance with the methods of the present invention. Such formats may be heterogeneous or homogeneous, sequential or simultaneous, competitive or noncompetitive.
  • U.S. Patent Nos. 5,563,036; 5,627,080; 5,633,141; 5,679,525; 5,691,147; 5,698,411; 5,747,352; 5,811,526; 5,851,778; and 5,976,822 illustrate several different assay formats and applications.
  • Such assays can be formatted to be quantitative, to measure the concentration or amount of an anti-S protein antibody, or they may be formatted to be qualitative, to measure the presence or absence of an anti-S protein antibody.
  • Heterogeneous immunoassay techniques typically involve the use of a solid phase material to which the reaction product becomes bound, but may be adapted to involve the binding of nonimmobilized antigens and antibodies (i.e., a solution- phase immunoassay). The reaction product is separated from excess sample, assay reagents, and other substances by removing the solid phase from the reaction mixture (e.g., by washing).
  • One type of solid phase immunoassay that may be used in accordance with the present invention is a sandwich immunoassay.
  • the more anti-S protein antibody present in the sample the greater the amount of label present on the solid phase.
  • This type of assay format is generally prefe ⁇ ed, especially for the visualization of low concentrations of antibody because the appearance of label on the solid phase is more readily detected.
  • S- protein antigen that is specifically reactive with anti-S-protein antibody is bound to a solid support (i.e., immobilized) and incubated in contact with the biological sample being tested for the presence of anti-S-protein antibody.
  • the S-protein antigen may be incubated with the biological sample in an unbound state and then subsequently bound to the solid support (i.e., immobilizable).
  • the supports are then preferably extensively treated (e.g., by washing, etc.) to substantially remove non-anti-S-protein antibodies that may be present but which failed to bind to the bound antigen. In consequence of such treatment, an immune complex forms between the antigen and anti-S-protein antibody.
  • a detectably labeled second antibody (e.g., an anti-human IgG antibody) is then preferably added and the support is incubated under conditions sufficient to permit the second antibody to bind to any anti-S-protein antibody that may be present.
  • the support is then preferably extensively treated (e.g., by washing, etc.) to substantially remove any unbound second antibody. If the anti-S-protein antibody is present in the test sample, then the two antibodies will form an immune complex with the S-protein antigen (e.g., anti-human IgG antibody/anti-S-protein antibody/S-protein antigen sandwich). In such an assay, the detection of second antibody bound to the support is indicative of anti-S-protein antibody in the biological sample being tested.
  • Sandwich assay formats are described by Schuurs et al.
  • the second antibody may be a natural immunoglobulin isolated from nonhuman primates (e.g., anti-human IgG murine antibody, anti- human IgG goat antibody, etc.), or can be produced recombinantly or synthetically. It may be an intact immunoglobulin, or an immunoglobulin fragment (e.g., FAb, F[Ab] 2 , etc.). As desired, other binding molecules (capable of binding to anti-S- protein antibodies) may be employed in concert with or in lieu of such second antibodies.
  • the anti-S-protein antibody can be biotinylated and the second antibody can be replaced with labeled avidin or steptavidin.
  • a homogeneous assay format may alternatively be employed.
  • one component of the binding pair may still be immobilized (e.g. the S-protein antigen); however, the presence of the second component of the binding pair is detected without a bound-free separation.
  • homogeneous optical methods are the EMIT method of Syva, Inc.
  • the binding assay of the present invention may be configured as a competitive assay. In a competitive assay, the more anti-S-protein antibody present in the test sample, the lower the amount of label present on the solid phase.
  • the competitive assay can be conducted by providing a defined amount of a labeled anti-S-protein antibody and determining whether the fluid being tested contains anti-S-protein antibody that would compete with the labeled antibody for binding to the support.
  • the amount of captured labeled antibody is inversely proportional to the amount of analyte present in the test sample.
  • Smith U.S. Patent No. 4,401,764 describes an alternative competitive assay format using a mixed binding complex that can bind analyte or labeled analyte but in which the analyte and labeled analyte cannot simultaneously bind the complex.
  • Clagett U.S. Patent No.
  • 4,746,631 describes an immunoassay method using a reaction chamber in which an analyte/ligand/marker conjugate is displaced from the reaction surface in the presence of test sample analyte and in which the displaced analyte/ligand/marker conjugate is immobilized at a second reaction site.
  • the conjugate includes biotin, bovine serum albumin, and synthetic peptides as the ligand component of the conjugate, and enzymes, chemiluminescent materials, enzyme inhibitors, and radionucleotides as the marker component of the conjugate.
  • 4,661,444 describes a competitive immunoassay using a conjugate of an anti-idiotype antibody and a second antibody, specific for a detectable label, in which the detectable response is inversely related to the presence of analyte in the sample.
  • Allen European Patent Appln. No. 177,191 describes a binding assay involving a conjugate of a ligand analog and a second reagent, such as fluorescein, in which the conjugate competes with the analyte (ligand) in binding to a labeled binding partner specific for the ligand, and in which the resultant labeled conjugate is then separated from the reaction mixture by means of solid phase carrying a binding partner for the second reagent.
  • This binding assay format combines the use of a competitive binding technique and a reverse sandwich assay configuration; i.e., the binding of conjugate to the labeled binding member prior to separating conjugate from the mixture by the binding of the conjugate to the solid phase.
  • the assay result is determined as in a conventional competitive assay in which the amount of label bound to the solid phase is inversely proportional to the amount of analyte in the test sample.
  • Chieregatt et al. (GB Patent No. 2,084,317) describe a similar assay format using an indirectly labeled binding partner specific for the analyte.
  • Mochida et al. U.S. Patent No.
  • At least one component of the assay reagents will preferably be labeled or otherwise detectable by the evolution or quenching of light.
  • Such component may be a second antibody, anti-S-protein antibody, or an antigen that binds to an anti-S-protein antibody, depending on the immunoassay format employed.
  • Radioisotopic-binding assay formats e.g., a radioimmunoassay, etc.
  • Enzymatic-binding assay formats e.g., an ELISA, etc.
  • Enzymatic-binding assay formats employ an enzyme as a label; the signal is detectable by the evolution of color or light in the presence of a chromogenic or fluorogenic moiety.
  • Other labels such as paramagnetic labels, materials used as colored particles, latex particles, colloidal metals such as selenium and gold, and dye particles (see U.S. Patent Nos. 4,313,734; 4,373,932, and 5,501,985) may also be employed.
  • enzymes especially alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase, or urease
  • EIA enzyme immunoassay
  • enzymatic labels may be detected through the use of chromogenic substrates (including those that evolve or adsorb fluorescent, UV, visible light, etc.) in response to catalysis by the enzyme label. More preferably, chemical labels may be employed (e.g., colloidal gold, latex bead labels, etc.). Detection of label can be accomplished using multiple detectors, multipass filters, gratings, or spectrally distinct fluors (see e.g., U.S. Patent No. 5,759,781), etc. It is particularly prefe ⁇ ed to employ peroxidase as an enzyme label, especially in concert with the chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB).
  • TMB trioxidase
  • Suitable materials for the solid support are synthetics such as polystyrene, polyvinyl chloride, polyamide, or other synthetic polymers, natural polymers such as cellulose, as well as derivatized natural polymers such as cellulose acetate or nitrocellulose, and glass, especially glass fibers.
  • the support can take the form of spheres, rods, tubes, and microassay or microtiter plates. Sheet-like structures such as paper strips, small plates, and membranes are likewise suitable.
  • the surface of the carriers can be permeable and impermeable for aqueous solutions.
  • the S-protein antigen may be indirectly or directly bound to the solid support.
  • the S protein is indirectly bound to the solid support via Galanthus nivalis lectin.
  • any fluidic biological sample e.g., tissue or biopsy extracts, extracts of feces, sputum, etc.
  • the biological sample being assayed will be semm or sputum.
  • Materials for use in the assay of the invention are ideally suited for the preparation of a kit.
  • kit may comprise a carrier means being compartmentalized to receive multiple samples in close confinement; one or more containers means vials, tubes and the like; each of the containers means comprising one of the separate elements to be used in the method.
  • one of the containers means may comprise a suitable S-protein antigen.
  • a second container may comprise soluble, detectably labeled second antibody, preferably in lyophilized form, or in solution.
  • the kit may also contain one or more containers, each of which comprises a (different) predetermined amount of S protein antigen or anti-S protein antibody, useful for the preparation of a standard curve.
  • the kit In using the kit, all the user has to do is add to a container a premeasured amount of a sample suspected of containing a measurable yet unknown amount of anti-S-protein antibodies, a premeasured amount of support-bound antigen present in the first container, and a premeasured amount of the detectably labeled second antibody present in the second container. After an appropriate time for incubation, an immune complex is formed and is separated from the supernatant fluid, and the immune complex or the supernatant fluid are detected, as by radioactive counting, addition of an enzyme substrate, and color development, or by inclusion of a chemical label (e.g., colloidal gold, latex beads, etc.).
  • the present invention particularly relates to the use of immuno- chromatographic assay formats to detect anti-S protein antibodies.
  • two contacting, but spatially distinct, porous carriers are employed.
  • the first such carrier will contain a non- immobilized, labeled S protein and the second such carrier will contain an immobilized, but unlabeled antibody that binds to IgG (e.g., where human anti-S protein antibodies are being assayed, the unlabeled antibody may be an anti-human IgG antibody).
  • the device will comprise a hollow casing constructed of, for example, a plastic material, etc., in which the first carrier will communicate indirectly with the interior of the casing via a multilayer filter system that is accessible from the device (e.g., by protruding therefrom or by being incompletely covered by the device), such that a semm, plasma, or whole blood test sample can be applied directly to the filter system and will permeate therefrom into the first porous carrier.
  • a multilayer filter system that is accessible from the device (e.g., by protruding therefrom or by being incompletely covered by the device), such that a semm, plasma, or whole blood test sample can be applied directly to the filter system and will permeate therefrom into the first porous carrier.
  • the permeation of fluid containing anti-S protein antibodies will cause the non-immobilized labeled S protein of the first carrier to become bound to the migrating antibodies, and will then permeate into the second carrier.
  • the second carrier contains immobilized antibody that binds human IgG, any labeled S protein entering the second ca ⁇ ier will be entrapped therein. Detection of labeled S protein in the ca ⁇ ier containing the immobilized unlabeled antibody thus indicates that anti-S protein antibodies are present in the sample being evaluated.
  • the assay can be made quantitative by measuring the quantity of labeled S protein that is bound within the second porous carrier.
  • the invention relates to viral vectors that have been pseudotyped with at least a portion of the SARS-CoV S protein and to methods of using the pseudotyped viral vectors to screen for inhibitors of SARS-CoV infection.
  • Viral vectors that may be employed include any vector that, when pseudotyped with the S protein, is able to support entry and fusion of the viral vector with a permissive host cell type for SARS-CoV infection such as, for example, Vero cells.
  • the viral vectors are retroviral vectors or lentiviral vectors.
  • the pseudotyped viral vectors are used to assay for neutralizing antibodies for SARS-CoV infection.
  • Such assays may be useful for diagnosing SARS-CoV infection or for monitoring the progress of the disease or the prognosis for the disease in a particular patient. It is contemplated as part of the invention that the pseudotyped viral vectors of the invention may be used to screen for agents that inhibit the entry of SARS-CoV into cells or that inhibit the cell mediated transfer of SARS-CoV.
  • the invention relates to a method of assaying a vaccine composition or an immunogenic compoud for antibody-dependent viras enhancing activity, wherein the method comprises the following steps: (a) generating an immune response to a vaccine composition or an immunogenic compound in an animal, preferably a non-human mammal or a human; (b) isolating immune sera or antibodies from the animal; (c) assaying the ability of the antibodies or the immune sera to enhance cellular entry of a strain of SARS-CoV viras or a pseudotype virus constructed with the SARS-CoV S protein; (d) identifying the vaccine composition or the immunogenic compound as having antibody-dependent virus enhancing activity if the antibodies or immune sera enhance cellular entry of the virus strain or the pseudotype viras.
  • the viras strain employed, or the co ⁇ esponding pseudotype viras would be an isolate from a non-human animal, preferably a non- human mammal, including, for example, an isolate from the Palm civt, the raccoon dog, or the Chinese fe ⁇ et badger.
  • Example 1 PH-Dependent Entry of SARS Coronavirus is Mediated by the Spike Glycoprotein and Enhanced by Dendritic Cell Mediated Transfer through DC-SIGN
  • Antibodies mouse immune serum and media Human semm from a recovered SARS patient can be obtained from Dr. William Bellini (CDC, U.S.A.). Antibodies to GDI lc , CD14 ,CD40, CD80, CD86 and HLA-DR are purchased from BD Pharmingen. Mouse anti-His (C- term)-FITC antibody is purchased from Invitrogen (Carlsbad, California). Media for human primary cell culture are purchased from Cambrex (East Rutherford, New Jersey). RPMI Media 1640 and Dulbecco's Modified Eagle Media are purchased from Invitrogen (Carlsbad, California).
  • Anti-SARS-CoV S mouse immune semm is generated by vaccinating 10-week old BALB/c mice with CMV/R plasmid DNA expression vectors, described below, encoding the S protein (25 ⁇ g at three week intervals, three times, bled after two months). Negative control antisera is obtained in a similar fashion by injecting the same plasmids with no insert (control)
  • Human primary cell lines are as follows: RPTEC (renal proximal tubule epithelial cell), HRE (renal epithelial cell), HRCE (renal cortex epithelial cell), SAEC (small airway epithelial cell), NHBE (bronchial epithelial cell), NHLF (lung fibroblast), HMVEC-L (lung microvascular endothehal cell), HUVEC (umbilical vein endothehal cell), HMVEC (microvascular endothehal cell), NHMEC (mammary epithelial cell), NHEK (keratinocyte), and hepatocytes. Human primary cell lines can be purchased from Cambrex.
  • Human and animal cell lines are as follows: ACHN (human kidney adenocarcinoma), 293 (human embryonic kidney), 786-0 (human kidney adenocarcinoma), A549 (human lung carcinoma), HeLa (human cervical adenocarcinoma), Colo205 (human colon adenocarcinoma), Jurkat (human T cell), CEM (human acute lymphoblast leukemia), M8166 (human CD4+ lymphoid cell), HL60 (human promyelocytic leukemia cell), THP-1 (human acute monocytic leukemia), Vero (African green monkey kidney epithelial cell), CRFK (cat kidney cortex epithelial cell), OK (opossum kidney cortex epithelial cell), M-l (mouse kidney cortex epithelial cell), FC2.Lu (cat lung fibroblast), FC28.1u (cat lung fibroblast), AK-D (cat lung epithelial cell), MLE12 (mouse lung epithelial cell), MM1
  • Human and animal cell lines can be purchased from the ATCC.
  • PBMCs human peripheral blood mononuclear cells
  • THP-1 human acute monocytic leukemia
  • THP-DC-SIGN THP- 1 expressing human DC-SIGN
  • THP-DC-SIGN ⁇ 35 THP-DC-SIGN ⁇ 35
  • Kwon et al. 2002, Immunity 16:135-144.
  • Human T cell leukemia cell lines A3R5 (a subline of CEM expressing both CCR5 and CXCR4)
  • MT-2 expressing CXCR4 and 293T can be obtained from Dr. John Mascola.
  • M8166 a human CD4+ lymphoid cell
  • Dr. Hayami a human CD4+ lymphoid cell
  • Gene synthesis and construction of expression vectors Genes encoding the SARS-CoV spike (S), membrane (M) and envelope (E) proteins are synthesized using human-prefe ⁇ ed codons. To synthesize these genes, protein sequences obtained from GenBank (AY278741) for the SARS-CoV Urbani strain are reverse translated using human-prefe ⁇ ed codons. Sets of 75 bp oligonucleotides, with 25 bp overlaps, covering each gene are synthesized and gel- purified. The oligonucleotides are assembled into DNA fragments 1 using Pfu Turbo Hotstart DNA polymerase (Stratagene, La Jolla, CA) at a 50-65°C gradient annealing temperature.
  • Pfu Turbo Hotstart DNA polymerase (Stratagene, La Jolla, CA) at a 50-65°C gradient annealing temperature.
  • DNA fragments are cloned into pCR-Blunt II-Topo vector (Invitrogen) and sequenced. Clones with the fewest mutations are picked, and further co ⁇ ected using the Quickchange kit (Stratagene) according to the manufacturer's protocol. Fully co ⁇ ected DNA fragments for each gene are cloned into mammalian expression vector CMV/R-mcs.
  • CMV/R-mcs contains the cytomegalovirus CMV) enhancer/promoter, splice donor, and the HTLV-1 R region.
  • COOH-terminal deletion mutants of the SARS-CoV S gene are generated using the Quickchange kit (Stratagene) and are cloned into the CMV/R-mcs expression vector.
  • mutants include the following constructs: (1) S ⁇ CD, in which the cytoplasmic domain is truncated, terminated at aa 1229 (residue 1229 of SEQ ID NO:3); (2) S ⁇ TM2, in which the transmembrane and cytoplasmic domains are deleted at aa 1190 (residue 1190 of SEQ ID NO:3; and (3) S ⁇ HRl, in which the transmembrane, cytoplasmic and heptad 2 domains are removed, with termination at aa 1153 (residue 1153 of SEQ ID NO:3).
  • the Myc-His tagged S(l 190) construct is truncated at aa 1190 to remove the transmembrane and cytoplasmic domains and tagged with a myc and his epitope at the COOH- terminus.
  • the constructs are sequenced on both strands to ensure that each gene is conect and the identity of the constructs is further confirmed using Western blot analysis of the gene expression products.
  • mDCs Myeloid dendritic cells
  • Myeloid DCs are isolated from the elutriated monocyte fraction with negative selection by depleting cells expressing BDCA-4 and CD9 using microbeads (Miltenyi Biotec, Auburn, CA), followed by positive selection using antibodies to CDlc (Miltenyi Biotech). Myeloid DCs are then cultured in media containing GM-CSF (PeproTech, 10 ng/ml), and induced to differentiate to mature mDC using poly (I:C) (50 ng/ml) (Sigma, St. Louis, MO) for 48 hours as described in Cella et al., 1999, J. Exp. Med. 189:821-829.
  • Antibodies to CD1 lc and CD14 are used to assess purity of mDCs and antibodies to CD40, CD80, CD86 and HLA-DR (BD Pharmingen) are used to characterize the differentiation of mDCs by flow cytometry.
  • the amount of plasmid DNA used for making different pseudotyped vectors is as follows: for lentiviral vectors, 7 ⁇ g of pCMV.R8.2 and 7 ⁇ g of pHR'CMV-Luc in combination with 400 ng CMV/R-SARS-S or 2 ⁇ g of pNGVL-4070A(Ampho), respectively; for retroviral vectors, 7 ⁇ g of pNGVL GagPol(MLV) and 7 ⁇ g of pLZR-Luc, in combination with 400 ng CMV/R-SARS- S or 2 ⁇ g of pNGVL- 4070A(Ampho), respectively. Cells are transfected overnight, washed, and replenished with fresh media.
  • GFP-Vpr-labeled SARS-S pseudotyped lentivims is produced by transfection of human embryonic kidney (HEK) 293 T cells with the an ercv-deleted pLAI provims (10 ⁇ g), CMV/R-SARS-S (1 ⁇ g) and the plasmid pEGFP-C3 (Clontech, Palo Alto, CA) containing the entire Vpr coding region fused to the carboxyterminus of eGFP (GFP-Vpr; 15 ⁇ g) as described in McDonald et al. (2002, J. CellBiol. 159:441- 452).
  • Viral titer is determined by infecting Vero cells in 96- well plate, and viral titers are calculated in TCIDso/ml four days after infection of Vero cells. The viral cytopathic effect is determined on days 3 and 4. Infection of mature and immature mDC is performed in 96-well plates (10,000 cells/well) with 50 ⁇ l of 1:10 diluted viral stock, and titered three days later, as described above.
  • Transfection and Western Blot Analysis 293T cells are transfected using calcium phosphate (Invitrogen, Carlsbad, CA). Transfected cells are harvested 48 hours after transfection. Cell lysates are resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel elecfrophoresis
  • Vero cells are plated in a 48-well-dish (30,000 cells/well) the day before the experiment is conducted. Cells are pre-incubated with indicated amounts of ammonium chloride or bafilomycin A (Sigma, St. Louis, MO) for one hour. Pseudovimses are mixed with the same concentrations of reagents in tubes and added to cells. Eight hours later, media containing the pseudovimses are removed and replaced with fresh media . Cells are harvested 48 hours after infection, and a luciferase assay is performed.
  • THP, THP-DC-SIGN or THP-DC-SIGN ⁇ 35 are incubated with SARS-CoV S pseudotyped lentiviral vector for 2 hours, and washed three times with tissue culture media. Cells are then added onto Vero, A3R5 or MT2 cells (30,000 cells/well) plated in 24-well-dishes. These cells are harvested 72 hours later for luciferase assay to assess THP, THP-DC-SIGN or THP-DC-SIGN ⁇ 35 cell-mediated transfer of pseudovims to the respective cells.
  • mature mDCs are incubated with SARS-CoV for 1 hr, washed, and detached with trypsin, and replated onto Vero cells in 96-well-plate (10,000 cells/well) in the presence of control or S specific mouse anti-serum (1:100 dilution).
  • Cell culture medium (DMEM+10%FBS) is collected 72 hours later, and SARS-CoV is titered in the cell culture medium as described above.
  • SARS-CoV envelope proteins are analyzed by cotransfection of expression vectors encoding either the S, M, or E glycoproteins with packaging plasmids for retroviral or lentiviral vectors into human 293T cells as described in Kinsella et al (1996, Hum.Gene Ther. 7:1405) andNaldini et al. (1996, Proc. Natl. Acad. Sci. USA 93:11382).
  • envelope glycoproteins from amphotropic murine leukemia viras and Ebola vims are substituted in place of the SARS-CoV envelope proteins.
  • Vero cells which support SARS-CoV replication (Ksiazek et al., 2003, N. Engl. J. Med. 348: 1953- 1966; Poutanen et al., 2003, N. Engl. J. Med. 348:1995-2005), are initially analyzed as a target cell.
  • SARS-CoV gene products only the S protein mediates entry into target cells, both with murine retroviral and human lentiviral vectors ( Figure 1, Panel A). Because both vectors can be pseudotyped with the S glycoprotein, further analyses are performed with the lentiviral vector, which can transduce and express recombinant genes in nondividing cells.
  • Viral glycoproteins typically mediate attachment, fusion and entry by one of two mechanisms. Virases such as the human immunodeficiency virus (HIV) or murine amphotropic retrovimses infect through a pH-independent cell fusion and entry process (Nussbaum et al., 1993, J. Virol. 67:7402-7405; Stein et al., Cell 49:659-668). In contrast, influenza and Ebola are prototypes for vimses that utilize a pH dependent endocytotic pathway (Wool-Lewis and Bates, 1998, J. Virol. 72:3155-3160).
  • HCV human immunodeficiency virus
  • Ebola are prototypes for vimses that utilize a pH dependent endocytotic pathway (Wool-Lewis and Bates, 1998, J. Virol. 72:3155-3160).
  • the cell specificity of the SARS-CoV S pseudotyped lentivims is analyzed by transducing different human cell types, including epithelial, endothehal and hematopoietic cells, and lung and renal cells from different species.
  • a renal epithelial cell line derived from the African green monkey, human renal epithelial cells (HPTRC, HRE, HRCE, ACHN, 786-0) are highly susceptible to infection with the SARS-CoV-S pseudotyped vims in comparison to the infectivity observed with a known positive control with broad host range, the 4070A amphotropic murine retroviral envelope.
  • Respiratory tract epithelial cells are also readily infected with the SARS-CoV-S pseudotyped viras.
  • a number of cells types including hepatocytes, lower airway fibroblasts, breast or colonic epithelial, vascular endothehal, or hematopoietic cells, are relatively resistant to infection (Figure 3, Panel A).
  • renal epithelial cell lines from humans, non-human primates, and felines, and to a lesser extent, lung cell lines derived from felines are susceptible to transduction, while similar cells from rodents, are resistant to transduction (Figure 3, Panel B).
  • the specificity of the pseudotyped vims transduction in these cell lines is confirmed by an analysis of the susceptibility of these cell lines to infection by SARS-CoV.
  • the susceptibility of these cells to pseudovims transduction co ⁇ elates well with their ability to support SARS-CoV replication. It therefore appears that a range of cell types and species are susceptible to infection mediated by the SARS-CoV S glycoprotein.
  • the S glycoprotein contains a number of N-linked glycosylation sites.
  • virases including HIV, the dengue vims, and the cytomegalo vims
  • glycosylation is known to affect binding of the vims to the dendritic cell DC-SIGN receptor that regulates cell-mediated transmission.
  • THP-1 a human acute monocytic leukemia cell line that expresses this gene product, THP-DC-SIGN, or a mutant form lacking the cytoplasmic domain required for intemalization and transfer
  • THP-DC-SIGN ⁇ 35 cells are incubated with purified His-tagged S protein. Binding is readily detected in the permissive Vero cell using flow cytometry, in contrast to two non-permissive T- cell leukemia cell lines, A3R5 and MT2 ( Figure 4, Panel A) or THP-1 cells lacking DC-SIGN (data not shown).
  • THP-DC-SIGN and ⁇ 35 cells interacted with purified S glycoprotein (Figure 4, Panel A), but unlike Vero cells, they could not be infected by S pseudotyped lentiviral vector ( Figure 4, Panel B- left).
  • Figure 4, Panel A purified S glycoprotein
  • Figure 4, Panel B- left S pseudotyped lentiviral vector
  • THP-DC-SIGN but not THP or THP- DC-SIGN ⁇ 35, which is unable to internalize vims (Kwon et al., Immunity 16:135- 144), readily transfened vims to Vero cells ( Figure 4, Panel B-right), indicating that DC-SIGN or a related lectin on dendritic cells might facilitate cell-mediated transfer of SARS-CoV. Both direct infection and DC-SIGN-mediated transfer are inhibited by SARS-CoV S-specific mouse immune sera ( Figure 4, Panel C), ⁇ onfirming that S is necessary and sufficient for infection in both cases.
  • a similar vector pseudotyped with HIV gp 160 has been shown to mediate the formation of an "infectious" synapse that facilitates HIV infection (McDonald et al., 2003, Science 300:1295-1297), but it was unknown whether similar structures could be formed by an unrelated virus whose target cell is non- lymphoid.
  • a culture of mDCs are incubated with vims for 30 minutes, trypsinized, and transfened to fresh 786-0 human renal cell cultures. Initially, vims distributes evenly throughout the mDCs (Figure 5, Panel A-uptake), but within minutes, immunofluorescent foci begin to form at the site of contact with Vero cells ( Figure 5, Panel A-polarization).
  • Viras is observed to transfer to the target cells through a structure analogous to the "synapse" previously described between mDC and lymphoid cells (Figure 5, Panel A-transfer) (McDonald et al., 2003, Science 300:1295-1297). After transfer, a characteristic streak of fluorescence is seen at the site of entry, suggesting a specific channeling of viral contents into cells (Figure 5, Panel A-post-transfer). This effect is seen consistently and is not caused by tunneling of DC beneath the 786-0 epithelial cell in culture. To confirm that mDCs mediate infection by vims, immature and poly (I:C) treated mDCs are incubated with SARS-CoV.
  • Plasmids encoding different versions of SARS-CoV spike (S) protein are synthesized using human-prefened codons as described in Example 1 herein. Protein expression is confirmed by western blot analysis (Kong et al., 2003, J. Virol. 77:12764-12772) with serum from a recovered patient (provided by W. Bellini, CDC).
  • Vaccination Female BALB/c mice (6-8 weeks old; Charles River Labs) are immunized with 25 ⁇ g of plasmid DNA in 200 ⁇ l of PBS (pH 7.4) at weeks 0, 3 and 6.
  • Flow cytometric analysis of intracellular cytokines CD4 + and CD8 + T-cell responses are evaluated by using intracellular cytokine flow cytometry (ICC) for IFN- ⁇ and TNF- ⁇ as described by Kong et al., (2003, J. Virol. 77, 12764-12772) with peptide pools (17-19 mers overlapping by 10 amino acids, 2.5 ⁇ g/ml each) covering the SARS-CoV S protein. Cells are then fixed, permeabilized, and stained using rat monoclonal antimouse CD3, CD4, CD8, IFN- ⁇ and TNF- ⁇ (BD-Pharmingen). The IFN- ⁇ - and TNF- ⁇ positive cells in the CD4 + and CD8 cell populations are analyzed with the program Flow Jo (Tree Star, Inc.).
