WO2004091524A2 - Respiratory virus vaccines - Google Patents

Abstract

The invention provides vaccines and methods for preventing, treating, and diagnosing respiratory virus infection.

Description

PATENT ATTORNEY DOCKET NO. 06132/080 O3

RESPIRATORY VIRUS VACCINES Background of the Invention

Severe Acute Respiratory Syndrome (SARS) is a life-threatening respiratory illness that has recently been reported in Asia, North America, and Europe. SARS is thought to have originated in the Guangdong Province of China, and then to have been transported to Hong Kong by an infected healthcare worker who, when visiting Hong Kong, was hospitalized and died. SARS is thought to be transmissible in droplet form. Thus, it may be transmitted when an infected individual coughs or sneezes droplets into the air, and someone else breathes them in. SARS may also be transmitted more broadly tlirough the air, or by the touching of objects that are contaminated. The illness usually begins with a fever, often accompanied by chills, headache, general discomfort, body aches, and/or mild respiratory symptoms. As the disease progresses, some patients develop a dry, non-productive cough. In addition, in some cases, the disease can progress to the point where mechanical ventilation is required to enable sufficient oxygen to enter a patient's bloodstream.

Viruses in the Coronaviradae family are characterized by a halo or crown-like (corona) appearance on their outer shell when viewed by microscopy. These viruses are a common cause of mild to moderate upper-respiratory illness in humans, and may account for up to one-third of cases of the common cold. Coronaviruses are also often found in animals, such as chickens, pigs, dogs, and cats, in which they can cause illnesses that range from diarrhea to respiratory infection. Further, coronaviruses have been found to survive in the environment for as long as three hours. It has been determined that a previously unrecognized coronavirus can be found in samples from patients with SARS.

Summary of the Invention The invention provides vaccines for inducing an immune response to a human coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS) in a patient. These vaccines can include a spike protein and/or a nucleocapsid protein of the virus, or immunogenic fragments of either or both of these proteins, and a pharmaceutically acceptable earner or diluent. Specific examples of spike protein fragments that can be included in the vaccine compositions of the invention are those including the SI domain, the SI domain and the S2 domain, in the absence of the coiled coil region, and the SI and S2 domains, including the coiled coil domain. Further, the spike protein (or fragment) can be present in the foπii of a monomer, a dimer, or a trimer.

Optionally, the vaccine compositions can also include an adjuvant, such as an adjuvant that stimulates a Thl-type immune response (e.g., an ISCOM, Ribi, DC-Choi, QS21, or MPL). Another example of an adjuvant that can be included in the vaccines of the invention is aluminum hydroxide (e.g., alum), ln one example, the proteins of the vaccines of the invention include an amino acid sequence that is substantially identical to the sequence of SEQ ID NO:37 or SEQ ID NO:35, or immunogenic fragments thereof.

The invention also includes additional vaccines for inducing an immune response to human coronaviruses that cause SARS. These vaccines include vectors (e.g., viral vectors) containing a nucleic acid sequence encoding a spike protein or a nucleocapsid protein of the virus, or an immunogenic fragment thereof, and a pharmaceutically acceptable earner or diluent. An example of a vector that can be used in such vaccines is a poxvirus, such as a Modified Vaccinia Ankara (MVA) vector. Another example of such a vector is adenovirus vectors.

The invention also provides methods for producing spike proteins or nucleocapsid proteins of human coronaviruses that cause SARS. These methods involve introducing into cells a vector that includes a nucleic acid sequence encoding the protein, under conditions in which the protein is expressed in the cells. These cells can be, for example, yeast cells, mammalian cells, insect, or bacterial cells.

The invention further provides methods of inducing an immune response to a human coronavirus that causes SARS in patients, by administration of the vaccines described above and elsewhere herein to the patients. The immune response can be prophylactic or therapeutic.

Also, the invention provides substantially pure spike proteins of human coronaviruses that cause SARS, or inmiunogenic fragments thereof. For example, such a protein can include a sequence that is substantially identical to or identical to the sequence of SEQ ID NO:37. or a fragment thereof. The spike proteins and fragments of the invention can be in the form of monomers, dimers. or trimers.

The invention also includes isolated nucleic acid molecules encoding spike proteins of human coronaviruses that cause SARS. Such a nucleic acid molecule can include the sequence of SEQ ID NO:36, or a sequence that hybridizes to the complement of the sequence of SEQ ID NO:36 under highly stringent conditions. The invention also includes nucleic acid molecule probes that include sequences that hybridize to the sequence of SEQ ID NO:36 or the complement thereof under highly stringent conditions.

In addition, the invention provides substantially pure nucleocapsid proteins of human coronaviruses that cause SARS, or immunogenic fragments thereof. For example, such a protein can include a sequence that is substantially identical to or identical to the sequence of SEQ ID NO:35, or a fragment thereof.

The invention also includes isolated nucleic acid molecules encoding nucleocapsid proteins of human coronaviruses that cause SARS. Such a nucleic acid molecule can include the sequence of SEQ ID NO:34, or a sequence that hybridizes to the complement of the sequence of SEQ ID NO:34 under highly stringent conditions. The invention also includes nucleic acid molecule probes that include sequences that hybridize to the sequence of SEQ ID NO:34 or the complement thereof under highly stringent conditions.

Further, the invention includes antibodies (e.g., monoclonal, monospecific, and polyclonal antibodies) that specifically bind to spike proteins or nucleocapsid proteins of human coronaviruses that cause SARS. These antibodies can be used in passive immunization methods, as described elsewhere herein.

By "polypeptide" or "polypeptide fragment" is meant a chain of two or more (e.g., 10, 15, 20, 30, 50, 100, or 200, or more) amino acids, regardless of any post- franslational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally or non-naturally occurring polypeptide. By "post-translational modification" is meant any change to a polypeptide or polypeptide fragment during or after synthesis. Post-translational modifications can be produced naturally (such as during synthesis within a cell) or generated artificially (such as by recombinant or chemical means). A "protein'^" can be made up of one or more polypeptides.

By "spike protein" or "spike polypeptide" is meant a polypeptide that has at least 45%, preferably at least 60%, more preferably at least 75%, 80%, or 85%, and most preferably at least 90%, 95%, 99%, or 100% amino acid sequence identity to the sequence of SEQ ID NO:37. These proteins and polypeptides (or fragments thereof, as well as coιτesponding nucleic acid molecules) can be used in vaccines as described herein, as well as for markers of infection by human coronaviruses that cause SARS.

By "SARS nucleocapsid protein" or "SARS nucleocapsid polypeptide" is meant a polypeptide that has at least 45%, preferably at least 60%, more preferably at least 75%, 80%, or 85%o, and most preferably at least 90%, 95%, 99%o, or 100% amino acid sequence identity to the sequence of SEQ ID NO:35. These proteins and polypeptides (or fragments thereof, as well as coιτesponding nucleic acid molecules) can be used in vaccines as described herein, as well as for markers of infection by human coronaviruses that cause SARS.

Useful polypeptide derivatives, e.g., polypeptide fragments, can be designed using computer-assisted analysis of amino acid sequences in order to identify sites in protein antigens having potential as surface-exposed, antigenic regions (see, e.g., Hughes et al., Infect. Immun. 60(9):3497, 1992). For example, the Laser Gene Program from DNA Star can be used to obtain hydrophilicity, antigenic index, and intensity index plots for the polypeptides of the invention. This program can also be used to obtain information about homologies of the polypeptides with known protein motifs. One skilled in the art can readily use the infonnation provided in such plots to select peptide fragments for use as vaccine antigens. For example, fragments spanning regions of the plots in which the antigenic index is relatively high can be selected. Fragments spanning regions in which both the antigenic index and the intensity plots are relatively high can also be selected, as well as fragments containing conserved sequences, particularly hydrophilic conserved sequences.

By a "spike nucleic acid molecule" is meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA) molecule, that encodes a spike protein (e.g.. a protein encoded by SEQ ID NO:36), a spike polypeptide, or a portion thereof, as defined above.

By a "SARS nucleocapsid protein nucleic acid molecule" is meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA) molecule, that encodes a spike protein (e.g.. a protein encoded by SEQ ID NO:34), a nucleocapsid polypeptide. or a portion thereof, as defined above.

The term "identity" is used herein to describe the relationship of the sequence of a particular nucleic acid molecule or polypeptide to the sequence of a reference molecule of the same type. For example, if a polypeptide or a nucleic acid molecule has the same amino acid or nucleotide residue at a given position, compared to a reference molecule to which it is aligned, there is said to be "identity" at that position. The level of sequence identity of a nucleic acid molecule or a polypeptide to a reference molecule is typically measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, BLAST, or PILEUP/PRETTYBOX programs). These software programs match identical or similar sequences by assigning degrees of identity to various substitutions, deletions, or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine. alanine, valine, isoleucine, and leucme; aspartic acid, glutamic acid, asparagine, and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

The sequence of a nucleic acid molecule or polypeptide is said to be "substantially identical" to that of a reference molecule if it exhibits at least 51%, preferably at least 55%, 60%, or 65%, and most preferably 75%, 85%, 90%, or 95% identity to the sequence of the reference molecule. For polypeptides, the length of comparison sequences is at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acid molecules, the length of comparison sequences is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 1 10 nucleotides. Of course, for polypeptides and nucleic acid molecules, the length of comparison can be any length up to and including full length.

