CN1791427A - 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

Design method and components of novel vaccines for prevention and treatment of SARS

This application claims priority to US Provisional Patent Application No. 60/474,507, filed May 31, 2003.

field of invention

The invention relates to virus vaccines, more particularly to SARS virus vaccines and methods for preventing or treating diseases caused by SARS virus infection.

Background of the invention

SARS is a respiratory disease first reported in Guangdong Province in southern China in November 2002. The disease has since spread, reaching more than a dozen countries and infecting more than 8,000 people worldwide. The overall mortality rate in SARS patients is estimated to be 14-15%, and in patients over 65 years, the overall mortality rate exceeds 50%.

The general symptoms of SARS include: fever exceeding 100.4F ⁰ , dry cough, body pain, and dyspnea. It is generally believed that SARS is transmitted through close contact between people, but it may also be caused by people touching contaminated items and then touching their nose and mouth.

Scientists at the U.S. Centers for Disease Control and other laboratories have detected a previously unseen coronavirus from SARS patients, and this new virus may be the main cause of SARS.

Contents of the invention

The invention provides a SARS virus vaccine comprising a nucleic acid sequence encoding a SARS coronavirus gene product. In a specific implementation, the nucleic acid sequence encoding one or more SARS virus gene products can be connected with the regulatory sequence on the pharmaceutically compatible carrier to form a recombinant vector.

In a specific application, the SARS coronavirus gene is selected from S protein, Orf8 protein, M protein, N protein, E protein, Orf1a protein, Orf1b protein and combinations thereof. The pharmaceutical composition includes a regulatory sequence, usually referred to as a promoter. For example, a CMV promoter is used as a promoter.

The pharmaceutically compatible carrier can be a viral vector, a plasmid vector or an RNA vector. For example, a replication-deficient adenovirus is used as the viral vector. In a particular application, the adenovirus used is a replication-deficient adenovirus type 5.

In one embodiment, the SARS virus vaccine consists of a nucleic acid molecule cloned on a pharmaceutically compatible carrier encoding two SARS coronavirus gene products, wherein the first gene product contains at least one SARS coronavirus gene extracellular domain , or its derivatives. Another gene product comprises an intracellular protein, or a derivative thereof. In a specific application, the first gene is selected from a SARS-CoV gene group including S, M, Orf8 and E, and the second gene is selected from a SARS-CoV gene group including N, Orf1a and Orf1b.

In another embodiment, the first gene product may be a part of the S protein of SARS coronavirus, or an antigenic determinant of its domain, or an analog thereof, wherein the S protein has a truncated C-terminus that removes the span Membrane anchors and intracellular domains.

In a related embodiment, the first gene product comprises two SARS coronavirus gene product fragments, and the first fragment comprises a part of SARS coronavirus S protein or an antigenic determinant of its domain, or an analog thereof, wherein The S protein has a truncated C-terminus to remove the membrane anchor and intracellular domain; the other fragment contains a part of the SARS coronavirus Orf8 protein, or an antigenic determinant of its domain, or its analogue, wherein the Orf8 protein Has a truncated C-terminus to remove the transmembrane anchor and intracellular domain.

In another embodiment, the nucleic acid molecule of the SARS vaccine is composed of optimized codons, which are optimized according to the frequency of usage of codons in highly expressed mammalian genes. In a specific embodiment, the optimized gene contains at least 50% GC bases. In another embodiment, at least 50% of the optimized codons have a G or C base in the third position.

In another embodiment, the SARS coronavirus gene product further comprises a secreted signal peptide fragment. In a specific application, the signal peptide fragment can be an Igk signal sequence.

The SARS vaccine is provided in the form of polypeptides. These polypeptides can be any of the above-mentioned gene products, and these peptides have been sufficiently purified in vitro.

The present invention also relates to a method of inducing an immune response against an antigen in an individual by providing the above-mentioned pharmaceutical composition to the individual.

In one embodiment, the pharmaceutical ingredients are administered for about 1 week, 2 weeks, 3 weeks, 4 weeks or 6 weeks, or, after the first administration, are administered in combination at different times.

In another embodiment, the pharmaceutical composition is administered in combination with an adjuvant.

In a specific application, the dosage of the pharmaceutical composition of the plasmid vector is 1 mg to 5 mg per injection. In another application, the dosage of the pharmaceutical composition of the virus vector is 1×10 ⁶ -1×10 ¹³ virus particles per injection.

In one embodiment, the pharmaceutical composition is administered by intramuscular injection. Alternatively, the pharmaceutical composition is administered intradermally, subcutaneously, intranasally or orally.

In another embodiment, the method for inducing an immune response against a certain antigen comprises administering a pharmaceutical composition, the first administration of which is a different carrier to elicit an immune response to the above-mentioned antigen.

Description of drawings

Figure 1 is a schematic diagram of the structure of the synthesized gene fragment.

Fig. 2 is the codon bias comparison of SARS coronavirus, HPV and human, and the G/C content of the optimized gene is the average value of the G/C percentage of the synthetic gene after two codon optimizations.

Figure 3 is a schematic diagram and full-length DNA sequence of a fusion peptide with a secretion signal. The fusion protein includes the S1 domain and the extracellular domain of the Orf8 protein. The two domains are linked by a flexible linker. The sequence encoding the fusion protein is optimized for optimal expression in primates.

Fig. 4 is a schematic diagram and amino acid sequence of S1-orf8 fusion protein with secretion signal.

Figure 5 is a schematic diagram of N protein and its full-length DNA sequence. The sequence encoding this fusion protein was optimized for optimal expression in primates.

Figure 6 is the amino acid sequence of N protein.

Fig. 7 is the antigen-specific T cell response of mice immunized with plasmid SV1000. Female Balb/c mice were immunized on days 0, 14 and 28, and total splenocytes were harvested on day 42. Splenocytes were stimulated for 20 hr with a polypeptide pool of 20 amino acids in length (each polypeptide overlapping the previous polypeptide by 10 amino acids) covering the entire gene 1 and gene 2. Each peptide library has about 12 peptides, and the final concentration of each peptide in the well is 1uM.

Figure 8 is the antibody response of mice after single injection of SV8000 plasmid immunization.

Determination of anti-SARS coronavirus IgG antibody titer uses inactivated SARS coronavirus particles to coat the ELISA plate for direct ELISA reaction.

Figure 9 is the mouse IFNg T cell response after immunization with recombinant adenovirus SV8000. Mice were immunized with increasing doses of SV8000 intramuscularly, and all lymphocytes were harvested 3 weeks later, and splenocytes were stimulated with a peptide library. Mice in the placebo group were used as controls (data not shown).

Figure 10 shows that the method of immunization induction combined with booster immunization greatly enhanced the antibody response. Mice were immunized with DNA, adenovirus alone, or a combination of DNA and adenovirus. Serum samples were collected before and after adenoviral challenge.

Figure 11 is a graph showing the effective induction of neutralizing antibody responses by a single injection of recombinant adenovirus vaccine in rhesus monkeys. Three monkeys were injected with placebo and 1E12 recombinant adenovirus particles (SV8000). Serum samples were collected every week, and Vero E6 cells were infected with serially diluted serum samples incubated with live SARS virus for standard neutralization test to determine the neutralization titer.

Fig. 12 is the time variation of antibody response in rhesus monkeys. Serum samples at different time points were diluted 40 times before the ELISA test. The enzyme activity of HRP was measured to represent the relative antibody titer.

Figure 13 shows the response of total IgG antibodies before and after virus challenge. Animal serum samples were collected 2 days before and 7 days after virus challenge, and the antibody assay was performed on a 96-well plate coated with inactivated SARS coronavirus particles.

Figure 14 is the situation of detecting SARS coronavirus in monkeys infected with PUMC-1 virus strain. Pharyngeal swab samples were collected on days 2, 5 and 7 after virus challenge. Each sample was first incubated with DMEM and filtered through a 0.22um filter before inoculating Vero cells. Negative values indicate no visible cytopathic effect (CPE) after 3 consecutive passages. Quantitative RT-PCR reactions were used to determine the amount of viral RNA present in throat swab samples. Follow the manual to draw a standard curve to determine the copy number of the genome.

