WO2005081716A2 - DNA VACCINES TARGETING ANTIGENS OF THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS (SARS-CoV) - Google Patents

DNA VACCINES TARGETING ANTIGENS OF THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS (SARS-CoV) Download PDF

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WO2005081716A2
WO2005081716A2 PCT/US2004/039579 US2004039579W WO2005081716A2 WO 2005081716 A2 WO2005081716 A2 WO 2005081716A2 US 2004039579 W US2004039579 W US 2004039579W WO 2005081716 A2 WO2005081716 A2 WO 2005081716A2
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polypeptide
nucleic acid
sars
protein
cov
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WO2005081716A3 (en
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Tzyy-Choou Wu
Chien-Fu Hung
Tae Woo Kim
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The Johns Hopkins University
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    • 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
    • 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
    • A61K39/0005—Vertebrate antigens
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention in the field of immunology, virology and medicine, provides immunogenic compositions and methods for inducing enhanced antigen-specific immune responses, particularly those mediated by cytotoxic T lymphocytes (CTL), using chimeric or hybrid nucleic acid molecules that encode an endoplasmic reticulum chaperone polypeptide, e.g., calreticulin, and a polypeptide or peptide antigen ofthe SARS coronavirus (SARS-CoV).
  • CTL cytotoxic T lymphocytes
  • SARS-CoV SARS coronavirus
  • Advantages of DNA is that it is relatively stable, and it can be easily prepared and harvested in large quantities.
  • naked plasmid DNA is relatively safe and therefore can be repeatedly administered as a vaccine (Donnelly et al, supra; Robinson, supra).
  • naked DNA lacks cell targeting specificity making it important to find an efficient route for delivery into appropriate target cells, such as professional antigen-presenting cells (APCs).
  • APCs professional antigen-presenting cells
  • Intradermal (i.d.) administration of DNA immunogens or vaccines using a gene gun represents a convenient form of delivery to professional APCs, such as dendritic cells (DCs), in vivo (Condon, C et al, 1996, Nat Med 2: 1122-8).
  • DCs are the most potent professional APCs for priming CD4+ T helper and CD8+ T cytotoxic or killer T cells in vivo (reviewed in Cella, M et al, 1997, Curr Opin Immunol 9:10-16; Hart, D ⁇ , 1997, Blood 0:3245-3287; Steinman, RM, 1991, Annu Rev Immunol 9:271-296).
  • D ⁇ A vaccines include "naked" D ⁇ A, such as plasmid D ⁇ A (U.S. Patent ⁇ os. 5,580,859; 5,589,466; 5,703,055), viral D ⁇ A, and the like.
  • plasmid D ⁇ A U.S. Patent ⁇ os. 5,580,859; 5,589,466; 5,703,055
  • viral D ⁇ A and the like.
  • a D ⁇ A molecule encoding a desired immunogenic protein or peptide is administered to an individual and the protein is generated in vivo.
  • Use of "naked" D ⁇ A vaccines has the advantages of being safe because, e.g.
  • the plasmid itself has low immunogenicity, it can be easily prepared with high purity and, compared to proteins or other biological reagents, it is highly stable.
  • D ⁇ A vaccines have limited potency.
  • Several strategies have been applied to increase the potency of DNA vaccines, including, e.g., targeting antigens for rapid intracellular degradation; directing antigens to APCs by fusion to ligands for APC receptors; fusing antigens to chemokines or to antigenic pathogenic sequences, co-injection with cytokines or co-stimulatory molecules or adjuvant compositions.
  • Antiviral and antitumor vaccines are an attractive approach for treatment of viral illnesses and cancer because they may have the potency to eradicate systemic virus (or virus- infected cells) or tumor cells in multiple sites in the body and the specificity to discriminate between neoplastic and non-neoplastic cells (Pardoll (1998) Nature Med. 4:525-531).
  • Effective anti- viral and most anti-tumor effects ofthe immune system are mediated by cellular immunity.
  • the cell-mediated component ofthe immune system is equipped with multiple effector mechanisms capable of eradicating virus-infected cells and tumors, and most of these responses are regulated by T cells.
  • HPN oncogenic proteins E6 and E7
  • HPN- 16 E7 and E6 are a well-characterized cytoplasmic/nuclear proteins.
  • Calreticulin and Related Proteins Calreticulin an abundant 46 kilodalton (kDa) protein located in the lumen ofthe cell's endoplasmic reticulum (ER), displays lectin activity and participates in the folding and assembly of nascent glycoproteins. See, e.g.,, ⁇ ash (1994) Mol. Cell. Biochem. 135:71-78; Hebert (1997) J Cell Biol. 139:613-623; Nassilakos (1998) Biochemistry 37:3480-3490; Spiro
  • Severe Acute Respiratory Syndrome Coronavirus SARS-CoV
  • the present invention is directed to compositions and methods for stimulating immunity specific for the coronavirus responsible for severe acute respiratory syndrome (SARS).
  • SARS-CoN coronavirus
  • SARS-CoN is the etiologic agent of SARS was demonstrated by experimental infection of macaques (Macacafascicularis), fulfilling Koch's postulates (Fouchier, RA, 2003. Nature 423:240).
  • Knowledge ofthe structure of SARS-CoN and characterization of its complete R ⁇ A genome have provided the basic information that enabled the present inventors to develop[ novel strategies for the prevention of SARS using vaccines.
  • SARS-CoN is a (+)-stranded R ⁇ A virus with a ⁇ 30kb genome encoding replicase (rep) gene products and structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid ( ⁇ ).
  • S protein is thought to be involved with receptor binding, E protein plays a role in viral assembly, M is important for virus budding, and ⁇ protein is associated with viral R ⁇ A packaging (reviewed in Holmes, KN, 2003, J Clin. Invest. 111:1605- 1609. Among these proteins, it was not evident a priori which contain useful SARS-CoN- specific T cell epitopes or epitopes for targeting by neutralizing or protective antibodies.
  • ⁇ protein was shown to generate coronavirus-specific CD8+ T cells, albeit in coronaviruses that infect non-human species (i.e., mouse hepatitis virus and infectious bronchitis virus) and have different tissue tropism (Bergmann, C et al, 1993, J Virol 67:7041-7049; Boots, AM et al, 1991, Immunology 74:8-13; Seo, SH et al, 1997, J Virol 71 :7889-789A; Stohlman, SAet al, 1992, Virology 189:217-22A; Stohlman, SAet al, 1993, J Virol 67:7050-7059).
  • non-human species i.e., mouse hepatitis virus and infectious bronchitis virus
  • SARS-CoN spike (S) protein has been found to bind to angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS CoN on susceptible cells (Dimitrov, DS, 2003 Cell 115:652-653; Li, W et al, 2003, Nature 426:450-454 ; Prabakaran, P et al, 2004, Biochem Biophys Res Commun. 374:235-241; Wang, P et al, 2004, Biochem Biophys Res Commun.
  • ACE2 angiotensin-converting enzyme 2
  • the receptor-binding domain SI is responsible for binding to the ACE2 receptor (Dimitrov, supra; Li et al, supra; Prabakaran et al, supra; Wang et al, supra).
  • innovative approaches interfering with the binding of S 1 to ACE2 such as the immunological approaches disclosed herein, may protect the host from SARS CoN infection.
  • D ⁇ A vaccines in general have the clear advantages of simplicity, stability and safety over viral or bacterial vectors, they suffer from lack of potency, since they do not have the intrinsic ability to amplify and spread as live viral vectors do.
  • the present invention is focused on improved D ⁇ A vaccines comprising epitopes of any one or more ofthe S, ⁇ , M and ⁇ proteins of SARS-CoN.
  • the invention provides a nucleic acid encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide.
  • the antigenic peptide can comprise an MHC Class I-binding peptide epitope.
  • the antigenic peptide e.g., the MHC class I-binding peptide epitope, can be between about 8 amino acid residues and about 11 amino acid residues in length.
  • the endoplasmic reticulum chaperone polypeptide includes any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides; or, analogues or mimetics thereof, or, functional fragments thereof. Such functional fragments can be screened using routine screening tests, e.g., as described in Examples 1 and 2, below.
  • the endoplasmic reticulum chaperone polypeptide comprises or consists of a calnexin polypeptide or an equivalent thereof, an ER60 polypeptide or an equivalent thereof, a GRP94/GP96 or a GRP94 polypeptide or an equivalent thereof, or, a tapasin polypeptide or an equivalent thereof.
  • the calreticulin polypeptide comprises a human calreticulin polypeptide.
  • the human calreticulin polypeptide sequence can comprise SEQ ID NO:l, or, it can consist essentially of a sequence from about residue 1 to about residue 180 of SEQ LD NO:l, or, it can consist essentially of a sequence from about residue 181 to about residue 417 of SEQ LD NO:l.
  • the isolated or recombinant nucleic acid molecule is operatively linked to a promoter, such as, e.g., a constitutive, an inducible or a tissue-specific promoter.
  • the promoter can be expressed in any cell, including cells ofthe immune system, including, e.g., antigen presenting cells (APCs), e.g., in a constitutive, an inducible or a tissue-specific manner.
  • APCs antigen presenting cells
  • the APCs are dendritic cells, keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like.
  • the invention also provides an expression cassette comprising a nucleic acid sequence encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide from a SARS-CoN.
  • the first domain comprises a calreticulin polypeptide and the second domain comprises an MHC class I-binding peptide epitope of a SARS-CoN antigen.
  • the expression cassette comprises an expression vector, a recombinant virus (e.g., an adenovirus, a retrovirus), a plasmid.
  • the expression cassette can comprise a self-replicating R ⁇ A replicon.
  • the self-replicating R ⁇ A replicon can comprise a Sindbis virus self-replicating R ⁇ A vector, such as, e.g., a Sindbis virus self-replicating R ⁇ A vector SI ⁇ rep5 (U.S. Patent No. 5,217,879).
  • the ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, 1, tapasin, or ER60 polypeptides; or, analogues or mimetics thereof, or, functional fragments thereof.
  • the invention also provides a particle comprising a nucleic acid encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide.
  • the isolated particle comprising an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising at least two domains, wherein the first domain comprises a calreticulin polypeptide and the second domain comprises an MHC class I-binding peptide epitope.
  • the isolated particle can comprise any material suitable for particle bombardment, such as, e.g., gold.
  • the ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides, as discussed herein.
  • the invention also provides a cell comprising a nucleic acid sequence encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide.
  • the cell comprises an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising at least two domains, wherein the first domain comprises a calreticulin polypeptide and the second domain comprises an MHC class I-binding peptide epitope.
  • the cell can be transfected, infected, transduced, etc., with a nucleic acid ofthe invention or infected with a recombinant virus ofthe invention.
  • the cell can be isolated from a non-human transgenic animal comprising cells comprising expression cassettes ofthe invention. Any cell can comprise an expression cassette ofthe invention, such as, e.g., cells ofthe immune system or antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • the APCs can be a dendritic cell, a keratinocyte, a macrophage, a monocyte, a B lymphocyte, an astrocyte, a microglial cell, or an activated endothelial cell.
  • the invention also provides a chimeric polypeptide comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide, preferably human CRT, and a second polypeptide domain comprising at least one antigenic peptide of SARS-CoN.
  • the antigenic peptide can comprise an MHC Class I-binding peptide epitope.
  • the ER chaperone polypeptide can be chemically linked to the antigenic peptide, e.g., as a fusion protein (e.g., a peptide bond), that can be, e.g., synthetic or recombinantly produced, in vivo or in vitro.
  • the polypeptide domains can be linked by a flexible chemical linker.
  • the first polypeptide domain ofthe chimeric polypeptide can be closer to the amino terminus than the second polypeptide domain, or, the second polypeptide domain can be closer to the amino terminus than the first polypeptide domain.
  • the ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides, as discussed herein.
  • the invention provides a pharmaceutical composition comprising a composition ofthe invention capable of inducing or enhancing an antigen specific immune response and a pharmaceutically acceptable excipient.
  • the composition comprises: a chimeric polypeptide comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain an antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; or, a cell comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising
  • the ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides, as discussed herein.
  • the invention provides a method of inducing or enhancing an antigen specific immune response comprising: (a) providing a composition comprising a composition ofthe invention capable of inducing or enhancing an antigen specific immune response, which, in alternative embodiments, can be: a cliimeric polypeptide comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain an antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first
  • the antigen specific immune response can comprise cellular response, such as a CD8 + CTL response.
  • the antigen specific immune response can also comprise an antibody-mediated response, or, a humoral and a cellular response.
  • the composition can administered ex vivo, or, the composition can be administered ex vivo to an antigen presenting cell (APC).
  • the APC is a dendritic cell, a keratinocyte, a macrophage, a, monocyte, a B lymphocyte, an astrocyte, a microglial cell, or an activated endothelial cell.
  • the APC can be a human cell.
  • the APC can be isolated from an in vivo or in vitro source.
  • the method can further comprise administering the ex vtvo-treated APC to a mammal, a human, a histocompatible individual, or to the same individual from which it was isolated.
  • the composition is administered directly in vivo to a mammal, e.g., a human.
  • the composition can be administered intramuscularly, mtradermally, or subcutaneously.
  • the composition e.g., the nucleic acid, expression cassette or particle, can be administered by biolistic injection.
  • the invention provides a method of increasing the numbers of CD8 CTLs specific for a desired SARS-CoN antigen in an individual comprising: (a) providing a composition comprising: a chimeric polypeptide comprising a first domain comprising an endoplasmic, reticulum chaperone polypeptide, preferably CRT, and a second domain comprising an antigenic peptide of SARS-CoN; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain the antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising the antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chap
  • the invention provides a method of inhibiting a SARS-CoN infection or spread ofthe virus in a subject comprising: (a) providing a composition comprising: a chimeric polypeptide comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising a S AR-CoN antigenic peptide; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain the antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising the antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising
  • composition can be co-administered with a second composition that has antiviral activity.
  • Figure 1 is a Western blot that characterizes recombinant SARS-CoN ⁇ protein expression in 293 cells transfected with pcD ⁇ A3.1/myc-His (-) encoding CRT, N, CRT/N, or no insert. Rabbit anti-GST-N sera was used at a 1 :100 dilution to detect N expression.
  • Lane 1 lysate from 293 cells transfected with pcDNA3.1/myc-His (-); Lane 2: lysate from 293 cells transfected with CRT DNA; Lane 3: lysate from 293 cells transfected with N DNA; Lane 4: lysate from 293 cells transfected with CRT/N DNA.
  • Figures 2A-2D are a gel, a blot and graphs showing the N-specific humoral immune response in mice vaccinated with various nucleic acid preparations.
  • Fig. 2A shows a
  • FIG. 2D shows results of an ⁇ LISA comparing the relative titers of N-specific IgGl and IgG2a antibodies in sera of DNA- vaccinated mice (5/group).
  • Figures 3A-3C are flow cytometric tracings and graphs showing SARS-CoN ⁇ -specific CD8+ T cell mediated immune responses in mice vaccinated with the various D ⁇ A compositions.
  • Fig. 3 A shows a representative flow cytometric analysis.
  • Fig. 3B depicts the number of SARS-CoN ⁇ peptide-specific ⁇ F ⁇ - ⁇ - secreting CD8+ T cell precursors (per 3xl0 5 splenocytes) stimulated by the indicated peptide in vitro after harvesting from spleens of mice vaccinated with CRT/ ⁇ D ⁇ A (5 per group).
  • the peptides derived from SARS-CoN ⁇ protein are defined in Table 3.
  • 3C is a graph depicting the number of ⁇ -specific LF ⁇ - ⁇ -secreting CD8+ T cell precursors/3xl0 5 splenocytes in spleen cells harvested from mice (5 per group) that had been vaccinated with various D ⁇ A constructs as indicated: plasmid D ⁇ A encoding ⁇ , CRT, CRT/ ⁇ or lacking any insert were cultured with MHC class I-restricted ⁇ peptide (aa 346-354, QFKD ⁇ VILL (S ⁇ Q LD ⁇ O:31 in vitro overnight and stained for CDS and LFN- ⁇ .
  • Figures 4A-4C shows SARS-CoN ⁇ protein expression in cells infected with recombinant ⁇ vaccinia .
  • Fig.43A shows a flow cytometric analysis.
  • Fig.4B shows immunofmorescence staining.
  • Fig. 4C shows a Western blot using cell lysate from 293 cells infected with either Nac-WT (Lane l)or Nac- ⁇ (Lane 2). Note: Lysate from 293 cells infected with Nac-N revealed a band approximately r 48,000 in size, corresponding to N protein of SARS-CoN.
  • Figures 5A-5B are graphs showing reduction ofthe viral titer of recombinant N vaccinia in mice vaccinated with the various DNA vaccines.
  • Mice (5 per group) were vaccinated with pcDNA3.1/myc-His (-) encoding CRT, N, CRT/N, or no insert as described in the Examples.
  • Fig. 1 A shows virus titers after intranasal challenge with vaccinia.
  • the immunized mice were infected with 2xl0 6 PFU/mouse of Nac-WT or Nac- ⁇ in 20 ⁇ l by intranasal instillation 1 week after the final immunization. Nac-WT infection was used as a negative control.
  • FIG. 5B shows results of i.v. challenge with vaccinia.
  • the immunized mice were infected with 10 7 PFU/mouse of Nac- ⁇ in 100 ⁇ l by intravenous injection 1 week after final immunization.
  • the titer of virus was determined by plaque assay 5 days after challenge.
  • Mice vaccinated with CRT/N DNA showed the greatest reduction in titer of Nac-N virus when challenged intranasally or intravenously.
  • Figure 6 is a schematic diagram of SARS-CoN S protein showing a domain structure.
  • Domain SI corresponds to residues 1-680 of SEQ ID ⁇ O:14; with residues 1-18 representing a signal sequence
  • S2 corresponds to residues 681-1225 of SEQ ID NO:14 and includes two helical regions (HR1 and HR2) as well as a transmembrane domain
  • Si represents an overlapping fragment of SI and S2, and includes residues 417-816 or SEQ ID NO:14; (polypeptide indicating and its recombmants used for immunization.
  • Recombinant nucleic acids comprising SI, S2 and Si were examined as immunogens.
  • Figure 7 A-7B show blots that represents expression and secretion of SARS-CoV S and its recombinant proteins after in vitro transfection .
  • SARS-CoN S and its recombinant proteins were determined in 293 cells transfected with a D ⁇ A molecule encoding S, SI, Si or S2 by Western blot analysis (Fig. 7A). Overnight after transfection, the cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of proteins (50 ⁇ g) were loaded and separated by 10% SDS-PAGE. Rabbit anti-S antibody at a 1 :2000 dilution was used to detect expression ofthe full length S polypeptide and its recombinant domains/ fragments. The presence of secreted SARS-CoV S proteins and recombinant domains confmned by Western blot analysis (Fig 7B).
  • FIG. 8A-8B shows results of an S-specific antibody responses in mice immunized with various recombinant SARS-CoV S DNA immunogens. Mice were immunized with the plasmid DNAs encoding S, SI, Si or S2 via gene gun. Serum samples were collected from one week after the last vaccination and tested for anti-S antibodies.
  • S-specific antibodies were detected in semm diluted to 1 :250 (in PBS) by Western blot analysis using 50 ⁇ g of transfected 293 lysates with DNA encoding S (Fig. 8A).
  • the end-point dilution titer of S-specific antibodies in the sera of DNA-immunized C57BL/6 mice were determined by ELISA in microplates coated with "TC-l/S” cells or "TC-l/No insert” cells (Fig. 8B). Absorbances >3-fold higher than negative controls were considered positive.
  • Figure 9A-9B show SARS-CoV S-specific CD8+ T cell responses in mice immunized with the various DNA immunogens.
  • Intracellular cytokine staining INF ⁇ was detemiined after flow cytometry to characterize the S-specific CD8 + T cell response.
  • Fig. 9A shows flow cytometric analysis and
  • Fig. 9B is a bar graph depicting the number of IFN ⁇ - secreting CD8 + T cell precursors /3xl0 5 splenocytes.
  • CD3 + cells (10 6 ) were harvested from spleens of immunized given S, SI, Si or S2-encoding DNA immunogens.
  • FIG. 10A-10B show expression and secretion of SI and CRT/SI chimeric polypeptide after in vitro transfection. Expression was delennined in 293 cells transfected with DNA constructs comprising no insert, CRT, SI or CRT/SI by Western blot analysis (Fig. 10A). After overnight incubation, transfected cells were lysed and equal amounts of proteins (50 ⁇ g) were loaded and separated by 10% SDS-PAGE.
  • mice were immunized with the plasmid DNAs encoding no insert, CRT, SI or CRT/SI via gene gun. Semm samples were collected and antibodies measure as described for Fig. 8A-8AB.
  • Figure 12A-12B shows that more potent SARS-CoV S-specific CD8+ T cell responses result from administration of DNA immunogens encoding the CRT/SI fusion protein. Methods are the same as described for Fig. 9A-9B.
  • Figure 13A-13B shows that mice vaccinated with DNA immunogens encoding the chimeric polypeptide CRT/S 1 have stronger in vivo protection against growth of a tumor expressing the SARS-CoV S protein.
  • FIG. 13 A shows a study in which transfected tumor cells expressing S (TC-l/S) were injected subcutaneously (5 ⁇ l0 s cells/mouse) into mice that had been immunized with a DNA constructs that encoded CRT, SI, CRT/SI or no insert (10 mice/group). Animals received the challenge in the right leg one week after the last vaccination and were monitored twice weekly for visible tumor.
  • Fig 13B shows results of tumor growth when various subsets of immune cells were depleted by antibody treatment in vivo. CD4, CD8, and Kl .1 depletion was initiated one week after last vaccination and the mice challenged one week later. The depletion treatment was terminated 32 days after tumor challenge.
  • Figure 14 is a Western blot that characterizes recombinant SARS-CoV M (membrane) protein expression in 293 cells transfected withpcDNA3.1/myc-His (-) encoding CRT, M or CRT/M. pcDNA3.1/myc-His (-) without insert was used as a negative control. The transfected cells were lysed 24 hours later and separated by SDS-PAGE. Mouse anti-myc antibody was used to detect M protein expression. Lanes 1-4 show lysates from 293 cells transfected with DNA without an insert and DNA encoding CRT, M or CRT/M, respectively.
  • FIG. 15A-15B show SARS-CoV M-specific CD8+ T cell responses in mice immunized with the various DNA immunogens encoding the M polypeptide.
  • Five mice per group were immunized with ⁇ cDNA3, ⁇ cDNA3-CRT, ⁇ cDNA3-M or pcDNA3-CRT/M.
  • CD3 + enriched T cells from spleens of immunized mice were stimulated in vitro with transfected dendritic cells, DC/S" dendritic cells or "DC/No insert", in vitro overnight and stained for both CD8 and intracellular IFN ⁇ .
  • Fig. 15A shows representative flow cytometry results for CD3 + enriched T cells from immunized or control mice.
  • FIG. 15B is a bar graph depicting the number of antigen-specific IFN ⁇ -secreting CD8 + T-cell precursors/3xl0 5 CD3 + enriched T cells (mean ⁇ SD) after DNA vaccination.
  • Figure 16A-16B presents flow cytometric analysis of IFN- ⁇ -secretmg M-specific CD4 + T-cells (Thl) in mice (five per group) immunized with pcDNA3, pcDNA3-CRT, pcDNA3-M or pcDN A3 -CRT/M.
  • CD3 + -enriched T cells from spleens of immunized mice were stimulated in vitro with DC-l/M or DC-l/no insert overnight, and stained for both CD4 and intracellular IFN ⁇ .
  • FIG. 16A presents representative flow cytometry data for splenocytes harvested from immunized mice.
  • Fig. 16B is a bar graph depicting the number of antigen-specific JJFN ⁇ - secreting CD4 + T-cells (Thl cells) per 3xl0 5 CD3 + enriched T cells (mean ⁇ SD).
  • Figure 17A-17B presents flow cytometry analysis of IL-4-secreting M-specific CD4 + T- cells (Tl 2) in mice (five per group) immunized with pcDNA3, pcDNA3-CRT, pcDNA3-M or pcDNA3-CRT/M.
  • FIG. 17A presents representative flow cytometry data for splenocytes harvested from immunized mice.
  • Fig. 17B presents a bar graph depicting the number of antigen-specific IL-4-secreting CD4 + T-cells (Th2 cells) per 3xl0 5 CD3+ enriched T cells (mean ⁇ SD).
  • FIG. 18A-18B shows that mice vaccinated with DNA immunogens encoding the chimeric polypeptide CRT/M are much better protected in vivo against growth of a tumor expressing the SARS-CoV M protein.
  • Fig. 18A shows a study in which transfected tumor cells expressing M (TC-l/M) were injected subcutaneously (5 ⁇ l0 4 cells/mouse) into mice that had been immunized with a plasmid DNA constructs that encoded (i) CRT, (ii) M, (iii) CRT/M or (iv) no insert (10 mice/group). Animals received the challenge in the right leg one week after the last vaccination and were monitored twice weekly for visible tumor.
  • Fig 18B shows results of tumor growth when various subsets of immune cells were depleted by antibody treatment in vivo.
  • CD4, CD8, and NKl.l depletion was initiated one week after last vaccination and the mice challenged one week later. The depletion treatment was terminated 32 days after tumor challenge. Both graphs show the percentage of tumor-free mice over time.
  • Figure 19 shows schematically SARS-CoV cDNA clones spanning the genome ofthe TW1 strain. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the invention provides compositions and methods for enhancing the immune responses, particularly cytotoxic T cell immune responses, induced in vivo administration of chimeric nucleic acids that encode (a) an endoplasmic reticulum chaperone polypeptide linked to (b) at least one antigenic polypeptide or peptide from SARS CoV.
  • chimeric polypeptides or fusion proteins can also be administered, although the preferred embodiment is a nucleic acid composition or expression plasmid for administration as an immunogen or vaccine.
  • the fusion polypeptide encoded by the nucleic acid immunogenic or vaccine composition comprises at least two "domains:" the first domain comprises a endoplasmic reticulum chaperone polypeptide, and the second domain comprises a full length polypeptide or a shorter fragment that comprises at least one epitope-comprising a SARS-CoV structural protein, most preferably the product ofthe S, E, M or N gene of SARS-CoV.
  • any endoplasmic reticulum chaperone polypeptide, or functional fragment or variation thereof can be used in the invention, such as calreticulin, tapasin, ER60 or calnexin polypeptides, human calreticulin (CRT) is prefened.
  • the antigenic domain ofthe chimeric molecule is preferably one that comprises an MHC class I-binding peptide epitope.
  • the chimeric nucleic acid or polypeptide are administered or applied to induce or enhance immune responses that are specific and anti-viral in their effect (e.g., that neutralize vims or result in damage and death of virus expressing cells) in vivo.
  • the experiments described herein demonstrate that the methods ofthe invention can enhance a cellular immune response, particularly, a CTL reactivity, induced by a DNA vaccine encoding various polypeptides ofthe SARS CoV. Initially, DNA encoding the nucleocapsid or N-protein was used. .
  • DNA vaccines comprising nucleic acid encoding a fusion protein comprising CRT linked to a N protein of SARS-CoV enhances the potency of DNA vaccines.
  • DNA vaccines ofthe invention containing chimeric CRT fusion genes were or will be administered to mice and other subjects by biolistic subcutaneous methods. They induced increased N-specific CD8+ CTL precursors, and are expected to improve immune protection against the vims. This increase in N-specific CD8+ T cell precursors was significant as compared to DNA vaccines containing N or CRT genes alone.
  • a potential mechanism for the enhanced antigen-specific CD8 T cell immune responses in vivo is the presentation of antigen through the MHC class I pathway by uptake of apoptotic bodies from cells expressing the antigen, also called "cross-priming".
  • antigen or immunogen refers to a compound or composition comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered (or expressed in vivo by an administered nucleic acid, e.g., a DNA vaccine) in an appropriate amount (an "immunogenically effective amount"), i.e., is capable of eliciting, augmenting or boosting a cellular and/or humoral immune response either alone or in combination or linked or fused to another substance (which can be administered at once or over several intervals).
  • Calnexin describes the well-characterized membrane protein ofthe endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component ofthe ER quality control machinery. Calreticulin is a soluble analogue of calnexin. In vivo, calreticulin and calnexin play important roles in quality control during protein synthesis, folding, and posttranslational modification. Calnexin polypeptides, and equivalents and analogues thereof, are species in the genus of ER chaperone polypeptides, as described herein (Wilson (2000) J. Biol. Chem. 275:21224-2132; Danilczyk (2000) J. Biol. Chem. 275:13089-13097; U.S. Patent Nos. 6,071,743 and 5,691,306). "Calreticulin” or “CRT” describes the well-characterized ⁇ 46 kDa resident protein ofthe
  • CRT acts as a "chaperoiie” polypeptide and a member ofthe MHC class I transporter TAP complex; CRT associates with TAP1 and TAP2 transporters, tapasin, MHC Class I heavy chain polypeptide and ⁇ 2 microglobulin to function in the loading of peptide epitopes onto nascent MHC class I molecules (Jorgensen (2000) Eur. J. Biochem. 267:2945- 2954).
  • CRT refers to polypeptides and nucleic acids molecules having substantial identity (defined herein) to the exemplary CRT sequences as described herein.
  • a CRT polypeptide is a polypeptides comprising a sequence identical to or substantially identical (defined herein) to the amino acid sequence of CRT.
  • An exemplary nucleotide and amino acid sequence for a CRT used in the present compositions and methods are SEQ ID NO:l and SEQ ID NO:2, respectively.
  • the terms “calreticulin” or “CRT” encompass native proteins as well as recombinantly produced modified proteins that induce an immune response, including a CTL response.
  • CRT calreticulin
  • the terms "calreticulin” or “CRT” encompass homologues and allelic variants of CRT, including variants of native proteins constructed by in vitro techniques, and proteins isolated from natural sources.
  • the CRT polypeptides ofthe invention, and sequences encoding them also include fusion proteins comprising non-CRT sequences, particularly MHC class I- binding peptides; and also further comprising other domains, e.g., epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals and the like.
  • endoplasmic reticulum chaperone polypeptide as used herein means any polypeptide having substantially the same ER chaperone function as the exemplary chaperone proteins CRT, tapasin, ER60 or calnexin. Thus, the term includes all functional fragments or variants or mimics thereof.
  • a polypeptide or peptide can be routinely screened for its activity as an ER chaperone using assays known in the art. While the invention is not limited by any particular mechanism of action, in vivo chaperones promote the conect folding and oligomerization of many glycoproteins in the ER, including the assembly ofthe MHC class I heterotrimeric molecule (heavy chain, ⁇ 2m, and peptide).
  • epitopes refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions used in the methods ofthe invention.
  • TCR T cell receptor
  • An "antigen” is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product of an immune response, such as an antibody or a CTL.
  • TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class II protein.
  • MHC major histocompatibility complex
  • ER60 or “GRP94” or “gp96” or “glucose regulated protein 94” as used herein describes the well-characterized ER chaperone polypeptide that is the ER representative ofthe heat shock protein-90 (HSP90) family of stress-induced proteins. These bind to a limited number of proteins in the secretory pathway, possibly by recognizing advanced folding intermediates or incompletely assembled proteins.
  • ER60 polypeptides and equivalents and analogues thereof, are species in the genus of ER chaperone polypeptides, as described herein (Argon (1999) Semin. CellDev. Biol. 10:495-505; Sastry (1999) J. Biol. Chem. 274:12023- 12035; Nicchitta (1998) Curr. Opin. Immunol. 10:103-109; U.S. Patent No. 5,981,706).
  • expression cassette or "expression vector” as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers. "Operably linked” refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription ofthe DNA sequence.
  • expression cassettes include plasmids, recombinant viruses, any form of a recombinant "naked DNA" vector, and the like.
  • a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell.
  • a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to replicons (e.g., RNA replicons), bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA, e.g., plasmids, virases, and the like (U.S. Patent No.
  • Such chemical linking includes the peptide bonds of a recombinantly or in vivo generated fusion protein.
  • the term "chimeric" or “fusion" polypeptide or protein refers to a composition comprising at least one polypeptide or peptide sequence or domain which is associated with a second polypeptide or peptide domain.
  • One embodiment of this invention is an isolated or recombinant nucleic acid molecule encoding a fusion protein comprising at least two domains, wherein the first domain comprises an endoplasmic reticulum chaperone, e.g., CRT, and the second domain comprising an antigenic epitope, e.g., an MHC class I-binding peptide epitope.
  • Additional domains can comprise a polypeptide, peptide, polysaccharide, or the like.
  • the "fusion” can be an association generated by a peptide bond, a chemical linking, a charge interaction (e.g., electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the "fusion protein" can be translated from a common message. Alternatively, the compositions ofthe domains can be linked by any chemical or electrostatic means.
  • the chimeric molecules ofthe invention can also include additional sequences, e.g., linkers, epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals, and the like.
  • additional sequences e.g., linkers, epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals, and the like.
  • a peptide can be linked to a carrier simply to facilitate manipulation or identification/ location ofthe peptide.
  • immunogen or “immunogenic composition” refers to a compound or composition comprising a peptide, polypeptide or protein which is "immunogenic,” i.e., capable of eliciting, augmenting or boosting a cellular and/or humoral immune response, either alone or in combination or linked or fused to another substance.
  • An immunogenic composition can be a peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a fragment 15 amino acids in length, a fragment 20 amino acids in length or greater; smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen.
  • the immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen' s coding sequence operably linked to a promoter, e.g., an expression cassette.
  • the immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids.
  • Epitopes of more than one SARS-CoV protein may be used in combination.
  • isolated when referring to a molecule or composition, such as, e.g., a CRT nucleic acid or polypeptide, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g., RNAs), or other contaminants with which it is associated in vivo or in its natural state.
  • a CRT composition is considered isolated when it has been isolated from any other component with which it is natively associated, e.g., cell membrane, as in a cell extract.
  • An isolated composition can, however, also be substantially pure.
  • An isolated composition can be in a homogeneous state and can be dry or in an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC). Thus, the isolated compositions of this invention do not contain materials normally associated with their in situ environment. Even where a protein has been isolated to a homogenous or dominant band, there are trace contaminants which co-purify with the desired protein.
  • polypeptide include compositions ofthe invention that also include “analogues ,” or “conservative variants” and “mimetics” or “peptidomimetics” with structures and activity that substantially conespond to the polypeptide from which the variant was derived, including, e.g., human CRT or a Class I-binding peptide epitope, such as from the SARS-CoV S, E, M or N proteins, as discussed in detail, below.
  • pharmaceutical composition refers to a composition suitable for pharmaceutical use, e.g., as a vaccine, in a subject.
  • compositions of this invention are formulations that comprise a pharmacologically effective amount of a composition comprising, e.g., a nucleic acid, or vector, or cell ofthe invention, and a pharmaceutically acceptable carrier.
  • promoter is an array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a “constitutive” promoter is a promoter which is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter which is under environmental or developmental regulation.
  • a “tissue specific” promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism.
  • the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription ofthe nucleic acid conesponding to the second sequence.
  • recombinant refers to (1) a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide”), (2) methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or (3) a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.
  • recombinant CRT or an MHC class I-binding peptide epitope can be recombinant as used to practice this invention.
  • Recombinant means also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of polypeptide coding sequences in the vectors used to practice this invention.
  • self-replicating RNA replicon refers to constructs based on RNA viruses, e.g., alphavirus genome RNAs (e.g., Sindbis virus, Semliki Forest vims, etc.), that have been engineered to allow expression of heterologous RNAs and proteins.
  • recombinant vectors are self-replicating (i.e., they are "replicons") and can be introduced into cells as naked RNA or DNA, as described in detail, below.
  • the self-replicating RNA replicon comprises a Sindbis virus self-replicating RNA vector SINrep5, which is described in detail in U.S. Patent No. 5,217,879.
  • systemic administration refers to administration of a composition or agent such as the molecular vaccine or the CRT-Class I-binding peptide epitope fusion protein described herein, in a manner that results in the introduction ofthe composition into the subject's circulatory system.
  • regional administration refers to administration of a composition into a specific anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ, and the like.
  • regional administration includes administration of the composition or drug into the hepatic artery.
  • local administration refers to administration of a composition or drag into a limited, or circumscribed, anatomic space, such as intrarumoral injection into a tumor mass, subcutaneous injections, intramuscular injections, and the like. Any one of skill in the art would understand that local administration or regional administration may also result in entry ofthe composition or drag into the circulatory system.
  • “Tapasin” is the Icnown ER chaperone polypeptide, as discussed above.
  • tapasin is a subunit ofthe TAP (transporter associated with antigen processing) complex and binds both to TAP1 and MHC class I polypeptides.
  • Tapasin polypeptides, and equivalents and analogues thereof, are species in the genus of ER chaperone polypeptides, as described herein (Bamden (2000) J. Immunol. 165:322-330; Li (2000) J. Biol. Chem. 275:1581-1586).
  • the methods ofthe invention provide for the administration of nucleic acids encoding a
  • CRT-SARS-CoV Class I epitope binding peptide fusion protein as described above.
  • Recombinant CRT-containing fusion proteins can be synthesized in vitro or in vivo.
  • Nucleic acids encoding these compositions can be in the form of "naked DNA” or they can be incorporated in plasmids, vectors, recombinant viruses (e.g., "replicons") and the like for in vivo or ex vivo administration.
  • Nucleic acids and vectors ofthe invention can be made and expressed in vitro or in vivo, a variety of means of making and expressing these genes and vectors can be used.
  • RNA, cDNA, genomic DNA, vectors, recombinant virases or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly.
  • any recombinant expression system can be used, including, in addition to bacterial cells, e.g., mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Eree Radic. Biol. Med. 79:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth.
  • Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Calreticulin Sequences The sequences of CRT, including human CRT, are well known in the art (McCauliffe (1990) J. Clin. Invest. 86:332-335; Bums (1994) Nature 367:476-480; Coppolino (1998) frit. J. Biochem. Cell Biol. 30:553-558). The nucleic acid sequence appears as GenBank Accession
  • the amino acid sequence ofhuman CRT protein (SEQ ID NO :2) is shown below 1 MLLSVPLLLG LLGLAVAEPA VYFKEQFLDG DG TSRWIES KHKSDFGKFV LSSGKFYGDE 61 EKDKGLQTSQ DARFYALSAS FEPFSNKGQT LVVQFTVKHE QNIDCGGGYV KLFPNSLDQT
  • polypeptides, peptides, other functional derivatives, including mimetics of CRT are preferably based on stracture and amino acid sequence of CRT, preferably human CRT, SEQ TD NO:2 above.
  • CRT preferably human CRT, SEQ TD NO:2 above.
  • SARS-CoV Genomic Sequences and Sequences of Polypeptides
  • the glycosylated spike protein (as well as the nucleocapsid protein) can be detected in infected cell culture supematants with antisera from SARS patients
  • the coding sequence for the M (membrane protein (SEQ ID NO: 8) is from nt 26348 to 26353 of SEQ ID NO:3, which comprises 666 nt encoding 221 aa + stop codon
  • the encoded amino acid sequence ofthe M polypeptide (SEQ ID NO:9) is:
  • the coding sequence for the N is from nt 28120 to 29388of SEQ ID NO:3, which comprises 1269 nt encoding 422 aa + stop codon.
  • the encoded aminei acid sequence ofthe E polypeptide (SEQ J-D NO:11) is:
  • N-terminal methionine encoded by the initiation ATG codon, is removed in the virion protein when it is processed, and all other methionines are oxidized, and the resulting N-terminal serine is acetylated.
  • Table 1 Summary of the 25 overlapping SARS-CoV TW-1 isolate cDNA clones sequenced and available. The cDNA sections are in the vector, between the BamHI and EcoRI cloning sites, orward and reverse se uencin rimers are shown.
  • the coding sequence for the S (spike) glycoprotein, SEQ ID NO:13 is from nt 21492 to 25259 ofSEQ ID NO:12, which comprises 3768 nt that encode 1255 residues + stop codon.
  • the encoded amino acid sequence ofthe S polypeptide (SEQ ID NO:14) is:
  • NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT REVFAQVKQM YKTPTLKYFG 780 GFNFSQILPD PLKPTKRSFI EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL 840 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE 900 NQKQIANQFN KAISQIQESL TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN 960 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK 1020 RVDFCGKGYH LMSFPQAAPH GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN 1080 GTSWFITQRN FFSPQIITTD NTFVSGNC
  • Domain SI - amino acids 1-680 of SEQ DD NO:14 which is shown below as SEQ ID NO:15:
  • SEQ ID NO:16 (residues 1-575):
  • NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN 480
  • Polypeptide Si overlaps domains SI and S2 and corresponds to residues 417-816 or SEQ ID NO:14. This polypeptide is shown below as SEQ 1D:17 (aa 1-400):
  • the present invention includes homologous sequences to the S polypeptide domains from any other strain of SARS-CoN. 2.
  • the coding sequence for the E (envelope, or "small envelope") protein (SEQ ID NO:18) is from nt 26117 to 26347 of SEQ ID NO: 12, which comprises 231 nt that encode 76 aa's + stop codon.
  • the coding sequence for the M is from nt 26398 to 27063 of SEQ ID NO: 12, which comprises 666 nt encoding 221 aa + stop codon.
  • N nucleocapsidprotein(SEQ IDNO:22) is from nt28120 to 29388 ofSEQ ID NO:12, which comprises 1269 nt encoding 422 aa+ stop codon.
  • HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYHGAIKL DDKDPQFKDN VILLNKHIDA 360
  • PCDNA3-S2 (SEO TD NO:29): vector pcDNA3.1(+) sequence (UPPER CASE) S2 - C-terminal domain of SARS-CoV S protein (lower case/bold/underscored) I 10 [ 20 I 30 I 40 I 50 I 60 I 70 I 80 I 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGT
  • the above SARS-CoV proteins may be substituted by homologues or analogues thereof from any viral isolate or strain, or with a sequence that has conservative substitutions such that the protein maintain their immunogenicity and antigenicity when administered in the form of a nucleic acid composition or polypeptide.
  • nucleic acids such as, e.g., generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Nols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Aus bel, ed.
  • Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g.
  • analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g.
  • Oligonucleotide primers can be used to amplify nucleic acids to generate fusion protein coding sequences used to practice the invention, to monitor levels of vaccine after in vivo administration (e.g., levels of a plasmid or virus), to confirm the presence and phenotype of activated CTLs, and the like.
  • the skilled artisan can select and design suitable oligonucleotide amplification primers using known sequences, e.g., SEQ ID NO:l.
  • Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide to Methods and Applications, ed. Innis, Academic Press, N.Y.
  • RNA polymerase mediated techniques NASBA, Cangene, Mississauga, Ontario; Berger (1987) Meth. Enzymol. 152:307-316; U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564).
  • Expression cassettes including plasmids, recombinant viruses (e.g., RNA viruses like the replicons described below) and other vectors encoding the fusion proteins described herein are used to express these polypeptides in vitro and in vivo.
  • Recombinant nucleic acids are expressed by a variety of conventional techniques (Roberts (1987) Nature 328:731; Schneider (1995) Protein Expr. Purif. 6435:10; Sambrook, supra Tijssen, supra; Ausubel, supra). Plasmids, vectors, etc., can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • the nucleic acids used to practice the invention can be stably or transiently expressed in cells such as episomal expression systems. Selection markers can be incorporated to confer a selectable phenotype on transformed cells. For example, selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
  • the marker may encode antibiotic resistance, e.g., chloramphenicol, kanamycin, G418, bleomycin, hygromycin) to permit selection of those cells transformed with the desired DNA sequences (Blondelet- Rouault (1997) Gene 190:315-317; Aubrecht (1997) J. Pharmacol. Exp. Ther. 281:992-997).
  • antibiotic resistance e.g., chloramphenicol, kanamycin, G418, bleomycin, hygromycin
  • a nucleic acid encoding a CRT-SARS peptide epitope chimeric polypeptide are cloned into expression cassettes such as plasmids or other vectors, viruses that can transfect or infect cells in vitro, ex vivo and/or in vivo.
  • expression cassettes such as plasmids or other vectors, viruses that can transfect or infect cells in vitro, ex vivo and/or in vivo.
  • a number of delivery approaches are known, including lipid or liposome based gene delivery (Ma iino (1988) BioTechniques 6:682- 691; U.S. Pat No. 5,279,833), replication-defective retroviral vectors with desired exogenous sequence as part ofthe retroviral genome (Miller (1990) Mol. Cell. Biol. 70:4239; Kolberg (1992) J. NIHRes.
  • Vectors which may be employed include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, examples of which are baculoviridae, parvoviridae, picornaviridae, herpesviridae, poxviridae, adenoviridae, picornaviridae or alphaviridae. Chimeric vectors may also be employed which exploit advantageous merits of each ofthe parent vector properties (Feng (1997) Nature Biotechnology 15 :866-870). Such viral genomes may be modified by recombinant DNA techniques to include the gene of interest and may be engineered to be replication-deficient, conditionally replicating or replication-competent.
  • Retroviral vectors can be derived from adenoviral, adeno-associated viral or retroviral genomes. Retroviral vectors can include those based upon murine leukemia virus (MuLN), gibbon ape leukemia virus (GaLN), simian immunodeficiency virus (SIN), human immunodeficiency virus (HIN), and combinations thereof (Buchscher
  • Adeno-associated virus (AAV)-based vectors can transduce cells for the in vitro production of nucleic acids and peptides, and be used in in vivo and ex vivo therapy procedures (Okada (1996) Gene Ther. 3:957-964; West (1987) Virology 160:38-47; Carter (1989) U.S. Patent No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) j. Clin. Invest. 94:1351).
  • AAV Adeno-associated virus
  • RNA replicons In addition to the above-described expression vectors and recombinant viruses, self- replicating RNA replicons can also be used to infect cells or tissues or whole organisms with a fusion protein-expressing nucleic acids ofthe invention.
  • the invention also incorporates RNA viruses, including alphavirus genome RNAs such as from Sindbis virus, Semliki Forest virus, Venezuelan equine encephalitis virus, and the like, that have been engineered to allow expression of heterologous RNAs and proteins. High levels of expression of heterologous sequences such as the fusion polypeptides ofthe invention, are achieved when the viral structural genes are replaced by the heterologous coding sequences.
  • RNAs are self-replicating ("replicons") and can be introduced into cells as naked RNA or DNA. However, they require trans complementation to be packaged and released from cells as infectious virion particles.
  • the defective helper RNAs contain the exacting sequences required for replication as well as an RNA promoter which drives expression of open reading frames.
  • viral nonstructural proteins translated from the replicon RNA allow replication and transcription ofthe defective helper RNA to produce the virion's structural proteins (Bredenbeek
  • RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (family Togaviridae) (Xiong (1989) Science 243:1188-1191), Semliki Forest virus (Ying (1999) Nat. Med. 5:823-827) or Venezuelan equine encephalitis virus (Pushko (1997) Virology 239:389-401) vectors.
  • Sindbis virus family Togaviridae
  • Semliki Forest virus Ying (1999) Nat. Med. 5:823-827)
  • Venezuelan equine encephalitis virus Pushko (1997) Virology 239:389-401 vectors.
  • These vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA, which is then transcribed into RNA replicons in transfected cells or in vivo (Berglund (1998) Nat. Biotechnol. 16:562-565).
  • the self-replicating RNA replicon comprises a Sindbis virus self-replicating RNA vector SINrep5, as described in detail by Bredenbeek, supra and Herrmann (1998) Biochem. Biophys. Res. Commun.
  • polypeptides in other embodiments, is directed to an isolated or recombinant polypeptide comprising at least two domains, wherein the first domain comprises a calreticulin (CRT) polypeptide; and, wherein the second domain comprises an MHC class I-binding peptide epitope of a SARS protein that is antigenic such that an immune response directed against such an epitope leads to any type of protective or prophylactic or therapeutic immunity' against the virus.
  • CRT calreticulin
  • polypeptide As noted above, the terms “polypeptide,” “protein,” and “peptide,” referring to polypeptides including the CRT, fragments of CRT that bind peptides, and MHC class I-binding peptide epitopes, SARS polypeptides, such as the S, E, M and N proteins to practice the invention. These proteins are disclosed in more detail, including amino acid sequence and encoding nucleic acid sequences, above.
  • the composition ofthe invention also include "analogues,” or “conservative variants” and “mimetics” or “peptidomimetics” with structures and activity that substantially correspond to CRT and SARS protein or epitope(s) thereof.
  • the terms “conservative variant” or “analogue” or “mimetic” also refer to a polypeptide or peptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity (ability to bind to "antigenic” peptides, to stimulate an immune response).
  • conservatively modified variations of an amino acid sequence i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
  • one exemplary guideline to select conservative substitutions includes (original residue/substitution): Ala/Gly or Ser; Arg/ Lys; Asn/ Gin or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; Gly/Ala or Pro; His/Asn or Gin; Ile/Leu or Nal; Leu/Ile or Nal; Lys/Arg or Gin or Glu; Met/Leu or Tyr or He; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or Phe; Nal Ile or Leu.
