WO2004110483A1 - Method and composition of a novel vaccine design for the prevention and treatment of sars - Google Patents

Abstract

The invention disclosed a pharmaceutical composition that can induce immune response against SARS virus, and a method for using such pharmaceutical composition. Vaccines were designed to express multiple SARS gene products, including S1 domain, extracellular domain of orf8, N protein and others. It was demonstrated that the vaccines could effectively induce immune reaction and protect primates from SARS virus infection.

Description

METHOD AND COMPOSITION OF A NOVEL VACCINE DESIGN FOR THE PREVENTION AND TREATMENT OF SARS

FIELD OF THE INVENTION

The present invention relates generally to viral vaccines and more particularly, to SARS virus vaccines and methods of protecting against or treating disease caused by infection with SARS virus.

BACKGROUND OF THE INVENTION

SARS is a respiratory illness that was first reported in November 2002 in the southern province of Guangdong, China. The disease has since spread to over a dozen countries and affected over 8,000 people worldwide. The overall death rate for SARS patients was estimated to be 14-15% with over 50% in patients over 65 years old.

The general symptoms of SARS include fever over 100.4F⁰, dry cough, body ache, and difficulty in breathing. It is believed that SARS spread via close person-to-person contact, although it is also possible that one can contract the disease by touching a contaminated object and then touching ones nose and mouth afterward. Scientists at US Center for Disease Control and other laboratories have detected a previously unrecognized coronavirus in patients with SARS. The new coronavirus is the leading hypothesis for the cause of SARS.

SUMMARY OF THE INVENTION

SARS virus vaccines comprising a nucleic acid molecule encoding SARS coronavirus gene product are provided. In one embodiment, the nucleic acid encodes one or more SARS coronavirus gene products operatively-linked to a control sequence, in a pharmaceutically acceptable vector.

Such SARS coronavirus gene, in a specific application, is selected from a group of S protein, Orf8 protein, M protein, N protein, E protein, Orfla, Orflb, and combination thereof.

The pharmaceutical composition comprises a control sequence that is commonly referred to as a promoter. In one application, the promoter is a CMV promoter.

The pharmaceutically acceptable vector can be a viral vector, a plasmid vector, or an RNA vector. In one embodiment, the viral vector is a replication-deficient adenovirus. In a specific application, the adenovirus used is a type 5 replication-deficient adenovirus.

In one embodiment, the SARS virus vaccine comprises a nucleic acid molecule encoding two SARS coronavirus gene products, with a first gene product comprising at least one extracellular domain of a SARS coronavirus gene, or a product derived therefrom, and a second gene product comprising an intracellular protein, or a product derived therefrom, in a pharmaceutically acceptable vector. In a specific application, such first gene is selected from a group of SARS coronavirus genes consisting of S, M, OrfS, and E, and the second gene is selected from a group of SARS-CoV genes consisting of N, Orfla, and Orflb.

In yet another embodiment, the first gene product comprises a portion of SARS coronavirus S protein or epitope-bearing domain thereof, or analog thereof, wherein the S protein has a truncation at the carboxy terminus to delete the transmembrane anchor and intracellular domain.

In a related embodiment, the first gene product comprises a first fragment comprising a portion of SARS coronavirus S protein or epitope-bearing domain thereof or analog thereof, wherein the S protein has a truncation at the carboxy terminus to delete the transmembrane anchor and intracellular domain, and a second fragment comprising a portion of SARS-CoV Orf8 protein or epitope-bearing domain thereof or analog thereof, wherein the Orf8 protein has a truncation at the carboxy terminus to delete the transmembrane anchor and intracellular domain.

The SARS vaccine is provided, in yet another embodiment, with a nucleic acid molecule comprising optimized codons for amino acids based on high expression mammalian condon usage rate. The optimized codons, in a specific application, consist of at least 50 % of GC contents. In yet another application, the third positions of the optimized condons consist of at least 50 % of GC contents.

In another embodiment, the SARS coronavirus gene product further comprises a signal peptide fragment for secretion. In a specific application, the signal peptide fragment for secretion is an Igk signal sequence.

The SARS vaccine is provided in polypeptide forms. Such polypeptide is a product of any of the gene product described above, wherein such peptide are substantially purified in vitro.

The invention further relates to a method of inducing an immune response to an antigen in an individual comprising providing to said individual the pharmaceutical compositions described above.

In one embodiment, the pharmaceutical composition is administered about 1 week, 2 weeks, 3 weeks, 4 weeks or 6 weeks, or a combination thereof, after the pharmaceutical composition is first administered. In yet another embodiment, the pharmaceutical composition is administered in combination with an adjuvant.