Fig. 15 is the histochemical study result of the autopsy lung tissue section of rhesus monkey infected with live SARS coronavirus. Animals were euthanized on day 7 post virus challenge and samples from various organs were processed as described in the Methods section.

Lung tissue sections are presented here as representative data, a. control group; b/c. high-dose group; d. low-dose group; e/f. placebo group. The bubbles indicated by the arrows are filled with proteinaceous liquid, and the arrows point to the hyaloid membranes of the placebo group.

Fig. 16 is the immunohistochemical analysis result of the autopsy lung tissue section of live SARS virus challenged monkey. Anti-SARS coronavirus monoclonal antibody is used to determine the antigen of lung tissue section, a. negative control; b. high-dose group; c. low-dose group; d. placebo group. Arrows point to cells positive for SARS antigen expression.

Detailed description of the invention

Before describing the use and methods of these protein and nucleic acid sequences in vaccination and therapy, it is to be understood that this invention is not limited to the methods, protocols, cell lines, vectors and reagents specifically described herein, as these may vary. The following detailed description should also be understood that the terminology used herein is only for describing specific embodiments, and its purpose is not to limit the scope of the present invention, and the scope of protection is only described in the appended claims.

It must be noted that, as used herein and in the appended claims, the singular forms include the plural unless the context clearly dictates otherwise. Therefore, for example, said one host cell includes plural forms of said host cell, said antibody refers to one or more antibodies, and its meaning is well known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. 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. The publications mentioned herein are intended to describe and illustrate the cell lines, vectors and methodologies which are reported in the publications and which are relevant to the present invention.

Nothing stated here should be taken as a reason that the present invention is not entitled to prior publication of the contents of an earlier application.

definition

As used herein, "nucleic acid sequence" refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or parts thereof, derived from DNA or RNA genome or synthetically single or double stranded, both It can be either the sense strand or the antisense strand.

Likewise, "amino acid sequence" as used herein refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or parts thereof, which may be naturally occurring or synthetic molecules.

The "amino acid sequence" mentioned here refers to the amino acid sequence of a naturally occurring protein molecule. "Amino acid sequence" and similar terms, such as "polypeptide" or "protein", are not limited to the protein molecule described The relevant complete, native amino acid sequence.

"SARS gene" as used herein refers to the gene or gene fragment encoded by SARS coronavirus. Some genes have been described by Marra, MA et al (2003) in Scienceexpress, 1-16. These genes include, but are not limited to, the genes listed below: Spike protein (S), membrane protein (M), nucleocapsid protein (N), replicase 1A, replicase 1B, small envelope E protein, orf3, orf4, orf6, orf8, orf9, orf10, orf11, orf13, and orf14.

The "SARS protein" used here refers to polypeptides encoded by SARS genes or gene fragments. These SARS proteins can be fully purified from various species, especially mammals, including cattle, sheep, pigs, mice, and horses. , especially humans, the source can be natural, synthetic, semi-synthetic or recombinant.

A "mutant" of a SARS protein as used herein refers to an amino acid sequence in which one or more amino acids are changed. The "mutant" may contain "conservative" changes, ie, substituted amino acids having similar structural and chemical properties, such as substitution of isoleucine for leucine. In rarer cases, this mutant can also contain "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor mutations can also be amino acid deletions or insertions, or both deletions and insertions. Methods for determining which amino acid residues should be substituted, inserted or deleted without affecting biological or immunological activity are available in existing computer software, eg, DNASTAR software.

As used herein, "deletion" refers to a change in amino acid or nucleic acid sequence, ie deletion of one or more amino acid or nucleic acid residues, respectively.

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

"Substitution" as used herein refers to the replacement of one or more amino acids or nucleic acids with different amino acids or nucleic acids, respectively.

The term "biological activity" means that the protein possesses the structural, regulatory, or biochemical functions of naturally occurring molecules. Similarly, "immunological activity" refers to the ability of a natural, recombinant or synthetic SARS protein or any related polypeptide to trigger a corresponding animal or cell-specific immune response and bind to a specific antibody.

The term "agonist" as used herein refers to a molecule that, when bound to the proteins encoded by the SARS genes, can cause changes in these proteins and thereby regulate the activity of these proteins. Agonists can be proteins, nucleic acids, carbohydrates or any other molecules that can bind to SARS proteins or protein fragments.

The term "antagonist" or "inhibitor" as used herein refers to a molecule that can inhibit or modulate the biological or immunological activity of the SARS protein when the molecule binds to the SARS protein or protein fragment. Antagonists and inhibitors can be proteins, nucleic acids, carbohydrates or any other molecules that can bind to SARS proteins.

The term "modulation" as used herein refers to the alteration or variation of the biological activity of the SARS protein. Modulation may be the enhancement or reduction of protein activity, or the change of binding properties, or any other change in the biological, functional or immunological properties of the SARS protein.

The term "mimetic" as used herein refers to a molecule whose structure is developed through the knowledge of the structure of the SARS protein or a part thereof to become a SARS-like protein molecule having the activity of part or all of the SARS protein.

The term "derivative" as used herein refers to a chemical modification that occurs on the nucleic acid encoding the SARS protein or the encoded SARS gene. Illustratively, such chemical modification may be the replacement of a hydrogen with a hydroxyl, acyl or amino group. The nucleic acid derivative may encode a polypeptide that retains the essential biological properties of the native molecule.

As used herein, the term "substantially purified" means that a nucleic acid or amino acid sequence isolated from its natural environment is separated or separated from other naturally It is best to reach 90%.

"Amplification" as used herein refers to the production of more copies of a nucleic acid sequence, usually by the well-known polymerase chain reaction (PCR) technique (Dieffenbach, C.W. and G.S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

As used herein, "hybridization" refers to the process by which any nucleic acid strand joins with a complementary strand through base pairing.

The term "hybridization complex" as used herein refers to a complex formed by the formation of hydrogen bonds between the complementary bases G and C and between the complementary bases A and T of two nucleic acid sequences. These hydrogen bonds can also be made more stable by base stacking effects. The hydrogen bonds of two complementary nucleic acid sequences have an antiparallel conformation. This hybrid complex can be formed in solution (such as C.sub.Ot or R.sub.Ot analysis), or one nucleic acid sequence can be in solution while the other nucleic acid sequence is immobilized on a solid support ( Such as membranes, filters, chips, needle tips or slides, cells are fixed for in situ hybridization).

As used herein, the terms "complementary" or "complementarity" refer to the natural association of polynucleotides through base pairing under appropriate conditions of salt concentration and temperature. For example, the sequence "A-G-T" is combined with the complementary sequence "T-C-A". The complementarity between two single-stranded molecules can be "partial", that is, only some nucleic acids are bound, or complete, that is, the two single-stranded molecules are completely complementary. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of their hybridization. This is especially important in amplification reactions, which rely on the association of two complementary strands.

The term "homology" as used herein refers to the degree of complementarity. It can be partial homology or complete homology (eg, identity). A partially complementary sequence is capable of at least partially inhibiting hybridization of an identical sequence to a target nucleic acid; such sequences may also be represented by the functional term "substantially homologous". Inhibition of hybridization of perfectly complementary sequences to the target sequence can be detected by low stringency hybridization assays (Southern or northern blot, solution hybridization, and other similar assays). Under low stringency conditions, a sufficiently homologous sequence or probe can compete with and inhibit binding (ie, hybridization) of a fully homologous sequence and probe. This is not to say that conditions of low stringency allow non-specific binding to occur; conditions of low stringency require that the binding between the two sequences be a specific (ie, selective) reaction. Whether there is no non-specific reaction needs to be detected with the sequence of the second item, the target sequence should be only a small part of complementarity (such as similarity below 30%); since there is no non-specific binding, then the probe will not Hybridizes to a second non-complementary target sequence.