  • substitutions are (i) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, He, Phe, Nal or Ala; (iii) substitution of a Cys residue for (or by) any other residue; (iv) substitution of a residue having an electropositive side chain, e.g.,, Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g.,, Glu or Asp; or (v) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.
  • a hydrophilic residue e.g., Ser or Thr
  • a hydrophobic residue e.g., Leu, He, Phe
  • substitutions are not the only possible conservative substitutions. For example, for some purposes, all charged amino acids may be considered conservative substitutions for each other whether they are positive or negative. Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered to yield “conservatively modified variants.”
  • the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has the necessary structural and/or functional characteristics of a peptide that permits use in the methods ofthe invention, such as mimicking CRT in interaction with peptides and MHC class I- proteins).
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a combination of partly natural amino acids and partly non-natural analogues.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetics' structure and/or activity. As with conservative variants, routine experimentation will determine whether a mimetic is within the scope ofthe invention, that its stereochemical structure and/or function is not substantially altered.
  • Peptide mimetics can contain any combination of "non-natural" structural components, typically from three groups: (a) residue linkage groups other than the natural amide bond ("peptide bond"); (b) non-natural residues in place of naturally occurring amino acids; or (c) residues which induce or stabilize a secondary structure, e.g., a ⁇ turn, ⁇ turn, ⁇ sheet, or ⁇ helix conformation.
  • a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical bonds other than peptide bonds.
  • peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'- dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • DCC dicyclohexylcarbodiimide
  • DIC N,N'-diisopropylcarbodiimide
  • the structure ofthe polypeptides, peptides, other functional derivatives, including mimetics ofthe present invention are preferably based on structure and amino acid sequence of CRT, preferably human CRT (SEQ ID NO:2, disclosed above) or a SARS-CoN protein such as S, E, M or ⁇ as disclosed herein for two viral isolates.
  • CRT preferably human CRT (SEQ ID NO:2, disclosed above) or a SARS-CoN protein such as S, E, M or ⁇ as disclosed herein for two viral isolates.
  • Individual synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies well known in the art, e.g., Organic Syntheses Collective Volumes, Gilman et al. (eds) John Wiley & Sons, Inc., Y.
  • Polypeptides incorporating mimetics can also be made using solid phase synthetic procedures (e.g., U.S. Pat. No.
  • Peptides and peptide mimetics ofthe invention can also be synthesized using combinatorial methodologies.
  • Various techniques for generation of peptide and peptidomimetic libraries are well known e.g., multipin, tea bag, and split-couple-mix techniques (al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234).
  • Modified polypeptide and peptides can be further produced by chemical modification (Belousov (1997) Nucleic Acids Res.
  • peptides can also be synthesized, whole or in part, using conventional chemical synthesis (Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol.
  • peptide-binding fragments or "sub-sequences" of CRT are used.
  • other peptides that bind to MHC proteins preferably MHC Class I proteins, are used.
  • Such peptides can be derived from any polypeptide, particularly, from a known pathogen, or it can be entirely synthetic).
  • compositions are administered to an individual as pharmacological compositions in amounts sufficient to induce an antigen-specific immune response (e.g., a CTL response, see Examples, below) in the individual.
  • an antigen-specific immune response e.g., a CTL response, see Examples, below
  • Pharmaceutically acceptable carriers and formulations for nucleic acids, peptides and polypeptides are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, PA ("Remington's"); Banga; Putney (1998) Nat. Biotechnol.
  • nucleic acids and polypeptides used in the methods ofthe invention can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally; by intraarterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa).
  • compositions can be administered by any protocol and in a variety of unit dosage forms depending upon the method and route and frequency of administration, whether other drugs are being administered, the individual's response, and the like. Dosages for typical nucleic acid, peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages may be adjusted depending on a variety of factors, e.g.
  • the initial responses e.g., number and activity of CTLs induced, tumor shrinkage, anti-viral activity measured as lysis of virus-infected cells or reduction of virus titer, and the like
  • the amount of pharmaceutical composition adequate to induce the desired response is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use, i.e., the "dosing regimen,” will depend upon a variety of factors, including, e.g., the diseases or conditions to be treated or prevented by the immunization, the general state ofthe patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of pharmaceutical composition, and the like.
  • the dosage regimen also takes into consideration pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailabihty, metabolism, clearance, and the like (Remington). Dosages can be determined empirically, e.g., by assessing the abatement or amelioration of symptoms, or, by objective criteria, e.g., measuring levels of antigen-specific CTLs. As noted above, a single or multiple administrations can be administered depending on the dosage and frequency as required and tolerated by the patient. The pharmaceutical compositions can be administered alone or in conjunction with other therapeutic treatments, or, as prophylactic immunization.
  • pharmacokinetics i.e., the pharmaceutical composition's rate of absorption, bioavailabihty, metabolism, clearance, and the like (Remington). Dosages can be determined empirically, e.g., by assessing the abatement or amelioration of symptoms, or, by objective criteria, e.g., measuring levels of antigen-specific CTLs. As noted above,
  • the nucleic acids and polypeptides ofthe invention are introduced into the individual by ex vivo treatment of antigen presenting cells (APCs), followed by administration ofthe manipulated APCs.
  • APCs are transduced (transfected) or infected with fusion protein-encoding nucleic acids ofthe invention; afterwards, the APCs are administered to the individual.
  • the APCs are stimulated with fusion proteins ofthe invention (purified or as a cell lysate from cells transfected and expressing a recombinant fusion protein in vivo). Afterward this "pulsing, the APCs are administered to the individual.
  • the fusion proteins can be in any form, e.g., as purified or synthetic polypeptides, as crude cell lysates (from transfected cells making recombinant fusion protein), and the like.
  • the APC can be an MHC-matched cell (a tissue-typed cell).
  • the APC can be a tissue-cultured cell or it can be an APC isolated from the individual to be treated and re-administered after ex vivo stimulation. Any APC can be used, as described above. Methods of isolating APCs, ex vivo treatment in culture, and re-administration are well known in the art (U.S. Patent Nos.
  • Kits The invention provides kits that contain the pharmaceutical or immunogenic compositions ofthe invention, as described above, to practice the methods ofthe invention.
  • kits can contain recombinant or synthetic chimeric polypeptides comprising a first domain comprising an ER chaperone polypeptide and a second domain comprising an antigenic peptide ofthe SARS CoV, e.g., a CRT-Class I-binding peptide epitope fusion protein; or, the nucleic acids encoding them, e.g., in the form of naked DNA (e.g., plasmids), viruses (e.g. alphavirus-derived "replicons” including Sindbis virus replicons) and the like.
  • naked DNA e.g., plasmids
  • viruses e.g. alphavirus-derived "replicons” including Sindbis virus replicons
  • the kit can contain instructional material teaching methodologies, e.g., means to administer the compositions used to practice the invention, means to inject or infect cells or patients or animals with the nucleic acids or polypeptides ofthe invention, means to monitor the resultant immune response and assess the reaction ofthe individual to which the compositions have been administered, and the like.
  • instructional material teaching methodologies e.g., means to administer the compositions used to practice the invention, means to inject or infect cells or patients or animals with the nucleic acids or polypeptides ofthe invention, means to monitor the resultant immune response and assess the reaction ofthe individual to which the compositions have been administered, and the like.
  • EXAMPLE 1 DNA Vaccines Targeting the Nucleocapsid Protein of SARS-CoV
  • CRT calreticulin
  • N SARS-CoV nucleocapsid
  • SARS-CoV N Protein cDNA encoding SARS nucleocapsid protein was generated by reverse transcription of SARS coronavirus TWl (18) (Hsueh, PR, 2003, Emerg Infect Dis 9:1163-1167;) (accession no. YA291451) using Superscript II (Invifrogen, Carlsbad, CA) followed by amplification using platinum T ⁇ q DNA polymerase (Invifrogen, Carlsbad, CA) as described previously (Huang et ⁇ l., supra).
  • the oligonucleotide primers for SARS-CoV N protein were generated by reverse transcription of SARS coronavirus TWl (18) (Hsueh, PR, 2003, Emerg Infect Dis 9:1163-1167;) (accession no. YA291451) using Superscript II (Invifrogen, Carlsbad, CA) followed by amplification using platinum T ⁇ q DNA polymerase (Invifrogen, Carlsbad, CA) as described previously (Huang e
  • SEQ ID NO : 100 The DNA fragment encoding N protein was cloned into pGEX-lplasmid (Amersham Pharmacia Biotech, Little Chalfont, England) to generate pGEX-l-NC-G3 (Huang et al, supra) for recombinant protein expression.
  • E. coli BL-21 were transformed with pGEX-1 or pGEX-1- NC-G3 plasmids and grown overnight in LB medium containing 50 ⁇ g/ml ampicillin to the midlog phase.
  • Lysates prepared from the large batch were incubated with TNE equilibrated glutathione resin. Bound protein was eluted by lOmM reduced glutathione in 50mM Tris (pH 8.0) buffer. The eluted and purified fractions were used for Western blot analysis and as the coating antigen for ELISA assay.
  • Membranes were probed with rabbit anti-GST-N sera (Huang et al, supra) at 1:1000 dilution in TTBS for 2 hr, washed four times with TTBS, and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Zymed, San Francisco, CA) at 1:1000 dilution in TTBS containing 5% nonfat milk. Membranes were washed four times with TTBS and developed using enhanced Hyperfilm-enhanced chemiluminescence (Amersham, Piscataway, NJ).
  • mice Six- to eight-week-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, Maryland) and kept in the oncology animal facility ofthe Johns Hopkins Hospital (Baltimore, Maryland). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals.
  • DNA Vaccination DNA-coated gold particles were prepared according to a previously described protocol (Chen et al, supra). DNA-coated gold particles were delivered to the shaved abdominal region of mice using a helium-driven gene gun (BioRad, Hercules, CA) with a discharge pressure of 400 p.s.i. C57BL/6 mice were immunized with 2 ⁇ g ofthe plasmid encoding no insert, CRT, N, or CRT/N protein. The mice received two boosters with the same dose at a one week interval.
  • Enzyme-Linked Immunoabsorbent Assay The presence of SARS-CoV N-specific antibodies in the sera from CRT/N DNA- vaccinated C57BL/6 mice (5 per group) were determined by ELISA using microwell plates coated with bacteria-derived recombinant GST-N protein. Purified GST-N protein was diluted to l ⁇ g/ml with o.o5 M carbonate buffer (pH 9.6), and 0.1 ml/well was added to 96-well microtiter plates. Purified GST protein was used as negative control.
  • the plates were incubated overnight at 4 °C, washed with phosphate buffered saline (PBS) - 0.05% Tween 20 (PT), incubated with (0.1 ml/well) PT-2% bovine serum albumin (PBT) for 60 minutes at 37°C and washed again with PT. Serial dilutions ofthe tested sera were added (0.1 ml/well) and the plates were incubated for 60 minutes at 37°C. The plates were washed with PT and were incubated with (0.1 ml/well) alkaline phosphatase-conjugated rabbit anti-mouse antibodies (Zymed, San Francisco, CA) for 30 minutes at 37°C.
  • PBS phosphate buffered saline
  • PBT bovine serum albumin
  • the plates were washed with PT and incubated with (0.1 ml/well) alkaline phosphatase substrate (according to Sigma instructions) for 60 minutes at 37 °C. Plates were read on a MicroElisa reader at a wavelength of 450 nm. Reading higher than 3 -fold negative controls were scored as positive reactions.
  • Splenocytes were harvested from mice one week after the last vaccination. Prior to intracellular cytokine staining, 4xl0 6 pooled splenocytes from the vaccinated mice were incubated for 16 hours with 1 ⁇ g/ml of each candidate peptide for detecting N-specific CD8 + T cell precursors. Intracellular IFN- ⁇ staining and flow cytometry analysis were performed as described previously. Flow cytometry analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA).
  • splenocytes from the various vaccinated mice were incubated with 1 ⁇ g/ml of N peptide (aa 346-354, QFKDNNILL; SEQ ID ⁇ O:31) for 16 hours. Intracellular IFN- ⁇ staining and flow cytometry analysis were performed as described above.
  • the recombinant vaccinia virus was generated using a protocol similar to that described previously Wu, T.-C, et al, 1995, Proc. Natl Acad. Sci. 92:11671-11675). Briefly, the DNA fragment encoding SARS-Co V nucleocapsid was amplified with PCR using a set of primers:
  • vaccinia-infected cells were incubated with rabbit anti-GST- ⁇ sera at 1:100 dilution in lx Perm (PharMingen, San Diego, CA) for 30 min after fixation with Cytofix/Cytoperm (PharMingen, San Diego, CA), washed four times with IX PBS, and then incubated with FITC-labeled goat anti-rabbit IgG (Jackson ImmunoReseach Laboratories, West Grove, PA) at 1 :1000 dilution. Western blot analysis was performed as described above.
  • the Nac-WT and Nac- ⁇ were amplified by infecting TK " cells in vitro according to a standard protocol. Titer was determined by plaque assay using BSC-1 cells. The viral stocks were preserved at — 70°C prior to vaccination. Before use, the virus was thawed, trypsinized with 1/10 volume of trypsin/EDTA in 37°C water bath for 30 min, and diluted with minimal essential medium (MEM) to the final concentration of 1 x 10 8 plaque-forming units (PFU)/ml.
  • MEM minimal essential medium
  • Immunofluorescence Staining for ⁇ Protein Expression Immunofluorescence staining was performed using a protocol similar to what has been described previously (Cheng, WF et al, 2002, Hum Gene Ther 13:553-568). Briefly, Tk " cells were cultured in 8-well culture chamber slides ( ⁇ alge ⁇ unc Int., ⁇ aperville, IL) until they reached 50% confluence. The cells were infected with Nac- ⁇ or Nac WT at 10 m.o.i. to evaluate the expression of ⁇ protein. After 24 hours of infection, cells were fixed and permeabilized with Cytofix/Cytoperm (Pharmingen) for 30 min.
  • Rabbit anti- ⁇ sera was added into the chamber at a dilution of 1:100 and incubated for 30 min.
  • the slides were mounted and observed immediately under a fluorescence microscope.
  • mice were anesthetized and infected with 2xl0 6 PFU/mouse of Vac-WT or Vac- ⁇ in 20 ⁇ l by intranasal instillation 1 week after the final immunization.
  • the immunized mice were infected with lxl0 7 PFU/mouse of Vac- ⁇ in 100 ⁇ l by intravenous injection 1 week after the final immunization. Five mice were used for each vaccinated group. To determine virus titers in lungs, mice were sacrificed 5 days after challenge. Both lungs were harvested, homogenized in 1 ml of MEM containing 2.5% fetal bovine serum, and subjected to three rounds of freezing and thawing before the titer of virus was determined by plaque assay.
  • N protein was not detected in lysates from 293 cells transfected with plasmid DNA with no insert (lane 1) or CRT DNA (lane 2).
  • mice vaccinated with CRT/N DNA generated the highest titer of N-specific antibody responses among mice vaccinated with the various DNA vaccines. Furthermore, ELISA to determine the subtype of IgG antibody showed significantly higher titer of N-specific IgGl Ab than N-specific IgG2a in serum from mice vaccinated with N or CRT/N DNA ( Figure 2D).
  • ELISA to determine the subtype of IgG antibody showed significantly higher titer of N-specific IgGl Ab than N-specific IgG2a in serum from mice vaccinated with N or CRT/N DNA (Figure 2D).
  • Vaccination with CRT/N DNA significantly improved SARS-CoV N-specific CD8+ T cell- mediated immune responses.
  • T cell mediated immunity has been shown to be important for control of viral infection.
  • N 3 ⁇ - 3 ⁇ 8 H-2K 8 311-318 SASAFFGM 39 11 18 We then synthesized these peptides and characterized their ability to activate N-specific CD8+ T cells using splenocytes harvested from mice vaccinated with the various DNA vaccines. As shown in Figure 3A, using intracellular cytokine staining followed by flow cytometry analysis, we showed that a D -restricted 9mer peptide positioned at aa 346-354 (QFKDNVILL; SEQ ID NO:31) of N protein was able to activate significantly more N-specific CD8+ T cells in splenocytes from mice vaccinated with CRT/N DNA than the other epitopes (p ⁇ 0.05).
  • the N peptide (aa 351-359, VILLNKHID; SEQ ID NO:34) only activated N- specific CD 8+ T cells in splenocytes from mice vaccinated with CRT/N DNA to a slightly higher level than the background level.
  • the other five peptides were not able to activate N- specific CD8+ T cells in splenocytes from mice vaccinated with CRT/N DNA ( Figure 3A).
  • the N peptide (aa 346-354, QFKDNVILL; SEQ ID NO:31) likely represents an H-2 D - restricted CTL epitope for SARS-CoV N protein.
  • mice vaccinated with CRT/N DNA generated significantly more N-specific CD8 T cells than mice vaccinated with N DNA ( Figure 3B) (p ⁇ 0.05).
  • Figure 3B shows that the linkage of CRT to N protein in a DNA vaccine enhances N-specific CD8+ T cell mediated immune responses in vaccinated mice.
  • Recombinant vaccinia expressing SARS-CoV N protein as surrogate virus for vaccine studies Certain factors preclude the usage of live SARS-CoN for our vaccine efficacy studies.
  • Vaccination with CRT/ ⁇ D ⁇ A results in the greatest reduction of titer of recombinant vaccinia virus expressing ⁇ protein.
  • the ability of a vaccine to successfully protect against viral challenge is an essential measure of its efficacy.
  • mice vaccinated with D ⁇ A encoding CRT/ ⁇ exhibited a significantly reduced titer of Vac- ⁇ in their lungs when compared to mice vaccinated with D ⁇ A encoding ⁇ (intranasal: p ⁇ 0.013; intravenous: p ⁇ 0.006).
  • vaccination with CRT/ ⁇ D ⁇ A can reduce titer of vaccinia expressing SARS-CoN ⁇ protein to a greater degree than vaccination with ⁇ D ⁇ A.
  • vaccination with CRT/ ⁇ D ⁇ A may generate the best protection against intranasal or intravenous challenge with viruses expressing SARS-CoN ⁇ protein.
  • SARS-CoN antigenic proteins of SARS- CoN
  • S, E, or M proteins antigenic proteins of SARS- CoN
  • the observed enhancement ofthe humoral immune response against the ⁇ protein of SARS-CoN in mice vaccinated with the chimeric CRT/ ⁇ D ⁇ A vaccine may not be useful for SARS-CoN neutralization given the location ofthe ⁇ protein inside the viral envelope.
  • ⁇ -specific antibodies may not be able to cross the envelope to bind with the nucleocapsid protein to abolish the infection.
  • SARS-CoN S, E, and M proteins are expressed on the envelope surface, and neutralizing antibodies against these proteins may thus be able to neutralize SARS-CoN infection.
  • ADE antibody-dependent enhancement
  • virus-specific antibodies have been shown to interact with the Fc and/or complement receptors to enhance viral entry into host immune cells, such as granulocytic cells and monocytes/macrophages.
  • the ADE phenomenon has been observed in at least one coronaviral system. It should therefore be considered when designing a vaccine against SARS- CoN.
  • ⁇ protein may be the logical choice for a target antigen, as antibodies against ⁇ will be unlikely to lead to ADE. This is due to the fact that the ⁇ protein is not expressed on the viral envelope and thus antibodies against ⁇ will probably not be able to facilitate viral entry.
  • the percentage of ⁇ -specific CD8+ T cells in CRT/ ⁇ D ⁇ A-vaccinated mice may potentially be further improved by coadministration with D ⁇ A encoding an antiapoptotic protein.
  • vaccinia virus expressing ⁇ protein of SARS-CoN as a surrogate virus for assaying the vaccine efficacy in our study because SARS-CoN, having mainly been isolated in Asia, is difficult to obtain in the United States. More importantly, the handling of live SARS-CoN is potentially extremely hazardous, whereas the handling of recombinant vaccinia is relatively safe. For these reasons, we generated vaccinia expressing SARS-CoN ⁇ protein for use as a surrogate viral challenge model. The development of such a model for testing of our vaccine strategy is not without precedent, as vaccinia virus has been previously used in several prior studies as a substitute viral challenge model.
  • a potential animal model is Macaca Fascicularis, which has been shown to be susceptible to live SARS-CoN infection and demonstrate pulmonary pathology similar to humans.
  • D ⁇ A vaccination can successfully elicit SARS-CoN ⁇ -specific humoral and CD8+ T cell responses in vaccinated mice, and vaccination with CRT/ ⁇ D ⁇ A can significantly enhance both humoral and cellular immune responses when compared to vaccination with ⁇ D ⁇ A.
  • the CRT D ⁇ A vaccination strategy is applicable to envelope-associated SARS-CoN proteins, such as S, E, or M proteins, for elicitation of both neutralizing antibodies against SARS-CoN and SARS-CoN antigen-specific CTLs.
  • S Spike Protein
  • the amplified product was cloned into the BamHI/EcoRI of pRSETA (Invifrogen, Carslbad, CA).
  • pcDNA3-S a DNA fragment encoding S was isolated from pRSETA-S and further cloned into the BamHI/EcoRI sites of pcDNA3.1(+) vector (Invifrogen, Carlsbad, CA).
  • pcDNA3 encoding SARS-CoV SI, Si or S2 the DNA fragments encoding SI, Si or S2 DNA fragments were amplified with PCR using the following set of primers:
  • pcDNA3-CRT has been described previously (Cheng, 2001, supra).
  • the CRT DNA fragment was amplified with PCR using a set of primers: 5 ' - ggtcttaagatgctgctccctgtgccgctg - 3 ' , (SEQ ID NO:48) 5 ' - caaagatctcagctcgtccttggcctggc - 3 ' (SEQ ID NO:49) and pcDNA3-CRT as a template.
  • the amplified CRT was cloned into the Aflll/BamH I sites of pcDNA3-Sl.
  • pMSCV-S a DNA fragment encoding S was isolated from pRSETA-S and further cloned into the Bglll/EcoRI sites of pMSCV vector (Invifrogen, Carlsbad, CA). The accuracy of these constructs was confirmed by DNA sequencing.
  • the DNA was amplified in E. coli DH5 ⁇ and purified as described previously. Cell Lines The production and maintenance of TC-1 cells has been described previously. In brief,
  • HPV-16 E6, E7 and ras oncogene were used to transform primary C57BL/6 mice lung epithelial cells to generate TC-1 cells.
  • DC-1 cells were generated from the dendritic cell line provided by
  • DC-1 DC-l/S and DC-l/S cells
  • the retroviral vector encoding the S protein of SARS-CoN was first generated.
  • the phoenix packaging cells were transfected with pMSCN-S or pMSCN using Lipofectamine 2000.
  • Supernatant from the transfected Phoenix ( ⁇ X ) cells was incubated with 50% confluent TC-1 or DC-1 cells in the presence of polybrene (8 ⁇ g/ml; Sigma).
  • the retroviral supematants were removed from the transduced cells, and DCs were propagated in culture medium containing 7.5 ⁇ g/ml of puromycin for selection.
  • the transduced TC-1 or DC-1 cells were further selected by growing in culture medium containing 10 ⁇ g/ml of puromycin for 5 days.
  • the expression of S antigen was confirmed by Western blbt analysis.
  • D ⁇ A 20 ⁇ g was transfected into 5xl0 6 293 cells using lipofectamine® 2000 (Life Technologies, Rockville, MD). After overnight transfection, the cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of proteins (50 ⁇ g) were loaded and separated on a 10% SDS-PAGE gel. The gels were elecfroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Blots were blocked with PBS/0.05% Tween 20 (TTBS) containing 5% nonfat milk overnight at 4°C.
  • TTBS Tween 20
  • Membranes were probed with rabbit anti- spike polyclonal antibody at 1 :2000 dilution in TTBS for 1 hr at room temperature, washed six times with TTBS, and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Zymed, San Francisco, CA) at 1 : 1000 dilution in TTBS containing 5% nonfat milk for 1 hr at room temperature. Membranes were washed four times with TTBS and developed using enhanced Hyperfilm-enhanced chemiluminescence (Amersham, Piscataway, ⁇ J). The presence of secreted SI and CRT/SI was confirmed by Western blot analysis.
  • the presence ofthe S-specific antibody in sera from the mice immunized with the various DNA vaccines was determined by Western blot analysis using TC-l/S lysates as a source of antigen.
  • the lysates from TC-l/No insert or TC-l/S were loaded and separated by SDS-10% PAGE gel before blotting.
  • Immune serum samples were collected from DNA- vaccinated mice two weeks after the last vaccination and were diluted to 1 :250 with PBS. Equal amounts of proteins (50 ⁇ g) from TC-l/No insert or TC-l/S lysates were probed with the diluted antisera from vaccinated mice.
  • mice were as described in Example 1. DNA Vaccination DNA-coated gold particles were prepared and used as described above. C57BL/6 mice were immunized with 2 ⁇ g ofthe plasmid which included either no insert, S, SI, Si, S2, CRT or
  • CD3 + cells were enriched from splenocytes, harvested from mice one week after the last vaccination.
  • DC cells (10 5 ) expressing S antigen (DC/S) were incubated with 10 6 ofthe isolated CD3 + T cells for 16 hours.
  • the DC cells not expressing S antigen (DC/No insert) served as a negative control.
  • T cells were stained for both surface CD8 and intracellular IFN- ⁇ , and analyzed with flow cytometry analysis as described before.
  • TC-1 cells expressing S antigen The production and maintenance of TC-1 cells has been described previously.
  • HPV-16 E6, E7 and ras oncogene were used to transform primary C57BL/6 mice lung epithelial cells to generate the TC-1 line.
  • supernatant from phoenix cells transfected with pMSCV- S was incubated with 50% confluent TC-1 cells in the presence of polybrene.
  • the transduced TC-1 cells were further selected by growing in culture medium containing 10 ⁇ g/ml of puromycin for 5 days.
  • the expression of S antigen was confirmed by Western analysis.
  • the immunized mice (10 per group) were subcutaneously challenged with 5xl0 5 cells/mouse in the right leg one week after last vaccination, and then monitored twice a week to check the formation of TC-l/S tumor.
  • In vivo antibody depletion was performed to determine the contribution of various lymphocyte subsets to the protection, as described previously.
  • the following mAbs were used: GK1.5 for CD4 depletion, mAb 2.43 for CD8 depletion, and mAb PK136 was used for NKl.l depletion. Depletions were started one week after final vaccination.
  • the immunized mice (10 per group) were challenged s.c.
  • S-specific antibody responses The presence ofthe S-specific antibody in sera from the mice immunized with the DNA vaccines encoding no insert, S, SI, Si, S2, CRT or CRT/SI via a gene gun was detected by
  • Plates coated with cells were incubated with (0.3 ml/well) PBS - 0.05% Tween 20 (PBT) containing 2% bovine serum albumin for 60 minutes at 37 °C and washed again with PBT. Serial dilutions of the tested sera were added (0.1 ml/well) and the plates were incubated for 60 minutes at 37 °C. The plates were washed with PBT and were incubated with (0.1 ml/well) peroxidase-conjugated rabbit anti-mouse IgG (Zymed, San Francisco, CA) for 30 minutes at 37 °C.
  • PBT PBS - 0.05% Tween 20
  • mice received pcDNA3-S, ⁇ cDNA3-Sl, PcDNA3, Si, pcDNA3-S2 or pcDNA3. Two weeks after the last booster, sera were collected and antibodies against S protein were measured. TC-l/S cell lysates were used as a source of S protein for Western Blot analysis as well as for ELISA.
  • Figure 8 A shows that sera diluted 1 :250 as probes in Western blots revealed that mice given the SI DNA construct generated the highest S-specific antibody immune response.
  • Vaccination with D ⁇ A encoding SARS CoV SI generates the higher numbers of S-specific CD8 + T cells in vivo
  • intracellular cytokine staining was done in conjunction with flow cytometric analysis using CD3 + cells enriched T cells from spleens of vaccinated mice one week after the last vaccination.
  • Enriched CD3 T cells enriched cells from immunized mice were stimulated in vitro with DCs transfected with D ⁇ A encoding
  • SARS CoV S protein (or as a control, D ⁇ A without an insert). After overnight incubation, cells were stained for both CD8 and intracellular JJF ⁇ . As shown in Figure 9A and 9B, pcD ⁇ A3-Sl induced the highest number of S-specific IFN ⁇ CD8 + T-cell precursors among all the DNA constructs tested (p ⁇ 0.01). Vaccination with pcDNA3-S or pcDNA3-Si also induced S-specific
  • SARS CoV S protein represents as a desirable target for generating SARS-CoV S specific antibodies as well as CD8+ T cell reactivity (likely cytotoxic T cells).
  • Cells transfected with the DNA encoding calreticulin linked to SI generate comparable levels of S protein as DNA encoding SI.
  • Some of th present inventors identified the use of DNA constructs comprising sequences encoding calreticulin (CRT) as an excellent strategy to enhance antigen-specific and T cell mediated immune responses to DNA vaccines that comprise DNA encoding an antigen.
  • CRT calreticulin
  • a DNA construct was made that encoded CRT linked to SI. Expression of such DNA was tested by transfecting 293 cells with the DNA constructs and performing Western blot analysis using rabbit anti-S polyclonal antibody.
  • DNA encoding CRT/SI is a potent stimulator of S-specific antibody responses in vaccinated mice
  • mice were immunized with pcDNA3-CRT/Sl, ⁇ cDNA3-Sl,'PcDNA3-CRT or ⁇ cDNA3. Two weeks after the last booster, sera were collected and assayed for antibodies against S protein.
  • TC-l/S cell lysates were used as a source of S protein for Western Blot analysis as well as in
  • mice vaccinated with the CRT/SI DNA generated the highest S-specific antibody i response.
  • Vaccination with DNA encoding SI also generated S-specific antibody responses, albeit lower than vaccination with the CRT/SI construct.
  • ELISA gave similar results in characterizing the S-specific antibody response.
  • mice vaccinated with the CRT/SI DNA generated the highest S-specific antibody i response.
  • Vaccination with DNA encoding SI also generated S-specific antibody responses, albeit lower than vaccination with the CRT/SI construct.
  • ELISA gave similar results in characterizing the S-specific antibody response.
  • mice vaccinated with the CRT/SI DNA generated the highest S-specific antibody i response.
  • Vaccination with DNA encoding SI also generated S-specific antibody responses, albeit lower than vaccination with the CRT/SI construct.
  • ELISA gave similar results in characterizing the S-specific antibody response.
  • mice vaccinated with the CRT/SI DNA generated the highest S-specific antibody i response
  • CRT/S 1 DNA generated the highest S-specific antibody response.
  • vaccination with DNA encoding CRT linked to a SARS antigen, the receptor-binding domain (SI) of SARS-CoV S protein generated enhanced S-specific antibody responses vs vaccination with DNA encoding the SI protein alone.
  • Vaccination with DNA encoding CRT/SI stimulates S-specific CD8 + T cells in vaccinated mice
  • S-specific CD8 + T-cell precursors generated by admimstration ofthe various DNA S protein constructs (pcDNA3 -CRT/SI, pcDNA3-Sl, PcDNA3-CRT or empty pcDNA3)
  • intracellular cytokine staining was performed with flow cytometric analysis using CD3 T cells enriched from spleens of vaccinated mice one week after the last vaccination. These T cells were stimulated in vitro with DCs transfected with DNA encoding S protein or control DNA, and stained for both CD8 and intracellular IFN ⁇ .
  • Vaccination with DNA encoding CRT/SI is generates preventive antitumor immunity against tumor cells that are engineered to express the SARS CoV S protein
  • a non-infectious model system was employed to determine a therapeutic outcome ofthe immunity generated by the present constructs and the enhancing effect ofthe CRT DNA on such immunity.
  • An antitumor response was examined using an in vivo tumor protection assay. TC-l/S tumor cells, transfected to express the S protein were the target ofthe immunity. As shown in Figur
  • mice receiving CRT/SI DNA remained tumor-free 35 days after TC-l/S challenge. Ii comparison, only 40% ofthe mice receiving SI DNA remained tumor-free at this time. All mice vaccinated with control CRT constructs or pcDNA3 plasmid controls grew tumors within two weeks after challenge. To confirm which subsets of lymphocytes were important for this therapeutic effect, an in vi ⁇ antibody depletion study was conducted. Its results appear in Figure 8B. All mice depleted of CD8 cells grew tumors within 10 days after TC-l/S challenge. In contrast, 100% of mice depleted of CD cells or NK cells remained tumor-free 35 days after challenge.
  • CD8 T cells are required for t therapeutic (antitumor) effect ofthe CRT/SI DNA vaccine.
  • the T cell-mediated immunity generated by immunization or vaccination with CRT/SI DNA can effect clinical-type therapeutic results, measured here as an antitumor effect.
  • Plasmid DNA Constructs and DNA Preparation In the current study we used the mammalian expression vector, pcDNA3.1/myc-His (-) (Invifrogen, Carlsbad, CA) for our DNA vaccine studies.
  • pcDNA3-M- myc the DNA fragment encoding SARS-Co V membrane antigen (M) was amplified with PCR using a set of primers: 5 ' -aaagaattcatggcagacaacggtactattac-3 ' , SEQ ID NO:50 5 ' -tttggtaccttactgtactagcaaagcaatat-3 ' SEQ ID NO:51 and pGEX-l-MG6 as a template.
  • the amplified product was further cloned into the EcoRI/Kpnl sites of pcDNA3.1/myc-His (-) vector.
  • CRT DNA segment was isolated from pcDNA3-CRT and cloned into the XhoI/EcoRI sites of pcDNA3.1/»zyc-His(-).
  • the amplified M DNA was cloned into the EcoRI/Kpnl sites of pcDNA3-CRT-myc. The accuracy of these constructs was confirmed by DNA sequencing.
  • the DNA was amplified in E. coli DH5 ⁇ and purified as described previously .
  • Supematants from the transfected phoenix cells were incubated with 50% confluent DC in the presence of polybrene (8ug/ml; Sigma). Following transduction, the retroviral supematants were removed, and DCs were propagated in culture medium containing 7.5 ⁇ g/ml of puromycin for selection. The expression of M antigen was confirmed by western blot analysis.
  • TC-l/M and DC-l/M cells we first generate retroviral vector encoding the M protein of SARS-CoN. The phoenix packaging cells were transfected with pMSCN-M or pMSCN using Lipofectamine 2000.
  • TC-1 or DC-1 cells were incubated with 50% confluent TC-1 or DC-1 cells in the presence of polybrene (8 ⁇ g/ml; Sigma). Following transduction, the retroviral supematants were removed from the transduced cells, and DCs were propagated in culture medium containing 7.5 ⁇ g/ml of puromycin for selection. The transduced TC-1 or DC-1 cells were further selected by growing in culture medium containing 10 ⁇ g/ml of puromycin for 5 days. The expression of M antigen was confirmed by Western blot analysis. All cells were maintained in supplemented RPMI medium as above.
  • mice were as described above. DNA Vaccination DNA-coated gold particles were prepared and used as described above. C57BL/6 mice were immunized with 2 ⁇ g ofthe plasmid encoding no insert, CRT, M, or CRT/M protein. Intracellular Cytokine Staining and Flow Cytometry Analysis This was described above. DC cells expressing M antigen (DC/M), 10 5 were incubated with 10 6 isolated CD3 + T cell for 16 hours. The DC cells not expressing M antigen (DC/No insert) were used as a negative control. After activation, T cells were stained for surface CD8 or CD4 and intracellular IFN ⁇ or IL-4 and analyzed flow cytometrically as described.
  • DC/M DC cells expressing M antigen
  • 10 5 were incubated with 10 6 isolated CD3 + T cell for 16 hours.
  • the DC cells not expressing M antigen (DC/No insert) were used as a negative control. After activation, T cells were stained for surface CD8 or CD4 and intracellular IFN
  • Cells transfected with M or CRT/M DNA vaccines generate comparable levels of M protein.
  • Western blot analysis was done using mouse anti-Myc antibody. 293 cells transfected with DNA encoding CRT or DNA without insert were used as controls. As shown in Figure 14, lysates from cells transfected with the various DNA constructs revealed protein bands having the expected sizes of M and CRT/M. 293 cells transfected with M and CRT/M DNA vaccines expressed comparable levels ofthe encoded proteins.
  • Vaccination with DNA encoding CRT M generates higher numbers of M-specific CD8 + T cells in vivo
  • To assess the quantity of M-specific CD 8 T-cell precursors generated by the pcDNA3, pcDNA3-CRT, pcDNA3-M or pcDNA3-CRT/M vaccine constructs in vaccinated mice To assess the numbers of M-specific CD8 + T-cell precursors that are triggered following administration of various ofthe DNA constructs (pcDNA3 control, pcDNA3-CRT control, pcDNA3-M and pcDNA3-CRT/M) to mice, intracellular cytokine staining was done in conjunction with flow cytometric analysis using spleen cells from the vaccinated mice one week after the last vaccination. Pooled spleen cells were stimulated in vitro with DCs transfected with
  • Vaccination with DNA encoding CRT/M generates high numbers of M-specific CD4 + T helper cells
  • intracellular cytokine staining and flow cytometric analysis was done on spleen cells from vaccinated mice harvested one week after the last vaccination. Pooled cells were stimulated in vitro with DCs transfected with DNA encoding M protein or, as a control, DNA with no insert. After overnight incubation, cells were stained for both CD4 and intracellular IFN ⁇ or IL-
  • IL-4-secreting M-specific CD4 + T helper cells ofthe Th2 class were measured after administering the two experimental and two control DNA vaccine preparations as assessed by intracellular cytokine staining followed by flow cytometric analysis.
  • vaccination with pcDNA3-CRT/M triggered higher numbers of IL-4-secreting M- specific CD4 + T cells compared to pcDNA3-M (p value ⁇ 0.05), although the absolute numbers of IL-4-secreting M-specific CD4 + T cells was lower than the number of IFN ⁇ -secreting, M- specific CD4 + Thl cells in CRT/M-vaccinated mice.
  • Immunization with pcDNA3-CRT/M generates protective antitumor immunity against tumor cells that are engineered to express the SARS CoV M protein.
  • a non-infectious model system was employed to determine a therapeutic outcome ofthe immunity generated by the present constructs and the enhancing effect ofthe CRT DNA on such immunity.
  • An antitumor response was examined using an in vivo tumor protection assay.
  • TC-l/M tumor cells, transfected to express the M protein, were the target ofthe immunity.
  • 100% of mice receiving pcDN A3 -CRT/M remained tumor-free six weeks after TC-l/M challenge.
  • the CRT M DNA construct was capable of generating not only a high number of M-specific T cells in vitro but also a protective antitumor effect against challenge with M-expressing tumor cells in vaccinated mice.
  • an in vivo antibody depletion study was conducted. Its results appear in Figure 18B. All mice depleted of CD8 + T cells grew tumors within 15 days of TC-l/M challenge. In contrast, 100% of mice depleted of CD4 + T cells or NK cells remained tumor-free.
  • CD8 + T cells are required for the therapeutic (antitumor) effect ofthe CRT/SI DNA vaccine.
  • the T cell-mediated immunity generated by immunization or vaccination with CRT/SI DNA can effect clinical-type therapeutic results, measured here as an antitumor effect.

Abstract

This invention provides compositions and methods for inducing and enhancing immune responses, particularly antigen-specific CD8+ T cell mediated responses, against antigens of the SARS coronavirus. These antigens include epitopes of the Membrane (M), Envelope (E), Spike (S) and Nucleocapsid (N) proteins of the virus. Such responses are induced using DNA constructs as an immunogens or vaccines, which encode chimeric polypeptides comprising endoplasmic reticulum chaperone polypeptides, such as human calreticulin (CRT) and an antigenic peptide or polypeptide. In particular, the invention provides compositions and methods for enhancing immune responses induced by polypeptides made in vivo by administered nucleic acid, such as naked DNA or expression vectors, encoding the chimeric molecules. Such enhanced immunity, whether T cell mediated or antibody-mediated protects an infected subject from infection or spread of the SARS CoV in vivo.

Description

DNA Vaccines Targeting Antigens of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) BACKGROUND OF THE INVENTION
Field ofthe Invention This invention, in the field of immunology, virology and medicine, provides immunogenic compositions and methods for inducing enhanced antigen-specific immune responses, particularly those mediated by cytotoxic T lymphocytes (CTL), using chimeric or hybrid nucleic acid molecules that encode an endoplasmic reticulum chaperone polypeptide, e.g., calreticulin, and a polypeptide or peptide antigen ofthe SARS coronavirus (SARS-CoV). Description of the Background Art DNA vaccines are known for their ability to induce both cellular and humoral antigen- specific immunity (reviewed in Donnelly, J et al, 1997. Annu Rev Immunol 15:617-6A8 ; Robinson, HL, 1997. Vaccine i5:785-787; Sin, JI et al. 2000, Intervirology 43:233-246). Advantages of DNA is that it is relatively stable, and it can be easily prepared and harvested in large quantities. In addition, naked plasmid DNA is relatively safe and therefore can be repeatedly administered as a vaccine (Donnelly et al, supra; Robinson, supra). However, naked DNA lacks cell targeting specificity making it important to find an efficient route for delivery into appropriate target cells, such as professional antigen-presenting cells (APCs). Intradermal (i.d.) administration of DNA immunogens or vaccines using a gene gun represents a convenient form of delivery to professional APCs, such as dendritic cells (DCs), in vivo (Condon, C et al, 1996, Nat Med 2: 1122-8). DCs are the most potent professional APCs for priming CD4+ T helper and CD8+ T cytotoxic or killer T cells in vivo (reviewed in Cella, M et al, 1997, Curr Opin Immunol 9:10-16; Hart, DΝ, 1997, Blood 0:3245-3287; Steinman, RM, 1991, Annu Rev Immunol 9:271-296). Thus, gene gun delivery of DΝA vaccines to DCs has become an important method for enhancing T cell-mediated immunity against viral infection. Forms of DΝA vaccines include "naked" DΝA, such as plasmid DΝA (U.S. Patent Νos. 5,580,859; 5,589,466; 5,703,055), viral DΝA, and the like. Basically, a DΝA molecule encoding a desired immunogenic protein or peptide is administered to an individual and the protein is generated in vivo. Use of "naked" DΝA vaccines has the advantages of being safe because, e.g. , the plasmid itself has low immunogenicity, it can be easily prepared with high purity and, compared to proteins or other biological reagents, it is highly stable. However, DΝA vaccines have limited potency. Several strategies have been applied to increase the potency of DNA vaccines, including, e.g., targeting antigens for rapid intracellular degradation; directing antigens to APCs by fusion to ligands for APC receptors; fusing antigens to chemokines or to antigenic pathogenic sequences, co-injection with cytokines or co-stimulatory molecules or adjuvant compositions. Antiviral and antitumor vaccines are an attractive approach for treatment of viral illnesses and cancer because they may have the potency to eradicate systemic virus (or virus- infected cells) or tumor cells in multiple sites in the body and the specificity to discriminate between neoplastic and non-neoplastic cells (Pardoll (1998) Nature Med. 4:525-531). Effective anti- viral and most anti-tumor effects ofthe immune system are mediated by cellular immunity. The cell-mediated component ofthe immune system is equipped with multiple effector mechanisms capable of eradicating virus-infected cells and tumors, and most of these responses are regulated by T cells. Therefore, there is a need in the art for antiviral or anticancer vaccines, particularly as DNA vaccines, that enhance virus-specific (or tumor-specific) T cell responses, to treat virus infections and to control tumors. HPN oncogenic proteins, E6 and E7, are co-expressed in most cervical cancers associated with HPN and are important in the induction and maintenance of cellular transformation. Therefore, in earlier studies, the present inventors and colleagues have described nucleic acid vaccines targeting E6 or E7 proteins as an approach to prevent and treat HPN-associated cervical malignancies. HPN- 16 E7 and E6 are a well-characterized cytoplasmic/nuclear proteins.
Calreticulin and Related Proteins Calreticulin (CRT), an abundant 46 kilodalton (kDa) protein located in the lumen ofthe cell's endoplasmic reticulum (ER), displays lectin activity and participates in the folding and assembly of nascent glycoproteins. See, e.g.,, Νash (1994) Mol. Cell. Biochem. 135:71-78; Hebert (1997) J Cell Biol. 139:613-623; Nassilakos (1998) Biochemistry 37:3480-3490; Spiro
(1996) J Biol. Chem. 27.7:11588-11594; Conway, EM et al, 1995. Heat shock-sensitive expression of calreticulin. In vitro and in vivo up-regulation. JBiol Chem 270:17011-17016) CRT is related to the family of heat shock proteins (HSPs) (Basu, S. et al, J. Exp. Med. 189:797-802; Conway et al, supra) and associates with peptides transported into the ER by transporters that are involved in antigen processing, such as TAP-1 and TAP-2 (Spee et αl,
(1997) Eur. J. Immunol. 27:2441-2449) and with MHC class I-β2m molecules to aid in antigen presentation Sadasivan, B et αl, 1996, Immunity 5:103-114; CRT also forms complexes with peptides in vitro. Upon administration to mice, such peptide-CRT complexes, elicited peptide- specific CD8+ T cell responses (Basu et al, supra; Nair, 1999, J. Immunol. .762:6426-6432). CRT purified from murine tumors elicited immunity specific for the tumor from which the CRT was taken, but not for an antigenically distinct tumor (Basu, supra). By pulsing mouse dendritic cells (DCs) in vitro with a CRT-peptide complex, the peptide was re-presented by MHC class I molecules on the DCs to stimulate a peptide-specific CTL response(Nair, supra). The present inventors and their colleagues have previously used the approach of fusing or combining, at the DNA (or RNA) level, a nucleotide sequence encoding an antigen to test several intracellular targeting strategies that enhance MHC class I and/or class II processing and antigen presentation (Hung, CF. et al. , 2003, Improving DNA vaccine potency via modification of professional antigen presenting cells. Curr Opin Mol Tter 5:20-24. Recently, several ofthe present inventors performed direct comparisons of these strategies for their ability to improve DNA vaccine potency. This comparison showed that linkage of antigen to CRT in a DNA vaccine resulted in the most marked enhancement ofthe humoral and T cell-mediated immune responses in vaccinated mice Kim, JW et al, 2004, Gene Ther. 11:1011-1018. Thus, DNA vaccines employing CRT in this manner have the ability to enhance antigen-specific immune responses (as was originally demonstrated with1 the HPN E7 oncoprotein (see above).
Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). The present invention is directed to compositions and methods for stimulating immunity specific for the coronavirus responsible for severe acute respiratory syndrome (SARS).