In a specific application, the pharmaceutical composition with plasmid vector is administered at a dose of 1 milligram to 5 milligrams per injection. In yet another application, the pharmaceutical composition with viral vector is administered at a dose of lxlO⁶ to lxlO¹³ particles per injection.

In one embodiment, the pharmaceutical composition is administered intramuscularly. The pharmaceutical composition is also administered intradermally, subcutaneously, intranasally, or orally.

In another embodiment, the method of inducing an immune response to an antigen comprises administering a pharmaceutical composition, upon first administering a pharmaceutical composition with different vector, to boost immune reaction to said antigen.

DESCRIPTION OF THE DRAWINGS

Fig. 1 shows schematic design of the synthetic gene structure

Fig. 2 shows Codon bias comparison between SARS-CoV, HPV, and human. The G/C content of the optimized gen is the mean G/C percentage of two codon optimized synthetic genes.

Fig. 3 shows schematic drawing and full DNA sequence of the fusion protein with secretion signal. The fusion protein is a combination of the SI domain and extracellular domain of orf8 protein. The two domains are linked through a flexible linker. The sequence encoding the fusion protein is optimized for optimal primate expression.

Fig. 4 shows chematic drawing and amino acid sequence of the Sl-orf8 fusion protein with secretion signal. Fig. 5 shows schematic drawing and full DNA sequence of N protein. The sequence encoding the fusion protein is optimized for optimal primate expression. Fig 6. shows amino acid sequence of N protein.

Fig. 7 shows Antigen-specific T cell response in mice immunized with plasmid SVIOOO. Female Balb/c mice were immunized on days 0, 14, and 28. Total splenocytes were harvested on day 42. 20-mer peptides overlapping by 10 amino acid covering the entire length of gene 1 and gene 2 were used to stimulate splenocytes for 20 hr. Each peptide pool contains approximately 12 peptides with a final concentration of 1 uM of each peptide in the wells.

Fig. 8 shows antibody response in mice immunized with a single injection of SV8000. Anti-SARS-CoV IgG titer was measured using direct ELIS A with plates coated with inactivated SARS-CoV particles.

Fig. 9 shows IFNg T cell response in mice immunized with recombinant adenovirus SV8000. Mice were immunized with increasing doses of SV8000 via intramuscular injection. Total splenocyted were harvested 3 weeks later and stimulated with peptide pools. Mice immunized with placebo were used as control (data not shown).

Fig. 10 shows prime/boost approach greatly enhances antibody response. Mice were immunized with either DNA or adenovirus alone, or with DNA plus adenovirus. Serum samples were collected both before and after the adenovirus boost.

Fig. 11 shows a single injection of recombinant adenovirus vaccine induced potent neutralizing antibody response in Rhesus Macaque. Three monkeys were injected with either placebo or 1E12 particles of recombinant adenovirus vaccine, SV8000. Serum samples were collected each week. Viral neutralization titer was determined with a standard neutralization assay using serial dilutions of serum incubated with live SARS virus before infecting Vero E6 cells.

Fig. 12 shows time course of antibody response in rhesus macaques. Serum samples from different time points were diluted 40-fold prior to ELIS A analysis. The enzymatic activity of HRP was measured as indicator of relative antibody titers

Fig. 13 shows total IgG response before and after the viral challenge. Serum samples from animals 2 days before and 7 days after the virus challenge were analyzed using 96-well plates coated with inactivated SARS-CoV particles

Fig 14 shows detection of SARS-CoV in monkeys infected with the PUMC-1 virus strain. Pharyngeal swab samples were collected 2, 5, and 7 days after viral challenge. Each sample was first incubated in DMEM media and passed through 0.22μm filter before being used to inoculate Vero cells. Negative value represents no visible cytopathic effect (CPE) after three consecutive passages. Quantitative RT-PCR reaction was used to quantify the presence of viral RNA in the pharyngeal swab samples. The genome copy number was determined according to a standard curve according to the Manufacturer's manual.

Fig. 15 shows histology study on postmortem lung sections from rhesus macaques infected with live SARS-CoV. All animals were euthanized 7 days after viral challenge, and tissues samples from multiple organs were processed as described in the Methods. Representative data from lung sections were shown, a. Control group, b/c. High-dose group. d. Low-dose group, e/f. Placebo group. The arrowheads point to alveoli space filled with proteinaceous fluid, and the arrows point to hyaline membranes in sections of the placebo group.

Fig. 16 shows immunohistochemical analysis of postmortem lung sections in monkeys challenged with live SARS-CoV. Anti-SARS-CoV monoclonal antibody was used to identify SARS antigens in the lung sections, a. Negative control, b. High-dose group, c. Low-does group, d. Placebo group. Arrows point to cells that were positive for SARS antigen expression.