It is well known that a number of equivalent conditional factors can be used to create conditions of low or high stringency. Many factors such as the length and nature of the sequence (DNA, RNA, base complexes), the nature of the target sequence (DNA, RNA, base complexes, in solution or immobilized, etc.), the concentration of salts and other components ( Such as the presence or absence of formamide, dextran sulfate, and/or polyethylene glycol) etc. should be taken into account, and the difference in hybridization solution will produce low stringency or high stringency, although different from the above conditions. degree conditions.

The term "stringency" as used herein refers to the "stringency" between -5°C below Tm (5°C below the melting temperature of the probe) to 20-25°C below Tm. It is understood that the stringency of hybridization can be varied to identify or detect identical or related polynucleotide sequences.

The term "antisense" as used herein refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand" as used herein refers to the nucleic acid strand that is complementary to the "sense" strand. Antisense molecules can be generated by any means, including synthesis by back-ligating the gene of interest to a viral promoter that supports complementary strand synthesis. Once inside the cell, this transcription chain binds to a dimer with the native sequence produced in the cell. These dimers then inhibit further transcription or translation. In this way a mutant phenotype can be generated. "Negative" is sometimes used to refer to the antisense strand, and "masculine" is sometimes used to refer to the sense strand.

The term "portion" as used herein refers to a protein (such as "a part of a specific protein") and refers to a fragment of the protein. The size of the fragment can range from 4 amino acid residues to only one amino acid less than the entire amino acid sequence. Therefore, the protein "containing at least a part of the amino acid sequence of the SARS protein" includes the full-length SARS protein and fragments thereof.

"Transformation" as defined herein refers to the process by which foreign DNA enters and changes recipient cells. This process can be achieved by well-known various means under natural or artificial conditions. Transformation may rely on any known method for the insertion of exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the conditions of the host cell being transformed and may include, but is not limited to, the following methods: virus infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells, ie, the inserted DNA is capable of replicating either as an autonomously replicating plasmid or as part of a chromosome. Also included are transformed cells that transiently express the inserted DNA or RNA only for a limited period of time.

The term "antigenic determinant" as used herein refers to a portion of a molecule (ie, an epitope) that is associated with a specific antibody. When a protein or part of a protein is used to immunize a host animal, many regions of the protein can elicit the production of antibodies that bind to specific regions or three-dimensional conformations of the protein; these regions or conformations are said antigenic determinants. An antigenic determinant can compete with the intact antigen (ie, the immunogen used to stimulate an immune response) for antibody binding.

The term "specific binding" or "specific binding" as used herein when referring to the interaction between an antibody and a protein or polypeptide specifically means that this interaction is dependent on the presence of a specific structure on the protein molecule /conformation (ie, antigenic determinant or epitope); in other words, the antibody is recognizing and binding to a specific protein structure/conformation rather than just the protein itself. For example, if an antibody is specific for epitope A, adding a protein with epitope A (unlabeled A) to a reaction containing labeled "A" and the antibody will reduce the amount of labeled "A". A Level of binding to antibody.

The term "sample" used here is a broad concept, which refers to any biological sample that may contain nucleic acid encoding SARS proteins and fragments thereof, including cells, chromosomes isolated from cells (such as chromosomes in the middle stage of cell division), genomic DNA ( in solution or bound to a solid support as in the Southern assay), RNA (in solution or bound to a solid support as in the Northern assay), cDNA (in solution or bound to a solid support substances), cell or tissue extracts, etc.

The term "related to polynucleotide expression" used here refers to the detection of ribonucleic acid similar to the polynucleotide encoded by SARS virus in the Northern analysis experiment, which indicates that there is mRNA encoding SARS protein in the sample, so as to be related to the protein encoding The expression of the transcript of the polynucleotide is related.

The "change" of the polynucleotide encoded by the SARS gene as used herein includes any alteration of the polynucleotide sequence encoded by the SARS gene, including deletions, insertions, and point mutations that can be detected by hybridization experiments. This definition also includes the detection of changes in the genomic DNA sequence encoding the SARS protein, the absence of the ability of the polynucleotide fragment encoded by the SARS virus to hybridize to the genomic DNA sample (such as the use of allele-specific oligonucleotides), and incorrect or Unexpected hybridization, such as hybridization occurs at a site other than the normal chromosomal site of the polynucleotide sequence encoding the SARS protein (such as fluorescence in situ hybridization for dividing metaphase chromosomes).

The term "antibody" as used herein refers to whole antibody molecules and fragments thereof, such as Fab fragments, F(ab') ₂ fragments and Fv fragments capable of binding antigenic determinants. Antibodies that bind to SARS proteins can be prepared using complete polypeptides or small peptides as immunogens. Polypeptides or peptides used to immunize animals can be synthesized by translation of corresponding RNA or directly synthesized by chemical methods, and can also be combined with carrier proteins if necessary. Commonly used carriers are chemically bonded to the polypeptide, including bovine serum albumin and thyroglobulin. The conjugated polypeptides can then be used to immunize animals (such as mice, rats or rabbits).

The term "humanized antibody" as used herein refers to an antibody molecule in which amino acids in non-antigen-binding regions have been substituted so as to make it more similar to a human antibody, but retain the original binding properties.

As used herein, the term "vector" refers to any nucleotide or nucleic acid sequence capable of expressing a polypeptide under suitable conditions such as cells and in vitro cell-free transcription/translation systems. Vectors include, but are not limited to, DNA plasmids, viral expression vectors, and RNA replicons.

Detailed description of the preferred embodiment

The present invention provides a novel vaccine design for preventing and treating SARS, including single or multiple genes and gene fragments, gene products, proteins and protein fragments of SARS coronavirus.

The genome of the SARS virus is single-stranded positive-strand RNA, and due to the low-fidelity of RNA-dependent RNA polymerase, the SARS genome may undergo rapid mutation. In fact, several research institutes around the world have sequenced the genome of the SARS coronavirus and submitted the results to databases such as GenBank. Standard sequence alignments revealed few sequence changes among different virus isolates.

For viruses with a high mutation rate, the development of effective vaccines is a huge challenge, because an enhanced immune response to one strain of the virus may not be effective against other mutant strains. The same problem occurs with the SARS virus. In order to minimize the impact of virus mutation, multiple genes or proteins of the SARS virus genome can be selected as the target of the vaccine at the same time.

In one embodiment of the present invention, a vector capable of expressing one or more SARS gene coding products, or a fragment thereof or a derivative thereof, can be used as a vaccine to prevent or treat SARS. These genes include spike glycoprotein (S), membrane glycoprotein (M), nucleocapsid protein (N), replicase 1A, replicase 1B, small envelope E protein, orf3, orf4, orf6, orf8, orf9, orf10, orf11, orf13 and orf14, but not limited thereto. These genes are called SARS genes (see definition). Some of these genes have been described (Marra, MA et al., 2003, Scienceexpress.1-5). Vaccine production methods are also described (Raz, E et al. 1994, Proc. Natl. Acad. Sci. USA, 91, 9519-9523; Donnelly, J.J et al., 1996, J. Infect. Dis., 173, 314-320). In a specific embodiment, a vector capable of expressing S and N, or fragments thereof, or derivatives thereof can be used as a vaccine for preventing or treating SARS.

In another embodiment, two or more SARS proteins (see definition) or fragments or derivatives thereof can be expressed in a suitable host and can be used as a vaccine for preventing and treating SARS.

In another embodiment, a vector capable of expressing a fusion protein of two or more SARS gene fragments or derivatives thereof can be used as a vaccine for preventing and treating SARS. These genes can be linked directly, or via a linker of one or more amino acids.

In another embodiment, a fusion protein vector capable of expressing the extracellular domain of the virus surface protein or its derivatives can be used as a vaccine for the prevention and treatment of SARS. These viral surface proteins include but are not limited to spike glycoprotein (S), membrane glycoprotein (M), small envelope protein (E), orf3 and orf8. In a specific embodiment, a fusion protein vector capable of expressing S and orf8 extracellular domains, or gene fragments, or derivatives thereof can be used as a vaccine for preventing or treating SARS.