By "probe" or "primer" is meant a single-stranded DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA molecule that contains a complementary sequence (a "target"). The stability of the resulting hybrid depends upon the extent of the base pairing that occurs. This stability is affected by parameters such as the degree of complementarity between the probe and target molecule, and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as the temperature, salt concentration, and concentration of organic molecules, such as formamide, and is determined by methods that are well known to those skilled in the art. Probes or primers specific for spike or nucleocapsid nucleic acid molecules, preferably, have greater than 45% sequence identity, more preferably at least 55-75%o sequence identity, still more preferably at least 75-85%) sequence identity, yet more preferably at least 85-99%) sequence identity, and most preferably 100% sequence identity to the sequences of genes encoding spike or nucleocapsid proteins of a SARS-causing human coronavirus (SEQ ID NOs:36 and 34, respectively). Probes can be detectably labeled, either radioactively or non-radioactively, by methods that are well lαiown to those skilled in the art. Probes can be used for methods involving nucleic acid hybridization, such as nucleic acid sequencing, nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMSA), and other methods that are well known to those skilled in the art.

A molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, a cDNA molecule, a polypeptide, or an antibody, can be said to be "detectably-labeled" if it is marked in such a way that its presence can be directly identified in a sample. Methods for detectably labeling molecules are well lαiown in the art and include, without limitation, radioactive labeling (e.g., with an isotope, such as P or S) and nonradioactive labeling (e.g., with a fluorescent label, such as fluorescein). By a "substantially pure polypeptide" is meant a polypeptide (or a fragment thereof) that has been separated from proteins and organic molecules that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least 60%, by weight, free from the prote: ins and naturally occuning organic molecules with which it is naturally associated. Prefe: rably, the polypeptide is a spike or nucleocapsid polypeptide that is at least 75%, 80%, o: 85%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure spike or nucleocapsid polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid molecule encoding a spike or nucleocapsid polypeptide, or by chemical synthesis. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated components when it is separated from those proteins and organic molecules that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system that is different from the cell in which it is naturally produced is substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only include those that are derived from coronaviruses, but also those synthesized in yeast systems, insect systems, mammalian systems. E. coli, other prokaryotes, or in other such systems (see below).

By "isolated nucleic acid molecule" is meant a nucleic acid molecule that is removed from the environment in which it naturally occurs. For example, a naturally- occuπing nucleic acid molecule present in the genome of cell or as part of a gene banlc is not isolated, but the same molecule, separated from the remaining part of the genome, as a result of, e.g., a cloning event (amplification), is "isolated." Typically, an isolated nucleic acid molecule is free from nucleic acid regions (e.g., coding regions) with which it is immediately contiguous, at the 5' or 3' ends, in the naturally occuning genome. Such isolated nucleic acid molecules can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment.

An antibody is said to "specifically bind" to a polypeptide if it recognizes and binds to the polypeptide (e.g., a spike or nucleocapsid polypeptide), but does not substantially recognize and bind to other molecules (e.g.. non-spike-related or non- nucleocapsid-related polypeptides) in a sample, e.g.. a biological sample, which naturally includes the polypeptide. Antibodies that specifically bind to the spike or nucleocapsid proteins of human coronaviruses causing SARS are also included in the invention.

By "high stringency conditions" is meant conditions that allow hybridization comparable with the hybridization that occurs using a DNA probe of at least 100, e.g., 200, 350, or 500. nucleotides in length, in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7%o SDS, 1 mM EDTA. and 1% BSA (fraction V), at a temperature of 65°C, or a buffer containing 48% fonnamide, 4.8 x SSC, 0.2 M Tris-Cl, pH 7.6, 1 x Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C. (These are typical conditions for high stringency northern or Southern hybridizations.) High stringency hybridization is also relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single sfrand conformational polymorphism analysis, and in situ hybridization. In contrast to northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in Ausubel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998, which is hereby incorporated by reference.

The invention provides several advantages. First, the invention provides approaches to preventing, treating, diagnosing a severe, life-threatening disease that has recently appeared in outbreaks around the world, in a short period of time. Further, the invention provides expression and vector systems that can be used to achieve high levels of expression and efficient delivery of SARS proteins, respectively.

Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims. Brief Description of the Drawings

Figures 1 -36 are schematic illustrations of constructs used in the expression of SARS spike proteins m Pichia pasioris, CHO cells, and Drosophila S2 cells.

In particular, Figure 1 provides the deduced amino acid sequence of pPICZ alpha 1190 clone P5-12 (SEQ ID NO:l); Figure 2 provides a linear map of the construct, including the AOX promoter, alpha signal sequence, spike amino acids 14-1190, and the AOX terminator sequence; Figure 3 provides a circular map of the construct; and Figure 4 provides the nucleotide sequence of this clone, based on the linear map (SEQ ID NO:2).

Figure 5 provides the deduced amino acid sequence of pPICZ alpha 709 clone Pl- 2 (SEQ ID NO:3); Figure 6 provides a linear map of the construct, including the AOX promoter, alpha signal sequence, spike amino acids 14-709, and the AOX terminator sequence; and Figure 7 provides the nucleotide sequence of the clone, based on the linear map (SEQ ID NO:4).

Figure 8 provides the deduced amino acid sequence of pPICZ alpha 719 clone Pl- 2 (SEQ ID NO:5); Figure 9 provides a linear map of the construct, including the AOX promoter, alpha signal sequence, spike amino acids 14-719, and the AOX terminator sequence: and Figure 10 provides the nucleotide sequence of the clone, based on the linear map (SEQ ID NO:6).

Figure 11 provides the deduced amino acid sequence of pPICZ alpha 883 clone P3-10 (SEQ ID NO:7); Figure 12 provides a linear map of the construct, including the AOX promoter, alpha signal sequence, spike amino acids 14-883, and the AOX terminator sequence; and Figure 13 provides the nucleotide sequence of the clone, based on the linear map (SEQ ID NO:8).

Figure 14 provides the deduced amino acid sequence of pPICZ alpha 883m clone P3-10 (SEQ ID NO:9); Figure 15 provides a linear map of the construct, including the AOX promoter, alpha signal sequence, spike amino acids 14-883, and the AOX teπninator sequence; and Figure 16 provides the nucleotide sequence of the clone, based on the linear map (SEQ ID NO: 10). Figure ] 7 provides a circular map of pGAPZ alpha 1 190 clone G5-14; Figure 18 provides the deduced amino acid sequence of the clone (SEQ ID NO:l 1): Figure 19 provides a linear map of the construct, including the GAP promoter, alpha signal sequence, spike ammo acids 14-1190, and the AOX terminator sequence; and Figure 20 provides the nucleotide sequence of the clone (SEQ ID NO:12).

Figure 21 provides a linear map of pGAPZ alpha 709 clone Gl-8, including the GAP promoter, alpha signal sequence, spike amino acids 14-709, and the AOX terminator sequence Figure 22 provides the nucleotide sequence of the clone (SEQ ID NO: 13); and Figure 23 provides the deduced amino acid sequence of the clone (SEQ ID NO: 14).

Figure 24 provides the deduced amino acid sequence of pGAPZ alpha 719 clone Gl-8 (SEQ ID NO: 15); Figure 25 provides a linear map of the construct, including the GAP promoter, alpha signal sequence, spike amino acids 14-719, and the AOX terminator sequence: and Figure 26 provides the nucleotide sequence of the clone (SEQ ID NO: 16).

Figure 27 provides the deduced amino acid sequence of pGAPZ alpha 883 clone G3-7 (SEQ ID NO: 17); Figure 28 provides a linear map of the construct, including the GAP promoter, alpha signal sequence, spike amino acids 14-883, and the AOX terminator sequence: and Figure 29 provides the nucleotide sequence of the clone (SEQ ID NO: 18).

Figure 30 provides the deduced amino acid sequence of pGAPZ alpha 883m clone G3-7 (SEQ ID NO: 19); Figure 31 provides a linear map of the construct, including the GAP promoter, alpha signal sequence, spike amino acids 14-883, and the AOX terminator sequence; and Figure 32 provides the nucleotide sequence of the clone (SEQ ID NO:20).

Figure 33 provides a linear map of pMT-Spike 1190 and the nucleotide (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences of this construct.

Figure 34 provides a linear map of pMT-Spike 719 and the nucleotide (SEQ ID NO:23) and amino acid (SEQ ID NO:24) sequences of this construct. Figure 35 provides a linear map of pMT-Spike 883 and the nucleotide (SEQ ID NO:25) and amino acid (SEQ ID NO:26) sequences of this construct.

Figure 36 provides a linear map of pSecl 190 and the nucleotide (SEQ ID NO:27) and amino acid (SEQ ID NO:28) sequences of this construct.

Figure 37 provides a linear map of pSec719 and the nucleotide (SEQ ID NO:29) and amino acid (SEQ ID NO:30) sequences of this construct.

Figure 38 provides a linear map of pSec883 and the nucleotide (SEQ ID NO:31) and amino acid (SEQ ID O:32) sequences of this construct.

Figure 39 is a schematic representation of the structure of SARS S protein and target antigenic domains selected for expression.

Figure 40 is a schematic representation of approaches described herein for obtaining S protein expression in the hosts Pichiapastoris, Drosophila S2 Schneider, and CHO cells.

Figure 41 is a schematic representation of a generalized strategy for constitutive (CHO) and inducible (S2) expression of recombinant spike protein.

Figure 42 shows PCR screening and Western blot analysis of transiently fransfected S2 cells.

Figure 43 shows RT-PCR confiimation of mRNA synthesis of S protein candidates 719. 883. and 1 190 in CHO cells.

Figure 44 is a schematic representation of a generalized strategy for expression of recombinant S protein in Pichia pastoris.

Figure 45 shows S gene specific PCR confinning integration into Pichia pastoris.