DESCRIPTION OF THE INVENTION

Before the use of present proteins and nucleotide sequences for vaccination and therapeutic purposes, and the methods for such uses are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a host cell" includes a plurality of such host cells, reference to the "antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

"Nucleic acid sequence", as used herein, refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. Similarly, "amino acid sequence" as used herein refers to an ohgopeptide, peptide. Polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms, such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

"SARS genes", as used herein, refers to genes or gene fragments that are encoded by the SARS coronavirus. Some of the genes have been described by Marra, MA et al (2003) Sciencexpress, 1-16. These genes include, but are not limited to, spike glycoprotein(S), membrane glycoprotein (M), nucelocapsid protein (N), replicase 1A, replicase IB, small envelope E protein, orβ, orf4, orf6, orf8, orf9, orflO, orfl 1, orfl3, and orfl4. "SARS proteins", as used herein, refers to polypeptides encoded by SARS genes or gene fragments substantially purified from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.

A "variant" of SARS proteins, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes. E.g., replacement of a glycine with a tiyptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

The term "biologically active", as used herein, refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" refers to the capability of the natural, recombinant, or synthetic SARS proteins or any polypeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, when bound to proteins encoded by the SARS genes, causes a change in these proteins which modulates the activity of these proteins. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to SARS proteins or protein fragments

The terms "antagonist" or "inhibitor", as used herein, refer to a molecule which, when bound to SARS proteins or protein fragments, blocks or modulates the biological or immunological activity of SARS proteins. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to SARS proteins.

The term "modulate", as used herein, refers to a change or an alteration in the biological activity of SARS proteins. Modulation may be an increase or a decrease in protein activity, a change in binding characteristics, or any other change in the biological, functional or immunological properties of SARS proteins.

The term "mimetic", as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of SARS proteins or portions thereof and, as such, is able to effect some or all of the actions of SARS protein-like molecules.

The term "derivative", as used herein, refers to the chemical modification of a nucleic acid encoding SARS proteins or the encoded SARS genes. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative would encode a polypeptide which (retains essential biological characteristics of the natural molecule.

The term "substantially purified", as used herein, refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 15% free, and most preferably 90% free from other components with which they are naturally associated.

"Amplification", as used herein, refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). The term "hybridization", as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

The term "hybridization complex", as used herein, refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., C.sub.O t or R.sub.01 analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which cells have been fixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, for the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30%) identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed to comprise either low or high stringency conditions. Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.), and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.

The term "stringent conditions", as used herein, is the "stringency" which occurs within a range from about Tm-5.degree. C. (5.degree. C. below the melting temperature I of the probe) to about 20.degree. C. to 25.degree. C. below Tm. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced b, the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand.

The term "portion", as used herein, with regard to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. Thus, a protein "comprising at least a portion of the amino acid sequence of SARS proteins" encompasses the full-length SARS proteins and fragments thereof.

"Transformation", as defined herein, describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

The term "antigenic determinant", as used herein, refers to that portion of a molecule that makes contact with a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein, in reference to the interaction of an antibody and a protein or peptide, mean that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein: in other words, the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "sample", as used herein, is used in its broadest sense. A biological sample suspected of containing nucleic acid encoding SARS proteins or fragments thereof may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), an extract from cells or a tissue, and the like.

The term "correlates with expression of a polynucleotide", as used herein, indicates that the detection of the presence of ribonucleic acid that is similar to the polynucleotides encoded by the SARS virus by northern analysis is indicative of the presence of mRNA encoding SARS proteins in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.

"Alterations", in the polynucleotides encoded by the SARS genes, as used herein, comprise any alteration in the sequence of polynucleotides encoding SARS proteins including deletions, insertions, and point mutations that may be detected using hybridization assays. Included within this definition is the detection of alterations to the genomic DNA sequence which encodes SARS proteins, the inability of a selected fragment of the polynucleotides encoded by the SARS virus to hybridize to a sample of genomic DNA (e.g., using allele- specific oligonucleotide probes), and improper or unexpected hybridization, such as hybridization to a locus other than the normal chromosomal locus for the polynucleotide sequence encoding SARS proteins (e.g., using fluorescent in situ hybridization [FISH] to metaphase chromosome spreads).

As used herein, the term "antibody" refers to intact molecules as well as fragments thereof, such as Fab F(ab').sub.2, and Fv, which are capable of binding the epitopic determinant. Antibodies that bind SARS proteins can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or peptide used to immunize an animal can be derived from the transition of RNA or synthesized chemically, and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.