  • mouse anti-SARS-CoV S IgG ELISA titre is measured using a modified lectin-capture method described by Kong et al., 2003, J. Virol. 77: 12764- 12772, except the Myc-tagged, transmembrane-domain truncated SARS-CoV S protein (SARS-SDTM-Myc) is used for capture.
  • SARS-SDTM-Myc Myc-tagged, transmembrane-domain truncated SARS-CoV S protein
  • SARS-CoV S pseudotyped lentivimses expressing a luciferase reporter gene are produced by teansfecting 293T cells with the following plasmids: 7 ⁇ g of pCMV ⁇ R8.2, 7 ⁇ g of pHR'CMV-Luc and 400 ng CMV/R-SARS-S. Cells are transfected overnight, washed and replenished with fresh media. Forty-eight hours later, supernatants are harvested, filtered through a 0.45 ⁇ m syringe filter, aliquotted and used immediately or frozen at 280°C. p24 levels are measured from different viral stocks using the Coulter HIV-1 p24 Antigen Assay kit (Beckman
  • Antisera are mixed with 100 ml of pseudovimses at various dilutions and added to Vero cells in 48-well dishes (30,000 cells per well). Plates are washed and fresh media are added 14-16 hours later. Forty-eight hours after infection, cells are lysed in mammalian cell lysis buffer (Promega). A standard quantity of cell lysate is used in a luciferase assay with luciferase assay reagent (Promega) according to the manufacturer's protocol.
  • mice Challenge of immunized mice with SARS-CoV Vaccinated mice are lightly anaesthetized with isoflurane and inoculated with 50 ml of diluted vims (10 4 TCID 50 of SARS-CoV; Urbani strain) intranasally according to institutional animal care and use guidelines in an ABSL3 facility. On day 2, mice are euthanized and lungs and nasal turbmates are removed and stored at 270°C until the end of the study.
  • the frozen tissues are thawed and homogenized in a 10% (lungs) or 5% (nasal turbinates) w/v suspension in Leibovitz 15 medium (Invitrogen), and vims titres are determined in Vero cell monolayers in 24- and 96-well plates. Vims titres are expressed as TCID 50 per g of tissue.
  • the lower limit of detection of infectious vims in 10% and 5% w/v suspensions is 1.5 for lung and 1.8 logio TCID 50 per g for nasal turbinate homogenates, respectively.
  • T cells from vaccinated (immune) or nonimmune (control) mice are enriched using a Pan T-Cell Isolation Kit (Miltenyi Biotec). Approximately 3 x 10 7 T cells in 0.5 ml PBS are administered into each recipient naive BALB/c mouse intravenously through the tail vein 24 hours before challenge. There are four recipient mice per group.
  • Two sets of cDNAs encoding the SARS-CoV S glycoproteins are prepared using modified codons to optimize expression and to minimize recombination with endogenous coronavimses.
  • the native leader sequence is retained in one set of vectors ( Figure 7, Panel A) and replaced in another set with a leader sequence derived from the interleukin-2 gene. Expression is not significantly altered by this leader sequence substitution.
  • Two S carboxy-terminal mutants, one that truncated the cytoplasmic domain (S ⁇ CD) and another that deleted the transmembrane and cytoplasmic domain (S ⁇ CD), are prepared and expression of these cDNAs by a mammalian expression vector suitable for human vaccination is confirmed (Figure 7, Panel B)
  • the plasmids encoding these modified S glycoproteins are analyzed for their ability to elicit antiviral immunity after intramuscular injection in BALB/c mice. Injection of S, S ⁇ TM and S ⁇ CD expression vectors induces a substantial immune response. A marked increase is observed in the number of SARS-CoV S- specific CD4 T cell immune responses ( Figure 8, Panel A), as measured by intracellular cytokine staining for interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor- ⁇ (TNF- ⁇ ). In addition, substantial SARS-CoV S-specific CD8 cellular immunity is detected at levels at least sevenfold above the background response.
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • Humoral immunity is initially assessed using an enzyme-linked immunosorbent assay (ELISA) with lectin-captured S ⁇ TM protein expressed in 293T cells. Substantial end-point dilution antibody titres are observed in all groups, ranging from approximately 1:400 to approximately 1:2,000 ( Figure 8, Panel B-left).
  • ELISA enzyme-linked immunosorbent assay
  • a pseudotyping assay is performed as described in Example 1 herein. Immunization of animals with the SARS-CoV S ⁇ CD expression vector induces substantial neutralizing antibody titees ranging from 1 :50 to 1 : 150, unlike the vector control ( Figure 8, Panel B-middle).
  • the S expression vector with the transmembrane domain deleted, S ⁇ TM also induces neutealizing r antibodies, but the titees are lower (from 1 :25 to 1:75).
  • the vector with the partial cytoplasmic domain deletion induced the optimal response ( Figure 8, Panel B- right). Similar neutralization is observed when the complete cytoplasmic domain is removed, suggesting that synthesis of the glycoprotein on the cell surface, without the cytoplasmic domain, is important for an optimal immune response. This is because it gives rise to a more native stracture relevant to the function of the virus.
  • the transmembrane region helps to form a more physiological form of the S protein by anchoring the protein on the membrane, preserving conformational determinants and/or stabilizing the formation of the putative trimer.
  • the same antisera are also used in a SARS-CoV microneutialization assay to assess the neutralization titre against SARS-CoV. Again, antisera from S ⁇ CD- vaccinated mice elicited the most potent neutealizing antibody responses (Figure 8, Panel B-right).
  • This model can be used to examine immune protection against viral replication in the respiratory tract as a measure of vaccme efficacy.
  • BALB/c mice are immunized with the various plasmid DNAs encoding S protein and challenged 30 days after the final boost. Animals are challenged inteanasally with 10 4 TCIDso units of SARS-CoV (Urbani strain), and viral replication in the respiratory tract is measured 2 days later.
  • T-cell depletion with specific monoclonal antibodies is performed, and depletion in the lung and spleen is confirmed ( Figures 11 and 12).
  • Depletion with CD4 or CD8, alone or in combination with CD90 does not affect vaccine-induced immunity (Figure 10, Panel A).
  • SARS-Co V expression vectors The protein sequences of S, N, M, and E from SARS-CoV Urbani strain (GenBank accession number AY278741) (Rota et al., 2003, Science 300, 1394- 1399) are reverse-translated by the GCG Package (Genetic Computer Group, Inc., Madison, WI) using human prefe ⁇ ed codons. Oligonucleotides covering these four genes are purchased from Sigma-Genosys Inc. Each of the oligonucleotides is 75 base pairs with 25 nt of overlap. The codon-modified genes are assembled by PCR PfuTurbo ® hotstart (Stratagene) high fidelity DNA polymerase.
  • PCR conditions are optimized with a PCR optimization kit (Stratagene) on a gradient Robocycler (Stratagene).
  • Full-length synthetic S, N, M, and E are cloned into the Xba I and Bam HI sites of the mammalian expression vector CMV/R derived from pNGVL-3 (Mantho ⁇ e et al., 1993, Hum. Gene Ther. 4, 419-431) and confirmed by DNA sequencing.
  • EM 293T cells are maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco-BRL), supplemented with 10% fetal bovine serum (FBS). Plasmid DNAs are purified using double cesium chloride sedimentation gradients. Approximately 3xl0 6 293 T cells are plated in a 10 cm dish one day before transfection. 3 ⁇ g of each plasmid (each containing one of the SARS-CoV genes) are mixed and used to teansfect 293T cells, using the calcium phosphate method (Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-2752). The vector backbone is used as filler DNA to maintain the same amount of DNA in each transfection.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • the cells are lifted from plates by resuspension in DMEM and then pelleted in a 15 ml conical tube by centrifugation at 1000 ⁇ m. The supernatant is removed and a 10-fold volume of fixing solution is added (3% glutaraldehyde and 3% formaldehyde, cacodylate buffer, pH 7.3; Tousimis Research Co ⁇ oration, Rockville, MD). The specimens are mixed gently, and analyzed in the electron microscopy core laboratory at the University of Michigan Health Center.
  • Viral capsid production and buoyant density gradient analysis 3xl0 6 293 T cells are transfected with 3 ⁇ g of each of pCMV/R-S, N, and M in a 10 cm tissue culture dish with DMEM medium. The cells are harvested after three days and freeze-thawed three times in PBS. The cleared lysates are pelleted onto 50% of an OptipreTM (IODLXANOL) medium (Invitrogen) at 20000 ⁇ m with a Sorvall Surespin 630 rotor, and the final concenteation of Optipre is adjusted to 30%.
  • OptipreTM IODLXANOL
  • Invitrogen Sorvall Surespin 630 rotor
  • a density gradient is formed by centrifugation at 75k for 3.5 hours with a NTI100 rotor (according to the manufacturer's instractions; Invitrogen).
  • the collected fractions are weighted at 100 ⁇ l of each fraction and plotted with density by fractions.
  • a 20 ⁇ l aliquot of each fraction is separated on a 4-15%) SDS-PAGE gel, transfened onto an ImmobilonTM-P membrane and blotted with human anti-SARS-CoV sera.
  • SARS genes are analyzed to define the SARS genes essential for viral assembly. These genes include the S, M, N and E proteins synthesized from the predicted amino acid sequence by reverse translation, with codon usage typical of human cells. The DNA sequence of each gene is confirmed, and expression is confirmed by in vitro transcription/translation and/or by Western blot analysis. Expression of the SARS S, M, N and E genes is analyzed in different combinations in transfected 293 cells to evaluate the contribution of these gene products to viral assembly. Table 1 below shows the results of a scanning TEM analysis of capsid formation in 293T cells transfected with plasmids containing the indicated genes.
  • Buoyant density gradient sedimentation analysis is performed to characterize these synthetic SARS-CoV particles. Because the capsid core is found in higher quantities within cells, lysates are prepared from transfected cells that are frozen and thawed three times. When fractions from a gradient of clarified cell lysates are analyzed by Western blot with human immune semm, the peak of viral protein expression, composed primarily of N and S proteins, is detected at a density of 1.18 g/ml, comparable to the buoyant density described for other coronavimses.
  • the interaction between these viral proteins is analyzed further to establish the biochemical basis for viras particle formation.
  • the major structural proteins are synthesized by transcription and translation with rabbit reticulocyte lysates in vitro, as are mutant forms of M.
  • the full length M protein is able to associate with the N protein when both are co-synthesized, and the COOH-terminal cytoplasmic domain is required for this association.
  • the COOH-terminal region of M bound more avidly to N than the full-length protein, suggesting that this domain may be less exposed during protein teanslation of the complete protein in vitro.
  • Co-expression of a mutant M gene that lacked the COOH terminal domain abolished its ability to form nucleocapsids.
  • Plasmids and ELISA A codon-modified spike (S) gene deleted of the transmembrane region to improve production and secretion, as well as a nucleocapsid (N) gene, are cloned from plasmids described in Example 1 herein using overlapping PCR.
  • the S and N genes are tagged with Myc and His, respectively, and inserted into plasmid mammalian expression vectors as described in Example 2 above.
  • the plasmids are transfected into the human renal epithelial 293 cell line. After cell culture, the S protein supernatant is filtered and the His-tagged N protein is isolated from transfected cell lysates by His-affinity column chromatography. The S protein filtered supernatant and the purified N protein are then applied to lectin-coated ELISA plates (Kong et al., 2003, J. Virol. 77:12764-12772).
  • the ELISA plates are first coated with Galanthus nivalis lectin by incubation of the wells with a 10 ⁇ g/ml solution overnight at 4°C. After blocking with 10% fetal bovine serum and washing twice with PBS containing 0.2% Tween 20 (PBS-T), S protein supernatant and purified N protein are added to the wells and incubated for one hour at room temperature. The plates are washed with PBS- T, and then the sera samples are added to the wells, with varying dilutions, and then incubated for 1 hour. The plates are washed with PBS-T and a horseradish peroxidase conjugated secondary antibody, at a 1:5,000 dilution, is added to the wells.
  • SARS-CoV neutealizing antibodies in patient sera are assayed using a lentiviral vector pseudotyped with the SARS-CoV S (Urbani) glycoprotein, which mediates gene delivery and shows similar specificity and sensitivity to neutralization as replication-competent SARS-CoV as shown in Examples 1 and 2 above.
  • Diluted semm samples are mixed at various dilutions with an S pseudovims that encodes a luciferase gene. The mixtures are incubated with 782- O human renal epithelial cells for 16 hours and then the cells are washed for 48 hours after infection. Cells are then lysed, and the luciferase assay is performed. High luminescence activity conelates directly with infection, whereas low luminescence activity indicates the presence of neutealizing antibodies in the test semm sample.
  • IgM antibodies against N are detected in only two samples collected (day 30), and those values decreased subsequently (Figure 13, Panel B).
  • IgA directed to N is detected at low levels early and increased with time ( Figure 13, Panel B).
  • the human renal adenocarcinoma cell line 786-0 is purchased from the ATCC (Manassas, VA) and maintained in DMEM+10% FBS.
  • the gene encoding SARS-CoV SfJJrbani) is synthesized using-human prefe ⁇ ed codons as previously described in the Examples herein.
  • Clones (cDNAs) encoding S proteins from various strains are constructed using the Quikchange XL kit (Stratagene) to introduce divergent amino acids into the gene encoding S(Urbani) according to the predicted translated sequence as disclosed in Genbank.
  • S genes co ⁇ esponding to following variant proteins are constructed: SfBJOl) (human isolate) (Genbank No. AY278488); S(FRA) (human isolate) (No. AY310120); S(GD01) (human isolate) (No. AY278489); S(GZ02) (human isolate) (No. AY390556);
  • lentivimses expressing a luciferase reporter gene are produced as described previously in the Examples herein. Briefly, 5 l0 6 293T cells are transfected overnight using calcium phosphate reagent (Invitrogen) with following plasmids: 7 ⁇ g of pCMV ⁇ R8.2, 7 ⁇ g of pHR'CMV-Luc, and 400 ng CMV/R-SARS-S. Supernatants are harvested 48 hours after transfection, filtered through a 0.45 ⁇ m syringe filter, aliquotted, and used immediately or frozen at - 80°C. Levels of p24 levels are measured from different viral stocks using "The Coulter HIV-1 p24 Antigen Assay" kit (Beckman Coulter) to estimate the viral titer.
  • mice Female Balb/c mice (6-8 weeks old) (Charles River) per group are immunized with 25 ⁇ g plasmid DNA (in 200 ⁇ l PBS pH 7.4) expressing co ⁇ esponding immunogen three times at weeks 0, 3 and 6.
  • mice Ten days after the last immunization, sera are collected, combined, and subsequently purified using "NabTM Spin Purification Kit” (Pierce) according to the manufacturer's protocol.
  • Neutralization and inhibition assays 786-0 cells (30,000 cells/well) are plated onto a 48-well dish the day before infection. The various pseudovimses, normalized for the amount of p24 added, are mixed with purified mouse IgG ,human monoclonal antibodies or recombinant human ACE-2 ectodomain (aa 1-740)(R&D Systems) at indicated concentrations, and incubated for 5 to 10 minutes before addition to 786-0 cells. Cells are infected for 14-16 hours, and collected for luciferase assay using "Luciferase assay reagent" (Promega) 48 hours later.
  • Biochemical and immunoprecipitation analyses 293T cells are transfected with 10 ⁇ g plasmid DNA overnight using calcium phosphate reagent (Invitrogen) and replenished with fresh media. 48 hours later, transfected cells are harvested, washed with PBS, and resuspended in lysis buffer (50 mM Hepes, 150 mM Nacl, 1% NP-40, pH 7.0, protease inhibitor cocktail) on ice for 45 minutes. Cell lysates are cleared by centrifugation at 13,000 ⁇ m for 10 minutes at 4°C.
  • lysis buffer 50 mM Hepes, 150 mM Nacl, 1% NP-40, pH 7.0, protease inhibitor cocktail
  • a 25 ⁇ g sample of cell lysate is incubated with mouse immune IgG (5 ⁇ g) , or human monoclonal antibodies (2 ⁇ g ) for 1 hour at room temperature, followed by incubation with 25 ⁇ l of agarose-protein G (Pierce) for another hour. Immunoprecipitates are washed three times with lysis buffer, resuspended in SDS-loading buffer (Quality Biological), and separated on 4-15% gradient SDS-PAGE (Bio-Rad), followed by Western blot analysis.
  • mice are injected with a DNA vaccine encoding full-length S from these respective isolates. Neither antisera can strongly inhibit cellular entry of pseudovims constructed with the homologous S, even after repeated immunization ( Figure 17, Panels B and C).
  • the genetic determinants of enhancement and neutralization are defined by expressing full length recombinant S protein from either SfJJrbani), S(SZ3), or relevant chimeras ( Figure 18, Panel A) and constructing the conesponding pseudovimses.
  • a central region, between amino acid 248 and 501, has been implicated previously in binding to the ACE-2 receptor (Babcock et al., 2004, J. Virol. 78:4552; Wong et al., 2004, J.Biol.Chem. 279:3197 9, 10).

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Abstract

The present invention relates to novel methods and compositions for the generation of a protective immune response in humans and other animals againts the coronavirus responsible dor Severe Acute Respiratory Syndrome (SARS-CoV).

Description

Title of the Invention: Methods and Compositions for the Generation of a Protective Immune Response Against SARS-CoV
Field of the Invention: The present invention relates to novel methods and compositions for the generation of a protective immune response in humans and other animals against the coronavirus responsible for Severe Acute Respiratory Syndrome (SARS-CoV).
Statement of Governmental Interest: This invention was funded by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. The United States Government has certain rights to this invention.
Cross-Refer ence to Related Applications: This application claims priority to United States Provisional Patent Applications Serial Nos. 60/503,508, filed September 15, 2003, and 60/550,317, filed March 8, 2004.
Background of the Invention: The severe acute respiratory syndrome (SARS), also termed "infectious atypical pneumonia," is a newly described and highly contagious respiratory infection that first occurred in late 2002 in Guangdong Province, China and spread to more than 30 countries in early 2003. The etiological agent of SARS has been identified as a novel coronavirus, named SARS-CoV (Ksiazek et al., 2003, N. Engl. J. Med. 348(20):1953-1966).
Coronavimses, a genus of the Coronaviridae family, are enveloped, positive-strand, RΝA viruses. In general, coronavimses cause respiratory and enteric diseases in humans and domestic animals (Holmes, 2001, Coronavimses, p. 1187-1203, In Knipe et al. (eds.), Fields Virology, (Lippincott Williams & Wilkins, Philadelphia); Lai and Holmes, 2001, Coronaviridae: The viruses and their replication, p. 1163-1185, In Knipe et al. (eds.), Fields Virology, (Lippincott Williams & Wilkins, Philadelphia)). Two previously known human corona vimses caused only mild upper respiratory infections. In contrast, SARS-CoV is a highly pathogenic vims that causes severe acute respiratory disease, especially in the elderly (World Health Organization. Consensus document on the epidemiology of severe acute respiratory sydrome (SARS). 10-17-2003).
Coronavimses can be divided into three serologically distinct groups (Holmes, 2001, Coronavimses, p. 1187-1203, In Knipe et al. (eds.), Fields Virology, (Lippincott Williams & Wilkins, Philadelphia). Phylogenetically, SARS-CoV is not closely related to any of the three groups (Marra et al., 2003, Science 300:1399-1404), although it is most similar to the group II coronavimses (Rest and Mindell, 2003, Infect. Genet. Evol. 3:219-225; Snijder et al., 2003, J. Mol. Biol. 331:991-1004).
Although the organization of the SARS-CoV genome is related to animal coronavimses, its genetic sequence is unique. Its genome encodes four structural proteins and two regulatory proteins. Based on a comparison to animal coronavimses, three structural gene products are predicted to be present on the viral envelope, the S, M, and E proteins (Lai and Holmes, 2001, supra). The structure of the SARS-CoV envelope differs in some respects from other budding, enveloped vimses, such as retrovimses and lentivimses, which contain one viral envelope protein.
Effective vaccines against SARS-CoV infection and methods for the early diagnosis of SARS-CoV infection are unmet public health needs. The present invention is directed to address this need.
Summary of the Invention: The present invention relates to novel compositions and methods for the inducement of an immunoprotective immune response against SARS-CoV. In one aspect, the invention relates to a vaccine composition comprising a nucleic acid molecule that comprises an S polynucleotide that encodes an extracellular domain portion of a SARS-CoV S protein.
In another aspect, the invention relates to a method of protecting a subject from infection by SARS-CoV comprising administering a nucleic acid molecule to the subject, wherein the nucleic acid molecule comprises an S polynucleotide that encodes an extracellular domain portion of a SARS-CoV S protein.
In another aspect, the invention relates to a method of treating a, subject for a SARS-CoV infection comprising administering to the subject a SARS-CoV neutralizing antibody.
In another aspect, the invention relates to a method of screening for inhibitors of SARS-CoV infection comprising: (a) incubating a cell susceptible to SARS-CoV infection with a SARS-CoV S pseudotyped viral vector in the presence or absence of a test inl ibitory agent; (b) assaying entry of said pseudotyped viral vector into said cell; (c) identifying the test inhibitory agent as an inhibitor of SARS-CoV infection if entry of said pseudotyped viral vector into said cell is reduced in the presence of the test inhibitory agent.
In another aspect, the invention relates to a method of measuring the presence or concentration of anti-SARS-CoV neutralizing antibodies in a biological sample of a mammal, wherein said method comprises the steps of: (a) incubating a cell susceptible to SARS-CoV infection with a SARS-CoV S pseudotyped viral vector in the presence or absence of the biological sample; (b) assaying entry of said pseudotyped viral vector into said cell; (c) identifying the biological sample as a positive for the presence of SARS-CoV neutralizing antibodies if entry of said pseudotyped viral vector into said cell is reduced in the presence of biological sample.
In another aspect, the invention relates to an immunoassay that measures the presence or concentration of an anti-SARS-CoV S protein antibody in a biological sample of a mammal, wherein said immunoassay comprises the steps of: (a) contacting said biological sample with mammalian expressed S protein antigen, said contacting being under conditions sufficient to permit anti-S-protein antibody if present in said sample to bind to said antigen and form an antigen-anti-S-protein antibody complex; (b) contacting said formed antigen-anti-S-protein antibody complex with an anti-S-protein antibody binding molecule under conditions sufficient to permit said anti-S-protein antibody binding molecule to bind to anti-S- protein antibody of said formed antigen-anti-S-protein antibody complex and form an extended complex; and (c) determining the presence or concentration of said anti-S-protein antibody in said biological sample by determining the presence or concentration of said formed extended complex.
In another aspect, the invention relates to a method of protecting a subject from infection by SARS-CoV comprising exposing the subject to isolated S protein.
In another aspect, the invention relates to a vaccine composition comprising at least a portion of the extracellular domain of isolated S protein.
In another aspect, the invention relates to a vaccme composition comprising a nucleic acid molecule that comprises an S polynucleotide that encodes an extracellular domain portion of a SARS-CoV S protein, wherein the vaccine composition does not exhibit antibody-dependent vims enhancing activity. In another aspect, the invention relates to a vaccme composition comprising at least a portion of the extracellular domain of isolated S protein, wherein the vaccine composition does not exhibit antibody-dependent vims enhancing activity.
In another aspect, the invention relates to a method of assaying a vaccine composition for antibody-dependent vims enhancing activity comprising the steps of: (a) generating an immune response to a vaccine composition in an animal; (b) isolating antibodies or antisera from the animal; (c) assaying the ability of the antibodies or antisera to enhance cellular entry of a SARS-CoV vims strain or a corresponding pseudovims; (d) identifying the vaccine composition as having an antibody-dependent vims enhancing activity if the antibodies or antisera enhance cellular entry of the SARS-CoV vims strain or the corresponding pseudotype.
Brief Description of the Figures: Figure 1 shows the infection of Vero (African green monkey kidney epithelial) cells retroviral and lentiviral vectors that have been pseudotyped with the SARS-CoV S protein. Panel A shows the infection of the Vero cell line with the S-pseudotyped lentiviral or retroviral vector expressing luciferase (Wood et al., 1984, Biochem. Biophys. Res. Commun. 124:592-596). Panel B shows that the S glycoprotein, but not the M or E glycoprotein, mediates viral entry by the lentiviral vector S pseudotype, and Panel C shows the requirement for the cytoplasmic domain of S for infection, as analyzed by the generation of pseudotyped vims using full-length (S) or carboxy-terminal deleted S proteins. Panel C (lower) shows a western blot analysis of the expression of the carboxy terminal deleted S variants. Figure 2 shows an analysis of the pH-dependent entry of the SARS-CoV pseudotyped lentiviral vectors. The pseudolentivimses are incubated in the presence of increasing amounts of ammonium chloride (Panel A) or bafilomycin (Sigma, St. Louis, MO) (Panel B). The experiment is performed in triplicate. Data is presented as percentage of activity at the indicated dose compared to no dmg treatment.
Figure 3 shows an analysis of the SARS-CoV S pseudotyped lentiviral vector tropism for human and animal cells and a correlation with SARS-CoV infectability. Panel A shows an analysis of the SARS-CoV S pseudo-lentivims tropism for different human cell types. All infections are performed in triplicate. Data is presented as the average ± standard deviation. The results of one of two independent experiments are shown. Panel B shows an analysis of the SARS-CoV S pseudo-lentivims infectivity of renal and lung cells from different animal species. All infections are performed in triplicate. Data is presented as the average ± standard deviation. The results of one of two independent experiments are shown. Panel C shows an analysis of infectivity of SARS-CoV (Urbani) on selected susceptible and resistant cells from Panels A and B above. SARS-CoV (Urbani) is titered on Vero cells as previously described in Subarrao et al., 2004, J. Virol. 78(7):3572-7. A dotted line indicates the detection limit of infectivity of SARS-CoV. Figure 4 shows an analysis of DC-SIGN receptor dependent uptake of
SARS-CoV S pseudotyped lentiviral vector and cell mediated transfer and infection of target cells. Panel A shows the binding of purified SARS-CoV S glycoprotein to cell lines. Cell cultures containing lxlO6 cells of African green monkey kidney cells (Vero), human T leukemia cells (A3R5 and MT2), or THP-1 myelomonocytic leukemia cells expressing wild type or mutant forms of DC-SIGN (THP-DC-SIGN and THP-DC-SIGNΔ35, respectively) are incubated with purified S protein, which is tagged with a myc and His epitope at amino acid 1190, for 20 minutes on ice. Binding of spike protein to the cells is detected using an FITC labeled anti-His (carboxy-terminal) antibody (Invitrogen, Carlsbad, CA). An FITC labeled IgG isotype is used as a control. Data is analyzed by flow cytometry. Panel B shows an analysis of direct viral entry of SARS-CoV S pseudotyped lentiviral vector (left panel) and cell mediated transfer of the pseudotyped lentiviral vector from THP-1, THP-DC-SIGN and THP-DC-SIGNΔ35 cells (right panel). Susceptibility of Vero, A3R5, MT2, THP-1, THP-DC-SIGN and THP-DC- SIGNΔ35 to SARS-CoV S pseudotyped lentiviral vector infection is measured after transduction using the luciferase reporter (left panel). Cell mediated pseudoviral transfer by THP-1, THP-DC-SIGN or THP-DC-SIGNΔ35 cells is assessed by incubating the particular THP cell type (3xl04 cells) with pseudotyped lentiviral vector for two hours at 37°C. After incubation, cells are washed three times before they are added to a cell culture containing 3xl04 target cells. Cells are collected 72 hours after coincubation for luciferase assays. Panel C shows the inhibition of direct infection and cell-mediated transfer of SARS-CoV S pseudo lentivirus by mouse anti-SARS-CoV S antisemm. SARS-CoV S pseudotyped lentiviral vector is exposed to mouse control or anti-S specific antisemm at mdicated dilutions for sixty minutes at 37°C before adding to Vero cells (left panel). For cell mediated transfer (right panel), the indicated THP-DC-SIGN cells are incubated with pseudovimses as described for Panel B above, followed by incubation with Vero cells in the presence or absence of mouse anti-SARS-CoV S specific antisemm for 48 hours. After the 48-hour incubation period, cells are collected for luciferase assays. Figure 5 shows the uptake and transfer of GFP-Vpr labeled SARS-CoV S pseudotyped lentiviral vector and SARS-CoV by mature and human myeloid dendritic cells (mDC). Panel A comprises confocal microscopic images of cells showing the uptake of GFP-Vpr labeled SARS-CoV S pseudotyped lentiviral vector by mDC, and the subsequent transfer of the pseudotyped lentiviral vector from mDC to renal epithelial cells. The mDC cells are infected with the GFP-Vpr labeled SARS-CoV S pseudotyped lentivims for 30 minutes at 37°C. The mDCs are then added onto human renal epithelial cells (786-0; 3xl04 cells per well, plated a day before) in eight well coverslip slides (Nalge Nunc, Naperville, IL) at a ratio of 1 : 1. Uptake, polarization and transfer are assessed by confocal microscopy with representative cells. The arrow indicates the transfer of labeled vims from mDCs to 786-0 cells. Panel B shows that human mature mDCs are not directly infected by SARS-CoV (Urbani strain) but instead promote cell-mediated infection of susceptible target cells. Vero cells and immature and mature dendritic cells are incubated with SARS-CoV (Urbani) for one hour in 96 well dishes (2xl04 cells/well). After incubation, cells are washed and maintained in culture medium for 72 hours before collection of culture supernatants for analysis of viral titer (left panel). Mature dendritic cells are also infected for one hour with SARS-CoV, washed, detached with trypsin, and replated onto 96-well-dishes with Vero cells (2xl04 cells/well; 1:1 ratio) in the presence or absence of mouse anti-SARS-CoV S antisemm (1 : 100 dilution) Cells are maintained in culture for 72 hours before supernatants are collected for analysis of viral titer (right panel). Viral titer is measured as described in Subarrao et al. ( 2004, J. Virol. 78(7):3572-7). Vims yield is expressed as TCID50 per ml. Dotted lines indicate the detection limit of SARS-CoV. Figure 6 shows the inhibition of viral pseudotyping using two types of inhibitors. Panel A shows the inhibitory activity of a peptide aptamer (Peptide 1 (HR2)) of the HR2 coiled-coil region of SARS-CoV S protein (residues 1154 to 1190 of SEQ ID NO:3) vs an unrelated peptide (Control). The Peptide 1 (HR2) sequence is LGDISGINASWNIQKEIDRL (SEQ ID NO:l) and the control peptide sequence is MFIFLLFLTLTSGSDLDR (SEQ ID NO:2). Both peptides are purified by high-pressure liquid chromatography. Panel B shows the inhibitory activity of human convalescent antisera from a patient infected with SARS (SARS) vs. non-immune control sera (Control).
