EP1893752A2 - Sras attenue: utilisation comme vaccin - Google Patents

Sras attenue: utilisation comme vaccin

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Publication number
EP1893752A2
EP1893752A2 EP06762172A EP06762172A EP1893752A2 EP 1893752 A2 EP1893752 A2 EP 1893752A2 EP 06762172 A EP06762172 A EP 06762172A EP 06762172 A EP06762172 A EP 06762172A EP 1893752 A2 EP1893752 A2 EP 1893752A2
Authority
EP
European Patent Office
Prior art keywords
sars
cov
nucleic acid
sequence
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06762172A
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German (de)
English (en)
Inventor
Luis Enjuanes Sanches
Marta Lopez De Diego
Enrique Alvarez Gomez
Fernando Almazan Toral
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Consejo Superior de Investigaciones Cientificas CSIC
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
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Application filed by Consejo Superior de Investigaciones Cientificas CSIC filed Critical Consejo Superior de Investigaciones Cientificas CSIC
Priority to EP06762172A priority Critical patent/EP1893752A2/fr
Publication of EP1893752A2 publication Critical patent/EP1893752A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20061—Methods of inactivation or attenuation

Definitions

  • the present invention is directed to nucleic acids encoding an attenuated SARS-CoV virus capable of producing a maximum viral titer in cell culture that is reduced when compared to the maximum viral titer of wild-type SARS-CoV virus in the same cell culture.
  • Gene therapy is a technique primarily for correcting defective genes responsible for disease development.
  • a carrier molecule also referred to as a vector is used to deliver the therapeutic gene to the patient's target cells.
  • the most common vector is a virus that has been genetically altered to carry human or animal genes . Viruses naturally have evolved in a way of encapsulating and delivering their genes to human and animal cells in a pathogenic manner. However, in the meantime scientists have taken advantage of this capability and manipulated the virus genome to remove disease-causing genes and insert therapeutic genes.
  • Target cells such as the patient's liver or lung cells are infected with the viral vector.
  • the vector then unloads its genetic material containing the therapeutic gene into the target cell.
  • the generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.
  • these viral vectors were used for expressing heterologous genes that cause an immunogenic response in the subject receiving the vector and thus immunize that subject. In that case the viral vector serves as a vaccine.
  • SARS-CoV coronavirus
  • Coronaviruses are ssRNA(+) viruses which have the largest genome so far found in RNA viruses with a length between 25 and 31 ki- lobases (kb; see Siddell S. G., 1995, The Coronaviridae) .
  • gRNA genomic RNA
  • sgRNA subgenomic RNAs
  • DI defective interfering
  • a respective system was used in the art to generate immune responses in an animal which received a composition containing a DI genome which amongst others contained sequences encoding a heterologous reporter gene or a gene derived from a different infectious agent (porcine reproductive and respiratory disease virus, PRRSV; see Alonso et al . , 2002a, 2002b).
  • coronavirus full-length cDNA clones (Al- mazan et al . , 2000; Casais et al . , 2001; Thiel et al . , 2001; Yount et al., 2000; Yount et al . , 2002; Yount et al . , 2003) provided the opportunity for the genetic manipulation of coronavirus genomes to study fundamental viral processes and to develop expression vectors.
  • the 3' one-third of the genome includes the genes encoding the structural and non-structural proteins, in the order 5'-S-3a- 3b-E-M-6-7a-7b-8a-8b-9b-N-3' . These proteins are expressed by a discontinuous transcription process that most probably takes place during the synthesis of the negative strand, leading to the generation of a 3' coterminal nested set of subgenomic mRNAs, each of which has at its 5' end a capped leader sequence derived from the 5' end of the genome (Sawicki and Sawicki, 1998; Z ⁇ niga et al . , 2004).
  • TRSs transcription- regulating sequences
  • S, E, M, and 3a are embedded in the membrane.
  • the M and E proteins are key factors for virus assembly and budding (Fischer et al . , 1998) .
  • expression of these proteins in cell lines results in the production of virus- like particles (Bau- doux et al., 1998; Ho et al . , 2004; Huang et al . , 2004; Mor- tola & Roy, 2004; Vennema et al . , 1996).