The term "vector", as used herein, refers to any nucleotide or nucleoside sequences that are capable of expressing polypeptides in an appropriate environment, such as cells and in vitro cell-free transcription/translation systems. Vector includes, but is not limited to, DNA plasmids, viral expression vectors, and RNA replicons.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention describes a novel vaccine design, which comprises single or multiple genes and gene fragments, gene products, or proteins and protein fragments from the SARS coronavirus for the prevention, or treatment of SARS.

The SARS virus has a single-stranded positive RNA genome. Due to the poor replication fidelity of RNA-dependent RNA polymerase, the SARS genome is predicted to undergo rapid mutations. In fact, several institutions all over the world have sequenced the genome of SARS coronavirus and deposited the result into data repositories such as GenBank. A standard sequence alignment revealed several sequence changes among different virus isolates.

Virus with high mutation rate creates a daunting challenge for the development of effective vaccines, since immune responses raised against one strain of the virus may not work effectively against a variant from the same strain. The same problem is believed to occur with SARS as well. To minimize the change of viral escape, multiple genes or proteins from the SARS genome can be simultaneously used as vaccine targets. In one embodiment of the invention, a vector capable of expressing one or more of genes encoded by the SARS genome, or a fragment or a derivative thereof, can be used as vaccine to prevent or treat SARS. The genes include, but are not limited to spike glycoprotein(S), membrane glycoprotein (M), nucelocapsid protein (N), replicase 1 A, replicase IB, small envelope E protein, orf3, orf4, orf6, orf8, orf9, orflO, orfl 1, orfl3, and orfl4. This group of genes is referred to as SARS genes (see definition). Some of the genes were described in Marra, MA et al. (2003) Sciencexpress, 1-5. One vaccine method is described in Raz, E. et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 9519-9523 and Donnelly, J. J. et al. (1996), J. Infect. Dis., 173, 314-320, among others. In a particular embodiment, a vector capable of expressing S and N, or fragments or derivatives thereof, can be used vaccine for the prevention or treatment of SARS.

In another embodiment, two or more SARS proteins (see definition), or fragments or derivatives thereof, can be expressed in an appropriate host and used as vaccine for the prevention and treatment of SARS.

In another embodiment, a vector capable of expressing fusions of two or more genes encoded by the SARS genome, or a fragment or a derivative thereof, can be used as a vaccine to prevent or treat SARS. The genes can be linked directly, or through a linker which has one or more amino acids.

In another embodiment, a vector capable of expressing fusions of extra-cellular domains of viral surface proteins, or a fragment or a derivative thereof from two or more SARS genes, can be used as a vaccine for the prevention and treatment of SARS. These viral surface proteins included, but are not limited to, include, spike glycoprotein(S), membrane glycoprotein (M), small envelope E protein, orf3, and orfδ. In a specific embodiment, a vector capable of expressing fusions of the extracellular domains of S and orf8, or fragments or derivatives thereof, can be used as a vaccine for the prevention or treatment of SARS.

In another embodiment, extra-cellular domains, or a fragment or a derivative thereof from two or more genes, including S, M, E, orf8, and orf3, can be fused and expressed as fusion proteins in an appropriate organism and used as vaccine for the prevention or treatment of SARS. In a particular embodiment, extra-cellular domains, or a fragment or a derivative thereof from S and orf8 can be fused and expressed as fusion proteins for the prevention or treatment of SARS.

In another embodiment, a vector capable of expressing a combination of fusions of SARS proteins, or fragments or derivatives thereof, and individual SARS proteins or protein fragments can be used vaccine for the prevention and treatment of SARS.

Any nucleic acid sequence, which encodes the amino acid sequence of SARS genes, or fragments thereof, can be used to generate recombinant molecule which express SARS genes, or fragments thereof. In a particular embodiment, the invention encompasses the use of polynucleotide encoding S, orf8, and N.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding SARS genes, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of nucleotide sequence that could be made by electing combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring SARS genes, and all such variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode SARS genes, and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring SARS genes, under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding these proteins or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding SARS genes, and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

DNA sequences, or portions thereof, which encode SARS genes, and its derivatives, can also be produced entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art at the time of the filing of this application. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding SARS genes, or arty portion thereof.

Altered nucleic acid sequences encoding SARS genes, which are encompassed by the invention include deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent proteins. The encoded protein may also contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent SARS genes. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the relevant biological activity, such as bout not limited to immunogenicity, of these proteins is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; and glutamine; serine and threonine; phenylalanine and tyrosine.