Eradication of SARS has become a priority for healthcare agencies around the world because of its communicability, associated mortality, and the potential for pandemic spread. As of July 31, 2003, 8098 cases had been identified worldwide and 774 had died, a mortality rate of about 9.6% (WHO statistics appear on the Web (at the URL ho.int/csr/sars/country/table2003_09_23/en/) ; SARS has been attributed to infection with a coronavirus (SARS-CoN) (Drosten, C et al, 2003, NEnglJMed 348:1967-76; Ksiazek, TG et al, 2003, NEnglJMed 348:1953-66; Peiris, JS et al, 2003, Lancet 367:1319-1225). Evidence that SARS-CoN is the etiologic agent of SARS was demonstrated by experimental infection of macaques (Macacafascicularis), fulfilling Koch's postulates (Fouchier, RA, 2003. Nature 423:240). Knowledge ofthe structure of SARS-CoN and characterization of its complete RΝA genome (Marra, MA et al, 2003, Science 300:1399- 404; Rota, PA et al, 2003, Science 300:1394-1399; Ruan, YJ et al, 2003, Lancet 361:1779- 1785) have provided the basic information that enabled the present inventors to develop[ novel strategies for the prevention of SARS using vaccines. Like its coronavirus relatives, SARS-CoN is a (+)-stranded RΝA virus with a ~30kb genome encoding replicase (rep) gene products and structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (Ν). S protein is thought to be involved with receptor binding, E protein plays a role in viral assembly, M is important for virus budding, and Ν protein is associated with viral RΝA packaging (reviewed in Holmes, KN, 2003, J Clin. Invest. 111:1605- 1609. Among these proteins, it was not evident a priori which contain useful SARS-CoN- specific T cell epitopes or epitopes for targeting by neutralizing or protective antibodies. Ν protein was shown to generate coronavirus-specific CD8+ T cells, albeit in coronaviruses that infect non-human species (i.e., mouse hepatitis virus and infectious bronchitis virus) and have different tissue tropism (Bergmann, C et al, 1993, J Virol 67:7041-7049; Boots, AM et al, 1991, Immunology 74:8-13; Seo, SH et al, 1997, J Virol 71 :7889-789A; Stohlman, SAet al, 1992, Virology 189:217-22A; Stohlman, SAet al, 1993, J Virol 67:7050-7059). Ν-specific CD8+ T cells were shown to generate protective effects in other coronaviral systems (Collisson, EW et al, 2000, Dev Comp Immunol 24:187-200; Seo et al, supra). SARS-CoN, spike (S) protein has been found to bind to angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS CoN on susceptible cells (Dimitrov, DS, 2003 Cell 115:652-653; Li, W et al, 2003, Nature 426:450-454 ; Prabakaran, P et al, 2004, Biochem Biophys Res Commun. 374:235-241; Wang, P et al, 2004, Biochem Biophys Res Commun. 375:439-444). Analysis ofthe S protein has identified the receptor-binding domain, SI (aa 1- 680), and the membrane fusion domain, S2 (aa 680-1225) (see Figure 6) and SEQ ID ΝO:14-17. The receptor-binding domain SI is responsible for binding to the ACE2 receptor (Dimitrov, supra; Li et al, supra; Prabakaran et al, supra; Wang et al, supra). Thus, innovative approaches interfering with the binding of S 1 to ACE2, such as the immunological approaches disclosed herein, may protect the host from SARS CoN infection. As a main surface antigen of SARS-CoN, was said to be one ofthe most important antigen candidates for vaccine design ((Zhao P et al, 2004, Acta Biochim Biophys Sin (Shanghai) 36:37-41). Vaccine strategies targeting the S protein of SARS-CoN have been developed. For instance, a highly attenuated modified vaccinia virus Ankara (MNA) has been engineered to express the S protein of SARS-CoN. Mice vaccinated with MNA-expressing S protein were capable of generating neutralizing antibodies (Bisht, H et al, 2004, Proc Natl Acad A Sci USA 101:6641-6). In addition, a recombinant attenuated parainfiuenza virus encoding SARS-CoN S protein has been shown to generate protective neutralizing antibodies in vaccinated mice (Buchholz, UJ et al, 2004, Proc Natl Acad Sci USA 707:9804-98) and African green monkeys (Bukreyev, A, 2004, Lancet 363:2122-2127). Furthermore, a naked DΝA vaccine encoding S protein generated protective neutralizing antibodies in vaccinated mice
(Zhao et al. , supra). Three fragments ofthe truncated S protein were expressed in E. coli , and analyzed with pooled sera of convalescence phase of SARS patients. The full length S gene DΝA vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoN was measured by ΕLIS A with E. coli expressed truncated S protein or SARS-CoN lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E. coli were able to react with sera of SARS patients and the S gene DΝA candidate vaccine could induce the production of specific IgG antibody against SARS-CoN efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. As indicated elsewhere, while naked DΝA vaccines in general have the clear advantages of simplicity, stability and safety over viral or bacterial vectors, they suffer from lack of potency, since they do not have the intrinsic ability to amplify and spread as live viral vectors do. The present invention is focused on improved DΝA vaccines comprising epitopes of any one or more ofthe S, Ε, M and Ν proteins of SARS-CoN. SUMMARY OF THE INVENTION The invention provides a nucleic acid encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide. The antigenic peptide can comprise an MHC Class I-binding peptide epitope. The antigenic peptide, e.g., the MHC class I-binding peptide epitope, can be between about 8 amino acid residues and about 11 amino acid residues in length. The endoplasmic reticulum chaperone polypeptide includes any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides; or, analogues or mimetics thereof, or, functional fragments thereof. Such functional fragments can be screened using routine screening tests, e.g., as described in Examples 1 and 2, below. Thus, in alternative embodiments, the endoplasmic reticulum chaperone polypeptide comprises or consists of a calnexin polypeptide or an equivalent thereof, an ER60 polypeptide or an equivalent thereof, a GRP94/GP96 or a GRP94 polypeptide or an equivalent thereof, or, a tapasin polypeptide or an equivalent thereof. In one embodiment, the calreticulin polypeptide comprises a human calreticulin polypeptide. In alternative embodiments, the human calreticulin polypeptide sequence can comprise SEQ ID NO:l, or, it can consist essentially of a sequence from about residue 1 to about residue 180 of SEQ LD NO:l, or, it can consist essentially of a sequence from about residue 181 to about residue 417 of SEQ LD NO:l. In one embodiment, the isolated or recombinant nucleic acid molecule is operatively linked to a promoter, such as, e.g., a constitutive, an inducible or a tissue-specific promoter. The promoter can be expressed in any cell, including cells ofthe immune system, including, e.g., antigen presenting cells (APCs), e.g., in a constitutive, an inducible or a tissue-specific manner. hi alternative embodiments, the APCs are dendritic cells, keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like. The invention also provides an expression cassette comprising a nucleic acid sequence encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide from a SARS-CoN. In alternative embodiments, the first domain comprises a calreticulin polypeptide and the second domain comprises an MHC class I-binding peptide epitope of a SARS-CoN antigen. In alternative embodiments, the expression cassette comprises an expression vector, a recombinant virus (e.g., an adenovirus, a retrovirus), a plasmid. The expression cassette can comprise a self-replicating RΝA replicon. The self-replicating RΝA replicon can comprise a Sindbis virus self-replicating RΝA vector, such as, e.g., a Sindbis virus self-replicating RΝA vector SIΝrep5 (U.S. Patent No. 5,217,879). As with all applicable embodiments ofthe invention, the ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, 1, tapasin, or ER60 polypeptides; or, analogues or mimetics thereof, or, functional fragments thereof. The invention also provides a particle comprising a nucleic acid encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide. In one embodiment, the isolated particle comprising an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising at least two domains, wherein the first domain comprises a calreticulin polypeptide and the second domain comprises an MHC class I-binding peptide epitope. The isolated particle can comprise any material suitable for particle bombardment, such as, e.g., gold. The ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides, as discussed herein. The invention also provides a cell comprising a nucleic acid sequence encoding a chimeric protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain comprising at least one antigenic peptide. In one embodiment, the cell comprises an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising at least two domains, wherein the first domain comprises a calreticulin polypeptide and the second domain comprises an MHC class I-binding peptide epitope. The cell can be transfected, infected, transduced, etc., with a nucleic acid ofthe invention or infected with a recombinant virus ofthe invention. The cell can be isolated from a non-human transgenic animal comprising cells comprising expression cassettes ofthe invention. Any cell can comprise an expression cassette ofthe invention, such as, e.g., cells ofthe immune system or antigen presenting cells (APCs). The APCs can be a dendritic cell, a keratinocyte, a macrophage, a monocyte, a B lymphocyte, an astrocyte, a microglial cell, or an activated endothelial cell. The invention also provides a chimeric polypeptide comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide, preferably human CRT, and a second polypeptide domain comprising at least one antigenic peptide of SARS-CoN. The antigenic peptide can comprise an MHC Class I-binding peptide epitope. The ER chaperone polypeptide can be chemically linked to the antigenic peptide, e.g., as a fusion protein (e.g., a peptide bond), that can be, e.g., synthetic or recombinantly produced, in vivo or in vitro. The polypeptide domains can be linked by a flexible chemical linker. In alternative embodiments, the first polypeptide domain ofthe chimeric polypeptide can be closer to the amino terminus than the second polypeptide domain, or, the second polypeptide domain can be closer to the amino terminus than the first polypeptide domain. The ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides, as discussed herein. The invention provides a pharmaceutical composition comprising a composition ofthe invention capable of inducing or enhancing an antigen specific immune response and a pharmaceutically acceptable excipient. In alternative embodiments, the composition comprises: a chimeric polypeptide comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain an antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; or, a cell comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide coding sequence and a second domain comprising an antigenic peptide. The ER chaperone polypeptide can include any ER polypeptide having chaperone functions similar to the exemplary chaperones calreticulin, calnexin, tapasin, or ER60 polypeptides, as discussed herein. The invention provides a method of inducing or enhancing an antigen specific immune response comprising: (a) providing a composition comprising a composition ofthe invention capable of inducing or enhancing an antigen specific immune response, which, in alternative embodiments, can be: a cliimeric polypeptide comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain an antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising an antigenic peptide; or, a cell comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide coding sequence and a second domain comprising an antigenic peptide; and, (b) administering an amount ofthe composition sufficient to induce or enhance an antigen specific immune response. The antigen specific immune response can comprise cellular response, such as a CD8+ CTL response. The antigen specific immune response can also comprise an antibody-mediated response, or, a humoral and a cellular response. In practicing the method the composition can administered ex vivo, or, the composition can be administered ex vivo to an antigen presenting cell (APC). In alternative embodiments, the APC is a dendritic cell, a keratinocyte, a macrophage, a, monocyte, a B lymphocyte, an astrocyte, a microglial cell, or an activated endothelial cell. The APC can be a human cell. The APC can be isolated from an in vivo or in vitro source. The method can further comprise administering the ex vtvo-treated APC to a mammal, a human, a histocompatible individual, or to the same individual from which it was isolated. Alternatively, the composition is administered directly in vivo to a mammal, e.g., a human. The composition can be administered intramuscularly, mtradermally, or subcutaneously. The composition ,e.g., the nucleic acid, expression cassette or particle, can be administered by biolistic injection. The invention provides a method of increasing the numbers of CD8 CTLs specific for a desired SARS-CoN antigen in an individual comprising: (a) providing a composition comprising: a chimeric polypeptide comprising a first domain comprising an endoplasmic, reticulum chaperone polypeptide, preferably CRT, and a second domain comprising an antigenic peptide of SARS-CoN; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain the antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising the antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising the antigenic peptide; or, a cell comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide coding sequence and a second domain comprising the antigenic peptide; wherein the MHC class I- binding peptide epitope is derived from a SARS-CoN antigen, preferably the S protein, the M protein, the Ν protein or the E protein , and, (b) administering an amount ofthe composition sufficient to increase the numbers of antigen-specific CD8+ CTL. The invention provides a method of inhibiting a SARS-CoN infection or spread ofthe virus in a subject comprising: (a) providing a composition comprising: a chimeric polypeptide comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising a S AR-CoN antigenic peptide; a nucleic acid molecule encoding a fusion protein comprising a first polypeptide domain comprising an endoplasmic reticulum chaperone polypeptide and a second polypeptide domain the antigenic peptide; an expression cassette comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising the antigenic peptide; a particle comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide and a second domain comprising the antigenic peptide; or, a cell comprising a nucleic acid sequence encoding a fusion protein comprising a first domain comprising an endoplasmic reticulum chaperone polypeptide coding sequence and a second domain comprising the antigenic peptide; and, (b) administering an amount ofthe composition sufficient to inhibit the infection or spread ofthe virus in vivo. The composition can be co-administered with a second composition that has antiviral activity. The details of one or more embodiments ofthe invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages ofthe invention will be apparent from the description and drawings, and from the claims. All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a Western blot that characterizes recombinant SARS-CoN Ν protein expression in 293 cells transfected with pcDΝA3.1/myc-His (-) encoding CRT, N, CRT/N, or no insert. Rabbit anti-GST-N sera was used at a 1 :100 dilution to detect N expression. Lane 1 : lysate from 293 cells transfected with pcDNA3.1/myc-His (-); Lane 2: lysate from 293 cells transfected with CRT DNA; Lane 3: lysate from 293 cells transfected with N DNA; Lane 4: lysate from 293 cells transfected with CRT/N DNA. Figures 2A-2D are a gel, a blot and graphs showing the N-specific humoral immune response in mice vaccinated with various nucleic acid preparations. Fig. 2A shows a
Coomassie blue-stained SDS-PAGE gel of N protein purified from E. coli. Lane 1 : marker; Lane 2: crude extract of E. coli expressing N protein; Lane 3: purified GST-N protein. Fig. 2B shows a Western blot confirming the presence of purified GST-N protein. Lane 1: lysate from 293 cells transfected with plasmid DNA without an insert (negative control) Lane 2: lysate from 293 cells transfected with plasmid DNA encoding N protein (positive control) Lane 3 : purified GST-N protein. Fig. 2C shows results of ΕLISA determining the titers of N-specific IgG antibodies in sera from vaccinated mice. Sera were collected from DNA- vaccinated mice (5/group) one week after the last vaccination and antibodies against bacteria-derived GST N protein were tested. Purified GST protein was used as a control. Sera from vaccinated mice only generated background level of color changes against GST (not shown). Fig. 2D shows results of an ΕLISA comparing the relative titers of N-specific IgGl and IgG2a antibodies in sera of DNA- vaccinated mice (5/group). Figures 3A-3C are flow cytometric tracings and graphs showing SARS-CoN Ν-specific CD8+ T cell mediated immune responses in mice vaccinated with the various DΝA compositions. Intracellular cytokine staining followed by flow cytometry analysis was used to characterize the Ν-specific CD8+ T cell response to vaccination. Fig. 3 A shows a representative flow cytometric analysis. Fig. 3B depicts the number of SARS-CoN Ν peptide-specific ΕFΝ-γ- secreting CD8+ T cell precursors (per 3xl05 splenocytes) stimulated by the indicated peptide in vitro after harvesting from spleens of mice vaccinated with CRT/Ν DΝA (5 per group). The peptides derived from SARS-CoN Ν protein are defined in Table 3. Fig. 3C is a graph depicting the number of Ν-specific LFΝ-γ-secreting CD8+ T cell precursors/3xl05 splenocytes in spleen cells harvested from mice (5 per group) that had been vaccinated with various DΝA constructs as indicated: plasmid DΝA encoding Ν, CRT, CRT/Ν or lacking any insert were cultured with MHC class I-restricted Ν peptide (aa 346-354, QFKDΝVILL (SΕQ LD ΝO:31 in vitro overnight and stained for CDS and LFN-γ. Figures 4A-4C shows SARS-CoN Ν protein expression in cells infected with recombinant Ν vaccinia . 293 cells were infected with either wild type vaccinia virus (Nac-WT) or vaccinia virus expressing SARS Ν protein (Nac-Ν). Rabbit anti-GST-Ν sera was used to identify Ν protein expression. Fig.43A shows a flow cytometric analysis. Fig.4B shows immunofmorescence staining. Fig. 4C shows a Western blot using cell lysate from 293 cells infected with either Nac-WT (Lane l)or Nac-Ν (Lane 2). Note: Lysate from 293 cells infected with Nac-N revealed a band approximately r 48,000 in size, corresponding to N protein of SARS-CoN. Figures 5A-5B are graphs showing reduction ofthe viral titer of recombinant N vaccinia in mice vaccinated with the various DNA vaccines. Mice (5 per group) were vaccinated with pcDNA3.1/myc-His (-) encoding CRT, N, CRT/N, or no insert as described in the Examples. Fig. 1 A shows virus titers after intranasal challenge with vaccinia. The immunized mice were infected with 2xl06 PFU/mouse of Nac-WT or Nac-Ν in 20 μl by intranasal instillation 1 week after the final immunization. Nac-WT infection was used as a negative control. Fig. 5B shows results of i.v. challenge with vaccinia. The immunized mice were infected with 107 PFU/mouse of Nac-Ν in 100 μl by intravenous injection 1 week after final immunization. The titer of virus was determined by plaque assay 5 days after challenge. Note: Mice vaccinated with CRT/N DNA showed the greatest reduction in titer of Nac-N virus when challenged intranasally or intravenously. Figure 6 is a schematic diagram of SARS-CoN S protein showing a domain structure. Domain SI corresponds to residues 1-680 of SEQ ID ΝO:14; with residues 1-18 representing a signal sequence), S2 corresponds to residues 681-1225 of SEQ ID NO:14 and includes two helical regions (HR1 and HR2) as well as a transmembrane domain. Si represents an overlapping fragment of SI and S2, and includes residues 417-816 or SEQ ID NO:14; (polypeptide indicating and its recombmants used for immunization. Recombinant nucleic acids comprising SI, S2 and Si were examined as immunogens. Figure 7 A-7B show blots that represents expression and secretion of SARS-CoV S and its recombinant proteins after in vitro transfection . The expression of SARS-CoN S and its recombinant proteins was determined in 293 cells transfected with a DΝA molecule encoding S, SI, Si or S2 by Western blot analysis (Fig. 7A). Overnight after transfection, the cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of proteins (50 μg) were loaded and separated by 10% SDS-PAGE. Rabbit anti-S antibody at a 1 :2000 dilution was used to detect expression ofthe full length S polypeptide and its recombinant domains/ fragments. The presence of secreted SARS-CoV S proteins and recombinant domains confmned by Western blot analysis (Fig 7B). Forty eight hours after transfection, 4 ml of culture supematants were collected, centrifuged to remove cellular debris and concentrated to 0.2 ml using Amicon Ultra centrifugal filter devices. Concentrated supematants (20 μl) were loaded and separated by 10% SDS-PAGE before blotting. The presence of S and its recombinant domains/fragments proteins was detected as above. Figure 8A-8B shows results of an S-specific antibody responses in mice immunized with various recombinant SARS-CoV S DNA immunogens. Mice were immunized with the plasmid DNAs encoding S, SI, Si or S2 via gene gun. Serum samples were collected from one week after the last vaccination and tested for anti-S antibodies. S-specific antibodies were detected in semm diluted to 1 :250 (in PBS) by Western blot analysis using 50 μg of transfected 293 lysates with DNA encoding S (Fig. 8A). The end-point dilution titer of S-specific antibodies in the sera of DNA-immunized C57BL/6 mice were determined by ELISA in microplates coated with "TC-l/S" cells or "TC-l/No insert" cells (Fig. 8B). Absorbances >3-fold higher than negative controls were considered positive. Figure 9A-9B show SARS-CoV S-specific CD8+ T cell responses in mice immunized with the various DNA immunogens. Intracellular cytokine staining (IFNγ = INFγ) was detemiined after flow cytometry to characterize the S-specific CD8+ T cell response. Fig. 9A shows flow cytometric analysis and Fig. 9B is a bar graph depicting the number of IFNγ- secreting CD8+ T cell precursors /3xl05 splenocytes. CD3+ cells (106) were harvested from spleens of immunized given S, SI, Si or S2-encoding DNA immunogens. These cells were stimulated with 105 "DC/S" dendritic cells or "DC/No insert" dendritic cells in vitro overnight and were stained for CD8 and IFNγ as measures of SARS-CoV S-specific CD8+ T cell immunity. Figure 10A-10B show expression and secretion of SI and CRT/SI chimeric polypeptide after in vitro transfection. Expression was delennined in 293 cells transfected with DNA constructs comprising no insert, CRT, SI or CRT/SI by Western blot analysis (Fig. 10A). After overnight incubation, transfected cells were lysed and equal amounts of proteins (50 μg) were loaded and separated by 10% SDS-PAGE. Rabbit anti-S antibody diluted 1:2000 was used to detect SI and the CRT/SI chimeric polypeptide. The presence of secreted SI and CRT/SI was also examined by Western blot analysis (Fig. 10B). Forty eight hours after transfection, 4 ml of culture supematants were obtained, centrifuged and concentrated as above. Samples (5, 10, 20 μl) ofthe concentrated supematants were separated by SDS- 10% PAGE before blotting. Detection was as above with rabbit anti-S antibody. Figure 11A-11B shows that immunization with DNA encoding CRT/SI induces a stronger antibody responses than DNA encoding alone. Mice were immunized with the plasmid DNAs encoding no insert, CRT, SI or CRT/SI via gene gun. Semm samples were collected and antibodies measure as described for Fig. 8A-8AB. Figure 12A-12B shows that more potent SARS-CoV S-specific CD8+ T cell responses result from administration of DNA immunogens encoding the CRT/SI fusion protein. Methods are the same as described for Fig. 9A-9B. Figure 13A-13B shows that mice vaccinated with DNA immunogens encoding the chimeric polypeptide CRT/S 1 have stronger in vivo protection against growth of a tumor expressing the SARS-CoV S protein. Fig. 13 A shows a study in which transfected tumor cells expressing S (TC-l/S) were injected subcutaneously (5χl0scells/mouse) into mice that had been immunized with a DNA constructs that encoded CRT, SI, CRT/SI or no insert (10 mice/group). Animals received the challenge in the right leg one week after the last vaccination and were monitored twice weekly for visible tumor. Fig 13B shows results of tumor growth when various subsets of immune cells were depleted by antibody treatment in vivo. CD4, CD8, and Kl .1 depletion was initiated one week after last vaccination and the mice challenged one week later. The depletion treatment was terminated 32 days after tumor challenge. For each time point shown, >99% ofthe appropriate cell subset was depleted with normal numbers of cells of other subsets. Figure 14. is a Western blot that characterizes recombinant SARS-CoV M (membrane) protein expression in 293 cells transfected withpcDNA3.1/myc-His (-) encoding CRT, M or CRT/M. pcDNA3.1/myc-His (-) without insert was used as a negative control. The transfected cells were lysed 24 hours later and separated by SDS-PAGE. Mouse anti-myc antibody was used to detect M protein expression. Lanes 1-4 show lysates from 293 cells transfected with DNA without an insert and DNA encoding CRT, M or CRT/M, respectively. Figure 15A-15B show SARS-CoV M-specific CD8+ T cell responses in mice immunized with the various DNA immunogens encoding the M polypeptide. Five mice per group were immunized with ρcDNA3, ρcDNA3-CRT, ρcDNA3-M or pcDNA3-CRT/M. CD3+ enriched T cells from spleens of immunized mice were stimulated in vitro with transfected dendritic cells, DC/S" dendritic cells or "DC/No insert", in vitro overnight and stained for both CD8 and intracellular IFNγ. Fig. 15A shows representative flow cytometry results for CD3+ enriched T cells from immunized or control mice. Fig. 15B is a bar graph depicting the number of antigen-specific IFNγ-secreting CD8+ T-cell precursors/3xl05 CD3+ enriched T cells (mean±SD) after DNA vaccination. Figure 16A-16B presents flow cytometric analysis of IFN-γ-secretmg M-specific CD4+ T-cells (Thl) in mice (five per group) immunized with pcDNA3, pcDNA3-CRT, pcDNA3-M or pcDN A3 -CRT/M. CD3+ -enriched T cells from spleens of immunized mice were stimulated in vitro with DC-l/M or DC-l/no insert overnight, and stained for both CD4 and intracellular IFNγ. Fig. 16A presents representative flow cytometry data for splenocytes harvested from immunized mice. Fig. 16B is a bar graph depicting the number of antigen-specific JJFNγ- secreting CD4+ T-cells (Thl cells) per 3xl05 CD3+ enriched T cells (mean±SD). Figure 17A-17B presents flow cytometry analysis of IL-4-secreting M-specific CD4+ T- cells (Tl 2) in mice (five per group) immunized with pcDNA3, pcDNA3-CRT, pcDNA3-M or pcDNA3-CRT/M. CD3+ enriched T cells from spleens of immunized mice were stimulated in vitro with DC-l/M or DC-l/no insert overnight, and stained for both CD4 and intracellular JX-4. Fig. 17A presents representative flow cytometry data for splenocytes harvested from immunized mice. Fig. 17B presents a bar graph depicting the number of antigen-specific IL-4-secreting CD4+ T-cells (Th2 cells) per 3xl05 CD3+ enriched T cells (mean±SD). Figure 18A-18B shows that mice vaccinated with DNA immunogens encoding the chimeric polypeptide CRT/M are much better protected in vivo against growth of a tumor expressing the SARS-CoV M protein. Fig. 18A shows a study in which transfected tumor cells expressing M (TC-l/M) were injected subcutaneously (5χl04cells/mouse) into mice that had been immunized with a plasmid DNA constructs that encoded (i) CRT, (ii) M, (iii) CRT/M or (iv) no insert (10 mice/group). Animals received the challenge in the right leg one week after the last vaccination and were monitored twice weekly for visible tumor. Fig 18B shows results of tumor growth when various subsets of immune cells were depleted by antibody treatment in vivo. CD4, CD8, and NKl.l depletion was initiated one week after last vaccination and the mice challenged one week later. The depletion treatment was terminated 32 days after tumor challenge. Both graphs show the percentage of tumor-free mice over time. Figure 19 shows schematically SARS-CoV cDNA clones spanning the genome ofthe TW1 strain. DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention provides compositions and methods for enhancing the immune responses, particularly cytotoxic T cell immune responses, induced in vivo administration of chimeric nucleic acids that encode (a) an endoplasmic reticulum chaperone polypeptide linked to (b) at least one antigenic polypeptide or peptide from SARS CoV. These chimeric polypeptides or fusion proteins can also be administered, although the preferred embodiment is a nucleic acid composition or expression plasmid for administration as an immunogen or vaccine. For descriptions of this general strategy as using chaperone polypeptides or other such polypeptides to enhance the potency of a vector carrying antigen-encoding DNA, see for example, Wu et al, WO 01/29233; Wu et al, WO 02/009645; Wu et al, WO 02/061113; Wu et al, WO 02/074920; Wu et al, WO 02/12281, all of which are incorporated by reference in their entirety. The fusion polypeptide encoded by the nucleic acid immunogenic or vaccine composition comprises at least two "domains:" the first domain comprises a endoplasmic reticulum chaperone polypeptide, and the second domain comprises a full length polypeptide or a shorter fragment that comprises at least one epitope-comprising a SARS-CoV structural protein, most preferably the product ofthe S, E, M or N gene of SARS-CoV. Although any endoplasmic reticulum chaperone polypeptide, or functional fragment or variation thereof, can be used in the invention, such as calreticulin, tapasin, ER60 or calnexin polypeptides, human calreticulin (CRT) is prefened. The antigenic domain ofthe chimeric molecule is preferably one that comprises an MHC class I-binding peptide epitope. In the methods ofthe invention, the chimeric nucleic acid or polypeptide are administered or applied to induce or enhance immune responses that are specific and anti-viral in their effect (e.g., that neutralize vims or result in damage and death of virus expressing cells) in vivo. The experiments described herein demonstrate that the methods ofthe invention can enhance a cellular immune response, particularly, a CTL reactivity, induced by a DNA vaccine encoding various polypeptides ofthe SARS CoV. Initially, DNA encoding the nucleocapsid or N-protein was used. . As described in Example 1, below, the results of these experiments demonstrate that DNA vaccines comprising nucleic acid encoding a fusion protein comprising CRT linked to a N protein of SARS-CoV enhances the potency of DNA vaccines. DNA vaccines ofthe invention containing chimeric CRT fusion genes were or will be administered to mice and other subjects by biolistic subcutaneous methods. They induced increased N-specific CD8+ CTL precursors, and are expected to improve immune protection against the vims. This increase in N-specific CD8+ T cell precursors was significant as compared to DNA vaccines containing N or CRT genes alone. A potential mechanism for the enhanced antigen-specific CD8 T cell immune responses in vivo is the presentation of antigen through the MHC class I pathway by uptake of apoptotic bodies from cells expressing the antigen, also called "cross-priming".
DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise. The term "antigen" or "immunogen" as used herein refers to a compound or composition comprising a peptide, polypeptide or protein which is "antigenic" or "immunogenic" when administered (or expressed in vivo by an administered nucleic acid, e.g., a DNA vaccine) in an appropriate amount (an "immunogenically effective amount"), i.e., is capable of eliciting, augmenting or boosting a cellular and/or humoral immune response either alone or in combination or linked or fused to another substance (which can be administered at once or over several intervals). "Calnexin" describes the well-characterized membrane protein ofthe endoplasmic reticulum (ER) that functions as a molecular chaperone and as a component ofthe ER quality control machinery. Calreticulin is a soluble analogue of calnexin. In vivo, calreticulin and calnexin play important roles in quality control during protein synthesis, folding, and posttranslational modification. Calnexin polypeptides, and equivalents and analogues thereof, are species in the genus of ER chaperone polypeptides, as described herein (Wilson (2000) J. Biol. Chem. 275:21224-2132; Danilczyk (2000) J. Biol. Chem. 275:13089-13097; U.S. Patent Nos. 6,071,743 and 5,691,306). "Calreticulin" or "CRT" describes the well-characterized ~46 kDa resident protein ofthe
ER lumen that has lectin activity and participates in the folding and assembly of nascent glycoproteins. CRT acts as a "chaperoiie" polypeptide and a member ofthe MHC class I transporter TAP complex; CRT associates with TAP1 and TAP2 transporters, tapasin, MHC Class I heavy chain polypeptide and β2 microglobulin to function in the loading of peptide epitopes onto nascent MHC class I molecules (Jorgensen (2000) Eur. J. Biochem. 267:2945- 2954). The term "calreticulin" or "CRT" refers to polypeptides and nucleic acids molecules having substantial identity (defined herein) to the exemplary CRT sequences as described herein. A CRT polypeptide is a polypeptides comprising a sequence identical to or substantially identical (defined herein) to the amino acid sequence of CRT. An exemplary nucleotide and amino acid sequence for a CRT used in the present compositions and methods are SEQ ID NO:l and SEQ ID NO:2, respectively. The terms "calreticulin" or "CRT" encompass native proteins as well as recombinantly produced modified proteins that induce an immune response, including a CTL response. The terms "calreticulin" or "CRT" encompass homologues and allelic variants of CRT, including variants of native proteins constructed by in vitro techniques, and proteins isolated from natural sources. The CRT polypeptides ofthe invention, and sequences encoding them, also include fusion proteins comprising non-CRT sequences, particularly MHC class I- binding peptides; and also further comprising other domains, e.g., epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals and the like. The term "endoplasmic reticulum chaperone polypeptide" as used herein means any polypeptide having substantially the same ER chaperone function as the exemplary chaperone proteins CRT, tapasin, ER60 or calnexin. Thus, the term includes all functional fragments or variants or mimics thereof. A polypeptide or peptide can be routinely screened for its activity as an ER chaperone using assays known in the art. While the invention is not limited by any particular mechanism of action, in vivo chaperones promote the conect folding and oligomerization of many glycoproteins in the ER, including the assembly ofthe MHC class I heterotrimeric molecule (heavy chain, β2m, and peptide). They also retain assembled MHC class I heterotrimeric complexes in the ER (Hauri (2000) FEBS Lett. 476:32-37). The term "epitope" as used herein refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., the MHC class I-binding peptide compositions used in the methods ofthe invention. An "antigen" is a molecule or chemical structure that either induces an immune response or is specifically recognized or bound by the product of an immune response, such as an antibody or a CTL. The specific conformational or stereochemical "domain" to which an antibody or a TCR bind is an "antigenic determinant" or "epitope." TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class II protein. The terms "ER60" or "GRP94" or "gp96" or "glucose regulated protein 94" as used herein describes the well-characterized ER chaperone polypeptide that is the ER representative ofthe heat shock protein-90 (HSP90) family of stress-induced proteins. These bind to a limited number of proteins in the secretory pathway, possibly by recognizing advanced folding intermediates or incompletely assembled proteins. ER60 polypeptides, and equivalents and analogues thereof, are species in the genus of ER chaperone polypeptides, as described herein (Argon (1999) Semin. CellDev. Biol. 10:495-505; Sastry (1999) J. Biol. Chem. 274:12023- 12035; Nicchitta (1998) Curr. Opin. Immunol. 10:103-109; U.S. Patent No. 5,981,706). The term "expression cassette" or "expression vector" as used herein refers to a nucleotide sequence which is capable of affecting expression of a protein coding sequence in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be included, e.g., enhancers. "Operably linked" refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription ofthe DNA sequence. Thus, expression cassettes include plasmids, recombinant viruses, any form of a recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons), bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA, e.g., plasmids, virases, and the like (U.S. Patent No. 5,217,879), and includes both the expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extrachromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous stracture, or is incoφorated within the host's genome. The term "chemically linked" refers to any chemical bonding of two moieties, e.g., as in one embodiment ofthe invention, where an ER chaperone polypeptide or CRT is chemically linked to an antigenic peptide. Such chemical linking includes the peptide bonds of a recombinantly or in vivo generated fusion protein. The term "chimeric" or "fusion" polypeptide or protein refers to a composition comprising at least one polypeptide or peptide sequence or domain which is associated with a second polypeptide or peptide domain. One embodiment of this invention is an isolated or recombinant nucleic acid molecule encoding a fusion protein comprising at least two domains, wherein the first domain comprises an endoplasmic reticulum chaperone, e.g., CRT, and the second domain comprising an antigenic epitope, e.g., an MHC class I-binding peptide epitope. Additional domains can comprise a polypeptide, peptide, polysaccharide, or the like. The "fusion" can be an association generated by a peptide bond, a chemical linking, a charge interaction (e.g., electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the "fusion protein" can be translated from a common message. Alternatively, the compositions ofthe domains can be linked by any chemical or electrostatic means. The chimeric molecules ofthe invention (e.g., CRT-class I-binding peptide fusion proteins) can also include additional sequences, e.g., linkers, epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals, and the like. Alternatively, a peptide can be linked to a carrier simply to facilitate manipulation or identification/ location ofthe peptide. The term "immunogen" or "immunogenic composition" refers to a compound or composition comprising a peptide, polypeptide or protein which is "immunogenic," i.e., capable of eliciting, augmenting or boosting a cellular and/or humoral immune response, either alone or in combination or linked or fused to another substance. An immunogenic composition can be a peptide of at least about 5 amino acids, a peptide of 10 amino acids in length, a fragment 15 amino acids in length, a fragment 20 amino acids in length or greater; smaller immunogens may require presence of a "carrier" polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the immunogen. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA comprising the immunogen' s coding sequence operably linked to a promoter, e.g., an expression cassette. The immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 amino acids. Epitopes of more than one SARS-CoV protein may be used in combination. The term "isolated" as used herein, when referring to a molecule or composition, such as, e.g., a CRT nucleic acid or polypeptide, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g., RNAs), or other contaminants with which it is associated in vivo or in its natural state. Thus, a CRT composition is considered isolated when it has been isolated from any other component with which it is natively associated, e.g., cell membrane, as in a cell extract. An isolated composition can, however, also be substantially pure. An isolated composition can be in a homogeneous state and can be dry or in an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC). Thus, the isolated compositions of this invention do not contain materials normally associated with their in situ environment. Even where a protein has been isolated to a homogenous or dominant band, there are trace contaminants which co-purify with the desired protein. The terms "polypeptide," "protein," and "peptide" include compositions ofthe invention that also include "analogues ," or "conservative variants" and "mimetics" or "peptidomimetics" with structures and activity that substantially conespond to the polypeptide from which the variant was derived, including, e.g., human CRT or a Class I-binding peptide epitope, such as from the SARS-CoV S, E, M or N proteins, as discussed in detail, below. The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use, e.g., as a vaccine, in a subject. The pharmaceutical compositions of this invention are formulations that comprise a pharmacologically effective amount of a composition comprising, e.g., a nucleic acid, or vector, or cell ofthe invention, and a pharmaceutically acceptable carrier. The term "promoter" is an array of nucleic acid control sequences which direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter which is active under most environmental and developmental conditions. An "inducible" promoter is a promoter which is under environmental or developmental regulation. A "tissue specific" promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription ofthe nucleic acid conesponding to the second sequence. The term "recombinant" refers to (1) a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide"), (2) methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or (3) a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide. For example, recombinant CRT or an MHC class I-binding peptide epitope can be recombinant as used to practice this invention. "Recombinant means" also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of polypeptide coding sequences in the vectors used to practice this invention. The term "self-replicating RNA replicon" refers to constructs based on RNA viruses, e.g., alphavirus genome RNAs (e.g., Sindbis virus, Semliki Forest vims, etc.), that have been engineered to allow expression of heterologous RNAs and proteins. These recombinant vectors are self-replicating (i.e., they are "replicons") and can be introduced into cells as naked RNA or DNA, as described in detail, below. In one embodiment, the self-replicating RNA replicon comprises a Sindbis virus self-replicating RNA vector SINrep5, which is described in detail in U.S. Patent No. 5,217,879. The term "systemic administration" refers to administration of a composition or agent such as the molecular vaccine or the CRT-Class I-binding peptide epitope fusion protein described herein, in a manner that results in the introduction ofthe composition into the subject's circulatory system. The term "regional" administration refers to administration of a composition into a specific anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ, and the like. For example, regional administration includes administration of the composition or drug into the hepatic artery. The term "local administration" refers to administration of a composition or drag into a limited, or circumscribed, anatomic space, such as intrarumoral injection into a tumor mass, subcutaneous injections, intramuscular injections, and the like. Any one of skill in the art would understand that local administration or regional administration may also result in entry ofthe composition or drag into the circulatory system. "Tapasin" is the Icnown ER chaperone polypeptide, as discussed above. While not limited by any particular mechanism of action, in vivo, tapasin is a subunit ofthe TAP (transporter associated with antigen processing) complex and binds both to TAP1 and MHC class I polypeptides. Tapasin polypeptides, and equivalents and analogues thereof, are species in the genus of ER chaperone polypeptides, as described herein (Bamden (2000) J. Immunol. 165:322-330; Li (2000) J. Biol. Chem. 275:1581-1586).
Generating and Manipulating Nucleic Acids The methods ofthe invention provide for the administration of nucleic acids encoding a
CRT-SARS-CoV Class I epitope binding peptide fusion protein, as described above. Recombinant CRT-containing fusion proteins can be synthesized in vitro or in vivo. Nucleic acids encoding these compositions can be in the form of "naked DNA" or they can be incorporated in plasmids, vectors, recombinant viruses (e.g., "replicons") and the like for in vivo or ex vivo administration. Nucleic acids and vectors ofthe invention can be made and expressed in vitro or in vivo, a variety of means of making and expressing these genes and vectors can be used. One of skill will recognize that desired gene activity can be obtained by modulating the expression or activity ofthe genes and nucleic acids (e.g., promoters) within vectors used to practice the invention. Any ofthe known methods described for increasing or decreasing expression or activity, or tissue specificity, of genes can be used for this invention. The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature. General Techniques \ The nucleic acid sequences used to practice this invention, whether RNA, cDNA, genomic DNA, vectors, recombinant virases or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to bacterial cells, e.g., mammalian, yeast, insect or plant cell expression systems. Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Eree Radic. Biol. Med. 79:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. Calreticulin Sequences The sequences of CRT, including human CRT, are well known in the art (McCauliffe (1990) J. Clin. Invest. 86:332-335; Bums (1994) Nature 367:476-480; Coppolino (1998) frit. J. Biochem. Cell Biol. 30:553-558). The nucleic acid sequence appears as GenBank Accession
No. NM 004343 and is SEQ ID NO:l.
1 gtccgtactg cagagccgct gccggagggt cgttttaaag ggccgcgttg ccgccccctc
61 ggcccgccat gctgctatcc gtgccgctgc tgctcggcct cctcggcctg gccgtcgccg
121 agcccgccgt ctacttcaag gagcagtttc tggacggaga cgggtggact tcccgctgga
181 tcgaatccaa acacaagtca gattttggca aattcgttct cagttccggc aagttctacg
241 gtgacgagga gaaagataaa ggtttgcaga caagccagga tgcacgcttt tatgctctgt
301 cggccagttt cgagcctttc agcaacaaag gccagacgct ggtggtgcag ttcacggtga
361 aacatgagca gaacatcgac tgtgggggcg gctatgtgaa gctgtttcct aatagtttgg
421 accagacaga catgcacgga gactcagaat acaacatcat gtttggtccc gacatctgtg
481 gccctggcac caagaaggtt catgtcatct tcaactacaa gggcaagaac gtgctgatca
541 acaaggacat ccgttgcaag gatgatgagt ttacacacct gtacacactg attgtgcggc
601 cagacaacac ctatgaggtg aagattgaca acagccaggt ggagtccggc tccttggaag
661 acgattggga cttcctgcca cccaagaaga taaaggatcc tgatgcttca aaaccggaag
721 actgggatga gcgggccaag atcgatgatc ccacagactc caagcctgag gactgggaca
781 agcccgagca tatccctgac cctgatgcta agaagcccga ggactgggat gaagagatgg
841 acggagagtg ggaaccccca gtgattcaga accctgagta caagggtgag tggaagcccc
901 ggcagatcga caacccagat tacaagggca cttggatcca cccagaaatt gacaaccccg
961 agtattctcc cgatcccagt atctatgcct atgataactt tggcgtgctg ggcctggacc
1021 tctggcaggt caagtctggc accatctttg acaacttcct catcaccaac gatgaggcat
1081 acgctgagga gtttggcaac gagacgtggg gcgtaacaaa ggcagcagag aaacaaatga
1141 aggacaaaca ggacgaggag cagaggctta aggaggagga agaagacaag aaacgcaaag
1201 aggaggagga ggcagaggac aaggaggatg atgaggacaa agatgaggat gaggaggatg
1261 aggaggacaa ggaggaagat gaggaggaag atgtccccgg ccaggccaag gacgagctgt
1321 agagaggcct gcctccaggg ctggactgag gcctgagcgc tcctgccgca gagcttgccg
1381 cgccaaataa tgtctctgtg agactcgaga actttcattt ttttccaggc tggttcggat
1441 ttggggtgga ttttggtttt gttcccctcc tccactctcc cccaccccct ccccgccctt
1501 tttttttttt tttttaaact ggtattttat cctttgattc tccttcagcc ctcacccctg
1561 gttctcatct ttcttgatca acatcttttc ttgcctctgt gccccttctc tcatctctta
1621 gctcccctcc aacctggggg gcagtggtgt ggagaagcca caggcctgag atttcatctg
1681 ctctccttcc tggagcccag aggagggcag cagaaggggg tggtgtctcc aaccccccag
1741 cactgaggaa gaacggggct cttctcattt cacccctccc tttctcccct gcccccagga
1801 ctgggccact tctgggtggg gcagtgggtc ccagattggc tcacactgag aatgtaagaa
1861 ctacaaacaa aatttctatt aaattaaatt ttgtgtctc 1899
The amino acid sequence ofhuman CRT protein (SEQ ID NO :2) is shown below 1 MLLSVPLLLG LLGLAVAEPA VYFKEQFLDG DG TSRWIES KHKSDFGKFV LSSGKFYGDE 61 EKDKGLQTSQ DARFYALSAS FEPFSNKGQT LVVQFTVKHE QNIDCGGGYV KLFPNSLDQT
121 DMHGDSEYNI MFGPDICGPG TKKVHVIFNY KGKNVLINKD IRCKDDEFTH LYTLIVRPDN
181 TYEVKIDNSQ VESGSLEDD DFLPPKKIKD PDASKPED D ERAKIDDPTD SKPEDWDKPE
241 HIPDPDAKKP ED DEEMDGE WEPPVIQNPE YKGEWKPRQI DNPDYKGTWI HPEIDNPEYS 301 PDPSIYAYDN FGVLGLDL Q VKSGTIFDNF LITNDEAYAE EFGNETWGVT KAAEKQMKDK
361 QDEEQRLKEE EEDKKRKEEE EAEDKEDDED KDEDEEDEED KEEDEEEDVP GQAKDEL 417
The structure of polypeptides, peptides, other functional derivatives, including mimetics of CRT are preferably based on stracture and amino acid sequence of CRT, preferably human CRT, SEQ TD NO:2 above. (See also, McCauliffe (1990) J. Clin. Invest. 86:332-335; Bums (1994) Nature 367:476-480; Coppolino (1998) Int. J. Biochem. Cell Biol. 30:553-558)
SARS-CoV Genomic Sequences, and Sequences of Polypeptides The genomic nucleotide sequence ofthe SARS coronavirus (nt 1 to 29751; SEQ TD NO:3), Tor2 strain , is deposited in Genbank under access no. NC_004718 (available at WWW URL ncbi.nlm.nih.qov/entrez/viewer.fcgi?db=nucleotide&val=30271926 . See, He, R. et al, Biochem. Biophys. Res. Commun. 3161. -476-483 (2004) ; Snijder, E.J. et al, J. Mol. Biol. 331 991-1004 (2003) ; Mana, MA et al, Science 300 : 1399-1404 (2003). The reference sequence was derived from AY274119. On May 1, 2003 this sequence version replaced gi:30124072.