In another embodiment, the extracellular domains of two or more genes of S, M, E, orf8 and orf3, or fragments thereof, or derivatives thereof can be fused together, in a suitable body as a fusion protein It can be used as a vaccine for preventing or treating SARS. In a detailed embodiment, the extracellular domain of S and orf8, or a fragment or derivative thereof can be fused together, and a fusion protein can be expressed for preventing or treating SARS.

In another embodiment, a vector capable of expressing a combination of SARS fusion proteins, or fragments, or derivatives thereof, and a single SARS protein or protein fragment can be used to prevent and treat SARS.

Any nucleic acid sequence encoding a SARS gene, or fragments thereof, can be used to generate recombinant molecules expressing a SARS gene or fragments thereof. In detailed embodiments, the present invention includes the use of polynucleotides encoding S, orf8 and N.

Those skilled in the art appreciate the degeneracy of the genetic code, because many nucleic acid sequences encoding SARS genes with little homology to any known and naturally occurring genes can be produced. Therefore, the present invention fully considers every possible nucleic acid sequence variation, and achieves the purpose of the invention by selecting different combinations of nucleic acid codons according to the possible codon selection. The codon combinations were designed according to the state of the standard triplet genetic code applied to the naturally occurring SARS gene, and the variations considered are described in detail below.

Under appropriate stringency conditions, the nucleic acid sequence encoding the SARS gene and its variants can hybridize well with the naturally occurring SARS gene fragments, but at some point it may be more advantageous to choose to use codons different from the naturally occurring codons. In individual prokaryotic and eukaryotic hosts, unique codons can be selected according to the frequency of host codon usage, thereby increasing their expression rate. Other reasons for altering the nucleic acid sequence of the SARS gene and its derivatives without altering the encoded amino acid sequence include that the RNA transcripts derived from it have better properties than those transcribed from naturally occurring sequences, such as longer half-lives.

The DNA sequence encoding the SARS gene or its partial sequence or its derivatives can be obtained by chemical synthesis, and the synthetic sequence can be inserted into any available expression vector and cell system. In addition, chemical synthesis is used to introduce mutations into the sequence encoding the SARS gene, including deletion, insertion, or replacement of different nucleosides in the present invention, resulting in proteins that encode the same or have the same function. The encoded protein may also contain deletions, insertions, or substitutions of amino acid residues, which will produce nonsense mutations, resulting in a functionally equivalent SARS gene. According to the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or characteristics of amphipathic molecules of amino acid residues, targeted amino acid substitutions can be carried out, as long as the corresponding biological activities of these proteins can be retained, such as immunogens , but not limited to. For example, the positively charged amino acids can be aspartic acid and glutamic acid, and the negatively charged amino acids can be lysine; the group of uncharged polar amino acids has similar hydrophilicity, including leucine, isoleucine Acine and Valine, Glycine and Alanine, Glutamate, Serine and Threonine, Phenylalanine and Tyrosine.

The scope of the present invention also includes the use of alleles encoding SARS genes. As used herein, an "allele" or "sequence of alleles" is an alternative form of a gene which may result from at least one mutation in the nucleic acid sequence. Alleles may result in changes in mRNA or polypeptide, which may or may not be altered in structure or function. Any known gene may be absent, or one or more allelic forms may be present. Routine mutations that result in alleles are usually natural deletions, additions, or substitutions of nucleosides. These mutant forms can occur alone or in combination with others, and can occur one or more times in a known sequence.

The method of DNA sequencing is a well-known and commonly used method in the industry, and can be applied to any embodiment of the present invention. The enzyme that this method uses has Klenow fragment DNA polymerase I, SEQUENCE DNA polymerase (U.S. BiochemicalCorp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago Ill), or combined application recombination Polymerases and exonucleases with proofreading functions, such as the ELONGASE Amplification System (GIBCO/BRL, Gaithersburg, Md). Preferably, the sequencing process can be carried out automatically on machines such as HamiltonMICROLAB 2200 (Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200, MJ Research, Watertown, Mass.) and ABI 377 DNA sequencer (Perkin Elmer).

Polynucleotide sequences or fragments thereof encoding SARS genes, gene products, fusion proteins or functional analogues thereof can be used in recombinant DNA molecules, and in appropriate host cells, can direct protein expression. Due to the degeneracy of the genetic code, it is possible to generate other DNA sequences encoding very similar or functionally equivalent amino acid sequences, which can be used to clone and express these proteins.

As understood by those skilled in the art, the use of non-naturally occurring codons may be helpful in generating the nucleotide sequence encoding the SARS gene. For example, a particular prokaryotic or eukaryotic host may prefer some codons to increase the efficiency of protein expression or to produce optimized recombinant RNA transcripts, eg, with longer half-lives than transcripts produced by naturally occurring sequences.

To change the sequence encoded by the SARS gene for different purposes, techniques well known to those skilled in the art can be used to modify the nucleotide sequence, including modification of clones, processing, and/or expression of gene products, but not limited thereto. Random fragment DNA rearrangement, PCR recombination of gene fragments and synthesis of oligonucleotides can be used to modify nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction endonuclease sites, change glycosylation patterns, change codon bias, generate splice variants or introduce mutations, etc.

In another embodiment of the invention, the natural, modified or recombinant nucleotide sequence encoding the SARS gene can be linked to a heterologous sequence to encode a fusion protein for the diagnosis, prevention and treatment of SARS. For example, screening a protein repressor from a polypeptide library, a SARS chimera gene may be useful because commercially available antibodies recognize the fusion protein. When constructing the fusion protein, a cleavage site can be added between the SARS gene coding sequence and the heterologous sequence, so that S or ORF8 can be cut and purified from the heterologous part.

Can adopt the chemical method well known in the art to synthesize the sequence of coding SARS gene fully or partially (referring to Caruthers, M H. etc., 1980, Nucl.Acids Res.Symp.7:2315-223; Horn, T. etc., 1980, Nucl AcidsRes. Symp, 7:225-232). The amino acid sequence of SARS gene or its part can also be selected chemically. For example, different solid-phase techniques (Roberge, JY., 1995, Science. 269: 202-204) and automatic synthesis techniques are used for peptide synthesis, for example, using ABI's peptide synthesizer (Perkin Elmer).

Newly synthesized peptides can be highly purified by high performance liquid chromatography (eg, Creighton, T. 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.). The composition of synthetic peptides can be confirmed by amino acid analysis or sequencing (eg Edman degradation, Creghton, supra). In addition, the amino acid sequence of the SARS gene or any part thereof may change during direct synthesis, and/or when it is chemically connected with the full or partial sequence of other proteins to produce a variant peptide.

In order to express a SARS gene with biological activity, the nucleotide sequence encoding the SARS gene or its functional analogue can be inserted into an appropriate expression vector, and the vector includes the necessary transcription and translation elements for the inserted sequence.

Using methods well known to those skilled in the art, an expression vector comprising a sequence encoding a SARS gene and appropriate transcriptional and translational control elements can be constructed. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination techniques. The above method can refer to 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.

There are many types of expression vectors and host systems that can accommodate and express sequences encoding SARS genes or gene products, including microorganisms such as bacteria transformed with recombinant phage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and infectious viruses Insect cell systems for expression vectors (such as adenovirus or baculovirus), plant cell systems for transformation of viral expression vectors (such as cauliflower mosaic virus, tobacco mosaic virus), or transformation of bacterial expression vectors (such as Ti or Pbr322 plasmids) A plant cell system, or an animal cell system, but not limited thereto.

"Control sequences" or "regulatory sequences" are vector enhancer, promoter, 5' and 3' untranslated regions that interact with host cell proteins for transcription and translation. These elements may vary in strength and specificity. Depending on the vector system and host used, suitable transcription and translation elements may be used, including basal and inducible promoters. For example, when cloning into bacterial systems, hybrid lacZ-inducible promoters like BLUESCRIPT phagemid (Stratagene, LaJolla, Calif) or PSPORT1 plasmids (Gibco BRL), or similar products can be used. In insect cells a baculovirus promoter can be used. Promoters, enhancers (such as heat shock, RUBISCO, and storage protein genes) or plant viruses (such as viral promoters or leader sequences) derived from plant cell genomes can be cloned into the vector. In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are preferred. If it is necessary to obtain cell lines containing multiple copies of the sequence encoding the SARS gene, SV40 or EBV-based vectors with appropriate selectable markers can be used.