Figure 46 shows constitutive expression of the S protein in Pichia pastori :

Figure 47 shows a scheme for fractionation of high molecular weight S glycoprotein, as well as analysis of the iimnunoreactivity of the high molecular weight complex.

Figure 48 shows a scheme for purification of high molecular weight S glycoprotein (1190), as well as immunoblot analysis of the purified material. Figure 49 shows Anti-SARS-CoV (hyperimmune) and Anti-SARS (human convalescent sera) analysis of pGAP-1 190 purified from Pichia pastoris supernatant (pre/post Endonuclease H treatment).

Figure 50 shows the results of mass spectroscopy (MALDI-ESI) of S glycoprotein expressed in Pichia pastoris (SEQ ID NO:33).

Figure 51 A shows the results of SDS-PAGE and Coomassie blue staining of fractionated Pichia pastoris-deήved rS glycoprotein (cAl) following diafilfr-ation tlirough a >300 kDa membrane cut-off. Ten μl of lOx concentrate was loaded. Figure 5 IB shows the iimnunoreactivity of clarified supernatant from a growing culture of cAl material 48 hours following conversion from batch to fed-batch fermentation with two conformational dependent monoclonal antibodies.

Figure 52 shows the results of size exclusion HPLC over TSK SW4000 _L (7.8 mm x 30 cm). The column was equilibrated with 0.1 M phosphate containing 0.25 M sodium chloride, pH 7.0 and appropriate size standards were included. Panel A shows a profile of diafiltered culture supernate harvested from cAl fermentation. Fractionated samples were harvested and their immunoreactivity against the anti-SARS polyclonal ( 1 :200) was evaluated in a dot blot format (5 μl/dot). Panel B shows the results of a refolding study on soluble aggregate. Samples were normalized for HMW soluble aggregate.

Figure 53 shows determination of the molecular mass of fractionated fennentation samples by size exclusion HPLC over TSK SW4000_XL coupled to a light scattering detector (Wyatt Technologies). The molar mass of selected peaks was calculated from the intensity of scattered light, times the square of the change in refractive index with respect to concentration. The separation range for this particular column is from 20,000 - 7,000,000 daltons.

Figure 54 shows Coomassie stain (SDS-PAGE; A) and Immunoblot (anti-SARS- CoV polyclonal; B) analysis of the expression of rS glycoprotein monomer in continuous culture.

Figure 55 shows native PAGE analysis of rS glycoprotein by Coomassie stain (PAGE; A) and Immunoblot (anti-SARS-CoV polyclonal hyperimmune). Figure 5b is a graph showing SE-HPLC analysis of rS glycoprotein HMW complexes.

Figure 57 shows native PAGE and immunoreactivity profiling with SARS- specific antibodies.

Figure 58 is a graph showing the fractionation and immunoreactivity profile of HMW rS glycoprotein.

Figure 59 is a schematic representation of the vaccinia insertion vector pTK53- gpt-Spike. Abbreviations: Spike - SARS Spike gene; gpt - dominant selectable marker E. coli guanine phosphoribosiltransferase; PI 1, P7.5 - Vaccinia virus promoters; pUC - plasmid replication origin; tk], and IIC_R - left and right shoulders of thymidine kinase (tk) gene; EcoRJ and BamHl - restriction endonuclease cleavage sites used for cloning.

Figure 60 is a schematic outline of the TDS approach used for generating rMVA- spike virus.

Figure 61 is a schematic outline of rMVA-spike studies.

Figure 62 shows Western blot analysis of rMVA-S (A, B, C, and D) and CEF/rMVA-N (1, 2, 3, and 4) cell lysates. MVA was grown in Chick Embryo Fibroblasts (CEF). The control is MVA-infected CEF.

Figure 63 provides a linear map of pTK53-N, as well as the nucleotide (SEQ ID NO:34) and amino acid (SEQ ID NO:35) sequences of the SARS nucleocapsid protein.

Figure 64 provides the nucleotide (SEQ ID NO:36) and amino acid (SEQ ID NO: 37) sequence of a SARS spike protein.

Figure 65 provides the nucleotide sequence of a SARS coronavirus genome (SEQ ID NO:38).

Detailed Description The invention relates to vaccines and methods that can be used to prevent or to treat Severe Acute Respiratory Syndrome (SARS) caused by human coronaviruses. Viruses causing this disease are lαiown as human coronavirus/SARS, CoV-SARS, TOR2, and Urbani SARS-associated coronavirus. Also included in the invention are metiiods of producing proteins (e.g., spike proteins and nucleocapsid proteins) of human coronaviruses causing SARS. as well as SARS spike and nucleocapsid proteins, and nucleic acid molecules encoding these proteins.

The vaccines of the invention can be used in methods to prevent SARS in patients, such as human patients. In these methods, one or more immunogenic agents derived from a human coronavirus causing SARS are administered to a patient. The agent(s) used can include, for example, an inactivated preparation of the virus or a fraction thereof, or an attenuated version of the virus. The agent(s) can also include an isolated protein (or fragment) from the virus or a nucleic acid molecule encoding such a protein. As a specific example, which is discussed in further detail below, the spike protein of a human coronavirus that causes SARS (or a nucleic acid molecule encoding such a protein) can be used in the vaccines of the invention. Also, the SARS nucleocapsid protein (or a nucleic acid molecule encoding such a protein) can be used. Further, these proteins or nucleic acid molecules (or immunogenic fragments thereof) can be used individually or together, optionally in combination with other agents, such as adjuvants.

The vaccines can also be used to treat patients that have already been exposed to or infected by a virus causing SARS. Optionally, such therapeutic vaccination can be caπied out in conjunction with antiviral therapy involving, for example, administration of antiviral agents, such as oseltamivir or ribavirin. The therapeutic vaccines can also be administered with steroids, in combination with ribavirin and other antimicrobial agents.

As is noted above, spike proteins from human coronaviruses causing SARS can be used in the vaccines of the present invention. The nucleotide and amino acid sequences of one example of such a protein are provided herein as SEQ ID NOs:36 and 37, respectively (also see Figure 64). In addition, SARS nucleocapsid proteins can be used, and the nucleotide and amino acid sequences of an example of such a protein are provided in Figure 63 (SEQ ID NO:34 and SEQ ID NO:35). These sequences and fragments and variants thereof (see above) are also included in the invention. These sequences were identified in a sequence of an entire genome of a human coronavirus causing SARS (SEQ ID NO:38). The proteins of the invention can be made, for example, using a eukaryotic or prokaryotic recombinant expression system. Eukaryotic hosts include, for example, yeasl cells (e.g., Pichia Fusions or Saccharomyces cerevisiae). mammalian cells (e.g., COS1. NIH3T3. HeLa. or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells, while an example of a prokaryotic host is E. coli. Eukaryotic and prokaryotic cells for use in the invention are available from a number of different sources that are lαiown to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Manassaε. Virginia; see also Ausubel et al., Cunent Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998, which is hereby incorporated by reference). The method of transformation and the choice of expression vehicle (e.g., expression vector) will depend on the host system selected. Transformation and transfection methods, as well as expression vehicles, are described, e.g., in Ausubel et al., supra; also see, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987. Specific examples of expression systems that can be used in the invention are described further as follows.

Preferred expression systems for use in making the antigens of the invention are those in which post-translational glycosylation takes place, and include, for example, yeast, mammalian, and insect systems. This is particularly important with respect to SARS spike proteins, which are glycosylated (see below). Examples of yeast hosts that can be used in the invention include Pichia pastoris, Pichia methanolica, Hansuneia polymorpha, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. In the case of P. pastoris, specific examples of host strains that can be used include X-33, GS115, KM71, KM71H, SMD1168, and SMD1168H. Examples of yeast vectors that can be used include pPIC vectors (Invitrogen), such as pPICZalpha for secretion using the alpha factor secretion signal. Also, pPIC vectors that allow multi-copy integrants can be used. These vectors allow multiple insertions into the genome. Use of methalymine or methanol-inducible expression systems can also be used. In another example of a yeast- based system that can be used in the invention, the yeast used to produce the proteins are engineered to make proteins so that they are glycosylated similarly to human proteins (see, e.g., Hamilton et al., Science 301:1244-1246, 2003). Transient transfection of a eukaryotic expression plasmid containing a spike or nucleocapsid protein gene into a mammalian host cell (e.g.. COS1. NIH3T3. HeLa. or .1EG3 cells) allows the transient production of the protein by the fransfected host cell. The proteins can also be produced by a stably-transfected eukaryotic (e.g., mammalian) cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public (see, e.g., Pouwels et al., supra), as are methods for constructing lines including such cells (see, e.g., Ausubel et al., supra). In one example, cDNA encoding a spike or nucleocapsid protein, fusion, mutant, or polypeptide fragment is cloned into an expression vector that includes the dihydrafolate reductase (DHFR) gene. Integration of the plasmid and. therefore, integration of the protein-encoding gene, into the host cell chromosome is selected for by inclusion of 0.01-300 μM methotrexate in the cell culture medium (Ausubel et al., supra). This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the fransfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al., supra. These methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. The most commonly used DHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A) (described, for example, in Ausubel et al., supra). The host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR- cells, ATCC Accession No. CRL 9096) are among those that are most prefened for DHFR selection of a stably fransfected cell line or DHFR-mediated gene amplification.

Another preferred eukaryotic expression system is the baculovirus system using, for example, the vector pBacPAK9, which is available from Clontech (Palo Alto, CA). If desired, this system can be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Molecular and Cellular Biology 5:3610-3616, 1985). Additional examples of insect systems that can be used are the Bac-to-Bac Baculovirus expression system, employing, e.g., pFastBacl vectors, as well as a Drosophila expression system employing S2 cells (see below). The latter system can employ, for example, the pMT/Bip/V5-His vector for regulated, secreted expression.