Also included within the scope of the present invention are the use of alleles of the genes encoding SARS genes. As used herein, an "allele" or "allelic sequence" is an alternative form of the gene which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered RNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. Methods for DNA sequencing which are well known and generally available in the art may be used to practice any embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENCE DNA polymerase (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago 111.), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE amplification system (GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automated with machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

Polynucleotide sequences or fragments thereof which encode SARS genes or gene products, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of proteins in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express these proteins.

As will be understood by those of skill in the art, it may be advantageous to produce SARS genes-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences can be engineered using methods generally known in the art in order to alter SARS genes encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments arid synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, site- directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth. In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding SARS genes, may be ligated to a heterologous sequence to encode a fusion protein for use in the diagnosis, prevention, and treatment of SARS. For example, to screen peptide libraries for inhibitors of protein activity, it may be useful to encode a chimeric SARS genes, protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the encoding sequence and the heterologous protein sequence, so that S or ORF 8 may be cleaved and purified away from the heterologous moiety.

Sequences encoding SARS genes may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 7 :2315-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of SARS genes, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A peptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Additionally, the amino acid sequence of SARS genes, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active SARS genes, the nucleotide sequences encoding SARS genes or functional equivalent, may be inserted into appropriate expression vector. I.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding SARS genes and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N. Y.

A variety of expression vector/host systems may be utilized to contain and express sequences encoding SARS genes or gene products. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., adenovirus or baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control sequence" or "regulatory sequences" are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif,) or PSPORTl plasmid (Gibco BRL) and the like may be used. The baculovirus romoter n promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding SARS genes, vectors based on SV40 or EBV may be used with an appropriate selectable marker. In bacterial systems, a number of expression vectors may be selected depending upon the use intended for SARS genes. For example, when large quantities of SARS genes are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT phagemid (Stratagene), in which the sequence encoding SARS genes may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of .beta. - galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster ( 1989) J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, fhrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast, Saccharomyce cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of sequences encoding SARS genes may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3 :1671-1680 ; Broglie, R. et al. (1984) Science 224:838- 843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook do Science and Technology (1992) McGraw Hill, New York, NT.; pp. 191-196). An insect system may also be used to express SARS genes. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding SARS genes may be cloned into a non-essential region of the virus, such as the romoter n gene, and placed under control of the romoter n promoter. Successful insertion of SARS genes will render the romoter n gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which S or ORF 8 may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding S or ORF 8 may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing SARS genes in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficient translation of sequences encoding SARS genes. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding SARS genes, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162). In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and WD 8, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express SARS genes may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. Et al. (1980) Cell 22:817-23) genes which can be employed in tk or aprt cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection: for example., dhfr which confers resistance to methotrexate (Wigler, M. et at. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin-acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptolphan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Nat. Acad. Sci. 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, .beta, glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding SARS genes is inserted within a marker gene sequence, recombinant cells containing sequences encoding SARS genes can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding S or ORF 8 under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequence encoding SARS genes and express SARS genes may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.

The presence of polynucleotide sequences encoding SARS genes can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of polynucleotides encoding SARS genes 8. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding S or ORF 8 to detect transformants containing DNA or RNA encoding S or ORF 8. As used herein "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer. A variety of protocols for detecting and measuring the expression of S or ORF 8, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies; reactive to two non-interfering epitopes on SARS genesis preferred, but a competitive birding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211- 1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SARS genes include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding SARS genes, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio). Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding SARS genes may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode SARS genesmay be designed to contain signal sequences which direct secretion of SARS genesthrough a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding SARS genes to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating pepitides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAG extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and SARS genes may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing SARS genesand a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides a means for purifying SARS genes from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of SARS genes may be produced by direct peptide synthesis using solid-phase techniques Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A peptide synthesizer (Perkin Elmer). Various fragments of SARS genes may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

Antibodies which are specific for SARS genes may be used as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express S or ORF 8.

In other embodiments, any of the therapeutic proteins, antagonists, antibodies, agonists, antisense sequences or vectors described above may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Genes encoding SARS genes can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes S or ORF 8. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of S or ORF 8, antibodies to S or ORF 8, mimetics, agonists, antagonists, or inhibitors of S or ORF 8. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including romot and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum romot, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hawks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipopbilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the-compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of S or ORF 8, such labeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active ingredient, for example SARS genes or fragments thereof, antibodies of SARS genes, agonists, antagonists or inhibitors of S or ORF 8, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50%o of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

In order to more fully demonstrate the advantages arising from the present invention, the following sample is set forth. It is to be understood that the following is by way of example only and is not intended as a limitation on the scope of the invention.

A synthetic gene was designed following two principals: 1) surface proteins potentially involved in viral attachment and infection are included; 2) At least one relatively conserved protein is included.