In the bacterial system, various expression vectors can be selected according to the purpose of using the SARS gene. For example, when a large number of SARS gene-induced antibodies are required, a vector that is easy to purify and directly affects the high-level expression of the fusion protein can be used. Similar vectors have multifunctional E.coli cloning expression vectors, such as BLUESCRIPT phagemid (Stragene), which can insert the sequence encoding the SARS gene into the vector. The N-terminal methionine of the SARS gene is in the reading frame of the vector, and the C-terminal is followed by 7 Residues of β-galactosidase. Also can select pIN carrier (Van Heeke, G.and S.M.Schuster (1989) J.Biol.Chem.264:5503-5509) and similar PGEX carrier (Promega, Madison, Wis.) to express with glutathione in the form of fusion protein A hybrid protein of glycidyl transferase (GST), but not limited thereto. In general, these proteins are soluble and can be conveniently obtained from lysed cells, which can be passed through a column, allowing them to adsorb to glutathione sepharose beads, which can be washed in the absence of glutathione. take off. The proteins produced by this system have heparin, thrombin, and factor XA protease cleavage sites, so that the target protein can be easily released from the hybrid.

In yeast, Saccharomyces cerevisiae, many vectors carrying constitutive or inducible promoters such as factor a, alcohol oxidase and PGH can be used. See Ausubel et al., (supra) and Grant et al., (1987), Methods Enzymol. 153:516-544.

In the example of using a plant expression vector, many promoters can be used to promote the expression of the gene sequence encoding SARS. For example, viral promoters, the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence of TMV (Takamatsu, N. (1987) EMBO J. 6: 307-311). Alternatively plant promoters, like RUBISCO's small subunit, or heat shock promoters can be used. (Coruzzi, G et al., 1984, EMBO J.3:1671-1680; Broglie, R. et al., 1984, Science 224:838-843; Winter, J. et al., 1991, Results Probl. Cell Differ.17:85-843 105). These constructed vectors can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. These techniques are described in several reviews (see eg Hobbs, S or Murry, L.E. in McGraw Hill Yearbook do Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

Insect systems can also be used to express SARS genes. For example, in such a system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express exogenous genes in Spodoptera frugiperda cells or Trichomela moth larvae. The sequence encoding the SARS gene can be cloned into non-essential parts of the virus, such as the romoten gene, and put it under the control of the romoten gene. The successful insertion of the SARS gene will inactivate the romoten gene, resulting in a recombinant virus lacking the envelope protein. The recombinant virus was used to infect Spodoptera frugiperda cells or Trichomus larvae to express S or ORF8 (Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a variety of viral-based expression systems are available. In the case of using adenovirus as a vector, the sequence encoding S or ORF8 can be linked to an adenoviral transcription/translation complex consisting of a late promoter and a tripartite leader sequence. An active virus can be obtained by inserting fragments at the non-essential E1 and E3 positions of the viral genome, which can express SARS genes (Logan, J.and Shenk, T, 1984, Proc.Natl.Acad. Sci. 81: 3655-3659). In addition, transcriptional enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Specific initiation signals, including ATG initiation codon and adjacent sequences, can improve the efficiency of translation of the gene sequence encoding SARS. In the case of the gene sequence encoding SARS, its initiation codon and upstream sequences are inserted into an appropriate expression vector, no other transcriptional or translational control signals are required. However, when only the coding sequence or part is inserted, it is necessary to provide exogenous translational control signals including the initiation codon ATG. In addition, the initiation codon must be in the correct reading frame to ensure correct translation of the entire inserted sequence. The source of exogenous translation elements and initiation codons may vary, but are either natural or synthetic. As described in the literature, the efficiency of expression can be increased by adding an enhancer to a specific expression system (Scharf, D. et al., 1994, Results Probl. Cell Differ. 20: 125-162).

In addition, host cell strains can be selected for their ability to regulate expression of the inserted sequence or to process the protein in the desired manner. Modifications of these polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which may be to cleave protein precursors, will also facilitate correction of insertion, folding and/or function. Different host cells, such as CHO, HeLa, MDCK, HEK293, and WI38, have their unique organelles and mechanism characteristics for post-translational modification activities, which can be selected according to needs to ensure the correct modification and processing of foreign proteins.

In the long run, high yield and stable expression of recombinant protein is the first choice. For example, cell lines stably expressing SARS genes can be obtained by transformation of expression vectors containing virally derived replication elements and/or internal expression elements, and a selectable marker gene, either on the same vector or in separate in the carrier. Following introduction of these vectors, cells are cultured in rich media for 1-2 days before being transferred to selective media. The purpose of the selectable marker is resistance selection, the presence of which allows the growth and acquisition of cells successfully expressing the introduced sequence. Resistant clones of stably transformed cells can be expanded in culture as long as tissue culture techniques appropriate to the cell type are used.

To obtain transformed cell lines, a number of selection systems can be used, including 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), respectively for tk or aprt cells, antimetabolites, antibiotics, or herbicides can also be used as resistance selection markers, such as dhfr resistance to methotrexate (Wigler , M. et al., 1980, Proc.Natl.Acad.Sci.77:3567-70), npt resistance to the aminoglycoside neomycin and G-418 (Colbere-Garapin, F. et al., 1981, J.Mol .Biol.150:1-14), als or pat resistance to chlorsulfuron and PPT acetyltransferase respectively (Murry, supra), but not limited thereto. Other genes that can be selected are also described, such as trpB gene, which can make cells replace tryptolphan with indole; hisD gene, which can make cells use histinol instead of histidine (Hartman, S.C. and R.C.Mulligan (1988) Proc. .85:8047-51). Recently, everyone likes to use visual markers, such as anthocyanins, β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin, which are widely used not only to identify transformants, but also To determine the amount of protein expressed transiently or stably by a particular vector system (Rhodes, C.A. et al., 1995, Methods Mol. Biol. 55:121-131).

Although the expression of the marker gene can indicate the presence of the gene of interest, its presence and expression also need to be confirmed. For example, if the sequence encoding the SARS gene is inserted into the marker gene, the loss of marker gene function can be used to identify whether the recombinant cell can express the SARS gene. Alternatively, if the S or ORF8 gene of SARS and the marker gene are arranged in tandem downstream of a promoter, then under the conditions of drug induction and selective culture, the expression of the marker gene usually also indicates the expression of the SARS gene.

In addition, the identification method for host cells expressing SARS genes comprising nucleotide sequences encoding SARS genes is well known to those skilled in the art, including DNA-DNA or DNA-RNA hybridization, protein bioassays or immunoassays such as membranes and solutions technology, and chip technology for nucleic acid or protein detection and/or quantification, but not limited thereto.

The polynucleotide encoding the SARS gene sequence can be detected by DNA-DNA hybridization, DNA-RNA hybridization, or amplification with a polynucleotide probe encoding the SARS gene, a partial sequence or a fragment thereof. Nucleic acid amplification assays are performed using oligonucleotides or oligos encoding S or ORF8 sequences to detect transformants comprising DNA or RNA encoding S or ORF8. As used herein, "oligonucleotide" or "oligomer" refers to a sequence comprising at least 10 nucleotides, up to about 60 nucleotides, preferably about 15-30 nucleotides, most preferably Of about 20-25 nucleosides, these "oligonucleotides" or "oligomers" can be used as probes or amplification primers.