Expression of foreign molecules in bacteria, such as Esche chia coli. requires the insertion of a foreign nucleic acid molecule, e.g.. a spike nucleic acid molecule or a nucleocapsid nucleic acid molecule, into a bacterial expression vector. Such plasmid vectors include several elements required for the propagation of the plasmid in bacteria, and for expression of foreign DNA contained within the plasmid. Propagation of only plasmid-bearing bacteria is achieved by introducing, into the plasmid, a selectable marker-encoding gene that allows plasmid-bearing bacteria to grow in the presence of an otherwise toxic drug. The plasmid also contains a transcriptional promoter capable of directing synthesis of large amounts of mRNA from the foreign DNA. Such promoters can be, but are not necessarily, inducible promoters that initiate transcription upon induction by culture under appropriate conditions (e.g., in the presence of a drug that activates the promoter). The plasmid also, preferably, contains a polylinker to simplify insertion of the gene in the conect orientation within the vector. An example of a prokaryotic system that can be used is E. coli, using BL21 lambda DE3 and pET vectors, pET26 with a pelB leader for expression to the periplasm. or pET24 for expression of native protein or overlapping fragments thereof.

Proteins of the invention can also be obtained using in vitro methods. For example, in vitro expression of the proteins, fusions, polypeptide fragments, or mutants encoded by cloned DNA can also be caπied out using the T7 late-promoter expression system. This system depends on the regulated expression of T7 RNA polymerase, an enzyme encoded in the DNA of bacteriophage T7. The T7 RNA polymerase initiates transcription at a specific 23 base pair promoter sequence called the T7 late promoter. Copies of the T7 late promoter are located at several sites on the T7 genome, but none are present in E. coli chromosomal DNA. As a result, in T7-infected E. coli, T7 RNA polymerase catalyzes transcription of viral genes, but notE. coli genes. In this expression system, recombinant E. coli cells are first engineered to carry the gene encoding T7 RNA polymerase next to the lac promoter. In the presence of IPTG, these cells transcribe the T7 polymerase gene at a high rate and synthesize abundant amounts of T7 RNA polymerase. These cells are then fransfoπned with plasmid vectors that carry a copy of the T7 late promoter protein. When IPTG is added to the culture medium containing these fransfoπned E. coli cells, large amounts of T7 RNA polymerase are produced. The polymerase then binds to the T7 late promoter on the plasmid expression vectors, catalyzing transcription of the inserted cDNA at a high rate. Since each E. coli cell contains many copies of the expression vector, large amounts of mRNA corresponding to the cloned cDNA can be produced in this system and the resulting protein can be radioactively labeled.

Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages, such as T3, T5, and SP6, can also be used for in vitro production of proteins from cloned DNA. E. coli can also be used for expression using an Ml 3 phage, such as mGPI-2. Furthermore, vectors that contain phage lambda regulatory sequences, or vectors that direct the expression of fusion proteins, for example, a maltose-binding protein fusion protein or a glutathione-S-transferase fusion protein, also can be used for expression in E. coli.

Polypeptides of the invention, particularly short fragments and longer fragments of the N-tenninus and C-terminus of the proteins, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2^nd ed., 1984, The Pierce Chemical Co., Rockford, IL). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful fragments or analogs, as described herein.

Once an appropriate expression vector containing a gene, or a fragment, fusion, or mutant thereof, is constructed, it can be introduced into an appropriate host cell using a transformation technique, such as, for example, calcium phosphate transfection, DEAE- dextran transfection, electroporation, microinjection, protoplast fusion, or liposome- mediated transfection. Host cells that can be fransfected with the vectors of the invention can include, but are not limited to, E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression), or cells derived from mice, humans, or other animals (see, e.g., above). Mammalian cells can also be used to express the proteins of the invention using a virus expression system (e.g., a vaccinia virus expression system) described, for example, in Ausubel et al.. supra. As a specific example of a vaccinia virus system that can be used. see. e.g.. Moore et al.. EMBO J.. 1 1 : 1973- 1980. 1992. erratum at EMBO J. 1 1 :3490. 1992: Skinner et al., J. Gen. Virol. 75:2495-2498. 1994: and Sroller et al., Arch. Virol. 143:131 1-1320, 1998, which describe the use of a Modified Vaccinia Ankara (MVA) strain. Also see, e.g., U.S. Patent No. 6,440,422.

Upon expression, a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the exfracellular medium or in the periplasmic space, or is embedded in the cellular membrane. Preferably, the polypeptide is secreted. The polypeptide can then be recovered in a substantially purified form from the cell extract or from the supernatant after ceπtrifugation of the cell culture. Typically, the recombinant polypeptide can be purified by antibody-based affinity purification or by any other method lαiown to a person skilled in the art, such as by genetic fusion to a small affinity- binding domain. Antibody-based affinity purification methods are also available for purifying a polypeptide of the invention. Antibodies useful for immunoaffinity purification of the polypeptides of the invention can be obtained using standard.

As is discussed further below, we have found that certain spike proteins produced using the methods described herein assemble into trim eric structures, which have been observed to form with certain spike proteins from animal coronaviruses. Thus, the invention includes human coronavirus spike proteins in this form, as well as monomeric and dimeric foπns, and the use of the proteins in such foπns in the methods described herein.

In addition to protein based antigens, the methods of the invention can employ nucleic acid (e.g., DNA or RNA)-based antigens, whether in the foπn of a vectpr delivering a gene to be expressed or admimsfration of a nucleic acid molecule itself. Polynucleotides of the invention can also be used in DNA vaccination methods, using either a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Typically, a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter can function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al., Molec. Cell Biol. 5:281, 1985). The desrnin promoter (Li et al., Gene 78:243, 1989; Li et al., J. Biol. Chem. 266:6562, 1991 ; Li et al., J. Biol. Chem. 268:10403. 1993) is tissue-specific and drives expression in muscle cells. More generally, useful promoters and vectors are described, e.g., in WO 94/21797 and by Hartikka et al. (Human Gene Therapy 7:1205, 1996).

Live vaccine vectors that can be used in the invention include viral vectors, such as adenoviruses and poxviruses (e.g., vaccinia virus vectors, such as MVA vectors), as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilie de Calmette-Guerin (BCG), and Streptococcus. An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a polynucleotide molecule of the invention, is described in U.S. Patent No. 4,920,209. Poxvirus vectors that can be used in the invention include, e.g., vaccinia and canary pox viruses, which are described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively (also see, e.g., Tartaglia et al., Virology 188:217, 1992, for a description of a vaccinia virus vector, and Taylor et al, Vaccine 13:539, 1995, for a description of a canary poxvirus vector). Poxvirus vectors capable of expressing a polynucleotide of the invention can be obtained by homologous recombination, as described in Kieny et al. (Nature 312:163, 1984) so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in maimnalian cells. Details of the use of a pox-based vector are provided in the Examples, below.

In addition to viral-based vectors, bacterial vectors can be used in the invention to administer SARS proteins. Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens, and their use as oral vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and in WO 92/11361. Prefened routes of admimsfration for these vectors include all mucosai routes (e.g., intranasal or oral routes). Others bacterial strains useful as vaccine vectors are described by High et al. (EMBO 11 :1991. 1992) and Sizemore et al. (Science 270:299. 1995: Shigella flexneri): Medaglini et al. (Proc. Natl. Acad. Sci. U.S.A. 92:6868. 1995: (Streptococcus gordoniϊ): Flynn (Cell. Mol. Biol. 40 (suppl. I):31, 1194), and in WO 88/6626. WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376 (Bacille Calmette Gυerin). In bacterial vectors, a polynucleotide of the invention can be inserted into the bacterial genome or it can remain in a free state, for example, earned on a plasmid. An adjuvant can also be added to a composition containing a bacterial vector vaccine.

Methods for administering vaccine compositions including the proteins, fragment, nucleic acid molecules, or vectors of the invention are described as follows.

Administration

As is noted above, the vaccines of the invention can include SARS spike or nucleocapsid polypeptides or iinmunogenic fragments, or nucleic acid molecules encoding such polypeptides or immunogenic fragments. The vaccines can be administered using routes, regimens, and formulations deteπnined to be appropriate by those of skill in this ail. Examples of these and other parameters for consideration in administering the vaccines of the invention are discussed as follows.

The vaccines of the invention can be administered by any conventional route in use in the vaccine field, for example, by a parenteral (e.g., subcutaneous, intradermal, infra epidermal, intramuscular, intravenous, or infraperitoneal) or a mucosai (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) route.

Appropriate amounts of vaccine to be administered can readily be determined by those of skill in the art, and can depend upon various parameters such as the nature of the vaccine vector itself, the route and frequency of admimsfration, the presence/absence of adjuvant, the desired effect (e.g., protection and/or freatment), and the condition of the mammal to be vaccinated (e.g., the weight, age, and general health of the mammal). In general, 0.1 μg - 1 mg, e.g., 1-500 μg, e.g., or 10-100 μg (e.g., 20-80, 30-70, 40-60 or about 50 μg), can be administered. For example, a vaccine of the invention can be administered mucosally in an amount ranging from about 10 μg to about 500 mg, e.g., from about 1 mg to about 200 mg. For a parenteral route of administration, the dose usually should not exceed about 1 mg. and can be, preferably, about 50-500. e.g.. 100- 250 μg.