Figure 7 shows a schematic representation of SARS-CoV S glycoprotein cDNAs and the expression of the encoded recombinant proteins. Panel A shows the stmcture of the cDNAs used. Panel B shows a western blot analysis of the expression of the recombinant SARS-CoV S proteins after transfection of the indicated plasmid expression vectors in 293T cells. Arrows indicate specific SΔCD (upper) and SΔTM (lower) bands.
Figure 8 shows immune responses to SARS-CoV DNA vaccination in BALB/c mice. Panel A shows intracellular cytokine staining is performed to response to stimulation with overlapping S peptide pools in CD4 (left) or CD8 (right) lymphocytes from mice (n = 5 per group) immunized with empty plasmid vector (control) or mice (n = 5 per group) immunized with the mdicated plasmid at weeks 0, 3 and 6. Immune responses are measured 10 days after the final boost. Non-stimulated cells gave responses similar to those of the control subjects, at background levels. Symbols indicate the response of each individual animal, and the median value is shown (horizontal bar). Panel B shows antibody responses induced by plasmid DNA vaccination against the SARS-CoV S protein. End-point dilution enzyme linked immunosorbent assay (ELISA) titres of SARS-CoV S- specific antibodies (left panel) in semm of vaccinated animals collected 10 days after the final boost are determined by optical density as described in the Materials and Methods of Example 2 herein. Neutralization by antisera from mice immunized with the relevant SARS-CoV S mutant or no insert (control) plasmid DNA vectors at the indicated concentrations is measured using the luciferase assay with S pseudotyped lentiviral vectors (middle panel). Reduction of gene transfer is observed with immune sera in a dose-dependent fashion. Two-fold dilutions of heat-inactivated sera are tested in a microneutralization assay for the presence of antibodies that neutralized the infectivity of 100 TCID50 of SARS-CoV in Vero cell monolayers, using four wells per dilution on a 96-well plate (right panel). The presence of viral cytopathic effect (cpe) is read on days 3 and 4. The dilution of semm that completely prevented cpe in 50% of the wells is calculated by the Reed Muench formula. Data are presented as the mean ± s.e. for each group. A non- parametric two-tailed t-test (Mann-Whitney) is used for statistical analysis, and the relevant P values are indicated (right panel)
Figure 9 shows that DNA vaccinated BALB/c mice are protected against pulmonary SARS-CoV replication after challenge. Immunization and challenge are performed in mice as described in Subbarao et al., 2004, J. Virol. 78, 3572-
3577, and viral replication (mean logioTCID5o per g tissue with standard error) in the lower respiratory tract (Panel A) and the upper respiratory tract (Panel B) respiratory tract after challenge with SARS-CoV is measured for five immunized animals inoculated with SΔCD, SΔTM or empty plasmid vector control. The lower limit for detection of SARS-CoV replication is 1.5 TCID50 per g in the lungs and 1.8 TCID50 per g in the nasal turbinates. A non-parametric two-tailed t-test
(Mann-Whitney) is used for statistical analysis. Log-transformed vims titres are compared, and statistical significance is assigned to the differences, both with a P value of 0.0079. 1 Figure 10 shows the results of a determination of the immune mechanism of protection, using the techniques of T-cell depletion, adoptive transfer and antibody passive transfer. In Panel A, monoclonal rat anti-mouse anti-CD4, CD8 or CD4/CD8/ CD90 are used to deplete T cells in SΔCD and control vaccinated mice (n=4) as described in Epstein et al., 2000,. Int. Immunol. 12, 91-101. Mice are then challenged with SARS-CoV 48 hours later. Viral replication in the lungs is measured as described in Figure 9. Data represent mean ± s.e., and no statistically significant difference is observed between groups depleted as shown. Panel B shows a lack of protection against SARS-CoV replication in the lungs after adoptive T-cell transfer from vaccinated mice. Each naive mouse (n = 4) received 3 x 107 purified T cells from a donor mouse confirmed to respond to the vaccine (immune) or from a non-immune mouse (control). The recipient mice are challenged 24 hours after adoptive T-cell transfer. Viral titre in lungs is measured as described in Figure 9. There is no statistically significant difference between these two groups using the non-parametric Mann-Whitney two-tailed t-test (P = 0.49). Panel C shows that protection against SARS-CoV replication in the lungs is provided by the passive transfer of immune IgG from vaccinated mice. Purified IgG from SΔCD or control vaccinated donor mice (n = 4) is passively transferred into recipient naive mice (n = 4). Semm from recipient mice is collected one day before challenge, and neutralization is confirmed using the S pseudotyped lentiviral vector (left panel). Recipient mice (n = 4 per group) are challenged 24 hours after IgG transfer with 104 TCID50 SARS-CoV and SARS-CoV replication is measured as described in Figure 9. The non-parametric two-tailed t-test (Mann- Whitney) revealed a statistically significant difference of P = 0.0286 between these two groups (right panel). Figure 11 shows the confirmation of T cell depletion in lung tissues. Flow cytometry with monoclonal antibodies to mouse CD4 (left column) or CD8 (right column) is performed on lymphocytes derived from the lungs of BALB/c mice depleted with rat anti-mouse monoclonal antibodies to CD4, CD8, and/or CD90 antibodies, individually or in the indicated combinations as described in the Materials and Methods section of Example 2 herein. Single-cell suspensions are prepared from lungs harvested at 48 hours after injection. Lung tissues are minced with scissors, resuspended in 5 ml of an enzyme mixture (RPMI 1640, 12 mg collagenase type I, and 100 μg DNAase) at 37°C for 30 min, followed by mincing and passage through a 40 μm nylon cell strainer. Cells are washed once with PBS, and resuspended in PBS. 2x 106 are used for staining on ice for 30 min using rat anti-mouse CD3-PE(clone 17A2) (BD-Pharmingen, San Diego, CA), rat anti- mouse CD4-PerCP(clone RM4-5) (BD-Pharmingen, San Diego, CA), rat anti- mouse CD8-APC(clone 53-6.7) (BD-Pharmingen, San Diego, CA), followed by washing with 1 ml ice cold PBS. The cells are finally resuspended in PBS+1% formaldehyde for fluorescence activated cell sorting analysis. 200,000 cells are collected and analyzed using Flowjo software. Figure 12 shows the confirmation of T cell depletion in spleen tissues. Flow cytometry with monoclonal antibodies to mouse CD4 (left column) or CD8 (right column) is performed on lymphocytes derived from the spleens of BALB/c mice depleted with rat anti-mouse monoclonal antibodies to CD4, CD8, and/or CD90 antibodies, individually or in the indicated combinations as described in the Materials and Methods section of Example 2 herein. Single-cell suspensions are prepared from spleens harvested at 48 hours after injection. Spleen tissues are homogenized, resuspended in PBS, and passed through a 40 μm nylon cell strainer (BD Falcon, Bedford, MA). Cells are washed once with PBS, and resuspended in PB S . 2 x 106 are used for staining on ice for 30 min using rat anti-mouse CD3 - PE(clone 17A2) (BD-Pharmingen, San Diego, CA), rat anti-mouse CD4- PerCP(clone RM4-5) (BD-Pharmingen, San Diego, CA), rat anti- mouse CD8- APC(clone 53-6.7) (BD-Pharmingen, San Diego, CA), followed by washing with 1 ml ice cold PBS. The cells are finally resuspended in PBS+1% formaldehyde for fluorescence activated cell sorting analysis. 200,000 cells are collected and analyzed using Flowjo software.
Figure 13 shows an ELISA analysis of semm samples from SARS patients. Eleven semm samples collected from five patients are analyzed for the presence of antibodies against SARS-CoV proteins using mammalian expressed S protein based ELISA. ELISA analyses of patient sera (1:133 dilution) for IgM, IgG and IgA against SARS-CoV spike (S) protein antigen (upper panel) or SARS-CoV nucleocapsid (N) protein antigen (lower panel) are shown at different time points after the onset of symptoms. Samples from each patient are represented by a different symbol. Solid black symbols represent samples that tested positive for SARS-CoV antibody as measured using the traditional vims-infected cell lysate based ELISA and immunofluorescence assays (IF A), whereas open symbols represent samples that tested negative using these methods.
Figure 14 shows an analysis of SARS-CoV neutralizing antibody levels in patient sera. Neutralizing antibody levels are measured using a SARS-CoV S pseudotyped lentiviral vector encoding the luciferase reporter as the neutralization target. Each patient sample is represented by a different symbol. Solid black symbols represent samples that tested positive for SARS-CoV antibody as measured using the traditional vims infected cell lysate based ELISA and IFA methods, whereas open symbols represent samples that tested negative using these methods. Lower luminescence values indicate a lower level of pseudotyped lentivims infection due to higher viral neutralization activity in the semm samples. Results at a semm dilution of 1 : 50 are shown.
Figure 15 shows results demonstrating the incorporation of diverse S proteins into lentiviral vectors with comparable efficiency. Plasmids encoding the indicated S proteins are cotransfected with lentiviral expression vectors into 293T cells. Forty-eight hours after transfection, supernatants are harvested and mn on sedimentation gradients as described in Example 3 herein. A representative Western blot analysis of gradient fractions for S(GD03T0013) is shown in Panel A. Panel B shows quantification of sedimentation gradient fractions for the indicated strains. Figure 16 shows an analysis of the sensitivity to antibody neutralization of lentivims vectors pseudotyped with alternative S proteins and an analysis of antibody dependent enhancement of vims entry using pseudovimses constructed with the S protein from two palm civet virases. In Panel A, lentivims vectors that are pseudotyped with the indicated SARS-CoV S glycoproteins from human isolates are incubated with purified IgG from mice vaccinated with a DNA expression vector encoding SfJrbani) or with a negative control sera from non- immune mice that was subtracted from immune sera. Percent inhibition, assessed by luciferase reporter gene expression, is calculated as the reduction in luciferase activity relative to values achieved in the absence of sera. Panel B shows an analysis of antibody-dependent enhancement of pseudovims entry with psuedovirases constructed with S-protein from two palm civet SARS-CoV isolates. Panel C shows an analysis of antibody neutralization of pseudovimses constructed with the S protein from the Urbani strain (SfJJrbani)), the resistant human strain (S (GD03T0013)), and two palm civet strains (S(SZ3) and S(SZ16)) using human neutralizing mabs (S3.1, S127, and SI 11) derived from EBV-transformed B lymphocytes. Figure 17 shows the differential sensitivity to ACE-2 inhibition of pseudovimses constructed with the S protein from the Urbani strain or the GD03T0013 strain and the relative resistance of S(GD03T0013) and S(SZ3) pseudotyped vimses to homologous neutralization. Panel A shows the results obtained when the indicated pseudovimses are incubated with increasing amounts of soluble recombinant hACE-2. Entry is assessed using the luciferase reporter gene..Panel B shows that immunization with homologous S protein (i.e S protein from GD03T001) does not result in sera with increased neutralization activity against pseudovims constructed with the S(GD03T0013). Panel C shows that sera from mice immunized with full length S(SZ3) neutralizes SfJJrbani) pseudotyped viras but does not neutralize S(SZ3) pseudotyped vims.
Figure 18 shows the results of experiments to define the genetic determinants of S glycoprotein sensitivity to antibody neutralilation or antibody enhancement. Panel A shows a schematic diagram of a neutralization sensitive S protein ( SfJJrbani)), a neutralization resistant S protein (palm civet S(SZ3)), and chimeric S proteins. Panel B shows an analysis of luciferase gene transfer efficiency into 786-0 cells of pseudovimses constructed with the indicated wild type and chimeric S proteins (left panel). The dashed line indicates the background levels of gene transfer in the absence of S. An analysis of the inhibition or enhancement of the indicated S pseudotypes using a neutralizing monoclonal antibody to S (right) shows the dependence on the hACE-2 binding domain (right panel). Panel C shows an analysis of anti-S antisera with SfJJrbani) protein or S(SZ3) protein. A western blot analysis compares the amount of S protein in transfected cell supernatants (left panel, Input) with the amount of protein immunoprecipitated with S-antisera (right panel, IP).
Figure 19 shows the results of experiments to identify immunogens and mabs that circumvent antibody-dependent enhancement of viras entry and the results of a biochemical analysis of the mechanism of enhancement. Panel A shows a neutralization profile of purified IgG from mice vaccinated with a DNA expression vector encoding a secreted form of S( Urbani) (S(l 153) - terminated at aa 1153) against the indicated human or civet pseudovimses. Panel B showsa Western blot analysis of the biochemical interaction of purified IgG from S(l 153) immunized mice with the following S proteins and chimeric S proteins: S(Urbani), SU, US, and S(SZ3). The amount of S protein in cell lysates from transfected cells (left panel, Input) is compared to the amount of protein immunoprecipitated with purified IgG from S(l 153) immunized mice (right panel, IP). Panel C shows results demonstrating that Mab SI 10 inhibits S (Urbani) pseudovims entry and does not enhance Palm Civet pseudovims entry. SI 10 antibody or an isotype control antibody is incubated with the indicated pseudovimses and inhibition is assessed as described in the legend for Figure 16 above.
Description of the Preferred Embodiments : In one aspect, the present invention relates to novel compositions and methods related to the development of a vaccine composition for providing immunity to SARS-CoV.
As used herein, the term "SARS-CoV" refers to the causal agent of SARS, and to evolutionarily derived variants of such coronavims that cause respiratory disease in humans and other animals. The methods and compositions of the present invention may be used in any species affected by SARS-CoV, including humans and non-human animals (e.g., non-human mammals and birds).
One aspect of the invention reflects the recognition that the SARS-CoV spike protein (" SARS-CoV S protein") mediates viral entry through pH dependent endocytosis and defines its cellular tropism. The invention further reflects the recognition that viral transmission can occur through cell-mediated transfer by dendritic cells. The invention further reflects the recognition that exposure of a subject to isolated SARS-CoV S protein via DNA vaccination induces a robust immune response. An immune response will typically comprise a humoral immune response, and will preferably also provide a cytotoxic cell response, and may involve B cells, T cells, and other types of lymphoid cells. Cytotoxic immunity complements the humoral system by eliminating the infected cells. The immune response to the compositions of the present invention will preferably be mediated by both CD4 and CD8 cells and will include the generation of significant antibody titres and neutralizing antibodies. As used herein, "neutralizing antibodies" refers to antibodies that bind to SARS-CoV viras particles and interfere with the ability of the vims particles to infect cells.
Immunogenic SARS-CoV Compounds As used herein, an "immunogenic SARS-CoV compound" is a proteinaceous molecule (i.e., a SARS-CoV protein, or a polypeptide derivative of a SARS-CoV protein) that: (A) possesses an epitope that binds to antibody that is immunologically reactive with a protein of the SARS-CoV; or (B) induces a recipient host to produce antibody that is immunologically reactive with the SARS- CoV. Such epitope may be a contiguous region of the protein, or may be discontinuous and formed through the three-dimensional conformation of the molecule. Most preferably, the immunogenic SARS-CoV compound will be a SARS-CoV S compound that will: (A) possess an epitope that binds to antibody that is immunologically reactive with SARS-CoV S protein; or (B) induce a recipient host to produce antibody that is immunologically reactive with SARS- CoV S protein.
As used herein, the term "polypeptide derivative" is intended to refer to a polypeptide of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more amino acid residues which has a sequence that is identical to a sequence of a SARS-CoV protein, or which contains insertions, deletions or substitutions from the sequence of a SARS-CoV protein that nevertheless permit such polypeptide derivative to function as an immunogenic SARS-CoV compound when administered into a recipient. Indeed, as is recognized in the art, some amino acid sequences of the polypeptides described herein can be varied without negatively affecting the immunogenic nature of the polypeptides and nucleic acids of the invention. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al. (1990) "DECIPHERING THE MESSAGE IN PROTEIN SEQUENCES: TOLERANCE TO AMINO ACID SUBSTITUTIONS," Science 247:1306-1310). The immunogenic SARS-CoV compounds of the present invention may be modified. Acceptable modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins— Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., 1990, Meth. Enzymol. 182:626-646; Rattan et al., 1992, Ann. NY Acad. Sci. 663:48-62).
Where present, the same type of modification may be present in the same or varying degrees at several sites in a given compound, and a given compound may contain many types of modifications. Compounds may have more than one type of modification. The immunogenic SARS-CoV compounds of the present invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods. Immunogenic SARS-CoV protein compounds can be prepared in any suitable manner (e.g., isolated from naturally occurring polypeptides, produced via recombinant techniques, produced synthetically or chemically, or produced by a combination of such methods.
SEQ ID NO:3 (GenBank AY278741) provides the amino acid sequence of the SARS-CoV S protein of the SARS-CoV (Urbani strain). (SEQ ID NO:3)
MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSS RGV YYPDEIFRSD TLYLTQDLFL PFYS VTGFH TINHTFGNPV IPFKDGIYFA ATEKSNWRG WVFGSTMKTK SQSVIIINMS T WIRACNF ΞLCDNPFFAV SKPMGTQTHT MIFDNAFNCT FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY QPIDWRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP AQDIWGTSAA AYFVGYLKPT TF LKYDENG TITDAVDCSQ NPLAELKCSV KSFEIDKGIY QTSNFRWPS GDWRFPNIT NLCPFGEVFN ATKFPSVYA ERKKISNCVA DYSVLYNSTF FSTFKCYGVS ATKL DLCFS VYADSFWK GDDVRQIAPG QTGVIADYY KLPDDFMGCV LAWNTRNIDA TSTGNYYKY RYLRHGKLRP FERDIS VPF SPDGKPCTPP ALNCY PLND YGFYTTTGIG YQPYRVWLS FELLNAPATV CGPKLSTDLI KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD SVRDP TSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD VNCTDVSTAI HADQLTPAWR IYSTGNWVFQ TQAGCLIGAE HVDTSYΞCDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS LGADSS1AYS N TIAIPTNF SISITTEVMP VSMAKTSVDC NMYICGDSTΞ CA LLLQYGS FCTQLNRALS GIAAEQDR T REVFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQ VLYE NQKQIA QFN KAISQIQESL TTTSTALGKL QDWNQNAQA LNTLVKQLSS NFGAISSVLN DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAΞI RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH GWFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASWN IQKEIDRLNE VAKNLNΞSLI DLQELGKYEQ YIKWP YV L GFIAGLIAIV MVTILLCC T SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV KLHYT
Prefeπed immunogenic SARS-CoV compounds of the invention comprise at least a portion of the extracellular domain of the S protein, which is composed of residues 14 to 1190 of SEQ ID NO:3. Preferably, such portion will comprise 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 300, 500, 1000 or more amino acid residues whose sequence is identical to a sequence of the extracellular domain of the S protein, or which contains one or more insertions, deletions or substitutions (e.g., >95% identical, >90% identical, >80% identical, >70% identical) from the sequence of the extracellular domain of the S protein, that nevertheless permit such immunogenic SARS-CoV compounds to function as an immunogenic SARS-CoV compound when administered into a recipient.
A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Bmtlag et al., 1990, Comp. App. Biosci.6:231 -245. In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. If the subject sequence is shorter than the query sequence due to N- or C- terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total residues of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%). In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual coπections are to be made for the purposes of the present invention.
The SARS-CoV S protein of SEQ ID NO:3 is encoded by residues 21,492 - 25,259 (SEQ ID NO:4) of the SARS-CoV genome (GenBank AY278741):
SEQ ID NO:4 atgtttatt ttcttattat ttcttactct cactagtggt agtgaccttg accggtgcac cacttttgat gatgttcaag ctcctaatta cactcaacat acttcatcta tgaggggggt ttactatcct gatgaaattt ttagatcaga cactctttat ttaactcagg atttatttct tccattttat tctaatgtta cagggtttca tactattaat catacgtttg gcaaccctgt catacctttt aaggatggta tttattttgc tgccacagag aaatcaaatg ttgtccgtgg ttgggttttt ggttctacca tgaacaacaa gtcacagtcg gtgattatta ttaacaattc tactaatgtt gttatacgag catgtaactt tgaattgtgt gacaaccctt tctttgctgt ttctaaaccc atgggtacac agacacatac tatgafcattc gataatgcat ttaattgcac tttcgagtac atatctgatg ccttttcgct tgatgtttca gaaaagtcag gtaattttaa acacttacga gagtttgtgt ttaaaaataa agatgggttt ctctatgttt ataagggcta tcaacctata gatgtagttc gtgatctacc ttctggtttt aacactttga aacctatttt taagttgcct cttggtatta acattacaaa ttttagagcc attcttacag ccttttcacc tgctcaagac atttggggca cgtcagctgc agcctatttt gttggctatt taaagccaac tacatttatg ctcaagtatg atgaaaatgg tacaatcaca gatgctgttg attgttctca aaatccactt gctgaactca aatgctctgt taagagcttt gagattgaca aaggaattta ccagacctct aatttcaggg ttgttccctc aggagatgtt gtgagattcc ctaatattac aaacttgtgt ccttttggag aggtttttaa tgctactaaa ttcccttctg tctatgcatg ggagagaaaa aaaatttcta attgtgttgc tgattactct gtgctctaca actcaacatt tttttcaacc tttaagtgct atggcgtttc tgccactaag ttgaatgatc tttgcttctc caatgtctat gcagattctt ttgtagtcaa gggagatgat gtaagacaaa tagcgccagg acaaactggt gttattgctg attataatta taaattgcca gatgatttca tgggttgtgt ccttgcttgg aatactagga acattgatgc tacttcaact ggtaattata attataaata taggtatctt agacatggca agcttaggcc ctttgagaga gacatatcta atgtgccttt ctcccctgat ggcaaacctt gcaccccacc tgctcttaat tgttattggc cattaaatga ttatggtttt tacaccacta ctggcattgg ctaccaacct tacagagttg tagtactttc ttttgaactt ttaaatgcac cggccacggt ttgtggacca aaattatcca ctgaccttat taagaaccag tgtgtcaatt ttaattttaa tggactcact ggtactggtg tgttaactcc ttcttcaaag agatttcaac catttcaaca atttggccgt gatgtttctg atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct cttttggggg tgtaagtgta attacacctg gaacaaatgc ttcatctgaa gttgctgttc tatatcaaga tgttaactgc actgatgttt ctacagcaat tcatgcagat caactcacac cagcttggcg catatattct actggaaaca atgtattcca gactcaagca ggctgtctta taggagctga gcatgtcgac acttcttatg agtgcgacat tcctattgga gctggcattt gtgctagtta ccatacagtt tctttattac gtagtactag ccaaaaatct attgtggctt atactatgtc tttaggtgct gatagttcaa ttgcttactc taataacacc attgctatac ctactaactt ttcaattagc attactacag aagtaatgcc tgtttctatg gctaaaacct ccgtagattg taatatgtac atctgcggag attctactga atgtgctaat ttgcttctcc aatatggtag cttttgcaca caactaaatc gtgcactctc aggtattgct gctgaacagg atcgcaacac acgtgaagtg ttcgctcaag tcaaacaaat gtacaaaacc ccaactttga aatattttgg tggttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaga ggtcttttat tgaggacttg ctctttaata aggtgacact cgctgatgct ggcttcatga agcaatatgg cgaatgccta ggtgatatta atgctagaga tctcatttgt gcgcagaagt tcaatggact tacagtgttg ccacctctgc tcactgatga tatgattgct gcctacactg ctgctctagt tagtggtact gccactgctg gatggacatt tggtgctggc gctgctcttc aaataccttt tgctatgcaa atggcatata ggttcaatgg cattggagtt acccaaaatg ttctctatga gaaccaaaaa caaatcgcca accaatttaa caaggcgatt agtcaaattc aagaatcact tacaacaaca tcaactgcat tgggcaagct gcaagacgtt gttaaccaga atgctcaagc attaaacaca cttgttaaac aacttagctc taattttggt gcaatttcaa gtgtgctaaa tgatatcctt tcgcgacttg ataaagtcga ggcggaggta caaattgaca ggttaattac aggcagactt caaagccttc aaacctatgt aacacaacaa ctaatcaggg ctgctgaaat cagggcttct gctaatcttg ctgctactaa aatgtctgag tgtgttcttg gacaatcaaa aagagttgac ttttgtggaa agggctacca ccttatgtcc ttcccacaag cagccccgca tggtgttgtc ttcctacatg tcacgtatgt gccatcccag gagaggaact tcaccacagc gccagcaatt tgtcatgaag gcaaagcata cttccctcgt gaaggtgttt ttgtgtttaa tggcacttct tggtttatta cacagaggaa cttcttttct ccacaaataa ttactacaga caatacattt gtctcaggaa attgtgatgt cgttattggc atcattaaca acacagttta tgatcctctg caacctgagc tcgactcatt caaagaagag ctggacaagt acttcaaaaa tcatacatca ccagatgttg atcttggcga catttcaggc attaacgctt ctgtcgtcaa cattcaaaaa gaaattgacc gcctcaatga ggtcgctaaa aatttaaatg aatcactcat tgaccttcaa gaattgggaa aatatgagca atatattaaa tggccttggt atgtttggct cggcttcatt gctggactaa ttgccatcgt catggttaca atcttgcttt gttgcatgac tagttgttgc agttgcctca agggtgcatg ctcttgtggt tcttgctgca agtttgatga ggatgactct gagccagttc tcaagggtgt caaattacat tacacataa SEQ ID NO:4 can therefore be employed to produce SARS-CoV vaccine compositions, that upon administration to a subject result in the production ofan immunogenic SARS-CoV S compound. Preferred nucleic acid molecules ofthe invention will also comprise a polynucleotide encoding a transmembrane polypeptide, refeπed to herein as a transmembrane polynucleotide. For example, the polynucleotide may encode a transmembrane portion ofa naturally occurring S protein. For example, the transmembrane portion ofthe S protein ofSEQ ID NO:3 comprises residues 1191 to 1216. The transmembrane polypeptide may comprise this polypeptide sequence or a variant or fragment thereof, or it may be an unrelated transmembrane polypeptide that serves the purpose of anchoring the extracellular domain portion in the cell membrane. It is of course understood by those of skill in the art that a transmembrane polynucleotide will be in frame with the extracellular domain polynucleotide described above. Nucleic acid molecules of the invention may also comprise a polynucleotide encoding a cytoplasmic domain. However, it is contemplated as part of this invention that a cytoplasmic domain is not necessary and that the inclusion of a cytoplasmic domain may be undesirable.