SEQ ID NO:3 1 atattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 61 ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 121 gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 181 tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 241 gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 301 cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 361 gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 421 ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 481 cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 541 gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 601 gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 661 ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 721 cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 781 ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 841 ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 901 tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 961 gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1021 acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1081 tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1141 actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1201 aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1261 acgtgcgact ttctgaaagc cacttgtgaa cattgtggca ctgaaaattt agttattgaa 1321 ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1381 tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1441 attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1501 tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1561 tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1621 atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1681 gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1741 agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1801 aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1861 ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1921 gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1981 atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2041 aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2101 ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2161 gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2221 attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2281 gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2341 gtcactatcg ctggcgcaaa gttgcgatca ctcaacttag gtgaagtctt catcgctcaa 2401 agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2461 cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2521 tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2581 ttcacaaatg gagctatcgt tggcacacca gtctgtgtaa atggcctcat gctcttagag 2641 attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2701 tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2761 gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2821 gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2881 gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2941 aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3001 ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3061 gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3121 acagaggatg attatcaagg tctccctctg gaatttggtg cctcagctga aacagttcga 3181 gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3241 ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3301 actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3361 atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3421 ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3481 ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3541 ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3601 tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3661 ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3721 attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3781 aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3841 gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3901 gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3961 gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4021 tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4081 acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4141 ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4201 tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4261 ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4321 gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4381 gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4441 gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4501 aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4561 tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4621 gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4681 tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4741 tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4801 cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4861 ctaaagagtc tcttatccct gcgggaggtt aagactataa aagtgttcac aactgtggac 4921 aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4981 ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5041 aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5101 catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5161 tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5221 ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5281 caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5341 gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5401 ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5461 ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5521 tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5581 tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5641 ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5701 tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5761 atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5821 accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5881 ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5941 ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6001 aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6061 tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6121 tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6181 caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6241 acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6301 atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6361 accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6421 atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6481 atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6541 gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6601 agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6661 tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6721 ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6781 acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6841 aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6901 ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6961 aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7021 gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7081 gactcccttg attcttatcc agctcttgaa accattcagg tgacgatttc atcgtacaag 7141 ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7201 aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7261 agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7321 cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7381 agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7441 aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7501 gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7561 gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7621 cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7681 gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7741 catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7801 ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7861 tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagct 7921 cttgtatcag acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7981 gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8041 gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8101 gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8161 aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8221 acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8281 gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8341 aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtagtgc tgccaagaag 8401 aacaacatac cttttagact aacttgtgct acaactagac aggttgtcaa tgtcataact 8461 actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8521 gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8581 ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8641 gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8701 gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8761 gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga 8821 gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8881 tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8941 gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9001 actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9061 cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9121 gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9181 atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9241 ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9301 caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9361 ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9421 catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9481 ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9541 ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9601 gtgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9661 ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9721 gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9781 gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9841 tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9901 aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9961 tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa
10021 gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg
10081 gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct
10141 aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat
10201 gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat
10261 acttctaacc ctaagacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt
10321 tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct
10381 aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt
10441 gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac
10501 gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag
10561 gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt
10621 atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt
10681 gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct
10741 ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg
10801 cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca
10861 ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt
10921 gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt
10981 caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact 11041 cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc 11101 ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg 11161 cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct 11221 ggttataggc ttaaggattg tgttatgtat gcttcagctt tagttttgct tattctcatg 11281 acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt 11341 acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc 11401 ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct 11461 agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc 11521 ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc 11581 cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc 11641 tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt 11701 gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt 11761 gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt 11821 cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac 11881 aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg 11941 tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc 12001 gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc 12061 gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc 12121 gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12181 gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12241 gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12301 atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12361 tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12421 gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12481 tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12541 atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12601 gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12661 gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12721 aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12781 ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12841 gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12901 aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12961 aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13021 cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13081 aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13141 atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13201 catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13261 tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13321 tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13381 gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13441 caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaagttgctg 13501 gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13561 atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13621 agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13681 ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13741 tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13801 aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13861 acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13921 aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13981 tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14041 aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14101 tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14161 cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14221 accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14281 ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14341 caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14401 ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14461 cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14521 ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14581 atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14641 tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14701 aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14761 gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14821 atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14881 tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14941 aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15001 ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15061 gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15121 gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15181 ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15241 gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15301 cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15361 gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15421 atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15481 taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15541 aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15601 agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15661 tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15721 cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15781 accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15841 atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15901 tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15961 ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16021 atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16081 ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16141 tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16201 cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16261 accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16321 ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16381 gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16441 tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16501 gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16561 ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16621 ccactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16681 ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16741 aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16801 gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16861 taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16921 tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16981 tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17041 ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17101 cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17161 gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17221 tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17281 tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17341 gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17401 tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17461 taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17521 tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17581 tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17641 aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17701 tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17761 ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17821 cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17881 ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17941 taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18001 gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18061 taaagttcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18121 accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18181 atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18241 tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18301 tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18361 cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18421 cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18481 gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18541 agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctgtgtg 18601 acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18661 tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18721 gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18781 gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18841 attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18901 aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gttcttcatg 18961 acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19021 acgatgctca gccatgtagt gacaaagctt acaaaataga ggaactcttc tattcttatg 19081 ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19141 gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agtcttgtca aacttgaact 19201 taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19261 tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19321 cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19381 ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19441 accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19501 acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19561 atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19621 tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19681 aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19741 aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19801 taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19861 tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19921 atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19981 cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20041 gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20101 gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20161 agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20221 gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20281 aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20341 aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20401 aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20461 agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20521 atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20581 aactacaagc aagtcaagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20641 aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20701 aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20761 ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20821 ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20881 cagatcttaa tgacttcgtc tccgacgcag attctacttt aattggagac tgtgcaacag 20941 tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21001 atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21061 agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21121 ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21181 atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21241 aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21301 agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21361 ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21421 gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca Gene S underscored-^
21481 actaaacgaa cATGtttatt ttcttattat ttcttactct cactaqtqqt aqtqaccttq 21541 accqqtqcac cacttttqat gatqttcaaq ctcctaatta cactcaacat acttcatcta
21601 tqaq qqqqt ttactatcct atqaaattt ttaqatcaqa cactctttat ttaactcaqq
21661 atttatttct tccattttat tctaatqtta caqqqtttca tactattaat catacgtttq
21721 qcaaccctqt catacctttt aaqqat qta tttattttgc tqccacaqaq aaatcaaatq
21781 ttqtccqtqq ttqqqttttt qqttctacca tqaacaacaa qtcaca tcq qtqattatta
21841 ttaacaattc tactaatqtt qttatacqaq catqtaactt tqaattqtqt qacaaccctt
21901 tctttqctqt ttctaaaccc atqqqtacac a acacatac tatqatattc qataatqcat
21961 ttaattqcac tttc agtac atatctgatg ccttttcgct tqatqtttca gaaaagtcaq
22021 qtaattttaa acacttacqa qaqtttqtqt ttaaaaataa agatqqqttt ctctat ttt
22081 ataaqqqcta tcaacctata qatqta ttc qtqatctacc ttctqqtttt aacactttqa
22141 aacctatttt taaqttqcct cttqqtatta acattacaaa ttttaqaqcc attcttacaq
22201 ccttttcacc tqctcaaqac atttqqgqca cqtcaqctqc aqcctatttt qttqqctatt
22261 taaaqccaac tacatttatq ctcaagtatq atqaaaatqq tacaatcaca qatqctqttq
22321 attgttctca aaatccactt qctqaactca aatgctctgt taagaqcttt qaqattgaca
22381 ^aaggaattta ccaqacctct aatttcag q ttqttccctc aggaqatgtt gtgaqattcc
22441 ctaatattac aaacttqtqt ccttttqqaq aqqtttttaa tqctactaaa ttcccttctq
22501 tctatqcatq qqaqaqaaaa aaaatttcta attqtqttqc tqattactct qtqctctaca
22561 actcaacatt tttttcaacc tttaaqt ct atqqcqtttc tqccactaa ttqaatqatc
22621 tttqcttctc caatqtctat qcaqattctt ttqtaqtcaa qqqaqatqat qtaaqacaaa
22681 tagcqccaqq acaaactggt qttatt ctq attataatta taaatt cca gatqatttca
22741 tqqqttqtqt ccttqcttqq aatacta qa acatt atgc tacttcaact qqtaattata
22801 attataaata taq tatctt aqacatqqca aqcttaqqcc ctttqaqaqa qacatatcta 22861 atqtqccttt ctcccctgat q caaacctt qcaccccacc t ctcttaat tqttattqqc
22921 cattaaatqa ttatqqtttt tacaccacta ctq cattqq ctaccaacct tacaqaqttg
22981 taqtactttc tttt aactt ttaaatqcac cqgccacqqt ttgtggacca aaattatcca
23041 ctqaccttat taaqaaccaq tqt tcaatt ttaattttaa tqqactcact qqtactqqtg
23101 tgttaactcc ttcttcaaaq aqatttcaac catttcaaca atttqqccqt qatqtttctg
23161 atttcactqa ttccqttcqa qatcctaaaa catctqaaat attaqacatt tcaccttqcg
23221 cttttqqqqq tqtaaqtqta attacacctq qaacaaatqc ttcatctqaa qttqctqttc
23281 tatatcaaqa tgttaactqc actqat ttt ctacaqcaat tcatgcaqat caactcacac
23341 caqcttggcq catatattct actqqaaaca atgtattcca qactcaaqca qqctgtctta
23401 tagqaqctqa qcatqtcqac acttcttatq aqtocqacat tcctattqqa qctqqcattt
23461 qtqctaqtta ccatacaqtt tctttattac qtaqtactaq ccaaaaatct attqtqqctt
23521 atactatqtc tttaqqtqct qataqttcaa ttqcttactc taataacacc attqctatac
23581 ctactaactt ttcaattaqc attactaca aaqtaat cc tqtttctatq qctaaaacct
23641 ccgtaqattq taatatqtac atctqcqqaq attctactga atgt ctaat ttqcttctcc
23701 aatatqqtaq cttttqcaca caactaaatc qtgcactctc aqgtattgct qctgaacagg
23761 atcqcaacac acgtqaagtq ttcqctcaaq tcaaacaaat qtacaaaacc ccaactttqa
23821 aatattttqq tqqttttaat ttttcacaaa tattacctqa ccctctaaa ccaactaaga
23881 qqtcttttat tqaqqacttq ctctttaata aqgtqacact cqctqatqct qqcttcatga
23941 aqcaatatqq cqaatqccta qqtqatatta atqctaqaqa tctcattt t qcqcaqaaqt
24001 tcaatqgact tacaqtgttq ccacctctgc tcactqatga tatgatt ct qcctacactg
24061 ctqctctaqt tagtqqtact qccactqctq qatqqacatt tqgtgctqgc qctqctcttc
24121 aaataccttt tgctatgcaa atgqcatata qqttcaatgg cattgqaqtt acccaaaatq
24181 ttctctatqa aaccaaaaa caaatcqcca accaatttaa caaqqcqatt aqtcaaattc
24241 aaqaatcact tacaacaaca tcaactqcat tqqqcaaqct qcaa acqtt qttaaccaga
24301 atqctcaaqc attaaacaca cttqttaaac aacttaqctc taattttqqt qcaatttcaa
24361 gtgtgctaaa tqatatcctt tcgcgacttq ataaaqtc a qqcqgaqqta caaattqaca
24421 ggttaattac aqqcaqactt caaaqccttc aaacctatqt aacacaacaa ctaatcaqgg
24481 ctgct aaat cagqqcttct qctaatcttq ctqctactaa aatqtctqaq tqtqttcttq
24541 gacaatcaaa aaqa ttqac ttttqtqqaa aqqqctacca ccttatqtcc ttcccacaag
24601 caqccccgca tggtqttqtc ttcctacat tcacqtatqt qccatcccaq qaqaqqaact
24661 tcaccacaqc qccaqcaatt tgtcatqaa qcaaaqcata cttccctcgt qaaqgtqttt
24721 ttgtgtttaa tqqcacttct tqgtttatta cacagaqgaa cttcttttct ccacaaataa
24781 ttactacaqa caatacattt qtctcaqgaa attgtqatgt cqttattqgc atcattaaca
24841 acacaqttta tgatcctctq caacctqaqc ttqactcatt caaaqaaqaq ctqqacaaqt
24901 acttcaaaaa tcatacatca ccaqat ttq atcttqqcqa catttcaqqc attaacqctt
24961 ctgtcqtcaa cattcaaaaa qaaattqacc qcctcaat a qqtcqctaaa aatttaaatq
25021 aatcactcat tqaccttcaa qaattqqqaa aatatqaqca atatattaaa tqgccttqgt
25081 atgtttggct cg cttcatt qctgqactaa ttqccatcqt catqqttaca atcttqcttt
25141 gttgcatgac tagttgttgc agttgcctca agq tqcatq ctcttqtqqt tcttqctqca
25201 aqtttqatqa qqatqactct qaqccaqttc tcaaqqqtgt caaattacat tacaca7Λ4a 25261 cgaacttatg gatttgttta tgagattttt tactcttaga tcaattactg cacagccagt 25321 aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25381 agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25441 cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25501 gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25561 tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25621 caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25681 attactttat gatgccaact actttgtttg ctggcacaca cataactatg actactgtat 25741 accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25801 aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25861 agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25921 aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25981 agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26041 aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga Gene E underscored-
26101 aagtgagtac qaacttATGt actcattcqt ttcqqaaqaa acaqqtacqt taata ttaa 26161 taqcqtactt ctttttcttq ctttcqtqqt attcttqcta qtcacactaq ccatccttac
26221 tqcqcttcqa ttgtqtgcgt actqctqcaa tattqttaac qtgaqtttaq taaaaccaac
26281 qqtttacqtc tactcqcqtq ttaaaaatct qaactcttct qaagqaqttc ctgatcttct
26341 qqtc7A4acq aactaactat tattattatt ctgtttggaa ctttaacatt gcttatcATG <-Gene M underscored- 26401 qcaqacaacq qtactattac cqttqaqqaq cttaaacaac tcctqqaaca atqqaaccta
26461 gtaataqqtt tcctattcct aqcctqqatt atqttactac aatttqccta ttctaatcqq
26521 aacaqqtttt tqtacataat aaaqcttqtt ttcctctq c tcttqtqqcc aqtaacactt
26581 qcttqttttq tqcttqctqc tqtctaca a attaattqqq tqactqqcqq qattqcqatt
26641 gcaatqgctt qtattqtagq cttqatqtqq cttaqctact tcqttqcttc cttcaqqctq 26701 tttgctcgta cccqctcaat gtqqtcattc aacccaqaaa caaacattct tctcaatqtg
26761 cctctccqqq qqacaattqt αaccaqaccq ι ctcatqqaaa qtqaacttqt cattqqtqct
26821 gtqatcattc qtgqtcactt qcqaatqqcc qgacactccc taqgqcqct tqacattaag
26881 qacctgccaa aaqaqatcac tqtqgctaca tcacgaacqc tttcttatta caaattaqqa
26941 qcqtcqca c qtqtaqqcac tqattcaqqt tttqctgcat acaaccqcta ccqtattqqa 27001 aactataaat taaatacaqa ccacqcc qt aqcaacgaca atattqcttt qctaqtacaq
_27061 TA4gtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat
27121 tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat
27181 agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga
27241 acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga
27301 ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac
27361 tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg
27421 ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg
27481 gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac
27541 aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat
27601 ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga
27661 cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt
27721 ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat
27781 gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca
27841 gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg
27901 gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat
27961 ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg
28021 gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta <-Gene N underscored-
28081 gagacgtact tgttgtttta aataaacgaa caaattaaaA TGtctqataa tqqaccccaa
28141 tcaaaccaac qtaqtqcccc ccqcattaca tttqqtqqac ccaca attc aactqacaat
28201 aaccagaatg qaqqacgcaa tqgqgcaaqq ccaaaacaqc qccqacccca aqqtttaccc
28261 aataatactg cqtcttqqtt cacagctctc actcaqcatq qcaaqqagga acttagattc
28321 cctcqagqcc aqqqcqttcc aatcaacacc aataqtqqtc cagatqacca aattqqctac
28381 taccqaagag ctacccgacg aqttcqtqgt qqtqacqqca aaatqaaaga qctcaqcccc
28441 aqatqqtact tctattacct aqqaactqqc ccaqaaqctt cacttcccta cgqcgctaac
28501 aaaqaaqqca tcqtatgqqt tqcaactgaq qqaqccttqa atacacccaa aqaccacatt
28561 qqcacccqca atcctaataa caatgct cc accqtgctac aacttcctca aqgaacaaca
28621 ttqccaaaaq qcttctacqc aqaqqqaaqc aqaqqcgqca qtcaa cctc ttctcqctcc
28681 tcatcac ta qtcqcqqtaa ttcaa aaat tcaactcctq qcagcagtaq qqqaaattct
28741 cctqctcqaa tqqctaqcqq aqqtgqtqaa actqccctc c ctattgct qctagacaga
28801 ttqaaccaqc ttqaqaqcaa aqtttctqqt aaaq ccaac aacaacaagq ccaaactqtc
28861 actaaqaaat ctqctqctqa gqcatctaaa aagcctcqcc aaaaac tac tqccacaaaa
28921 caqtacaacq tcactcaaqc atttgggaga cgtggtccag aacaaaccca aqqaaatttc
28981 qqqqaccaag acctaatcaq acaag aact gattacaaac attgqccqca aattqcacaa
29041 tttqctccaa qtqcctctqc attctttqga atgtcacqca ttq catqga agtcacacct
29101 tcqqgaacat gqctqactta tcatq aqcc attaaatt q at acaaaga tccacaattc
29161 aaaqacaacq tcatactqct qaacaaqcac attqacqcat acaaaacatt cccaccaaca
29221 qaqcctaaaa aq acaaaaa qaaaaaqact qatgaaqctc aqccttt cc qcaqaqacaa 29281 aaqaagcagc ccactqtqac tcttcttcct qcqqctqaca tqqat attt ctccaqacaa
29341 cttcaaaatt ccatqagtgq aqcttctqct qattcaactc aggc&TAAac actcatgatg <-3 'UTR
29401 accacacaag gcaga tgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29461 tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29521 atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29581 cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29641 ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29701 attttaatag cttcttagga gaatgacaaa aaaaaaaaaa aaaaaaaaaa The following subsequences are shown and annotated above by underscoring the coding sequences of interest with the initiation codon ATG in uppercase characters, and the stop codon in uppercase italic characters. The individual coding sequences and translated amino acid sequences are provided below: 1. The coding sequence for the S (spike) protein, SEQ LD NO:4, is from nt 21492 to
25259 of SEQ ID NO:3, which comprises 3768 nt that encode 1255 residues + stop codon As established by Krokhin et al. (2003), the glycosylated spike protein (as well as the nucleocapsid protein) can be detected in infected cell culture supematants with antisera from SARS patients
SEO ID NO:4
ATG ttt att ttc tta tta ttt ctt act etc act agt ggt agt gac ctt gac egg tgc acc act ttt gat gat gtt caa get cct aat tac act caa cat act tea tct atg agg ggg gtt tac tat cct gat gaa att ttt aga tea gac act ctt tat tta act cag gat tta ttt ctt cca ttt tat tct aat gtt aca ggg ttt cat act att aat cat acg ttt ggc aac cct gtc ata cct ttt aag gat ggt att tat ttt get gcc aca gag aaa tea aat gtt gtc cgt ggt tgg gtt ttt ggt tct acc atg aac aac aag tea cag teg gtg att att att aac aat tct act aat gtt gtt ata cga gca tgt aac ttt gaa ttg tgt gac aac cct ttc ttt get gtt tct aaa ccc atg ggt aca cag aca cat act atg ata ttc gat aat gca ttt aat tgc act ttc gag tac ata tct gat gcc ttt teg ctt gat gtt tea gaa aag tea ggt aat ttt aaa cac tta cga gag ttt gtg ttt aaa aat aaa gat ggg ttt etc tat gtt tat aag ggc tat caa cct ata gat gta gtt cgt gat cta cct tct ggt ttt aac act ttg aaa cct att ttt aag ttg cct < ctt ggt att aac att aca aat ttt aga gcc att ctt aca gcc ttt tea cct get caa gac att tgg ggc acg tea get gca gcc tat ttt gtt ggc tat tta aag cca act aca ttt atg etc aag tat gat gaa aat ggt aca ate aca gat get gtt gat tgt tct caa aat cca ctt get gaa etc aaa tgc tct gtt aag age ttt gag att gac aaa gga att tac cag acc tct aat ttc agg gtt gtt ccc tea gga gat gtt gtg aga ttc cct aat att aca aac ttg tgt cct ttt gga gag gtt ttt aat get act aaa ttc cct tct gtc tat gca tgg gag aga aaa aaa att tct aat tgt gtt get gat tac tct gtg etc tac aac tea aca ttt ttt tea acc ttt aag tgc tat ggc gtt tct gcc act aag ttg aat gat ctt tgc ttc tec aat gtc tat gca gat tct ttt gta gtc aag gga gat gat gta aga caa ata gcg cca gga caa act ggt gtt att get gat tat aat tat aaa ttg cca gat gat ttc atg ggt tgt gtc ctt get tgg aat act agg aac att gat get act tea act ggt aat tat aat tat aaa tat agg tat ctt aga cat ggc aag ctt agg ccc ttt gag aga gac ata tct aat gtg cct ttc tec cct gat ggc aaa cct tgc acc cca cct get ctt aat tgt tat tgg cca tta aat gat tat ggt ttt tac acc act act ggc att ggc tac caa cct tac aga gtt gta gta ctt tct ttt gaa ctt tta aat gca ccg gcc acg gtt tgt gga cca aaa tta tec act gac ctt att aag aac cag tgt gtc aat ttt aat ttt aat gga etc act ggt act ggt gtg tta act cct tct tea aag aga ttt caa cca ttt caa caa ttt ggc cgt gat gtt tct gat ttc act gat tec gtt cga gat cct aaa aca tct gaa ata tta gac att tea cct tgc get ttt ggg ggt gta agt gta att aca cct gga aca aat get tea tct gaa gtt get gtt cta tat caa gat gtt aac tgc act gat gtt tct aca gca att cat gca gat caa etc aca cca get tgg cgc ata tat tct act gga aac aat gta ttc cag act caa gca ggc tgt ctt ata gga get gag cat gtc gac act tct tat gag tgc gac att cct att gga get ggc att tgt get agt tac cat aca gtt tct tta tta cgt agt act age caa aaa tct att gtg get tat act atg tct tta ggt get gat agt tea att get tac tct aat aac acc att get ata cct act aac ttt tea att age att act aca gaa gta atg cct gtt tct atg get aaa acc tec gta gat tgt aat atg tac ate tgc gga gat tct act gaa tgt get aat ttg ctt etc caa tat ggt age ttt tgc aca caa cta aat cgt gca etc tea ggt att get get gaa cag gat cgc aac aca cgt gaa gtg ttc get caa gtc aaa caa atg tac aaa acc cca act ttg aaa tat ttt ggt ggt ttt aat ttt tea caa ata tta cct gac cct cta aag cca act aag agg tct ttt att gag gac ttg etc ttt aat aag gtg aca etc get gat get ggc ttc atg aag caa tat ggc gaa tgc cta ggt gat att aat get aga gat etc att tgt gcg cag aag ttc aat gga ctt aca gtg ttg cca cct ctg etc act gat gat atg att get gcc tac act get get cta gtt agt ggt act gcc act get gga tgg aca ttt ggt get ggc get get ctt caa ata cct ttt get atg caa atg gca tat agg ttc aat ggc att gga gtt acc caa aat gtt etc tat gag aac caa aaa caa ate gcc aac caa ttt aac aag gcg att agt caa att caa gaa tea ctt aca aca aca tea act gca ttg ggc aag ctg caa gac gtt gtt aac cag aat get caa gca tta aac aca ctt gtt aaa caa ctt age tct aat ttt ggt gca att tea agt gtg cta aat gat ate ctt teg cga ctt gat aaa gtc gag gcg gag gta caa att gac agg tta att aca ggc aga ctt caa age ctt caa acc tat gta aca caa caa cta ate agg get get gaa ate agg get tct get aat ctt get get act aaa atg tct gag tgt gtt ctt gga caa tea aaa aga gtt gac ttt tgt gga aag ggc tac cac ctt atg tec ttc cca caa gca gcc ccg cat ggt gtt gtc ttc cta cat gtc acg tat gtg cca tec cag gag agg aac ttc acc aca gcg cca gca att tgt cat gaa ggc aaa gca tac ttc cct cgt gaa ggt gtt ttt gtg ttt aat ggc act tct tgg ttt att aca cag agg aac ttc ttt tct cca caa ata att act aca gac aat aca ttt gtc tea gga aat tgt gat gtc gtt att ggc ate att aac aac aca gtt tat gat cct ctg caa cct gag ctt gac tea ttc aaa gaa gag ctg gac aag tac ttc aaa aat cat aca tea cca gat gtt gat ctt ggc gac att tea ggc att aac get tct gtc gtc aac att caa aaa gaa att gac cgc etc aat gag gtc get aaa aat tta aat gaa tea etc att gac ctt caa gaa ttg gga aaa tat gag caa tat att aaa tgg cct tgg tat gtt tgg etc ggc ttc att get gga cta att gcc ate gtc atg gtt aca ate ttg ctt tgt tgc atg act agt tgt tgc agt tgc etc aag ggt gca tgc tct tgt ggt tct tgc tgc aag ttt gat gag gat gac tct gag cca gtt etc aag ggt gtc aaa tta cat tac aca TAA Glycosylation sites of this protein include residues encoded by codons at the following positions: 21843-21845; 21846-21848; 22170-22172; 22296-22298; and 23838-23840. The encoded amino acid sequence ofthe S polypeptide (SEQ TD NO:5) is:
MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGV YYPDEIFRSD TLYLTQDLFL 60 PFYSNVTGFH TINHTFGNPV IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS 120 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT FEYISDAFSL DVSEKSGNFK 180 HLREFVFKNK DGFLYVYKGY QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP 240 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ NPLAELKCSV KSFEIDKGIY 300 QTSNFRVVPS GDVVRFPNIT NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF 360 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG QTGVIADYNY KLPDDFMGCV 420 LA NTRNIDA TSTGNYNYKY RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND 480 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI KNQCVNFNFN GLTGTGVLTP 540 SSKRFQPFQQ FGRDVSDFTD SVRDPKTSEI LDISPCAFGG VSVITPGTNA SSEVAVLYQD 600 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE HVDTSYECDI PIGAGICASY 660 HTVSLLRSTS QKSIVAYTMS LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC 720 N YICGDSTE CA LLLQYGS FCTQLNRALS GIAAEQDRNT REVFAQVKQM YKTPTLKYFG 780 GFNFSQILPD PLKPTKRSFI EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL 840 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE 900 NQKQIANQFN KAISQIQESL TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN 960 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASA LAATK MSECVLGQSK 1020 RVDFCGKGYH LMSFPQAAPH GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN 1080 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN 1140 HTSPDVDLGD ISGINASVVN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL 1200 GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV KLHYT 1255 2. The coding sequence for the E (envelope, or "small envelope") protein (SEQ ID NO:6) is from nt 26117 to 26347 ofSEQ ID NO:3, which comprises 231 nt that encode 76 aa's + stop codon
SEO ID NO:6
ATG tac tea ttc gtt teg gaa gaa aca ggt acg tta ata gtt aat age gta ctt ctt ttt ctt get ttc gtg gta ttc ttg cta gtc aca cta gcc ate ctt act gcg ctt cga ttg tgt gcg tac tgc tgc aat att gtt aac gtg agt tta gta aaa cca acg gtt tac gtc tac teg cgt gtt aaa aat ctg aac tct tct gaa gga gtt cct gat ctt ctg gtc
TAA The encoded amino acid sequence ofthe E polypeptide (SEQ ID NO:7) is:
MYSFVSEETG TLIVNSVLLF LAFVVFLLVT LAILTALRLC AYCCNIVNVS LVKPTVYVYS 60 RVKNLNSSEG VPDLLV 76 3. The coding sequence for the M (membrane protein (SEQ ID NO: 8) is from nt 26348 to 26353 of SEQ ID NO:3, which comprises 666 nt encoding 221 aa + stop codon
SEO ID NO:8
ATG gca gac aac ggt act att acc gtt gag gag ctt aaa caa etc ctg gaa caa tgg aac cta gta ata ggt ttc cta ttc cta gcc tgg att atg tta cta caa ttt gcc tat tct aat egg aac agg ttt ttg tac ata ata aag ctt gtt ttc etc tgg etc ttg tgg cca gta aca ctt get tgt ttt gtg ctt get get gtc tac aga att aat tgg gtg act ggc ggg att gcg att gca atg get tgt att gta ggc ttg atg tgg ctt age tac ttc gtt get tec ttc agg ctg ttt get cgt acc cgc tea atg tgg tea ttc aac cca gaa aca aac att ctt etc aat gtg cct etc egg ggg aca att gtg acc aga ccg etc atg gaa agt gaa ctt gtc att ggt get gtg ate att cgt ggt cac ttg cga atg gcc gga cac tec cta ggg cgc tgt gac att aag gac ctg cca aaa gag ate act gtg get aca tea cga acg ctt tct tat tac aaa tta gga gcg teg cag cgt gta ggc act gat tea ggt ttt get gca tac aac cgc tac cgt att gga aac tat aaa tta aat aca gac cac gcc ggt age aac gac aat att get ttg cta gta cag TAA
The encoded amino acid sequence ofthe M polypeptide (SEQ ID NO:9) is:
MADNGTITVE ELKQLLEQWN LVIGFLFLAW IMLLQFAYSN RNRFLYIIKL VFLWLLWPVT 60
LACFVLAAVY RINWVTGGIA lAMACIVGLM WLSYFVASFR LFARTRSMWS FNPETNILLN 120
VPLRGTIVTR PLMESELVIG AVIIRGHLRM AGHSLGRCDI KDLPKEITVA TSRTLSYYKL 180
GASQRVGTDS GFAAYNRYRI GNYKLNTDHA GSNDNIALLV Q 221
4. The coding sequence for the N (nucleocapsid protein (SEQ ID NO:10) is from nt 28120 to 29388of SEQ ID NO:3, which comprises 1269 nt encoding 422 aa + stop codon. SEO ID NO: 10
ATG tct gat aat gga ccc caa tea aac caa cgt agt gcc ccc cgc att aca ttt ggt gga ccc aca gat tea act gac aat aac cag aat gga gga cgc aat ggg gca agg cca aaa cag cgc cga ccc caa ggt tta ccc aat aat act gcg tct tgg ttc aca get etc act cag cat ggc aag gag gaa ctt aga ttc cct cga ggc cag ggc gtt cca ate aac acc aat agt ggt cca gat gac caa att ggc tac tac cga aga get acc cga cga gtt cgt ggt ggt gac ggc aaa atg aaa gag etc age ccc aga tgg tac ttc tat tac cta gga act ggc cca gaa get tea ctt ccc tac ggc get aac aaa gaa ggc ate gta tgg gtt gca act gag gga gcc ttg aat aca ccc aaa gac cac att ggc acc cgc aat cct aat aac aat get gcc acc gtg cta caa ctt cct caa gga aca aca ttg cca aaa ggc ttc tac gca gag gga age aga ggc ggc agt caa gcc tct tct cgc tec tea tea cgt agt cgc ggt aat tea aga aat tea act cct ggc age agt agg gga aat tct cct get cga atg get age gga ggt ggt gaa act gcc etc gcg cta ttg ctg cta gac aga ttg aac cag ctt gag age aaa gtt tct ggt aaa ggc caacaa caa caa ggc caa act gtc act aag aaa tct get get gag gca tct aaa aag cct cgc caa aaa cgt act gcc aca aaa cag tac aac gtc act caa gca ttt ggg aga cgt ggt cca gaa caa acc caa gga aat ttc ggg gac caa gac cta ate aga caa gga act gat tac aaa cat tgg ccg caa att gca caa ttt get cca agt gcc tct gca ttc ttt gga atg tea cgc att ggc atg gaa gtc aca cct teg gga aca tgg ctg act tat cat gga gcc att aaa ttg gat gac aaa gat cca caa ttc aaa gac aac gtc ata ctg ctg aac aag cac att gac gca tac aaa aca ttc cca cca aca gag cct aaa aag gac aaa aag aaa aag act gat gaa get cag cct ttg ccg cag aga caa aag aag cag ccc act gtg act ctt ctt cct gcg get gac atg gat gat ttc tec aga caa ctt caa aat tec atg agt gga get tct get gat tea act cag gca TAA
The encoded aminei acid sequence ofthe E polypeptide (SEQ J-D NO:11) is:
MSDNGPQSNQ RSAPRITFGG PTDSTDNNQN GGRNGARPKQ RRPQGLPNNT ASWFTALTQH 60
GKEELRFPRG QGVPINTNSG PDDQIGYYRR ATRRVRGGDG KMKELSPRWY FYYLGTGPEA 120
SLPYGANKEG IVWVATEGAL NTPKDHIGTR NPNNNAATVL QLPQGTTLPK GFYAEGSRGG 180
SQASSRSSSR SRGNSRNSTP GSSRGNSPAR MASGGGETAL ALLLLDRLNQ LESKVSGKGQ 240
QQQGQTVTKK SAAEASKKPR QKRTATKQYN VTQAFGRRGP EQTQGNFGDQ DLIRQGTDYK 300 HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYHGAIKL DDKDPQFKDN VILLNKHIDA 360 YKTFPPTEPK KDKKKKTDEA QPLPQRQKKQ PTVTLLPAAD MDDFSRQLQN SMSGASADST 420 QA 422 As established by Krokhin, O. et al, 2003, Mol Cell Proteomics 2:346-56, the N-terminal methionine (encoded by the initiation ATG codon, is removed in the virion protein when it is processed, and all other methionines are oxidized, and the resulting N-terminal serine is acetylated.
CLONING OF THE GENOME OF THE TW1 STRAIN OF SARS-CoV The presently exemplified and prefened sequences are based on the Taiwanese strain, TW1, of SARS-CoN. The Superscript cDΝA system (Invifrogen, Carlsbad, CA, USA) was used to reverse transcribe the RΝA template into cDΝA (Hsueh, PR et al,. Emerg Infect Dis, 9: 1163-1167,
2003). To sequence the viral genome, 25 primer sets were designed based on the cDΝA sequence data from the Tor2 SARS isolate (accession no. ΝC_004718, supra). See Figure 19 and Table 1.
After PCR amplification, products were analyzed by agarose gel electrophoresis and then processed for direct sequencing reactions. Sequences were assembled and edited to obtain the sequence ofthe genome ofthe TW1 strain of SARS-CoN, which was subsequently deposited in GenBank (as accession number AY291451; available at WWW URL \ ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=πucleotide8tval=30698326V data from the Tor2 SARS isolate (accession no. ΝC_004718, supra). See Figure 19 and Table 1. After PCR amplification, products were analyzed by agarose gel electrophoresis and then processed for direct sequencing reactions. Sequences were assembled and edited to obtain the sequence ofthe genome ofthe TW1 strain of SARS-CoN, which was subsequently deposited in GenBank (as accession number AY291451 ; available at WWW URL
Table 1. Summary of the 25 overlapping SARS-CoV TW-1 isolate cDNA clones sequenced and available. The cDNA sections are in the vector, between the BamHI and EcoRI cloning sites, orward and reverse se uencin rimers are shown.
Figure imgf000039_0001