The vaccines of the invention can be administered in regimens that can be deteπnined to be appropriate by those of skill in this art. For example, the administration can be achieved in a single dose or repeated at intervals. As a specific example, the vaccines can be administered in three doses biweekly, 1 month apart, or on days 0, 28, and 56 of a multi-dose regimen, ln another example, a priming dose is followed by 1-3 booster doses at weekly or monthly intervals (e.g., a boost within 1-6 months), with follow-up boosting every 1-5 (e.g., 3) years, if needed. As yet another example, a subject can initially be primed with a vaccine vector of the invention, such as a pox virus (e.g., MVA or adenovirus) by, e.g., a parenteral route, and then boosted (e.g., 2-4 times) with a polypeptide encoded by the vaccine vector by the parenteral or mucosai route. Alternatively, a polypeptide can be used in a priming step, and boosting can be canied out using a vaccine vector, such as a pox virus or an adenovirus. In another example, liposomes associated with a polypeptide or polypeptide of the invention can be used for priming, with boosting being canied out mucosally using a soluble polypeptide or polypeptide derivative of the invention, in combination with a mucosai adjuvant (e.g., LT). In a further example, the antigen is administered mucosally (e.g., intranasally) in a priming step, and boosting is by parenteral administration. Further, the vaccines described herein can be used in combination with each other or other vaccines against SARS, by co-administration or in prime/boost methods in which a vaccine as described herein is used in either the prime or a boosting step, and the other vaccine is used in a step in which a vaccine as described herein is not used.

The vaccines of the invention can be foπnulated using standard methods (see, e.g., in Remington 's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA), ln addition to the antigenic agent(s), the vaccines can optionally also include an adjuvant. Examples of adjuvants that can be included in the vaccines of the invention include alum and other aluminum compounds (e.g., aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate), DC-Choi, QS-21, MPL. Ribi. as well as other parenteral adjuvants that are known in the art. Additional foimulations that can be used can include the use of liposomes. such as neutral or anionic liposomes. microspheres. or virus-like particles (VLPs), to facilitate delivery and/or enhance the immune response. Another example of an adjuvant that can be used is ISCOMs, which can be used, e.g., in mucosai (e.g., intranasal or oral) administration of. e.g., the polypeptide antigens described herein. These compounds are readily available to those skilled in the art; for example, see Liposomes: A Practical Approach (supra).

Additional adjuvants that can be used for mucosai administration include, for example, bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A, the pertussis toxin (PT), and combinations, subunits, toxoids, or mutants thereof. For example, a purified preparation of native cholera toxin subunit B (CTB) can be used. Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that can be used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69- Gly, Glu-1 10-Asp, and Glu-112-Asp mutants. Other adjuvants, such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota. Salmonella typhimurium. or Shigella flexneri; saponins, and polylactide glycolide (PLGA) microspheres, can also be used in mucosai administration. Adjuvants useful for both mucosai and parenteral adminisfrations, such as polyphosphazene (WO 95/2415), can also be used.

As is noted above, the vaccination methods of the invention can also include the use of polynucleotide molecules, which can, optionally, be administered in a vector. A polynucleotide of the invention can be used in a naked foπn, free of any delivery vehicles, such as anionic liposomes, cationic lipids, microparticles, e.g., gold microparticles, precipitating agents, e.g., calcium phosphate, or any other fransfection- facilitating agent. In this case, the polynucleotide can simply be diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without a carrier. When present, the carrier preferably is isotonic. hypotonic. or weakly hypertonic. and has a relatively low ionic strength, such as provided by a sucrose solution, e.g.. a solution containing 20% sucrose.

Alternatively, a polynucleotide can be associated with agents that assist in cellular uptake. It can be, e.g., (i) complemented with a chemical agent that modifies cellular peπneability. such as bupivacaine (see, e.g., WO 94/16737), (ii) encapsulated into liposomes. or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles. Anionic and neutral liposomes are well-known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL Press, 1990, for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.

Cationic lipids can also be used for gene delivery. Such lipids include, for example, Lipofectin^{1 M}, which is also lαiown as DOTMA (N-[l-(2,3-dioleyloxy)propyl]- N,N,N-trimethylaιmnonium chloride), DOTAP (l,2-bis(oleyloxy)-3- (frimethy]ammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadeeylamidologlycyl spennine), and cholesterol derivatives. A description of these cationic lipids can be found in EP 187,702, WO 90/1 1092, U.S. Patent No. 5,283,185, WO 91/15501. WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neufral lipid such as DOPE (dioleyl phosphatidylethanolamine; WO 90/11092). Other transfection- facilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, e.g., WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. They include, e.g., spennine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles can also be used for gene delivery, as described in WO 91/359, WO 93/17706, and by Tang et al. (Nature 356:152, 1992). In this case, the microparticle-coated polynucleotides can be injected via intradermal or intraepideπnal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patent No. 4.945.050. U.S. Patent No. 5.015.580. and WO 94/24263.

The amount of DNA to be used in a vaccine recipient depends, e.g.. on the sfrength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of admimsfration, and the type of formulation, ln general, a therapeutically or prophylactically effective dose from about 1 μg to about 1 mg, preferably, from about 10 μg to about 800 μg. and, more preferably, from about 25 μg to about 250 μg. can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals.

A preferred approach for vaccination according to the present invention involves the use of a live vector, such as a live viral vector. For example, nucleotide sequences encoding SARS spike proteins or immunogenic fragments thereof, as described elsewhere herein, can be inserted into a live vector, such as a pox vector, which is administered in vaccination methods. Additional viral and bacterial vectors that can be used in the invention are lαiown in the art (also, see above). As a specific example, the attenuated vaccinia virus Modified Vaccinia Ankara (MVA) can be used as a viral delivery vehicle in the invention. Details of the use of such a viral vector are provided below in the Examples, ln general, the dose of a viral vector vaccine, for therapeutic or prophylactic use, can be from about lxlO⁴ to about lxl O^{1 1}, e.g., 1x10' to lxl θ'°, or 1x10' to about lxl 0⁹, plaque-forming units per kilogram. Such vectors can be administered, e.g., parenterally, for example, in 3 doses that are 4 weeks apart.

Also included in the invention are passive immunization methods for preventing or treating SARS infection. In these methods, antibodies against the SARS virus, or one or more components thereof, are administered to patients to prevent or treat infection. As a specific example, polyclonal hyperimmune globulin that is obtained from plasma donors that have been actively immunized with a SARS antigen (e.g., a spike protein antigen or a nucleocapsid protein antigen; see, e.g., above) can be used. Routes of administration include, for example, mucosai and parenteral routes. For example, in the case of mucosai administration, the antibody preparation can be administered in the foπn of nose drops or by inhalation, using standard methods in the art. Other mucosai routes, such as those listed above, can also be used. In the case of parenteral administration, subcutaneous injection or any other parenteral route (see, e.g., those listed above) can be used. The passive immunization methods can be used as sole approaches to prevention or freatment, or can be used in combination with active vaccination approaches, such as those described herein (see, e.g., WO 99/20304 for additional details on passive immunization approaches).

Examples

Example 1 - Expression Constructs

Constructs for expressing the SARS coronavirus spike proteins in three different eukaryotic systems, the yeast Pichia pastoris, mammalian CHO cells, and drosophila S2 cells, were made and characterized. The details of these constructs are summarized in the following table, and are illustrated in Figures 1-38. The constructs each lack the native N-teπninal spike signal sequence (amino acids 1-13), in favor of those provided by the vectors used in each of the systems. The vector-provided signal sequences ensure that the proteins are secreted in the relevant systems.

As is illustrated in Figure 39, the SARS spike protein can be divided up into an extracellular domain, a transmembrane domain, and a cytoplasmic tail. We tested constructs that include different combinations of these regions. For example, the 14-719 (or 709) constructs include the exfracellular domain (i.e., the putative SI domain, which represents the receptor binding domain and the region including neutralization determinants); the 14-883 constructs include the extracellular domain and the S2 domain, but not the intracellular coiled coil domain, while the 14-1190 constructs include the extracellular domain, but not the fransmembrane domain, and the cytoplasmic tail. The table set forth below provides information as to the vectors used, the construct names, and the spike protein amino acids included in the constructs, for each of the three systems. Constructs including amino acids 14-1190, 14-883, and 14-709 are alternatively refened to herein as clones Al, A2, and A3, respectively. Pichia pastoris expression constructs

Expression system Vector Construct name Spike protein limits ( aa)

I nducible pPICZalpha P l -2 14-709 pPICZalpha Pl -2.2 14-719 pPlCZalpha P3- 1 0 mutant (H 641 R) 14-883 pPICZalpha P3- 10.2 14-883 pPlCZalpha P5- 12--¹ 14- 1 190

* contains 2 sil em mutations within aa C133. C1 I 08

Constitutive pGAPZalpha Gl -S 14-709 pGAPZalpha G l -8.2 14-719 pGAPZalpha G3-7 mutant (Y484C) 14-883 pPAGZalpha G3-7.2 14-883 pPAGZalpha G5- 14** 14- 1 190

** contains 2 silent mutations within aa Y723, G739 pSEC expression constructs (CHO cells)

Expression system Vector Construct name Spike protein limits (aa)

Constitutive pSEC pSEC-719 14-719 pSEC pSEC-883 14-883 pSEC pSEC- 1 190 14-1 190

DES constructs (Drosophila S2 cells)

Expression system Vector Construct name Spike protein limits (aa)

Inducible p T pMT-719 14-719 p T pMT-883 14-883 pMT pMT-1 190 14- 1 190

Additional details as to the construction of these constructs are as follows. Crude RNA was extracted from Vero E6 cells infected with SARS-CoV (provided by the CDC) and reverse transcriptase-polymerase chain reaction (RT-PCR) was perfonned for cloning. cDNA clones representing all structural genes in their entirety were constructed and characterized by DNA sequencing. Clones A1-A3 were constructed by PCR in the pPICZalpha and pGAPZalpha expression vectors for inducible and constitutive expression in Pichia pastoris, respectively. Fragments were engineered in-frame at the N-terminus with the alpha factor signal sequence allowing for export from a growing culture and were prematurely terminated at the C-terminus with a stop codon. Clones A1-A3 were then electroporated into competent X-33 Pichiapastoris and fransfonnants were evaluated for integration by PCR, copy number by enhanced resistance to the selectable marker canied on the integration vector, and expression by, e.g., immunoreactivity with SARS-specific antisera using a dot blot foπnat (see below). Example 2 - Expression Studies

The constructs described above were analyzed for expression in the relevant systems, with the goal being to analyze the systems for yield, purity, solubility, and glycosylation. After such initial characterization, virus neutralization studies in, e.g., mice, can be earned out to determine appropriate regimens, doses, scheduling, adjuvants, and formulations, and then efficacy can be confirmed, if desired, in an appropriate non- human primate model, ln each system, clones were generated by infroduction of the constructs noted above into cells by lipofection or electroporation (Figure 40).