Preferred nucleic acid molecules of the invention will also comprise a polynucleotide encoding a signal polypeptide, referred to herein as a signal polynucleotide, which directs the secretion of the polypeptide encoded by the polynucleotide or polynucleotide construct of the present invention. Those of ordinary skill in the art are aware that polypeptides secreted by mammalian cells normally have a signal peptide which is cleaved from the complete polypeptide to produce a secreted "mature" form of the polypeptide. It is of course understood by those of skill in the art that a signal polynucleotide will be in frame with the extracellular domain polynucleotide described above. In one embodiment, a naturally occurring leader sequence, or a functional derivative thereof, is employed. Alternatively, a completely synthetic (i.e., an amino acid sequence not occurring in nature) amino acid coding sequence that functions as a leader sequence can be constructed by those skilled in the art utilizing recombinant DNA techniques, and may be employed. If naturally occurring, the signal polynucleotide may be that which is ordinarily associated with the SARS-CoV S protein in nature or it may be a heterologous sequence derived from another secreted protein. In a preferred embodiment of this invention, nucleic acid molecules of the invention encode immunogenic compounds using human preferred codons. Human preferred codons are well known in the art and are described for example, in Haas et al., 1996, Curr. Biol. 6:315-324.
In prefeπed embodiments, the nucleic acid molecules of the invention will be optionally operatively linked to one or more regulatory elements (promoters, translation initiation sites, etc.) so as to permit the expression of the encoded polypeptides in a recipient cell. Alternatively, the nucleic acid molecules will not contain such regulatory elements, and will require cellular processes (such as recombination, integration into nuclear or mitochondrial DNA, etc.) in order to produce the encoded polypeptides.
Alterations in the coding region of the SARS-CoV genome, or of SEQ ID NO:4 in particular, may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not substantially alter the three dimensional stracture of the extracellular domain of the S protein or that do not alter the ability of the nucleic acid constract encoding the S protein, or the S protein, to induce SARS-CoV neutralizing antibodies. Also especially preferred in this regard are conservative substitutions. For example, aromatic amino acids that can be conservatively substituted for one another include phenylalanine, tryptophan, and tyrosine. Hydrophobic amino acids that can be conservatively substituted for one another include leucine, isoleucine, and valine. Polar amino acids that can be conservatively substituted for one another include glutamine and asparagine. Basic amino acids that can be conservatively substituted for one another include arginine, lysine, and histidine. Acidic amino acids that can be conservatively substituted for one another include aspartic acid and glutamic acid. Small amino acids that can be conservatively substituted for one another include alanine, serine, threonine, methionine, and glycine.
In a preferred embodiment of the invention, immunogenic SARS-CoV compounds comprise polynucleotides encoding at least two SARS CoV proteins, or portions thereof, capable of forming synthetic nucleocapsids upon co- transfection. For example, one aspect of the invention relates to the recognition that co-transfection of polynucleotides encoding the following combinations of SARS CoV proteins results in the formation of nucleocapsids: N, M and S proteins; N, M and E proteins; N and M proteins; and N, M, S and E proteins. Thus, the invention relates to the following: co-transfection of polynucleotides encoding the N, M and S proteins, or portions of these proteins; co-transfection of polyncleotides encoding the N, M and E proteins, or portions of these proteins; co- transfection of polyncleotides encoding the M and N proteins, or portions of these proteins; and co-transfection of polyncleotides encoding the N, M, S and E proteins, or portions of these proteins. The invention also relates to the recognition that co-transfection of polynucleotides encoding the N, M, and S proteins, or portions of these proteins, results in the formation of budding virus or corona-like structures. Preferrably, co-transfected polynucleotides comprise polynucleotides that encode the N, M and S proteins, or portions of these proteins, wherein co- transfection results in the formation of budding vims or the formation of corona- like stractures.
Preferred immunogenic SARS-CoV compounds of the invention will comprise polynucleotides encoding 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 300, 500, 1000 or more amino acid residues of the SARS CoV S, M, N or E whose sequence is identical to a sequence of the S, M, N or E protein, or which contains one or more insertions, deletions or substitutions (e.g., >95% identical, >90%> identical, >80% identical, >70% identical) from the sequence of the S, M, N or E proteins, that nevertheless permit such immunogenic SARS-CoV compounds to function form nucleocapsids, preferably corona-like stractures, when administered into a recipient. The protein sequences of S, M, N, and E proteins, and the nucleic acid sequences encoding those proteins may be found at Genbank Accession No. AY278741.
The protein sequences of the M, N or E SARS-CoV proteins may be readily ascertained by one of skill in the art. For example, the protein sequences for SARS CoV (Urbani strain) are as follows:
E protein (Genbank Accession No. AAP13443.1) (SEQ ID NO: 5) YSFVSEETG TLIVNSVLLF LAFWFLLVT LAILTALRLC AYCCNIV VS LVKPTVYVYS RVKNLNSSEG VPDLLV
M protein (Genbank Accession No. AAP 13444.1 ) (SEQ ID NO: 6)
MADNGTITVE ELKQLLΞQ N LVIGFLFLA IMLLQFAYSN RNRFLYIIKL VFLWLLWPVT LACFVLAAVY RI WVTGGIA IA ACIVGLM LSYFVASFR LFARTRSMWS FNPETNILLN
VPLRGTIVTR PLMESΞLVIG AVIIRGHLRM AGHPLGRCDI KDLPKEITVA TSRTLSYYKL GASQRVGTDS GFAAYNRYRI G Y LNTDHA GS DNIALLV Q
N protein (Genbank Accession No. AAP 13445.1) (SEQ ID NO: 7)
MSDNGPQSNQ RSAPRITFGG PTDSTDNNQN GGRNGARPKQ RRPQGLPNNT AS FTALTQH GKEELRFPRG QGVPINTNSG PDDQIGYYRR ATRRVRGGDG KMKELSPR Y FYYLGTGPEA SLPYGANKEG IVWVATEGAL NTPKDHIGTR NPNNNAATVL QLPQGTTLPK GFYAEGSRGG SQASSRSSSR SRGKTSR STP GSSRGNSPAR MASGGGETAL ALLLLDRLNQ LESKVSGKGQ QQQGQTVTKK SAAEASKKPR QKRTATKQYN VTQAFGRRGP EQTQGNFGDQ DLIRQGTDYK HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYHGAIKL DDKDPQFKDN VILLNKHIDA YKTFPPTEPK DKKKKTDEA QPLPQRQKKQ PTVTLLPAAD MDDFSRQLQN SMSGASADST QA It is understood that co-transfection of polynucleotides refers to situations wherein the polynucleotides are contained as part of the same nucleic acid molecule upon transfection (i.e. administration to the subject) and wherein the polynucleotides are contained on different nucleic acid molecules upon transfection (i.e. administration to the subject). It is also contemplated as part of the invention that co-tranfection may occur via serial transfection (i.e. administration to the subject) of the polynucleotides, wherein one or more polynucleotides encoding at least one nucleocapsid forming protein (M, N, S or E), or portions thereof, are administered initially, with a second administration of at least one polynucleotide encoding at least one additional nucleocapsid forming protein.
SARS-CoV Vaccine Compositions
As used herein, a "SARS-CoV vaccine composition" is a pharmaceutically acceptable composition that, when administered to a subject, exposes the subject to an immunogenic SARS-CoV compound, and induces the formation of an immune response against SARS-CoV. In one embodiment, the SARS-CoV vaccine composition will comprise the immunogenic SARS-CoV compound. In an alternative embodiment, the SARS-CoV vaccine composition will comprise one or more nucleic acid molecules that upon administration to the subject directs the synthesis or expression of an immunogenic SARS-CoV compound. In a prefeπed embodiment of the invention, the vaccine compositions of the present invention will comprise one or more nucleic acid molecules that encode at least a portion of the S protein, and most preferably at least a portion of the extracellular domain of the S protein. The nucleic acid molecules of such vaccme compositions may be single stranded or double-stranded, and may be circular or linear. Such molecules may be DNA or RNA, or composed of both DNA and RNA. The nucleic acid molecules of such vaccine compositions may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). Two or more such nucleic acid molecules can be present in a single construct, e.g., on a single plasmid, or in separate constructs, e.g., on separate plasmids. Such nucleic acid molecules will preferably additionally comprise a regulatory element such as a promoter, a transcription terminator, etc. or a secretory or other signal peptide or domain. Vaccination via the introduction of a nucleic acid molecule encoding the S protein reduces SARS-CoV replication by six orders of magnitude in the lungs of vaccinated subjects.
In one embodiment, the administration of such vaccine compositions will mediate the induction of neutralizing antibodies for SARS-CoV in a human or non- human animal (e.g., canine, feline, porcine, ovine, avine, equine, bovine, simian, etc.). The immune response induced is preferably, but need not be, clinically immunoprotective. As used herein an "immunoprotective" immune response is an immune response that reduces the growth, infectivity, pathogenicity, or viability of an infectious agent in a human or a non-human animal. A clinically immunoprotective immune response is an immunoprotective response that significantly attenuates, and more preferably prevents, the growth, infectivity, pathogenicity, or viability of an infectious agent in a human or a non-human animal.
In preferred embodiments, the methods and compositions of the present invention will serve to protect an uninfected subject so as to substantially prevent or attenuate symptoms or SARS-CoV infection. Such symptoms include, but are not limited to high fever (>38°C), dry cough, shortness of breath or breathing difficulties, pneumonia, headache, muscular stiffness, loss of appetite, malaise, confusion, rash and diarrhea.
In one embodiment, the methods and compositions of the invention will serve to prevent an individual from becoming infected with SARS-CoV. In another embodiment, the methods and compositions of the invention will serve to reduce the time frame for persistence of the viras in infected individuals (referred to herein as "persistence time"). For example, as is known in the art, the persistence time of the viras in infected individuals can be assayed in a number of different ways. For example, persistence time of the vims can be detected via the use of samples obtained from mouth, nose or throat swabs or from nasal washings. Samples may be analyzed for the presence of infectious particles using, for example, techniques that are described in the Examples herein. In preferred embodiments, vaccine compositions of the invention are effective in reducing the persistence time of the vims, as detected by any selected sample procedure and assay procedures, by about 15 %, 30%, 45%, 60%, 75% or 90% in vaccinated subjects as compared to unvaccinated control subjects.
Preferred SARS-CoV Svaccine compositions and immunogenic compounds of the present invention will elicit a robust immune response but will not possess an antibody-dependent vims enhancing activity. As used herein, an "antibody-dependent vims enhancing activity" with respect to a vaccine composition or an immunogenic compound, refers to the ability of a particular immunogenic compound or vaccine composition to elicit an antibody response that enhances cellular entry of any strain of the SARS-CoV viras, particularly strains of SARS-CoV that are isolated from animals, preferably non-human mammals, such as, for example, the Palm Civet (Paguma larvata), the raccoon dog, or the Chinese ferret badger. For any particular immunogenic compound or vaccine composition, antibody-dependent virus enhancing activity can be assessed, for examples, using the techniques described in Example 5 herein.
In one embodiment of the invention, vaccine compositions or immunogenic compounds that that do not possess an antibody depedent viras enhancing activity will comprise a DNA molecule that encodes the SARS-CoV S protein that is truncated at or near the juncture of the extracellular domain and the transmembrane domain (i.e. within 10 amino acids, preferably within 5 amino acids, upstream or downstream, of amino acid 1191 of SEQ ID NO:3), to result in cellular secretion of the extracellular domain portion, or a portion thereof, of the SARS-CoV S protein.
Special Considerations Relating to Nucleic Acid SARS-CoV Vaccine Compositions In one embodiment, the vaccme composition will comprise an RNA molecule that encodes an immunogenic SARS-CoV compound. Preferably in this embodiment, the RNA is in the form of messenger RNA (mRNA). Methods for introducing RNA sequences into vertebrate cells are described in U.S. Patent No. 5,580,859. Alternatively, the RNA may be in the form of an RNA vims genome. Preferably an RNA virus genome of the present invention is noninfectious, (i.e., does not result in the production of infectious viras particles in vertebrate cells). Suitable RNA viras genomes include, but are not limited to, alphaviras genomes, picornaviras genomes, and retroviras genomes. Methods for the in vivo introduction of non-infectious viral genomes to vertebrate tissues are well known to those of ordinary skill in the art and are described, e.g., in Altman-Hamamdzic et al. , 1997, EXPRESSION OF BETA-GALACTOSIDASE IN MOUSE BRAIN: UTILIZATION ' OF A NOVEL NONREPLICATΓVΈ SINDBIS VIRUS VECTOR As A NEURONAL GENE DELIVERY SYSTEM, Gene Ηierapy 4:815-822, in U.S. Patent No. 4,980,289, and in Miller et al. , 1993 , USE OF RETROVIRAL VECTORS FOR GENE TRANSFER AND EXPRESSION, Meth. Enzymol. 217:581-599. Viral replicons, i.e., non-infectious RNA viras genomes packaged in a viral coat, e.g., a picornaviras coat or an alphaviras coat, are also useful for efficient administration of RNA. See, e.g., U.S. Patents Nos. 5,766,602; 5,614,413, and PCT Publication No. WO 95/07994.
In a preferred embodiment, the vaccine composition will comprise a DNA molecule that encodes an immunogenic SARS-CoV compound. Such a molecule is typically a component of an expression vector. A typical expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from retrovimses, e.g., RSV, HTLVI, HIVI, MPSV and the immediate early promoter of the cytomegalovirus (CMV IEP). Suitable DNA viras genomes include heφesviras genomes, adenoviras genomes, adeno-associated vims genomes, and poxvims genomes. However, cellular elements can also be used (e.g., the human actin promoter, metallothionem promoter). In humans, CMV IEP is preferred. Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109), VR1051, VR1055, and pcDNA3 (Invitrogen, San Diego, Calif). In a preferred embodiment, such cytomegalovirus (CMV)-derived vectors, such as VRC8100, VRC 8103, VRC8106, VRC 8107, or VRC8108 are employed. Other preferred transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
All forms of DNA, whether replicating or non-replicating, preferably which do not become integrated into the genome, and which are expressible, are within the methods and compositions contemplated by the invention.
Promoters may be a cell-specific promoter and direct substantial transcription of the DNA only in predetermined cells or the promoter may not have cell type specificity. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the S polynucleotide or polynucleotide construct to direct cell-specific transcription. Preferably, a nucleic acid molecule of a SARS-CoV vaccine composition of the present invention is part of a circular or linearized plasmid, which is preferably non-infectious (i.e., does not result in the production of infectious vims particles in vertebrate cells), and noninitegrating (i.e., does not integrate into the genome of vertebrate cells). A linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease.
As used herein, the term "non-infectious" means that the vector is not capable of eliciting the production of infectious vims particles upon its entry into a recipient cell. Thus, a non-infectious vector containing a nucleic acid molecule of a SARS-CoV vaccine composition of the present invention can contain functional sequences from non-mammalian (e.g., viral or bacterial) species, but does not contain at least one viral sequence necessary for the production of infectious virion particles.
As used herein, the term "non-integrating" is intended to denote that the nucleic acid molecule of the SARS-CoV vaccine composition does not functionally integrate into the genome of recipient cells. The construct can, for example, be a non-replicating polynucleotide, or a specific replicating polynucleotide genetically engineered to lack the ability to mediate integration into the genome of a recipient cell. A non-integrating nucleic acid molecule lacks a functional sequence required for the integration of a polynucleotide into the genome of a recipient cell.
The choice of nucleic acid molecule form depends in part on the desired kinetics and duration of expression. When long-term expression of the vaccine composition is desired, DNA is preferred, especially DNA plasmids. Conversely, when short-term expression of the vaccine composition is desired, RNA is preferred, especially messenger RNA, since RNA is rapidly translated into polypeptide, but is degraded more quickly than DNA.
An "operable association" is achieved when a nucleic acid molecule encoding a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the molecule under the influence or control of the regulatory sequence(s). Two DNA molecules (such as an immunogenic SARS- CoV compound-encoding molecule and a promoter associated with the 5' end of such molecule) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA molecules does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with an immunogenic SARS-CoV compound- encoding molecule if the promoter is capable of effecting transcription of that molecule.
Special Considerations Relating to the Administration of Nucleic Acid Vaccine Compositions of the Present Invention Numerous methods for delivering nucleic acid molecules are known in the art. The most convenient way to delivery the polynucleotide constructs is in a plasmid (DNA) vector. Alternatively, a viral vector can be used. A number of viral based systems have been developed for transfecting mammalian cells. For example, nucleic acid molecules of the invention can be inserted into a vector and packaged as retroviral particles using techniques known in the art. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller et al., 1989, BioTechniques 7:980-990; Miller, 1990, Human Gene Therapy 1:5-14; and Burns et al., 1993, Proc. Natl. Acad. Sci. USA 90:8033-8037. A number of adenoviras vectors have also been described (Haj -Ahmad et al. (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;
Mittereder et al. (1994) Human Gene Therapy 5:717-729; and Rich et al. (1993) Human Gene Therapy 4:461-476). Additionally, various adeno-associated vims (AAV) vector systems have been developed. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801. Additional viral vectors, which will find use for delivering the recombinant nucleic acid molecules of the present invention, include, but are not limited to, those derived from the pox family of vimses, including vaccinia viras and avian poxvirus.
DNA vaccines and methods of their manufacture and delivery that may be used in accordance with the present invention are disclosed in US Patents Nos. 5,589,466; 5,620,896; 5,641,665; 5,703,055; 5,707,812; 5,846,946; 5,861,397; 5,891,718; 6,022,874; 6,147,055; 6,214,804; 6,228,844; 6,399,588; 6,413,942; 6,451,769, European Patent Documents EP 1165140A2; EP1006796A1 and EP0929536A1; and PCT Patent Publications WO00/57917; WO00/73263; WO01/09303; WO03/028632; W094/29469; WO95/29703; and W098/14439. Administration may be by needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., "gene guns" or pneumatic "needle less" injectors) Med-E-Jet (Vahlsing, H., et al. (1994) "IMMUNIZATION WITH PLASMID DNA USING A PNEUMATIC GUN," J. Immunol. Methods 171:11-22), Pigjet (Schrijver, R.S. et al. (1997) "IMMUNIZATION OF CATTLE WITH A BHV1 VECTOR VACCINE OR A DNA VACCINE BOTH CODING FOR THE G PROTEIN OF BRSV," Vaccine 15:1908-1916), Biojector (Davis, H.L. etal. (1994) "DIRECT GENE TRANSFER IN SKELETAL MUSCLE: PLASMID DNA-BASED IMMUNIZATION AGAINST THE HEPATITIS B VIRUS SURFACE ANTIGEN," Vaccine 12:1503-1509; Gramzinski, R., et al. (1998) "IMMUNE RESPONSE TO A HEPATITIS B DNA VACCINE IN AOTUS MONKEYS: A COMPARISON OF VACCINE FORMULATION, ROUTE, AND METHOD OF ADMINISTRATION," Mol Med 4: 109-118), AdvantaJet (Lindmayer, I., et al. (1986) "DEVELOPMENT OF NEW JET INJECTOR FOR INSULIN THERAPY," Diabetes Care 9:294-297), Medi-jector (Martins, J.K. et al. (1979) "MEDIJECTOR-A NEW METHOD OF CORTICOSTEROID-ANESTHETIC DELIVERY," J. Occup. Med. 21:821- 824), gelfoam sponge depots, other commercially available depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, use of polynucleotide coated suture (Qin, J.Y. et al. (1999) "GENE SUTURE~A NOVEL METHOD FOR INTRAMUSCULAR GENE TRANSFER AND ITS APPLICATION IN HYPERTENSION THERAPY," Life Sciences 65:2193-2203) or topical applications during surgery. The preferred modes of administration are intramuscular needle- based injection and pulmonary application via catheter infusion.
Thus, in one embodiment, administration is into muscle tissue, i.e., skeletal muscle, smooth muscle, or myocardium. Most preferably, the muscle is skeletal muscle. For polynucleotide constructs in which the polynucleotide or polynucleotide constract is DNA, the DNA can be operably linked to a cell- specific promoter that directs substantial transcription of the DNA only in predetermined cells. In certain embodiments, a polynucleotide construct, or composition comprising a polynucleotide or polynucleotide construct, is delivered to any tissue including, but not limited to those disclosed herein, such that the polynucleotide or polynucleotide construct is free from association with liposomal formulations and charged lipids. Alternatively, the polynucleotide, polynucleotide construct, or composition is delivered to a tissue other than brain or nervous system tissue, for example, to muscle, skin, or blood, in any composition as described herein. Preferably, the vaccine composition is delivered to the interstitial space of a tissue. "Interstitial space" comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels.
The DNA vaccine, preferably in the form of plasmid DNA, may be administered (especially by injection) into tissue and voltage pulses applied between electrodes disposed in the tissue, thus applying electric fields to cells of the tissue. The electrically-mediated enhancement covers administration using either iontophoresis or electroporation in vivo. Suitable techniques of electroporation and iontophoresis are provided by Singh, J. et al. (1989) "TRANSDERMAL DELIVERY OF DRUGS BY IONTOPHORESIS: A REVIEW," Drug Des. Deliv. 4:1-12; Theiss, U. et al. (1991) "IONTOPHORESIS-IS THERE A FUTURE FOR CLINICAL APPLICATION?," Methods Find. Exp. Clin. Pharmacol. 13:353-359; Singh and Maibach (1993) "TOPICAL IONTOPHORETIC DRUG DELIVERY IN VIVO: HISTORICAL DEVELOPMENT, DEVICES AND FUTURE PERSPECTIVES," Dermatology. 187:235-238; Singh, P. et al. (1994) "IONTOPHORESIS IN DRUG DELIVERY: BASIC PRINCIPLES AND APPLICATIONS," Crit. Rev. Ther. Drag Carrier Syst. 11:161-213; Su, Y. et al. (1994) "SPHINGOSINE 1 -PHOSPHATE, A NOVEL SIGNALING MOLECULE, STIMULATES DNA BINDING ACTIVITY OF AP- 1 IN QUIESCENT SWISS 3T3 FIBROBLASTS," J. Pharm. Sci. 83:12-17; Costello, CT. etal. (1995) "IONTOPHORESIS: APPLICATIONS IN TRANSDERMAL MEDICATION DELIVERY," Phys. Ther. 75:554-563; Howard, J.p. et al. (1995) "EFFECTS OF ALTERNATING CURRENT IONTOPHORESIS ON DRUG DELIVERY," Arch. Phys. Med. Rehabil. 76:463-466; Kassan, D.G. et al. (1996) "PHYSICAL ENHANCEMENT OF DERMATOLOGIC DRUG DELIVERY: IONTOPHORESIS AND PHONOPHORESIS," J. Amer. Acad. Dermatol. 34:657-666; Riviere et al. (1997) "ELECTRICALLY-ASSISTED TRANSDERMAL DRUG DELIVERY," Pharm. Res. 14:687-697; Zempsky, W.T. et al. (1998) "IONTOPHORESIS: NONINVASIVE DRUG DELIVERY," Amer. J. Anesthesiol. 25:158- 162; Muramatsu, T. et al. (1998) "IN Vivo ELECTROPORATION: A POWERFUL AND CONVENIENT MEANS OF NONVIRAL GENE TRANSFER To TISSUES OF LIVING ANIMALS," Int. J. Mol. Med. 1:55-62; Garrison J. (1998) "IONTOPHORESIS: AN ALTERNATIVE DRUG-DELIVERY SYSTEM," Med. Device Technol. 9:32-36; Banga A.K. et al. (1998) "ASSESSING THE POTENTIAL OF SKIN ELECTROPORATION FOR THE DELIVERY OF PROTEIN- AND GENE-BASED DRUGS," Trends Biotechnol. 16:408-412; Banga A.K. etal. (1999) "IONTOPHORESIS AND ELECTROPORATION: COMPARISONS AND CONTRASTS," Int. J. Pharm. 179:1-19; Neumann E. et al. (1999) "FUNDAMENTALS OF ELECTROPORATIVE DELIVERY OF DRUGS AND GENES," Bioelectiochem. Bioenerg. 48:3-16; and Heiser, W.C. (2000)
"OPTIMIZING ELECTROPORATION CONDITIONS FOR THE TRANSFORMATION OF MAMMALIAN CELLS," Methods Mol. Biol. 130: 117-134. The nature of the electric field generated in accordance with such methods is determined by the nature of the tissue, the size of the selected tissue and its location. The use of insufficient or excessive field strength is to be avoided. As used herein, a field strength is excessive if it results in the lysing of cells. A field strength is insufficient if it results in a reduction of efficacy of 90% relative to the maximum efficacy obtainable. The electrodes may be mounted and manipulated in many ways known in the art. The waveform of the electrical signal provided by the pulse generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train or a bipolar oscillating pulse train. The waveform, electric field strength and pulse duration are dependent upon the type of cells and the DNA that are to enter the cells via electrical-mediated delivery and thus are determined by those skilled in the art in consideration of these criteria. Any number of known devices may be used for delivering the DNA vaccine and generating the desired electric field. Examples of suitable devices include, but are not limited to, a single needle probe, a bipolar probe and a combination needle and plate probe. Alternatively, methods such as continuous-flow electroporation may be employed (See, U.S. Patents Nos. 6,485,961; 6,090,617; 6,074,605; 5,720,921; 5,612,207; and 5,098,843).
Nucleic acid molecules of the present invention, e.g., plasmid DNA, ' derivatives of plasmid DNA, mRNA, linear DNA, viral genomes, or polynucleotide fragments contained therein may be formulated into any of the various compositions and may be used in any of the methods disclosed herein. For aqueous compositions used in vivo, use of sterile pyrogen-free water is preferred. Such formulations will contain an effective amount of a polynucleotide or polynucleotide constract together with a suitable salt and/or auxiliary agent as disclosed herein, in order to prepare pharmaceutically acceptable compositions suitable for optimal administration to a vertebrate. Insoluble polynucleotides or polynucleotide constructs may be solubilized in a weak acid or weak base, and then diluted to the desired volume, for example, with an aqueous solution of the present invention. The pH of the solution may be adjusted as appropriate. In addition, a pharmaceutically acceptable additive can be used to provide an appropriate osmolarity.
The compositions of the present invention may include one or more transfection facilitating materials that facilitate delivery of polynucleotides or polynucleotide constructs to the interior of a cell, and/or to a desired location within a cell. Examples of the transfection facilitating materials include, but are not limited to lipids, preferably cationic lipids; inorganic materials such as calcium phosphate, and metal (e.g., gold or tungsten) particles (e.g., "powder" type delivery solutions); peptides, including cationic peptides, targeting peptides for selective delivery to certain cells or intracellular organelles such as the nucleus or nucleolus, and amphipathic peptides, i.e. helix forming or pore forming peptides; basic proteins, such as histories; asialoproteins; viral proteins (e.g., Sendai vims coat protein); pore-forming proteins; and polymers, including dendrimers, star- polymers, "homogenous" poly-amino acids (e.g., poly-lysine, poly-arginine), "heterogeneous" poly-amino acids (e.g., mixtures of lysine & glycine), copolymers, polyvinylpyrcolidinone (PVP), and polyethylene glycol (PEG). Furthermore, those auxiliary agents of the present invention that facilitate and enhance the entry of a polynucleotide or polynucleotide construct into vertebrate cells in vivo, may also be considered "transfection facilitating materials." Certain embodiments of the present invention may include lipids as a transfection facilitating material, including cationic lipids (e.g., DMRIE, DOSPA, DC-Choi, GAP-DLRIE), basic lipids (e.g., steryl amine), neutral lipids (e.g., cholesterol), anionic lipids (e.g., phosphatidyl serine), and zwitterionic lipids (e.g., DOPE, DOPC). Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide
(DOGS) and dipalmitoyl-phophatidylethanolamine-5-carboxy- spermylamide (DPPES). Cationic cholesterol derivatives are also useful, including {3.beta.-[N- N',N'-dimethylamino)ethane]-carbomoyl}-cholesterol (DC-Chol). Dimethyldioctdecyl-ammonium bromide (DDAB), N-(3-aminopropyl)-N,N-(bis- (2-tetradecyloxyethyl))-N-methyl-ammonium bromide (PADEMO), N-(3- aminopropyl)-N,N-(bis-(2-dodecyloxyethyl))-N-methy- l-ammonium bromide (PADELO), N,N,N-tris-(2-dodecyloxy)ethyl-N-(3-amino)pro- pyl-ammonium bromide (PATELO), and N.sup.l-(3-ammopropyl)((2-dodecyloxy)e- thyl)- N.sup.2-(2-dodecyloxy)ethyl-l-piperazinaminium bromide (GALOE-BP) can also be employed in the present invention.