There are many schemes for detecting and measuring the expression of S or ORF8, usually using polyclonal or monoclonal antibodies well known in the industry, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA ), flow cytometry (FACS). The first choice is to use two monoclonal antibodies to recognize two SARS gene epitopes that do not interfere with each other. The method of competitive birding experiment can also be used. The above analytical methods are described 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 labeling and coupling techniques, well known to those skilled in the art, can be used for different nucleic acid and amino acid assays. Probe labeling methods include labeling oligomers, nick translation, end labeling or PCR amplification with a labeled nucleotide, these probes are hybridization or PCR probes, and are used to detect polynucleotides related to the gene encoding SARS related sequence. In addition, SARS gene or any other protein gene can be cloned into the vector to obtain mRNA probe. Such vectors are commercially available, and RNA probes can be synthesized in vitro after administration of a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. Preparation methods can be found in commercial kit instructions (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio). Suitable labels and reporter molecules include radionuclides, enzymes, fluoresceins, chemiluminescent substances, chromophores and substrates, cofactors, inhibitors, magnetic beads, and the like.

The culture conditions of the host cells transferred to the nucleotide sequence encoding the SARS gene should be suitable for the expression of the protein and for harvesting the protein from the cell culture fluid. Depending on the sequence and/or vector used, proteins produced by recombinant cells may exist in secreted form or as inclusion bodies. As well known to those skilled in the art, the polynucleotide expression vector encoding the SARS gene mostly contains a signal sequence, which can guide protein secretion to the outside of the prokaryotic or eukaryotic cell membrane. Other recombinants can link the sequence encoding the SARS gene with a sequence encoding a polypeptide domain that facilitates the purification of soluble proteins, and these domains include metal-chelating peptides, such as histidine-tryptophan molecules can Purified on immobilized metal, protein A can be purified on immobilized immunoglobulin, or using the FLAG extension/affinity system (Immunex Corp., Seattle, Wash.), but is not limited thereto. Purification is facilitated by cleavable linker sequences between the purified domain and the SARS gene, such as specific factor XA and enterokinase (Invitrogen, San Diego, Calif.). Similar expression vectors can provide expression of a fusion protein comprising the SARS gene and a encoding of six histidine residues that can precede the thioredoxin or enterokinase cleavage site. The histidine residue facilitates the purification of IMIAC (immobilized metal ion affinity chromatography, see Porath, J. et al., 1992, Prot. Exp. Purif. 3: 263-281), however, the enterokinase cleavage site provides A method for purifying SARS gene from fusion protein. For a discussion of fusion protein vectors see Kroll, D J. et al. (1993, DNA Cell Biol. 12:441-453).

In addition to the production of recombinant proteins, the SARS gene fragments can be directly synthesized into peptides using solid-phase synthesis technology (Merrifield J., 1963, J. Am. Chem. Soc. 85: 2149-2154). Protein synthesis can use the methods provided in the manual, or automated synthesis techniques. There are many kinds of automated systems, such as using the ABI 43 IA peptide synthesizer (PerkinElmer), different SARS gene fragments can be chemically synthesized separately and connected using chemical methods to produce full-length molecules.

Antibodies specific to SARS genes can be used as targets or delivery tools to guide pharmaceutical agents to cells or tissues expressing S or ORF8.

In another embodiment, any of the therapeutic proteins, antagonists, antibodies, agonists, antisense sequences, or vectors as described above, may be used in combination with other appropriate therapeutic agents. In combination therapy, the selection of appropriate reagents is one of ordinary skill in the industry according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically on different disorders in the treatment or prevention phase. Using this approach, lower doses of each agent may be therapeutically effective, thereby reducing potential side effects.

By transforming cells or tissues with vectors that can express S or ORF8 fragments at a high level, the expression of SARS genes may be shut down. Such vectors will likely introduce non-translated sense or antisense sequences, and even if no DNA sequences are integrated, the vectors can continue to transcribe RNA molecules until they are inactivated by endogenous nucleases. Transient expression may last for a month or more with non-replicating vectors, or longer if the vector system has appropriate replication elements in it.

There are many methods for introducing vectors into cells or tissues, and these methods are equally applicable in vivo, in vitro, or ex vivo. For in vitro therapy, the vector can be introduced into the stem cells of the patient, cloned and proliferated, and then reintroduced into the same patient by autologous transplantation. Delivery can be accomplished by transfection and liposome injection using methods well known in the industry.

Any of the above methods of treatment can be applied to any subject in need of treatment, including mammals such as dogs, cats, cows, horses, rabbits, monkeys, preferably humans.

For any of the above therapeutic effects, another embodiment of the invention relates to the combined administration of the pharmaceutical composition and the pharmaceutical carrier. These pharmaceutical compositions include S or ORF8, antibodies, analogs, agonists, antagonists, or inhibitors of S or ORF8. These ingredients can be administered alone, or in combination with at least one other agent, such as a stabilizer. These stabilizing ingredients can be administered in any sterile, biologically compatible carrier including, but not limited to, saline, buffered saline, dextrose and water. These may be administered alone, or in combination with other agents, drugs or hormones.

There are many routes of administration for the pharmaceutical ingredients used in the present invention, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, intradermal, subcutaneous, intraperitoneal, intranasal, intestinal, Methods of topical, sublingual, or rectal administration, without limitation.

In addition to the active ingredients, these pharmaceutical ingredients may include suitable pharmaceutically compatible carriers, including excipients, auxiliaries, which facilitate the processing of the pharmaceutical ingredients into pharmaceutically usable preparations. Specific details regarding formulation and administration techniques can be found in Remington Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Pharmaceutical ingredients for oral administration can be formulated using pharmaceutically compatible carriers well known in the industry, and administered in an appropriate dosage for oral administration. These pharmaceutical ingredients can be designed into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, or the like, for easy ingestion by patients.

Oral pharmaceutical preparations can be obtained by combining the active ingredient with a solid excipient, optionally grinding into the form of a mixture, processing the mixture into granules, adding suitable auxiliaries, and processing, if desired, into tablets or dragee cores . Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannose, or sorbitol, corn, wheat, rice, potato or other vegetable starches, celluloses such as methylcellulose, hydroxypropyl Methylcellulose or sodium carboxymethylcellulose, rubber including romot and tragacanth, protein including gelatin and collagen. If necessary, a decomposition or dissolution agent such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate may be added.

Dragee cores may be used with a suitable coating, such as a concentrated sugar solution, which may also be impregnated with gum romot, talc, polyvinylpyrrolidone, carbomer, polyethylene glycol and/or titanium dioxide, lacquer solution and suitable Organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablet or dragee coating, for identification of the drug or to indicate the quantity of active ingredient, eg dosage.

Pharmaceutical preparations which can be used orally include easy-to-take capsules of a gel, such as glycerol or sorbitol, as well as capsules of a soft, airtight gel, such as glycerol or sorbitol. Capsules, ready to take, may contain the active ingredients with fillers or binders, such as lactose or starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fats, liquids, or polyethylene glycol liquids, with or without stabilizers.

Pharmaceutical formulations for parenteral administration can be in the form of aqueous solutions, especially those that are physiologically compatible, such as Hawks' solution, Ringer's solution or physiologically buffered saline. Viscosity-increasing substances such as sodium carboxycellulose, sorbitol or dextran can also be added to the aqueous solution. Additionally, active ingredient suspensions may be prepared as appropriate oily injection suspensions, or as synthetic fatty acid esters, such as ethane oleate, triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or soluble substances may be added to facilitate the preparation of a more concentrated solution.

For topical or nasal administration, formulation design allows for the use of appropriate penetrants for specific barriers. These penetrants are commonly used in the industry.

The pharmaceutical composition of the present invention can be prepared according to a method well known in the industry, such as conventional mixing, dissolving, granulating, dragee making, grinding, emulsifying, filling into capsules, embedding or freeze-drying.

The pharmaceutical ingredients may be provided in the form of salts, which may be combined with a number of acids including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts are more soluble in aqueous or otherwise protic solutions than the corresponding free radical forms. In other examples, the preferred preparation is a lyophilized powder comprising any/or all of the following: 1-50 mM histidine, 0.1%-2% sucrose and 2-7% mannitol, pH range 4.5- 5.5, used in combination with the aforementioned buffer.