A generalized strategy for constitutive (CHO) and inducible (S2) expression of recombinant spike proteins is illustrated in Figure 41. Briefly, the spike gene is cloned into an appropriate vector (e.g., pMT/BiP for S2 cells or pSec/FRT TOPO for Flp-In CHO cells), positive fransfonnants are selected and sequenced, and then the constructs are integrated into the S2 or CHO cells by use of a co-fransfected recombination plasmid and selection with hygromycin (CHO) or Blasticidine (S2). The integrants are then screened for high level expression, a candidate is selected, expression is optimized, and production is then scaled up, if desired. Figure 42 shows the results of PCR screening of genomic DNA purified from transiently fransfected S2 cells 24 and 48 hours after transfection with pMT-719, pMT-883. and pMT-1190 consfructs, as well as Western blot analysis of these cells. Figure 43 presents RT-PCR data showing that the spike 719, 883. and 1 190 genes are expressed in CHO cells.

The generalized strategy for expression of recombinant spike proteins in the yeast Pichia pastoris is illustrated in Figure 44. Briefly, consfructs are sequenced, midi- prepped, and subcloned, and then are integrated into P. pastoris by linearization, electroporation, and Zeocin selection. The integrants are then screened for high copy numbers, fermented, and a candidate is selected and optimized. Figure 45 shows spike gene-specific PCR of cliromosomal DNA, confinning integration for constructs encoding 1190 and 883 amino acids of the SARS spike protein, as described above. In particular, Figure 45 shows a sample set of PCR positive (N-tenninal fragment) integrants for Al (1190) and A2 (883) constructs for both inducible and constitutive expression. Small- scale expression studies were then performed on integrants to identify clones for bench- scale fermentation. Figure 45 further shows the iimnunoreactivity of a panel of Al ( ] 190) integrants engineered to produce full-length ectodomain following inducible expression, that are immunoreactive with a neutralizing, murine hyperimmune polyclonal antibody raised against gamma-irradiated SARS-CoV. Clone 64 (identified by the anow) was observed to react strongly with the SARS polyclonal serum and was selected for further study. Similar studies identified a clone that expressed immunoreactive product following constitutive expression.

Figure 46 shows the results of analysis of clones constitutively expressing the 1 190 (lanes 2 and 6) and 883 (lanes 3 and 7) amino acid versions of recombinant spike. Panel A is a glycostain; panel B is an immunoblot with an anti-SARS coronavirus murine polyclonal antibody; panel C is a dot blot using such an antibody; panel D is an immunoblot using human convalescent sera; and panel E is a dot blot using the latter sera. In lanes 2 and 3, the proteins are glycosylated, while in lanes 6 and 7, the proteins have been treated with Endonuclease H, resulting in deglycosylation. These data show that high molecular weight material is immunoreactive with anti-spike antibodies, and that this material breaks down upon Endonuclease H freatment, yielding the expected 139 kDa (full ectodomain, 1 190) and 98 kDa (bulbar head, 883) products. The dot blot results show the maintenance of at least some conformational integrity of the recombinant proteins.

The spike protein was purified from fermentor bulk material by successive diafilfr-ation steps (>300 kDa, 15x; 100-300 kDa, 15x; and <100 kDa), and the fractions were tested for iimnunoreactivity with an anti-SARS coronavirus antibody (Figure 47). Most of the iimnunoreactivity was found in the >300 kDa fraction. No reactivity was observed with lower molecular weight material that could represent monomer or similarly treated material expressed from the confrol strain X-33 lacking the S gene.

The partially purified retentate material (1190) was then purified further by lectin affinity chromatography in batch mode by binding to Concanavalin A-Sepharose 4B and eluting with sugar (methyl a-D-Mannopyranoside, 750 mM) (Figure 48). The eluted material (glycosylated and deglycosylated samples) was fractionated by SDS-PAGE, and detected by Western blot analysis as high molecular weight material at about 180-250 kDa (glycosylated). while Endo-H freated material (deglycosylated) was detected at about 138 kDa. as was expected. These studies show that the spike protein secreted from P. pastoris through the secretory pathway was glycosylated.

The results of additional studies showing the purification of pGAP-1190 from P. pastoris supematants are shown in Figure 49. SDS-PAGE and removal of DTT from the sample buffer suggests the presence of monomeric, pichia glycosylated spike protein of >250 kDa. Figure 50 shows that mass spectrometry (MALDI-ESI) confirms expression of the S glycoprotein in pichia.

In other studies, fed-batch fermentation (2 L) of Pichia pastoris integrants expressing full-length rS ectodomain (cAl and iAl) was perfonned in a confrolled environment with basal salts medium (BSM) in the absence of selection. The following illustrates the methodology employed when perfonning constitutive expression of cAl expressing full- length rS glycoprotein. Briefly a vial of cAl research cell bank (RCB) was seeded into 100 ml of BSM plus PTMi frace salts and grown for 18 hours at 28°C. On day 2, a 5% inoculum was added to the fermentor containing BSM (pH5) containing 4% glycerol and grown at 30°C, with dissolved O₂ (DO) active control maintained at 35%, agitation set 100-1000 rpm, with airflow at 3.0 L/minute. Fermentation was monitored using BioCommand software (New Brunswick). At carbon exhaustion, the feed program was initiated were 50% glycerol solution was added with PTM_t trace salts at a rate of 0.5%/liter/hour. After 2 additional days of fermentation, the culture supemate was harvested and EDTA added to 5 mM. Using fed-batch fermentation, we observed dramatic increases in production of target protein (Figure 51) with yields of monomer calculated at 100 mg/L by densitomefry. Fermentation at low pH is known to be optimal for yeast growth and likely limited proteolytic breakdown of expressed product. We have achieved cell densities (OD₆Q_O) of > 400 using both our inducible and constitutive expression systems. Gel extraction of the 180 kDa monomer was confinned as Spike protein following Mass Spectroscopy and in gel digestion and sequencing of generated fragments. Over 90% of the harvested sequence spanned the entire length of the protein confinning high-level expression of monomeric S glycoprotein. Size exclusion, high-pressure liquid chromatography (HPLC) of diafiltered (> 300 kDa) material supported expression of several HMW species ranging in size from 100 to >1000 kDa (Figure 52A). Isolated fractions representing peaks i & ii and that ran near the void volume were demonstrated to be immunoreactive with the polyclonal anti-SARS hyperimmune raised against SARS-CoV (Figure 52A). Fractions 13-18 were subsequently freated with endo-deglycosidase H and in every case immunoreactivity with the mouse polyclonal was observed with a circa 130 kDa deglycosylated protein, as expected. Denaturation of the diafiltered material with Guanidine Hydrocholoride (GuHCl) plus DTT resolved much of the void volume peaks to the lower molecular weight species, peak iii (Figure 52B). When the denatured sample was dialyzed against citrate buffer (pH 4). the lower molecular weight component appeared to re-associate preferentially to the higher molecular weight peak ii (Figure 52B). This material was soluble and stable at +4°C. Dot blots of isolated fractions with both the mouse polyclonal and conformational dependent monoclonal antibodies, previously demonstrated to neutralize SARS-CoV, confirmed the iimnunoreactivity and structure of re- associated peak ii.

Different fractions representing peaks i, ii, and iii were then re-injected and further analyzed by light scattering with accurate molecular weight determinations of 160. 322. and 623 kDa. supporting monomeric. dimeric, and trimeric forms (Figure 53). Importantly, the trimeric structure was quite stable. The ability to correctly re- fold trimer as determined by iimnunoreactivity provides us with a method enabling enrichment of a preferred S glycoprotein structure.

Additional data supporting fermentation and expression of full-length rS ectodomain using continuous culture are described as follows. Constitutive expression of rS glycoprotein has been achieved for 40 days and the effect of temperature and pH on production of target protein monitored. The data supports expression of a soluble, rS glycoprotein at pH 7.0. To mitigate effects of enhanced proteolysis at the elevated pH due to intrinsic proteases produced by Pichiapastoris, fermentation is carried out at 15°C. Figure 54 shows expression of a circa 180 kDa monomer in neat culture supematants and its immunoreactivity profile with the murine polyclonal antibody raised against γ-frrdadiated SARS- CoV under denaturing/reducing conditions (Figures 54A and 54B). Two time- points are represented. Yields of rS glycoprotein using a constitutive expression system seem to favor increased production levels and is therefore the method of choice for production pmposes. Performing continuous culture with this construct will ensure production of very respectable levels of rS glycoprotein for manufacturing purposes.