Non-diether cationic lipids, such as DL-l,2-dioleoyl-3-dimethylamin- opropyl-.beta.-hydroxyethylammonium (DORI diester), l-0-oleyl-2-oleoyl-3- dimethylaminopropyl-.beta.-hydroxyethylammonium (DORI ester/ether), and their salts promote in vivo gene delivery. Preferred cationic lipids comprise groups attached via a heteroatom attached to the quaternary ammonium moiety in the head group. A glycyl spacer can connect the linker to the hydroxyl group.
Cationic lipids for use in certain embodiments of the present invention include DMRIE ((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2- ,3-bis(tetradecyloxy)-l- propanaminium bromide), and GAP-DMORIE ((+)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-l-pro- panaminium bromide), as well as (±)-N,N-dimethyl-N-[2-(sperminecarboxamido)et- hyl]-2,3-bis(dioleyloxy)-l- propaniminium pentahydrochloride (DOSPA), (±)-N-(2-aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanimini- um bromide (.beta.-aminoethyl- DMRIE or .beta.AE-DMRIE) (Wheeler, et al., Biochim. Biophys. Acta 1280:1-11 (1996)), and (±)-N-(3-aminoρropyl)-N,- N-dimethyl-2,3-bis(dodecyloxy)-l- propaniminium bromide (GAP-DLRIE) (Wheeler, et al., Proc. Natl. Acad. Sci. USA 93:11454-11459 (1996)), which have been developed from DMRIE. Other examples of DMRIE-derived cationic lipids that are useful for the present invention are (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-decyloxy)-l- propanaminium bromide (GAP-DDRIE), (±)-N-(3-aminopropyl)-N,- N-dimethyl- 2,3 -(bis-tettadecyloxy)-l -propanaminium bromide (GAP-DMRIE), (±)-N-((N"- methyl)-N'-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GMU-DMRIE), (±)-N-(2-hydrpxyethyl)-N,N-dimeth- yl-2,3- bis(dodecyloxy)-l -propanaminium bromide (DLRIE), and (±)-N-(2-hydroxyethyl)- N,N-dimethyl-2,3-bis-([Z]-9-octadecenyloxy)prop- yl-1-propaniminium bromide (HP-DORIE). A cationic lipid that may be used in concert with the vaccine compositions of the present invention is a "cytofectin." As used herein, a "cytofectin" refers to a subset of cationic lipids that incorporate certain structural features including, but not limited to, a quaternary ammonium group and/or a hydrophobic region (usually with two or more alkyl chains), but which do not require amine protonation to develop a positive charge. Examples of cytofectins may be found, for example, in U.S. Patent No. 5,861,397. Cytofectins that may be used in the present invention, include DMRIE ((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- pr- opanaminium bromide), GAP-DMORIE ((±)-N-(3-aminopropyl)-N,N- dimethyl-2,- 3 -bis(syn-9-tetradeceneyloxy)-l -propanaminium bromide), and GAP- DLRIE ((±)-N-(3-aminoproρyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-l- propanamini- um bromide).
The cationic lipid may be mixed with one or more co-lipids. The term "co- lipid" refers to any hydrophobic material that may be combined with the cationic lipid component and includes amphipathic lipids, such as phospholipids, and neutral lipids, such as cholesterol. Cationic lipids and co-lipids may be mixed or combined in a number of ways to produce a variety of non-covalently bonded macroscopic stractures, including, for example, liposomes, multilamellar vesicles, unilamellar vesicles, micelles, and simple films. A prefeπed class of co-lipids is the zwitterionic phospholipids, which include the phosphatidylethanolamines and the phosphatidylcholines. Most preferably, the co-lipids are phosphatidylethanolamines, such as, for example, DOPE, DMPE and DPyPE. DOPE and DPyPE are particularly preferred. For immunization, the most preferred co-lipid is DPyPE, which comprises two phytanoyl substituents incoφorated into the diacylphosphatidylethano- lamine skeleton. The cationic lipidxo-lipid molar ratio may range from about 9: 1 to about 1 :9, or from about 4: 1 to about 1 :4, or from about 2: 1 to about 1 :2, or about 1:1. In order to maximize homogeneity, such cationic lipid and co-lipid components may be dissolved in a solvent such as chloroform, followed by evaporation of the cationic lipid/co-lipid solution under vacuum to dryness as a film on the inner surface of a glass vessel (e.g., a Rotovap round-bottomed flask). Upon suspension in an aqueous solvent, the amphipathic lipid component molecules self-assemble into homogenous lipid vesicles. These lipid vesicles may subsequently be processed to have a selected mean diameter of uniform size prior to combining with, for example, plasmid DNA according to methods known to those skilled in the art. For example, the sonication of a lipid solution is described in Feigner, P.L., et al. (1987) "LIPOFECTION: A HIGHLY EFFICIENT, LIPID-MEDIATED DNA-TRANSFECTION PROCEDURE," Proc Natl. Acad. Sci. USA 84:7413-7417 and in U.S. Patent No. 5,264,618.
In the embodiments, the polynucleotide or polynucleotide constructs) are combined with lipids by mixing, for example, a plasmid DNA solution and a solution of cationic lipid:co-lipid liposomes. Preferably, the concentration of each of the constituent solutions is adjusted prior to mixing such that the desired final plasmid DNA/cationic lipid: co-lipid ratio and the desired plasmid DNA final concentration will be obtained upon mixing the two solutions. For example, if the desired final solution is to be 2.5 mM sodium phosphate, the various components of the composition, e.g., plasmid DNA, cationic lipid: co-lipid liposomes, and any other desired auxiliary agents, transfection facilitating materials, or additives are each prepared in 2.5 mM sodium phosphate and then simply mixed to afford the desired complex. Alternatively, if the desired final solution is to be, e.g., 2.5 mM sodium phosphate, certain components of the composition, e.g., the auxiliary agent and/or cationic lipid:co-lipid liposomes, is prepared in a volume of water which is less than that of the final volume of the composition, and certain other components of the composition, e.g., the plasmid DNA, is prepared in a solution of sodium phosphate at a higher concentration than 2.5 mM, in a volume such that when the components in water are added to the components in the sodium phosphate solution, the final composition is in an aqueous solution of 2.5 mM sodium phosphate. For example, the plasmid DNA could be prepared in 5.0 mM sodium phosphate at one half the final volume, the auxiliary agent and/or cationic lipidxo- lipid liposome is prepared in water at one half the final volume, and then these two elements are mixed together to produce the final composition. The cationic lipid: co-lipid liposomes are preferably prepared by hydrating a thin film of the mixed lipid materials in an appropriate volume of aqueous solvent by vortex mixing at ambient temperatures for about 1 minute. The thin films are prepared by admixing chloroform solutions of the individual components to afford a desired molar solute ratio followed by aliquoting the desired volume of the solutions into a suitable container. The solvent is removed by evaporation, first with a stream of dry, inert gas (e.g. argon) followed by high vacuum treatment.
A transfection facilitating material can be used alone or in combination with one or more other transfection facilitating materials. Two or more transfection facilitating materials can be combined by chemical bonding (e.g., covalent and ionic such as in lipidated polylysine, PEGylated polylysine) (Toncheva, V., et al. (1998) "NOVEL VECTORS FOR GENE DELIVERY FORMED BY SELF-ASSEMBLY OF DNA WITH POLY(L-LYSINE) GRAFTED WITH HYDROPHILIC POLYMERS," Biochim. Biophys. Acta 1380(3):354-368), mechanical mixing (e.g., free moving materials in liquid or solid phase such as "polylysine+cationic lipids") (Gao, X. et al. (1996) "POTENTIATION OF CATIONIC LIPOSOME-MEDIATED GENE DELIVERY BY POLYCATIONS," Biochemistry 35:1027-1036); Trabetskoy, V.S., et al. (1992) "CATIONIC LIPOSOMES ENHANCE TARGETED DELIVERY AND EXPRESSION OF EXOGENOUS DNA MEDIATED BY N-TERMINAL MODIFIED POLY(L-LYSINE)- ANTIBODY CONJUGATE IN MOUSE LUNG ENDOTHELIAL CELLS," Biochem. Biophys. Acta 1131:311-313), and aggregation (e.g., co-precipitation, gel forming such as in cationic lipids+poly-lactide co-galactide, and polylysine+gelatin).
Other hydrophobic and amphiphilic additives, such as, for example, sterols, fatty acids, gangliosides, glycolipids, lipopeptides, liposaccharides, neobees, niosomes, prostaglandins and sphingolipids, may also be included in the compositions of the present invention. In such compositions, these additives may be included in an amount between about 0.1 mol % and about 99.9 mol % (relative to total lipid). Preferably, these additives comprise about 1-50 mol % and, most preferably, about 2-25 mol %. Preferred additives include lipopeptides, liposaccharides and steroids. General Considerations Relating to the Administration of the Vaccine Compositions of the Present Invention The SARS-CoV vaccine compositions of the present invention can be administered either prior to infection with SARS-CoV or after infection with SARS-CoV. For the methods of the present invention, a vaccme composition may comprise multiple copies of a single polynucleotide constract or several different polynucleotide constructs. Additionally, when the vaccine composition comprises immunogenic polypeptides, the composition may comprise multiple copies of a single immunogenic polypeptide, or several different immunogenic polypeptides. Additionally, the vaccine composition may comprise a combination of polynucleotide constructs and immunogenic polypeptides. It is also contemplated as part of the invention that vaccine compositions of the invention may be coadministered or sequentially administered, and administered in conjunction with other vaccine compositions. It is also contemplated as an aspect of this invention that the nucleic acid molecules may be introduced ex vivo into cells, which cells are then introduced into the subject to be vaccinated. In preferred embodiments of ex vivo administration, the cells are derived from the subject to be vaccinated. As used herein, "ex vivo" cells are cells into which the polynucleotide construct is introduced, for example, by transfection, lipofection, electroporation, bombardment, or microinjection. The cells contaimng the polynucleotide constract are then administered in vivo into mammalian tissue (see, for example, see Belldegrun, A., et al. (1993) "HUMAN RENAL CARCINOMA LINE TRANSFECTED WITH INTERLEUKIN-2 AND/OR INTERFERON ALPHA GENE(S): IMPLICATIONS FOR LIVE CANCER VACCINES," J. Natl. Cancer Inst. 85: 207-216; Ferrantini, M. et al. ( 1993) "ALPHA 1 -INTERFERON GENE TRANSFER INTO METASTATIC FRIEND LEUKEMIA CELLS ABROGATED TUMORIGENICITY IN IMMUNOCOMPETENT MICE: ANTITUMOR THERAPY BY MEANS OF INTERFERON-PRODUCING CELLS," Cancer Research 53:1107-1112; Ferrantini, M. et al. (1994) "IFN-ALPHA 1 GENE EXPRESSION INTO A METASTATIC MURINE ADENOCARCINOMA (TS/A) RESULTS IN CD8+ T CELL-MEDIATED TUMOR REJECTION AND DEVELOPMENT OF ANTITUMOR IMMUNITY. COMPARATIVE STUDIES WITH IFN-GAMMA-PRODUCING TS/A CELLS," J. Immunology 153:4604-4615; Kaido, T. et al. (1995) "IFN-ALPHA 1 GENE TRANSFECTION COMPLETELY ABOLISHES THE TUMOPJGENICITY OF MURINE B 16 MELANOMA CELLS IN ALLOGENEIC DBA/2 MICE AND DECREASES THEIR TUMORIGENICITY IN SYNGENEIC C57BL/6 MICE," Int. J. Cancer 60: 221-229; Ogura, H. et al. (1990) "IMPLANTATION OF GENETICALLY MANIPULATED FIBROBLASTS INTO MICE AS ANTITUMOR ALPHA-INTERFERON THERAPY," Cancer Research 50:5102-5106; Santodonato, L. et al. (1996) "CURE OF MICE WITH ESTABLISHED METASTATIC FRIEND LEUKEMIA CELL TUMORS BY A COMBINED THERAPY WITH TUMOR CELLS EXPRESSING BOTH INTERFERON-ALPHA 1 AND HERPES SIMPLEX THYMIDINE KINASE FOLLOWED BY GANCICLOVTR," Human Gene Therapy 7: 1-10; Santodonato, L., et al. (1997) "LOCAL AND SYSTEMIC ANTITUMOR RESPONSE AFTER COMBINED THERAPY OF MOUSE METASTATIC TUMORS WITH TUMOR CELLS EXPRESSING IFN-ALPHA AND HSVTK: PERSPECTIVES FOR THE GENERATION OF CANCER VACCINES," Gene Therapy 4: 1246-1255; and Zhang, J.F. et al. (1996) "GENE THERAPY WITH AN ADENO-ASSOCIATED VIRUS CARRYING AN INTERFERON GENE RESULTS IN TUMOR GROWTH SUPPRESSION AND REGRESSION," Cancer Gene Therapy 3:31-38.
In the "local delivery" embodiment of the present invention, a vaccine composition is administered in vivo, such that the vaccine is incoφorated into the local cells at the site of administration. The local cells subsequently express the immunogenic compound in an amount sufficient to provide an immunoprotective effect against SARS-CoV. The vaccine composition can be administered either within ex vivo cells or free of ex vivo cells or ex vivo cellular material. Preferably, the polynucleotide construct is administered free of ex vivo cells or ex vivo cellular material. The vaccines of the present invention may thus be administered by inhalation, or interdermally, intracavity (e.g., oral, vaginal, rectal, nasal, peritoneal, ventricular, or intestinal), intradermally, intramuscularly, intranasally, intraocularly, intraperitoneally, intrarectally, intratracheally, intravenously, orally, subcutaneously, transdermally, or transmucosally (i.e., across a mucous membrane) in a dose effective for the production of neutralizing antibody and resulting in protection from infection or disease. The present vaccine can generally be administered in the form of a spray for intranasal administration, or by nose drops, inhalants, swabs on tonsils, or a capsule, liquid, suspension or elixirs for oral administration. The vaccine may be in the form of single dose preparations or in multi-dose flasks, which can be used for mass vaccination programs. Reference is made to Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980); and New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), for methods of preparing and using vaccines.
Any mode of administration can be used so long as the mode results in the expression of SARS-CoV immunogenic compounds in the desired tissue, in an amount sufficient to be detectable, and/or prophylactically or therapeutically effective. Methods to detect such a response include serological methods to detect the polypeptide in semm, e.g., western blotting, staining tissue sections by immunohistochemical methods, measuring an immune response generated by the mammalian against the polypeptide, and measuring the activity of the polypeptide.
The vaccine compositions of the present invention can be lyophilized to produce a vaccine composition in a dried form for ease in transportation and storage. The vaccine compositions of the present invention may be stored in a sealed vial, ampule or the like. In the case where the vaccine is in a dried form, the vaccine is dissolved or suspended (e.g., in sterilized distilled water) before administration. An inert carrier such as saline or phosphate buffered saline or any such carrier in which the vaccine composition has suitable solubility, may be used.
Further, the vaccine composition may be prepared in the form of a mixed vaccine that contains one or more additional antigens so long as such additional antigens do not interfere with the effectiveness of the SARS-CoV vaccine of the invention, and the side effects and adverse reactions are not increased additively or synergistically. The vaccine compositions of the present invention can be associated with chemical moieties, which may improve the vaccine's solubility, absoφtion, biological half-life, etc. The moieties may alternatively decrease the toxicity of the vaccine, eliminate or attenuate any undesirable side effect of the vaccine, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.
The immunogenic SARS-CoV vaccine compositions of the invention can be formulated according to known methods for preparing pharmaceutical compositions, whereby the substance to be delivered is combined with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their preparation are described, for example, in Remington's Pharmaceutical Sciences, 16.th Edition, A. Osol, Ed., Mack Publishing Co., Easton, Pa. (1980), and Remington's Pharmaceutical Sciences, 19.sup.th Edition, A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995).
The compositions of the present invention may be in the form of an emulsion, gel, solution, suspension, etc. In addition, the pharmaceutical composition can also contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Administration of pharmaceutically acceptable salts of the polynucleotides described herein is preferred. Such salts can be prepared from pharmaceutically acceptable non-toxic bases including organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, basic amino acids, and the like.
The amount of polypeptides or nucleic acid molecules in a vaccine composition of the present invention depends on many factors, including the age and weight of the subject, the delivery method and route, the type of treatment desired, and the type of nucleic acid molecule being administered. The optimal amount of polypeptides or nucleic acid molecules of the invention that should be administered to a subject to induce a protective immune response or neutralizing antibodies in a subject can be readily determined by one of skill in the art. In general, a composition of the present invention containing nucleic acids contains from about 1 ng to about 30 mg of an immunogenic SARS-CoV nucleic acid molecule or polynucleotide constract, more preferably, from about 100 ng to about 10 mg of an immunogenic SARS-CoV nucleic acid molecule or polynucleotide construct. Certain preferred compositions of the present invention may include about 1 ng of an immunogenic SARS-CoV nucleic acid molecule, about 5 ng of an immunogenic SARS-CoV nucleic acid molecule, about 10 ng of an immunogenic SARS-CoV nucleic acid molecule, about 50 ng of an immunogenic SARS-CoV nucleic acid molecule, about 100 ng of an immunogenic SARS-CoV nucleic acid molecule, about 500 ng of an immunogenic SARS-CoV nucleic acid molecule, about 1 μg of an immunogenic SARS-CoV nucleic acid molecule, about 5 μg of an immunogenic SARS-CoV nucleic acid molecule, about 10 μg of an immunogenic SARS-CoV nucleic acid molecule, about 50 μg of an immunogenic SARS-CoV nucleic acid molecule, about 100 μg of an immunogenic SARS-CoV nucleic acid molecule, about 150 μg of an immunogenic SARS-CoV nucleic acid molecule, about 200 μg of an immunogenic SARS-CoV nucleic acid molecule, about 250 μg of a polynucleo-tide, about 300 μg of an immunogenic SARS-CoV nucleic acid molecule, about 350 μg of an immunogenic SARS-CoV nucleic acid molecule, about 400 μg of an immunogenic SARS-CoV nucleic acid molecule, about 450 μg of an immunogenic SARS-CoV nucleic acid molecule, about 500 μg of an immunogenic SARS-CoV nucleic acid molecule, about 550 μg of an immunogenic SARS-CoV nucleic acid molecule, about 600 μg of a polynucleo-tide, about 650 μg of an immunogenic SARS-CoV nucleic acid molecule, about 700 μg of an immunogenic SARS-CoV nucleic acid molecule, about 750 μg of an immunogenic SARS-CoV nucleic acid molecule, about 800 μg of an immunogenic SARS-CoV nucleic acid molecule, about 850 μg of an immunogenic SARS-CoV nucleic acid molecule, about 900 μg of an immunogenic SARS-CoV nucleic acid molecule, about 950 μg of an immunogenic SARS-CoV nucleic acid molecule, about 1 mg of an immunogenic SARS-CoV nucleic acid molecule, about 5 mg of an immunogenic SARS-CoV nucleic acid molecule, about 10 mg of an immunogenic SARS-CoV nucleic acid molecule, about 15 mg of an immunogenic SARS-CoV nucleic acid molecule, about 20 mg of an immunogenic SARS-CoV nucleic acid molecule, about 25 mg of an immunogenic SARS-CoV nucleic acid molecule, and about 30 mg of an immunogenic SARS-CoV nucleic acid molecule.
Likewise, a composition of the present invention containing an immunogenic SARS-CoV polypeptide contains from about 1 ng to about 30 mg of an immunogenic SARS-CoV polypeptide, more preferably, from about 100 ng to about 10 mg of an immunogenic SARS-CoV polypeptide. Certain preferred compositions of the present invention may include about 1 ng of an immunogenic SARS-CoV polypeptide, about 5 ng of an immunogenic SARS-CoV polypeptide, about 10 ng of an immunogenic SARS-CoV polypeptide, about 50 ng of an immunogenic SARS-CoV polypeptide, about 100 ng of an immunogenic SARS- CoV polypeptide, about 500 ng of an immunogenic SARS-CoV polypeptide, about 1 μg of an immunogenic SARS-CoV polypeptide, about 5 μg of an immunogenic SARS-CoV polypeptide, about 10 μg of an immunogenic SARS-CoV polypeptide, about 50 μg of an immunogenic SARS-CoV polypeptide, about 100 μg of an immunogenic SARS-CoV polypeptide, about 150 μg of an immunogenic SARS- CoV polypeptide, about 200 μg of an immunogenic SARS-CoV polypeptide, about 250 μg of an immunogenic SARS-CoV polypeptide, about 300 μg of an immunogenic SARS-CoV polypeptide, about 350 μg of an immunogenic SARS- CoV polypeptide, about 400 μg of an immunogenic SARS-CoV polypeptide, about 450 μg of an immunogenic SARS-CoV polypeptide, about 500 μg of an immunogenic SARS-CoV polypeptide, about 550 μg of an immunogenic SARS- CoV polypeptide, about 600 μg of an immunogenic SARS-CoV polypeptide, about 650 μg of an immunogenic SARS-CoV polypeptide, about 700 μg of an immunogenic SARS-CoV polypeptide, about 750 μg of an immunogenic SARS- CoV polypeptide, about 800 μg of an immunogenic SARS-CoV polypeptide, about 850 μg of an immunogenic SARS-CoV polypeptide, about 900 μg of an immunogenic SARS-CoV polypeptide, about 950 μg of an immunogenic SARS- CoV polypeptide, about 1 mg of an immunogenic SARS-CoV polypeptide, about 5 mg of an immunogenic SARS-CoV polypeptide, about 10 mg of an immunogenic SARS-CoV polypeptide, about 15 mg of an immunogenic SARS-CoV polypeptide, about 20 mg of an immunogenic SARS-CoV polypeptide, about 25 mg of an immunogenic SARS-CoV polypeptide, and about 30 mg of an immunogenic SARS-CoV polypeptide.
The vaccine compositions of the present invention may be used in concert with adjuvants and other compounds to enhance their immunologic effect.
Adjuvants that enhance production of SARS-CoV-specifϊc antibodies include, but are not limited to, various oil formulations such as stearyl tyrosine (ST, see U.S. Patent No. 4,258,029), the dipeptide MDP, saponin, aluminum hydroxide, and lymphatic cytokine. Mucosal adjuvants include cholera toxin B subunit (CTB), a heat labile enterotoxin (LT) from E. coli, and Emulsomes (Pharmos, LTD.,
Rehovot, Israel). The adjuvant alum (aluminum hydroxide) or ST may be used for administration to humans.
For aqueous pharmaceutical compositions used in vivo, sterile pyrogen-free water is preferred. Such formulations will contain an effective amount of the substance together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions suitable for administration to a human or animal. Insoluble polynucleotides or polynucleotide constructs may be solubilized in a weak acid or weak base, and then diluted to the desired volume, for example, with an aqueous solution of the present invention. The pH of the solution may be adjusted as appropriate. In addition, a pharmaceutically acceptable additive can be used to provide an appropriate osmolarity.
As used herein a "salt" is a substance produced from the reaction between acids and bases, which comprises a metal (cation) and a nonmetal (anion). Salt crystals may be "hydrated" i.e., contain one or more water molecules. Such hydrated salts, when dissolved in an aqueous solution at a certain molar concentration, are equivalent to the corresponding anhydrous salt dissolved in an aqueous solution at the same molar concentration. For the present invention, salts that are readily soluble in an aqueous solution are preferred. The terms "saline" or "normal saline" as used herein refer to an aqueous solution of about 145 mM to about 155 mM sodium chloride, preferably about 154 mM sodium chloride. The terms "phosphate buffered saline" or PBS" refer to an aqueous solution of about 145 mM to about 155 mM sodium chloride, preferably about 154 sodium chloride, and about 10 mM sodium phosphate, at a pH ranging from about 6.0 to 8.0, preferably at a pH ranging from about 6.5 to about 7.5; most preferably at pH 7.2.
Certain embodiments of the present invention are drawn to vaccme compositions comprising nucleic acid molecules dissolved in a salt solution which improves entry of the polynucleotide or polynucleotide construct into vertebrate cells in vivo. Preferred salts in which to dissolve a polynucleotide or polynucleotide construct of the present invention include but are not limited to sodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium pyruvate, potassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium pyruvate, disodium DL-.alpha.-glycerol-phosphate, and disodium glucose-6-phosphate. "Phosphate" salts of sodium or potassium can be either the monobasic form, e.g., NaHP04, or the dibasic form, e.g., Na2HP04, but a mixture of the two, resulting in a desired pH, is most preferred. The most prefeπed salts are sodium phosphate or potassium phosphate. As used herein, the terms "sodium phosphate" or "potassium phosphate," refer to a mixture of the dibasic and monobasic forms of each salt to present at a given pH.
Salts of the present invention are preferably dissolved in aqueous solution at concentrations which enhance entry of nucleic acids of the invention into mammalian cells in vivo, and/or enhance polypeptide expression, relative to saline, PBS, or water. For example, in certain embodiments, nucleic acid molecules of the invention are dissolved in a salt solution of about 150 mM NaHP04, Na2HP04, or NaHC03.
Additional embodiments of the present invention are drawn to vaccine compositions comprising an immunogenic nucleic acid molecules or polypeptides of the invention and an auxiliary agent. The present invention is further drawn to methods to use such compositions, methods to make such compositions, and pharmaceutical kits. As used herein, an "auxiliary agent" is a substance mcluded in a composition for its ability to enhance, relative to a composition which is identical except for the inclusion of the auxiliary agent, the entry of polynucleotides or polynucleotide constructs into vertebrate cells in vivo, and/or the in vivo expression of polypeptides encoded by such polynucleotides or polynucleotide constructs. Auxiliary agents of the present invention include nonionic, anionic, cationic, or zwitterionic surfactant or detergents, with nonionic, anionic, cationic, or zwitterionic surfactant or detergents, with nonionic surfactant or detergents being preferred, chelators, Dnase inhibitors, agents that aggregate or condense nucleic acids, emulsifying or solubilizing agents, wetting agents, gel- forming agents, and buffers.
Suitable auxiliary agents include non-ionic detergents and surfactant such as poloxamers. Poloxamers are a series of non-ionic surfactant that are block copolymers of ethylene oxide and propylene oxide. The poly(oxyethylene) segment is hydrophilic and the poly(oxypropylene) segment is hydrophobic. The physical forms are liquids, pastes or solids. The molecular weight ranges from 1000 to greater than 16000. The basic structure of a poloxamer is HO-- [CH2CH20]x-[CH2CHO(CH3)]y~[CH2CH20]x-H, where x and y represent repeating units of ethylene oxide and propylene oxide respectively. Thus, the propylene oxide (PO) segment is sandwiched between two ethylene oxide (EO) segments, (EO--PO--EO). The number of x's and y's distinguishes individual poloxamers. If the ethylene oxide segment is sandwiched between two propylene oxide segments, (PO--EO--PO), then the resulting stracture is a reverse poloxamer. The basic stracture of a reverse poloxamer is HO~[CH(CH3)CH20)]x~ [CH2CH20]y~[CH2C-HO(CH3)]x~H.
Poloxamers that may be used in concert with the methods and compositions of the present invention include, but are not limited to commercially available poloxamers such as Pluronic L121 (avg. MW:4400), Pluronic L101 (avg. MW:3800), Pluronic L81 (avg. MW:2750), Pluronic L61 (avg. MW:2000), Pluronic L31 (avg. MW: 1100), Pluronic L122 (avg. MW:5000), Pluronic L92 (avg. MW:3650), Pluronic L72 (avg. MW:2750), Pluronic L62 (avg. MW:2500), Pluronic L42 (avg. MW:1630), Pluronic L63 (avg. MW:2650), Pluronic L43 (avg. MW: 1850), Pluronic L64 (avg. MW:2900), Pluronic L44 (avg. MW:2200), Pluronic L35 (avg. MW:1900), Pluronic P123 (avg. MW:5750), Pluronic P103 (avg. MW:4950), Pluronic P104 (avg. MW:5900), Pluronic P84 (avg.