data from the Tor2 SARS isolate (accession no. NC_004718, supra). See Figure 19 and Table 1. After PCR amplification, products were analyzed by agarose gel electrophoresis and then processed for direct sequencing reactions. Sequences were assembled and edited to obtain the sequence ofthe genome ofthe TW1 strain of SARS-CoV, which was subsequently deposited in GenBank (as accession number AY291451; available at WWW URL ncbi.nlm.nih.qov/entrez/viewer.fcqi?db=nucleotide&val=30698326). This data is based on Yeh, S-H et al, Proc. Natl. Acad. Sci. U.S.A. 101 :2542-2547 (2004) and later deposits by the same group (see URL). The genomic sequence ofthe TW1 strain, nt 1-29729 is shown below fSEO ID NO: 12) Annotation is as in SEQ ID NO:3 above (the TOR2 strain) SEO ID NO: 12 1 atattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttg tagatctgtt 61 ctctaaacga actttaaaat ctgtgtagct gtcgctcggc tgcatgccta gtgcacctac 121 gcagtataaa caataataaa ttttactgtc gttgacaaga aacgagtaac tcgtccctct 181 tctgcagact gcttacggtt tcgtccgtgt tgcagtcgat catcagcata cctaggtttc 241 gtccgggtgt gaccgaaagg taagatggag agccttgttc ttggtgtcaa cgagaaaaca 301 cacgtccaac tcagtttgcc tgtccttcag gttagagacg tgctagtgcg tggcttcggg 361 gactctgtgg aagaggccct atcggaggca cgtgaacacc tcaaaaatgg cacttgtggt 421 ctagtagagc tggaaaaagg cgtactgccc cagcttgaac agccctatgt gttcattaaa 481 cgttctgatg ccttaagcac caatcacggc cacaaggtcg ttgagctggt tgcagaaatg 541 gacggcattc agtacggtcg tagcggtata acactgggag tactcgtgcc acatgtgggc 601 gaaaccccaa ttgcataccg caatgttctt cttcgtaaga acggtaataa gggagccggt 661 ggtcatagct atggcatcga tctaaagtct tatgacttag gtgacgagct tggcactgat 721 cccattgaag attatgaaca aaactggaac actaagcatg gcagtggtgc actccgtgaa 781 ctcactcgtg agctcaatgg aggtgcagtc actcgctatg tcgacaacaa tttctgtggc 841 ccagatgggt accctcttga ttgcatcaaa gattttctcg cacgcgcggg caagtcaatg 901 tgcactcttt ccgaacaact tgattacatc gagtcgaaga gaggtgtcta ctgctgccgt 961 gaccatgagc atgaaattgc ctggttcact gagcgctctg ataagagcta cgagcaccag 1021 acacccttcg aaattaagag tgccaagaaa tttgacactt tcaaagggga atgcccaaag 1081 tttgtgtttc ctcttaactc aaaagtcaaa gtcattcaac cacgtgttga aaagaaaaag 1141 actgagggtt tcatggggcg tatacgctct gtgtaccctg ttgcatctcc acaggagtgt 1201 aacaatatgc acttgtctac cttgatgaaa tgtaatcatt gcgatgaagt ttcatggcag 1261 aegtgegact ttetgaaage caettgtgaa eattgtggca etgaaaattt agttattgaa 1321 ggacctacta catgtgggta cctacctact aatgctgtag tgaaaatgcc atgtcctgcc 1381 tgtcaagacc cagagattgg acctgagcat agtgttgcag attatcacaa ccactcaaac 1441 attgaaactc gactccgcaa gggaggtagg actagatgtt ttggaggctg tgtgtttgcc 1501 tatgttggct gctataataa gcgtgcctac tgggttcctc gtgctagtgc tgatattggc 1561 tcaggccata ctggcattac tggtgacaat gtggagacct tgaatgagga tctccttgag 1621 atactgagtc gtgaacgtgt taacattaac attgttggcg attttcattt gaatgaagag 1681 gttgccatca ttttggcatc tttctctgct tctacaagtg cctttattga cactataaag 1741 agtcttgatt acaagtcttt caaaaccatt gttgagtcct gcggtaacta taaagttacc 1801 aagggaaagc ccgtaaaagg tgcttggaac attggacaac agagatcagt tttaacacca 1861 ctgtgtggtt ttccctcaca ggctgctggt gttatcagat caatttttgc gcgcacactt 1921 gatgcagcaa accactcaat tcctgatttg caaagagcag ctgtcaccat acttgatggt 1981 atttctgaac agtcattacg tcttgtcgac gccatggttt atacttcaga cctgctcacc 2041 aacagtgtca ttattatggc atatgtaact ggtggtcttg tacaacagac ttctcagtgg 2101 ttgtctaatc ttttgggcac tactgttgaa aaactcaggc ctatctttga atggattgag 2161 gcgaaactta gtgcaggagt tgaatttctc aaggatgctt gggagattct caaatttctc 2221 attacaggtg tttttgacat cgtcaagggt caaatacagg ttgcttcaga taacatcaag 2281 gattgtgtaa aatgcttcat tgatgttgtt aacaaggcac tcgaaatgtg cattgatcaa 2341 gtcaetateg etggcgcaaa gttgegatca cteaaettag gtgaagtett eatcgeteaa 2401 agcaagggac tttaccgtca gtgtatacgt ggcaaggagc agctgcaact actcatgcct 2461 cttaaggcac caaaagaagt aacctttctt gaaggtgatt cacatgacac agtacttacc 2521 tctgaggagg ttgttctcaa gaacggtgaa ctcgaagcac tcgagacgcc cgttgatagc 2581 ttcacaaatg gagctatcgt tggcacacca gtctgtgtaa atggcctcat gctcttagag 2641 attaaggaca aagaacaata ctgcgcattg tctcctggtt tactggctac aaacaatgtc 2701 tttcgcttaa aagggggtgc accaattaaa ggtgtaacct ttggagaaga tactgtttgg 2761 gaagttcaag gttacaagaa tgtgagaatc acatttgagc ttgatgaacg tgttgacaaa 2821 gtgcttaatg aaaagtgctc tgtctacact gttgaatccg gtaccgaagt tactgagttt 2881 gcatgtgttg tagcagaggc tgttgtgaag actttacaac cagtttctga tctccttacc 2941 aacatgggta ttgatcttga tgagtggagt gtagctacat tctacttatt tgatgatgct 3001 ggtgaagaaa acttttcatc acgtatgtat tgttcctttt accctccaga tgaggaagaa 3061 gaggacgatg cagagtgtga ggaagaagaa attgatgaaa cctgtgaaca tgagtacggt 3121 acagaggatg attatcaagg tctccctctg gaatttggtg cctcggctga aacagttcga 3181 gttgaggaag aagaagagga agactggctg gatgatacta ctgagcaatc agagattgag 3241 ccagaaccag aacctacacc tgaagaacca gttaatcagt ttactggtta tttaaaactt 3301 actgacaatg ttgccattaa atgtgttgac atcgttaagg aggcacaaag tgctaatcct 3361 atggtgattg taaatgctgc taacatacac ctgaaacatg gtggtggtgt agcaggtgca 3421 ctcaacaagg caaccaatgg tgccatgcaa aaggagagtg atgattacat taagctaaat 3481 ggccctctta cagtaggagg gtcttgtttg ctttctggac ataatcttgc taagaagtgt 3541 ctgcatgttg ttggacctaa cctaaatgca ggtgaggaca tccagcttct taaggcagca 3601 tatgaaaatt tcaattcaca ggacatctta cttgcaccat tgttgtcagc aggcatattt 3661 ggtgctaaac cacttcagtc tttacaagtg tgcgtgcaga cggttcgtac acaggtttat 3721 attgcagtca atgacaaagc tctttatgag caggttgtca tggattatct tgataacctg 3781 aagcctagag tggaagcacc taaacaagag gagccaccaa acacagaaga ttccaaaact 3841 gaggagaaat ctgtcgtaca gaagcctgtc gatgtgaagc caaaaattaa ggcctgcatt 3901 gatgaggtta ccacaacact ggaagaaact aagtttctta ccaataagtt actcttgttt 3961 gctgatatca atggtaagct ttaccatgat tctcagaaca tgcttagagg tgaagatatg 4021 tctttccttg agaaggatgc accttacatg gtaggtgatg ttatcactag tggtgatatc 4081 acttgtgttg taataccctc caaaaaggct ggtggcacta ctgagatgct ctcaagagct 4141 ttgaagaaag tgccagttga tgagtatata accacgtacc ctggacaagg atgtgctggt 4201 tatacacttg aggaagctaa gactgctctt aagaaatgca aatctgcatt ttatgtacta 4261 ccttcagaag cacctaatgc taaggaagag attctaggaa ctgtatcctg gaatttgaga 4321 gaaatgcttg ctcatgctga agagacaaga aaattaatgc ctatatgcat ggatgttaga 4381 gccataatgg caaccatcca acgtaagtat aaaggaatta aaattcaaga gggcatcgtt 4441 gactatggtg tccgattctt cttttatact agtaaagagc ctgtagcttc tattattacg 4501 aagctgaact ctctaaatga gccgcttgtc acaatgccaa ttggttatgt gacacatggt 4561 tttaatcttg aagaggctgc gcgctgtatg cgttctctta aagctcctgc cgtagtgtca 4621 gtatcatcac cagatgctgt tactacatat aatggatacc tcacttcgtc atcaaagaca 4681 tctgaggagc actttgtaga aacagtttct ttggctggct cttacagaga ttggtcctat 4741 tcaggacagc gtacagagtt aggtgttgaa tttcttaagc gtggtgacaa aattgtgtac 4801 cacactctgg agagccccgt cgagtttcat cttgacggtg aggttctttc acttgacaaa 4861 ctaaagagtc tcttatccct gcgggaggtt aagactataa aagtgttcac aactgtggac 4921 aacactaatc tccacacaca gcttgtggat atgtctatga catatggaca gcagtttggt 4981 ccaacatact tggatggtgc tgatgttaca aaaattaaac ctcatgtaaa tcatgagggt 5041 aagactttct ttgtactacc tagtgatgac acactacgta gtgaagcttt cgagtactac 5101 catactcttg atgagagttt tcttggtagg tacatgtctg ctttaaacca cacaaagaaa 5161 tggaaatttc ctcaagttgg tggtttaact tcaattaaat gggctgataa caattgttat 5221 ttgtctagtg ttttattagc acttcaacag cttgaagtca aattcaatgc accagcactt 5281 caagaggctt attatagagc ccgtgctggt gatgctgcta acttttgtgc actcatactc 5341 gcttacagta ataaaactgt tggcgagctt ggtgatgtca gagaaactat gacccatctt 5401 ctacagcatg ctaatttgga atctgcaaag cgagttctta atgtggtgtg taaacattgt 5461 ggtcagaaaa ctactacctt aacgggtgta gaagctgtga tgtatatggg tactctatct 5521 tatgataatc ttaagacagg tgtttccatt ccatgtgtgt gtggtcgtga tgctacacaa 5581 tatctagtac aacaagagtc ttcttttgtt atgatgtctg caccacctgc tgagtataaa 5641 ttacagcaag gtacattctt atgtgcgaat gagtacactg gtaactatca gtgtggtcat 5701 tacactcata taactgctaa ggagaccctc tatcgtattg acggagctca ccttacaaag 5761 atgtcagagt acaaaggacc agtgactgat gttttctaca aggaaacatc ttacactaca 5821 accatcaagc ctgtgtcgta taaactcgat ggagttactt acacagagat tgaaccaaaa 5881 ttggatgggt attataaaaa ggataatgct tactatacag agcagcctat agaccttgta 5941 ccaactcaac cattaccaaa tgcgagtttt gataatttca aactcacatg ttctaacaca 6001 aaatttgctg atgatttaaa tcaaatgaca ggcttcacaa agccagcttc acgagagcta 6061 tctgtcacat tcttcccaga cttgaatggc gatgtagtgg ctattgacta tagacactat 6121 tcagcgagtt tcaagaaagg tgctaaatta ctgcataagc caattgtttg gcacattaac 6181 caggctacaa ccaagacaac gttcaaacca aacacttggt gtttacgttg tctttggagt 6241 acaaagccag tagatacttc aaattcattt gaagttctgg cagtagaaga cacacaagga 6301 atggacaatc ttgcttgtga aagtcaacaa cccacctctg aagaagtagt ggaaaatcct 6361 accatacaga aggaagtcat agagtgtgac gtgaaaacta ccgaagttgt aggcaatgtc 6421 atacttaaac catcagatga aggtgttaaa gtaacacaag agttaggtca tgaggatctt 6481 atggctgctt atgtggaaaa cacaagcatt accattaaga aacctaatga gctttcacta 6541 gccttaggtt taaaaacaat tgccactcat ggtattgctg caattaatag tgttccttgg 6601 agtaaaattt tggcttatgt caaaccattc ttaggacaag cagcaattac aacatcaaat 6661 tgcgctaaga gattagcaca acgtgtgttt aacaattata tgccttatgt gtttacatta 6721 ttgttccaat tgtgtacttt tactaaaagt accaattcta gaattagagc ttcactacct 6781 acaactattg ctaaaaatag tgttaagagt gttgctaaat tatgtttgga tgccggcatt 6841 aattatgtga agtcacccaa attttctaaa ttgttcacaa tcgctatgtg gctattgttg 6901 ttaagtattt gcttaggttc tctaatctgt gtaactgctg cttttggtgt actcttatct 6961 aattttggtg ctccttctta ttgtaatggc gttagagaat tgtatcttaa ttcgtctaac 7021 gttactacta tggatttctg tgaaggttct tttccttgca gcatttgttt aagtggatta 7081 gactcccttg attcttatcc agctcttgaa accattcagg tgacgatttc atcgtacaag 7141 ctagacttga caattttagg tctggccgct gagtgggttt tggcatatat gttgttcaca 7201 aaattctttt atttattagg tctttcagct ataatgcagg tgttctttgg ctattttgct 7261 agtcatttca tcagcaattc ttggctcatg tggtttatca ttagtattgt acaaatggca 7321 cccgtttctg caatggttag gatgtacatc ttctttgctt ctttctacta catatggaag 7381 agctatgttc atatcatgga tggttgcacc tcttcgactt gcatgatgtg ctataagcgc 7441 aatcgtgcca cacgcgttga gtgtacaact attgttaatg gcatgaagag atctttctat 7501 gtctatgcaa atggaggccg tggcttctgc aagactcaca attggaattg tctcaattgt 7561 gacacatttt gcactggtag tacattcatt agtgatgaag ttgctcgtga tttgtcactc 7621 cagtttaaaa gaccaatcaa ccctactgac cagtcatcgt atattgttga tagtgttgct 7681 gtgaaaaatg gcgcgcttca cctctacttt gacaaggctg gtcaaaagac ctatgagaga 7741 catccgctct cccattttgt caatttagac aatttgagag ctaacaacac taaaggttca 7801 ctgcctatta atgtcatagt ttttgatggc aagtccaaat gcgacgagtc tgcttctaag 7861 tctgcttctg tgtactacag tcagctgatg tgccaaccta ttctgttgct tgaccaagct 7921 cttgtatcag acgttggaga tagtactgaa gtttccgtta agatgtttga tgcttatgtc 7981 gacacctttt cagcaacttt tagtgttcct atggaaaaac ttaaggcact tgttgctaca 8041 gctcacagcg agttagcaaa gggtgtagct ttagatggtg tcctttctac attcgtgtca 8101 gctgcccgac aaggtgttgt tgataccgat gttgacacaa aggatgttat tgaatgtctc 8161 aaactttcac atcactctga cttagaagtg acaggtgaca gttgtaacaa tttcatgctc 8221 acctataata aggttgaaaa catgacgccc agagatcttg gcgcatgtat tgactgtaat 8281 gcaaggcata tcaatgccca agtagcaaaa agtcacaatg tttcactcat ctggaatgta 8341 aaagactaca tgtctttatc tgaacagctg cgtaaacaaa ttcgtagtgc tgccaagaag 8401 aacaacatac cttttagact aacttgtgct acaactagac aggttgtcaa tgtcataact 8461 actaaaatct cactcaaggg tggtaagatt gttagtactt gttttaaact tatgcttaag 8521 gccacattat tgtgcgttct tgctgcattg gtttgttata tcgttatgcc agtacataca 8581 ttgtcaatcc atgatggtta cacaaatgaa atcattggtt acaaagccat tcaggatggt 8641 gtcactcgtg acatcatttc tactgatgat tgttttgcaa ataaacatgc tggttttgac 8701 gcatggttta gccagcgtgg tggttcatac aaaaatgaca aaagctgccc tgtagtagct 8761 gctatcatta caagagagat tggtttcata gtgcctggct taccgggtac tgtgctgaga 8821 gcaatcaatg gtgacttctt gcattttcta cctcgtgttt ttagtgctgt tggcaacatt 8881 tgctacacac cttccaaact cattgagtat agtgattttg ctacctctgc ttgcgttctt 8941 gctgctgagt gtacaatttt taaggatgct atgggcaaac ctgtgccata ttgttatgac 9001 actaatttgc tagagggttc tatttcttat agtgagcttc gtccagacac tcgttatgtg 9061 cttatggatg gttccatcat acagtttcct aacacttacc tggagggttc tgttagagta 9121 gtaacaactt ttgatgctga gtactgtaga catggtacat gcgaaaggtc agaagtaggt 9181 atttgcctat ctaccagtgg tagatgggtt cttaataatg agcattacag agctctatca 9241 ggagttttct gtggtgttga tgcgatgaat ctcatagcta acatctttac tcctcttgtg 9301 caacctgtgg gtgctttaga tgtgtctgct tcagtagtgg ctggtggtat tattgccata 9361 ttggtgactt gtgctgccta ctactttatg aaattcagac gtgtttttgg tgagtacaac 9421 catgttgttg ctgctaatgc acttttgttt ttgatgtctt tcactatact ctgtctggta 9481 ccagcttaca gctttctgcc gggagtctac tcagtctttt acttgtactt gacattctat 9541 ttcaccaatg atgtttcatt cttggctcac cttcaatggt ttgccatgtt ttctcctatt 9601 gtgccttttt ggataacagc aatctatgta ttctgtattt ctctgaagca ctgccattgg 9661 ttctttaaca actatcttag gaaaagagtc atgtttaatg gagttacatt tagtaccttc 9721 gaggaggctg ctttgtgtac ctttttgctc aacaaggaaa tgtacctaaa attgcgtagc 9781 gagacactgt tgccacttac acagtataac aggtatcttg ctctatataa caagtacaag 9841 tatttcagtg gagccttaga tactaccagc tatcgtgaag cagcttgctg ccacttagca 9901 aaggctctaa atgactttag caactcaggt gctgatgttc tctaccaacc accacagaca 9961 tcaatcactt ctgctgttct gcagagtggt tttaggaaaa tggcattccc gtcaggcaaa
10021 gttgaagggt gcatggtaca agtaacctgt ggaactacaa ctcttaatgg attgtggttg
10081 gatgacacag tatactgtcc aagacatgtc atttgcacag cagaagacat gcttaatcct
10141 aactatgaag atctgctcat tcgcaaatcc aaccatagct ttcttgttca ggctggcaat
10201 gttcaacttc gtgttattgg ccattctatg caaaattgtc tgcttaggct taaagttgat
10261 acttctaacc ctaagacacc caagtataaa tttgtccgta tccaacctgg tcaaacattt
10321 tcagttctag catgctacaa tggttcacca tctggtgttt atcagtgtgc catgagacct
10381 aatcatacca ttaaaggttc tttccttaat ggatcatgtg gtagtgttgg ttttaacatt
10441 gattatgatt gcgtgtcttt ctgctatatg catcatatgg agcttccaac aggagtacac
10501 gctggtactg acttagaagg taaattctat ggtccatttg ttgacagaca aactgcacag
10561 gctgcaggta cagacacaac cataacatta aatgttttgg catggctgta tgctgctgtt
10621 atcaatggtg ataggtggtt tcttaataga ttcaccacta ctttgaatga ctttaacctt
10681 gtggcaatga agtacaacta tgaacctttg acacaagatc atgttgacat attgggacct
10741 ctttctgctc aaacaggaat tgccgtctta gatatgtgtg ctgctttgaa agagctgctg
10801 cagaatggta tgaatggtcg tactatcctt ggtagcacta ttttagaaga tgagtttaca
10861 ccatttgatg ttgttagaca atgctctggt gttaccttcc aaggtaagtt caagaaaatt
10921 gttaagggca ctcatcattg gatgctttta actttcttga catcactatt gattcttgtt
10981 caaagtacac agtggtcact gtttttcttt gtttacgaga atgctttctt gccatttact
11041 cttggtatta tggcaattgc tgcatgtgct atgctgcttg ttaagcataa gcacgcattc
11101 ttgtgcttgt ttctgttacc ttctcttgca acagttgctt actttaatat ggtctacatg
11161 cctgctagct gggtgatgcg tatcatgaca tggcttgaat tggctgacac tagcttgtct
11221 ggttataggc ttaaggattg tgttatgtat gcttcagctt tagttttgct tattctcatg
11281 acagctcgca ctgtttatga tgatgctgct agacgtgttt ggacactgat gaatgtcatt
11341 acacttgttt acaaagtcta ctatggtaat gctttagatc aagctatttc catgtgggcc
11401 ttagttattt ctgtaacctc taactattct ggtgtcgtta cgactatcat gtttttagct
11461 agagctatag tgtttgtgtg tgttgagtat tacccattgt tatttattac tggcaacacc
11521 ttacagtgta tcatgcttgt ttattgtttc ttaggctatt gttgctgctg ctactttggc
11581 cttttctgtt tactcaaccg ttacttcagg cttactcttg gtgtttatga ctacttggtc
11641 tctacacaag aatttaggta tatgaactcc caggggcttt tgcctcctaa gagtagtatt
11701 gatgctttca agcttaacat taagttgttg ggtattggag gtaaaccatg tatcaaggtt
11761 gctactgtac agtctaaaat gtctgacgta aagtgcacat ctgtggtact gctctcggtt
11821 cttcaacaac ttagagtaga gtcatcttct aaattgtggg cacaatgtgt acaactccac
11881 aatgatattc ttcttgcaaa agacacaact gaagctttcg agaagatggt ttctcttttg
11941 tctgttttgc tatccatgca gggtgctgta gacattaata ggttgtgcga ggaaatgctc
12001 gataaccgtg ctactcttca ggctattgct tcagaattta gttctttacc atcatatgcc
12061 gcttatgcca ctgcccagga ggcctatgag caggctgtag ctaatggtga ttctgaagtc
12121 gttctcaaaa agttaaagaa atctttgaat gtggctaaat ctgagtttga ccgtgatgct 12181 gccatgcaac gcaagttgga aaagatggca gatcaggcta tgacccaaat gtacaaacag 12241 gcaagatctg aggacaagag ggcaaaagta actagtgcta tgcaaacaat gctcttcact 12301 atgcttagga agcttgataa tgatgcactt aacaacatta tcaacaatgc gcgtgatggt 12361 tgtgttccac tcaacatcat accattgact acagcagcca aactcatggt tgttgtccct 12421 gattatggta cctacaagaa cacttgtgat ggtaacacct ttacatatgc atctgcactc 12481 tgggaaatcc agcaagttgt tgatgcggat agcaagattg ttcaacttag tgaaattaac 12541 atggacaatt caccaaattt ggcttggcct cttattgtta cagctctaag agccaactca 12601 gctgttaaac tacagaataa tgaactgagt ccagtagcac tacgacagat gtcctgtgcg 12661 gctggtacca cacaaacagc ttgtactgat gacaatgcac ttgcctacta taacaattcg 12721 aagggaggta ggtttgtgct ggcattacta tcagaccacc aagatctcaa atgggctaga 12781 ttccctaaga gtgatggtac aggtacaatt tacacagaac tggaaccacc ttgtaggttt 12841 gttacagaca caccaaaagg gcctaaagtg aaatacttgt acttcatcaa aggcttaaac 12901 aacctaaata gaggtatggt gctgggcagt ttagctgcta cagtacgtct tcaggctgga 12961 aatgctacag aagtacctgc caattcaact gtgctttcct tctgtgcttt tgcagtagac 13021 cctgctaaag catataagga ttacctagca agtggaggac aaccaatcac caactgtgtg 13081 aagatgttgt gtacacacac tggtacagga caggcaatta ctgtaacacc agaagctaac 13141 atggaccaag agtcctttgg tggtgcttca tgttgtctgt attgtagatg ccacattgac 13201 catccaaatc ctaaaggatt ctgtgacttg aaaggtaagt acgtccaaat acctaccact 13261 tgtgctaatg acccagtggg ttttacactt agaaacacag tctgtaccgt ctgcggaatg 13321 tggaaaggtt atggctgtag ttgtgaccaa ctccgcgaac ccttgatgca gtctgcggat 13381 gcatcaacgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca ccgtgcggca 13441 caggcactag tactgatgtc gtctacaggg cttttgatat ttacaacgaa aaagttgctg 13501 gttttgcaaa gttcctaaaa actaattgct gtcgcttcca ggagaaggat gaggaaggca 13561 atttattaga ctcttacttt gtagttaaga ggcatactat gtctaactac caacatgaag 13621 agactattta taacttggtt aaagattgtc cagcggttgc tgtccatgac tttttcaagt 13681 ttagagtaga tggtgacatg gtaccacata tatcacgtca gcgtctaact aaatacacaa 13741 tggctgattt agtctatgct ctacgtcatt ttgatgaggg taattgtgat acattaaaag 13801 aaatactcgt cacatacaat tgctgtgatg atgattattt caataagaag gattggtatg 13861 acttcgtaga gaatcctgac atcttacgcg tatatgctaa cttaggtgag cgtgtacgcc 13921 aatcattatt aaagactgta caattctgcg atgctatgcg tgatgcaggc attgtaggcg 13981 tactgacatt agataatcag gatcttaatg ggaactggta cgatttcggt gatttcgtac 14041 aagtagcacc aggctgcgga gttcctattg tggattcata ttactcattg ctgatgccca 14101 tcctcacttt gactagggca ttggctgctg agtcccatat ggatgctgat ctcgcaaaac 14161 cacttattaa gtgggatttg ctgaaatatg attttacgga agagagactt tgtctcttcg 14221 accgttattt taaatattgg gaccagacat accatcccaa ttgtattaac tgtttggatg 14281 ataggtgtat ccttcattgt gcaaacttta atgtgttatt ttctactgtg tttccaccta 14341 caagttttgg accactagta agaaaaatat ttgtagatgg tgttcctttt gttgtttcaa 14401 ctggatacca ttttcgtgag ttaggagtcg tacataatca ggatgtaaac ttacatagct 14461 cgcgtctcag tttcaaggaa cttttagtgt atgctgctga tccagctatg catgcagctt 14521 ctggcaattt attgctagat aaacgcacta catgcttttc agtagctgca ctaacaaaca 14581 atgttgcttt tcaaactgtc aaacccggta attttaataa agacttttat gactttgctg 14641 tgtctaaagg tttctttaag gaaggaagtt ctgttgaact aaaacacttc ttctttgctc 14701 aggatggcaa cgctgctatc agtgattatg actattatcg ttataatctg ccaacaatgt 14761 gtgatatcag acaactccta ttcgtagttg aagttgttga taaatacttt gattgttacg 14821 atggtggctg tattaatgcc aaccaagtaa tcgttaacaa tctggataaa tcagctggtt 14881 tcccatttaa taaatggggt aaggctagac tttattatga ctcaatgagt tatgaggatc 14941 aagatgcact tttcgcgtat actaagcgta atgtcatccc tactataact caaatgaatc 15001 ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc tctatctgta 15061 gtactatgac aaatagacag tttcatcaga aattattgaa gtcaatagcc gccactagag 15121 gagctactgt ggtaattgga acaagcaagt tttacggtgg ctggcataat atgttaaaaa 15181 ctgtttacag tgatgtagaa actccacacc ttatgggttg ggattatcca aaatgtgaca 15241 gagccatgcc taacatgctt aggataatgg cctctcttgt tcttgctcgc aaacataaca 15301 cttgctgtaa cttatcacac cgtttctaca ggttagctaa cgagtgtgcg caagtattaa 15361 gtgagatggt catgtgtggc ggctcactat atgttaaacc aggtggaaca tcatccggtg 15421 atgctacaac tgcttatgct aatagtgtct ttaacatttg tcaagctgtt acagccaatg 15481 taaatgcact tctttcaact gatggtaata agatagctga caagtatgtc cgcaatctac 15541 aacacaggct ctatgagtgt ctctatagaa atagggatgt tgatcatgaa ttcgtggatg 15601 agttttacgc ttacctgcgt aaacatttct ccatgatgat tctttctgat gatgccgttg 15661 tgtgctataa cagtaactat gcggctcaag gtttagtagc tagcattaag aactttaagg 15721 cagttcttta ttatcaaaat aatgtgttca tgtctgaggc aaaatgttgg actgagactg 15781 accttactaa aggacctcac gaattttgct cacagcatac aatgctagtt aaacaaggag 15841 atgattacgt gtacctgcct tacccagatc catcaagaat attaggcgca ggctgttttg 15901 tcgatgatat tgtcaaaaca gatggtacac ttatgattga aaggttcgtg tcactggcta 15961 ttgatgctta cccacttaca aaacatccta atcaggagta tgctgatgtc tttcacttgt 16021 atttacaata cattagaaag ttacatgatg agcttactgg ccacatgttg gacatgtatt 16081 ccgtaatgct aactaatgat aacacctcac ggtactggga acctgagttt tatgaggcta 16141 tgtacacacc acatacagtc ttgcaggctg taggtgcttg tgtattgtgc aattcacaga 16201 cttcacttcg ttgcggtgcc tgtattagga gaccattcct atgttgcaag tgctgctatg 16261 accatgtcat ttcaacatca cacaaattag tgttgtctgt taatccctat gtttgcaatg 16321 ccccaggttg tgatgtcact gatgtgacac aactgtatct aggaggtatg agctattatt 16381 gcaagtcaca taagcctccc attagttttc cattatgtgc taatggtcag gtttttggtt 16441 tatacaaaaa cacatgtgta ggcagtgaca atgtcactga cttcaatgcg atagcaacat 16501 gtgattggac taatgctggc gattacatac ttgccaacac ttgtactgag agactcaagc 16561 ttttcgcagc agaaacgctc aaagccactg aggaaacatt taagctgtca tatggtattg 16621 ccactgtacg cgaagtactc tctgacagag aattgcatct ttcatgggag gttggaaaac 16681 ctagaccacc attgaacaga aactatgtct ttactggtta ccgtgtaact aaaaatagta 16741 aagtacagat tggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtgtaca 16801 gaggtactac gacatacaag ttgaatgttg gtgattactt tgtgttgaca tctcacactg 16861 taatgccact tagtgcacct actctagtgc cacaagagca ctatgtgaga attactggct 16921 tgtacccaac actcaacatc tcagatgagt tttctagcaa tgttgcaaat tatcaaaagg 16981 tcggcatgca aaagtactct acactccaag gaccacctgg tactggtaag agtcattttg 17041 ccatcggact tgctctctat tacccatctg ctcgcatagt gtatacggca tgctctcatg 17101 cagctgttga tgccctatgt gaaaaggcat taaaatattt gcccatagat aaatgtagta 17161 gaatcatacc tgcgcgtgcg cgcgtagagt gttttgataa attcaaagtg aattcaacac 17221 tagaacagta tgttttctgc actgtaaatg cattgccaga aacaactgct gacattgtag 17281 tctttgatga aatctctatg gctactaatt atgacttgag tgttgtcaat gctagacttc 17341 gtgcaaaaca ctacgtctat attggcgatc ctgctcaatt accagccccc cgcacattgc 17401 tgactaaagg cacactagaa ccagaatatt ttaattcagt gtgcagactt atgaaaacaa 17461 taggtccaga catgttcctt ggaacttgtc gccgttgtcc tgctgaaatt gttgacactg 17521 tgagtgcttt agtttatgac aataagctaa aagcacacaa ggataagtca gctcaatgct 17581 tcaaaatgtt ctacaaaggt gttattacac atgatgtttc atctgcaatc aacagacctc 17641 aaataggcgt tgtaagagaa tttcttacac gcaatcctgc ttggagaaaa gctgttttta 17701 tctcacctta taattcacag aacgctgtag cttcaaaaat cttaggattg cctacgcaga 17761 ctgttgattc atcacagggt tctgaatatg actatgtcat attcacacaa actactgaaa 17821 cagcacactc ttgtaatgtc aaccgcttca atgtggctat cacaagggca aaaattggca 17881 ttttgtgcat aatgtctgat agagatcttt atgacaaact gcaatttaca agtctagaaa 17941 taccacgtcg caatgtggct acattacaag cagaaaatgt aactggactt tttaaggact 18001 gtagtaagat cattactggt cttcatccta cacaggcacc tacacacctc agcgttgata 18061 taaagttcaa gactgaagga ttatgtgttg acataccagg cataccaaag gacatgacct 18121 accgtagact catctctatg atgggtttca aaatgaatta ccaagtcaat ggttacccta 18181 atatgtttat cacccgcgaa gaagctattc gtcacgttcg tgcgtggatt ggctttgatg 18241 tagagggctg tcatgcaact agagatgctg tgggtactaa cctacctctc cagctaggat 18301 tttctacagg tgttaactta gtagctgtac cgactggtta tgttgacact gaaaataaca 18361 cagaattcac cagagttaat gcaaaacctc caccaggtga ccagtttaaa catcttatac 18421 cactcatgta taaaggcttg ccctggaatg tagtgcgtat taagatagta caaatgctca 18481 gtgatacact gaaaggattg tcagacagag tcgtgttcgt cctttgggcg catggctttg 18541 agcttacatc aatgaagtac tttgtcaaga ttggacctga aagaacgtgt tgtctgtgtg 18601 acaaacgtgc aacttgcttt tctacttcat cagatactta tgcctgctgg aatcattctg 18661 tgggttttga ctatgtctat aacccattta tgattgatgt tcagcagtgg ggctttacgg 18721 gtaaccttca gagtaaccat gaccaacatt gccaggtaca tggaaatgca catgtggcta 18781 gttgtgatgc tatcatgact agatgtttag cagtccatga gtgctttgtt aagcgcgttg 18841 attggtctgt tgaataccct attataggag atgaactgag ggttaattct gcttgcagaa 18901 aagtacaaca catggttgtg aagtctgcat tgcttgctga taagtttcca gt cttcatg 18961 acattggaaa tccaaaggct atcaagtgtg tgcctcaggc tgaagtagaa tggaagttct 19021 acgatgctca gccatgtagt gacaaagctt acaaaataga ggaactcttc tattcttatg 19081 ctacacatca cgataaattc actgatggtg tttgtttgtt ttggaattgt aacgttgatc 19141 gttacccagc caatgcaatt gtgtgtaggt ttgacacaag agtcttgtca aacttgaact 19201 taccaggctg tgatggtggt agtttgtatg tgaataagca tgcattccac actccagctt 19261 tcgataaaag tgcatttact aatttaaagc aattgccttt cttttactat tctgatagtc 19321 cttgtgagtc tcatggcaaa caagtagtgt cggatattga ttatgttcca ctcaaatctg 19381 ctacgtgtat tacacgatgc aatttaggtg gtgctgtttg cagacaccat gcaaatgagt 19441 accgacagta cttggatgca tataatatga tgatttctgc tggatttagc ctatggattt 19501 acaaacaatt tgatacttat aacctgtgga atacatttac caggttacag agtttagaaa 19561 atgtggctta taatgttgtt aataaaggac actttgatgg acacgccggc gaagcacctg 19621 tttccatcat taataatgct gtttacacaa aggtagatgg tattgatgtg gagatctttg 19681 aaaataagac aacacttcct gttaatgttg catttgagct ttgggctaag cgtaacatta 19741 aaccagtgcc agagattaag atactcaata atttgggtgt tgatatcgct gctaatactg 19801 taatctggga ctacaaaaga gaagccccag cacatgtatc tacaataggt gtctgcacaa 19861 tgactgacat tgccaagaaa cctactgaga gtgcttgttc ttcacttact gtcttgtttg 19921 atggtagagt ggaaggacag gtagaccttt ttagaaacgc ccgtaatggt gttttaataa 19981 cagaaggttc agtcaaaggt ctaacacctt caaagggacc agcacaagct agcgtcaatg 20041 gagtcacatt aattggagaa tcagtaaaaa cacagtttaa ctactttaag aaagtagacg 20101 gcattattca acagttgcct gaaacctact ttactcagag cagagactta gaggatttta 20161 agcccagatc acaaatggaa actgactttc tcgagctcgc tatggatgaa ttcatacagc 20221 gatataagct cgagggctat gccttcgaac acatcgttta tggagatttc agtcatggac 20281 aacttggcgg tcttcattta atgataggct tagccaagcg ctcacaagat tcaccactta 20341 aattagagga ttttatccct atggacagca cagtgaaaaa ttacttcata acagatgcgc 20401 aaacaggttc atcaaaatgt gtgtgttctg tgattgatct tttacttgat gactttgtcg 20461 agataataaa gtcacaagat ttgtcagtga tttcaaaagt ggtcaaggtt acaattgact 20521 atgctgaaat ttcattcatg ctttggtgta aggatggaca tgttgaaacc ttctacccaa 20581 aactacaagc aagtcaagcg tggcaaccag gtgttgcgat gcctaacttg tacaagatgc 20641 aaagaatgct tcttgaaaag tgtgaccttc agaattatgg tgaaaatgct gttataccaa 20701 aaggaataat gatgaatgtc gcaaagtata ctcaactgtg tcaatactta aatacactta 20761 ctttagctgt accctacaac atgagagtta ttcactttgg tgctggctct gataaaggag 20821 ttgcaccagg tacagctgtg ctcagacaat ggttgccaac tggcacacta cttgtcgatt 20881 cagatcttaa tgacttcgtc tccgacgcag attctacttt aattggagac tgtgcaacag 20941 tacatacggc taataaatgg gaccttatta ttagcgatat gtatgaccct aggaccaaac 21001 atgtgacaaa agagaatgac tctaaagaag ggtttttcac ttatctgtgt ggatttataa 21061 agcaaaaact agccctgggt ggttctatag ctgtaaagat aacagagcat tcttggaatg 21121 ctgaccttta caagcttatg ggccatttct catggtggac agcttttgtt acaaatgtaa 21181 atgcatcatc atcggaagca tttttaattg gggctaacta tcttggcaag ccgaaggaac 21241 aaattgatgg ctataccatg catgctaact acattttctg gaggaacaca aatcctatcc 21301 agttgtcttc ctattcactc tttgacatga gcaaatttcc tcttaaatta agaggaactg 21361 ctgtaatgtc tcttaaggag aatcaaatca atgatatgat ttattctctt ctggaaaaag 21421 gtaggcttat cattagagaa aacaacagag ttgtggtttc aagtgatatt cttgttaaca Gene S underscored-^
21481 actaaacgaa cATGtttatt ttcttattat ttcttactct cactaqtqqt aqtqaccttq 21541 accqqtqcac cacttttqat qatqttcaaq ctcctaatta cactcaacat acttcatcta
21601 tqaqqqqgqt ttactatcct gatqaaattt ttaqatcaqa cactctttat ttaactcaqq 21661 atttatttct tccattttat tctaatqtta caqqqtttca tactattaat catacgtttq 21721 qcaaccct t catacctttt aaqqatqqta tttattttqc tqccaca aq aaatcaaatq 21781 ttqtcc tqq ttqqqttttt qqttctacca t aacaacaa qtcacaqtcq gt attatta 21841 ttaacaattc tactaatgtt gttatacqaq catqtaactt tqaattqtqt qacaaccctt 21901 tctttqctqt ttctaaaccc atqqqtacac aqacacatac tatqatattc qataatqcat 21961 ttaattqcac tttcqagtac atatctqatq ccttttcqct tqatqtttca qaaaaqtcaq 22021 taattttaa acacttacqa qaqtttgtgt ttaaaaataa agatqqqttt ctctatqttt 22081 ataaq qcta tcaacctata gatqtaqttc qtqatctacc ttctqqtttt aacactttqa 22141 aacctatttt taaqttgcct cttggtatta acattacaaa ttttagaqcc attcttacag 22201 ccttttcacc tgctcaaqac atttqqqqca cqtcaqctqc aqcctatttt qttqqctatt 22261 taaaqccaac tacatttat ctcaaqtatq atqaaaatqq tacaatcaca qatqctqttq 22321 attgttctca aaatccactt qctqaactca aatqctct t taagaqcttt qaqattgaca 22381 aaggaattta ccaqacctct aatttcaqqq ttqttccctc aqqagatqtt qtqagattcc 22441 ctaatattac aaacttgtqt ccttttgqaq agqtttttaa tqctactaaa ttcccttctq 22501 tctatqcatq qgagaqaaaa aaaatttcta attgtgttqc tqattactct qtgctctaca 22561 actcaacatt tttttcaacc tttaaqtqct atqqcqtttc tqccactaaq ttqaatgatc 22621 tttgcttctc caatgtctat gcaqattctt ttqtaqtcaa qqqaqatqat qtaaqacaaa 22681 taqcqccaqq acaaactqqt gttattqctq attataatta taaattqcca qatqatttca 22741 tqqqttqtgt ccttqcttqq aatactaqqa acattqatqc tacttcaact qqtaattata 22801 attataaata taqqtatctt aqacatqqca aqcttaqqcc ctttqaqaqa qacatatcta 22861 at tqccttt ctcccctqat qqcaaacctt gcaccccacc tqctcttaat tqttattqqc 22921 cattaaatqa ttatqqtttt tacaccacta ctqqcattqq ctaccaacct tacaqaqttq 22981 taqtactttc ttttqaactt ttaaatqcac cqqccacqqt ttqt qacca aaattatcca 23041 ctgaccttat taaqaaccaq tqtqtcaatt ttaattttaa tqqactcact qgtactggtg 23101 tgttaactcc ttcttcaaaq agatttcaac catttcaaca atttggccgt gatgtttctg 23161 atttcactga ttccgttcga gatcctaaaa catctgaaat attagacatt tcaccttgct 23221 cttttggggq tqtaaqtqta attacacctq qaacaaatqc ttcatctqaa qttqctqttc 23281 tatatcaa a tqttaact c actqatqttt ctacaqcaat tcatqcaqat caactcacac 23341 caqctt qcq catatattct actqqaaaca atgtattcca gactcaaqca qqctqtctta 23401 taq aqctqa qcatqtcqac acttcttatq aqt cqacat tcctattqqa qctqqcattt 23461 qtqctagtta ccatacagtt tctttattac gtagtactaq ccaaaaatct attqtgqctt 23521 atactatgtc tttaqqtqct qataqttcaa ttqcttactc taataacacc attqctatac 23581 ctactaactt ttcaattaqc attactacag aaqtaat cc tqtttctatq qctaaaacct 23641 ccqtaqattq taatatqtac atctqcqqaq attctactqa atqt ctaat ttqcttctcc 23701 aatatqqtaq cttttqcaca caactaaatc qtqcactctc aqqtattqct qctqaacaqq 23761 atcqcaacac acqtqaaqtq ttcqctcaa tcaaacaaat tacaaaacc ccaactttqa 23821 aatattttqq t gttttaat ttttcacaaa tattacctga ccctctaaag ccaactaaqa 23881 qgtcttttat tgagqacttq ctctttaata aqqtqacact cqctgatqct qgcttcatqa 23941 aqcaatatqq cqaatqccta qqtqatatta atqctaqaga tctcatttgt gcqcaqaaqt 24001 tcaatqgact tacagtgttg ccacctctqc tcactqatqa tatqattqct qcctacactq 24061 ctgctctaqt ta tggtact qccactqctq qatqoacatt tqqtqctqqc qctqctcttc 24121 aaataccttt tqctatqcaa atqqcatata q ttcaatqq cattqqaqtt acccaaaatq 24181 ttctctatqa qaaccaaaaa caaatcqcca accaatttaa caaqqcqatt aqtcaaattc 24241 aaqaatcact tacaacaaca tcaactqcat tqqqcaaqct qcaaqacqtt ttaaccaoa 24301 atqctcaaqc attaaacaca cttqttaaac aactta ctc taatttt qt qcaatttcaa 24361 qtqtqctaaa tqatatcctt tcqcqacttq ataaaqtcqa qqcqqaqqta caaattqaca 24421 qqttaattac aqqcaoactt caaa ccttc aaacctatqt aacacaacaa ctaatcaqqq 24481 ctqctqaaat caqqocttct qctaatcttq ctgctactaa aatqtctqaq tqtgttcttq 24541 qacaatcaaa aaoaottqac ttttqtq aa aqqqctacca ccttatqtcc ttcccacaao 24601 caqccccqca tqqtqttgtc ttcctacatg tcacqtat t qccatcccaq qaqaqqaact 24661 tcaccacaqc ccagcaatt tqtcatqaaq qcaaa cata cttccctc t qaaqqtqttt 24721 ttqtqtttaa tqqcacttct tqqtttatta cacaqaqqaa cttcttttct ccacaaataa 24781 ttactacaqa caatacattt qtctcaqqaa attqtqatqt cqttattqqc atcattaaca 24841 acacaottta tqatcctctg caacctqa c tt actcatt caaaqaaqaq ctqqacaaqt 24901 acttcaaaaa tcatacatca ccaqatqttq atcttqgcqa catttcaqqc attaacqctt 24961 ctqtcqtcaa cattcaaaaa qaaattqacc qcctcaat a qqtcqctaaa aatttaaatg 25021 aatcactcat tqaccttcaa qaattqqqaa aatatqa ca atatattaaa tqqccttggt 25081 atqtttq ct cqqcttcatt gctq actaa ttqccatcqt catqgttaca atcttqcttt 25141 qttgcatqac taqttottgc aqttgcctca aqggtgcatq ctcttqtqqt tcttqctqca 25201 aqtttgatqa qqatqactct qaqccaqttc tcaaqggtqt caaattacat tacacaFA4a
25261 cgaacttatg gatttgttta tgagattttt tactcttgga tcaattactg cacagccagt 25321 aaaaattgac aatgcttctc ctgcaagtac tgttcatgct acagcaacga taccgctaca 25381 agcctcactc cctttcggat ggcttgttat tggcgttgca tttcttgctg tttttcagag 25441 cgctaccaaa ataattgcgc tcaataaaag atggcagcta gccctttata agggcttcca 25501 gttcatttgc aatttactgc tgctatttgt taccatctat tcacatcttt tgcttgtcgc 25561 tgcaggtatg gaggcgcaat ttttgtacct ctatgccttg atatattttc tacaatgcat 25621 caacgcatgt agaattatta tgagatgttg gctttgttgg aagtgcaaat ccaagaaccc 25681 attactttat gatgccaact actttgtttg ctggcacaca cataactatg actactgtat 25741 accatataac agtgtcacag atacaattgt cgttactgaa ggtgacggca tttcaacacc 25801 aaaactcaaa gaagactacc aaattggtgg ttattctgag gataggcact caggtgttaa 25861 agactatgtc gttgtacatg gctatttcac cgaagtttac taccagcttg agtctacaca 25921 aattactaca gacactggta ttgaaaatgc tacattcttc atctttaaca agcttgttaa 25981 agacccaccg aatgtgcaaa tacacacaat cgacggctct tcaggagttg ctaatccagc 26041 aatggatcca atttatgatg agccgacgac gactactagc gtgcctttgt aagcacaaga Gene E underscored-
26101 aagtgagtac gaacttATGt actcattcqt ttcqqaaqaa acaqqtacqt taataqttaa 26161 taqcqtactt ctttttcttg ctttcqtqqt attcttqcta qtcacactaq ccatccttac
26221 tqcqcttcqa ttqtqtqcgt actqctqcaa tattqttaac qtqaqtttaq taaaaccaac
26281 qgtttacqtc tactcqcqtg ttaaaaatct gaactcttct gaaqgagttc ctgatcttct
26341 ggtc7Α4acg aactaactat tattattatt ctgtttggaa ctttaacatt qcttatcat <-Gene underscored-*
26401 qcaqacaacq qtactattac cqttqaqqaq cttaaacaac tcctqqaaca atqqaaccta
26461 qtaataqqtt tcctattcct aqcctqqatt at ttactac aattt ccta ttctaatcqq
26521 aacaqqtttt t tacataat aaaqctt tt ttcctctqqc tcttqtqqcc aqtaacactt
26581 qcttgttttq tqcttqctqc tqtctacaga attaattqq tqact qcqq qattqcqatt
26641 gcaatqgctt qtattqtagq cttqatqtqq cttaqctact tcqttqcttc cttcaqqctq
26701 tttqctcqta cccqctcaat qtqqtcattc aaccca aaa caaacattct tctcaatqtq
26761 cctctccqqq qqacaattqt qaccaqaccq ctcatqqaaa qtqaacttqt cattqqtqct
26821 qtqatcattc qtqgtcactt qcgaatggcc ggacactccc taqqqcqctq tqacattaag
26881 qacctgccaa aaqaqatcac tqtqqctaca tcacqaacqc tttcttatta caaattaqqa
26941 qcqtcqcaqc qtqtagqcac tqattcaqqt tttqctqcat acaacc cta ccqtattqqa
27001 aactataaat taaatacaqa ccacqccqqt aqcaacqaca atattqcttt qctaqtacaq
27061 TAAqtgacaa cagatgtttc atcttgttga cttccaggtt acaatagcag agatattgat
27121 tatcattatg aggactttca ggattgctat ttggaatctt gacgttataa taagttcaat 27181 agtgagacaa ttatttaagc ctctaactaa gaagaattat tcggagttag atgatgaaga 27241 acctatggag ttagattatc cataaaacga acatgaaaat tattctcttc ctgacattga 27301 ttgtatttac atcttgcgag ctatatcact atcaggagtg tgttagaggt acgactgtac 27361 tactaaaaga accttgccca tcaggaacat acgagggcaa ttcaccattt caccctcttg 27421 ctgacaataa atttgcacta acttgcacta gcacacactt tgcttttgct tgtgctgacg 27481 gtactcgaca tacctatcag ctgcgtgcaa gatcagtttc accaaaactt ttcatcagac 27541 aagaggaggt tcaacaagag ctctactcgc cactttttct cattgttgct gctctagtat 27601 ttttaatact ttgcttcacc attaagagaa agacagaatg aatgagctca ctttaattga 27661 cttctatttg tgctttttag cctttctgct attccttgtt ttaataatgc ttattatatt 27721 ttggttttca ctcgaaatcc aggatctaga agaaccttgt accaaagtct aaacgaacat 27781 gaaacttctc attgttttga cttgtatttc tctatgcagt tgcatatgca ctgtagtaca 27841 gcgctgtgca tctaataaac ctcatgtgct tgaagatcct tgtaaggtac aacactaggg 27901 gtaatactta tagcactgct tggctttgtg ctctaggaaa ggttttacct tttcatagat 27961 ggcacactat ggttcaaaca tgcacaccta atgttactat caactgtcaa gatccagctg 28021 gtggtgcgct tatagctagg tgttggtacc ttcatgaagg tcaccaaact gctgcattta <-Gene N underscored-*
28081 gagacgtact tgttgtttta aataaacgaa caaattaaaΛ TGtctqataa tqqaccccaa 28141 tcaaaccaac qtaqtqcccc ccqcattaca tttqqtqqac ccacaqattc aactqacaat 28201 aaccaqaatq qaqqacqcaa tqqqqcaaqq ccaaaacaqc qccqacccca aqqtttaccc 28261 aataatactq cqtcttqqtt cacaqctctc actcaqcatq qcaaqqaqqa acttaqattc 28321 cctcqaqqcc aqqαcqttcc aatcaacacc aataqtqgtc cagatqacca aattqqctac 28381 taccqaa aq ctacccqacq aqttcgtgqt ggtqacqqca aaatqaaaqa qctcaqcccc 28441 aqatqqtact tctattacct aqqaactqqc ccaqaa ctt cacttcccta cqqcgctaac 28501 aaaqaaqqca tcqtatqqqt tqcaact aq gqaqccttqa atacacccaa aqaccacatt 28561 qqcaccc ca atcctaataa caatqctqcc accqtqctac aacttcctca aqqaacaaca 28621 ttqccaaaaq qcttctacgc aqaqqqaaqc aqaq cqqca qtcaa cctc ttctcgctcc 28681 tcatcac ta qtc cqqtaa ttcaagaaat tcaactcctq qcagcagtag gqqaaattct 28741 cctqctc aa tqqctaqcqq aqqtqgtqaa actqccctcq cqctattqct gctagacaqa 28801 ttqaaccaqc ttqaqa caa aqtttctgqt aaaqqccaac aacaacaaqq ccaaactqtc 28861 actaaqaaat ctgctqctqa qqcatctaaa aaqcctcqcc aaaaacqtac tqccacaaaa 28921 caqtacaacq tcactcaaqc atttqgqaqa cqtqqtccaq aacaaaccca aqqaaatttc 28981 qqqqaccaaq acctaatcaq acaaqqaact qattacaaac attgqccqca aattqcacaa 29041 tttqctccaa qtqcctctgc attctttqqa atqtcacqca ttqqcatqqa aqtcacacct 29101 tcqqqaacat qqctqactta tcatq aqcc attaaatt q atqacaaaqa tccacaattc 29161 aaagacaacq tcatactqct qaacaagcac attqacqcat acaaaacatt cccaccaaca 29221 qaqcctaaaa aqqacaaaaa qaaaaaqact qat aaqctc aqcctttqcc qcaqaqacaa 29281 aaqaaqcaqc ccactqtqac tcttcttcct qcqqctqaca tqqatqattt ctccaαacaa 29341 cttcaaaatt ccatqaqtqq aqcttctqct gattcaactc aqqca7A4ac actcatgatg 29401 accacacaag gcagatgggc tatgtaaacg ttttcgcaat tccgtttacg atacatagtc 29461 tactcttgtg cagaatgaat tctcgtaact aaacagcaca agtaggttta gttaacttta 29521 atctcacata gcaatcttta atcaatgtgt aacattaggg aggacttgaa agagccacca 29581 cattttcatc gaggccacgc ggagtacgat cgagggtaca gtgaataatg ctagggagag 29641 ctgcctatat ggaagagccc taatgtgtaa aattaatttt agtagtgcta tccccatgtg 29701 attttaatag cttcttagga gaatgacaa The following subsequences are shown and annotated above by underscoring the coding sequences ofinterest with the initiation codonATG in uppercase characters, and the stop codon in uppercase italic characters. The individual coding sequences and translated amino acid sequences are provided below:
1. The coding sequence for the S (spike) glycoprotein, SEQ ID NO:13, is from nt 21492 to 25259 ofSEQ ID NO:12, which comprises 3768 nt that encode 1255 residues + stop codon.
SEOIDNO:13
ATG ttt att ttc tta tta ttt ctt act etc act agt ggt agt gac ctt gac egg tgc acc act ttt gat gat gtt caa get cct aat tac act caa cat act tea tct atg agg ggg gtt tac tat cct gat gaa att ttt aga tea gac act ctt tat tta act cag gat tta ttt ctt cca ttt tat tct aat gtt aca ggg ttt cat act att aat cat acg ttt ggc aac cct gtc ata cct ttt aag gat ggt att tat ttt get gcc aca gag aaa tea aat gtt gtc cgt ggt tgg gtt ttt ggt tct acc atg aac aac aag tea cag teg gtg att att att aac aat tct act aat gtt gtt ata cga gca tgt aac ttt gaa ttg tgt gac aac cct ttc ttt get gtt tct aaa ccc atg ggt aca cag aca cat act atg ata ttc gat aat gca ttt aat tgc act ttc gag tac ata tct gat gcc ttt teg ctt gat gtt tea gaa aag tea ggt aat ttt aaa cac tta cga gag ttt gtg ttt aaa aat aaa gat ggg ttt etc tat gtt tat aag ggc tat caa cct ata gat gta gtt cgt gat cta cct tct ggt ttt aac act ttg aaa cct att ttt aag ttg cct ctt ggt att aac att aca aat ttt aga gcc att ctt aca gcc ttt tea cct get caa gac att tgg ggc acg tea get gca gcc tat ttt gtt ggc tat tta aag cca act aca ttt atg etc aag tat gat gaa aat ggt aca ate aca gat get gtt gat tgt tct caa aat cca ctt get gaa etc aaa tgc tct gtt aag age ttt gag att gac aaa gga att tac cag acc tct aat ttc agg gtt gtt ccc tea gga gat gtt gtg aga ttc cct aat att aca aac ttg tgt cct ttt gga gag gtt ttt aat get act aaa ttc cct tct gtc tat gca tgg gag aga aaa aaa att tct aat tgt gtt get gat tac tct gtg etc tac aac tea aca ttt ttt tea acc ttt aag tgc tat ggc gtt tct gcc act aag ttg aat gat ctt tgc ttc tec aat gtc tat gca gat tct ttt gta gtc aag gga gat gat gta aga caa ata gcg cca gga caa act ggt gtt att get gat tat aat tat aaa ttg cca gat gat ttc atg ggt tgt gtc ctt get tgg aat act agg aac att gat get act tea act ggt aat tat aat tat aaa tat agg tat ctt aga cat ggc aag ctt agg ccc ttt gag aga gac ata tct aat gtg cct ttc tec cct gat ggc aaa cct tgc acc cca cct get ctt aat tgt tat tgg cca tta aat gat tat ggt ttt tac acc act act ggc att ggc tac caa cct tac aga gtt gta gta ctt tct ttt gaa ctt tta aat gca ccg gcc acg gtt tgt gga cca aaa tta tec act gac ctt att aag aac cag tgt gtc aat ttt aat ttt aat gga etc act ggt act ggt gtg tta act cct tct tea aag aga ttt caa cca ttt caa caa ttt ggc cgt gat gtt tct gat ttc act gat tec gtt cga gat cct aaa aca tct gaa ata tta gac att tea cct tgc tct ttt ggg ggt gta agt gta att aca cct gga aca aat get tea tct gaa gtt get gtt cta tat caa gat gtt aac tgc act gat gtt tct aca gca att cat gca gat caa etc aca cca get tgg cgc ata tat tct act gga aac aat gta ttc cag act caa gca ggc tgt ctt ata gga get gag cat gtc gac act tct tat gag tgc gac att cct att gga get ggc att tgt get agt tac cat aca gtt tct tta tta cgt agt act age caa aaa tct att gtg get tat act atg tct tta ggt get gat agt tea att get tac tct aat aac acc att get ata cct act aac ttt tea att age att act aca gaa gta atg cct gtt tct atg get aaa acc tec gta gat tgt aat atg tac ate tgc gga gat tct act gaa tgt get aat ttg ctt etc caa tat ggt age ttt tgc aca caa cta aat cgt gca etc tea ggt att get get gaa cag gat cgc aac aca cgt gaa gtg ttc get caa gtc aaa caa atg tac aaa acc cca act ttg aaa tat ttt ggt ggt ttt aat ttt tea caa ata tta cct gac cct cta aag cca act aag agg tct ttt att gag gac ttg etc ttt aat aag gtg aca etc get gat get ggc ttc atg aag caa tat ggc gaa tgc cta ggt gat att aat get aga gat etc att tgt gcg cag aag ttc aat gga ctt aca gtg ttg cca cct ctg etc act.gat gat atg att get gcc tac act get get cta gtt agt ggt act gcc act get gga tgg aca ttt ggt get ggc get get ctt caa ata cct ttt get atg caa atg gca tat agg ttc aat ggc att gga gtt acc caa aat gtt etc tat gag aac caa aaa caa ate gcc aac caa ttt aac aag gcg att agt caa att caa gaa tea ctt aca aca aca tea act gca ttg ggc aag ctg caa gac gtt gtt aac cag aat get caa gca tta aac aca ctt gtt aaa caa ctt age tct aat ttt ggt gca att tea agt gtg cta aat gat ate ctt teg cga ctt gat aaa gtc gag gcg gag gta caa att gac agg tta att aca ggc aga ctt caa age ctt caa acc tat gta aca caa caa cta ate agg get get gaa ate agg get tct get aat ctt get get act aaa atg tct gag tgt gtt ctt gga caa tea aaa aga gtt gac ttt tgt gga aag ggc tac cac ctt atg tec ttc cca caa gca gcc ccg cat ggt gtt gtc ttc cta cat gtc acg tat gtg cca tec cag gag agg aac ttc acc aca gcg cca gca att tgt cat gaa ggc aaa gca tac ttc cct cgt gaa ggt gtt ttt gtg ttt aat ggc act tct tgg ttt att aca cag agg aac ttc ttt tct cca caa ata att act aca gac aat aca ttt gtc tea gga aat tgt gat gtc gtt att ggc ate att aac aac aca gtt tat gat cct ctg caa cct gag ctt gac tea ttc aaa gaa gag ctg gac aag tac ttc aaa aat cat aca tea cca gat gtt gat ctt ggc gac att tea ggc att aac get tct gtc gtc aac att caa aaa gaa att gac cgc etc aat gag gtc get aaa aat tta aat gaa tea etc att gac ctt caa gaa ttg gga aaa tat gag caa tat att aaa tgg cct tgg tat gtt tgg etc ggc ttc att get gga cta att gcc ate gtc atg gtt aca ate ttg ctt tgt tgc atg act agt tgt tgc agt tgc etc aag ggt gca tgc tct tgt ggt tct tgc tgc aag ttt gat gag
The encoded amino acid sequence ofthe S polypeptide (SEQ ID NO:14) is:
MFIFLL-FLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGV YYPDEIFRSD TLYLTQDLFL 60
PFYSNVTGFH TINHTFGNPV IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS 120
TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT FEYISDAFSL DVSEKSGNFK 180 HLREFVFKNK DGFLYVYKGY QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP 240
AQDI GTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ NPLAELKCSV KSFEIDKGIY 300
QTSNFRVVPS GDWRFPNIT NLCPFGEVFN ATKFPSVYA ERKKISNCVA DYSVLYNSTF 360
FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG QTGVIADYNY KLPDDFMGCV 420
LA NTRNIDA TSTGNYNYKY RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCY PLND 480 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI KNQCVNFNFN GLTGTGVLTP 540
SSKRFQPFQQ FGRDVSDFTD SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD 600
VNCTDVSTAI HADQLTPA R IYSTGNNVFQ TQAGCLIGAE HVDTSYECDI PIGAGICASY 660
HTVSLLRSTS QKSIVAYTMS LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC 720
NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT REVFAQVKQM YKTPTLKYFG 780 GFNFSQILPD PLKPTKRSFI EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL 840 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE 900 NQKQIANQFN KAISQIQESL TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN 960 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK 1020 RVDFCGKGYH LMSFPQAAPH GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN 1080 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN 1140 HTSPDVDLGD ISGINASVVN IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL 1200 GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV KLHYT 1255
Sequences of domains ofthe S polypeptide (see Figure 6) are set forth below: Domain SI: - amino acids 1-680 of SEQ DD NO:14 which is shown below as SEQ ID NO:15:
MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGVt YYPDEIFRSD TLYLTQDLFL 60 PFYSNVTGFH TINHTFGNPV IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS 120 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT FEYISDAFSL DVSEKSGNFK 180 HLREFVFKNK DGFLYVYKGY QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP 240 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ NPLAELKCSV KSFEIDKGIY 300 QTSNFRVVPS GDWRFPNIT NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF 360 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG QTGVIADYNY KLPDDFMGCV 420 LAWNTRNIDA TSTGNYNYKY RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND 480 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI KNQCVNFNFN GLTGTGVLTP 540 SSKRFQPFQQ FGRDVSDFTD SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD 600 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE HVDTSYECDI PIGAGICASY 660 HTVSLLRSTS QKSIVAYTMS 680
Domain S2 - aa 680-1225 ofSEQ TD NO:14 which is shown below as SEQ ID NO:16 (residues 1-575):
LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC NMYICGDSTE CANLLLQYGS 60
FCTQLNRALS GIAAEQDRNT REVFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI 120
EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL TVLPPLLTDD MIAAYTAALV 180
SGTATAGWTF GAGAALQIPF AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL 240 TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN DILSRLDKVE AEVQIDRLIT 300
GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH 360
GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN GTSWFITQRN FFSPQIITTD 420
NTFVSGNCDV VIGIINNTVY DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN 480
IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL GFIAGLIAIV MVTILLCCMT 540 SCCSCLKGAC SCGSCCKFDE DDSEPVLKGV KLHYT 575
Polypeptide Si overlaps domains SI and S2 and corresponds to residues 417-816 or SEQ ID NO:14. This polypeptide is shown below as SEQ 1D:17 (aa 1-400):
MGCVLAWNTR NIDATSTGNY NYKYRYLRHG KLRPFERDIS NVPFSPDGKP CTPPALNCYW 60 PLNDYGFYTT TGIGYQPYRV VVLSFELLNA PATVCGPKLS TDLIKNQCVN FNFNGLTGTG 120 VLTPSSKRFQ PFQQFGRDVS DFTDSVRDPK TSEILDISPC SFGGVSVITP GTNASSEVAV 180 LYQDVNCTDV STAIHADQLT PAWRIYSTGN NVFQTQAGCL IGAEHVDTSY ECDIPIGAGI 240 CASYHTVSLL RSTSQKSIVA YTMSLGADSS IAYSNNTIAI PTNFSISITT EVMPVSMAKT 300 SVDCNMYICG DSTECANLLL QYGSFCTQLN RALSGIAAEQ DRNTREVFAQ VKQMYKTPTL 360 KYFGGFNFSQ ILPDPLKPTK RSFIEDLLFN KVTLADAGFM 400
The present invention includes homologous sequences to the S polypeptide domains from any other strain of SARS-CoN. 2. The coding sequence for the E (envelope, or "small envelope") protein (SEQ ID NO:18) is from nt 26117 to 26347 of SEQ ID NO: 12, which comprises 231 nt that encode 76 aa's + stop codon.