Previous data supported the expression of a high molecular weight (HMW) complex (> 300 kDa) that was preferentially immunoreactive with both anti-SARS-CoV polyclonal and monoclonal neutralizing antibodies. Polyacrylamide gel electrophoresis (PAGE) under native conditons supports the existence of a HMW complex and at least two isoforms of the rS glycoprotein. To better observe this phenomenon, culmre supernatant was diafiltered through a 100 kDa membrane and concentrated 10-fold prior to running native PAGE (Figure 55, lanes 2). In the presence of reducing agents (DTT, lane 4; β- mercaptoethanol, lane 5) the higher of the two protein complexes is reduced to a single species, and presumably represents the monomeric foi of the rS glycoprotein ectodomain. Both isomers are immunoreactive with the anti-SARS polyclonal antibody. The existence of the higher molecular weight isomer was also confirmed following re-folding of a gel-extracted 180 kDa monomer.

Following the concentration step over a 100 kDa membrane, recent studies have focused on separation of the HMW immunoreactive protein complexes by gel filtration to better define the immunoreactive products. Concentrated culture supernatant (Figure 55, lane 2) was first separated over Sephacryl S-500 (Phaπnacia; Figure 55, lane 3) and both the void volume and one isolated peak further characterized by size exclusion high pressure liquid chromatography (SE- HPLC; Figure 56) over TSKSW 4000. Both samples were fractionated to discrete peaks, and harvested samples prepared for native PAGE, iimnunoreactivity profiling, and size deteπnination by light scattering. The initial S-500 gel filtration step successfully separated HMW protein complex from the lower molecular weight products (double headed anow). Further separation of the low molecular weight proteins (B) using TSK SW4000 confirmed our ability to successfully fractionate the majority of the lower molecular weight products (fractions 12-19; Figure 57A) and enabled us to identify those protein complexes that were more immunoreactive (Figures 57B and 57C). A circa 300 kDa protein was isolated from fractions 12 and 13 that appeared to be preferentially recognized by both the polyclonal antibody raised against SARS-Coλ⁷ and a monospecific polyclone (directed to a linear determinant on the C-teπninus of the protein). Results are presented in a dot blot foπnat and a Western blot of native PAGE.

Fractions 14 through 19 also appeared to be recognized with Spike protein-specific antibodies but to a lesser degree, possibly suggesting proteolytic cleavage to lower molecular weight products. Recent data supports this hypothesis through mass spectoscopy and peptide sequencing of gel extracted fragments representing these lower molecular weight peaks. Samples representing fractions 12 and 13 were then re-injected, SE-HPLC perfonned over TSK SW4000, and molecular weight determinations made by light scattering. A molecular weight of circa 300 kDa was assigned to the protein present in fraction 12 and probably represents a dimer. Fraction 13 was determined to have 2 proteins sizing at 300 and 177 kDa, likely representing the dimeric and monomeric foπns, respectively.

Similar studies were perfonned on concentrated culture medium fractionated using Sephacryl S-300 to characterize the HMW complex. The first of two peaks harvested was further fractionated under pressure using TSK SW4000 (Figure 58). Samples were analyzed for their iimnunoreactivity against SARS-CoV and Spike protein specific antibodies and molecular weight deteπninations were made by light scattering. Immunoblot data confirmed the immunoreactivity of the dimer as determined in the LMW fractionation study, but also confirmed the existence of a third protein complex (peak 2) that was even more immunoreactive with the anti-SARS-CoV antibody. Molecular weight determinations by light scattering supports peak 2 ranging in size from 450 - 750 kDa. Our previous studies support the existence of a trimer. Cunently we believe the majority of protein in the medium to be Spike protein with total protein concentrations of approximately 400 mg/L culture. Based on the areas under the curve and the protein concentration of the fractionated material we are cunently estimating yields of 50 mg protein for each isoform per liter of culture medium.

Example 3 - Delivery of Spike Proteins using Live Virus Vectors

A live virus approach using Modified Vaccinia vims Ankara (MVA) as a vector is now described. Mλ<A has been proven to be extremely attenuated when compared to wild-type Vaccinia virus strain (Mayr et al, Infection 3:6-14, 1975; Werner et al., Arch.Nirol. 64:247-256, 1980) and was established as exceptionally safe viral vector (Moss et al., In S. Cohen and A. Shaffeπnan (eds.), Novel Strategies in the Design and Production of Vaccines, Plenum Press, New York, 1996, p. 7-13; Stittelaar et al., Vaccine 19:3700-3709, 2001; Sutter et al, Dev. Biol. Stand. 84:195-200, 1995). The following is a description of the construction of rMVAs expressing full-length recombinant SARS spike proteins, which can be used in vaccination methods against SARS, as described above.

For generating recombinant MVA a strategy called "transient dominant selection" (TDS) (Falkner et al., J. Virol. 64:3108-3111, 1990) can be used. The spike gene is amplified by PCR from a source clone (ACAM 250-0013; also see SEQ ID NO:38, Figure 65) and cloned into the BamHI-EcoRI sites of the insertion vector pTK53-gpt (Falkner et al, J. Virol. 64:3108-3111, 1990). The resulting plasmid, pTK-53-gpt-Spike (Figure 59), contains the Spike protein gene flanked by left (TK_L) and right (TK_R) shoulders of the vaccinia, thymidine kinase (TK) gene, and is confrolled by a powerful late Vaccinia PI 1 promoter. A schematic outline of the TDS approach is shown in Figure 60. When the resulting plasmid is fransfected to the cells that have been infected with MVA virus, homologous recombination occurs due to homology between virus and plasmid TK gene sequences. As a result of a single crossover event, an unstable intermediate vims containing the whole plasmid will be generated. Because of the presence of direct repeats, a second crossover event occurs and results in the fonnation of either wild type or recombinant virus containing the spike gene. All three types of genomes can be packaged separately into particles and are infectious, but only virus containing the gpt gene can form plaques under selective conditions.

Thus, the first two rounds of plaque isolation are done in presence of mycophenolic acid, xanthine. and hypoxanthine, which only allow the growth of viruses that express E. coli gpt (RM 2026 #7). The next two rounds are canied out without selection, and in the final plaque assay, the isolated virus can be checked for the expression of gpt and t/ by the use selective media (RM 2026 #10). All gpt+tk- viruses should contain the spike gene, and this can be confirmed by PCR.

The viruses can be grown in chick embryo fibroblast (CEF) cells (Sutter et al, Proc. Natl. Acad. Sci. U.S.A 89:10847-10851, 1992) and/or baby hamster kidney (BHK) cells (Drexler et al, J. Gen. Virol. 79 (Pt 2):347-352, 1998). As an additional cell substrate for propagating MVA, a spontaneously immortalized chicken cell line, DF1. derived from 10 day old East Lancing Line (ELL-0) eggs (19) (ATCC # CRL- 12203) can be used.

CEF, BHK, or DF-1 cells are infected with MVA as described in Gomez et al. Arch. Virol. 146:875-892, 2001. Briefly, 0.1 PFU/cell MVA or MVA recombinant in serum free medium can be used as infective dose. After 1 hour of virus adsoiption, the inocula are removed and cells are supplemented with medium containing 2%o serum and antibiotics. After 3-4 days of culture (depending on the type of cells), the cells are collected by centrifugation, washed and resuspended in medium, and sonicated; cell extracts are centrifuged at 2K/10 minutes, the supernatant collected, and the pellet resuspended in 1 mM of Na₂HPO .then re-extracted as described previously. Pooled supematants are centrifuged 15K/30 minutes, the pellet resuspended by sonication in 1 mM of Na₂HPO₄, applied over 20-45 % (w/v) sucrose gradient in the same solution, and centrifuged at 15K/20 minutes. The virus band iscollected, diluted in 1 mM Na₂HPO , and sedimented at 15K/30 minutes; the vims pellet is then resuspended in a small volmne of 1 mM Na₂HPO₄ and stored in aliquots at -70°C. To titrate or plaque purify the vimses (Falkner et al.. .1. Virol. 62:1849-1854. 1988). a layer of semisolid medium (incubation medium + 1 % agarose) can be added to the infected cells. After incubation for one day at 37^UC. a second layer of semisolid medium with 0.2 % of neutral red can be added, and after another 8-12 hours of incubation plaques are counted or collected by aspiration into glass pasteur pipettes. Virus can be released from agar by sonication or repetitive freezing-melting rounds.

An exemplary recombination protocol is described below. Note that although the term "cotransfection" is used in Figure 61, for the sake of simplicity, virus infection actually precedes plasmid transfection by 2-3 hours (Falkner et al, J. Virol. 64:3108- 31 11. 1990). After 2 hours of infection with MVA, CEF or DF-1 cells can be fransfected with plasmid pTK53-gpt-spike and placed in gpt+ selective medium MXHAT (Boyle et al. Gene 65:123-128, 1988) (Dullbeco's modified Eagle Medium, 2.5 % fetal bovine serum, 25 μg of MPA per ml, 250 μg of xanthine per ml, 15 μg of hypoxanthine per ml). After 14 to 24 hours of incubation, plaques can be detected by staining with neutral red. Ten-twenty plaques of normal size and shape can be picked and then reassayed a second time under the same selective conditions. Three more plaque purification rounds are carried out under nonselective conditions. The gpftk^' phenotypes are then detennined by plaque assay in the presence of MXHAT and 5-bromodeoxyuridine overlay as described by others (Chakrabarti et al.. Mol. Cell Biol. 5:3403-3409, 1985; Mackett et al.. .1. Virol. 49:857-864, 1984). TK^" selection is then canied out as described previously.