Based on sequence predictions from the SARS-CoV genome, two surface proteins were chosen to be included: SI and orf8. The SI domain of the Spike protein is the primary mediator of viral attachment to the cell surface, whereas the orf8 protein has unknown function but has an extended extra-cellular domain that might be involved viral infection as well. The N protein is relatively well conserved and has been shown to provide strong T cell help, therefore it is included in the gene package. In order to express three proteins at the same time, the SI and the extracellular domain of orf8 were fused with a flexible linker designed to help each protein to fold independently. The N protein was transcriptionally fused to the Sl-orf8 fusion protein via an Internal Ribosomal Entry Site (IRES). As shown in Fig. 1, both proteins can be expressed at the same time via a single CMV promoter.

As discussed above, there is significant difference in the codon bias between viruses and human. Many viruses, such as HIV and HPV, have lower G-C content in their coding sequences. In contrast, the G-C content of human genes has much higher G-C content, which is especially the case when the 3^rd base position was considered. The bias towards codons with higher G-C content may be due to the higher intracellular tRNA concentration of the respective codons so that proteins can be translated more efficiently. Furthermore, higher G-C content is also associated with higher mRNA stability, which can increase target protein expression.

The coding sequence for target antigens was optimized using optimal human codons to ensure efficient transcription. As shown in Fig. 2, the SARS-CoV S and N gene showed a typical viral bias toward lower G-C content, with less than 40%» G-C content at the 3^rd base position. In contrast, the G-C percentage for highly expressed human proteins is 77%> percent. The optimized genes have an average G-C content of 66% at the 3 ^rd base, making it in line with the average human percentage. To further optimize the synthetic genes, potential alternative mRNA splicing sites were identified using an intron-exon junction prediction algorithm. Removal of these sites via silent mutagenesis will ensure that only the correct target proteins will be expressed.

In one preferred embodiment, the S 1 domain and orfS extracellular domain are fused in a structure as shown in Fig. 3. An IgK secretion signal is added to the N-terminal of the fusion protein so that the fusion protein will be secreted and generate more potent antibody reaction. The sequence of such fusion protein, which is shown in Fig. 3, has been optimized for optimal expression in primates. The amino acid sequence of such fusion protein is shown in Fig. 4.

In another preferred embodiment, N protein sequence is optimized and is shown in Fig. 5. Corresponding amino acid sequence is shown in Fig. 6. Such synthetic genes are cloned into various vectors, including viral and plasmid vectors.

In one experiment demonstrating the efficacy of the vaccine in a DNA plasmid vector (SVIOOO), six-to-eight week old female Balb/c mice were immunized with lOOug endotoxin- free DNA plasmid SVIOOO on week 0, 2, and 5. Blood samples were collected on week 3 and 6. Total splenocytes were collected at week 6 when the experiment was terminated. Most mice have sera-converted by week 3, although the antibody titer was still relatively low. The antibody titer has climbed to around 1 :640 by week 6, which is within the range of antibody response induced by other DNA vaccines as reported in the literature.

One of the serum samples was also tested for its ability to neutralize live SARS virus to infect Vero E6 cells. Serial 2-fold dilutions of the serum were pre-incubated with virus before being used to infect Vero E6 cells. The final neutralizing titer was calculated to be around 1:300.

IFNg specific ELISPOT assay was carried out to quantify antigen-specific T cell responses. 20-mer peptides overlapping by 10 amino acids covering the entire length of both synthetic genes were synthesized. Pools of 12 peptides each were used to stimulate whole splenocytes from immunized mice. As shown in Fig. 7, SVIOOO can induce a potent Thl T cell response against multiple regions of the vaccine. Importantly, regions from both Sl-orf8 and N were recognized, indicating that both genes were expressed in vivo and were involved in generating specific immune responses.

A recombinant type V adenovirus carrying the synthetic gene, termed SV8000, was also constructed. As shown in Fig. 8, a single injection with SV8000 induced strong antibody responses in mice, with antibody titer exceeding 1:1000. The response is also dose-dependent in that 1E9 viral particles induced stronger antibody response than 1E8 viral particles.

IFNg T cell response was also analyzed via ELISOPT. As shown in Fig. 9, a single injection of SV8000 induced potent T cell response across multiple regions of the synthetic genes. The magnitude of response is also significantly greater than that induced by SVIOOO DNA vaccine. Interestingly, there is no linear correlation between vaccine dosage and T cell response. 1E8 particles induced the strongest T cell response, whereas mice immunized with either 1E9 or 1E7 particles have lower response.