SEO ID NO: 18 ATG tac tea ttc gtt teg gaa gaa aca ggt acg tta ata gtt aat age gta ctt ctt ttt ctt get ttc gtg gta ttc ttg cta gtc aca cta gcc ate ctt act gcg ctt cga ttg tgt gcg tac tgc tgc aat att gtt aac gtg agt tta gta aaa cca acg gtt tac gtc tac teg cgt gtt aaa aat ctg aac tct tct gaa gga gtt cct gat ctt ctg gtc TAA The encoded amino acid sequence ofthe Epolypeptide (SEQIDNO:19) is:
MYSFVSEETG TLIVNSVLLF LAFVVFLLVT LAILTALRLC AYCCNIVNVS LVKPTVYVYS 60 RVKNLNSSEG VPDLLV 76
3. The coding sequence for the M (membrane protein (SEQ ID NO:20) is from nt 26398 to 27063 of SEQ ID NO: 12, which comprises 666 nt encoding 221 aa + stop codon. SEO ID NO:20
ATG gca gac aac ggt act att acc gtt gag gag ctt aaa caa etc ctg gaa caa tgg aac cta gta ata ggt ttc cta ttc cta gcc tgg att atg tta cta caa ttt gcc tat tct aat egg aac agg ttt ttg tac ata ata aag ctt gtt ttc etc tgg etc ttg tgg cca gta aca ctt get tgt ttt gtg ctt get get gtc tac aga att aat tgg gtg act ggc ggg att gcg att gca atg get tgt att gta ggc ttg atg tgg ctt age tac ttc gtt get tec ttc agg ctg ttt get cgt acc cgc tea atg tgg tea ttc aac cca gaa aca aac att ctt etc aat gtg cct etc egg ggg aca att gtg acc aga ccg etc atg gaa agt gaa ctt gtc att ggt get gtg ate att cgt ggt cac ttg cga atg gcc gga cac tec cta ggg cgc tgt gac att aag gac ctg cca aaa gag ate act gtg get aca tea cga acg ctt tct tat tac aaa tta gga gcg teg cag cgt gta ggc act gat tea ggt ttt get gca tac aac cgc tac cgt att gga aac tat aaa tta aat aca gac cac gcc ggt age aac gac aat att get ttg cta gta cag TAA The encoded amino acid sequence oftheMpolypeptide (SEQ ID NO:21) is:
MADNGTITVE ELKQLLEQWN LVIGFLFLAW IMLLQFAYSN RNRFLYIIKL VFLWLLWPVT 60
LACFVLAAVY RINWVTGGIA lAMACIVGLM WLSYFVASFR LFARTRSMWS FNPETNILLN 120
VPLRGTIVTR PLMESELVIG AVIIRGHLRM AGHSLGRCDI KDLPKEITVA TSRTLSYYKL 180
GASQRVGTDS GFAAYNRYRI GNYKLNTDHA GSNDNIALLV Q 221 4. The coding sequence forthe N (nucleocapsidprotein(SEQ IDNO:22) is from nt28120 to 29388 ofSEQ ID NO:12, which comprises 1269 nt encoding 422 aa+ stop codon. SEO ID NO:22
ATG tct gat aat gga ccc caa tea aac caa cgt agt gcc ccc cgc att aca ttt ggt gga ccc aca gat tea act gac aat aac cag aat gga gga cgc aat ggg gca agg cca aaa cag cgc cga ccc caa ggt tta ccc aat aat act gcg tct tgg ttc aca get etc act cag cat ggc aag gag gaa ctt aga ttc cct cga ggc cag ggc gtt cca ate aac acc aat agt ggt cca gat gac caa att ggc tac tac cga aga get acc cga cga gtt cgt ggt ggt gac ggc aaa atg aaa gag etc age ccc aga tgg tac ttc tat tac cta gga act ggc cca gaa get tea ctt ccc tac ggc get aac aaa gaa ggc ate gta tgg gtt gca act gag gga gcc ttg aat aca ccc aaa gac cac att ggc acc cgc aat cct aat aac aat get gcc acc gtg cta caa ctt cct caa gga aca aca ttg cca aaa ggc ttc tac gca gag gga age aga ggc ggc agt caa gcc tct tct cgc tec tea tea cgt agt cgc ggt aat tea aga aat tea act cct ggc age agt agg gga aat tct cct get cga atg get age gga ggt ggt gaa act gcc etc gcg cta ttg ctg cta gac aga ttg aac cag ctt gag age aaa gtt tct ggt aaa ggc caa caa caa caa ggc caa act gtc act aag aaa tct get get gag gca tct aaa aag cct cgc caa aaa cgt act gcc aca aaa cag tac aac gtc act caa gca ttt ggg aga cgt ggt cca gaa caa acc caa gga aat ttc ggg gac caa gac cta ate aga caa gga act gat tac aaa cat tgg ccg caa att gca caa ttt get cca agt gcc tct gca ttc ttt gga atg tea cgc att ggc atg gaa gtc aca cct teg gga aca tgg ctg act tat cat gga gcc att aaa ttg gat gac aaa gat cca caa ttc aaa gac aac gtc ata ctg ctg aac aag cac att gac gca tac aaa aca ttc cca cca aca gag cct aaa aag gac aaa aag aaa aag act gat gaa get cag cct ttg ccg cag aga caa aag aag cag ccc act gtg act ctt ctt cct gcg get gac atg gat gat ttc tec aga caa ctt caa aat tec atg agt gga get tct get gat tea act cag gca TAA The encoded amino acid sequence ofthe Npolypeptide (SEQ ID NO:23) is:
MSDNGPQSNQ RSAPRITFGG PTDSTDNNQN GGRNGARPKQ RRPQGLPNNT ASWFTALTQH 60
GKEELRFPRG QGVPINTNSG PDDQIGYYRR ATRRVRGGDG KMKELSPRWY FYYLGTGPEA 120
SLPYGANKEG IVWVATEGAL NTPKDHIGTR NPNNNAATVL QLPQGTTLPK GFYAEGSRGG 180
SQASSRSSSR SRGNSRNSTP GSSRGNSPAR MASGGGETAL ALLLLDRLNQ LESKVSGKGQ 240 QQQGQTVTKK SAAEASKKPR QKRTATKQYN VTQAFGRRGP EQTQGNFGDQ DLIRQGTDYK 300
HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYHGAIKL DDKDPQFKDN VILLNKHIDA 360
YKTFPPTEPK KDKKKKTDEA QPLPQRQKKQ PTVTLLPAAD MDDFSRQLQN SMSGASADST 420 QA 422
pcDNA3-CRT/N (SEQ TD NO:24) Vector sequence (UPPERCASE) CRT: lower case/italic N protein: lower case/bold/underscored
Figure imgf000052_0001
I 10 I 20 I 30 I 40 1 50 I 60 I 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CC 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA AC 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CA 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TG 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA AT 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CT 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT AT 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CG 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TG 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GT 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA AT 881 GGAGACCCAA GCTGGCTAGC GTTTAAACGG GCCCTCTAGA atgctgctcc ctgtgccgct gc 961 tggccgccgc cgagcccgtc gtctacttca aggagcagtt tctggacgga gatgggtgga cc 1041 aaacacaagt ccgattttgg caaattcgtc ctcagttcgg gcaagttcta cggcgatcag ga 1121 gaccagccag gacgcccgct tctacgccct gtcggcccga ttcgagccgt tcagcaacaa gg 1201 agttcaccgt gaaacacgag cagaacattg actgcggggg cggctacgtg aagctgtttc cg 1281 gacatgcacg gggactctga gtacaacatc atgtttggtc ctgacatctg tggccccggc ac 1361 cttcaactac aagggcaaga acgtgctgat caacaaggac atccgttgca aggacgacga gt 1441 tgatcgtgcg gccggacaac acgtatgagg tgaagattga caacagccag gtggagtcgg gc 1521 gacttcctac cccccaagaa gataaaggac ccagatgcct cgaagcctga agactgggac ga 1601 ccccacggac tccaagcccg aggactggga caagcccgag ca cat ccc eg acccggacgc ga 1681 acgaagaaat ggacggagag tgggagccgc cggtgattca gaaccccgag tacaagggtg ag 1761 gacaaccccg attacaaagg cacctggatc caccccgaaa tcgacaaccc cgagtactcg cc 1841 ctacgacagc tttgccgtgc tgggcttgga cctctggcag gtcaagtcgg gcaccatctt cg 1921 acgatgaggc gtacgcagag gagtttggca acgagacgtg gggcgtcacc aagacggccg ag 2001 caggacgagg agcagcggct gaaggaggag gaggaggaga agaagcggaa ggaggaggag ga 2081 ggacaaggac gacaaggagg acgaggatga ggacgaggag gacaaggacg aggaggagga gg 2161 ccaaggacga gctgtagGAA TTCatqtctq ataatqqacc ccaatcaaac caacqtaqtq cc 2241 qqacccacaq attcaactqa caataaecaq aatqqaqqac geaatqqqge aaqqccaaaa ca 2321 aeccaataat actgegtett qgttcacaqc tctcactcaq catqqeaagq aqqaacttaq at 2401 ttccaatcaa caccaataqt qgtccaqatq accaaattq ctaetaccqa agaqctaccc qa 2481 qqeaaaatqa aaqaqctcaq ccccaqatqq tacttctatt acctaqqaac tqqeccaqaa c 2561 taaeaaaqaa qqeatcqtat qqgttqeaac tqaqqqaqec ttqaatacac ccaaaqacca ca 2641 ataacaatqe tqccaccqtq etacaacttc ctcaaqqaac aacattqeca aaaqqettet ac 2721 qqeaqtcaaq cctcttctcq ctcctcatca cqtaqtcqeq qtaattcaag aaattcaact cc
2801 tteteetqet eqaatqqeta qcqqaqqtqq tgaaactgcc ctcgcqctat tqetqctaqa caqattqaac cagcttqaga 2880
2881 qcaaaqtttc tqqtaaaqqc caacaacaac aagqccaaac tqtcactaaq aaatctqctq ctqaqqcatc taaaaaqcct 2960
2961 cqccaaaaac qtactqccac aaaacaqtac aacqtcactc aaqcatttqq qaqacqtqqt ccaqaacaaa cccaaqqaaa 3040
3041 tttcqqqqac caaqacctaa tcagacaaq aactqattac aaacattqqc cgcaaattgc acaatttqct ccaaqtqcct 3120
3121 ctqcattctt tqqaatqtca cqcattqqca tqqaaqtcac accttcqqqa acatqqctqa cttatcatqq aqccattaaa 3200
3201 ttqgatqaca aaqatccaca attcaaaqac aacqtcatac tqctqaacaa qcacattqac qcatacaaaa cattcccacc 3280
3281 aacaqa cct aaaaaqqaca aaaa aaaaa gactqatgaa qctca cctt tqccqcaqaq acaaaaqaaq caqcccactq 3360
3361 tqactcttct tcctqcg ct qacatqqatq atttctccaq acaacttcaa aattccatqa qtqqaqcttc tgctgattca 3440 3441 actcaqqcaG GTACCAAGCT TGGGCCCGAA CAAAAACTCA TCTCAGAAGA GGATCTGAAT AGCGCCGTCG ACCATCATCA 3520
3521 TCATCATCAT TGAGTTTAAA CGGTCTCCAG CTTAAGTTTA AACCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC 3600 3601 CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG 3680 3681 GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG GGTGGGGCAG GACAGCAAGG GGGAGGATTG 3760 3761 GGAAGACAAT AGCAGGCATG CTGGGGATGC GGTGGGCTCT ATGGCTTCTG AGGCGGAAAG AACCAGCTGG GGCTCTAGGG 3840 3841 GGTATCCCCA CGCGCCCTGT AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG GTTACGCGCA GCGTGACCGC TACACTTGCC 3920 3921 AGCGCCCTAG CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC GTTCGCCGGC TTTCCCCGTC AAGCTCTAAA 4000 4001 TCGGGGGCTC CCTTTAGGGT TCCGATTTAG TGCTTTACGG CACCTCGACC CCAAAAAACT TGATTAGGGT GATGGTTCAC 4080 4081 GTAGTGGGCC ATCGCCCTGA TAGACGGTTT TTCGCCCTTT GACGTTGGAG TCCACGTTCT TTAATAGTGG ACTCTTGTTC 4160 4161 CAAACTGGAA CAACACTCAA CCCTATCTCG GTCTATTCTT TTGATTTATA AGGGATTTTG CCGATTTCGG CCTATTGGTT 4240 4241 AAAAAATGAG CTGATTTAAC AAAAATTTAA CGCGAATTAA TTCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC 4320 4321 AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC 4400 4401 CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC 4480 4481 CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG AGGCCGCCTC 4560 4561 TGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCI lllll GGAGGCCTAG GCTTTTGCAA AAAGCTCCCG GGAGCTTGTA 4640 4641 TATCCATTTT CGGATCTGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT 4720 4721 CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG 4800 4801 GCTGTCAGCG CAGGGGCGCC CGGTTCI I I I TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAG GACGAGGCAG 4880 4881 CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG 4960 4961 CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA 5040 5041 TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC 5120 5121 GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC 5200 5201 GCCAGGCTCA AGGCGCGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT 5280 5281 GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA 5360 5361 CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG 5440 5441 CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG 5520 5521 CCCAACCTGC CATCACGAGA TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC 5600 5601 CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCCAACTT GTTTATTGCA GCTTATAATG 5680 5681 GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCA I I I I I I TCACTGCATT CTAGTTGTGG TTTGTCCAAA 5760 5761 CTCATCAATG TATCTTATCA TGTCTGTATA CCGTCGACCT CTAGCTAGAG CTTGGCGTAA TCATGGTCAT AGCTGTTTCC 5840 5841 TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT 5920 5921 GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA 6000 6001 TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC 6080 6081 GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG 6160 6161 GAAAGAACAT GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCG lllll CCATAGGCTC 6240 6241 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC 6320
6321 GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT 6400 6401 CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT 6480 6481 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA 6560 6561 CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT 6640 6641 GGTGGCCTAA CTACGGCTAC ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 6720 6721 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG 6800 6801 AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA 6880 6881 TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT 6960 6961 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC 7040 10 7041 ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA 7120 7121 TACCGCGAGA CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT 7200 7201 CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT 7280 7281 GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC 7360 7361 AACGATCAAG GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA 7440 15 7441 AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG 7520 7521 CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT 7600 7601 CAATACGGGA TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC 7680 7681 TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT 7760 7761 CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA 7840 20 7841 TACTCATACT CTTCCIT I I I CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 7920 7921 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTC 7983 10 I 20 I 30 40 I 50 I 60 I
Ul 70 I 80 I KJ pcDNA3-S (Spike) (SEQ ID NO:25) Vector sequence, pcDNA3.1 (+) in UPPERCASE 25 Spike(S) protein sequence (lower case/ bold/underscored) I 10 I 20 I 30 I 40 I 50 I 60 I 70 I 80 I 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 30 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 35 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTGGTA CCGAGCTCGG ATCCat ttt attttcttat tatttcttac 960
961 tcteaetaqt qqtaqtqacc ttqaecqqtq eaecactttt qatqatqtte aaqcteetaa ttacaetcaa catacttcat 1040
1041 ctatqaqqqq qqtttactat cctqatqaaa tttttaqatc aqacactctt tatttaacte aqgatttatt tcttccattt 1120
1121 tattetaatq ttaeaqqqtt teatactatt aatcatacqt ttqqeaacec tqteatacct tttaaqqatg qtatttattt 1200
1201 tqctqceaea qaqaaateaa atqttqtccq tqqttgggtt tttqqttcta ccatqaaeaa caaqtcacaq tcqqtqatta 1280 1281 ttattaacaa ttctactaat qttqttatac qaqcatqtaa ctttqaattq tgtqacaacc ctttctttqc tqtttctaaa 1360
1361 cccatqqqta cacaqacaca tactatqata ttcqataatq catttaattq cactttcqaq tacatatctq atqccttttc 1440
1441 qcttqatgtt teagaaaaqt caqqtaattt taaacactta cqaqagtttq tqtttaaaaa taaagatqgg tttctctatq 1520
1521 tttataaqqq ctatcaacct ataqatqtaq ttcqtqatct accttctqqt tttaacactt tqaaacctat ttttaagttq 1600
1601 cctcttqqta ttaacattac aaattttaqa qccattctta cagccttttc acctqctcaa qacatttqgq qcacgtcaqc 1680 1681 tqcaqcctat tttqttqqct atttaaaqcc aactacattt atqctcaaqt atqatqaaaa tqqtacaatc acaqatqctq 1760
1761 ttqattqttc tcaaaatcca cttqctqaac tcaaatgctc tgttaagaqc tttqaqattg acaaaqqaat ttaccagacc 1840
1841 tctaatttca qqqttqttcc ctcaqqaqat gttqtqaqat tccctaatat tacaaacttq tqtccttttq qaqaqqtttt 1920
1921 taatqctact aaattccctt ctqtctatqc atqgqagaga aaaaaaattt ctaattqtqt tqctqattac tctgtgctct 2000
2001 acaactcaac atttttttca acctttaaqt qctatqqcqt ttctqccact aaqttqaatq atctttqctt ctccaatqtc 2080 2081 tatqcaqatt ettttqtaqt caaqgqaqat qatqtaagac aaataqcqcc agqacaaact qqtqttattq ctqattataa 2160
2161 ttataaattq ccaqatqatt teatgqqttq tqtccttqct tggaatacta qqaacattqa tqctacttca actqgtaatt 2240
2241 ataattataa atataqqtat cttaqacatq qcaaqcttaq gccctttqaq aqa acatat ctaatqtqcc tttctcccct 2320
2321 qatqqcaaac cttqcacccc acctgctctt aattqttatt qccattaaa tqattatgqt ttttacacca ctactqqcat 2400
2401 tqqctaccaa ccttacaqaq ttqtaqtact ttctttt aa cttttaaat caccqqccac qttt tqqa ccaaaattat 2480 2481 ccactqacct tattaaqaac caqtqtqtca attttaattt taatqqactc actqqtactq tgtqttaac tccttcttca 2560
2561 aaqaqatttc aaccatttca acaatttqqc cqtqatqttt ct atttcac tgattccgtt cgaqatccta aaacatctqa 2640
2641 aatattaqac atttcacctt qctcttttqq qq tqtaaqt gtaattacac ctqqaacaaa tqcttcatct qaagttqctq 2720
2721 ttctatatca aqatgttaac tqcactqatq tttctacaqc aattcatgca qatcaactca caccaqctt gcqcatatat 2800
2801 tctactqqaa acaatqtatt cca actcaa gcaqqctqtc ttataq aqc tqaqcatgtc qacacttctt atqagtqcqa 2880 2881 cattcctatt qqagctggca tttgtgctaq ttaccataca gtttctttat tacqtaqtac tagccaaaaa tctattqtqq 2960
2961 cttatactat qtctttaqqt qctqataqtt caattgctta ctctaataac accattqcta tacctactaa cttttcaatt 3040
3041 aqcattacta caqaa taat qcctqtttct atqqctaaaa cctccqtaqa ttqtaatatq tacatctqcq qaqattctac 3120
3121 tqaatqtqct aatttqcttc tccaatatqq taqcttttqc acacaactaa atcqtqcact ctcaqqtatt qctqctqaac 3200
3201 a qatcqcaa cacacqtqaa qtqttcqctc aa tcaaaca aat tacaaa accccaactt tqaaatattt tqqtgqtttt 3280 3281 aatttttcac aaatattacc tqaccctcta aaqccaacta aqaqqtcttt tattqaqqac ttgctcttta ataaggtgac 3360
3361 actcgctqat qctqqcttca tqaaqcaata tqqcqaatgc ctaqqtgata ttaatgctag a atctcatt tqtgcgcaqa 3440
3441 agttcaatqq acttacaqtq ttqccacctc tqctcactqa tqatatqatt qctqcctaca ctqctqctct aqttaqtqqt 3520
3521 actqccactq ctqqatqqac atttqqtqct qqcqctqctc ttcaaatacc ttttqctatq caaatqqcat ataqqttcaa 3600
3601 tqqcattqqa qttacccaaa atqttctcta tqaqaaccaa aaacaaatc ccaaccaatt taacaaqqcg attagtcaaa 3680 3681 ttcaaqaatc acttacaaca acatcaact catt qqcaa qctqcaaqac qttqttaacc aqaatqctca a cattaaac 3760
3761 acacttqtta aacaacttaq ctctaatttt gqtqcaattt caaqtqtgct aaatqatatc ctttcqcqac ttqataaaqt 3840
3841 cqaqqcgqag qtacaaattq acaqgttaat tacaqqcaqa cttcaaaqcc ttcaaaccta tqtaacacaa caactaatca 3920
3921 gqgctqctga aatcaqqqct tct ctaatc ttqctgctac taaaatqtct qagtqtqttc ttggacaatc aaaaagagtt 4000
4001 qacttttqtq qaaagqqcta ccaccttat tccttcccac aagcaqcccc qcatqqtgtt qtcttcctac atqtcacqta 4080 4081 tqtqccatcc caqqaqaqqa acttcaccac aqcqccaqca atttqtcat aaggcaaagc atacttccct cqtqaaqqtq 4160
4161 tttttqtqtt taat qcact tcttggttta ttacacaqaq aacttcttt tctccacaaa taattactac aqacaataca 4240
4241 ttt tctcag qaaattqtqa tqtc ttatt qqcatcatta acaacaca t ttatqatcct ctgcaacctq aqcttqactc 4320
4321 attcaaaqaa qaqctqqaca aqtacttcaa aaatcataca tcaccaqatq ttqatcttgg cqacatttca qqcattaacq 4400
4401 cttctqtcgt caacattcaa aaaqaaattq accqcctcaa tqaqqtcgct aaaaatttaa atgaatcact cattqacctt 4480
4481 caaqaattqq qaaaatatqa qcaatatatt aaatqqcctt gqtatqtttq qctcqqcttc attqetqqac taattqccat 4560 4561 cqtcatqqtt acaatcttqc tttqttqcat qactagttgt tqcaqttqcc tcaaqqqtqc atgctcttqt qqttcttqct 4640 4641 qcaaqtttqa tqaqqatqac tctqaqccaq ttctcaaqqq tqtcaaatta cattacacat aaGAATTCTG CAGATATCCA 4720 4721 GCACAGTGGC GGCCGCTCGA GTCTAGAGGG CCCGTTTAAA CCCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC 4800 5 4801 ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT CCTTTCCTAA TAAAATGAGG 4880 4881 AAATTGCATC GCATTGTCTG AGTAGGTGTC ATTCTATTCT GGGGGGTGGG GTGGGGCAGG ACAGCAAGGG GGAGGATTGG 4960 4961 GAAGACAATA GCAGGCATGC TGGGGATGCG GTGGGCTCTA TGGCTTCTGA GGCGGAAAGA ACCAGCTGGG GCTCTAGGGG 5040 5041 GTATCCCCAC GCGCCCTGTA GCGGCGCATT AAGCGCGGCG GGTGTGGTGG TTACGCGCAG CGTGACCGCT ACACTTGCCA 5120 5121 GCGCCCTAGC GCCCGCTCCT TTCGCTTTCT TCCCTTCCTT TCTCGCCACG TTCGCCGGCT TTCCCCGTCA AGCTCTAAAT 5200 10 5201 CGGGGGCTCC CTTTAGGGTT CCGATTTAGT GCTTTACGGC ACCTCGACCC CAAAAAACTT GATTAGGGTG ATGGTTCACG 5280 5281 TAGTGGGCCA TCGCCCTGAT AGACGGTTTT TCGCCCTTTG ACGTTGGAGT CCACGTTCTT TAATAGTGGA CTCTTGTTCC 5360 5361 AAACTGGAAC AACACTCAAC CCTATCTCGG TCTATTCTTT TGATTTATAA GGGATTTTGC CGATTTCGGC CTATTGGTTA 5440 5441 AAAAATGAGC TGATTTAACA AAAATTTAAC GCGAATTAAT TCTGTGGAAT GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA 5520 5521 GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC 5600 15 5601 AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC 5680 5681 TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAAT llll TTTATTTATG CAGAGGCCGA GGCCGCCTCT 5760 5761 GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG GAGCTTGTAT 5840 5841 ATCCATTTTC GGATCTGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC 5920 5921 CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG 6000 20 6001 CTGTCAGCGC AGGGGCGCCC GGTTClllll GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC 6080 6081 GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC 6160 6161 TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT 6240
'JI 6241 GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG 6320
4~ 6321 TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG 6400 25 6401 CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG 6480 6481 GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC 6560 6561 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC 6640 6641 AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC 6720 6721 CCAACCTGCC ATCACGAGAT TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT CCGGGACGCC 6800 30 6801 GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT TCTTCGCCCA CCCCAACTTG TTTATTGCAG CTTATAATGG 6880 6881 TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCAIITI III CACTGCATTC TAGTTGTGGT TTGTCCAAAC 6960 6961 TCATCAATGT ATCTTATCAT GTCTGTATAC CGTCGACCTC TAGCTAGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT 7040 7041 GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG GTGCCTAATG 7120 7121 AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT 7200 35 7201 GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG 7280 7281 GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG 7360 7361 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGI I I I IC CATAGGCTCC 7440 7441 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG 7520 7521 TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC 7600 40 7601 GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG 7680 7681 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC 7760 7761 TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 7840 7841 GTGGCCTAAC TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG 7920 7921 TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGG I l"T I I I I GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 8000
8001 AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT 8080 8081 GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT 8160 8161 ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 8240 8241 ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC 8320 8321 GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG 8400 8401 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC 8480 8481 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG 8560 8561 ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA 8640 8641 AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT 8720 10 8721 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT 8800 8801 ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA 8880 8881 GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC 8960 8961 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT 9040 9041 CATACTCTTC Cl IT I ICAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT 9120 15 9121 AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTC 9179 10 I 20 I 30 I 40 I 50 I 60 I 70 80 pcDNA3-Sl comprises the first domain of the S (spike) protein (SEQ ID NO:26): Vector pcDNA3.1(+) (UPPERCASE nt's) SI: lower case/bold/underscored
'Jl Ul 20 10 I 20 I 30 I 40 I 50 60 I 70 I 80 I 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 25 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720 30 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTGGTA CCGAGCTCGG ATCCatqttt attttcttat tatttcttac 960 961 tcteaetaqt ggtagtgace ttqaccggtq eaecactttt qatqatqtte aaqcteetaa ttacaetcaa catacttcat 1040 1041 ctatqaqqqq qqtttactat cctqatqaaa tttttaqatc aqacactctt tatttaacte aqgatttatt tcttccattt 1120 35 1121 tattetaatq ttaeaqqqtt teatactatt aatcatacqt ttqqeaacec tqteatacct tttaaqqatg qtatttattt 1200 1201 tqctqceaea qaqaaateaa atqttqtccq tqqttgqqtt tttqqttcta ccatqaaeaa caaqtcacaq tcqqtqatta 1280 1281 ttattaacaa ttctactaat qttqttatac qaqcatqtaa ctttqaattq tgtqacaacc ctttctttqc tqtttctaaa 1360 1361 cccatqqqta cacaqacaca tactatqata ttcqataatq catttaattq cactttcgag tacatatctq atqccttttc 1440 1441 qcttqatgtt teagaaaaqt caqqtaattt taaacactta cqaqagtttq tqtttaaaaa taaaqatqqq tttctctatq 1520
1521 tttataaqqq ctatcaacct ataqatqtaq ttcqtqatct accttctqqt tttaacactt tqaaacctat ttttaaqttq 1600
1601 cctcttqqta ttaacattac aaattttaqa qccattctta cagccttttc acctqctcaa qacatttqgq qcacgtcaqc 1680
1681 tqcaqcctat tttqttqqct atttaaaqcc aactacattt atqctcaaqt atgatqaaaa tqqtacaatc acaqatqctq 1760
1761 ttqattqttc tcaaaatcca cttqctqaac tcaaatgctc tgttaagaqc tttqaqattg acaaaqqaat ttaccagacc 1840
1841 tctaatttca qqqttqttcc ctcaqqaqat gttqtqaqat tccctaatat tacaaacttq tqtccttttq aqaggtttt 1920
1921 taatqctact aaattccctt ctqtctatqc atqqqaqaqa aaaaaaattt ctaattqtqt tqctqattac tctgtgctct 2000
2001 acaactcaac atttttttca acctttaaqt qctatggcgt ttctqccact aagttgaatq atctttqctt ctccaatqtc 2080
2081 tatqcaqatt ettttqtaqt caaqggagat qatqtaagac aaataqcqcc agqacaaact ggtqttattg ctqattataa 2160
2161 ttataaattq ccaqatqatt teatgqqttq tqtccttqct tggaatacta qqaacattqa tqctacttca actqgtaatt 2240
2241 ataattataa atataqqtat cttaqacatq qcaaqcttaq qccctttqaq agaqacatat ctaat tqcc tttctcccct 2320
2321 gatqqcaaac cttqcacccc acctgctctt aattgttatt ggccattaaa tgattatqqt ttttacacca ctactqqcat 2400
2401 tqqctaccaa ccttacaqag ttqtagtact ttcttttqaa cttttaaatq caccqqccac qqtttqtqqa ccaaaattat 2480
2481 ccactqacct tattaaqaac caqtgtqtca attttaattt taatqqactc actqqtactq qtqtqttaac tccttcttca 2560
2561 aaqaqatttc aaccatttca acaatttqqc cqtqatqttt ctqatttcac tqattcc tt cqagatccta aaacatctqa 2640
2641 aatatta ac atttcacctt qctcttttqq qqqtqtaaqt qtaattacac ctgqaacaaa t cttcatct aaqttqctq 2720
2721 ttctatatca aqatqttaac tqcactqatq tttctacagc aattcatqca qatcaactca caccaqcttq qcqcatatat 2800
2801 tctactqqaa acaatqtatt ccaqactcaa qcaqqctqtc ttataqqaqc tqa catqtc qacacttctt atqa tqcqa 2880
2881 cattcctatt qqaqctqqca tttqtqctaq ttaccataca qtttctttat tacqtaqtac tagccaaaaa tctattqtqq 2960 2961 cttatactat qtcttaaGAA TTCTGCAGAT ATCCAGCACA GTGGCGGCCG CTCGAGTCTA GAGGGCCCGT TTAAACCCGC 3040
3041 TGATCAGCCT CGACTGTGCC TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG 3120 3121 TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG 3200 3201 GTGGGGTGGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT 3280 3281 TCTGAGGCGG AAAGAACCAG CTGGGGCTCT AGGGGGTATC CCCACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT 3360 3361 GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG 3440 3441 CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC 3520 3521 GACCCCAAAA AACTTGATTA GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG G I I I I I CGCC CTTTGACGTT 3600 3601 GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT TCTTTTGATT 3680 3681 TATAAGGGAT TTTGCCGATT TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT 3760 3761 GGAATGTGTG TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG 3840 3841 TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC 3920 3921 CATAGTCCCG CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA 4000 4001 I I I I I I I I AT TTATGCAGAG GCCGAGGCCG CCTCTGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC 4080 4081 CTAGGCTTTT GCAAAAAGCT CCCGGGAGCT TGTATATCCA TTTTCGGATC TGATCAAGAG ACAGGATGAG GATCGTTTCG 4160 4161 CATGATTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC TGGGCACAAC 4240 4241 AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA GACCGACCTG 4320 4321 TCCGGTGCCC TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT 4400 4401 GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG TCATCTCACC 4480 4481 TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC 4560 4561 GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA 4640 4641 AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC GCATGCCCGA CGGCGAGGAT CTCGTCGTGA 4720 4721 CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT 4800 4801 GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT 4880 4881 CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG 4960 4961 GACTCTGGGG TTCGAAATGA CCGACCAAGC GACGCCCAAC CTGCCATCAC GAGATTTCGA TTCCACCGCC GCCTTCTATG 5040
5041 AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC 5120 5121 GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT 5200 5201 TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT ATCATGTCTG TATACCGTCG ACCTCTAGCT 5280 5281 AGAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC 5360 5361 GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT 5440 5441 CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT 5520 5521 CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA 5600 5601 ATACGGTTAT CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA 5680 5681 AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT 5760 10 5761 GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG 5840 5841 CCGCTTACCG GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG 5920 5921 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC TTATCCGGTA 6000 6001 ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG 6080 6081 AGGTATGTAG GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGAACAGTAT TTGGTATCTG 6160 15 6161 CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT GGTAGCGGTT 6240 6241 I ITT IGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC 6320 6321 GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA 6400 6401 TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 6480 6481 CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG 6560 20 6561 GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA 6640 6641 GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG 6720 6721 CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC ACGCTCGTCG 6800
Ul 6801 TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT 6880 -4 6881 TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA 6960 25 6961 ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT 7040 7041 ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT 7120 7121 CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG 7200 7201 CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA 7280 7281 AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA 7360 30 7361 TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 7440 7441 TGCCACCTGA CGTC 7454 10 I 20 I 30 40 50 I 60 I 70 I 80 pcDNA3-CRT/Sl construct comprising the human CRT sequence and SI domain of the SARS-CoV S protein: (SEQ TD NO:27) pcDNA3.1(+) vector (from Invitrogen) - sequence both 5' and 3' of the CRT and SI sequences: UPPERCASE nt's 35 CRT sequence : lower case/italic SI sequence — lower case, bold/underscored 10 20 30 40 I - 50 I 60 I 70 80 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160
161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 10 881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGatgctgc tccctgtgcc gctgctgctc ggcctgctcg gcctggccgc 960 961 cgccgagccc gtcgtctact tcaaggagca gtttctggac ggagatgggt ggaccgagcg ctggatcgaa tccaaacaca 1040 1041 agtccgattt tggcaaattc gtcctcagtt cgggcaagtt ctacggcgat caggagaaag ataaagggct gcagaccagc 1120 1121 caggacgccc gcttctacgc cctgtcggcc cgattcgagc cgttcagcaa caagggccag ccactggtgg tgcagttcac 1200 1201 cgtgaaacac gagcagaaca ttgactgcgg gggcggctac gtgaagctgt ttccggccgg cctggaccag aaggacatgc 1280 15 1281 acggggactc tgagtacaac atcatgtttg gtcctgacat ctgtggcccc ggcaccaaga aggttcacgt catcttcaac 1360 1361 tacaagggca agaacgtgct gatcaacaag gacatccgtt gcaaggacga cgagttcaca cacctgtaca cgctgatcgt 1440 1441 gcggccggac aacacgtatg aggtgaagat tgacaacagc caggtggagt cgggctccct ggaggatgac tgggacttcc 1520 1521 taccccccaa gaagataaag gacccagatg cctcgaagcc tgaagactgg gacgagcggg ccaagatcga cgaccccacg 1600 1601 gactccaagc ccgaggactg ggacaagccc gagcacatcc ccgacccgga cgcgaagaag cccgaagact gggacgaaga 1680 20 1681 aatggacgga gagtgggagc cgccggtgat tcagaacccc gagtacaagg gtgagtggaa gccgcggcag atcgacaacc 1760 1761 ccgattacaa aggcacctgg atccaccccg aaatcgacaa ccccgagtac tcgcccgacg ctaacatcta tgcctacgac 1840 1841 agctttgccg tgctgggctt ggacctctgg caggtcaagt cgggcaccat cttcgacaac ttcctcatca ccaacgatga 1920
Ul 1921 ggcgtacgca gaggagtttg gcaacgagac gtggggcgtc accaagacgg ccgagaagca gatgaaagac aagcaggacg 2000 ce 2001 aggagcagcg gctgaaggag gaggaggagg agaagaagcg gaaggaggag gaggaggccg aggaggacga ggaggacaag 2080 25 2081 gacgacaagg aggacgagga tgaggacgag gaggacaagg acgaggagga ggaggaggcg gccgccggcc aggccaagga 2160 2161 cgagctgAGA TCCatqttta ttttettatt atttcttaet ctcactaqtq qtaqtqacct tqaccqqtqc accacttttq 2240 2241 atqatqttca a ctectaat tacactcaae atactteatc tatqaqqqqq qtttactate ctqatqaaat ttttaqatea 2320 2321 qacactcttt atttaactca ggatttattt cttccatttt attetaatgt taeagqgttt catactatta atcatacgtt 2400 2401 tgqcaaccct qtcatacctt ttaagqatqg tatttatttt qctqccacaq agaaatcaaa tqttqtccqt qqttqqqttt 2480 30 2481 ttgqttctac catqaacaac aaqtcacaqt cq tqattat tattaacaat tctactaatg ttqttatacg aqcatgtaac 2560 2561 tttgaattqt qtqacaaccc tttctttqct qtttctaaac ccatqqqtac acaqacacat actatqatat tcqataatqc 2640 2641 atttaattqc actttcqaqt acatatctqa tqccttttcq cttqatgttt caqaaaa tc aqqtaatttt aaacacttac 2720 2721 qaqaqtttgt qtttaaaaat aaaqatqqqt ttctctatqt ttataaqqqc tatcaaccta taqatqtaqt tcqtqatcta 2800 2801 ccttctqqtt ttaacacttt qaaacctatt tttaaqttqc ctcttqqtat taacattaca aattttaqaq ccattcttac 2880 35 2881 aqccttttca cctqctcaaq acatttq qq cacqtcagct gcagcctatt ttqttqqcta tttaaaqcca actacattta 2960 2961 tqctcaaqta tqatqaaaat qqtacaatca cagatgctgt t attqttct caaaatccac ttqct aact caaatqctct 3040 3041 gttaagaqct ttgaqattqa caaaq aatt taccaqacct ctaatttcaq qgttqttccc tcaqqaqatq ttqtqaqatt 3120 3121 ccctaatatt acaaacttqt qtccttttqq aqaqqttttt aatqctacta aattcccttc tqtctat ca tqqqaqaqaa 3200 3201 aaaaaatttc taattqtqtt qctqattact ctqt ctcta caactcaaca tttttttcaa cctttaaqt ctatq cqtt 3280 40 3281 tctqccacta aqttqaatqa tctttqcttc tccaatqtct atqca attc ttttqtaqtc aaqq aqatq atqtaaqaca 3360 3361 aataqcqcca qqacaaactg gtqttattqc tqattataat tataaatt c caqat attt catqq ttgt qtccttgctt 3440 3441 qqaatactaq qaacattqat qctacttcaa ctqqtaatta taattataaa tatagqtatc ttaqacatqg caaqcttaqq 3520 3521 ccctttqa a qaqacatatc taatqtqcct ttctcccctq atqqcaaacc ttqcacccca cctqctctta attqttattq 3600 3601 qccattaaat qattatqqtt tttacaccac tactqqcatt qqctaccaac cttacaqa t tqtaqtactt tcttttqaac 3680
3681 ttttaaatqc aecqqccacq qtttqtggac caaaattatc caetgacctt attaagaacc aqtgtqtcaa ttttaatttt 3760 3761 aatqqaetca etqgtaetqq tqtqttaact ecttettcaa aqaqatttca aecatttcaa eaatttqqec gtqatgtttc 3840 3841 tqatttcact qatteeqttc qaqatcetaa aaeatctqaa atattagaca ttteaeettg ctettttqqq qqtqtaaqtq 3920 3921 taattacacc tqqaacaaat qcttcatctq aaqttgctqt tctatatcaa qatqttaact qcactqatgt ttctacaqca 4000 4001 attcatqcaq atcaactcac accagcttqq cqcatatatt ctactqgaaa caatqtattc cagactcaaq caqqctqtct 4080 4081 tataqqaqct qaqcatqtcq acacttctta tqaqtgcgac attcctatt qa ctqqcat ttgt ctaqt taccatacaq 4160 4161 tttctttatt acqtaqtact aqccaaaaat ctattgtqqc ttatactatg tcttaaGAAT TCTGCAGATA TCCAGCACAG 4240 4241 TGGCGGCCGC TCGAGTCTAG AGGGCCCGTT TAAACCCGCT GATCAGCCTC GACTGTGCCT TCTAGTTGCC AGCCATCTGT 4320 4321 TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG 4400 10 4401 CATCGCATTG TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA TTGGGAAGAC 4480 4481 AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCTT CTGAGGCGGA AAGAACCAGC TGGGGCTCTA GGGGGTATCC 4560 4561 CCACGCGCCC TGTAGCGGCG CATTAAGCGC GGCGGGTGTG GTGGTTACGC GCAGCGTGAC CGCTACACTT GCCAGCGCCC 4640 4641 TAGCGCCCGC TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC CACGTTCGCC GGCTTTCCCC GTCAAGCTCT AAATCGGGGG 4720 4721 CTCCCTTTAG GGTTCCGATT TAGTGCTTTA CGGCACCTCG ACCCCAAAAA ACTTGATTAG GGTGATGGTT CACGTAGTGG 4800 15 4801 GCCATCGCCC TGATAGACGG lllll CGCCC TTTGACGTTG GAGTCCACGT TCTTTAATAG TGGACTCTTG TTCCAAACTG 4880 4881 GAACAACACT CAACCCTATC TCGGTCTATT CTTTTGATTT ATAAGGGATT TTGCCGATTT CGGCCTATTG GTTAAAAAAT 4960 4961 GAGCTGATTT AACAAAAATT TAACGCGAAT TAATTCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC 5040 5041 CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG 5120 5121 CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC 5200 20 5201 CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT 1 I I I I I IATT TATGCAGAGG CCGAGGCCGC CTCTGCCTCT 5280 5281 GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTC CCGGGAGCTT GTATATCCAT 5360 5361 TTTCGGATCT GATCAAGAGA CAGGATGAGG ATCGTTTCGC" ATGATTGAAC AAGATGGATT GCACGCAGGT TCTCCGGCCG 5440
'Jl 5441 CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA 5520
VO 5521 GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG CAGCGCGGCT 5600 25 5601 ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC TGGCTGCTAT 5680 5681 TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG 5760 5761 CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG 5840 5841 GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG TTCGCCAGGC 5920 5921 TCAAGGCGCG CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT 6000 30 6001 GGCCGCTTTT CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA 6080 6081 TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT TCGCAGCGCA 6160 6161 TCGCCTTCTA TCGCC1 ΓCTT GACGAGTTCT TCTGAGCGGG ACTCTGGGGT TCGAAATGAC CGACCAAGCG ACGCCCAACC 6240 6241 TGCCATCACG AGA1 TCGAT TCCACCGCCG CCTTCTATGA AAGGTTGGGC TTCGGAATCG TTTTCCGGGA CGCCGGCTGG 6320 6321 ATGATCCTCC AGCGCGGGGA TCTCATGCTG GAGTTCTTCG CCCACCCCAA CTTGTTTATT GCAGCTTATA ATGGTTACAA 6400 35 6401 ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC AAACTCATCA 6480 6481 ATGTATCTTA TCATGTCTGT ATACCGTCGA CCTCTAGCTA GAGCTTGGCG TAATCATGGT CATAGCTGTT TCCTGTGTGA 6560 6561 AATTGTTATC CGCTCACAAT TCCACACAAC ATACGAGCCG GAAGCATAAA GTGTAAAGCC TGGGGTGCCT AATGAGTGAG 6640 6641 CTAACTCACA TTAATTGCGT TGCGCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT TAATGAATCG 6720 6721 GCCAACGCGC GGGGAGAGGC GGTTTGCGTA TTGGGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG CTCGGTCGTT 6800 40 6801 CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA 6880 6881 CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC 6960 6961 CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC 7040 7041 CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG 7120 7121 CGTGGCGCTT TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG 7200
7201 AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA CGACTTATCG 7280 7281 CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC 7360 7361 TAACTACGGC TACACTAGAA GAACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA 7440 7441 GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTTT TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA 7520 7521 TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT 7600 7601 GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT 7680 7681 AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT 7760 7761 GCCTGACTCC CCGTCGTGTA GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA 7840 7841 CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT CCTGCAACTT 7920 7921 TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT 8000 8001 GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG 8080 8081 GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA AGTAAGTTGG 8160 8161 CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG 8240 8241 ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA 8320 8321 TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC TCAAGGATCT 8400 8401 TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT 8480 8481 TCTGGGTGAG CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT 8560 8561 CTTCC lllll CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA 8640 8641 ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC GTC 8693 10 20 I 30 I 40 I 50 I 60 I 70 I 80 DNA3-Si: (SEQ ID NO:28) Vector pcDNA3.1(+) (UPPER CASE) Si polypeptide coding sequence: lower case/bold/underscored 10 I 20 I 30 I 40 I 50 60 I 70 I 80 I 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTGGTA CCGAGCTCGG ATCCatqggt tgtgtccttg cttqqaatac 960 961 taqqaacatt qatqetactt eaactqqtaa ttataattat aaatataqqt atcttaqaca tqgcaaqctt aqqccctttq 1040
1041 aqaqaqacat atctaatqtq cctttctccc ctqatqgcaa accttgcacc ccacctgctc ttaattgtta ttqqccatta 1120 1121 aatqattatq qtttttacac cactactqqc attqqctacc aaccttacaq aqttqtaqta ctttcttttq aacttttaaa 1200
1201 tgcaccgqcc acgqtttqtg gaceaaaatt atccactqac cttattaaqa accaqtqtqt caattttaat tttaatqqac 1280
1281 tcactqqtac tqqtqtqtta actccttctt caaaqaqatt tcaaccattt caacaatttg gccqtqatgt ttctgatttc 1360
1361 actqattceq ttcqaqatcc taaaacatct qaaatattaq acatttcacc ttqctctttt qqgqqtqtaa qtgtaattac 1440
1441 acctqqaaca aatqcttcat ctqaaqttqc tqttctatat caagatqtta actgcactga tgtttctaca qcaattcatq 1520
1521 caqatcaact cacaccaqct tqqcqcatat attctactqq aaacaatqta ttccaqactc aaqcaqqctq tcttataq a 1600
1601 qctqaqcatq tcqacacttc ttatqaqtqc qacattccta ttqqaqctqq catttqtqct a ttaccata caqtttcttt 1680
1681 attacqtaqt actaqccaaa aatctattqt qqcttatact atqtctttaq qtqctqataq ttcaattqct tactctaata 1760
1761 acaccattqc tatacctact aacttttcaa ttaqcattac tacaqaa ta atqcctqttt ctatqqctaa aacctccqta 1840
1841 qattqtaata tqtacatct cqqaqattct actqaat tq ctaatttqct tctccaatat qqtaqctttt qcacacaact 1920
1921 aaatcqtqca ctctcaqqta ttqctqctqa acaqqatcqc aacacacqtq aagtqttcqc tcaaqtcaaa caaatqtaca 2000
2001 aaaccccaac tttqaaatat tttqgtqqtt ttaatttttc acaaatatta cctqaccctc taaaqccaac taaqaggtct 2080
2081 tttattgagg acttqctctt taataaqqt acactcgctq atqctqqctt catqtaaGAA TTCTGCAGAT ATCCAGCACA 2160
2161 GTGGCGGCCG CTCGAGTCTA GAGGGCCCGT TTAAACCCGC TGATCAGCCT CGACTGTGCC TTCTAGTTGC CAGCCATCTG 2240 2241 TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT 2320 2321 GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA 2400 2401 CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG AAAGAACCAG CTGGGGCTCT AGGGGGTATC 2480 2481 CCCACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC 2560 2561 CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG 2640 2641 GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT TCACGTAGTG 2720 2721 GGCCATCGCC CTGATAGACG Gi i i ΓΓCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT 2800 2801 GGAACAACAC TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGCCTATT GGTTAAAAAA 2880 2881 TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT CCCCAGGCTC 2960 2961 CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG 3040 3041 GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC CGCCCATCCC GCCCCTAACT 3120 3121 CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA I I I I I I I IAT TTATGCAGAG GCCGAGGCCG CCTCTGCCTC 3200 3201 TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT CCCGGGAGCT TGTATATCCA 3280 3281 TTTTCGGATC TGATCAAGAG ACAGGATGAG GATCGTTTCG CATGATTGAA CAAGATGGAT TGCACGCAGG TTCTCCGGCC 3360 3361 GCTTGGGTGG AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC 3440 3441 AGCGCAGGGG CGCCCGGTTC lllll GTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT GCAGGACGAG GCAGCGCGGC 3520 3521 TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA CTGGCTGCTA 3600 3601 TTGGGCGAAG TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT 3680 3681 GCGGCGGCTG CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC 3760 3761 GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT GTTCGCCAGG 3840 3841 CTCAAGGCGC GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA 3920 3921 TGGCCGCTTT TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG 4000 4001 ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA TTCGCAGCGC 4080 4081 ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTCTGGGG TTCGAAATGA CCGACCAAGC GACGCCCAAC 4160 4161 CTGCCATCAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG 4240 4241 GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA 4320 4321 AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC 4400 4401 AATGTATCTT ATCATGTCTG TATACCGTCG ACCTCTAGCT AGAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG 4480 4481 AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA 4560 4561 GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC 4640 4641 GGCCAACGCG CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT 4720
4721 TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC GCAGGAAAGA 4800 4801 ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC 4880 4881 CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC 4960 4961 CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC CCTTCGGGAA 5040 5041 GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC 5120 5121 GAACCCCCCG TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC 5200 5201 GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC 5280 5281 CTAACTACGG CTACACTAGA AGAACAGTAT TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT 5360 5361 AGCTCTTGAT CCGGCAAACA AACCACCGCT GGTAGCGGTT IIII ΓGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG 5440 5441 ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 5520 5521 TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG 5600 5601 TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT 5680 5681 TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG 5760 5761 ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT 5840 5841 TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT 5920 5921 TGTTGCCATT GCTACAGGCA TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 6000 6001 GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG 6080 6081 GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT 6160 6161 GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG 6240 6241 ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC 6320 6321 TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT 6400 6401 TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC 6480 6481 TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA 6560 6561 AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTC 6614 I 10 I 20 I 30 I 40 I 50 I 60 I 70 I 80
PCDNA3-S2 (SEO TD NO:29): vector pcDNA3.1(+) sequence (UPPER CASE) S2 - C-terminal domain of SARS-CoV S protein (lower case/bold/underscored) I 10 [ 20 I 30 I 40 I 50 I 60 I 70 I 80 I 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720
721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTGGTA CCGAGCTCGG ATCCat atq ttaqqtqctq ataqttcaat 960 961 tqcttactct aataacacca ttgctatacc tactaacttt tcaattaqca ttactaeaqa aqtaatqcct gtttctatqg 1040
1041 ctaaaacetc cqtaqattqt aatatqtaca tctqcqqaqa ttctactqaa tgtqetaatt tqettctcca atatgqtaqe 1120
1121 ttttqcacac aaetaaatcq tqcactctca qqtattqctq ctqaacaqqa tcqcaacaca eqtqaaqtgt tcqctcaaqt 1200
1201 caaacaaatg tacaaaacec caactttqaa atattttqqt qqttttaatt tttcacaaat attacetqac cctctaaaqc 1280
1281 caactaaqaq qtettttatt gaqqacttqc tctttaataa qqtqacactc qctgatqctq qcttcatqaa qcaatatqqc 1360
1361 qaatqcctaq qtqatattaa tqctaqaqat ctcatttqtq cgcaqaaqtt caatqqactt aeagtqttqc caectctqct 1440
1441 cactqatqat atqattqctq cctacactqc tqctctaqtt aqtqqtactq ccactqctqq atqqacattt qqtgct qcq 1520
1521 ctgctcttca aatacctttt qctatqcaaa tqqcatataq qttcaatqqc attgqaqtta cccaaaat t tctctatqaq 1600
1601 aaccaaaaac aaatcgccaa ccaatttaac aaggcqatta tcaaattca agaatcactt acaacaacat caactgcatt 1680
1681 gqqcaaqctq caagacqttg ttaaccaqaa tqctcaaqca ttaaacacac ttqttaaaca acttaqctct aattttqqtq 1760
1761 caatttcaaq tqtgctaaat qatatccttt cqcqacttqa taaaqtcqaq qcggaqqtac aaattqacaq qttaattaca 1840
1841 gqcaqacttc aaagccttca aacctatqta acacaacaac taatcaq qo tqctqaaatc aqqqcttctq ctaatctt c 1920
1921 tqctactaaa atqtctqaqt gtqttcttqq acaatcaaaa aqaqtt act tttgtgqaaa qqgctaccac cttat tcct 2000
2001 tcccacaagc aqccccqcat ggtgttqtct tcctacatqt cacqtatqtq ccatcccagg agaqqaactt caccacaqcq 2080
2081 ccagcaattt qtcatqaaqq caaaqcatac ttccctcqtq aaqqtqtttt tqtqtttaat qqcacttctt qqtttattac 2160
2161 acaqaqqaac ttcttttctc cacaaataat tactacaqac aatacatttq tctcaqqaaa ttqt atqtc qttatt qca 2240
2241 tcattaacaa cacaqtttat qatcctctqc aacctqaqct tqactcattc aaaqaa aqc tqgacaa ta cttcaaaaat 2320
2321 catacatcac caqatqttqa tcttqqcqac atttcaqqca ttaacqcttc tgtcqtcaac attcaaaaaq aaattqaccq 2400
2401 cctcaatqaq qtcgctaaaa atttaaatga atcactcatt gaccttcaag aattgggaaa atatqaqcaa tatattaaat 2480
2481 qqccttqqta tqtttqqctc q cttcattq ctqqactaat tqccatcqtc atqqttacaa tcttqctttq ttqcatqact 2560
2561 aqttqttqca qttqcctcaa qqqtqcatqc tcttqtqqtt cttqctqcaa qtttqatqaq qatqactctg agccaqttct 2640
2641 caaqqqtqtc aaattacatt acacataaGA ATTCTGCAGA TATCCAGCAC AGTGGCGGCC GCTCGAGTCT AGAGGGCCCG 2720
2721 TAAACCCG CTGATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT GCCTTCCTTG 2800
2801 ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT TGCATCGCAT TGTCTGAGTA GGTGTCATTC 2880
2881 TATTCTGGGG GGTGGGGTGG GGCAGGACAG CAAGGGGGAG GATTGGGAAG ACAATAGCAG GCATGCTGGG GATGCGGTGG 2960
2961 GCTCTATGGC TTCTGAGGCG GAAAGAACCA GCTGGGGCTC TAGGGGGTAT CCCCACGCGC CCTGTAGCGG CGCATTAAGC 3040
3041 GCGGCGGGTG TGGTGGTTAC GCGCAGCGTG ACCGCTACAC TTGCCAGCGC CCTAGCGCCC GCTCCTTTCG CTTTCTTCCC 3120
3121 TTCCTTTCTC GCCACGTTCG CCGGCTTTCC CCGTCAAGCT CTAAATCGGG GGCTCCCTTT AGGGTTCCGA TTTAGTGCTT 3200
3201 TACGGCACCT CGACCCCAAA AAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC CCTGATAGAC GG I I I I I CGC 3280
3281 CCTTTGACGT TGGAGTCCAC GTTCTTTAAT AGTGGACTCT TGTTCCAAAC TGGAACAACA CTCAACCCTA TCTCGGTCTA 3360
3361 TTCTTTTGAT TTATAAGGGA TTTTGCCGAT TTCGGCCTAT TGGTTAAAAA ATGAGCTGAT TTAACAAAAA TTTAACGCGA 3440
3441 ATTAATTCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC 3520
3521 ATCTCAATTA GTCAGCAACC AGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT 3600
3601 TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TCCGCCCATT CTCCGCCCCA 3680
3681 TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCTGCCT CTGAGCTATT CCAGAAGTAG TGAGGAGGCT 3760
3761 I I I ITGGAGG CCTAGGCTTT TGCAAAAAGC TCCCGGGAGC TTGTATATCC ATTTTCGGAT CTGATCAAGA GACAGGATGA 3840
3841 GGATCGTTTC GCATGATTGA ACAAGATGGA TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GAGAGGCTAT TCGGCTATGA 3920