  • WO 04/092360 discloses nucleic acids and proteins from the
  • SARS coronavirus which can be used in the preparation and manufacture of vaccine formulations, diagnostic reagents and kits.
  • SARS-CoV first broke out in humans in late 2002 and spread within a few months from its origin in Guangdong (China) to more than 30 countries.
  • the rapid transmission and high mortality rate made SARS-CoV a global threat for which no efficacious therapy is available.
  • the problem underlying the present invention thus resides in providing vaccines for protection against SARS-CoV with good safety and immunogenicity .
  • the attenuated virus is capable of producing a maximum viral titer in cell culture that is reduced at least by a factor of 2 when compared to the maximum viral titer of wild-type SARS-CoV virus in the same cell culture.
  • the present invention is directed to nucleic acids encoding a SARS-CoV virus, which are obtainable by a method comprising steps, wherein the genome of a SARS-CoV virus is modified by amending the sequence of the gene encoding the SARS-CoV E protein so that the nucleic acid cannot express a functional E protein.
  • a functional E protein is a protein that:
  • SEQ ID NO: 6 is encoded by SEQ ID NO: 6 or encoded by a gene having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NO: 6; and is capable to associate with other SARS-CoV proteins into a viral envelope.
  • a nucleic acid encoding a modified E protein as described above will generate a virus particle that does not contain an E protein in its envelope.
  • the present invention is further directed to nucleic acids encoding a SARS-CoV virus, wherein the nucleic acid does not encode a sequence of a functional protein E of the SARS-CoV virus as described above.
  • the nucleic acids may encode an attenuated SARS-CoV virus and comprise the sequences encoding the viral proteins replicase and S and M and N.
  • the nucleic acid may contain other SARS derived proteins, such as the 3a protein.
  • the nucleic acids as defined above do not contain any other SARS derived proteins.
  • the nucleic acids preferably are not capable of expressing a protein, which protein is encoded by:
  • the nucleic acids preferably are not capable of expressing a protein, which protein is encoded by:
  • the nucleic acid only encodes the viral proteins replicase and S and M and N.
  • the nucleic acids of the present invention encode a SARS-CoV virus, wherein the nucleic acid comprises the sequences of the viral proteins replicase and S and M and N and/or E.
  • the nucleic acid may contain other SARS derived proteins, such as sequences of viral protein 3a, but preferably does not contain any other SARS derived proteins as defined above.
  • the nucleic acid of the SARS-CoV virus does not encode the viral proteins 6, 7a, 7b, 8a, 8b, and 9b.
  • the nucleic acid may be RNA or DNA.
  • the present invention further relates to host cells and SARS- CoV virus particles comprising one of the above nucleic acids.
  • the SARS-CoV virus particle may for example be obtainable by introducing one of the above nucleic acids into a host cell.
  • the present invention further relates to vaccines comprising a respective SARS-CoV virus particle or a respective nucleic acid or a respective host cell.
  • the virus particle may be present in an attenuated form, i.e. replication competent and infectious, but may also be present in an inactivated form. Inactivating the attenuated virus using heat or chemicals according to well known methods for viral inactiva- tion will further increase the safety of the use of the vaccine.
  • the vaccine may further comprise a pharmaceutically acceptable adjuvant, carrier or excipient.
  • the vaccine is preferably used to vaccinate a mammal, preferably a human, and gives rise to an immune response that reduces or eliminates the disease symptoms caused by infection with the SARS-CoV virus .
  • the present invention relates to methods for preparing a SARS-CoV vaccine, comprising modifying a nucleic acid comprising the genome of a SARS-CoV virus by amending the nucleic acid coding for SARS-CoV proteins.
  • the methods for preparing a SARS-CoV vaccine comprising modifying a nucleic acid comprising the genome of a SARS-CoV virus by amending the sequence of the gene encoding the SARS-CoV E protein so that the gene cannot express a functional E protein.
  • the method further comprises amending the sequence of the genome of a SARS-CoV such that the nucleic acid is expressing the viral proteins replicase, S, M and N and further expresses or does not express the sequences encoding protein 3a.
  • the method further comprises amending the sequence of the genome of a SARS-CoV such that the nucleic acid is not capable of expressing a protein having a homology of at least 80%, preferably a homology of at least 90% or a homology of at least 95% to the protein encoded by SARS-CoV genes E, 6, 7a, 7b, 8a, 8b and 9b (corresponding to SEQ ID NO: 6, 14 to 19) .