Recent research has demonstrated that heterologous prime-boost approach can vastly enhance the immune response generated by either single vaccine component. This is especially true when DNA vaccine was used as the priming antigen. It was thought that the DNA priming could activate and expand antigen-specific T cells with high avidity during the priming phase, due to the low concentration of antigen available and the lack of "noise" antigens normally contained in recombinant viruses. The viral boost further expands the memory pool, creating superior response than either vaccine alone.

As shown in Fig. 10, the prime boost approach induced at least 10-fold higher antibody response than either DNA alone or recombinant adenovirus alone.

It has been reported in the literature that a single injection of recombinant adenovirus vaccine can completely protect non-human primate from Ebola virus challenge. This is potentially useful for dealing with public health emergency such as SARS outbreak when prolonged immunization schedule is not feasible. A single injection, rapid acting vaccine can offer fast protection and reduces the potential danger faced by the emergency response team.

Two Rhesus Macaque monkeys were immunized with 1E12 particles of the SV8000 vaccine. A third monkey was immunized with just media. Blood samples were collected prior to the immunization, and every week after the immunization for 10 weeks. PBMCs were isolated and stored in liquid nitrogen for batch analysis later. The serum samples were used in a standard virus neutralization assay. A clear trend of immune response can be observed from the data. As shown in Fig. 11, the neutralizing antibody appears to peak at around week four and then stay relatively constant for the next 3 weeks. It is worth noting that the mean neutralizing antibody titer in convalescent patients is only around 1:64. Although it's tempting to suggest that a single injection of SV8000 has the potential to offer protection, it is not clear whether neutralizing antibody alone is sufficient. T cells, especially CTLs, have been implicated to play an important role in other animal models of coronavirus infection, such as IBV and MHV. Furthermore, due the relative short history of SARS-CoV, it is not known whether this virus will come back with a serologically different S protein, in which case T cell activation will be crucial for cross-strain protection.

Efficacy of the vaccine was examined in monkeys. Rhesus Macaque monkeys were immunized twice with S V8000 intramuscularly at week 0 and 4. Antibody response was relatively low in the first 4 weeks. It rose quickly after the booster injection (Fig. 12) By week 8, all immunized onkeys produced high titers of IgG antibody.

To test the protective efficacy of the vaccine, monkeys in the high-dose, low-dose, and placebo group were challenged intranasally with 10⁵TCID₅₀ live virus. Two monkeys in the control group were housed in the same animal facility but were not infected with live virus, serving as negative control for the experiment. Three days after virus inoculation, all four monkeys in the placebo group became lethargic, and had markedly reduced food intake. Two animals displayed symptoms of respiratory distress. In contrast, none of the immunized animals, or the control animals, displayed visible clinical symptoms.

The IgG concentration specific for SARS conoravirus for the immunized groups increased significantly compared to the pre-challenge concentration (Fig. 13). The increase ranges from 3 folds to almost 30 folds 7 days after viral challenge, indicating that he humoral response has been effectively primed by the vaccine injection.

Pharyngeal swab ad serum samples were collected on day 2, 5 and 7 after challenge. The presence of SARS coronavirus was detected via either RT-PCR or viral culture. Tow out of four animals from the high dose group had detectable viral RNA two days after the challenge (Fig. 14). Viral RNA disappeared by day 5. The other two animals in the same group did not have detectable viral RNA any time point. None of the four animals from the low-dose had detectable viral RNA on either day 2 or day 5, and there is only a low level of viral RNA detected on day 7 from one animal. In contrast, all four animals in the placebo group are positive for viral RNA after challenge. Pharyngeal swab samples were also used to inoculate Vero cells in order to re-isolate live virus after the challenge. As shown in Fig. 14, live virus can be isolated from three out of four animals in the placebo group. Both high dose and low does group were protected from viral infection.

Animals in the placebo group suffered severe pulmonary damage, characterized by extensive disruption of alveoli walls and the accumulation of proteinaceous edema fluid in the . alveoli space (Fig.l5e). The alveoli wall was thickened by heavy infiltration of mononuclear cells, and hyaline membrane could be identified lining the alveoli walls (Figl5f). In the most severe cases, extensive destruction of the epithelial cell layer and local hemorrhage could be observed. Histology for the high-dose animals (Fig. 15b) were overall comparable to that of the control animals (Fig.l5a), although in rare instances minor edma and mononuclear cell infiltration in the alveoli wall could be observed (Fig.l5c). The phenotype for the low-dose group was somewhat in between the high-dose and the placebo group (Fig.l5d).

In order to correlate the disease pathology with viral replication, sections of the lung were also analyzed by immunohistochemistry, as shown in Fig. 16. There was mimmal, if any, staining in the sections for the high-dose group, which was similar to that of negative control. In contrast, sections for the placebo group showed numerous positive cells, with predommantly cytosolic staining, indicating continuing antigen expression in these cells. The low-dose group, again, showed intermediate phenotype, with antigen positive cells both in the alveoli wall and the epithelial cells.