3921 CTGGGCACAA CAGACAATCG GCTGCTCTGA TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG GCGCCCGGTT Cl I ITTGTCA 4000
4001 AGACCGACCT GTCCGGTGCC CTGAATGAAC TGCAGGACGA GGCAGCGCGG CTATCGTGGC TGGCCACGAC GGGCGTTCCT 4080
4081 TGCGCAGCTG TGCTCGACGT TGTCACTGAA GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GTGCCGGGGC AGGATCTCCT 4160
4161 GTCATCTCAC CTTGCTCCTG CCGAGAAAGT ATCCATCATG GCTGATGCAA TGCGGCGGCT GCATACGCTT GATCCGGCTA 4240
4241 CCTGCCCATT CGACCACCAA GCGAAACATC GCATCGAGCG AGCACGTACT CGGATGGAAG CCGGTCTTGT CGATCAGGAT 4320 4321 GATCTGGACG AAGAGCATCA GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG CGCATGCCCG ACGGCGAGGA 4400 4401 TCTCGTCGTG ACCCATGGCG ATGCCTGCTT GCCGAATATC ATGGTGGAAA ATGGCCGCTT TTCTGGATTC ATCGACTGTG 4480 4481 GCCGGCTGGG TGTGGCGGAC CGCTATCAGG ACATAGCGTT GGCTACCCGT GATATTGCTG AAGAGCTTGG CGGCGAATGG 4560 4561 GCTGACCGCT TCCTCGTGCT TTACGGTATC GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TATCGCCTTC TTGACGAGTT 4640 4641 CTTCTGAGCG GGACTCTGGG GTTCGAAATG ACCGACCAAG CGACGCCCAA CCTGCCATCA CGAGATTTCG ATTCCACCGC 4720 4721 CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC 4800 4801 TGGAGTTCTT CGCCCACCCC AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA 4880 4881 AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TATCATGTCT GTATACCGTC 4960 4961 GACCTCTAGC TAGAGCTTGG CGTAATCATG GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA 5040 5041 ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA 5120 5121 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG GCGGTTTGCG 5200 5201 TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT 5280 5281 CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC 5360 5361 AGGAACCGTA AAAAGGCCGC GTTGCTGGCG lllll CCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC 5440 5441 AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG 5520 5521 TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT 5600 5601 AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC 5680 5681 CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA 5760 5761 TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGAACAGTA 5840 5841 TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC 5920 5921 TGGTAGCGGT I I II I I GTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA 6000 6001 CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG 6080 6081 ATCCTTTTAA ATTAAAAATG AAG llllAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT 6160 6161 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA 6240 6241 CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA 6320 6321 GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG 6400 6401 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT 6480 6481 CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC 6560 6561 AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC 6640 6641 AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT 6720 6721 GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA 6800 6801 AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA 6880 6881 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA 6960 6961 ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT 7040 7041 TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT 7120 7121 TCCCCGAAAA GTGCCACCTG ACGTC 7145 10 I 20 I 30 I 40 I 50 I 60 I 70 I 80
pcDNA3-CRT/M (SEQ ID NO:30)
Vector sequence pcDNA3.1 (-)mycHisA (UPPERCASE) CRT Sequence (lower case/italic) Sequence of M protein (lower case/bold/underscored)
I 10 I 20 I 30 I 40 I 50 I 60 I 70 I 80 1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT 80 81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA 160 161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT 240 241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA 320 321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT 400 401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT 480 481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA 560 561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA 640 641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC 720 721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG 800 801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 880 881 GGAGACCCAA GCTGGCTAGC GTTTAAACGG GCCCTCTAGA atgctgctcc ctgtgccgct gctgctcggc ctgctcggcc 960 961 tggccgccgc cgagcccgtc gtctacttca aggagcagtt tctggacgga gatgggtgga ccgagcgctg gatcgaatcc 1040
1041 aaacacaagt ccgattttgg caaattcgtc ctcagttcgg gcaagttcta cggcgatcag gagaaagata aagggctgca 1120
1121 gaccagccag gacgcccgct tctacgccct gtcggcccga ttcgagccgt tcagcaacaa gggccagcca ctggtggtgc 1200
1201 agttcaccgt gaaacacgag cagaacattg actgcggggg cggctacgtg aagctgtttc cggccggcct ggaccagaag 1280
1281 gacatgcacg gggactctga gtacaacatc atgtttggtc ctgacatctg tggccccggc accaagaagg ttcacgtcat 1360
1361 cttcaactac aagggcaaga acgtgctgat caacaaggac atccgttgca aggacgacga gttcacacac ctgtacacgc 1440
1441 tgatcgtgcg gccggacaac acgtatgagg tgaagattga caacagccag gtggagtcgg gctccctgga ggatgactgg 1520
1521 gacttcctac cccccaagaa gataaaggac ccagatgcct cgaagcctga agactgggac gagcgggcca agatcgacga 1600
1601 ccccacggac tccaagcccg aggactggga caagcccgag cacatccccg acccggacgc gaagaagccc gaagactggg 1680
1681 acgaagaaat ggacggagag tgggagccgc cggtgattca gaaccccgag tacaagggtg agtggaagcc gcggcagatc 1760
1761 gacaaccccg attacaaagg cacctggatc caccccgaaa t cga caa ccc cgagtactcg cccgacgcta aca t cta tgc 1840
1841 ctacgacagc tttgccgtgc tgggcttgga cctctggcag gtcaagtcgg gcaccatctt cgacaacttc ctcatcacca 1920
1921 acgatgaggc gtacgcagag gagtttggca acgagacgtg gggcgtcacc aagacggccg agaagcagat gaaagacaag 2000
2001 caggacgagg agcagcggct gaaggaggag gaggaggaga agaagcggaa ggaggaggag gaggccgagg aggacgagga 2080
2081 ggacaaggac gacaaggagg acgaggatga ggacgaggag gacaaggacg aggaggagga ggaggcggcc gccggccagg 2160
2161 ccaaggacga gctgtagGAA TTCatqgcaq acaacqqtac tattaecqtt gaggagctta aacaaetcct qqaacaatqq 2240
2241 aacctaqtaa taqqtttcct attcctaqce tggattatqt tactacaatt tgcctattct aatcggaaca qqtttttqta 2320
2321 eataataaaq cttgttttce tetggctctt qtqqccaqta acaettqctt qttttqtqct tqctqctqtc taeaqaatta 2400
2401 attq qtqac tqqcqqqatt qcqatt caa tqqcttgtat tqtaqqcttq atqtqqctta qctacttcqt tqcttccttc 2480
2481 aggctgtttq ctcgtacecg ctcaatgtqg tcattcaace caqaaacaaa cattcttcte aatqtqcctc tccq qqqac 2560
2561 aattgtqacc aqaccqctca t qaaaqtqa acttqtcatt qqtqctgtqa tcattcqtqq tcacttqcqa atqqccqqac 2640
2641 actccctaq qcqctgtqac attaagqacc tqccaaaaqa qatcactqtq qctacatcac qaacqctttc ttattacaaa 2720
2721 ttaggaqcqt cqca cqtqt agqcactqat tcaqqttttq ctgcatacaa ccqctaccgt attqqaaact ataaattaaa 2800
2801 tacaqaccac qcc qtaqca acqacaatat tqctttgcta gtacagGGTA CCAAGCTTGG GCCCGAACAA AAACTCATCT 2880
2881 CAGAAGAGGA TCTGAATAGC GCCGTCGACC ATCATCATCA TCATCATTGA GTTTAAACGG TCTCCAGCTT AAGTTTAAAC 2960
2961 CGCTGATCAG CCTCGACTGT GCCTTCTAGT TGCCAGCCAT CTGTTGTTTG CCCCTCCCCC GTGCCTTCCT TGACCCTGGA 3040 3041 AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA ATTGCATCGC ATTGTCTGAG TAGGTGTCAT TCTATTCTGG 3120 3121 GGGGTGGGGT GGGGCAGGAC AGCAAGGGGG AGGATTGGGA AGACAATAGC AGGCATGCTG GGGATGCGGT GGGCTCTATG 3200 3201 GCTTCTGAGG CGGAAAGAAC CAGCTGGGGC TCTAGGGGGT ATCCCCACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG 3280 3281 TGTGGTGGTT ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC 3360 3361 TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT TTAGGGTTCC GATTTAGTGC TTTACGGCAC 3440 3441 CTCGACCCCA AAAAACTTGA TTAGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG ACGG I I I I I C GCCCTTTGAC 3520 3521 GTTGGAGTCC ACGTTCTTTA ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG 3600 3601 ATTTATAAGG GATTTTGCCG ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA AATTTAACGC GAATTAATTC 3680 3681 TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT 3760 3761 TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC 3840 3841 AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC 3920 3921 TAA I M I N I TATTTATGCA GAGGCCGAGG CCGCCTCTGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA 4000 4001 GGCCTAGGCT TTTGCAAAAA GCTCCCGGGA GCTTGTATAT CCATTTTCGG ATCTGATCAA GAGACAGGAT GAGGATCGTT 4080 4081 TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT GACTGGGCAC 4160 4161 AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTC I I I I I GT CAAGACCGAC 4240 4241 CTGTCCGGTG CCCTGAATGA ACTGCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC 4320 4321 TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG GCAGGATCTC CTGTCATCTC 4400 4401 ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA 4480 4481 TTCGACCACC AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA 4560 4561 CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGCGCATGCC CGACGGCGAG GATCTCGTCG 4640 4641 TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG 4720 4721 GGTGTGGCGG ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG 4800 4801 CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT TCTTGACGAG TTCTTCTGAG 4880 4881 CGGGACTCTG GGGTTCGAAA TGACCGACCA AGCGACGCCC AACCTGCCAT CACGAGATTT CGATTCCACC GCCGCCTTCT 4960 4961 ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC GGGACGCCGG CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC 5040 5041 TTCGCCCACC CCAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC 5120 5121 ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCATGT CTGTATACCG TCGACCTCTA 5200 5201 GCTAGAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA 5280 5281 GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC 5360 5361 TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC 5440 5441 GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG 5520 5521 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 5600 5601 TAAAAAGGCC GCGTTGCTGG CG I I I I I CCA TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA 5680 5681 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 5760 5761 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 5840 5841 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG 5920 5921 GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA 6000 6001 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 6080 6081 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG 6160 6161 GTGG I I I I I I TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG 6240 6241 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 6320 6321 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA 6400 6401 GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 6480
6481 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT 6560 6561 AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC 6640 6641 GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC 6720 6721 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA 6800 6801 AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC 6880 6881 TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 6960 6961 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT 7040 7041 GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA 7120 7121 CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 7200 10 7201 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CC I I I TTCAA TATTATTGAA GCATTTATCA 7280 7281 GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC 7360 7361 GAAAAGTGCC ACCTGACGTC 7380 I 10 I 20 I 30 I 40 50 60 70 80
-4
Figure imgf000069_0001
In the DNA constructs ofthe present invention, the above SARS-CoV proteins may be substituted by homologues or analogues thereof from any viral isolate or strain, or with a sequence that has conservative substitutions such that the protein maintain their immunogenicity and antigenicity when administered in the form of a nucleic acid composition or polypeptide. In view ofthe information provided above and in the examples, it is within the skill ofthe art, without undue experimentation, to combine various SARS-CoN proteins or fragments thereof with a CRT sequence, preferably a human CRT sequence, or a functional variant or fragment thereof that enhances immunogenicity, or the sequence of another endoplasmic reticulum chaperone polypeptide that has similar activity to CRT, to generate a composition that is useful, as, e.g., a chimeric nucleic acid immunogen or vaccine to enhance immunity to a linked antigenic peptide or polypeptide. Table 2 below shows nucleotide base differences among the TW-1, TOR-2, HKU-39849, CUHK-W1 , and the Urbani sequences of SARS-CoN TABLE 1
Figure imgf000070_0001
Indicates a base difference resulting in an amino acid change between TW1 and Urbani.
Techniques for the manipulation of nucleic acids, such as, e.g., generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Nols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Aus bel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Tijssen, ed. Elsevier, N.Y. (1993). Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g. , analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g. fluid or gel precipitin reactions, immunodiffusion, im uno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno- fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography. Amplification of Nucleic Acids Oligonucleotide primers can be used to amplify nucleic acids to generate fusion protein coding sequences used to practice the invention, to monitor levels of vaccine after in vivo administration (e.g., levels of a plasmid or virus), to confirm the presence and phenotype of activated CTLs, and the like. The skilled artisan can select and design suitable oligonucleotide amplification primers using known sequences, e.g., SEQ ID NO:l. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide to Methods and Applications, ed. Innis, Academic Press, N.Y. (1990) and PCR Strategies (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh (1989) Proc. Natl. Acad. Sci. USA 55:1173); and, self-sustained sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:187 A); Qβ replicase amplification (Smith (1997) J Clin. Microbiol. 35:1477-1491; Burg (1996) Mol. Cell. Probes 70:257-271) and other RNA polymerase mediated techniques (NASBA, Cangene, Mississauga, Ontario; Berger (1987) Meth. Enzymol. 152:307-316; U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564). Clonins and construction of expression cassettes Expression cassettes, including plasmids, recombinant viruses (e.g., RNA viruses like the replicons described below) and other vectors encoding the fusion proteins described herein are used to express these polypeptides in vitro and in vivo. Recombinant nucleic acids are expressed by a variety of conventional techniques (Roberts (1987) Nature 328:731; Schneider (1995) Protein Expr. Purif. 6435:10; Sambrook, supra Tijssen, supra; Ausubel, supra). Plasmids, vectors, etc., can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods. The nucleic acids used to practice the invention can be stably or transiently expressed in cells such as episomal expression systems. Selection markers can be incorporated to confer a selectable phenotype on transformed cells. For example, selection markers can code for episomal maintenance and replication such that integration into the host genome is not required. For example, the marker may encode antibiotic resistance, e.g., chloramphenicol, kanamycin, G418, bleomycin, hygromycin) to permit selection of those cells transformed with the desired DNA sequences (Blondelet- Rouault (1997) Gene 190:315-317; Aubrecht (1997) J. Pharmacol. Exp. Ther. 281:992-997). In Vivo Nucleic Acid Administration Preferred methods of administration are exemplified herein and are well-known in the art. In one embodiment, a nucleic acid encoding a CRT-SARS peptide epitope chimeric polypeptide are cloned into expression cassettes such as plasmids or other vectors, viruses that can transfect or infect cells in vitro, ex vivo and/or in vivo. A number of delivery approaches are known, including lipid or liposome based gene delivery (Ma iino (1988) BioTechniques 6:682- 691; U.S. Pat No. 5,279,833), replication-defective retroviral vectors with desired exogenous sequence as part ofthe retroviral genome (Miller (1990) Mol. Cell. Biol. 70:4239; Kolberg (1992) J. NIHRes. 4:A3; Cornetta (1991) Hum. Gene Thar. 2: 215; Zhang (1996) Cancer Metastasis Rev. 75:385-401; Anderson, Science ("1992) 256: 808-813; Nabel (1993) TIBTECH 11: 211-217; Mitani (1993) TIBTECH 11: 162-166; Mulligan (1993) Science 2504:926-932; Dillon (1993) TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460). Expression cassettes can also be derived from viral genomes. Vectors which may be employed include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, examples of which are baculoviridae, parvoviridae, picornaviridae, herpesviridae, poxviridae, adenoviridae, picornaviridae or alphaviridae. Chimeric vectors may also be employed which exploit advantageous merits of each ofthe parent vector properties (Feng (1997) Nature Biotechnology 15 :866-870). Such viral genomes may be modified by recombinant DNA techniques to include the gene of interest and may be engineered to be replication-deficient, conditionally replicating or replication-competent. Vectors can be derived from adenoviral, adeno-associated viral or retroviral genomes. Retroviral vectors can include those based upon murine leukemia virus (MuLN), gibbon ape leukemia virus (GaLN), simian immunodeficiency virus (SIN), human immunodeficiency virus (HIN), and combinations thereof (Buchscher
(1992) J Virol. 55:2731-2739; Johann (1992) J. Virol. 55:1635-1640 (1992); Sommerfelt (1990) Nirol. 176:58-59; Wilson (1989) J. Virol. 63:2374-2378; Miller (1991) J. Virol. 65:2220-2224. Adeno-associated virus (AAV)-based vectors can transduce cells for the in vitro production of nucleic acids and peptides, and be used in in vivo and ex vivo therapy procedures (Okada (1996) Gene Ther. 3:957-964; West (1987) Virology 160:38-47; Carter (1989) U.S. Patent No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) j. Clin. Invest. 94:1351).
In vivo administration using self-replicating: RNA replicons In addition to the above-described expression vectors and recombinant viruses, self- replicating RNA replicons can also be used to infect cells or tissues or whole organisms with a fusion protein-expressing nucleic acids ofthe invention. Thus, the invention also incorporates RNA viruses, including alphavirus genome RNAs such as from Sindbis virus, Semliki Forest virus, Venezuelan equine encephalitis virus, and the like, that have been engineered to allow expression of heterologous RNAs and proteins. High levels of expression of heterologous sequences such as the fusion polypeptides ofthe invention, are achieved when the viral structural genes are replaced by the heterologous coding sequences. These recombinant RNAs are self-replicating ("replicons") and can be introduced into cells as naked RNA or DNA. However, they require trans complementation to be packaged and released from cells as infectious virion particles. The defective helper RNAs contain the exacting sequences required for replication as well as an RNA promoter which drives expression of open reading frames. In cells co-transfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from the replicon RNA allow replication and transcription ofthe defective helper RNA to produce the virion's structural proteins (Bredenbeek
(1993) J. Virol. 67:6439-6446). RNA replicon vaccines may be derived from alphavirus vectors, such as Sindbis virus (family Togaviridae) (Xiong (1989) Science 243:1188-1191), Semliki Forest virus (Ying (1999) Nat. Med. 5:823-827) or Venezuelan equine encephalitis virus (Pushko (1997) Virology 239:389-401) vectors. These vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA, which is then transcribed into RNA replicons in transfected cells or in vivo (Berglund (1998) Nat. Biotechnol. 16:562-565). Self-replicating RNA infects a diverse range of cell types and allows the expression ofthe antigen of interest at high levels (Huang (1996) Curr. Opin. Biotechnol. 7:531-535). Additionally, self-replicating RNA eventually causes lysis of transfected cells because viral replication is toxic to infected host cells (Frolov (1996) J. Virol. 70:1182-1190). These vectors therefore do not raise the concern associated with naked DNA vaccines of integration into the host genome. In one embodiment, the self-replicating RNA replicon comprises a Sindbis virus self-replicating RNA vector SINrep5, as described in detail by Bredenbeek, supra and Herrmann (1998) Biochem. Biophys. Res. Commun. 253:524-531. Polypeptides In other embodiments, the invention is directed to an isolated or recombinant polypeptide comprising at least two domains, wherein the first domain comprises a calreticulin (CRT) polypeptide; and, wherein the second domain comprises an MHC class I-binding peptide epitope of a SARS protein that is antigenic such that an immune response directed against such an epitope leads to any type of protective or prophylactic or therapeutic immunity' against the virus. As noted above, the terms "polypeptide," "protein," and "peptide," referring to polypeptides including the CRT, fragments of CRT that bind peptides, and MHC class I-binding peptide epitopes, SARS polypeptides, such as the S, E, M and N proteins to practice the invention. These proteins are disclosed in more detail, including amino acid sequence and encoding nucleic acid sequences, above. The composition ofthe invention also include "analogues," or "conservative variants" and "mimetics" or "peptidomimetics" with structures and activity that substantially correspond to CRT and SARS protein or epitope(s) thereof. Thus, the terms "conservative variant" or "analogue" or "mimetic" also refer to a polypeptide or peptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity (ability to bind to "antigenic" peptides, to stimulate an immune response). These include conservatively modified variations of an amino acid sequence, i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select conservative substitutions includes (original residue/substitution): Ala/Gly or Ser; Arg/ Lys; Asn/ Gin or His; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; Gly/Ala or Pro; His/Asn or Gin; Ile/Leu or Nal; Leu/Ile or Nal; Lys/Arg or Gin or Glu; Met/Leu or Tyr or He; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/Tyr; Tyr/Trp or Phe; Nal Ile or Leu. An alternative exemplary guideline uses the groups shown in the Table below. For a detailed description of protein chemistry and structure, see Schulz, GE et al, Principles of Protein Structure, Springer-Nerlag, New York, 1978, and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions that may be made in the polypeptides of this invention may be based on analysis ofthe frequencies of amino acid changes between a homologous protein of different species, defined herein as exchanges within one ofthe following five groups:
Figure imgf000075_0001
The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking a side chain and thus imparts flexibility to the chain. Pro, because of its unusual geometry, tightly constrains the chain. Cys can participate in disulfide bond formation, which is important in protein folding. More substantial changes in biochemical, functional (or immunological) properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above five groups. Such changes will differ more significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk ofthe side chain. Examples of such substitutions are (i) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (ii) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g.,, Leu, He, Phe, Nal or Ala; (iii) substitution of a Cys residue for (or by) any other residue; (iv) substitution of a residue having an electropositive side chain, e.g.,, Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g.,, Glu or Asp; or (v) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly. One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, all charged amino acids may be considered conservative substitutions for each other whether they are positive or negative. Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered to yield "conservatively modified variants." The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound that has the necessary structural and/or functional characteristics of a peptide that permits use in the methods ofthe invention, such as mimicking CRT in interaction with peptides and MHC class I- proteins). The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a combination of partly natural amino acids and partly non-natural analogues. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetics' structure and/or activity. As with conservative variants, routine experimentation will determine whether a mimetic is within the scope ofthe invention, that its stereochemical structure and/or function is not substantially altered. Peptide mimetics can contain any combination of "non-natural" structural components, typically from three groups: (a) residue linkage groups other than the natural amide bond ("peptide bond"); (b) non-natural residues in place of naturally occurring amino acids; or (c) residues which induce or stabilize a secondary structure, e.g., a β turn, γ turn, β sheet, or α helix conformation. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical bonds other than peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'- dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that are alternatives to peptide bonds include, ketomethylene ( -C(=O)-CH2- for -C(=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole, retroamide, thioamide, or ester (Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, Peptide Backbone Modifications, Marcell Dekker, NY). The structure ofthe polypeptides, peptides, other functional derivatives, including mimetics ofthe present invention are preferably based on structure and amino acid sequence of CRT, preferably human CRT (SEQ ID NO:2, disclosed above) or a SARS-CoN protein such as S, E, M or Ν as disclosed herein for two viral isolates. Individual synthetic residues and polypeptides incorporating mimetics can be synthesized using a variety of procedures and methodologies well known in the art, e.g., Organic Syntheses Collective Volumes, Gilman et al. (eds) John Wiley & Sons, Inc., Y. Polypeptides incorporating mimetics can also be made using solid phase synthetic procedures (e.g., U.S. Pat. No. 5,422,426). Peptides and peptide mimetics ofthe invention can also be synthesized using combinatorial methodologies. Various techniques for generation of peptide and peptidomimetic libraries are well known e.g., multipin, tea bag, and split-couple-mix techniques (al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234). Modified polypeptide and peptides can be further produced by chemical modification (Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896). The peptides can also be synthesized, whole or in part, using conventional chemical synthesis (Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis, e.g., using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the manufacturer' instructions. In one embodiment ofthe invention, peptide-binding fragments or "sub-sequences" of CRT are used. In another embodiment, other peptides that bind to MHC proteins, preferably MHC Class I proteins, are used. Such peptides can be derived from any polypeptide, particularly, from a known pathogen, or it can be entirely synthetic). Methods for determining whether, and to what extent, a peptide binds to a CRT or a CRT fragment, or an MHC protein are routine in the art (Jensen (1999; Immunol. Rev. 172:229-238; Zhang (1998; J. Mol. Biol. 281:929-947; Morgan (1997) Protein Sci 6:1771-1773; Fugger (1996) Mol. Med. 2:181-188; Sette (1994) Mol. Immunol. 31:813-822; Elvin (1993) J. Immunol. Meth. 158:161-171; U.S. Patent Nos. 6,048,530; 6,037,135; 6,033,669; 6,007,820).
Formulation and Administration of Pharmaceutical or Immunological Compositions hi various embodiments ofthe invention, polypeptides, nucleic acids, expression cassettes, cells, and particles, are administered to an individual as pharmacological compositions in amounts sufficient to induce an antigen-specific immune response (e.g., a CTL response, see Examples, below) in the individual. Pharmaceutically acceptable carriers and formulations for nucleic acids, peptides and polypeptides are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, PA ("Remington's"); Banga; Putney (1998) Nat. Biotechnol. 16:153-157; Patton (1998) Biotechniques 16:141-143; Edwards (1997) Science 276: 1868-1871; U.S. Patent Nos. 5,780,431; 5,770,700; 5,770,201. The nucleic acids and polypeptides used in the methods ofthe invention can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally; by intraarterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa). Actual methods for delivering compositions will be known or apparent to those skilled in the art and are described in detail in the scientific and patent literature, see e.g., Remington's. The pharmaceutical compositions can be administered by any protocol and in a variety of unit dosage forms depending upon the method and route and frequency of administration, whether other drugs are being administered, the individual's response, and the like. Dosages for typical nucleic acid, peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages may be adjusted depending on a variety of factors, e.g. , the initial responses (e.g., number and activity of CTLs induced, tumor shrinkage, anti-viral activity measured as lysis of virus-infected cells or reduction of virus titer, and the like), the particular therapeutic context, patient health and tolerance. The amount of pharmaceutical composition adequate to induce the desired response is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use, i.e., the "dosing regimen," will depend upon a variety of factors, including, e.g., the diseases or conditions to be treated or prevented by the immunization, the general state ofthe patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of pharmaceutical composition, and the like. The dosage regimen also takes into consideration pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailabihty, metabolism, clearance, and the like (Remington). Dosages can be determined empirically, e.g., by assessing the abatement or amelioration of symptoms, or, by objective criteria, e.g., measuring levels of antigen-specific CTLs. As noted above, a single or multiple administrations can be administered depending on the dosage and frequency as required and tolerated by the patient. The pharmaceutical compositions can be administered alone or in conjunction with other therapeutic treatments, or, as prophylactic immunization. Ex vivo treatment and re-administration of APCs In various embodiments ofthe invention, the nucleic acids and polypeptides ofthe invention are introduced into the individual by ex vivo treatment of antigen presenting cells (APCs), followed by administration ofthe manipulated APCs. In one embodiment, APCs are transduced (transfected) or infected with fusion protein-encoding nucleic acids ofthe invention; afterwards, the APCs are administered to the individual. In another embodiment, the APCs are stimulated with fusion proteins ofthe invention (purified or as a cell lysate from cells transfected and expressing a recombinant fusion protein in vivo). Afterward this "pulsing, the APCs are administered to the individual. The fusion proteins can be in any form, e.g., as purified or synthetic polypeptides, as crude cell lysates (from transfected cells making recombinant fusion protein), and the like. The APC can be an MHC-matched cell (a tissue-typed cell). The APC can be a tissue-cultured cell or it can be an APC isolated from the individual to be treated and re-administered after ex vivo stimulation. Any APC can be used, as described above. Methods of isolating APCs, ex vivo treatment in culture, and re-administration are well known in the art (U.S. Patent Nos. 5,192,537; 5,665,350; 5,728,388; 5,888,705; 5,962,320; 6,017,527; 6,027,488). Kits The invention provides kits that contain the pharmaceutical or immunogenic compositions ofthe invention, as described above, to practice the methods ofthe invention. In alternative embodiments, the kits can contain recombinant or synthetic chimeric polypeptides comprising a first domain comprising an ER chaperone polypeptide and a second domain comprising an antigenic peptide ofthe SARS CoV, e.g., a CRT-Class I-binding peptide epitope fusion protein; or, the nucleic acids encoding them, e.g., in the form of naked DNA (e.g., plasmids), viruses (e.g. alphavirus-derived "replicons" including Sindbis virus replicons) and the like. The kit can contain instructional material teaching methodologies, e.g., means to administer the compositions used to practice the invention, means to inject or infect cells or patients or animals with the nucleic acids or polypeptides ofthe invention, means to monitor the resultant immune response and assess the reaction ofthe individual to which the compositions have been administered, and the like. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope ofthe appended claims.
EXAMPLES The following examples are offered to illustrate, but not to limit the claimed invention. EXAMPLE 1 DNA Vaccines Targeting the Nucleocapsid Protein of SARS-CoV This Example is built upon the prior discovery ofthe present inventors that DNA vaccination with antigen linked to calreticulin (CRT) dramatically enhances MHC class I presentation of a linked antigen to CD8+ T cells. In this study, they employed a CRT-based enhancement strategy to create effective DNA vaccines using SARS-CoV nucleocapsid (N) protein as a target antigen. Vaccination with naked CRT/N DNA generated the most potent N- specific humoral and T cell-mediated immune responses in vaccinated C57BL/6 mice among all ofthe DNA constructs compared here. Animals vaccinated with CRT/N DNA were capable of significantly reducing the titer of challenging vaccinia expressing the N protein ofthe SARS virus. These results show that a DNA composition encoding CRT linked to a SARS-CoN antigen Ν can generate strong Ν-specific humoral and cellular immunity that can control infection with SARS-CoN. Materials and Methods Plasmid DΝA Constructs and DΝA Preparation The current study employed the mammalian expression vector, pcDNA3.1/myc-His (-) (Invifrogen, Carlsbad, CA). For the generation of pcDNA3-N-myc, the DNA fragment encoding SARS-Co V nucleocapsid was amplified with PCR using a set of primers:
5 ' -AAAGAATTCATGTCTGATAATGGACCCCAATC-3 ' , SEQ ID NO : 97
5 ' -TTTGGTACCTGCCTGAGTTGAATCAGCAGA-3 ' SEQ ID NO: 98 and pGEX-l-NC-G3 (Huang, LR et al, 2004, J Med Virol. 73:338-346) as a template. The amplified product was further cloned into the EcoRI/Kpnl sites of pcDNA3.1/røyc-His (-) vector. To generate pcDNA3-CRT-myc, CRT DNA segment was isolated from pcDNA3-CRT (Cheng, W.-F. et al, 2001, J. Clinical Invest. 108:669-678) and cloned into the XhoI/EcoRI sites of pcDNA3.1/7«yc-His (-). For the generation of pcDNA3-CRT/N-myc, the amplified N DNA was cloned into the EcoRI/Kpnl sites of pcDNA3-CRT-myc. The accuracy of these constructs was confirmed by DNA sequencing. The DNA was amplified in E. coli DH5α and purified as described previously (Chen, C.-H. et al, 2000, Cancer Research 60:1035-1042; Wu et al, PCT Publication WO 01/29233).
Generation of Bacteria-Derived SARS-CoV N Protein cDNA encoding SARS nucleocapsid protein was generated by reverse transcription of SARS coronavirus TWl (18) (Hsueh, PR, 2003, Emerg Infect Dis 9:1163-1167;) (accession no. YA291451) using Superscript II (Invifrogen, Carlsbad, CA) followed by amplification using platinum Tαq DNA polymerase (Invifrogen, Carlsbad, CA) as described previously (Huang et αl., supra). The oligonucleotide primers for SARS-CoV N protein were
5 ' -ATGTCTGATAATGGACCCCA-3 ' (forward, nt28120-nt28139) SEQ ID NO : 99 ; and
5 ' -TTATGCCTGAGTTGAATCAG-3 ' (reversed, nt29369-nt29388). SEQ ID NO : 100 The DNA fragment encoding N protein was cloned into pGEX-lplasmid (Amersham Pharmacia Biotech, Little Chalfont, England) to generate pGEX-l-NC-G3 (Huang et al, supra) for recombinant protein expression. E. coli BL-21 were transformed with pGEX-1 or pGEX-1- NC-G3 plasmids and grown overnight in LB medium containing 50μg/ml ampicillin to the midlog phase. Cells transformed with GST or GST-N fusion constructs were directly induced with 0.25 mM IPTG (isopropyl-β-D-thiogalactoside) for 3 hours at 30 °C. Cells were collected by centrifugation and then resuspended in TNE buffer (50mM Tris, pH 8.0, 0.15M NaCl, lmM EDTA, and lmM PMSF), about 1ml per 25OD600 cells. The fusion protein solubility was determined by sonication, and centrifugation followed by SDS-PAGE separation of both the supernatant and pellet fractions. In larger volume of culture (~3 liters), cells were lysed by ' microfluidizer. Lysates prepared from the large batch were incubated with TNE equilibrated glutathione resin. Bound protein was eluted by lOmM reduced glutathione in 50mM Tris (pH 8.0) buffer. The eluted and purified fractions were used for Western blot analysis and as the coating antigen for ELISA assay.
Western Blot Analysis The expression of N protein in 293 cells transfected with pcDNA3.1/røye-His (-) encoding no insert, CRT, N, or CRT/N DNA was characterized by western blot analysis. 20 μg of DNA were transfected into 5xl06 293 cells using lipofectamine 2000 (Life Technologies, Rockville, MD). 24 hr after transfection, cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of proteins (50 μg) were loaded and separated by SDS-PAGE using a 10% polyacrylamide gel. For the characterization of bacteria-derived N protein, 1 μg of purified GST-N fusion protein was loaded and separated by SDS-PAGE using a 10% polyacrylamide gel. The gels were electroblotted to a polyvinylidene difluoride membrane (BioRad, Hercules, CA). Blots were blocked with PBS/0.05% Tween 20 (TTBS) containing 5% nonfat milk for 2 hr at room temperature. Membranes were probed with rabbit anti-GST-N sera (Huang et al, supra) at 1:1000 dilution in TTBS for 2 hr, washed four times with TTBS, and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Zymed, San Francisco, CA) at 1:1000 dilution in TTBS containing 5% nonfat milk. Membranes were washed four times with TTBS and developed using enhanced Hyperfilm-enhanced chemiluminescence (Amersham, Piscataway, NJ).
Mice Six- to eight-week-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, Maryland) and kept in the oncology animal facility ofthe Johns Hopkins Hospital (Baltimore, Maryland). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. DNA Vaccination DNA-coated gold particles were prepared according to a previously described protocol (Chen et al, supra). DNA-coated gold particles were delivered to the shaved abdominal region of mice using a helium-driven gene gun (BioRad, Hercules, CA) with a discharge pressure of 400 p.s.i. C57BL/6 mice were immunized with 2 μg ofthe plasmid encoding no insert, CRT, N, or CRT/N protein. The mice received two boosters with the same dose at a one week interval.
Enzyme-Linked Immunoabsorbent Assay (ELISA) The presence of SARS-CoV N-specific antibodies in the sera from CRT/N DNA- vaccinated C57BL/6 mice (5 per group) were determined by ELISA using microwell plates coated with bacteria-derived recombinant GST-N protein. Purified GST-N protein was diluted to lμg/ml with o.o5 M carbonate buffer (pH 9.6), and 0.1 ml/well was added to 96-well microtiter plates. Purified GST protein was used as negative control. The plates were incubated overnight at 4 °C, washed with phosphate buffered saline (PBS) - 0.05% Tween 20 (PT), incubated with (0.1 ml/well) PT-2% bovine serum albumin (PBT) for 60 minutes at 37°C and washed again with PT. Serial dilutions ofthe tested sera were added (0.1 ml/well) and the plates were incubated for 60 minutes at 37°C. The plates were washed with PT and were incubated with (0.1 ml/well) alkaline phosphatase-conjugated rabbit anti-mouse antibodies (Zymed, San Francisco, CA) for 30 minutes at 37°C. The plates were washed with PT and incubated with (0.1 ml/well) alkaline phosphatase substrate (according to Sigma instructions) for 60 minutes at 37 °C. Plates were read on a MicroElisa reader at a wavelength of 450 nm. Reading higher than 3 -fold negative controls were scored as positive reactions.
Intracellular Cytokine Staining and Flow Cytometry Analysis In order to assess the ability of our DNA vaccine encoding SARS-CoV N protein to elicit an N-specific CD8+ T cell response, we sought to identify the MHC class I-restricted CTL epitope ofthe SARS-CoN Ν protein. Using the Biolnformatics & Molecular Analysis Section (BEVIAS) for Db and K peptide binding predictions (URL is bimas.cit.nih.gov/molbio/hla bind/) and the SYFPEITHI database of MHC ligands and peptide motifs (URL is syipeithi.bmi- heidelberg.com/), we analyzed various peptides of eight, nine, or ten residues and determined their sequences, positions, and scores, and eventually generated 7 potential peptides for our studies (see Table 3). We used splenocytes from C57BL/6 mice vaccinated with CRT/Ν DΝA for the characterization of these candidate peptides. Splenocytes were harvested from mice one week after the last vaccination. Prior to intracellular cytokine staining, 4xl06 pooled splenocytes from the vaccinated mice were incubated for 16 hours with 1 μg/ml of each candidate peptide for detecting N-specific CD8+ T cell precursors. Intracellular IFN-γ staining and flow cytometry analysis were performed as described previously. Flow cytometry analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA). To characterize the various DNA vaccines in eliciting an N-specific CD8+ T cell response, splenocytes from the various vaccinated mice (5 per group) were incubated with 1 μg/ml of N peptide (aa 346-354, QFKDNNILL; SEQ ID ΝO:31) for 16 hours. Intracellular IFN- γ staining and flow cytometry analysis were performed as described above.
Generation and Characterization of Recombinant Vaccinia The recombinant vaccinia virus was generated using a protocol similar to that described previously Wu, T.-C, et al, 1995, Proc. Natl Acad. Sci. 92:11671-11675). Briefly, the DNA fragment encoding SARS-Co V nucleocapsid was amplified with PCR using a set of primers:
5 '-AAAGCATGCATGTCTGATAATGGACCCCAATC-3 ' (SEQ ID NO:32)
5 '-TTTGGTACCTTATGCCTGAGTTGAATCAGCAGA-3 ' (SEQ TD NO:32)and ρGEX-l-NC-G3 as a template. The amplified product was further cloned into sphl/Kpnl sites of pSCIIMCS2. This construct was transfected into Vac-WT infected CV-1 using Lipofectamine 2000. The recombinant vaccinia viruses were isolated as in Wu et al, supra. Plaque-purified recombinant vaccinia viruses were checked for the expression of N protein by flow cytometry analysis, immunofluorescence staining, and Western blot analysis using rabbit anti-GST-N sera (Huang et al, supra). For the detection ofthe expression of SARS-CoN Ν protein in TK" cells infected with Nac-Ν by flow cytometry analysis, the vaccinia-infected cells were incubated with rabbit anti-GST-Ν sera at 1:100 dilution in lx Perm (PharMingen, San Diego, CA) for 30 min after fixation with Cytofix/Cytoperm (PharMingen, San Diego, CA), washed four times with IX PBS, and then incubated with FITC-labeled goat anti-rabbit IgG (Jackson ImmunoReseach Laboratories, West Grove, PA) at 1 :1000 dilution. Western blot analysis was performed as described above. The Nac-WT and Nac-Ν were amplified by infecting TK" cells in vitro according to a standard protocol. Titer was determined by plaque assay using BSC-1 cells. The viral stocks were preserved at — 70°C prior to vaccination. Before use, the virus was thawed, trypsinized with 1/10 volume of trypsin/EDTA in 37°C water bath for 30 min, and diluted with minimal essential medium (MEM) to the final concentration of 1 x 108 plaque-forming units (PFU)/ml.
Immunofluorescence Staining for Ν Protein Expression Immunofluorescence staining was performed using a protocol similar to what has been described previously (Cheng, WF et al, 2002, Hum Gene Ther 13:553-568). Briefly, Tk" cells were cultured in 8-well culture chamber slides (Νalge Νunc Int., Νaperville, IL) until they reached 50% confluence. The cells were infected with Nac-Ν or Nac WT at 10 m.o.i. to evaluate the expression of Ν protein. After 24 hours of infection, cells were fixed and permeabilized with Cytofix/Cytoperm (Pharmingen) for 30 min. Rabbit anti-Ν sera was added into the chamber at a dilution of 1:100 and incubated for 30 min. Diluted FITC goat anti-rabbit IgG (10 μg/ml, Jackson ImmunoReseach Laboratories, West Grove, PA), was added and incubated for 30 min. The slides were mounted and observed immediately under a fluorescence microscope.
In Vivo Challenge with Recombinant Vaccinia Virus For the local challenge experiment, the immunized mice were anesthetized and infected with 2xl06 PFU/mouse of Vac-WT or Vac-Ν in 20 μl by intranasal instillation 1 week after the final immunization. For the systemic challenge experiment, the immunized mice were infected with lxl07PFU/mouse of Vac-Ν in 100 μl by intravenous injection 1 week after the final immunization. Five mice were used for each vaccinated group. To determine virus titers in lungs, mice were sacrificed 5 days after challenge. Both lungs were harvested, homogenized in 1 ml of MEM containing 2.5% fetal bovine serum, and subjected to three rounds of freezing and thawing before the titer of virus was determined by plaque assay.
Statistical Analysis All data expressed as means ± SEM are from one experiment of at least two experiments performed. Data for intracellular cytokine staining with flow cytometry analysis and in vivo viral challenge experiments were evaluated by analysis of variance (AΝOVA). Comparisons between individual data points were made using a student's t-test. Results
Characterization of N protein in cells transfected with the various DNA vaccines. In order to characterize the expression ofthe SARS-CoV N protein in 293 cells transfected with the various DNA constructs, we performed a Western blot analysis, using cell lysates derived from DNA-transfected cells. Rabbit anti-GST-N sera were used for Western blot analysis. As shown in Figure 1, lysate from 293 cells transfected with N DNA revealed a protein band with a size of approximately Mτ 48,000 corresponding to N protein in Lane 3. Lysate from 293 cells transfected with CRT/N DNA revealed a protein band with a size of approximately Mτ 90,000 corresponding to the chimeric CRT/N protein in Lane 4. hi contrast, N protein was not detected in lysates from 293 cells transfected with plasmid DNA with no insert (lane 1) or CRT DNA (lane 2). Our data indicated that N DNA-transfected cells exhibited levels of N protein expression comparable to CRT/N DNA-transfected cells.
Vaccination with CRT N DNA significantly enhances N-specific antibody responses. To evaluate the humoral immune response to DNA vaccines encoding SARS-CoV N protein, we performed ELISA analysis using bacteria-derived GST-N fusion protein and sera from mice vaccinated with the various DNA vaccines. As shown in Figures 2A and 2B, recombinant GST-N protein was purified from bacteria. The purification of bacteria-derived GST-N protein was demonstrated by gel electrophoresis (Figure 2A). The confirmation of GST-N protein was demonstrated by Western blot analysis with rabbit anti-GST-N sera (Figure 2B). We used the bacteria-derived GST-N protein for our ELISA. As shown in Figure 2C, mice vaccinated with CRT/N DNA generated the highest titer of N-specific antibody responses among mice vaccinated with the various DNA vaccines. Furthermore, ELISA to determine the subtype of IgG antibody showed significantly higher titer of N-specific IgGl Ab than N-specific IgG2a in serum from mice vaccinated with N or CRT/N DNA (Figure 2D). We also used purified GST protein as a control for our ELISA. Sera from vaccinated mice only generated background level of color changes against GST (data not shown). These data show that vaccination with CRT/N DNA elicits a significantly stronger N-specific humoral immune response than vaccination with N DNA. This suggests that the linkage of CRT to N protein in a DNA vaccine enhances N-specific antibody production in vaccinated mice.