After the pharmaceutical composition is prepared, it can be placed in a suitable container and labeled to indicate the indication. For administration of S or ORF8, these labels should include the amount, frequency, and method of administration.

The pharmaceutical compositions used in the present invention contain effective doses of the active ingredients. Determination of effective doses is well known to those skilled in the art.

For any component, initial estimates of the therapeutically effective dose can be made in cell culture assays (eg, tumor cells) or animal models (usually mice, rabbits, dogs or pigs). Animal models can also be used to determine appropriate concentration ranges or routes of administration. These data can also be used as a reference to determine the effective dose and route of administration in humans.

The effective dose for treatment refers to the quantity of active ingredients, including SARS gene or its fragments, antibodies, agonists, antagonists of SARS genes, inhibitors of S or ORF8, these active ingredients will improve symptoms. Therapeutic effect and toxicity can be carried out through cell culture and experimental animal experiments by standard drug test methods, such as ED50 (half effective dose) and LD50 (half lethal dose). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as LD50/ED50. Drug ingredients with a high therapeutic index are usually selected. The data obtained from cell culture assays and animal assays can be used in planning a range of dosage for use in humans. An effective dose of a drug in circulation should achieve the ED50 amount with little or no toxicity. The effective dose will generally vary depending on the dosage form used, the sensitivity of the patient, and the route of administration.

The exact dose will be determined by a physician according to the specific conditions of the subject to be treated. Dosage and administration are appropriately adjusted in order to obtain sufficient active moiety or the desired therapeutic effect. The following factors should be considered: severity of disease, general health status, age, weight and sex of the subject, diet, time and frequency of administration, drug combination, reaction sensitivity and tolerance/response to treatment. Depending on the half survival and clearance rates of specific formulations, long-acting drugs can be administered every 3-4 days, every other week or every two weeks.

Due to the different routes of administration, the normal dosage ranges from 0.1-100,000ug, sometimes as high as 1g. Specific dosages and methods of administration are available to physicians in the art from the general literature. Those skilled in the art will use different nucleoside drug formulations than proteins or inhibitors. Likewise, the way of administering polynucleosides or polypeptides should be determined according to specific cells, states and locations.

In order to more fully illustrate the advantages of the present invention, the following examples are given. It should be understood that the following examples are only used to illustrate the present invention, not to limit the scope of the invention.

The synthetic gene is designed according to the following two principles: 1) include surface proteins that may be related to virus adsorption and infection; 2) include at least one relatively conserved protein.

According to the predicted genome sequence of SARS-CoV, two surface proteins, S1 and orf8, were selected. The S1 domain of the spike protein regulates virus adhesion to the cell surface, and the function of the orf8 protein is unknown, but it has a deep extracellular domain that may also be involved in viral infection. The N protein is a relatively well conserved protein that has been shown to strongly activate T helper cell function, therefore, this gene is included in the gene package.

To express the three proteins simultaneously, the domains of S1 and orf8 are fused with a flexible linker that facilitates protein-independent folding.

Through a ribosome entry site (IRES), the N protein is fused to the S1-orf8 fusion protein during transcription. As shown in Figure 1, two proteins can be expressed simultaneously through a CMV promoter.

As mentioned above, viruses have very different codon biases than humans. Many viruses, like HIV and HPV, have low G-C content in their coding sequences. On the contrary, the G-C content of human genes is higher, especially the third base of the codon. The preference for codons with higher G-C content may be due to the higher concentration of intracellular tRNA for the respective codons, so that the protein can be translated more efficiently. In addition, high G-C content is associated with high mRNA stability, which can increase the expression of the target protein.

The target antigen coding sequence was optimized using preferred human codons to ensure efficient transcription, as shown in Figure 2, the SARS-CoV S and N genes have low G-C content, and the G-C content at the third base position is less than 40% , with a typical viral bias; in contrast, the G-C percentage was 77% in the highly expressed human protein.

At the 3rd base position, the preferred gene has an average G-C content of 66%, making it in line with the human average percentage. To further optimize the synthetic genes, potential alternative mRNA splice sites were identified using the intron-exon junction region rule. These sites are removed by silent mutagenesis to ensure that only the correct protein of interest is expressed.

In a preferred embodiment, the S1 domain is fused with the extracellular domain of orf8, as shown in FIG. 3 . An IgK secretion signal is added to the N-terminus of the fusion protein so that the fusion protein can be secreted and generate more potential antibody responses. As shown in Figure 3, the sequences of these fusion proteins have been optimized for optimal expression in primates. The amino acid sequences of these fusion proteins are shown in FIG. 4 .

In another preferred embodiment, the sequence of the N protein is optimized, as shown in FIG. 5 . The corresponding amino acid sequence is shown in FIG. 6 .

These synthetic genes are cloned into different vectors, including viral and plasmid vectors.

The effectiveness of the DNA plasmid vector (SV1000) vaccine was shown in one trial. 100ug of endotoxin-free DNA plasmid SV1000 was used to immunize female Balb/c mice of 6-8 weeks at week 0, week 2, and week 5, and serum samples were collected at week 3 and week 6. Total lymphocytes were collected at week 6 at the end of the experiment. Most mice seroconverted by 3 weeks, despite relatively low antibody titers. At week 6, antibody titers rose to 1:640, which was consistent with the range of antibody responses induced by other DNA vaccines reported in the literature.

A serum sample was also tested for its ability to neutralize live SARS virus infection in Vero E6 cells. Before infecting Vero E6 cells, the sera were serially diluted 2-fold and incubated with the virus. After calculation, the final neutralization titer was about 1:300.

Antigen-specific T cell responses were quantified using an IFNg-specific ELISPOT assay. Synthesize a 20-mer peptide covering 2 full-length genes and overlapping by 10 amino acids. The total lymphocyte responses of immunized mice were stimulated with 12 peptide pools respectively. As shown in Figure 7, targeting multiple regions of the vaccine, SV1000 was able to induce potential Th1 T cell responses. Importantly, regions of S1-orf8 and N were identified, suggesting that both genes are expressed in vivo and involved in generating specific immune responses.

At the same time, a recombinant type 5 adenovirus with a synthetic gene, namely SV8000, was constructed. As shown in Figure 8, a single injection of SV8000 elicited a strong immune response in mice, with antibody titers exceeding 1:1000. This response was also dose-dependent, with strong antibody responses induced in 1E9 virions over 1E8 virions.

IFNg T cell responses were also analyzed by ELISOPT. As shown in Figure 9, a single injection of SV8000 induced latent T cell responses across numerous regions of the synthetic gene. The magnitude of the response was significantly greater than that induced by the SV1000 DNA vaccine. Interestingly, there was no linear correlation between vaccine dose and T cell responses. The T cell response induced by IE8 particles was the strongest, however, whether it was 1E9 or 1E7 particles, after immunizing mice, the immune response was relatively weak.

Recent studies have shown that different primer-boosting patterns can greatly enhance the immune response generated by any single vaccine component. This is especially true when DNA vaccines are used as priming antigens. It is said that DNA priming can activate and amplify antigen-specific T cell responses, with high affinity during the priming phase, because DNA vaccines have lower antigen concentrations than recombinant viruses and lack the “noisy” antigens typically found in recombinant viruses. Viral boosting further expanded the reservoir, generating a strong immune response compared to a single vaccine.

As shown in Figure 10, the prime boost approach induced at least a 10-fold higher antibody response compared to single DNA or recombinant adenovirus.

According to literature reports, a single injection of recombinant adenovirus vaccine can completely protect non-human primates from Ebola virus challenge. This is especially beneficial in dealing with public health emergencies, such as the SARS outbreak, when long-term immunization programs are not feasible. A single shot, fast-acting vaccine can provide immediate protection, reducing potential danger to emergency response teams.