Claims

CLAIMS What is claimed is:

1. A pharmaceutical composition comprising a nucleic acid molecule encoding one or more SARS coronavirus gene products operatively-linked to a control sequence, in a pharmaceutically acceptable vector.

2. The pharmaceutical composition of Claim 1, wherein the SARS coronavirus gene is selected from a group of S protein, OrfS protein, M protein, N protein, E protein, Orfla, Orflb, and combination thereof.

3. The pharmaceutical composition of Claim 1, wherein the control sequence is a promoter.

4. The pharmaceutical composition of Claim 3, wherein the promoter is a CMV promoter.

5. The pharmaceutical composition of Claim 1 , wherein the pharmaceutically acceptable vector is either a viral vector, a plasmid vector, or an RNA vector.

6. The pharmaceutical composition of Claim 5, wherein the viral vector is a replication- deficient adenovirus.

7. The pharmaceutical composition of Claim 6, wherein the viral vector is a type 5 replication-deficient adenovirus.

8. The pharmaceutical composition of Claim 1 , wherein the nucleic acid molecule encoding two SARS coronavirus gene products, with a first gene product comprising at least one extracellular domain of a SARS-CoV gene, or a product derived therefrom, and a second gene product comprising an intracellular protein, or a product derived therefrom, in a pharmaceutically acceptable vector.

9. The pharmaceutical composition of Claim 8, wherein the first gene is selected from a group of SARS-CoV genes consisting of S, M, Orf8, and E, and the second gene is selected from a group of SARS-CoV genes consisting of N, Orfla, and Orflb.

10. The pharmaceutical composition of Claim 8, wherein the first gene product comprising a portion of SARS-CoV S protein or epitope-bearing domain thereof, or analog thereof, wherein the S protein has a truncation at the carboxy terminus to delete the transmembrane anchor and intracellular domain.

11. The pharmaceutical composition of Claim 8 wherein the first gene product comprising a first fragment comprising a portion of SARS-CoV S protein or epitope-bearing domain thereof or analog thereof, wherein the S protein has a truncation at the carboxy terminus to delete the transmembrane anchor and intracellular domain, and a second fragment comprising a portion of SARS-CoV OrfS protein or epitope-bearing domain thereof or analog thereof, wherein the OrfS protein has a truncation at the carboxy terminus to delete the transmembrane anchor and intracellular domain.

12. The pharmaceutical composition of Claim 1, wherein the nucleic acid molecule comprising optimized codons for amino acids based on high expression mammalian condon usage rate.

13. The pharmaceutical composition of Claim 12, wherein the optimized codons consist of at least 50 % of GC contents.

14. The pharmaceutical composition of Claim 12, wherein the third base positions of the optimized condons consist of at least 50 % of GC contents.

15. The pharmaceutical composition of Claim 1 , wherein the SARS-CoV gene product further comprising a signal peptide fragment for secretion.

16. The pharmaceutical composition of Claim 8, wherein the first gene product further comprising a signal peptide fragment for secretion.

17. The pharmaceutical composition of Claim 16, wherein the signal peptide fragment for secretion is an Igk signal sequence.

18. A peptide or a combination of peptides selected from the gene product according to any of claims 1- 17, wherein such peptides are purified in vitro.

19. A method of inducing an immune response to an antigen in an individual comprising providing to said individual the pharmaceutical composition according to any of claims 1-17.

20. The method of inducing an immune response to an antigen according to Claim 19, wherein the pharmaceutical composition is administered about 1 week, 2 weeks, 3 weeks, 4 weeks or 6 weeks, or a combination thereof, after the pharmaceutical composition is first administered.

21. The method of inducing an immune response to an antigen according to Claim 19, wherein the pharmaceutical composition is administered in combination with an adjuvant.

22. The method of inducing an immune response to an antigen according to Claim 19, wherein the pharmaceutical composition with plasmid vector is administered at a dose of 1 milligram to 5 milligrams per injection.

23. The method of inducing an immune response to an antigen according to Claim 19, wwhheerreeiinn tthhee pphhaarrmmaacceeuuttiiccaall cccomposition with viral vector is administered at a dose of 1x10 to lxlO¹³ particles per injection.

24. The method of inducing an immune response to an antigen according to Claim 19, wherein the pharmaceutical composition is administered intramuscularly, intradermally, subcutaneously, intranasally, or orally.

25. The method of inducing an immune response to an antigen according to Claim 19, further comprising, after administering a first pharmaceutical composition, administering a pharmaceutical composition with different vector to boost immune reaction to said antigen.