  • the methods further comprises amending the sequence of the genome of a SARS-CoV such that the nucleic acid is not capable of expressing a protein having a homology of at least 80%, preferably a homology of at least 90% or a homology of at least 95% to the protein encoded by SARS-CoV genes 3a, 3b, 6, 7a, 7b, 8a and 8b (corresponding to SEQ ID NO: 12 to 19) .
  • the method further comprises amending the sequence of the genome of a SARS-CoV such that the nucleic acid is expressing the viral proteins replicase, S, M, N and E and further expresses or does not express the sequences encoding protein 3a.
  • the method further comprises amending the sequence of the genome of a SARS-CoV such that the nucleic acid is not capable of expressing a protein having a homology of at least 80%, preferably a homology of at least 90% or a homology of at least 95% to the protein encoded by SARS-CoV genes 6, 7a, 7b, 8a, 8b and 9b (corresponding to SEQ ID NO: 14 to 19) .
  • the method further comprises amending the sequence of the genome of a SARS-CoV such that the nucleic acid is not capable of expressing any SARS-CoV derived protein coding sequences other than replicase S, M, N, and/or E and further expresses or does not express the sequences encoding protein 3a.
  • the method further comprises introducing the modified nucleic acid into a host cell and isolating a nucleic acid encoding the amended genome or SARS- CoV virus particles comprising the modified nucleic acid.
  • the method for preparing a SARS-CoV vaccine further comprises the mixing of the nucleic acid or of the SARS-CoV virus particle with a pharmaceutically acceptable adjuvants, carrier or excipient .
  • the present invention relates to nucleic acids encoding attenuated SARS-CoV viruses and respective viruses as well as their medical use.
  • the attenuated virus is preferably capable of producing a maximum viral titer in cell culture that is reduced at least by a factor of 2 when compared to the maximum viral titer of wild-type SARS-CoV virus in the same cell culture .
  • the attenuated SARS-CoV virus is capable of producing a maximum viral titer in cell culture that is reduced at least by a factor of 5, preferably at least by a factor of 10 or 20.
  • the attenuated SARS-CoV virus is capable of producing a maximum viral titer in cell culture that is reduced preferably by a factor between 2 and 100, more preferred by a factor between 5 and 50 and most preferred by a factor between 10 and 25.
  • the nucleic acids do not encode a sequence of a functional protein E of the SARS-CoV virus.
  • the nucleic acids are for example obtainable by a method comprising steps, wherein the genome of a SARS-CoV virus is modified by amending the sequence of the gene encoding the SARS-CoV E protein so that the nucleic acid cannot express a functional E protein.
  • the modifications may include a deletion and/or a substitution of the coding sequences or of the regulatory sequences . It is for example possible, but may not be necessary to remove the entire coding sequence of the E protein.
  • sending a sequence is used to refer to the modification of a nucleic acid sequence with techniques well known in the art. When researchingamending a sequence" said sequence is modified to render it different from the wild type sequence by applying techniques including, but not limited to nucleotide or sequence deletion, nucleotide substitution or nucleotide addition. These and further techniques are described by Sambrook & Russel (2001) .
  • the terms ,,E ⁇ precede or ,, ⁇ E" will be used interchangeably in this application to refer to attenuated SARS-CoV virus lacking (at least) gene E or to nucleic acids etc. coding for an attenuated SARS-CoV virus lacking (at least) gene E.
  • the modification of the E gene comprises the preparation of a SARS-CoV replicon or a full length cDNA clone of SARS-CoV in a BAC and the subsequent modification of that BAC.
  • a vaccine comprising a nucleic acid of the present invention is safe and can initiate an immune response against SARS-CoV viruses.
  • the vaccine comprises a nucleic acid or an attenuated virus particle, which contains all elements necessary to infect human or animal cells and which may have been inactivated or not.
  • the attenuated virus as a vaccine, infection of a cell will lead to replication of the virus and production of further infective virus particles.
  • the titer of the attenuated virus will be significantly lower when compared to the wild-type SARS virus.
  • the vaccine strain will only be able to grow to reduced maximal titers in a vaccinated host.