Two rhesus macaques were immunized with 1E12 of the SV8000 vaccine. A third monkey was immunized with medium only. Serum samples were collected before immunization and weekly for 10 weeks after immunization. PBMCs were isolated and stored in liquid nitrogen for subsequent batch analysis. Serum samples were used for standard virus neutralization experiments. A clear trend in the immune response can be observed from the data. As shown in Figure 11, the neutralizing antibody peaked at the 4th week, and remained relatively stable for the next 3 weeks. It is worth noting that the average neutralizing antibody titer of convalescent patients is only about 1:64. Although this may suggest that SV8000 alone may provide protection, it is unclear whether neutralizing antibodies alone are protective. T cells, especially CTLs, play an important role in other animal models of coronavirus infection, such as IBV and MHV. Moreover, due to the short history of SARS-CoV, it is not known whether this virus will invade again with a different S protein. In this particle, T cell activation is critical to the protection of cross strains.

The efficacy of the vaccine was tested in monkeys. At week 0 and week 4, rhesus monkeys were immunized with SV8000 intramuscularly. During the first 4 weeks, antibody responses were relatively low. Antibody responses increased rapidly after the booster immunization (Figure 12). At week 8, all immunized monkeys produced high titers of IgG antibodies.

In order to test the protective efficiency of the vaccine, the monkeys in the high-dose, low-dose and placebo groups were challenged intranasally with 10 ⁵ TCID ₅₀ live virus, and the two monkeys in the control group were raised in the same animal breeding facility, but they were not infected with live virus. Virus, as the negative control of the experiment. Three days after inoculation with the virus, four monkeys in the placebo group became lethargic, ate significantly less, and two monkeys showed symptoms of respiratory distress. In contrast, neither the immunized animals nor the control animals showed visible clinical signs.

Compared with before the challenge, the concentration of SARS-CoV-specific IgG in the immune group increased significantly after the challenge (Fig. 13). Seven days after virus challenge, the increase ranged from 3-fold to nearly 30-fold, indicating that the vaccine had effectively elicited a humoral immune response after injection.

Pharyngeal swabs and serum samples were collected on days 2, 5 and 7 after virus challenge. Detect the presence of SARS coronavirus by RT-PCR or virus culture. Of the 4 animals in the high dose group, 2 were able to detect viral RNA 2 days after virus challenge (Figure 14), and the viral RNA disappeared by 5 days. The other two had no detectable viral RNA at any time. Four animals in the low-dose group had no detectable viral RNA on day 2 or day 5, and only one animal had detectable low levels of RNA on day 7. In contrast, all four animals in the placebo group tested positive for viral RNA after virus challenge.

To re-isolate live virus after challenge, Vero cells were inoculated with throat swab samples. As shown in Figure 14, live virus could be isolated from 3 of 4 animals in the placebo group. Both the high-dose and low-dose groups protected animals from viral infection.

In animals in the placebo group, the lungs were severely damaged, with extensive damage to the alveolar walls and accumulation of proteinaceous fluid in the alveolar spaces (Fig. 15e). The alveolar walls are thickened due to severe infiltration of monocytes, and the hyaloid membranes line the alveolar walls. (Fig. 15f). In the most severe cases, extensive destruction of the epithelial layer and localized hemorrhages can be observed. Although in rare cases less edema and mononuclear cell infiltration were found in the alveolar walls (Fig. 15c), the high dose group (Fig. 15b) was histologically distinct from the control group (Fig. 15a). The phenotype of the low-dose group was between that of the high-dose group and the placebo group (Fig. 15d).

In order to find the relationship between disease pathology and virus replication, lung tissue sections were analyzed by immunohistochemical method, as shown in Figure 16. There was a very small amount of positive staining in the slices of the high-dose group, which was very similar to that of the negative control group. In contrast, lung tissue sections from the placebo group showed many positive cells with prominent cytoplasmic staining, indicating continuous antigen expression in these cells. The low-dose group also showed an intermediate type, with antigen-positive cells appearing in alveolar walls and epithelial cells.

Claims (25)

1. A pharmaceutical composition comprising a nucleic acid molecule encoding one or more SARS coronavirus gene products, which is linked to a regulatory sequence and present in a drug-compatible carrier. 2. The pharmaceutical composition according to claim 1, wherein the SARS coronavirus gene is selected from the group consisting of S protein, Orf8 protein, M protein, N protein, E protein, Orf1a protein, Orf1b protein and combinations thereof. 3. The pharmaceutical composition according to claim 1, wherein the regulatory sequence is a promoter. 4. The pharmaceutical composition according to claim 3, wherein the promoter is a CMV promoter. 5. The pharmaceutical composition according to claim 1, wherein the pharmaceutically compatible carrier is a viral vector, a plasmid vector or an RNA vector. 6. The pharmaceutical composition according to claim 5, wherein the viral vector is a replication-deficient adenovirus. 7. The pharmaceutical composition according to claim 6, wherein the viral vector is a type 5 replication-defective adenovirus. 8. The pharmaceutical composition according to claim 1, wherein the nucleic acid molecule encodes two SARS coronavirus gene products, and in a pharmaceutically compatible carrier, the first gene product comprises at least one SARS-CoV gene extracellular domain , or a derivative thereof, the second gene product comprises an intracellular protein, or a derivative thereof. 9. The pharmaceutical composition according to claim 8, wherein the first gene is selected from a SARS-CoV gene group including S, M, Orf8 and E, and the second gene is selected from SARS-CoV including N, Orf1a and Orf1b - the genetic group of the CoV. 10. The pharmaceutical composition according to claim 8, wherein the first gene product comprises a part of the SARS-CoV S protein, or an antigenic determinant binding domain thereof, or an analog thereof. Among them, the S protein has a truncated C-terminus that removes the transmembrane anchor and intracellular domain. 11. The pharmaceutical composition according to claim 8, wherein the first gene product comprises a first gene fragment, a part of the SARS coronavirus S protein or its antigenic determinant binding domain, or an analog thereof, wherein the S protein There is a truncated C-terminus to remove the transmembrane anchor and the intracellular domain; the second gene segment contains a part of the SARS-CoV Orf8 protein or its epitope-binding domain or its analogue, wherein the Orf8 protein Has a truncated C-terminus that removes the transmembrane anchor and intracellular domain. 12. The pharmaceutical composition of claim 1, wherein the nucleic acid molecule consists of amino acid codons that are preferred based on highly expressed mammalian codon usage. 13. The pharmaceutical composition according to claim 12, wherein the preferred codons have a GC content of at least 50%. 14. The pharmaceutical composition according to claim 12, wherein the G-C content of the third base position of the preferred codon is at least 50%. 15. The pharmaceutical composition according to claim 1, wherein the SARS-CoV gene product further comprises a secretion signal peptide fragment. 16. The pharmaceutical composition of claim 8, wherein the first gene product further comprises a secretion signal peptide fragment. 17. The pharmaceutical composition according to claim 16, wherein the secretory signal peptide fragment is an Igk signal sequence. 18. A peptide or peptide combination selected from the gene products of any one of claims 1-17, wherein the peptides are purified in vitro. 19. A method of inducing an immune response against an antigen in a body, comprising providing the individual with the pharmaceutical composition of any one of claims 1-17. 20. A method of inducing an immune response against an antigen as claimed in claim 19, wherein the drug composition is administered at about 1 week, 2 weeks, 3 weeks, 4 weeks or 6 weeks after the first administration of the drug composition , or in combination. 21. A method of inducing an immune response against an antigen as claimed in claim 19, wherein the pharmaceutical composition is administered in combination with an adjuvant. 22. A method for inducing an immune response against an antigen as claimed in claim 19, wherein the dosage of the plasmid vector pharmaceutical composition is 1-5 mg per injection. 23. A method for inducing an immune response against an antigen as claimed in claim 19, wherein the dosage of the viral vector drug composition is 1×10 ⁶ -1×10 ¹³ virus particles per injection. 24. A method of inducing an immune response against an antigen as claimed in claim 19, wherein the pharmaceutical composition is administered intramuscularly, intradermally, subcutaneously, intranasally or orally. 25. A method for inducing an immune response against an antigen as claimed in claim 19, further comprising administering the pharmaceutical composition with a different carrier after the first administration of the pharmaceutical composition, so as to trigger the immune response to the above-mentioned antigen.

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