MW:4200),Pluronic P105 (avg. MW:6500), Pluronic P85 (avg. MW:4600), Pluronic P75 (avg. MW:4150), Pluronic P65 (avg. MW:3400), Pluronic F127 (avg. MW: 12600), Pluronic F98 (avg. MW: 13000), Pluronic F87 (avg. MW:7700), Pluronic F77 (avg. MW:6600), Pluronic F 108 (avg. MW: 14600), Pluronic F98 (avg. MW: 13000), Pluronic F88 (avg. MW:11400), Pluronic F68 (avg. MW:8400), and Pluronic F38 (avg. MW:4700).
Reverse poloxamers of the present invention include, but are not limited to Pluronic R31R1 (avg. MW:3250), Pluronic R 25R1 (avg. MW:2700), Pluronic R17R1 (avg. MW:1900), Pluronic R31R2 (avg. MW:3300), Pluronic R25R2 (avg. MW:3100), Pluronic Rl 7R2 (avg. MW:2150), Pluronic Rl 2R3 (avg.
MW:1800),Pluronic R31R4 (avg. MW:4150), Pluronic R25R4 (avg. MW:3600), Pluronic R22R4 (avg. MW:3350), Pluronic R17R4 (avg. MW:3650), Pluronic R25R5 (avg. MW:4320), Pluronic R10R5 (avg. MW:1950), Pluronic R25R8 (avg. MW:8850), Pluronic R17R8 (avg. MW:7000), Pluronic R10R8 (avg. MW:4550). Other commercially available poloxamers include compounds that are block copolymer of polyethylene and polypropylene glycol such as Synperonic L121, Synperonic L122, Synperonic P104, Synperonic P105, Synperonic P123, Synperonic P85, and Synperonic P94; and compounds that are nonylphenyl polyethylene glycol such as Synperonic NP10, Synperonic NP30, and Synperonic NP5.
Suitable auxiliary agents include non-ionic detergents and surfactants such as Pluronic F68, Pluronic F77, Pluronic F108, Pluronic F127, Pluronic P65, Pluronic P85, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic P123, Pluronic L31, Pluronic L43, Pluronic L44, Pluronic L61, Pluronic L62, Pluronic L64, Pluronic L81, Pluronic L92, Pluronic LlOl, Pluronic L121, Pluronic R17R4, Pluronic R25R4, Pluronic R25R2, IGEPAL CA 630, NONIDET NP-40, Nonidet P40, Tween-20, Tween-80, Triton X-100, Triton X-l 14, Thesit; the anionic detergent sodium dodecyl sulfate (SDS); the sugar stachyose; the condensing agent DMSO; and the chelator/DNAse inhibitor EDTA. Even more preferred are the auxiliary agents Nonidet P40, Triton X-100, Pluronic F68, Pluronic F77, Pluronic F108, Pluronic P65, Pluronic P103, Pluronic L31, Pluronic L44, Pluronic L61, Pluronic L64, Pluronic L92, Pluronic R17R4, Pluronic R25R4 and Pluronic R25R2. A most preferred auxiliary agent is Pluronic R25R2.
Optimal concentrations of auxiliary agents of the present invention are disclosed in U.S. Patent Application Publication No. 20020019358 and PCT Publication WO0180897A3. For example, in certain embodiments, pharmaceutical compositions of the present invention comprise about 5 ng to about 30 mg of a suitable polynucleotide or a polynucleotide construct, or an active fragment or variant thereof, and about 0.001% (w/v) to about 2.0% (w/v) of Pluronic R 25R4, preferably about 0.002% (w/v) to about 1.0% (w/v) of Pluronic R 25R4, more preferably about 0.01% (w/v) to about 0.01% (w/v) of Pluronic R 25R4, with about 0.01% (w/v) of Pluronic R 25R4 being the most prefeπed; about 0.001% (w/v) to about 2.0% (w/v) of Pluronic R 25R2, preferably about 0.001% (w/v) to about 1.0% (w/v) of Pluronic R 25R2, more preferably about 0.001% (w/v) to about 0.1% (w/v) of Pluronic R 25R2, with about 0.01 % (w/v) of Pluronic R 25R2 being the most preferred.
The vaccme composition can be solubilized in a buffer prior to administration. Suitable buffers include, for example, phosphate buffered saline (PBS), normal saline, Tris buffer, and sodium phosphate vehicle (100-150 mM preferred). Insoluble polynucleotides can be solubilized in a weak acid or base, and then diluted to the desired volume with a neutral buffer subh as PBS. The pH of the buffer is suitably adjusted, and moreover, a pharmaceutically acceptable additive can be used in the buffer to provide an appropriate osmolarity within the lipid vesicle. Preferred salt solutions and auxiliary agents are disclosed herein. A systemic delivery embodiment is particularly preferred for treating non- localized disease conditions. A local delivery embodiment can be particularly useful for treating disease conditions that might be responsive to high local concentration. When advantageous, systemic and local delivery can be combined. U.S. Patents Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and PCT publication W094/29469 provide methods for delivering compositions comprising naked DNA, or DNA cationic lipid complexes to mammals.
Compositions used in of the present invention can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol, ed., Mack Publishing Co., Easton, Pa. (1980), and Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), both of which are incoφorated herein by reference in their entireties. Although the composition is preferably administered as an aqueous solution, it can be formulated as an emulsion, gel, solution, suspension, lyophilized form, or any other form known in the art. According to the present invention, if the composition is fonnulated other than as an aqueous solution, it will require resuspension in an aqueous solution prior to administration. In addition, the composition may contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives.
For aqueous compositions used in vivo, the use of sterile pyrogen-free water is preferred. Such formulations will contain an effective amount of a polynucleotide or polynucleotide constract together with a suitable amount of an aqueous solution in order to prepare pharmaceutically acceptable compositions suitable for administration
The present invention also provides kits for use in treating SARS-CoV comprising an administration means and a container means containing a vaccine composition of the present invention. Preferably, the polynucleotide or polynucleotide constract of such composition is in the amount of 1 ng to 30 mg, more preferably in the amount of 100 ng to 20 mg. The container in which the composition is packaged prior to use can comprise a hermetically sealed container enclosing an amount of the lyophilized formulation or a solution containing the formulation suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose. The composition is packaged in a sterile container, and the hermetically sealed container is designed to preserve sterility of the pharmaceutical formulation until use.
It is specifically contemplated as one aspect of the invention that vaccine compositions of the invention may be used as part of a prime-boost protocol wherein a first vaccine composition that comprises a SARS-CoV antigen, a prime vaccine composition, is administered in conjunction with a boost vaccine composition that comprises a SARS-CoV antigen that differs in form from the SARS-CoV antigen of the prime vaccine composition. Either the prime vaccine composition or the boost vaccine composition, or both, may be vaccme compositions of the invention. When both the prime and boost vaccine compositions are vaccine compositions of the invention they expose the subject to the SARS-CoV S protein in different forms.
The boost vaccine composition may be administered at the same time as the prime vaccme composition or it may be administered at some time following the initial administration of the prime vaccine composition. The prime and boost vaccine compositions may be administered via the same route or they may be administered via different routes. If the prime and boost vaccine compositions are administered at the same time they may be administered as part of the same formulation or as different formulations. Both the prime vaccine composition and the boost vaccine composition may be administered one or several times. Thus some doses of the prime vaccine may be administered after the administration of a dose of the boost vaccine. It is within the skill of one with ordinary skill in the art to optimize prime boost combinations, including the optimization of routes of vaccine administration and timing for vaccine administrations. Prefeπed prime-boost combinations of the invention include, but are not limited to, the following exemplary combinations: (i) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccine composition comprising live attenuated SARS-CoV viras; (ii) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccme composition comprising live viral vectors that expresses SARS-CoV S proteins, wherein prefeπed viral vectors include the expression of the SARS-CoV S or N protein; (iii) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccine composition comprising isolated SARS- CoV S protein; (iv) a vaccine composition comprising a plasmid encoding a SARS-CoV S protein and a vaccine composition comprising isolated SARS- CoV N protein or a plasmid that encodes SARS-CoV N protein.
It is understood that, for the above listed combinations, either of the vaccine compositions may be the prime or the boost vaccine. In a prefeπed embodiment of the invention, a vaccine composition comprising a plasmid encoding a SARS-CoV S protein is used as the prime vaccine. In prefeπed embodiments of the invention viral vectors used to deliver either the prime or the boost vaccine composition are advenovirus vectors and vaccinia vims vectors (e.g. MVA).
Other Uses Of The Compositions Of The Present Invention
In another embodiment, the present invention relates to methods of reducing the effect or symptoms of SARS-CoV infection in a subject via the administration of SARS-CoV neutralizing antibodies. The subject may be already infected with the viras or may be at risk for potential infection with the virus. In accordance with this embodiment of the invention, the methods and compositions of the present invention are used to elicit antisera and antibodies reactive against SARS-CoV vims, and an effective amount of an antibody reactive with SARS- CoV is administered to such patient. The employed antibodies will preferably not possess an antibody-dependent vims enhancing activity. As used herein, an "antibody-dependent vims enhancing activity", with respect to an antibody refers to the ability of the antibody to enhance cellular entry of any strain of SARS-CoV or to enhance cellular entry of a pseudotype vims constructed with a SARS-CoV S protein.
The employed antibodies may be derived from human antisera, from non- human mammalian origin, or may be monoclonal, recombinant, single-chain, or humanized. Antigen-binding fragments of such antibodies (e.g., Fab and F(ab)2 fragments) may alternatively be employed. If desired, such administration can be provided in concert with administration of the vaccine compositions of the invention in order to provoke a long-term immunity to SARS-CoV infection. When providing a patient with such antibodies, the dosage administered will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of the above compounds that is in the range of from about 1 pg/kg to 500 mg/kg (body weight of patient), although a lower or higher dosage may be administered.
In another aspect, the invention relates to an immunoassay that measures the presence or concentration of an anti-SARS-CoV S protein antibody (refeπed to herein as an "anti-S-protein antibody") in a biological sample of a mammal. The immunoassay comprises the following step: (a) contacting said biological sample with mammalian expressed SARS-CoV S protein antigen (refeπed to herein as an "S-protein antigen"), said contacting being under conditions sufficient to permit anti-S-protein antibody if present in said sample to bind to said S-protein antigen and form an S-protein antigen-anti-S-protein antibody complex; (b) contacting said formed S-protein antigen-anti-S-protein antibody complex with an anti-S-protein antibody binding molecule, said contacting being under conditions sufficient to permit said anti-S-protein antibody binding molecule to bind to anti-S-protein antibody of said formed S-protein antigen-anti-S-protein antibody complex and form an extended complex; and (c) determining the presence or concentration of said anti-S-protein antibody in said biological sample by determining the presence or concentration of said formed extended complex.
As used herein, a "mammalian expressed SARS-CoV S-protein antigen" refers to any isolated protein or polypeptide that can specifically react with anti- SARS-CoV-S-protein antibodies and that is expressed in mammalian cells. S- protein antigens may comprise the entire S protein or portions thereof. Prefeπed S protein antigens comprise at least a portion of the extracellular domain of the S- protein (i.e. at least a portion of residues 14 to 1190 of SEQ ID NO:3. S-protein antigens may comprise homologs of the S protein, or portions thereof. Prefeπed S protein homologs are highly homologous to at least a portion of the extracellular domain of the S protein of a SARS-CoV vims. For example, prefeπed S protein homologs will comprise a polypeptide sequence that is at least 20, preferably at least 30 , more preferably at least 40 amino acids long and that is at least 80% identical to a portion of the extracellular domain of the S protein, more preferably at least 90% identical, and more preferably at least 95% identical to at least a portion of the extracellular domain of the S protein.
Methods of producing mammalian expressed S-protein antigens are well known in the art. Mammalian expressed S-protein antigens are preferably produced using the techniques of recombinant DNA technology wherein a mammalian expression vector comprising a nucleic acid sequence that encodes an S-protein antigen is introduced into a mammalian cell. The mammalian cell is then cultured under conditions that allow for the production of the S-protein antigen. Various mammalian expression systems are well known in the art and an artisan of ordinary skill in the art could readily design and operate such a system. As used herein, anti SARS-CoV S protein antibody refers to any of IgM,
IgG, or IgA, molecules. In a prefeπed embodiment of the invention, a biological sample is assayed for the presences of anti-SARS-CoV S protein antibodies that are IgG molecules. Any of a wide variety of assay formats may be used in accordance with the methods of the present invention. Such formats may be heterogeneous or homogeneous, sequential or simultaneous, competitive or noncompetitive. U.S. Patent Nos. 5,563,036; 5,627,080; 5,633,141; 5,679,525; 5,691,147; 5,698,411; 5,747,352; 5,811,526; 5,851,778; and 5,976,822 illustrate several different assay formats and applications. Such assays can be formatted to be quantitative, to measure the concentration or amount of an anti-S protein antibody, or they may be formatted to be qualitative, to measure the presence or absence of an anti-S protein antibody. Heterogeneous immunoassay techniques typically involve the use of a solid phase material to which the reaction product becomes bound, but may be adapted to involve the binding of nonimmobilized antigens and antibodies (i.e., a solution- phase immunoassay). The reaction product is separated from excess sample, assay reagents, and other substances by removing the solid phase from the reaction mixture (e.g., by washing). One type of solid phase immunoassay that may be used in accordance with the present invention is a sandwich immunoassay. In the sandwich assay, the more anti-S protein antibody present in the sample, the greater the amount of label present on the solid phase. This type of assay format is generally prefeπed, especially for the visualization of low concentrations of antibody because the appearance of label on the solid phase is more readily detected.
In accordance with a prefeπed embodiment of the present invention, S- protein antigen that is specifically reactive with anti-S-protein antibody is bound to a solid support (i.e., immobilized) and incubated in contact with the biological sample being tested for the presence of anti-S-protein antibody. As will be appreciated, the S-protein antigen may be incubated with the biological sample in an unbound state and then subsequently bound to the solid support (i.e., immobilizable). The supports are then preferably extensively treated (e.g., by washing, etc.) to substantially remove non-anti-S-protein antibodies that may be present but which failed to bind to the bound antigen. In consequence of such treatment, an immune complex forms between the antigen and anti-S-protein antibody.
A detectably labeled second antibody (e.g., an anti-human IgG antibody) is then preferably added and the support is incubated under conditions sufficient to permit the second antibody to bind to any anti-S-protein antibody that may be present. The support is then preferably extensively treated (e.g., by washing, etc.) to substantially remove any unbound second antibody. If the anti-S-protein antibody is present in the test sample, then the two antibodies will form an immune complex with the S-protein antigen (e.g., anti-human IgG antibody/anti-S-protein antibody/S-protein antigen sandwich). In such an assay, the detection of second antibody bound to the support is indicative of anti-S-protein antibody in the biological sample being tested. Sandwich assay formats are described by Schuurs et al. U.S. Patent Nos. 3,791,932 and 4,016,043, and by Pankratz, et al., U.S. Patent No. 5,876,935. The second antibody may be a natural immunoglobulin isolated from nonhuman primates (e.g., anti-human IgG murine antibody, anti- human IgG goat antibody, etc.), or can be produced recombinantly or synthetically. It may be an intact immunoglobulin, or an immunoglobulin fragment (e.g., FAb, F[Ab]2, etc.). As desired, other binding molecules (capable of binding to anti-S- protein antibodies) may be employed in concert with or in lieu of such second antibodies. For example, the anti-S-protein antibody can be biotinylated and the second antibody can be replaced with labeled avidin or steptavidin.
To eliminate the bound-free separation step and reduce the time and equipment needed for a chemical binding assay, a homogeneous assay format may alternatively be employed. In such assays, one component of the binding pair may still be immobilized (e.g. the S-protein antigen); however, the presence of the second component of the binding pair is detected without a bound-free separation. Examples of homogeneous optical methods are the EMIT method of Syva, Inc. (Sunnyvale, CA), which operates through detection of fluorescence quenching; the laser nephelometry latex particle agglutination method of Behringwerke (Marburg, Germany), which operates by detecting changes in light scatter; the LPIA latex particle agglutination method of Mitsubishi Chemical Industries (Tokyo, Japan); the TDX fluorescence depolarization method of Abbott Laboratories (Abbott Park, IL); and the fluorescence energy transfer method of Cis Bio International (Paris, France). Any of such assays may be adapted for use in accordance with the objectives of the present invention. The binding assay of the present invention may be configured as a competitive assay. In a competitive assay, the more anti-S-protein antibody present in the test sample, the lower the amount of label present on the solid phase.
In a manner similar to the sandwich assay, the competitive assay can be conducted by providing a defined amount of a labeled anti-S-protein antibody and determining whether the fluid being tested contains anti-S-protein antibody that would compete with the labeled antibody for binding to the support. In such a competitive assay, the amount of captured labeled antibody is inversely proportional to the amount of analyte present in the test sample. Smith (U.S. Patent No. 4,401,764) describes an alternative competitive assay format using a mixed binding complex that can bind analyte or labeled analyte but in which the analyte and labeled analyte cannot simultaneously bind the complex. Clagett (U.S. Patent No. 4,746,631) describes an immunoassay method using a reaction chamber in which an analyte/ligand/marker conjugate is displaced from the reaction surface in the presence of test sample analyte and in which the displaced analyte/ligand/marker conjugate is immobilized at a second reaction site. The conjugate includes biotin, bovine serum albumin, and synthetic peptides as the ligand component of the conjugate, and enzymes, chemiluminescent materials, enzyme inhibitors, and radionucleotides as the marker component of the conjugate. Li (U.S. Patent No. 4,661,444) describes a competitive immunoassay using a conjugate of an anti-idiotype antibody and a second antibody, specific for a detectable label, in which the detectable response is inversely related to the presence of analyte in the sample. Allen (European Patent Appln. No. 177,191) describes a binding assay involving a conjugate of a ligand analog and a second reagent, such as fluorescein, in which the conjugate competes with the analyte (ligand) in binding to a labeled binding partner specific for the ligand, and in which the resultant labeled conjugate is then separated from the reaction mixture by means of solid phase carrying a binding partner for the second reagent. This binding assay format combines the use of a competitive binding technique and a reverse sandwich assay configuration; i.e., the binding of conjugate to the labeled binding member prior to separating conjugate from the mixture by the binding of the conjugate to the solid phase. The assay result, however, is determined as in a conventional competitive assay in which the amount of label bound to the solid phase is inversely proportional to the amount of analyte in the test sample. Chieregatt et al. (GB Patent No. 2,084,317) describe a similar assay format using an indirectly labeled binding partner specific for the analyte. Mochida et al. (U.S. Patent No. 4, 185,084) also describe the use of a double-antigen conjugate that competes with an antigen analyte for binding to an immobilized antibody and that is then labeled. This method also results in the detection of label on a solid phase in which the amount of label is inversely proportional to the amount of analyte in the test sample. Sadeh et al. (U.S. Patent No. 4,243,749) describe a similar enzyme immunoassay in which a hapten conjugate competes with analyte for binding to an antibody immobilized on a solid phase. Any of such variant assays may be used in accordance with the present invention.
In all such assay formats, at least one component of the assay reagents will preferably be labeled or otherwise detectable by the evolution or quenching of light. Such component may be a second antibody, anti-S-protein antibody, or an antigen that binds to an anti-S-protein antibody, depending on the immunoassay format employed. Radioisotopic-binding assay formats (e.g., a radioimmunoassay, etc.) employ a radioisotope as such label; the signal is detectable by the evolution of light in the presence of a fluorescent or fluorogenic moiety (see Lucas et al. [U.S. Patent No. 5,698,411] and Landram et al. [U.S. Patent No. 5,976,822]). Enzymatic-binding assay formats (e.g., an ELISA, etc.) employ an enzyme as a label; the signal is detectable by the evolution of color or light in the presence of a chromogenic or fluorogenic moiety. Other labels, such as paramagnetic labels, materials used as colored particles, latex particles, colloidal metals such as selenium and gold, and dye particles (see U.S. Patent Nos. 4,313,734; 4,373,932, and 5,501,985) may also be employed. The use of enzymes (especially alkaline phosphatase, β-galactosidase, horse radish peroxidase, or urease) as the detectable label (i.e., an enzyme immunoassay or EIA) is prefeπed.
The presence of enzymatic labels may be detected through the use of chromogenic substrates (including those that evolve or adsorb fluorescent, UV, visible light, etc.) in response to catalysis by the enzyme label. More preferably, chemical labels may be employed (e.g., colloidal gold, latex bead labels, etc.). Detection of label can be accomplished using multiple detectors, multipass filters, gratings, or spectrally distinct fluors (see e.g., U.S. Patent No. 5,759,781), etc. It is particularly prefeπed to employ peroxidase as an enzyme label, especially in concert with the chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB). In the case of labeling of the antibodies with peroxidase as enzyme, it is possible to use the periodate technique (Nakane. et al., 1974, J Histochem Cytochem. 22:1084- 90) or a method reported in which the partners are linked with a heterobifunctional reagent (Ishikawa et al, 1983, J Immunoassay. 4[3]:209-327). Any of a wide variety of solid supports may be employed in the immunoassays of the present invention. Suitable materials for the solid support are synthetics such as polystyrene, polyvinyl chloride, polyamide, or other synthetic polymers, natural polymers such as cellulose, as well as derivatized natural polymers such as cellulose acetate or nitrocellulose, and glass, especially glass fibers. The support can take the form of spheres, rods, tubes, and microassay or microtiter plates. Sheet-like structures such as paper strips, small plates, and membranes are likewise suitable. The surface of the carriers can be permeable and impermeable for aqueous solutions.
The S-protein antigen may be indirectly or directly bound to the solid support. In a prefeπed embodiment of the invention the S protein is indirectly bound to the solid support via Galanthus nivalis lectin.
Although the foregoing description pertains to assaying for the presence of anti-S-protein antibodies in biological samples that are fluids (e.g., sera, blood, urine, saliva, pancreatic juice, cerebrospinal fluid, semen, etc.), it will be appreciated that any fluidic biological sample (e.g., tissue or biopsy extracts, extracts of feces, sputum, etc.) may likewise be employed in the assays of the present invention. Most preferably, the biological sample being assayed will be semm or sputum. Materials for use in the assay of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive multiple samples in close confinement; one or more containers means vials, tubes and the like; each of the containers means comprising one of the separate elements to be used in the method. For example, one of the containers means may comprise a suitable S-protein antigen. A second container may comprise soluble, detectably labeled second antibody, preferably in lyophilized form, or in solution. In addition, the kit may also contain one or more containers, each of which comprises a (different) predetermined amount of S protein antigen or anti-S protein antibody, useful for the preparation of a standard curve.
In using the kit, all the user has to do is add to a container a premeasured amount of a sample suspected of containing a measurable yet unknown amount of anti-S-protein antibodies, a premeasured amount of support-bound antigen present in the first container, and a premeasured amount of the detectably labeled second antibody present in the second container. After an appropriate time for incubation, an immune complex is formed and is separated from the supernatant fluid, and the immune complex or the supernatant fluid are detected, as by radioactive counting, addition of an enzyme substrate, and color development, or by inclusion of a chemical label (e.g., colloidal gold, latex beads, etc.). The present invention particularly relates to the use of immuno- chromatographic assay formats to detect anti-S protein antibodies. In a prefeπed immunochromatographic assay format, two contacting, but spatially distinct, porous carriers are employed. The first such carrier will contain a non- immobilized, labeled S protein and the second such carrier will contain an immobilized, but unlabeled antibody that binds to IgG (e.g., where human anti-S protein antibodies are being assayed, the unlabeled antibody may be an anti-human IgG antibody).
Preferably, the device will comprise a hollow casing constructed of, for example, a plastic material, etc., in which the first carrier will communicate indirectly with the interior of the casing via a multilayer filter system that is accessible from the device (e.g., by protruding therefrom or by being incompletely covered by the device), such that a semm, plasma, or whole blood test sample can be applied directly to the filter system and will permeate therefrom into the first porous carrier. In such a device, the permeation of fluid containing anti-S protein antibodies will cause the non-immobilized labeled S protein of the first carrier to become bound to the migrating antibodies, and will then permeate into the second carrier. Because the second carrier contains immobilized antibody that binds human IgG, any labeled S protein entering the second caπier will be entrapped therein. Detection of labeled S protein in the caπier containing the immobilized unlabeled antibody thus indicates that anti-S protein antibodies are present in the sample being evaluated. The assay can be made quantitative by measuring the quantity of labeled S protein that is bound within the second porous carrier.
In another embodiment, the invention relates to viral vectors that have been pseudotyped with at least a portion of the SARS-CoV S protein and to methods of using the pseudotyped viral vectors to screen for inhibitors of SARS-CoV infection. Viral vectors that may be employed include any vector that, when pseudotyped with the S protein, is able to support entry and fusion of the viral vector with a permissive host cell type for SARS-CoV infection such as, for example, Vero cells. In prefeπed embodiments, the viral vectors are retroviral vectors or lentiviral vectors. In prefeπed embodiments, the pseudotyped viral vectors are used to assay for neutralizing antibodies for SARS-CoV infection. Such assays may be useful for diagnosing SARS-CoV infection or for monitoring the progress of the disease or the prognosis for the disease in a particular patient. It is contemplated as part of the invention that the pseudotyped viral vectors of the invention may be used to screen for agents that inhibit the entry of SARS-CoV into cells or that inhibit the cell mediated transfer of SARS-CoV.
In another embodiment, the invention relates to a method of assaying a vaccine composition or an immunogenic compoud for antibody-dependent viras enhancing activity, wherein the method comprises the following steps: (a) generating an immune response to a vaccine composition or an immunogenic compound in an animal, preferably a non-human mammal or a human; (b) isolating immune sera or antibodies from the animal; (c) assaying the ability of the antibodies or the immune sera to enhance cellular entry of a strain of SARS-CoV viras or a pseudotype virus constructed with the SARS-CoV S protein; (d) identifying the vaccine composition or the immunogenic compound as having antibody-dependent virus enhancing activity if the antibodies or immune sera enhance cellular entry of the virus strain or the pseudotype viras. Such a method would be useful for assessing the therapeutic viability of any particular immunogenic compound or vaccine composition. In prefeπed embodiments of the above-described method, the viras strain employed, or the coπesponding pseudotype viras, would be an isolate from a non-human animal, preferably a non- human mammal, including, for example, an isolate from the Palm civt, the raccoon dog, or the Chinese feπet badger. Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified.
Example 1 PH-Dependent Entry of SARS Coronavirus is Mediated by the Spike Glycoprotein and Enhanced by Dendritic Cell Mediated Transfer through DC-SIGN
Materials and Methods Antibodies, mouse immune serum and media Human semm from a recovered SARS patient can be obtained from Dr. William Bellini (CDC, U.S.A.). Antibodies to GDI lc , CD14 ,CD40, CD80, CD86 and HLA-DR are purchased from BD Pharmingen. Mouse anti-His (C- term)-FITC antibody is purchased from Invitrogen (Carlsbad, California). Media for human primary cell culture are purchased from Cambrex (East Rutherford, New Jersey). RPMI Media 1640 and Dulbecco's Modified Eagle Media are purchased from Invitrogen (Carlsbad, California). Anti-SARS-CoV S mouse immune semm is generated by vaccinating 10-week old BALB/c mice with CMV/R plasmid DNA expression vectors, described below, encoding the S protein (25 μg at three week intervals, three times, bled after two months). Negative control antisera is obtained in a similar fashion by injecting the same plasmids with no insert (control)
Cell lines Human primary cell lines are as follows: RPTEC (renal proximal tubule epithelial cell), HRE (renal epithelial cell), HRCE (renal cortex epithelial cell), SAEC (small airway epithelial cell), NHBE (bronchial epithelial cell), NHLF (lung fibroblast), HMVEC-L (lung microvascular endothehal cell), HUVEC (umbilical vein endothehal cell), HMVEC (microvascular endothehal cell), NHMEC (mammary epithelial cell), NHEK (keratinocyte), and hepatocytes. Human primary cell lines can be purchased from Cambrex. Human and animal cell lines are as follows: ACHN (human kidney adenocarcinoma), 293 (human embryonic kidney), 786-0 (human kidney adenocarcinoma), A549 (human lung carcinoma), HeLa (human cervical adenocarcinoma), Colo205 (human colon adenocarcinoma), Jurkat (human T cell), CEM (human acute lymphoblast leukemia), M8166 (human CD4+ lymphoid cell), HL60 (human promyelocytic leukemia cell), THP-1 (human acute monocytic leukemia), Vero (African green monkey kidney epithelial cell), CRFK (cat kidney cortex epithelial cell), OK (opossum kidney cortex epithelial cell), M-l (mouse kidney cortex epithelial cell), FC2.Lu (cat lung fibroblast), FC28.1u (cat lung fibroblast), AK-D (cat lung epithelial cell), MLE12 (mouse lung epithelial cell), MM14.1u (mouse lung), LA-4 (mouse lung adenoma), LH4 (guinea pig lung fibroblast), and CHL-11 (Chinese hamster lung fibroblast). Human and animal cell lines can be purchased from the ATCC. PBMCs (human peripheral blood mononuclear cells) are prepared from whole blood by Ficoll gradient centrifugation. THP-1 (human acute monocytic leukemia), THP-DC-SIGN ( THP- 1 expressing human DC-SIGN) and THP-DC-SIGNΔ35 (THP-1 expressing a 35 amino acid cytoplasmic domain deleted DCSIGN) are constructed as described by Kwon et al. (2002, Immunity 16:135-144). Human T cell leukemia cell lines A3R5 (a subline of CEM expressing both CCR5 and CXCR4), MT-2 expressing CXCR4 and 293T can be obtained from Dr. John Mascola. M8166 (a human CD4+ lymphoid cell) can be obtained from Dr. Hayami.