As widespread smallpox vaccination is again considered (Abramson et al. Pediatrics 111 : 1431 - 1432, 2003), the prevalence of immunity to vaccinia vims could increase substantially. A possible preceding immunity to vaccinia virus could reduce its ability to serve as a vector for the delivery of recombinant genes used for other infectious deceases (Cooney et al. Lancet 337:567-572, 1991). An approach to alleviating this is by using either mucosai route (Belyakov et al, Proc. Natl. Acad. Sci. U.S.A 96:4512- 4517, 1999) or DNA priming before vector boosting (Yang et al, J. Virol. 77:799-803, 2003).

The mucosai administration approach is based on the fact that migration of immune T cells between the mucosai and systemic immune systems is asymmetrically restricted in the sense that cells traffic from mucosai system to the systemic system but not vice versa. Thus, systemic infection with vaccinia virus does not induce CTL that migrate to mucosai immune system, and apparently the virus does not infect mucosai tissues sufficiently under these circumstances to induce immunity (Belyakov et al, .1. Clin. Invest 102:2072-2081 , 1998; Belyakov et al, Proc. Natl. Acad. Sci. U.S.A 95:1709- 1714, 1998; Belyakov et al, J. Virol. 72:8264-8272, 1998). On this basis, the mucosai system remains naϊ^'ve to vaccinia virus. In contrast, mucosai infection with recombinant vaccinia virus induces not only CTL in the mucosa, but CTL that traffic out to the systemic immune system.

DNA priming has been shown to be highly effective in stimulating a primary immune response based on T-cell recognition of diverse subdominant epitopes (Barouch et al, J. Virol. 75:2462-2467, 2001). The response is presumably based on the ability of antigen-presenting cells to take up and present endogenously synthesized antigens. In the absence of proteins from viral vector, the primary immune response presumably can focus on the antigen of interest and facilitate the generation of memory T cells specific for the relevant antigen. Once these memory cells are present, viral vector proteins do not interfere with recall response, allowing a robust immune response to develop (Yang et al, J. Virol. 77:799-803, 2003).

Two rMVA constructs have been generated. rMVA-S and rMVA-N contain SARS spike (S) and nucleocapsid (N) genes, respectively. Expression cassettes canying these target genes under the control of a late vaccinia virus promoter have been cloned into the thymidine kinase gene. Stable rMVA strains expressing both structural genes separately have been identified and the iimnunoreactivity of the expressed product determined by immunoblot with the polyclonal anti-SARS-CoV hyperimmune antibody (Figure 62). Both vimses have been plaque purified and amplified to a high titer. These viruses can be used, e.g., in the methods described above. As a specific example, they can be used in a prime boost strategy, in which they are administered mucosally in a priming step, which is followed by a parenteral boost with the recombinant protein. Other examples of regimens and routes that can be used are lαiown in the art and discussed above.

Claims

AU of the references cited herein are nicoiporated herein by reference in their entiren Othei embodiments are present m the following claims. What is claimed is:

1. A vaccine for inducing an immune response to a human coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS) in a patient, said vaccine comprising a spike protein or a nucleocapsid protein of said virus, or an inmiunogenic fragment of either of these proteins, and a phaimaceutically acceptable carrier or diluent.

2. The vaccine of claim 1, wherein said iimnunogenic fragment of said spike protein comprises the S 1 domain of said spike protein.

3. The vaccine of claim 2, wherein said iimnunogenic fragment of said spike protein further comprises the S2 domain of said spike protein, but not the coiled coil region of said spike protein.

4. The vaccine of claim 3, wherein said inmiunogenic fragment of said spike protein further comprises the coiled coil region of said spike protein.

5. The vaccine of claim 4, wherein said iimnunogenic fragment of said spike protein is in the form of a trimer.

6. The vaccine of claim 1, further comprising an adjuvant.

7. The vaccine of claim 6, wherein said adjuvant preferentially stimulates a Thl -type immune response.

8. The vaccine of claim 7, wherein said adjuvant is selected from the group consisting of an ISCOM, Ribi, DC-Choi, QS21, and MPL.

9. The vaccine of claim 6, wherein said adjuvant is alum.

10. The vaccine of claim 1. wherein said spike protein comprises an amino acid sequence that is substantially identical to the sequence of SEQ ID NO:37. or an iimnunogenic Iragment thereof.

11. The vaccine of claim 1, wherein said nucleocapsid protein comprises an amino acid sequence that is substantially identical to the sequence of SEQ ID NO:35, or an iimnunogenic fragment thereof.

12. A vaccine for inducing an immune response to a human coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS) in a patient, said vaccine comprising a vector comprising a nucleic acid sequence encoding a spike protein or a nucleocapsid protein of said virus, or an iimnunogenic fragment of either of these proteins, and a phaimaceutically acceptable canϊer or diluent.

13. The vaccine of claim 12, wherein said iimnunogenic fragment of said spike protein comprises the SI domain of said spike protein.

14. The vaccine of claim 13, wherein said iimnunogenic fragment of said spike protein further comprises the S2 domain of said spike protein, but not the coiled coil region of said spike protein.

15. The vaccine of claim 14, wherein said iimnunogenic fragment of said spike protein further comprises the coiled coil region of said spike protein.

16. The vaccine of claim 12, wherein said vector is a viral vector.

17. The vaccine of claim 16, wherein said vector comprises a poxvirus or an adenovirus.

1 8. The vaccine of claim 17. wherein said poxvirus is a modified vaccinia akara virus.

19. A method for producing a spike protein or a nucleocapsid protein of a human coronavirus. or an immunogenic fragment thereof, said method comprising introducing into cells a vector comprising a nucleic acid sequence encoding said protein or said fragment, under conditions in which said protein or fragment is expressed in said cells.

20. The method of claim 19, wherein said iimnunogenic fragment of said spike protein comprises the S 1' domain of said spike protein.

21. The method of claim 20, wherein said iimnunogenic fragment of said spike protein further comprises the S2 domain of said spike protein, but not the coiled coil region of said spike protein.

22. The method of claim 21 , wherein said immunogenic fragment of said spike protein further comprises the coiled coil region of said spike protein.

23. The method of claim 19, wherein said cells are yeast cells, maimnalian cells, insect cells, or bacterial cells.

24. The method of claim 23, wherein said yeast cells are Pichia pastoris cells.

25. A method of inducing an immune response to a human coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS) in a patient, said method comprising administering the vaccine of claim 1 or claim 12 to said patient.

26. A substantially pure spike protein of a human coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS). or an immunogenic fragment thereof.

27. The protein of claim 26. wherein said iimnunogenic fragment of said spike protein comprises S 1 domain of said spike protein.

28. The protein of claim 27, wherein said immunogenic fragment of said spike protein further comprises the S2 domain of said spike protein, but not the coiled coil region of said spike protein.

29. The protein of claim 28, wherein said iimnunogenic fragment of said spike protein further comprises the coiled coil region of said spike protein.

30. The protein of claim 26, wherein said spike protein or fragment comprises a sequence that is substantially identical to the sequence of SEQ ID NO:37, or a fragment thereof.

31. The protein of claim 26. wherein said spike protein or fragment comprises the sequence of SEQ ID NO:37, or a fragment thereof.

32. The protein of claim 26, wherein said spike protein or fragment is in the foim of a trimer.

33. An isolated nucleic acid molecule encoding a spike protein of a human coronavirus or an iimnunogenic fragment thereof.

34. The nucleic acid molecule of claim 33, wherein said immunogenic fragment of said spike protein comprises the SI domain of said spike protein.

35. The nucleic acid molecule of claim 34. wherein said iimnunogenic fragment of said spike protein further comprises the S2 domain of said spike protein, but not the coiled coil region of said spike protein.

36. The nucleic acid molecule of claim 35, wherein said immunogenic fragment of said spike protein further comprises the coiled coil region of said spike protein.

37. The nucleic acid molecule of claim 33, wherein said nucleic acid molecule comprises the sequence of SEQ ID NO:36.

38. The nucleic acid molecule of claim 33, wherein said nucleic acid molecule hybridizes to the complement of the sequence of SEQ ID NO:36 under highly stringent conditions.

39. A nucleic acid molecule probe comprising a sequence that hybridizes to the sequence of SEQ ID NO:36 or the complement thereof under highly stringent conditions.

40. An antibody that specifically binds to the protein or iimnunogenic fragment of claim 26.

41. A substantially pure nucleocapsid protein of a human coronavirus that is the causative agent of Severe Acute Respiratory Syndrome (SARS), or an immunogenic fragment thereof.

42. The protein of claim 41, wherein said spike protein or fragment comprises a sequence that is substantially identical to the sequence of SEQ ID NO:37, or a fragment thereof.

43. The protein of claim 41. wherein said spike protein or fragment comprises the sequence of SEQ ID NO:37. or a fragment thereof.

44. An isolated nucleic acid molecule encoding a nucleocapsid protein of a human coronavirus or an iimnunogenic fragment thereof.

45. The nucleic acid molecule of claim 44, wherein said nucleic acid molecule comprises the sequence of SEQ ID NO:34.

46. The nucleic acid molecule of claim 44, wherein said nucleic acid molecule hybridizes to the complement of the sequence of SEQ ID NO:34 under highly stringent conditions.

47. A nucleic acid molecule probe comprising a sequence that hybridizes to the sequence of SEQ ID NO:34 or the complement thereof under highly stringent conditions.

48. An antibody that specifically binds to the protein or immunogenic fragment of claim 41.

49. A method of preventing or treating infection by a human coronavirus that is the causative agent of Severe Acute Respiratoiy Syndrome (SARS) in a patient, comprising administering to the patient the antibody of claim 40 or claim 48.

50. The method of claim 49, wherein said antibody is a polyclonal hyperimmune globulin preparation.