Vaccination with CRT/N DNA significantly improved SARS-CoV N-specific CD8+ T cell- mediated immune responses. T cell mediated immunity has been shown to be important for control of viral infection. In order to develop quantitative assays for characterizing N-specific CD8+ T cell mediated immune responses, we sought to identify the MHC class I-restricted CTL epitope ofthe SARS- CoV N protein. Using the Biolnformatics & Molecular Analysis Section (BEvIAS) for Db and Kb peptide binding predictions (htto:/^imas.cit.nih.gov/molbio/hla bind/) and the SYFPEITHI database of MHC ligands and peptide motifs (htto://svφeitM.bmi-heidelberg.com/), we identified several potential candidate peptides for SARS-CoV N protein in C57BL/6 mice. Table 3 shows their sequences, positions, and scores. Table 3. Candidate CTL epitopes for SARS coronavirus nucleocapsid protein
Peptide MHC length Peptide Peptide SEQ ID BIMAS SYFPEITHI name Class I position sequence NO: score score
N 346-354 H-2DD 9 346-354 QFKDNVILL 31 60 20
N 351-359 H-2D 9 351-359 VILLNKHID 34 33 11
N 352-36Q H-2Db 9 352-360 ILLNKHIDA 35 n/a 2 202-2n H-2D 10 202-211 SSRGNSPAR 36 n/a 24
N 122-131 H-2D 10 122-131 LPYGANKEGI 37 200 n/a
N 50-5 H-2Kb 8 50-57 TASWFTAL 38 11 22
N 3π-3ι8 H-2K 8 311-318 SASAFFGM 39 11 18 We then synthesized these peptides and characterized their ability to activate N-specific CD8+ T cells using splenocytes harvested from mice vaccinated with the various DNA vaccines. As shown in Figure 3A, using intracellular cytokine staining followed by flow cytometry analysis, we showed that a D -restricted 9mer peptide positioned at aa 346-354 (QFKDNVILL; SEQ ID NO:31) of N protein was able to activate significantly more N-specific CD8+ T cells in splenocytes from mice vaccinated with CRT/N DNA than the other epitopes (p<0.05). In comparison, the N peptide (aa 351-359, VILLNKHID; SEQ ID NO:34) only activated N- specific CD 8+ T cells in splenocytes from mice vaccinated with CRT/N DNA to a slightly higher level than the background level. The other five peptides were not able to activate N- specific CD8+ T cells in splenocytes from mice vaccinated with CRT/N DNA (Figure 3A). Thus, the N peptide (aa 346-354, QFKDNVILL; SEQ ID NO:31) likely represents an H-2 D - restricted CTL epitope for SARS-CoV N protein. Our results also showed that mice vaccinated with CRT/N DNA generated significantly more N-specific CD8 T cells than mice vaccinated with N DNA (Figure 3B) (p<0.05). Thus, our data suggest that the linkage of CRT to N protein in a DNA vaccine enhances N-specific CD8+ T cell mediated immune responses in vaccinated mice. Recombinant vaccinia expressing SARS-CoV N protein as surrogate virus for vaccine studies Certain factors preclude the usage of live SARS-CoN for our vaccine efficacy studies.
Thus, we generated vaccinia virus expressing SARS-CoN Ν protein as a surrogate virus for our vaccine efficacy studies. To demonstrate the expression of SARS-CoN Ν protein expression, we infected 293 cells with vaccinia virus encoding Ν (Nac-Ν) and confirmed Ν expression via flow cytometry analysis, immunofluorescence staining, and Western blot analysis using rabbit anti-
GST-Ν sera (Figure 4). 293 cells infected with wild-type vaccinia (Nac-WT) were used as a negative control. All three assays determined that 293 cells infected with Nac-Ν expressed significant levels of Ν protein and that 293 cells infected with Nac-WT did not express Ν protein.
Vaccination with CRT/Ν DΝA results in the greatest reduction of titer of recombinant vaccinia virus expressing Ν protein. The ability of a vaccine to successfully protect against viral challenge is an essential measure of its efficacy. To test the ability of our DΝA vaccines encoding SARS-COV Ν protein to protect against viral challenge, we vaccinated mice with DΝA encoding CRT/Ν, Ν, CRT or no insert and challenged these mice with Vac-Ν or Vac-WT intranasally or intravenously one week after the last vaccination. As shown in Figure 5A, while no difference in Vac-WT titer was observed among mice vaccinated with any ofthe DΝA vaccines, we found significantly lower titers of Vac-Ν in lungs of mice vaccinated with DΝA encoding Ν than in lungs of mice vaccinated with DΝA encoding CRT, or no insert (intranasal: p<0.009; intravenous: p<0.033). More importantly, mice vaccinated with DΝA encoding CRT/Ν exhibited a significantly reduced titer of Vac-Ν in their lungs when compared to mice vaccinated with DΝA encoding Ν (intranasal: p<0.013; intravenous: p<0.006). These data indicate that vaccination with CRT/Ν DΝA can reduce titer of vaccinia expressing SARS-CoN Ν protein to a greater degree than vaccination with Ν DΝA. Thus, vaccination with CRT/Ν DΝA may generate the best protection against intranasal or intravenous challenge with viruses expressing SARS-CoN Ν protein. Discussion Vaccination with CRT/Ν DΝA can elicit SARS-CoV nucleocapsid-specific humoral and cellular immune responses, and our results suggest that these responses can significantly reduce the titer of challenging vaccinia virus expressing Ν protein. These results also indicate that the linkage of CRT DΝA to Ν DΝA leads to enhanced DΝA vaccine potency against a virus expressing a SARS-CoV protein. This is consistent with our previous studies using a different antigen (HPV-16 E7). Thus, the ability ofthe CRT strategy to enhance cellular and humoral immune responses has been confirmed in two distinct antigenic systems. This indicates that a similar DNA vaccine strategy may prove effective against other antigenic proteins of SARS- CoN, such as the S, E, or M proteins. The observed enhancement ofthe humoral immune response against the Ν protein of SARS-CoN in mice vaccinated with the chimeric CRT/Ν DΝA vaccine may not be useful for SARS-CoN neutralization given the location ofthe Ν protein inside the viral envelope. Thus, Ν- specific antibodies may not be able to cross the envelope to bind with the nucleocapsid protein to abolish the infection. In comparison, SARS-CoN S, E, and M proteins are expressed on the envelope surface, and neutralizing antibodies against these proteins may thus be able to neutralize SARS-CoN infection. This raises the possibility that a DΝA vaccine strategy employing CRT linked to the S, E, or M proteins may elicit effective neutralizing antibodies as well as potent T cell responses against infection by live SARS-CoN (see following Examples). While the humoral immune response may represent an effective means of generating protection from SARS-CoN infection, it may also lead to an antibody-dependent enhancement (ADE) reaction. In ADE, virus-specific antibodies have been shown to interact with the Fc and/or complement receptors to enhance viral entry into host immune cells, such as granulocytic cells and monocytes/macrophages. The ADE phenomenon has been observed in at least one coronaviral system. It should therefore be considered when designing a vaccine against SARS- CoN. If the ADE phenomenon is observed in SARS-CoN infection or vaccination, Ν protein may be the logical choice for a target antigen, as antibodies against Ν will be unlikely to lead to ADE. This is due to the fact that the Ν protein is not expressed on the viral envelope and thus antibodies against Ν will probably not be able to facilitate viral entry. We observed significant enhancement ofthe Ν-specific CD8+ T cell response as a result of linkage of Ν protein to CRT in a DΝA vaccine. The percentage of Ν-specific CD8+ T cells in CRT/Ν DΝA-vaccinated mice may potentially be further improved by coadministration with DΝA encoding an antiapoptotic protein. Coadministration of DΝA encoding BCL-xL with DΝA encoding E7 HSP70, CRT/E7, or Sig/E7/LAMP-1 resulted in further enhancement ofthe E7-specific CD8+ T cell response for.all three constructs. Because intracellular targeting and anti-apoptotic strategies modify DCs via different mechanisms, it is potentially feasible to combine anti-apoptotic strategies for prolonging DC life with CRT for enhancing MHC class I processing and presentation of SARS-CoN antigen by DCs to further enhance DΝA vaccine potency. In this study we used vaccinia virus expressing Ν protein of SARS-CoN as a surrogate virus for assaying the vaccine efficacy in our study because SARS-CoN, having mainly been isolated in Asia, is difficult to obtain in the United States. More importantly, the handling of live SARS-CoN is potentially extremely hazardous, whereas the handling of recombinant vaccinia is relatively safe. For these reasons, we generated vaccinia expressing SARS-CoN Ν protein for use as a surrogate viral challenge model. The development of such a model for testing of our vaccine strategy is not without precedent, as vaccinia virus has been previously used in several prior studies as a substitute viral challenge model. While these studies may show a good correlation between the reduction of vaccinia titer and vaccine potency, it would preferable for our research to explore vaccine efficacy against live SARS-CoN virus in a near-human model. A potential animal model is Macaca Fascicularis, which has been shown to be susceptible to live SARS-CoN infection and demonstrate pulmonary pathology similar to humans. DΝA vaccination can successfully elicit SARS-CoN Ν-specific humoral and CD8+ T cell responses in vaccinated mice, and vaccination with CRT/Ν DΝA can significantly enhance both humoral and cellular immune responses when compared to vaccination with Ν DΝA. These enhanced immune responses resulting from linkage of antigen to CRT correlate with a strong reduction of titer of challenging vaccinia expressing Ν protein in mice vaccinated with CRT/Ν DΝA. While Ν protein may not be able to elicit an effective neutralizing antibody response against live SARS-CoN, we have shown that it is capable of eliciting a SARS-CoN antigen- specific CD 8+ T cell response that results in a significant reduction of titer of challenging vaccinia when linked to CRT in a DΝA vaccine. This makes the present CRT/Ν DΝA vaccine a potential candidate for future clinical translation. Furthermore, the CRT DΝA vaccination strategy is applicable to envelope-associated SARS-CoN proteins, such as S, E, or M proteins, for elicitation of both neutralizing antibodies against SARS-CoN and SARS-CoN antigen- specific CTLs. EXAMPLE 2 DΝA Vaccines Targeting the Spike Protein (S) of SARS -CoV
Materials and Methods
Plasmid DΝA Constructs and DΝA Preparation For the generation of pRSETA-S, the DNA fragment encoding the full-length S protein of SARS-CoV was amplified using a set of primers 5 ' - cggatccatgtttattttcttattatttct -3 ' (SEQ ID NO:40) and 5 ' - cagaattcttatgtgtaatgtaatttgaca -3 ' (SEQ JJD NO:41) and cDNA from TW-1 strain of SARS- CoV. The amplified product was cloned into the BamHI/EcoRI of pRSETA (Invifrogen, Carslbad, CA). For the generation of pcDNA3-S, a DNA fragment encoding S was isolated from pRSETA-S and further cloned into the BamHI/EcoRI sites of pcDNA3.1(+) vector (Invifrogen, Carlsbad, CA). For the generation of pcDNA3 encoding SARS-CoV SI, Si or S2, the DNA fragments encoding SI, Si or S2 DNA fragments were amplified with PCR using the following set of primers:
51 5 ' -ccggatccatgtttattttcttattat-3 ' , (SEQ ID NO:42) 5 ' -ccgaattcttaagacatagtataagccacaatag-3 ' ) , (SEQ ID NO:43)
Si 5 ' -cttggatccatgggttgtgtccttgcttgg-3 ' , (SEQ ID NO:44) 5 ' -ccgaattcttacatgaagccagcatcagcgag) and (SEQ ID NO:45)
52 5 ' -ccggatccatgatgttaggtgctgatagttcaattg-3 ' , (SEQ ID NO:46) 5 ' -gccgaatt cttatgtgtaatgtaatttg- 3 ' ) , (SEQ ID NO:47) and pRSETA-S as a template. The amplified products were further cloned into the
BamHI/EcoRI sites of pcDNA3.1 (+) vector. pcDNA3-CRT has been described previously (Cheng, 2001, supra). For the generation of pcDNA3 -CRT/SI, the CRT DNA fragment was amplified with PCR using a set of primers: 5 ' - ggtcttaagatgctgctccctgtgccgctg - 3 ' , (SEQ ID NO:48) 5 ' - caaagatctcagctcgtccttggcctggc - 3 ' (SEQ ID NO:49) and pcDNA3-CRT as a template. The amplified CRT was cloned into the Aflll/BamH I sites of pcDNA3-Sl. For the generation of pMSCV-S, a DNA fragment encoding S was isolated from pRSETA-S and further cloned into the Bglll/EcoRI sites of pMSCV vector (Invifrogen, Carlsbad, CA). The accuracy of these constructs was confirmed by DNA sequencing. The DNA was amplified in E. coli DH5α and purified as described previously. Cell Lines The production and maintenance of TC-1 cells has been described previously. In brief,
HPV-16 E6, E7 and ras oncogene were used to transform primary C57BL/6 mice lung epithelial cells to generate TC-1 cells. DC-1 cells were generated from the dendritic cell line provided by
Dr. Kenneth Rock, University of Massachusetts. With continued passage, subclones of DCs (DC-1) were generated that are easy to transfect (Kim, TW et al, 2004, Gene Tier. 77:1011- 1018). For the generation of TC-l/S and DC-l/S cells, the retroviral vector encoding the S protein of SARS-CoN was first generated. The phoenix packaging cells were transfected with pMSCN-S or pMSCN using Lipofectamine 2000. Supernatant from the transfected Phoenix (φΝX ) cells was incubated with 50% confluent TC-1 or DC-1 cells in the presence of polybrene (8 μg/ml; Sigma). Following transduction, the retroviral supematants were removed from the transduced cells, and DCs were propagated in culture medium containing 7.5 μg/ml of puromycin for selection. The transduced TC-1 or DC-1 cells were further selected by growing in culture medium containing 10 μg/ml of puromycin for 5 days. The expression of S antigen was confirmed by Western blbt analysis. All cells were maintained in RPMI medium (Invifrogen, Carlsbad, CA) supplemented with 2mM glutamine, lmM sodium pyruvate, 20mM HEPES, 50μM β-mercaptoethanol, 100 IU/ml penicillin, lOOμg/ml streptomycin and 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA). Western Blot Analysis The expression ofthe full length protein S and its recombinant polypeptide fragments was examined in 293 cells transfected with various ofthe present DΝA vectors encoding either no insert (control), S, SI, Si, S2, CRT or CRT/SI was characterized by Western blot analysis. DΝA, 20 μg, was transfected into 5xl06 293 cells using lipofectamine® 2000 (Life Technologies, Rockville, MD). After overnight transfection, the cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of proteins (50μg) were loaded and separated on a 10% SDS-PAGE gel. The gels were elecfroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Blots were blocked with PBS/0.05% Tween 20 (TTBS) containing 5% nonfat milk overnight at 4°C. Membranes were probed with rabbit anti- spike polyclonal antibody at 1 :2000 dilution in TTBS for 1 hr at room temperature, washed six times with TTBS, and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Zymed, San Francisco, CA) at 1 : 1000 dilution in TTBS containing 5% nonfat milk for 1 hr at room temperature. Membranes were washed four times with TTBS and developed using enhanced Hyperfilm-enhanced chemiluminescence (Amersham, Piscataway, ΝJ). The presence of secreted SI and CRT/SI was confirmed by Western blot analysis. Forty eight hours after transfection as above with 20 μg of DΝA encoding either no insert, S, SI, Si, S2, CRT or CRT/SI, 4 ml of culture supematants were collected, centrifuged to remove cellular debris and then was concentrated to 0.2 ml using an Amicon Ultra centrifugal filter device. Varying volumes (5, 10, 20 μl) ofthe concentrated supematants were loaded and separated by SDS-10% PAGE before blotting. The presence of S polypeptides was detected by probing with Rabbit anti-S antibody at a 1:2000 dilution. The presence ofthe S-specific antibody in sera from the mice immunized with the various DNA vaccines was determined by Western blot analysis using TC-l/S lysates as a source of antigen. The lysates from TC-l/No insert or TC-l/S were loaded and separated by SDS-10% PAGE gel before blotting. Immune serum samples were collected from DNA- vaccinated mice two weeks after the last vaccination and were diluted to 1 :250 with PBS. Equal amounts of proteins (50 μg) from TC-l/No insert or TC-l/S lysates were probed with the diluted antisera from vaccinated mice.
Mice were as described in Example 1. DNA Vaccination DNA-coated gold particles were prepared and used as described above. C57BL/6 mice were immunized with 2 μg ofthe plasmid which included either no insert, S, SI, Si, S2, CRT or
Intracellular Cytokine Staining and Flow Cytometry Analysis Using CD3 negative selection kit (Miltenyi Biotec, Auburn, CA), CD3+ cells were enriched from splenocytes, harvested from mice one week after the last vaccination. DC cells (105) expressing S antigen (DC/S) were incubated with 106 ofthe isolated CD3+ T cells for 16 hours. The DC cells not expressing S antigen (DC/No insert) served as a negative control. After activation, T cells were stained for both surface CD8 and intracellular IFN-γ, and analyzed with flow cytometry analysis as described before.
ELISA The end-point dilution titer of S-specific antibodies in the sera from DNA- vaccinated C57BL/6 mice were determined by ELISA using 96 microwell plates coated with TC-l/S or TC-l/No insert cells. After overnight incubation, the cells (5xl04/well) were washed once in phosphate buffered saline (PBS), then fixed and permeabilized using Cytofix/Cytoperm Kit (Pharmingen). Plates coated with cells were incubated with lxPBS (0.3 ml/well) with 0.05% Tween 20 (PBT) containing 2% bovine serum albumin for 60 minutes at 37°C and washed again with PBT. Serial dilutions ofthe tested sera were added (0.1 ml/well) and the plates were incubated for 60 minutes at 37°C. The plates were washed with PBT and were incubated with (0.1 ml/well) peroxidase-conjugated rabbit anti-mouse IgG (Zymed, San Francisco, CA) for 30 minutes at 37°C. The plates were washed with PT and incubated with (0.1 ml well) peroxidase substrate according to the manufacturer's instructions for 15 minutes at 37 °C. Plates were read on a MicroElisa reader at a wavelength of 450 nm. Absorbance >3-fold above the absorbance from the negative controls were scored as positive reactions.
In Vivo Challenge with TC-1 cells expressing S antigen The production and maintenance of TC-1 cells has been described previously. In brief, HPV-16 E6, E7 and ras oncogene were used to transform primary C57BL/6 mice lung epithelial cells to generate the TC-1 line. For the construction of TC-l/S, supernatant from phoenix cells transfected with pMSCV- S was incubated with 50% confluent TC-1 cells in the presence of polybrene. The transduced TC-1 cells were further selected by growing in culture medium containing 10 μg/ml of puromycin for 5 days. The expression of S antigen was confirmed by Western analysis. For the challenge experiment, the immunized mice (10 per group) were subcutaneously challenged with 5xl05 cells/mouse in the right leg one week after last vaccination, and then monitored twice a week to check the formation of TC-l/S tumor. In vivo antibody depletion was performed to determine the contribution of various lymphocyte subsets to the protection, as described previously. The following mAbs were used: GK1.5 for CD4 depletion, mAb 2.43 for CD8 depletion, and mAb PK136 was used for NKl.l depletion. Depletions were started one week after final vaccination. The immunized mice (10 per group) were challenged s.c. (5xl05 cells/mouse) with TC-l/S cells one week after initiation of Ab depletion. The depletion was terminated on day 32 after challenge. The completeness of depletion was examined by flow cytometry. For each time point of analysis, >99% depletion of the appropriate subset was achieved while retaining normal levels of cells ofthe other subsets.
S-specific antibody responses The presence ofthe S-specific antibody in sera from the mice immunized with the DNA vaccines encoding no insert, S, SI, Si, S2, CRT or CRT/SI via a gene gun was detected by
Western blot analysis. Immune serum samples were collected from DNA- vaccinated mice two weeks after the last vaccination and were diluted to 1 :250 with PBS. Equal amounts of proteins
(50 μg) from TC-l/No insert or TC-l/S lysates were probed with the diluted antisera. The end- point dilution titer of S-specific antibodies in the sera from DNA- vaccinated C57BL/6 mice were determined by ELISA using 96 microwell plates coated with TC-l/S or TC-l/No insert cells. After overnight, the cells (5xl04/well) were washed once in phosphate buffered saline (PBS), then fixed and permeabilized using Cyofix/Cytoperm Kit (Pharmingen). Plates coated with cells were incubated with (0.3 ml/well) PBS - 0.05% Tween 20 (PBT) containing 2% bovine serum albumin for 60 minutes at 37 °C and washed again with PBT. Serial dilutions of the tested sera were added (0.1 ml/well) and the plates were incubated for 60 minutes at 37 °C. The plates were washed with PBT and were incubated with (0.1 ml/well) peroxidase-conjugated rabbit anti-mouse IgG (Zymed, San Francisco, CA) for 30 minutes at 37 °C. The plates were washed with PT and incubated with (0.1 ml/well) peroxidase substrate (according to Sigma instructions) for 15 minutes at 37 °C. Plates were read on a MicroElisa reader at a wavelength of 450 nm. Reading higher than 3-fold negative controls were scored as positive reactions. Statistical Analysis All results expressed as means ± SD are representative of at least two different experiments. Data for intracellular cytokine staining with flow cytometry analysis and in vivo viral challenge experiments were evaluated by analysis of variance (ANOVA). Comparisons between individual data points were made using a student's t-test. In the tumor protection experiment, the principal outcome of interest was time to tumor development. The event time distributions for different mice were compared using the method of Kaplan and Meier and the log-rank statistic. p< 0.05 was considered significant.
Results
Cells transfected with the various S DNA immunogenic constructs expressed comparable levels of S protein In order to characterize protein expression in cells (293 line) transfected with DNA constructs encoding the various domains of SARS-CoN S protein, Western blot analysis was done using rabbit anti-S polyclonal antibody. As shown in Figure 7A, lysates from 293 cells transfected with the various DΝA constructs revealed protein bands correlated with the expected sizes of S, SI, Si and S2. Furthermore, levels of protein expression by 293 cells transfected with the various DΝA constructs appeared to be comparable. As shown in Figure 7B, only cells transfected with the SI DΝA construct were able to secrete SI protein. In contrast, cells transfected with S, Si or S2 DΝA did not secrete the encoded proteins. DNA encoding SI generates the highest S-specific antibody immune response in vaccinated mice.
To determine the antibody immune response induced by immunization with the various DNA constructs encoding the domains of S protein, a study was done in which mice received pcDNA3-S, ρcDNA3-Sl, PcDNA3, Si, pcDNA3-S2 or pcDNA3. Two weeks after the last booster, sera were collected and antibodies against S protein were measured. TC-l/S cell lysates were used as a source of S protein for Western Blot analysis as well as for ELISA. Figure 8 A shows that sera diluted 1 :250 as probes in Western blots revealed that mice given the SI DNA construct generated the highest S-specific antibody immune response. Immunization with DNA encoding the full length S protein also resulted in an S-specific antibody responses, albeit lower. Similar results were observed when testing these sera in ELISA. As shown in Figure 8B, mice given SI DNA generated the greatest S-specific antibody responses. Thus, administration of DNA that encodes the receptor-binding domain (SI) of SARS-CoN S protein is capable of generating stronger S-specific antibody responses than does administration of DΝA encoding the full length S protein. SI is therefore an excellent target for development of preventive SARS-CoN DΝA vaccines ofthe type disclosed herein.
Vaccination with DΝA encoding SARS CoV SI generates the higher numbers of S-specific CD8+ T cells in vivo To assess the numbers of S-specific CD8+ T-cell precursors that are triggered following administration of various ofthe DΝA constructs to mice, intracellular cytokine staining was done in conjunction with flow cytometric analysis using CD3+ cells enriched T cells from spleens of vaccinated mice one week after the last vaccination. Enriched CD3 T cells enriched cells from immunized mice were stimulated in vitro with DCs transfected with DΝA encoding
SARS CoV S protein (or as a control, DΝA without an insert). After overnight incubation, cells were stained for both CD8 and intracellular JJFΝγ. As shown in Figure 9A and 9B, pcDΝA3-Sl induced the highest number of S-specific IFNγ CD8+ T-cell precursors among all the DNA constructs tested (p<0.01). Vaccination with pcDNA3-S or pcDNA3-Si also induced S-specific
CD8+ T cells to a larger extent that did pcDNA3-S2 (ρ<0.05), but less than did SI DNA. These results indicate that pcDNA3-Sl is the more potent immunogen for S-specific CD8+ T cell immune responses. Taken together, the results argue in favor or the receptor binding domain of
SARS CoV S protein represents as a desirable target for generating SARS-CoV S specific antibodies as well as CD8+ T cell reactivity (likely cytotoxic T cells).. Cells transfected with the DNA encoding calreticulin linked to SI generate comparable levels of S protein as DNA encoding SI. Some of th present inventors identified the use of DNA constructs comprising sequences encoding calreticulin (CRT) as an excellent strategy to enhance antigen-specific and T cell mediated immune responses to DNA vaccines that comprise DNA encoding an antigen. In the present, a DNA construct was made that encoded CRT linked to SI. Expression of such DNA was tested by transfecting 293 cells with the DNA constructs and performing Western blot analysis using rabbit anti-S polyclonal antibody. As shown in Figure 10 A, lysates from 293 cells transfected with the CRT/SI or SI DNA revealed protein bands correlated with the expected sizes ofthe fusion polypeptide CRT/SI or of SI alone. Furthermore, the level of protein expression by 293 cells transfected with the these DNA constructs appeared to be comparable. As shown in Figure 10B, cells transfected with CRT/SI DNA and with SI DNA construct could secrete SI protein.
DNA encoding CRT/SI is a potent stimulator of S-specific antibody responses in vaccinated mice
Mice were immunized with pcDNA3-CRT/Sl, ρcDNA3-Sl,'PcDNA3-CRT or ρcDNA3. Two weeks after the last booster, sera were collected and assayed for antibodies against S protein.
TC-l/S cell lysates were used as a source of S protein for Western Blot analysis as well as in
ELISA. As shown in Figure 11 A, examining sera diluted at 1:250 in Western blot analysis, it was found that mice vaccinated with the CRT/SI DNA generated the highest S-specific antibody i response. Vaccination with DNA encoding SI also generated S-specific antibody responses, albeit lower than vaccination with the CRT/SI construct. ELISA gave similar results in characterizing the S-specific antibody response. As shown in Figure 1 IB, mice vaccinated with
CRT/S 1 DNA generated the highest S-specific antibody response. Thus, vaccination with DNA encoding CRT linked to a SARS antigen, the receptor-binding domain (SI) of SARS-CoV S protein, generated enhanced S-specific antibody responses vs vaccination with DNA encoding the SI protein alone.
Vaccination with DNA encoding CRT/SI stimulates S-specific CD8+ T cells in vaccinated mice To assess the quantity of S-specific CD8+ T-cell precursors generated by admimstration ofthe various DNA S protein constructs (pcDNA3 -CRT/SI, pcDNA3-Sl, PcDNA3-CRT or empty pcDNA3), intracellular cytokine staining was performed with flow cytometric analysis using CD3 T cells enriched from spleens of vaccinated mice one week after the last vaccination. These T cells were stimulated in vitro with DCs transfected with DNA encoding S protein or control DNA, and stained for both CD8 and intracellular IFNγ. As shown in Figure 12A and 12B, vaccination with pcDNA3-CRT/Sl was the most potent in generating S-specific IFNγ+ CD8+ T-cell (compared to vaccination with pcDNA3-Sl) (p <0.005). Vaccination with either ofthe two controls (pcDNA3-CRT or pcDNA3) resulted in only background levels of S- specific CD8+ T cells. These results indicate that vaccination with pcDNA3 -CRT/SI chimeric construct generates higher numbers of antigen-specific CD8 T cells in vivo compared to vaccination with pcDNA3-Sl. Thus, in addition to some ofthe present inventors' successes using the CRT strategy with human papillomavirus vaccines (the E6 and E7 protein; see, for example WO02/012281) the present results show that SI DNA vaccines employing the CRT strategy are potent in generating SARS-CoV S specific humoral and CD8+ T cell-mediated immune responses.
Vaccination with DNA encoding CRT/SI is generates preventive antitumor immunity against tumor cells that are engineered to express the SARS CoV S protein A non-infectious model system was employed to determine a therapeutic outcome ofthe immunity generated by the present constructs and the enhancing effect ofthe CRT DNA on such immunity. An antitumor response was examined using an in vivo tumor protection assay. TC-l/S tumor cells, transfected to express the S protein were the target ofthe immunity. As shown in Figur
13A, 100% of mice receiving CRT/SI DNA remained tumor-free 35 days after TC-l/S challenge. Ii comparison, only 40% ofthe mice receiving SI DNA remained tumor-free at this time. All mice vaccinated with control CRT constructs or pcDNA3 plasmid controls grew tumors within two weeks after challenge. To confirm which subsets of lymphocytes were important for this therapeutic effect, an in vi\ antibody depletion study was conducted. Its results appear in Figure 8B. All mice depleted of CD8 cells grew tumors within 10 days after TC-l/S challenge. In contrast, 100% of mice depleted of CD cells or NK cells remained tumor-free 35 days after challenge. Thus, CD8 T cells are required for t therapeutic (antitumor) effect ofthe CRT/SI DNA vaccine. Thus, the T cell-mediated immunity generated by immunization or vaccination with CRT/SI DNA can effect clinical-type therapeutic results, measured here as an antitumor effect.
EXAMPLE 3 DNA Vaccines Targeting the Membrane Protein (M) of SARS-CoV Materials and Methods
Plasmid DNA Constructs and DNA Preparation In the current study we used the mammalian expression vector, pcDNA3.1/myc-His (-) (Invifrogen, Carlsbad, CA) for our DNA vaccine studies. For the generation of pcDNA3-M- myc, the DNA fragment encoding SARS-Co V membrane antigen (M) was amplified with PCR using a set of primers: 5 ' -aaagaattcatggcagacaacggtactattac-3 ' , SEQ ID NO:50 5 ' -tttggtaccttactgtactagcaaagcaatat-3 ' SEQ ID NO:51 and pGEX-l-MG6 as a template. The amplified product was further cloned into the EcoRI/Kpnl sites of pcDNA3.1/myc-His (-) vector. To generate pcDNA3-CRT-myc, CRT DNA segment was isolated from pcDNA3-CRT and cloned into the XhoI/EcoRI sites of pcDNA3.1/»zyc-His(-). For the generation of pcDNA3-CRT/N-myc, the amplified M DNA was cloned into the EcoRI/Kpnl sites of pcDNA3-CRT-myc. The accuracy of these constructs was confirmed by DNA sequencing. The DNA was amplified in E. coli DH5α and purified as described previously .
Cell Lines: Construction of DC expressing M The production and maintenance of TC-1 cells and DC-1 cells was described above. To generate SARS CoV membrane antigen presenting cell, the immortalized DC line, which was kindly provided by Dr. Kenneth Rock (University of Massachusetts, Worcester, MA), was genetically manipulated by retroviral system. For this, the cDNA of M was isolated from pGEX-l-MG6 after BamHI/EcoRI restriction and further cloned into the Bglll/EcoRI sites of pMSCV vector (Invifrogen). Phoenix (φNX ) packaging cells were transfected with pMSCV-M or pMSCV using Lipofectamine 2000. Supematants from the transfected phoenix cells were incubated with 50% confluent DC in the presence of polybrene (8ug/ml; Sigma). Following transduction, the retroviral supematants were removed, and DCs were propagated in culture medium containing 7.5 μg/ml of puromycin for selection. The expression of M antigen was confirmed by western blot analysis. For the generation of TC-l/M and DC-l/M cells, we first generate retroviral vector encoding the M protein of SARS-CoN. The phoenix packaging cells were transfected with pMSCN-M or pMSCN using Lipofectamine 2000. Supernatant from the transfected Phoenix (φΝX ) cells was incubated with 50% confluent TC-1 or DC-1 cells in the presence of polybrene (8 μg/ml; Sigma). Following transduction, the retroviral supematants were removed from the transduced cells, and DCs were propagated in culture medium containing 7.5 μg/ml of puromycin for selection. The transduced TC-1 or DC-1 cells were further selected by growing in culture medium containing 10 μg/ml of puromycin for 5 days. The expression of M antigen was confirmed by Western blot analysis. All cells were maintained in supplemented RPMI medium as above. Western Blot Analysis The expression of M protein in TC-l/M, DC-l/M or 293 cells transfected with pcDNA3.1/m) c-His (-) encoding no insert, CRT, M, or CRT/M DNA was characterized by Western blot analysis. 5xl06 293 cells were transfected with 20 μg of DNA using lipofectamine 2000 (Life Technologies, Rockville, MD). The remaining methods were as in the previous Examples.
Mice - were as described above. DNA Vaccination DNA-coated gold particles were prepared and used as described above. C57BL/6 mice were immunized with 2 μg ofthe plasmid encoding no insert, CRT, M, or CRT/M protein. Intracellular Cytokine Staining and Flow Cytometry Analysis This was described above. DC cells expressing M antigen (DC/M), 105 were incubated with 106 isolated CD3+ T cell for 16 hours. The DC cells not expressing M antigen (DC/No insert) were used as a negative control. After activation, T cells were stained for surface CD8 or CD4 and intracellular IFNγ or IL-4 and analyzed flow cytometrically as described. In Vivo Challenge with TC-1 expressing M antigen The production and maintenance of TC-1 cells has been described previously. For the construction of TC-l/M cells, supernatant from the transfected phoenix cells with pMSCV-M was incubated with 50% confluent TC-1 as described in the earlier Examples. The expression of M antigen was confirmed by Western blot. Tumor Challenge experiments were as above. In vivo antibody depletions was performed as above. Statistical Analysis - as above
RESULTS
Cells transfected with M or CRT/M DNA vaccines generate comparable levels of M protein. In order to characterize M protein expression in cells (293 line) transfected with DNA constructs encoding SARS-CoV M or CRT/M, Western blot analysis was done using mouse anti-Myc antibody. 293 cells transfected with DNA encoding CRT or DNA without insert were used as controls. As shown in Figure 14, lysates from cells transfected with the various DNA constructs revealed protein bands having the expected sizes of M and CRT/M. 293 cells transfected with M and CRT/M DNA vaccines expressed comparable levels ofthe encoded proteins.
Vaccination with DNA encoding CRT M generates higher numbers of M-specific CD8+ T cells in vivo To assess the quantity of M-specific CD 8 T-cell precursors generated by the pcDNA3, pcDNA3-CRT, pcDNA3-M or pcDNA3-CRT/M vaccine constructs in vaccinated mice, To assess the numbers of M-specific CD8+ T-cell precursors that are triggered following administration of various ofthe DNA constructs (pcDNA3 control, pcDNA3-CRT control, pcDNA3-M and pcDNA3-CRT/M) to mice, intracellular cytokine staining was done in conjunction with flow cytometric analysis using spleen cells from the vaccinated mice one week after the last vaccination. Pooled spleen cells were stimulated in vitro with DCs transfected with
DNA encoding M protein or, as a control, DNA with no insert and stained for both CD8 and intracellular IFNγ. As shown in Figure 15A and 15B, pcDNA3-CRT/M induced the highest number of M-specific IFNγ+ CD8+ T-cell precursors when compared to pcDNA3-M (p <0.005).
Vaccination with pcDNA3-CRT or pcDNA3 only generated background levels of M-specific
CD8+ T cells. These results indicate that vaccination with pcDNA3 -CRT/M is the more potent immunogen for M-specific CD8+ T cells immune responses. Thus M protein DNA vaccines employing the CRT strategy are effective in stimulating strong SARS-CoV M-specific CD 8+ T cell reactivity (likely to include cytotoxic T cells).
Vaccination with DNA encoding CRT/M generates high numbers of M-specific CD4+ T helper cells To assess the numbers of M-specific CD4+ T cells generated by the same DNA constructs, intracellular cytokine staining and flow cytometric analysis was done on spleen cells from vaccinated mice harvested one week after the last vaccination. Pooled cells were stimulated in vitro with DCs transfected with DNA encoding M protein or, as a control, DNA with no insert. After overnight incubation, cells were stained for both CD4 and intracellular IFNγ or IL-
4. As shown in Figure 16A and 16B, pcDNA3-CRT/M induced the higher number of M- specific IFNγ+ CD4+ T helper type 1 (Thl) cells compared to pcDNA3-M (p<0.005). Control vaccination (pcDNA3-CRT or pcDNA3) generated only background levels of M-specific CD4+ Thl cells. These results further support the success ofthe CRT strategy in generating greater numbers of M-specific CD4+ Thl as compared to immunization with DNA encoding antigen alone (e.g., pcDNA3-M). IL-4-secreting M-specific CD4+ T helper cells ofthe Th2 class were measured after administering the two experimental and two control DNA vaccine preparations as assessed by intracellular cytokine staining followed by flow cytometric analysis. As shown in Figure 17A and 17B, vaccination with pcDNA3-CRT/M triggered higher numbers of IL-4-secreting M- specific CD4+ T cells compared to pcDNA3-M (p value<0.05), although the absolute numbers of IL-4-secreting M-specific CD4+ T cells was lower than the number of IFNγ-secreting, M- specific CD4+ Thl cells in CRT/M-vaccinated mice. The two control plasmids, pcDNA3-CRT and pcDNA3 resulted in only background levels of M-specific CD4+ Th2 cells. Taken together, the results indicate that M DNA vaccines employing the CRT strategy are potent stimuli for SARS-CoV M-specific IFN-γ-secreting, CD4+ and CD8+ T cells.
Immunization with pcDNA3-CRT/M generates protective antitumor immunity against tumor cells that are engineered to express the SARS CoV M protein. As discussed in Example 2, a non-infectious model system was employed to determine a therapeutic outcome ofthe immunity generated by the present constructs and the enhancing effect ofthe CRT DNA on such immunity. An antitumor response was examined using an in vivo tumor protection assay. TC-l/M tumor cells, transfected to express the M protein, were the target ofthe immunity. As shown in Figure 18 A, 100% of mice receiving pcDN A3 -CRT/M remained tumor-free six weeks after TC-l/M challenge. In contrast, all animals vaccinated with the control plasmid (no insert) or the pcDNA3-CRT plasmid, developed tumors within 10 days after the tumor challenge. Therefore, the CRT M DNA construct was capable of generating not only a high number of M-specific T cells in vitro but also a protective antitumor effect against challenge with M-expressing tumor cells in vaccinated mice. To confirm which subsets of lymphocytes were important for this therapeutic effect, an in vivo antibody depletion study was conducted. Its results appear in Figure 18B. All mice depleted of CD8+ T cells grew tumors within 15 days of TC-l/M challenge. In contrast, 100% of mice depleted of CD4+ T cells or NK cells remained tumor-free. Thus, CD8+ T cells are required for the therapeutic (antitumor) effect ofthe CRT/SI DNA vaccine. Thus, the T cell-mediated immunity generated by immunization or vaccination with CRT/SI DNA can effect clinical-type therapeutic results, measured here as an antitumor effect.
The references cited above are all incorporated by reference herein, whether specifically incorporated or not. Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope ofthe invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations ofthe invention following, in general, the principles ofthe 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 as follows in the scope ofthe appended claims.

Claims

WHAT IS CLAIMED IS:
1. A nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises: (a) a first nucleic acid sequence encoding a first polypeptide that comprises an endoplasmic reticulum chaperone polypeptide; (b) optionally, fused in frame with the first nucleic acid sequence, a linker nucleic acid sequence encoding a linker peptide; and (c) a second nucleic acid sequence that is linked in frame to said first nucleic acid sequence or to said linker nucleic acid sequence and that encodes an antigenic polypeptide or peptide from a SARS-CoV, said SARS-CoV antigenic polypeptide or peptide being one that is the target of a protective or neutralizing immune response.
2. The nucleic acid molecule of claim 1, wherein the antigenic peptide comprises an epitope that binds to a MHC class I protein.
3. The nucleic acid molecule of claim 2, wherein said epitope is between about 8 amino acid residues and about 11 amino acid residues in length.
4. The nucleic acid molecule of claim 1 wherein the chaperone polypeptide comprises calreticulin or an immunologically active fragment or variant thereof.
5. The nucleic acid molecule of claim 4, wherein said calreticulin is human calreticulin having the amino acid sequence SEQ ID NO:2 and wherein the active fragment or variant is a fragment or variant of SEQ ID NO:2.
6. The nucleic acid molecule of claim 4, wherein the first nucleic acid sequence comprises the coding portion of SEQ ID NO: 1 , or of a fragment or variant thereof.
7. The nucleic acid molecule of claim 5 wherein the calreticulin consists essentially of a sequence from about residue 1 to about residue 180 of SEQ ID NO:2.
8. The nucleic acid molecule of claim 5, wherein the calreticulin consists essentially of a sequence from about residue 181 to about residue 417 of SEQ ID NO:2.
9. The nucleic acid molecule of claim 1, wherein the chaperone polypeptide comprises (a) a calnexin polypeptide or an equivalent thereof; (b) an ER60 polypeptide or an equivalent thereof; (c) a tapasin polypeptide or an equivalent thereof; or (d) a GRP94/GP96 polypeptide, a GRP94 polypeptide or an equivalent thereof.
10. The nucleic acid molecule of any of claims 1 -9 wherein the antigen is one which is present on, or cross-reactive with an epitope of a SARS-CoV structural protein
11. The nucleic acid molecule of claim 10 wherein the antigen is from a strain or isolate of SARS-CoV selected from the group consisting of TOR2 and TWl.
12. The nucleic acid molecule of claim 10 wherein the structural protein is selected from the group consisting ofthe Spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein.
13. The nucleic acid molecule of claim 10 wherein the structural protein is the S protein having an amino acid sequence SEQ ID NO: 14 or a domain or fragment thereof.
14. The nucleic acid molecule of claim 13 wherein the domain or fragment is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17
15. The nucleic acid molecule of claim 10 wherein the structural protein is the E protein having an amino acid sequence SEQ ID NO: 19 or a fragment thereof.
16. The nucleic acid molecule of claim 10 wherein the structural protein is the M protein having an amino acid sequence SEQ ID NO:21 or a fragment thereof.
17. The nucleic acid molecule of claim 10 wherein the structural protein is the N protein having an amino acid sequence SEQ ID NO:23 or a fragment thereof.
18. The nucleic acid molecule of claim 10 having a sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:27 or SEQ ID NO:30.
19. An expression vector or cassette comprising the nucleic acid molecule of any of claims 1-9 operatively linked to (a) a promoter; and (b) optionally, additional regulatory seqμences that regulate expression of said nucleic acid in a eukaryotic cell.
20. The expression vector or cassette of claim 19 wherein the antigen is one which is present on, or cross-reactive with an epitope of a SARS-CoV structural protein.
21. The expression vector or cassette of claim 20 wherein the structural protein is selected from the group consisting ofthe Spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein.
22. The expression vector or cassette of claim 20 which is a viral vector or a plasmid.
23. The expression vector or cassette of claim 20, wherein the chaperone polypeptide comprises a calreticulin polypeptide or an active fragment thereof.
24 The expression vector or cassette of claim 23 wherein the calreticulin polypeptide: (i) comprises amino acid sequence SEQ ID NO:2 ; or (ii) is encoded by the coding portion ofthe nucleic acid molecule having the sequence SEQ ID NO: 1.
25. The expression vector or cassette of claim 20, wherein the chaperone polypeptide comprises any one or more of a tapasin, an ER60, an ERP94 or a calnexin polypeptide, or an equivalent thereof.
26. A cell which has been modified to express the nucleic acid molecule of any of claims 1-9.
27. A cell which has been modified to comprise the expression vector or cassette of claim 19.
28. A particle suitable for introduction into a cell or an animal by particle bombardment comprising the nucleic acid of any of claims 1-9.
29. A particle suitable for introduction into a cell or an animal by particle bombardment comprising expression cassette or vector of any of claims 20.
30. The particle of claim 29 wherein the particle comprises gold.
31. A fusion or chimeric polypeptide comprising (a) a first polypeptide comprising an endoplasmic reticulum chaperone polypeptide; and (b) a second polypeptide comprising an antigenic polypeptide or peptide from a SARS-CoV, said SARS-CoV antigenic polypeptide or peptide being one that is the target of an anti- viral immune response.
32. The fusion or chimeric polypeptide of claim 31 wherein the chaperone polypeptide comprises a calreticulin polypeptide, an active fragment thereof, or a homologue thereof.
33. The fusion or chimeric polypeptide of claim 32 wherein the calreticulin polypeptide is a human calreticulin polypeptide that:: (i) comprises amino acid sequence SEQ ID NO:2 ; or (ii) is encoded by a coding portion ofthe nucleic acid molecule having the sequence SEQ ID NOT.
34. The fusion or chimeric polypeptide of claim 31 , wherein the antigenic peptide or polypeptide conesponds to a SARS-CoV structural protein is a selected from the group consisting ofthe Spike (S) protein, the envelope (E) protein, the membrane (M) protein, and the nucleocapsid (N) protein.
35. The fusion or chimeric polypeptide of claim 31 wherein the chaperone polypeptide and the antigenic polypeptide or peptide are linked by a chemical linker.
36. The fusion polypeptide of any of claims 31-35 wherein the first polypeptide is N- terminal to the second polypeptide.
37. The fusion polypeptide of any of claims 31-35 wherein the second polypeptide is N-terminal to the first polypeptide.
38. The fusion or chimeric polypeptide of claim 31 wherein the chaperone polypeptide comprises any one or more of a tapasin, an ER60, an ERP94 or a calnexin polypeptide, or an equivalent thereof.
39. A pharmaceutical composition capable of inducing or enhancing a SARS-CoV antigen-specific immune response, comprising: (a) pharmaceutically and immunologically acceptable excipient in combination with; (b) the nucleic acid molecule of claim 1-9.
40. A pharmaceutical composition capable of inducing or enhancing a SARS-CoV antigen-specific immune response, comprising: (a) pharmaceutically and immunologically acceptable excipient in combination with; (b) the expression vector or cassette of claim 19.
41. A pharmaceutical composition capable of inducing or enhancing a SARS-CoV antigen-specific immune response, comprising: (a) pharmaceutically and immunologically acceptable excipient in combination with; (b) the expression vector or cassette of claim 20.
42. A pharmaceutical composition capable of inducing or enhancing a SARS-CoV antigen-specific immune response, comprising: (a) pharmaceutically and immunologically acceptable excipient in combination with; (b) , the expression vector or cassette of claim 21.
43. A pharmaceutical composition capable of inducing or enhancing a SARS-CoV antigen-specific immune response, comprising: (a) phannaceutically and immunologically acceptable excipient in combination with; (b) the fusion or chimeric polypeptide of claim 31.
44. A pharmaceutical composition capable of inducing or enhancing a SARS-CoV antigen-specific immune response, comprising: (a) pharmaceutically and immunologically acceptable excipient in combination with; (b) the particle of claim 29.
45. A method of inducing or enhancing a SARS-CoV antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim 39, thereby inducing or enhancing said response.
46. A method of inducing or enhancing a SARS-CoV antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim 40, thereby inducing or enhancing said response.
47. A method of inducing or enhancing a SARS-CoV antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim 41, thereby inducing or enhancing said response.
48. A method of inducing or enhancing a SARS-CoV antigen specific immune response in a subject comprising admimstering to the subject an effective amount ofthe pharmaceutical composition of claim 42, thereby inducing or enhancing said response.
49. A method of inducing or enhancing a SARS-CoV antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim 43, thereby inducing or enhancing said response.
50. A method of inducing or enhancing a SARS-CoV antigen specific immune response in a subject comprising administering to the subject an effective amount ofthe pharmaceutical composition of claim 44, thereby inducing or enhancing said response.
51. The method of claim 45, wherein the response is mediated at least in part by CD8+ cytotoxic T lymphocytes (CTL).
52. The method of claim 45, wherein the response is mediated at least in part by antibodies.
53. The method of claim 45 wherein said administering is by a intramuscular, intradermal, or subcutaneous route.
54. The method of claim 45 wherein administering is by biolistic injection of said nucleic acid molecule.
55. A method of inducing or enhancing an antigen specific lymphocyte response or immune response in cells or in a subject comprising providing to said cells or to said subject an effective amount ofthe pharmaceutical composition of claim 39-44, thereby inducing or enhancing said response.
56. A method of increasing the numbers or lytic activity of CD8+ T cells specific for a selected SARS-CoV antigen in a subject, comprising administering to said subject an effective amount ofthe pharmaceutical composition of claim 45, wherein (i) said nucleic acid molecule encodes said selected antigen, and (ii) said selected SARS-CoV antigen comprises an epitope that binds to, and is presented on the cell surface by, MHC class I proteins, thereby increasing the numbers or activity of said CTLs.
57. A method of inhibiting a viral infection by a SARS-CoV or preventing or diminishing spread of said virus in a subject, comprising administering to said subject an effective amount of a pharmaceutical composition of claim 45, wherein said nucleic acid molecule encodes one or more SARS-CoV epitopes present on said virus or on virus infected cells in said subject, thereby inhibiting said infection or preventing or diminishing said spread.
58. The method of claim 57, further comprising before, together with or after said administration of said pharmaceutical composition, administering to said subject a second composition having effective SARS-CoV-directed anti- viral activity.
PCT/US2004/039579 2003-11-24 2004-11-24 DNA VACCINES TARGETING ANTIGENS OF THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS (SARS-CoV) WO2005081716A2 (en)

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US8128922B2 (en) 1999-10-20 2012-03-06 Johns Hopkins University Superior molecular vaccine linking the translocation domain of a bacterial toxin to an antigen
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