Gene synthesis and construction of expression vectors Genes encoding the SARS-CoV spike (S), membrane (M) and envelope (E) proteins are synthesized using human-prefeπed codons. To synthesize these genes, protein sequences obtained from GenBank (AY278741) for the SARS-CoV Urbani strain are reverse translated using human-prefeπed codons. Sets of 75 bp oligonucleotides, with 25 bp overlaps, covering each gene are synthesized and gel- purified. The oligonucleotides are assembled into DNA fragments1 using Pfu Turbo Hotstart DNA polymerase (Stratagene, La Jolla, CA) at a 50-65°C gradient annealing temperature. DNA fragments are cloned into pCR-Blunt II-Topo vector (Invitrogen) and sequenced. Clones with the fewest mutations are picked, and further coπected using the Quickchange kit (Stratagene) according to the manufacturer's protocol. Fully coπected DNA fragments for each gene are cloned into mammalian expression vector CMV/R-mcs. CMV/R-mcs contains the cytomegalovirus CMV) enhancer/promoter, splice donor, and the HTLV-1 R region. COOH-terminal deletion mutants of the SARS-CoV S gene are generated using the Quickchange kit (Stratagene) and are cloned into the CMV/R-mcs expression vector. These mutants include the following constructs: (1) SΔCD, in which the cytoplasmic domain is truncated, terminated at aa 1229 (residue 1229 of SEQ ID NO:3); (2) SΔTM2, in which the transmembrane and cytoplasmic domains are deleted at aa 1190 (residue 1190 of SEQ ID NO:3; and (3) SΔHRl, in which the transmembrane, cytoplasmic and heptad 2 domains are removed, with termination at aa 1153 (residue 1153 of SEQ ID NO:3). The Myc-His tagged S(l 190) construct is truncated at aa 1190 to remove the transmembrane and cytoplasmic domains and tagged with a myc and his epitope at the COOH- terminus. The constructs are sequenced on both strands to ensure that each gene is conect and the identity of the constructs is further confirmed using Western blot analysis of the gene expression products.
Purification and differentiation of human myeloid DCs Myeloid dendritic cells (mDCs) are purified from elutriated monocytes from healthy adult donors by a two-step procedure consisting of automated leukapheresis and counterflow centrifugal elutriation at the Transfusion Medicine Department of Waπen Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD as described in Abrahamsen et al. (1991, J. Clin. Apheresis 6:48-53). Myeloid DCs are isolated from the elutriated monocyte fraction with negative selection by depleting cells expressing BDCA-4 and CD9 using microbeads (Miltenyi Biotec, Auburn, CA), followed by positive selection using antibodies to CDlc (Miltenyi Biotech). Myeloid DCs are then cultured in media containing GM-CSF (PeproTech, 10 ng/ml), and induced to differentiate to mature mDC using poly (I:C) (50 ng/ml) (Sigma, St. Louis, MO) for 48 hours as described in Cella et al., 1999, J. Exp. Med. 189:821-829. Antibodies to CD1 lc and CD14 (BD Pharmingen) are used to assess purity of mDCs and antibodies to CD40, CD80, CD86 and HLA-DR (BD Pharmingen) are used to characterize the differentiation of mDCs by flow cytometry.
Production of pseudotyped lentivims and retrovirus Recombinant lentivimses and retrovimses expressing a luciferase reporter gene are produced essentially as described in Kinsella et al (1996, Hum. Gene Ther. 7:1405) andNaldini et al. (1996, roc. Natl. Acad. Sci. USA 93:11382). Briefly, 5xl06293 T cells are plated in 10 cm tissue culture dishes the day before transfection. The cells are transfected the next day using calcium phosphate reagent (Invitrogen). The amount of plasmid DNA used for making different pseudotyped vectors is as follows: for lentiviral vectors, 7 μg of pCMV.R8.2 and 7 μg of pHR'CMV-Luc in combination with 400 ng CMV/R-SARS-S or 2 μg of pNGVL-4070A(Ampho), respectively; for retroviral vectors, 7 μg of pNGVL GagPol(MLV) and 7μg of pLZR-Luc, in combination with 400 ng CMV/R-SARS- S or 2 μg of pNGVL- 4070A(Ampho), respectively. Cells are transfected overnight, washed, and replenished with fresh media. After 48 hours, supernatants are harvested, filtered through a 0.45 μm syringe filter, aliquotted, and used immediately or frozen at -80°C. Levels of p24 are measured from different viral stocks (S3) using "The Coulter HIV-1 p24 Antigen Assay" kit (Beckman Coulter, Somerset, NJ). The same amount of p24 is used with each pseudotyped vims to standardize the amount used for infection for comparison. Cell lines are infected for 16-18 hours for SARS-S pseudotyped vimses and for 3-4 hours for Ampho and Eco ENV pseudotyped vimses. Forty-eight hours after infection, cells are lysed in "mammalian cell lysis buffer" (Promega, Madison, WI). Equal amounts of each cell lysate are used in luciferase assays with "Luciferase assay reagent" (Promega, Madison, WI), according to the manufacturer's suggestion.
Production of GFP-Vpr-labeled SARS-S pseudotyped lentivims GFP-Vpr-labeled SARS lentivims is produced by transfection of human embryonic kidney (HEK) 293 T cells with the an ercv-deleted pLAI provims (10 μg), CMV/R-SARS-S (1 μg) and the plasmid pEGFP-C3 (Clontech, Palo Alto, CA) containing the entire Vpr coding region fused to the carboxyterminus of eGFP (GFP-Vpr; 15 μg) as described in McDonald et al. (2002, J. CellBiol. 159:441- 452). Cells are washed at 16-20 hours post-transfection and replenished with fresh media. Forty-eight hours later, supernatants are harvested, filtered through a 0.45 μm syringe filter, and concentrated. Briefly, 32 ml of supernatant is layered on 5 ml of Optiprep (Iodoxinal) medium (Invitrogen, Carlsbad CA) and centrifuged at 50,000 x g for 1.5 hours with a Surespin 630 rotor (Sorvall, Newtown, CT). The last 3 ml of supernatant remaining above the Optiprep interface is collected and frozen at -80°C in 500 μl aliquots. Infection of cells with SARS-CoV and titration of SARS-CoV Cells in six- well dishes are infected in each well with 100 μl of a 1:10 dilution of SARS-CoV (Urbani stram)(CDC, U.S.A)(106'25 TCID50/ml) under appropriate containment in a BSL3 laboratory. After one hour of adsoφtion, the cells are washed three times with medium, replenished with 3 ml of fresh medium, and maintained at 37°C in a 5% C02 incubator. Seventy-two hours after infection, 0.5 ml tissue culture medium is harvested, and viral titer in the medium is determined. Viral titer is determined by infecting Vero cells in 96- well plate, and viral titers are calculated in TCIDso/ml four days after infection of Vero cells. The viral cytopathic effect is determined on days 3 and 4. Infection of mature and immature mDC is performed in 96-well plates (10,000 cells/well) with 50 μl of 1:10 diluted viral stock, and titered three days later, as described above.
Infection of cells with pseudovirus An aliquot of 30,000 cells are plated into each well of a 48-well dish the day before infection. Cells are infected with 150 μl of viral supernatant for 16-18 hours for SARS-CoV-S pseudotyped viruses, and for 3-4 hours for Ampho Env and Ebola glycoprotein pseudotyped virases. Viral supernatant is replaced with fresh medium at the end of the infection period. Forty-eight hours after infection, cells are lysed in "mammalian cell lysis buffer" (Promega, Madison, WI). The same amount of cell lysate is used in a luciferase assay with "Luciferase assay reagent" (Promega, Madison, WI) according to the manufacturer's suggestion.
Transfection and Western Blot Analysis 293T cells are transfected using calcium phosphate (Invitrogen, Carlsbad, CA). Transfected cells are harvested 48 hours after transfection. Cell lysates are resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel elecfrophoresis
(PAGE) and then transfened onto nitrocellulose membrane (BIO-RAD, Hercules, CA). The membrane is incubated with convalescent human sera from a SARS patient at a 1 :2500 dilution for 1 hour at room temperature in blocking buffer (TBS + 1% BSA + 5% skim milk + 0.3% Tween-20). After incubation, the membrane is washed three times in washing buffer (TBS + 0.3% Tween-20). The blot is further incubated in blocking buffer with HRP conjugated donkey anti-human IgG (Chemicon, Temecula, CA), at a 1:5000 dilution, for 30 minutes and then washed four times in washing buffer. Antibody binding is detected using an ECL reagent (Amersham, Piscataway, NJ).
Cell staining with S(1190)-myc-his An aliquot of 500 ng of purified S(l 190)-myc-his glycoprotein is incubated with lxlO6 cells in 100 μl of PBS containing 2% fetal bovine semm for 20 min on ice and washed once with 1 ml cold PBS. Each cell line is split into two aliquots and then stained using either an FITC conjugated mouse anti-his tag monoclonal antibody (Invitrogen, Carlsbad, CA)(1 : 100 dilution) or an FITC labeled isotype control, followed by flow cytometric analysis.
Confocal Microscopy Myeloid DCs (l lO5), isolated from human elutriated monocytes, are plated onto a 12- well dish. After 24-48 hrs, the cells are infected with 100 μl Vpr- GFP labeled SARS-CoV S pseudo-lentivims for 30 min. Cells are washed, detached with Trypsin/EDTA, washed again, added to human renal epithelial cells (786-0; 3χ104 cells/well) and plated onto 8 well cover-slip slides (Nalge Nunc, Naperville, IL). Sequential images of live cells are recorded every 3 min under confocal microscopy, and uptake, polarization and transfer is assessed with representative cells.
pH-dependent entry of the SARS-CoV-S pseudotyped lentiviral vectors Vero cells are plated in a 48-well-dish (30,000 cells/well) the day before the experiment is conducted. Cells are pre-incubated with indicated amounts of ammonium chloride or bafilomycin A (Sigma, St. Louis, MO) for one hour. Pseudovimses are mixed with the same concentrations of reagents in tubes and added to cells. Eight hours later, media containing the pseudovimses are removed and replaced with fresh media . Cells are harvested 48 hours after infection, and a luciferase assay is performed. Cell-mediated transfer of SARS-CoV and SARS-CoV-S pseudotyped lentiviral vector THP, THP-DC-SIGN or THP-DC-SIGNΔ35 (30,000 cells/well) are incubated with SARS-CoV S pseudotyped lentiviral vector for 2 hours, and washed three times with tissue culture media. Cells are then added onto Vero, A3R5 or MT2 cells (30,000 cells/well) plated in 24-well-dishes. These cells are harvested 72 hours later for luciferase assay to assess THP, THP-DC-SIGN or THP-DC-SIGNΔ35 cell-mediated transfer of pseudovims to the respective cells.
To measure the transfer of SARS-CoV by mature mDC and to assess whether anti-S ARS-CoV S protein immune semm can block the transfer of SARS- CoV by mDC, mature mDCs are incubated with SARS-CoV for 1 hr, washed, and detached with trypsin, and replated onto Vero cells in 96-well-plate (10,000 cells/well) in the presence of control or S specific mouse anti-serum (1:100 dilution). Cell culture medium (DMEM+10%FBS) is collected 72 hours later, and SARS-CoV is titered in the cell culture medium as described above.
Results
Pseudotyping SARS-CoV envelope proteins are analyzed by cotransfection of expression vectors encoding either the S, M, or E glycoproteins with packaging plasmids for retroviral or lentiviral vectors into human 293T cells as described in Kinsella et al (1996, Hum.Gene Ther. 7:1405) andNaldini et al. (1996, Proc. Natl. Acad. Sci. USA 93:11382). For comparison, envelope glycoproteins from amphotropic murine leukemia viras and Ebola vims (Yang et al., 1998, Science 279:1034-1037) are substituted in place of the SARS-CoV envelope proteins. Vero cells, which support SARS-CoV replication (Ksiazek et al., 2003, N. Engl. J. Med. 348: 1953- 1966; Poutanen et al., 2003, N. Engl. J. Med. 348:1995-2005), are initially analyzed as a target cell. Among the SARS-CoV gene products, only the S protein mediates entry into target cells, both with murine retroviral and human lentiviral vectors (Figure 1, Panel A). Because both vectors can be pseudotyped with the S glycoprotein, further analyses are performed with the lentiviral vector, which can transduce and express recombinant genes in nondividing cells. Neither M nor E alone is able to support viral entry in the absence of S, suggesting that they serve other functions in the virus (Figure 1, Panel B). Cotransfection of M together with the S glycoprotein inhibits the generation of functional lentiviral vector, in contrast to E, which does not alter the efficacy of gene transfer (Figure 1, Panel B). To confirm the specificity of the effect and demonstrate that full-length S is required for gene transfer, deletion mutants of S are prepared with various carboxy terminal deletions. Although these mutants show comparable levels of expression (Figure 1, Panel C-Iower), the progressive COOH-terminal deletions show markedly lower levels of recombinant gene transfer (Figure 1, Panel C-upper), demonstrating that the cytoplasmic domain of S is required for viral entry.
pH dependent fusion mediated by SARS-CoV S protein Viral glycoproteins typically mediate attachment, fusion and entry by one of two mechanisms. Virases such as the human immunodeficiency virus (HIV) or murine amphotropic retrovimses infect through a pH-independent cell fusion and entry process (Nussbaum et al., 1993, J. Virol. 67:7402-7405; Stein et al., Cell 49:659-668). In contrast, influenza and Ebola are prototypes for vimses that utilize a pH dependent endocytotic pathway (Wool-Lewis and Bates, 1998, J. Virol. 72:3155-3160). To determine the pathway utilized by the SARS-CoV, pH dependence of the SARS-CoV S pseudotyped lentiviral vector is analyzed. Addition of ammonium chloride, which prevents acidification of the endosome, causes a dose-dependent reduction in viral entry (Figure 2, Panel A), similar to that described for other pH dependent viral glycoproteins (Bullough et al., 1994, Nature 371:37-43; Eckert and Kim, 2001, Annu. Rev. Biochem. 70:777-810; Wool- Lewis and Bates, 1998, supra). This effect is also observed with another inhibitor of endosomal acidification, bafilomycin, also in a dose-dependent fashion (Figure 2, Panel B). Susceptibility of infection of human and animal cells The cell specificity of the SARS-CoV S pseudotyped lentivims is analyzed by transducing different human cell types, including epithelial, endothehal and hematopoietic cells, and lung and renal cells from different species. Like Vero cells, a renal epithelial cell line derived from the African green monkey, human renal epithelial cells (HPTRC, HRE, HRCE, ACHN, 786-0) are highly susceptible to infection with the SARS-CoV-S pseudotyped vims in comparison to the infectivity observed with a known positive control with broad host range, the 4070A amphotropic murine retroviral envelope. Respiratory tract epithelial cells are also readily infected with the SARS-CoV-S pseudotyped viras. In contrast, a number of cells types, including hepatocytes, lower airway fibroblasts, breast or colonic epithelial, vascular endothehal, or hematopoietic cells, are relatively resistant to infection (Figure 3, Panel A). Interestingly, renal epithelial cell lines from humans, non-human primates, and felines, and to a lesser extent, lung cell lines derived from felines, are susceptible to transduction, while similar cells from rodents, are resistant to transduction (Figure 3, Panel B). The specificity of the pseudotyped vims transduction in these cell lines is confirmed by an analysis of the susceptibility of these cell lines to infection by SARS-CoV. A number of human cell lines, particularly of renal and pulmonary origin, yield high titer vims and were not previously recognized as susceptible to infection by the vims because they did not show cytopathic effect (Figure 3, Panel C). The susceptibility of these cells to pseudovims transduction coπelates well with their ability to support SARS-CoV replication. It therefore appears that a range of cell types and species are susceptible to infection mediated by the SARS-CoV S glycoprotein.
Binding of SARS-CoV spike proteins to DC-SIGN The S glycoprotein contains a number of N-linked glycosylation sites. For a number of virases, including HIV, the dengue vims, and the cytomegalo vims, glycosylation is known to affect binding of the vims to the dendritic cell DC-SIGN receptor that regulates cell-mediated transmission. To determine whether SARS- CoV S protein could bind to the DC-SIGN receptor, THP-1, a human acute monocytic leukemia cell line that expresses this gene product, THP-DC-SIGN, or a mutant form lacking the cytoplasmic domain required for intemalization and transfer, THP-DC-SIGNΔ35 cells (Kwon et al., 2002, Immunity 16:135-144), are incubated with purified His-tagged S protein. Binding is readily detected in the permissive Vero cell using flow cytometry, in contrast to two non-permissive T- cell leukemia cell lines, A3R5 and MT2 (Figure 4, Panel A) or THP-1 cells lacking DC-SIGN (data not shown). In contrast, THP-DC-SIGN and Δ35 cells interacted with purified S glycoprotein (Figure 4, Panel A), but unlike Vero cells, they could not be infected by S pseudotyped lentiviral vector (Figure 4, Panel B- left). To determine whether DC-Sign could nonetheless promote cell-mediated transfer of vims, the ability of these cells to transfer the SARS-CoV S pseudotyped lentiviral vector to Vero cells is analyzed. THP-DC-SIGN, but not THP or THP- DC-SIGNΔ35, which is unable to internalize vims (Kwon et al., Immunity 16:135- 144), readily transfened vims to Vero cells (Figure 4, Panel B-right), indicating that DC-SIGN or a related lectin on dendritic cells might facilitate cell-mediated transfer of SARS-CoV. Both direct infection and DC-SIGN-mediated transfer are inhibited by SARS-CoV S-specific mouse immune sera (Figure 4, Panel C), ςonfirming that S is necessary and sufficient for infection in both cases.
Cell mediated transfer of GFP-Vpr labeled pseudotyped lentivims by human mDC To determine whether cell-mediated transfer could be mediated by primary human mDCs, mature mDCs are isolated and incubated with GFP-Vpr labeled SARS-CoV-S pseudotyped lentivims (McDonald et al., 2002, J. CellBiol. 159:441-452). A similar vector pseudotyped with HIV gp 160 has been shown to mediate the formation of an "infectious" synapse that facilitates HIV infection (McDonald et al., 2003, Science 300:1295-1297), but it was unknown whether similar structures could be formed by an unrelated virus whose target cell is non- lymphoid. A culture of mDCs are incubated with vims for 30 minutes, trypsinized, and transfened to fresh 786-0 human renal cell cultures. Initially, vims distributes evenly throughout the mDCs (Figure 5, Panel A-uptake), but within minutes, immunofluorescent foci begin to form at the site of contact with Vero cells (Figure 5, Panel A-polarization). Viras is observed to transfer to the target cells through a structure analogous to the "synapse" previously described between mDC and lymphoid cells (Figure 5, Panel A-transfer) (McDonald et al., 2003, Science 300:1295-1297). After transfer, a characteristic streak of fluorescence is seen at the site of entry, suggesting a specific channeling of viral contents into cells (Figure 5, Panel A-post-transfer). This effect is seen consistently and is not caused by tunneling of DC beneath the 786-0 epithelial cell in culture. To confirm that mDCs mediate infection by vims, immature and poly (I:C) treated mDCs are incubated with SARS-CoV. No direct infection is observed (Figure 5, Panel B- left); however, mature mDCs readily transfer viras that infects Vero cells (Figure 5, Panel B-right). Transfer is inhibited by specific anti-S mouse antiseram (Figure 5, Panel B-right), documenting that cell mediated transfer of SARS-CoV is mediated by mDCs and is dependent on the interaction of S glycoprotein with mDCs.
SARS-CoV Entry Neutralization with Peptide Mimetics Mimetics of the coiled-coil region are analyzed for their ability to inhibit viral vector gene expression. A peptide derived from the distal portion of the heptad repeat 2 showed a reproducible and dose-dependent inhibition of viral entry (Figure 6, Panel A)
Example 2 Virus Neutralization and Protective Immunity with a DNA Vaccine for the SARS-coronavirus
Material and Methods
Immunogen and plasmid construction Plasmids encoding different versions of SARS-CoV spike (S) protein (Urbani strain, GenBank AY278741) are synthesized using human-prefened codons as described in Example 1 herein. Protein expression is confirmed by western blot analysis (Kong et al., 2003, J. Virol. 77:12764-12772) with serum from a recovered patient (provided by W. Bellini, CDC). Vaccination Female BALB/c mice (6-8 weeks old; Charles River Labs) are immunized with 25 μg of plasmid DNA in 200 μl of PBS (pH 7.4) at weeks 0, 3 and 6.
Flow cytometric analysis of intracellular cytokines CD4+ and CD8+ T-cell responses are evaluated by using intracellular cytokine flow cytometry (ICC) for IFN-γ and TNF-α as described by Kong et al., (2003, J. Virol. 77, 12764-12772) with peptide pools (17-19 mers overlapping by 10 amino acids, 2.5 μg/ml each) covering the SARS-CoV S protein. Cells are then fixed, permeabilized, and stained using rat monoclonal antimouse CD3, CD4, CD8, IFN-γ and TNF-α (BD-Pharmingen). The IFN-γ- and TNF-α positive cells in the CD4+ and CD8 cell populations are analyzed with the program Flow Jo (Tree Star, Inc.).
ELISA for mouse anti-SARS-s IgG The mouse anti-SARS-CoV S IgG ELISA titre is measured using a modified lectin-capture method described by Kong et al., 2003, J. Virol. 77: 12764- 12772, except the Myc-tagged, transmembrane-domain truncated SARS-CoV S protein (SARS-SDTM-Myc) is used for capture.
Inhibition of viral gene transfer to measure mouse antibody titre SARS-CoV S pseudotyped lentivimses expressing a luciferase reporter gene are produced by teansfecting 293T cells with the following plasmids: 7 μg of pCMVΔR8.2, 7 μg of pHR'CMV-Luc and 400 ng CMV/R-SARS-S. Cells are transfected overnight, washed and replenished with fresh media. Forty-eight hours later, supernatants are harvested, filtered through a 0.45 μm syringe filter, aliquotted and used immediately or frozen at 280°C. p24 levels are measured from different viral stocks using the Coulter HIV-1 p24 Antigen Assay kit (Beckman
Coulter). Antisera are mixed with 100 ml of pseudovimses at various dilutions and added to Vero cells in 48-well dishes (30,000 cells per well). Plates are washed and fresh media are added 14-16 hours later. Forty-eight hours after infection, cells are lysed in mammalian cell lysis buffer (Promega). A standard quantity of cell lysate is used in a luciferase assay with luciferase assay reagent (Promega) according to the manufacturer's protocol.
Neutralization of SARS-CoV by mouse immune antisera Two-fold dilutions of heat-inactivated sera are tested in a microneutralization assay for the presence of antibodies that neutralize the infectivity of 100 TCID50 of SARS-CoV on Vero cell monolayers, using four wells per dilution on a 96-well plate. The presence of viral cytopathic effect (cpe) is tested on days 3 and 4. The dilution of semm that completely prevents cpe in 50% of the wells is calculated by the Reed Muench formula (Reed & Muench, 1938, Am. J. Hyg. 27:493^197).
Challenge of immunized mice with SARS-CoV Vaccinated mice are lightly anaesthetized with isoflurane and inoculated with 50 ml of diluted vims (104 TCID50 of SARS-CoV; Urbani strain) intranasally according to institutional animal care and use guidelines in an ABSL3 facility. On day 2, mice are euthanized and lungs and nasal turbmates are removed and stored at 270°C until the end of the study. The frozen tissues are thawed and homogenized in a 10% (lungs) or 5% (nasal turbinates) w/v suspension in Leibovitz 15 medium (Invitrogen), and vims titres are determined in Vero cell monolayers in 24- and 96-well plates. Vims titres are expressed as TCID50 per g of tissue. The lower limit of detection of infectious vims in 10% and 5% w/v suspensions is 1.5 for lung and 1.8 logio TCID50 per g for nasal turbinate homogenates, respectively.
Depletion of T-cell subsets in vivo To deplete specific T-cell subsets, known rat monoclonal antibodies (anti- mouse CD4 (GK1.5), anti-mouse CD8(2.43) or anti-mouse CD90(30-H12)), prepared as described by Epstein et al., 2000, Int. Immunol. 12:91-101, are administered by intraperitoneal injection (1 mg each in 1ml PBS) 48 hours before challenge. The depletion is confirmed before challenge, as described in the descriptions of Figures 11 and 12 herein. Passive transfer of immunoglobulins IgG from mice immunized with plasmid DNA encoding SΔCD (immune) or no insert (controls) is purified from sera using a Protein A Antibody Purification Kit (Sigma), and neutralization activity is confirmed using the mouse pseudotyping assay. Briefly, 0.3 ml of purified IgG (from approximately 1 ml of semm) is administered intravenously into each recipient naive mouse (n = 4 per group) by tail vein injection 24 hours before challenge. Sera are collected from recipient BALB/c mice 3 hours post transfer to confirm the neutralizing antibody titre in recipient animals.
Adoptive T-cell transfer studies T cells from vaccinated (immune) or nonimmune (control) mice are enriched using a Pan T-Cell Isolation Kit (Miltenyi Biotec). Approximately 3 x 107 T cells in 0.5 ml PBS are administered into each recipient naive BALB/c mouse intravenously through the tail vein 24 hours before challenge. There are four recipient mice per group.
Results
Two sets of cDNAs encoding the SARS-CoV S glycoproteins are prepared using modified codons to optimize expression and to minimize recombination with endogenous coronavimses. The native leader sequence is retained in one set of vectors (Figure 7, Panel A) and replaced in another set with a leader sequence derived from the interleukin-2 gene. Expression is not significantly altered by this leader sequence substitution. Two S carboxy-terminal mutants, one that truncated the cytoplasmic domain (SΔCD) and another that deleted the transmembrane and cytoplasmic domain (SΔCD), are prepared and expression of these cDNAs by a mammalian expression vector suitable for human vaccination is confirmed (Figure 7, Panel B)
The plasmids encoding these modified S glycoproteins are analyzed for their ability to elicit antiviral immunity after intramuscular injection in BALB/c mice. Injection of S, SΔTM and SΔCD expression vectors induces a substantial immune response. A marked increase is observed in the number of SARS-CoV S- specific CD4 T cell immune responses (Figure 8, Panel A), as measured by intracellular cytokine staining for interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In addition, substantial SARS-CoV S-specific CD8 cellular immunity is detected at levels at least sevenfold above the background response. Humoral immunity is initially assessed using an enzyme-linked immunosorbent assay (ELISA) with lectin-captured SΔTM protein expressed in 293T cells. Substantial end-point dilution antibody titres are observed in all groups, ranging from approximately 1:400 to approximately 1:2,000 (Figure 8, Panel B-left). To analyze the ability of these S glycoprotein plasmids to elicit a neutralizing antibody response, a pseudotyping assay is performed as described in Example 1 herein. Immunization of animals with the SARS-CoV SΔCD expression vector induces substantial neutralizing antibody titees ranging from 1 :50 to 1 : 150, unlike the vector control (Figure 8, Panel B-middle). The S expression vector with the transmembrane domain deleted, SΔTM, also induces neutealizing r antibodies, but the titees are lower (from 1 :25 to 1:75). The vector with the partial cytoplasmic domain deletion induced the optimal response (Figure 8, Panel B- right). Similar neutralization is observed when the complete cytoplasmic domain is removed, suggesting that synthesis of the glycoprotein on the cell surface, without the cytoplasmic domain, is important for an optimal immune response. This is because it gives rise to a more native stracture relevant to the function of the virus. Possibly, the transmembrane region helps to form a more physiological form of the S protein by anchoring the protein on the membrane, preserving conformational determinants and/or stabilizing the formation of the putative trimer. The same antisera are also used in a SARS-CoV microneutialization assay to assess the neutralization titre against SARS-CoV. Again, antisera from SΔCD- vaccinated mice elicited the most potent neutealizing antibody responses (Figure 8, Panel B-right).
Infection of non-human primates with SARS-CoV has been reported, with evidence of pulmonary pathology and seroconversion (Fouchier et al., 2003,
Nature 423:240). More recently, an animal model that may better reproduce the signs and time course of the human disease has been described. In this model, which uses feπets, the development of pneumonitis and increased viral replication have been observed (Martina et al., 2003, Nature 425:915). A murine model of SARS-CoV replication has also been described recently, in which inteanasal administration of 104 50%> tissue culture infectious dose (TCID50) units of SARS- CoV leads to virus replication in the lungs and nasal turbinates within 1-2 days (Subbarao et al., 2004, J. Virol. 78:3572-3577). This model can be used to examine immune protection against viral replication in the respiratory tract as a measure of vaccme efficacy. BALB/c mice are immunized with the various plasmid DNAs encoding S protein and challenged 30 days after the final boost. Animals are challenged inteanasally with 104 TCIDso units of SARS-CoV (Urbani strain), and viral replication in the respiratory tract is measured 2 days later. This analysis shows that the most potent immunogen, SARS SΔCD, provides a >106- fold reduction in viral load in the lungs compared with a control group injected with vector alone, in which mean viral titees of >108 are observed (Figure 9, Panel A; P = 0.0079). Although the S plasmid with the deleted transmembrane domain elicits a lower neutealizing antibody response, it is equally effective in reducing viral replication in the lungs. A 60- to 300-fold reduction of vims titre in the nasal turbinates is also observed (Figure 9, Panel B; P = 0.0079). In both cases, evidence of productive SARS-CoV replication is not observed in mice vaccinated with plasmid DNAs encoding SΔCD or SΔTM.
To define the mechanism of protection, T-cell depletion with specific monoclonal antibodies is performed, and depletion in the lung and spleen is confirmed (Figures 11 and 12). Depletion with CD4 or CD8, alone or in combination with CD90, does not affect vaccine-induced immunity (Figure 10, Panel A). This finding is con-firmed when the role of T-cell immunity is further analyzed by adoptive T-cell transfer; donor immune T cells are unable to reduce pulmonary viral replication in recipient animals (Figure 10, Panel B; P = 0.49). By contrast, passive transfer of purified IgG from immunized mice, but not control mice, provides immune protection (Figure 10, Panel C-right; P = 0.0286) comparable to that observed in DNA-vaccinated animals (Figure 9, Panel A). These findings indicate that immune T cells do not control pulmonary viras replication in this animal model, although it remains possible that T cells contribute to viral clearance if replication persists.
Example 3 Regulation and Assembly of Synthetic SARS Coronavirus
Materials and Methods
Development of synthetic SARS-Co V expression vectors The protein sequences of S, N, M, and E from SARS-CoV Urbani strain (GenBank accession number AY278741) (Rota et al., 2003, Science 300, 1394- 1399) are reverse-translated by the GCG Package (Genetic Computer Group, Inc., Madison, WI) using human prefeπed codons. Oligonucleotides covering these four genes are purchased from Sigma-Genosys Inc. Each of the oligonucleotides is 75 base pairs with 25 nt of overlap. The codon-modified genes are assembled by PCR PfuTurbo® hotstart (Stratagene) high fidelity DNA polymerase. The PCR conditions are optimized with a PCR optimization kit (Stratagene) on a gradient Robocycler (Stratagene). Full-length synthetic S, N, M, and E are cloned into the Xba I and Bam HI sites of the mammalian expression vector CMV/R derived from pNGVL-3 (Manthoφe et al., 1993, Hum. Gene Ther. 4, 419-431) and confirmed by DNA sequencing.
Transient transfection and electron microscopy (EM) 293T cells are maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco-BRL), supplemented with 10% fetal bovine serum (FBS). Plasmid DNAs are purified using double cesium chloride sedimentation gradients. Approximately 3xl06293 T cells are plated in a 10 cm dish one day before transfection. 3 μg of each plasmid (each containing one of the SARS-CoV genes) are mixed and used to teansfect 293T cells, using the calcium phosphate method (Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-2752). The vector backbone is used as filler DNA to maintain the same amount of DNA in each transfection. At 63 hours after transfection, the cells are lifted from plates by resuspension in DMEM and then pelleted in a 15 ml conical tube by centrifugation at 1000 φm. The supernatant is removed and a 10-fold volume of fixing solution is added (3% glutaraldehyde and 3% formaldehyde, cacodylate buffer, pH 7.3; Tousimis Research Coφoration, Rockville, MD). The specimens are mixed gently, and analyzed in the electron microscopy core laboratory at the University of Michigan Health Center.
Viral capsid production and buoyant density gradient analysis 3xl06293 T cells are transfected with 3 μg of each of pCMV/R-S, N, and M in a 10 cm tissue culture dish with DMEM medium. The cells are harvested after three days and freeze-thawed three times in PBS. The cleared lysates are pelleted onto 50% of an Optipre™ (IODLXANOL) medium (Invitrogen) at 20000 φm with a Sorvall Surespin 630 rotor, and the final concenteation of Optipre is adjusted to 30%. A density gradient is formed by centrifugation at 75k for 3.5 hours with a NTI100 rotor (according to the manufacturer's instractions; Invitrogen). The collected fractions are weighted at 100 μl of each fraction and plotted with density by fractions. A 20 μl aliquot of each fraction is separated on a 4-15%) SDS-PAGE gel, transfened onto an Immobilon™-P membrane and blotted with human anti-SARS-CoV sera.
Results Mammalian expression vectors encoding different viral genes are analyzed to define the SARS genes essential for viral assembly. These genes include the S, M, N and E proteins synthesized from the predicted amino acid sequence by reverse translation, with codon usage typical of human cells. The DNA sequence of each gene is confirmed, and expression is confirmed by in vitro transcription/translation and/or by Western blot analysis. Expression of the SARS S, M, N and E genes is analyzed in different combinations in transfected 293 cells to evaluate the contribution of these gene products to viral assembly. Table 1 below shows the results of a scanning TEM analysis of capsid formation in 293T cells transfected with plasmids containing the indicated genes.
TABLE 1 Summary of SARS-CoV capsid formation after co-transfection of viral genes by scanning TEM
Figure imgf000083_0001
Coexpression of all four gene products results in the assembly of electron- dense stractures that resemble nucleocapsids of ~100 nm, characteristic of coronavimses. No single viral gene is able to support the formation of viral capsids within these cells (Table 1). These structures are localized primarily to endosomal vesicles and are similar in size and appearance to replication-competent viras (Ksiazek et al., 2003, N. Engl. J. Med. 348, 1953-1966). The absence of negative-stranded viral genomic RΝA, protease, or the viral polymerase indicates that they are not essential for the formation of the SARS core particles, though the central lucency raised the possibility that RΝA is taken up nonspecifically by the nucleocapsids.
Different combinations of these viral genes are analyzed systematically by co-transfection to examine the minimum requirements for pseudoparticle formation. No gene product alone supports assembly, indicating that at least two proteins are necessary for capsid formation. Additionally, the nucleocapsid does not form in the absence of the M and N protein (Table 1). Though co-transfection of the N and M proteins allowed detection of viral capsids intracellularly, no budding vims or corona-like structures are visible in these transfected cells. The addition of the spike (S) glycoprotein allowed for budding and formation of a corona-like stracture, indicating that it is likely to be important not only for viral fusion but also for maturation and egress through cells. Buoyant density gradient sedimentation analysis is performed to characterize these synthetic SARS-CoV particles. Because the capsid core is found in higher quantities within cells, lysates are prepared from transfected cells that are frozen and thawed three times. When fractions from a gradient of clarified cell lysates are analyzed by Western blot with human immune semm, the peak of viral protein expression, composed primarily of N and S proteins, is detected at a density of 1.18 g/ml, comparable to the buoyant density described for other coronavimses.
The interaction between these viral proteins is analyzed further to establish the biochemical basis for viras particle formation. The major structural proteins are synthesized by transcription and translation with rabbit reticulocyte lysates in vitro, as are mutant forms of M. The full length M protein is able to associate with the N protein when both are co-synthesized, and the COOH-terminal cytoplasmic domain is required for this association. In fact, the COOH-terminal region of M bound more avidly to N than the full-length protein, suggesting that this domain may be less exposed during protein teanslation of the complete protein in vitro. Co-expression of a mutant M gene that lacked the COOH terminal domain abolished its ability to form nucleocapsids. In contrast, deletion of the NH2- teπninal putative extracellular region had no effect on particle formation or their association. In contrast, the S glycoprotein is unable to interact with N in this assay, though it did bind to M and E. Together, these findings suggest that M plays a pivotal role in nucleocapsid assembly through its ability to interact with N through its COOH-terminal cytoplasmic domain and with S through other regions. It thus serves as a critical bridge between essential internal and external components of the viras. Example 4 Characterization of the Humoral Response during SARS Coronavirus Infection
Materials and Methods Plasmids and ELISA A codon-modified spike (S) gene deleted of the transmembrane region to improve production and secretion, as well as a nucleocapsid (N) gene, are cloned from plasmids described in Example 1 herein using overlapping PCR. The S and N genes are tagged with Myc and His, respectively, and inserted into plasmid mammalian expression vectors as described in Example 2 above. The plasmids are transfected into the human renal epithelial 293 cell line. After cell culture, the S protein supernatant is filtered and the His-tagged N protein is isolated from transfected cell lysates by His-affinity column chromatography. The S protein filtered supernatant and the purified N protein are then applied to lectin-coated ELISA plates (Kong et al., 2003, J. Virol. 77:12764-12772).
The ELISA plates are first coated with Galanthus nivalis lectin by incubation of the wells with a 10 μg/ml solution overnight at 4°C. After blocking with 10% fetal bovine serum and washing twice with PBS containing 0.2% Tween 20 (PBS-T), S protein supernatant and purified N protein are added to the wells and incubated for one hour at room temperature. The plates are washed with PBS- T, and then the sera samples are added to the wells, with varying dilutions, and then incubated for 1 hour. The plates are washed with PBS-T and a horseradish peroxidase conjugated secondary antibody, at a 1:5,000 dilution, is added to the wells. Plates are incubated with the secondary antibody for 1 hour and then washed with PBS-T. Substeate (Sigma Fast o-phenylenediamine dihydrochloride; catalog no. P-9187) is then added to each well for 30 minutes. The reaction is stopped by adding 1 N H2S04, and the optical density (OD) reading is taken at 450 nm.
Neutralization Assays SARS-CoV neutealizing antibodies in patient sera are assayed using a lentiviral vector pseudotyped with the SARS-CoV S (Urbani) glycoprotein, which mediates gene delivery and shows similar specificity and sensitivity to neutralization as replication-competent SARS-CoV as shown in Examples 1 and 2 above. Diluted semm samples are mixed at various dilutions with an S pseudovims that encodes a luciferase gene. The mixtures are incubated with 782- O human renal epithelial cells for 16 hours and then the cells are washed for 48 hours after infection. Cells are then lysed, and the luciferase assay is performed. High luminescence activity conelates directly with infection, whereas low luminescence activity indicates the presence of neutealizing antibodies in the test semm sample.
Results
Analysis of immunoglobulin responses by ELISA Semm samples from 5 SARS patients are collected at different times after the onset of symptoms by investigators from the Centers for Disease Control (CDC). Antibody levels are quantitated by ELISA using γ-iπadiated (106 Rads) semm samples (Figure 13). While IgM antibody responses against the S protein are detected in only one patient at two time points (days 10 and 11), a substantial IgG response to S is detected in all samples measured (Figure 13, Panel A). There is a small increase in the IgA response to S in one patient that is not observed in other subjects (Figure 13, Panel A). The antibody response to N differs in time and magnitude from that observed for S. IgM antibodies against N are detected in only two samples collected (day 30), and those values decreased subsequently (Figure 13, Panel B). Two patients initially have undetectable IgG responses (days 7 and 14) but show a substantial increase on days 26 and 41, respectively. Several patients have moderately positive IgG levels at early time points (days 5 and 10) (Figure 13, Panel B). In contrast to the S response, IgA directed to N is detected at low levels early and increased with time (Figure 13, Panel B). Two alternative assays, an ELISA and an immunofluorescence assay (IF A), using SARS-CoV infected Vero cell lysates as the antigen, failed to detect vims-specific immunoglobulins in three subjects where they are readily detected by the ELISA method developed here (Figure 13, open symbols). The results demonstrate that this mammalian recombinant protein ELISA, particularly the measurement of the IgG response to S, provides sensitive and specific detection of infection early after suspected disease.
Evolution of neutralizing antibody responses during infection To characterize the neutralizing antibody response, a replication-defective lentiviral vector encoding the luciferase reporter is pseudotyped with S and used as the neutealization target as described in Example 1 and Example 2 herein. Nine of eleven samples contain neutealizing antibodies (Figure 14). The two negative samples are from the earliest time points (days 5 and 7). These results suggest that neutealizing antibodies to SARS-CoV are not present in the early phases of infection but develop over time and coπelate with recovery from disease.
Example 5 Evasion of Antibody Neutralization in Emerging SARS Coronaviruses
Materials and Methods Plasmids and cell lines The human renal adenocarcinoma cell line 786-0 is purchased from the ATCC (Manassas, VA) and maintained in DMEM+10% FBS. The gene encoding SARS-CoV SfJJrbani) is synthesized using-human prefeπed codons as previously described in the Examples herein. Clones (cDNAs) encoding S proteins from various strains are constructed using the Quikchange XL kit (Stratagene) to introduce divergent amino acids into the gene encoding S(Urbani) according to the predicted translated sequence as disclosed in Genbank. S genes coπesponding to following variant proteins are constructed: SfBJOl) (human isolate) (Genbank No. AY278488); S(FRA) (human isolate) (No. AY310120); S(GD01) (human isolate) (No. AY278489); S(GZ02) (human isolate) (No. AY390556);
S(HGZ8L1-A) (human isolate) (No. AY394981); S(Tor2) (human isolate) (No. AY274119); S(ZS-A) (human isolate) (No. AY394997); S(GD03T0013) (human isolate) (No. AY525636), S(SZ3) (palm civet isolate) (No. AY304486), S(SZ16) (palm civet isolate) (No. AY304488) according to the manufacturer's protocol. A stop codon is introduced at amino acid 1153 of SfJJrbani) also using Quikchange XL kit to make the relevant S(l 153) truncations. All cDNAs are cloned into CMV/R vector as described in the Examples herein as a Sal I BamH I fragment, and expression is confirmed by Western blot analysis using rabbit anti-S polyclonal antibody (IMG-541) (Imgenex).
Production of pseudotyped lentivims Recombinant lentivimses expressing a luciferase reporter gene are produced as described previously in the Examples herein. Briefly, 5 l06 293T cells are transfected overnight using calcium phosphate reagent (Invitrogen) with following plasmids: 7 μg of pCMVΔR8.2, 7 μg of pHR'CMV-Luc, and 400 ng CMV/R-SARS-S. Supernatants are harvested 48 hours after transfection, filtered through a 0.45 μm syringe filter, aliquotted, and used immediately or frozen at - 80°C. Levels of p24 levels are measured from different viral stocks using "The Coulter HIV-1 p24 Antigen Assay" kit (Beckman Coulter) to estimate the viral titer.
Vaccination and purification of immune IgG Five female Balb/c mice (6-8 weeks old) (Charles River) per group are immunized with 25 μg plasmid DNA (in 200 μl PBS pH 7.4) expressing coπesponding immunogen three times at weeks 0, 3 and 6. Ten days after the last immunization, sera are collected, combined, and subsequently purified using "Nab™ Spin Purification Kit" (Pierce) according to the manufacturer's protocol.
Neutralization and inhibition assays 786-0 cells (30,000 cells/well) are plated onto a 48-well dish the day before infection. The various pseudovimses, normalized for the amount of p24 added, are mixed with purified mouse IgG ,human monoclonal antibodies or recombinant human ACE-2 ectodomain (aa 1-740)(R&D Systems) at indicated concentrations, and incubated for 5 to 10 minutes before addition to 786-0 cells. Cells are infected for 14-16 hours, and collected for luciferase assay using "Luciferase assay reagent" (Promega) 48 hours later. Biochemical and immunoprecipitation analyses 293T cells are transfected with 10 μg plasmid DNA overnight using calcium phosphate reagent (Invitrogen) and replenished with fresh media. 48 hours later, transfected cells are harvested, washed with PBS, and resuspended in lysis buffer (50 mM Hepes, 150 mM Nacl, 1% NP-40, pH 7.0, protease inhibitor cocktail) on ice for 45 minutes. Cell lysates are cleared by centrifugation at 13,000 φm for 10 minutes at 4°C. A 25 μg sample of cell lysate is incubated with mouse immune IgG (5 μg) , or human monoclonal antibodies (2 μg ) for 1 hour at room temperature, followed by incubation with 25 μl of agarose-protein G (Pierce) for another hour. Immunoprecipitates are washed three times with lysis buffer, resuspended in SDS-loading buffer (Quality Biological), and separated on 4-15% gradient SDS-PAGE (Bio-Rad), followed by Western blot analysis.
Results
Analysis of the sensitivity of SARS-CoV variants to antibody neutralization S sequences from alternative human isolates and from two palm civet vims isolates are synthesized as human codon modified cDNAs and inserted into expression vectors as described in Example 2 above. Pseudovimses constructed with the S proteins from these variant strains show similar incoφoration of diverse S proteins into virions (Figure 15). Each pseudovims is incubated independently with immune antisera from mice vaccinated with S(Urbani) that inhibited cellular entry of this prototype strain. Similar patterns of inhibition are observed with pseudovimses from all early 2003 human isolates, which show minor differences in neutralization by this polyclonal antisera (Figure 16, Panel A). In contrast, the S(GD03T0013) pseudotype is qualitatively different in that it is markedly resistant to antibody inhibition (Figure 16, Panel A, bottom right panel). Unexpectedly, these antisera enhance cellular entry of two pseudovimses constructed with palm civet S glycoproteins (Figure 16, Panel B). Identification of antibodies that enhance virus entry in humans To determine whether antibodies that enhance entry can be detected in humans, neutealizing monoclonal antibodies (mab) derived from Epstein-Ban virus transformation of memory B cells from a recovered patient are analyzed (Traggiai et al., 2004, Nat.Med.). Several mab with similar specificities are defined. These antibodies inhibit cellular entry of pseudotyped viras constructed with S (Urbani) (Figure 16, Panel C, left), which is representative of the eight early 2003 human isolates (Figure 16, Panel A). While they strongly inhibit S(Urbani) pseudotyped vims, minimal effects were seen on S(GD03T0013) and several mab enhance palm civet viras entry (Figure 16, Panel C, middle and right), similar to the results effects observed with polyclonal antisera. This finding confirms that enhancing antibodies can be detected in humans and further suggest that enhancement is mediated by a specific epitope on S.
The affinity of S pseudotyped viruses for the hACE-2 receptor is assessed by using inhibition with soluble recombinant bACE-2. Cellular entry of pseudovims constructed with SfJJrbani) is substantially inhibited by hACE-2 (Figure 17, Panel A, left). In contrast, cellular entry of pseudovims constructed with S(GD03T0013) is much less sensitive to hACE-2 inhibition (Figure 17, Panel A, right). To determine whether the palm civet and GD03T0013 vimses can be neutralized by vaccination with the homologous S, mice are injected with a DNA vaccine encoding full-length S from these respective isolates. Neither antisera can strongly inhibit cellular entry of pseudovims constructed with the homologous S, even after repeated immunization (Figure 17, Panels B and C).
Definition of the genetic determinants of sensitivity antibody neutralization sensitivity and antibody enhancement sensitivity The genetic determinants of enhancement and neutralization are defined by expressing full length recombinant S protein from either SfJJrbani), S(SZ3), or relevant chimeras (Figure 18, Panel A) and constructing the conesponding pseudovimses. A central region, between amino acid 248 and 501, has been implicated previously in binding to the ACE-2 receptor (Babcock et al., 2004, J. Virol. 78:4552; Wong et al., 2004, J.Biol.Chem. 279:3197 9, 10). This region from SfJJrbani) mediates more efficient entry of viras into 786-0 renal epithelial cells (Figure 18, Panel B, left). Importantly, insertion of this region from S(Urbani) into S(SZ3) rendered the chimeric pseudovims sensitive to neutralization (Figure 18, Panel B, right, SU). Conversely, intioduction of the hACE-2 receptor binding region from S(SZ3) into SfJJrbani) switches its sensitivity from neutealization to antibody-dependent enhancement (Figure 18, Panel B, right, US). The five amino acid differences in this region therefore mediate both the differential ACE-2 affinity and sensitivity to antibodies. The interaction of S antisera is nearly 20-fold higher with S(Urbani) compared to
S(SZ3) (Figure 18, Panel C), suggesting that these lower affinity interactions with SZ3 may contribute to enhancement, consistent with the finding that the hACE-2 receptor binding region determines this effect.
Development ofS immunogens that avoid antibody dependent enhancement of viral entry A variety of S-immunogens are evaluated to identify S-immunogens that avoid antibody dependent enhancement of viral entry are developed. A series of teansmembrane-deleted immunogens are prepared. One of these mutants, truncated at amino acid 1153, induces neutralizing antibodies to human isolates, wherein the neutealizing antibodies do not cause antibody-dependent enhancement of viras entry (Figure 19, Panel A). Biochemical analysis reveals that this antisera reacts poorly with S(SZ3) and US but reacts strongly with SfJJrbani) and SU (Figure 19, Panel B), consistent with the patterns of neutealization and enhancement. This finding demonstrates that immunogens can be developed that elicit neutralizing antibodies for human isolates that do not enhance civet pseudovims entry. Monoclonal antibodies that have vims neutralization activity for SARS-CoV (Urbani) in the absence of antibody-dependent enhancement of viral entry activity are also identified. Though most mab showed enhancement, some mabs, such as SI 10, were identified that failed to enhance entry while mediating potent neutealization (Figure 19, Panel C). All publications and patents mentioned in this specification are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

What Is Claimed Is:
Claiml : A vaccine composition comprising a nucleic acid molecule that comprises a polynucleotide that encodes an extracellular domain of a SARS-CoV S protein, or a fragment or portion of such extracellular domain.
Claim 2: The vaccine composition of claim 1 , wherein codons of the polynucleotide have been optimized for expression in human cells.
Claim 3: A vaccine composition according to claim 1 wherein the amino acid sequence of the encoded extracellular domain is at least 90% identical to the amino acid sequence of residues 14 to 1190 of SEQ ID NO:3.
Claim 4: A vaccine composition according to claim 1 , wherein the nucleic molecule further comprises a vector polynucleotide that is replicable in mammalian cells.
Claim 5: A vaccine composition according to claim 1 , wherein the nucleic molecule additionally encodes a transmembrane domain or a fragment or portion of such transmembrane domain.
Claim 6: A vaccine composition according to claim 1, wherein the nucleic molecule additionally encodes a signal polypeptide domain or a fragment or portion of such signal polypeptide domain.
Claim 7: A vaccine composition according to claim 5, wherein the nucleic molecule additionally encodes a signal polypeptide domain or a fragment or portion of such signal polypeptide domain.
Claim 8: A method of protecting a subject from infection by SARS-CoV comprising administering a nucleic acid molecule to the subject, wherein the nucleic acid molecule comprises a polynucleotide that encodes an extracellular domain of a SARS-CoV S protein, or a fragment or portion of such extracellular domain.
Claim 9: A method according to claim 8, wherein the extracellular domain portion is expressed on the surface of cells in said subject.
Claim 10: A method according to claim 8, wherein said vaccine is sufficient to cause the production of neutealizing antibodies to SARS-CoV in said subject.
Claim 11: A method according to claim 8, wherein the subject is a human or a non-human mammal.
Claim 12: A method according to claim 8, wherein the subject is a human.
Claim 13: A method of treating a subject for infection from SARS-CoV comprising administering to the subject a SARS-CoV neutralizing antibody.
Claim 14: A method according to claim 13, wherein the antibody binds to the extracellular domain of the S protein.
Claim 15: A method according to claim 14, wherein the antibody is a monoclonal antibody.
Claim 16: A method of screening for inhibitors of SARS-CoV infection comprising: (a) incubating a cell susceptible to SARS-CoV infection with a SARS-CoV S pseudotyped viral vector, in the presence or absence of a test inhibitory agent; (b) assaying entry of said pseudotyped viral vector into said cell; (c) identifying the test inhibitory agent as an inhibitor of SARS- CoV infection if entry of said pseudotyped viral vector into said cell is reduced in the presence of the test inhibitory agent. ,
Claim 17: A method of measuring the presence or concentration of an anti- SARS-CoV neutralizing antibodies in a biological sample of a mammal, wherein said method comprises the steps of: (a) incubating a cell susceptible to SARS-CoV infection with a SARS-CoV S pseudotyped viral vector, in the presence or absence of the biological sample; (b) assaying entry of said pseudotyped viral vector into said cell; (c) identifying the biological sample as a positive for the presence of SARS-CoV neutralizing antibodies if entry of said pseudotyped viral vector into said cell is reduced in the presence of biological sample.
Claim 18: An immunoassay that measures the presence or concenteation of an anti-SARS-CoV S protein antibody in a biological sample of a mammal, wherein said immunoassay comprises the steps of: (a) contacting said biological sample with mammalian expressed S protein antigen under conditions sufficient to permit anti-S-protein antibody, if present in said sample, to bind to said antigen and form an antigen-anti-S-protein antibody complex; (b) contacting said formed antigen-anti-S-protein antibody complex with an anti-S-protein antibody binding molecule under conditions sufficient to permit said anti-S-protein antibody binding molecule to bind to anti-S-protein antibody of said formed antigen-anti-S-protein antibody complex and form an extended complex; and (c) determining the presence or concentration of said anti-S- protein antibody in said biological sample by determining the presence or concentration of said formed extended complex..
Claim 19: A method of protecting a subject from infection by SARS-CoV comprising exposing the subject to isolated S protein.
Claim 20: A method according to claim 19, wherein the isolated S protein is mammalian expressed isolated S protein.
Claim 21: A vaccine composition comprising at least a portion of the extracellular domain of isolated S protein.
Claim 22: A vaccine composition according to claim 1, wherein the vaccine composition does not exhibit antibody-dependent viras enhancing activity.
Claim 23 : A vaccine composition according to claim 21 , wherein the vaccine composition does not exhibit antibody-dependent viras enhancing activity.
Claim 24: A vaccine composition according to claim 22, wherein the vaccine compostion does not exhibit antibody-dependent viras enhancing activity with respect to a Palm civet SARS-CoV isolate selected from the group consisting SZ3 or SZ16.
Claim 25 : A method of assaying a vaccine composition for antibody- dependent virus enhancing activity comprising the steps of: (a) generating an immune response to a vaccine composition in an animal; ( ) isolating antibodies or antisera from the animal; 00 assaying the ability of the antibodies or antisera to enhance cellular entry of a SARS-CoV viras strain or a coπesponding pseudovims; (d) identifying the vaccine composition as having an antibody- dependent vims enhancing activity if the antibodies or antisera enhance cellular entry of the SARS-CoV viras strain or the conesponding pseudotype.
Claim 26: A method according to claim 25, wherein the SARS-CoV viras steain is selected from the group consisting of a Palm civet isolate, a raccoon dog isolate, and a Chinese fenet badger isolate.
Claim 27: A vaccine composition according to claim 1 , where the vaccine composition further comprises a polynucleotide encoding the SARS CoV M protein, or a portion or fragment thereof, and a polynucleotide encoding a SARS CoV N protein, or a fragment or portion thereof, wherein the encoded S, M and N proteins are capable of forming corona-like structures when present in the same cell.
Claim 28: The method of claim 8, further comprising administering to the subject a polynucleotide encoding the SARS CoV M protein, or a portion or fragment thereof, and a polynucleotide encoding the SARS CoV N protein, or a fragment or portion thereof, wherein corona-like stractures are generated in transfected cells of said subject.
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