  • the immune system of the host will produce an immune response directed against the virus which reduces or eliminates the disease symptoms caused by infection with a wild-type SARS-CoV.
  • a collection of recombinant vaccines based on a SARS-CoV defective virus able to replicate and propagate both in cultured cells and in vivo have been generated.
  • the nucleic acid encodes SARS-CoV replicase and some, but not all of the struc- tural and non-structural proteins.
  • One of the main characteristics of the SARS-CoV engineered vaccine viruses is that they are infectious but highly attenuated in vivo.
  • the attenuated virus is obtained by expression of the nucleic acids of the present invention and subsequently inactivated using well known methods for virus in- activation, such as chemical or heat treatment.
  • This embodiment has the specific advantage that the virus is disarmed twice and thus safer than the vaccines previously suggested in this field.
  • the term "attenuated” is used to refer to replication competent and infectious viruses, which viruses produce a reduced maximum viral titer in mammals or in a cell culture when compared to the maximum viral titer produced by the wild-type SARS-CoV virus in mammals or in a culture of the same cells under the same conditions (temperature, medium, incubation titer, incubation time, etc.).
  • This evaluation step may thus be based on cell cultures or non-human mammals, preferably birds or Hamsters (such as the Golden Syrian Hamster used in Example 15) .
  • Attenuated SARS-CoV virus in cell culture for the production of vaccines is limited or even impossible in cases where the growth rate of the attenuated virus in cell culture is too low.
  • the growth rate of attenuated SARS-CoV virus in cell culture can be improved up to a maximum viral titer even higher than the maximum titer achieved by the wild- type virus in that culture by adapting the virus to the culture media. Since virus are able to adapt to changing environments, long-term culture with repeated passaging of the virus will improve the growth rate of attenuated virus in cell culture. However, this approach is very time consuming.
  • Attenuated virus Another approach for improving in vitro growth of attenuated virus is the use of host cells for infection that provide the essential SARS-CoV proteins not encoded by the nucleic acid of the attenuated SARS-CoV in trans. Further, in a slightly different approach for providing such factors in trans the host cells can be infected by the attenuated SARS-CoV virus and a helper virus or other nucleic acid expressing the essential SARS-CoV proteins.
  • SARS-CoV virus that is capable to grow to a maximum viral titer in cell culture, which exceeds the maximum viral titer of the wild-type virus in the same culture, and still is an attenuated virus that will grow in vivo with maximal titers that are markedly reduced compared to wild type SARS but still sufficient to induce both a systemic immune response and a mucosal immune response against SARS-CoV.
  • replication competent viral nucleic acid is used to refer to a nucleic acid comprising sequences encoding a viral replicase and sequences which represent an origin of replication, i.e. sequences which will trigger replication of the nucleic acid by the replicase.
  • infectious is used to refer to a virus which is cable to grow by infection of cells, replication of the viral nucleic acid, expression of viral proteins, assembly of the viral nucleic acid with the viral proteins to a virus particle and infection of further cells by the newly produced virus particles.
  • nucleic acid which encodes an attenuated virus is used to refer to a nucleic acid which encodes all sequences of an attenuated virus. Respective nucleic acids can be used to obtain virus particles by transfecting a cell culture which will lead to growth of the attenuated virus in the culture. The nucleic acid can also be used as a vaccine.
  • SARS-CoV coat proteins are proteins, such as the SARS-CoV E, M or S (spike) protein or proteins.
  • the proteins may have a sequence as observed in a SARS-CoV isolate or may be derived therefrom by methods known in the art. Using methods of recombinant expression it is for example possible to modify protein sequences by addition, deletion or substitution of one or several amino acids.
  • the present invention thus covers viruses comprising or encoding coat proteins with a sequence homology of at least 60%, preferably at least 75% and most preferably at least 95% to the wild type SARS-CoV proteins.
  • SARS-CoV Repla SEQ ID NO : 1
  • SARS-CoV Replb SEQ ID NO: 2
  • SARS-CoV N SEQ ID NO: 3
  • SARS-CoV S SEQ ID NO : 4
  • SARS-CoV M SEQ ID NO: 5
  • SARS-CoV E SEQ ID NO: 6
  • SARS-CoV Full length clone SARS-CoV E: SEQ ID NO: 6
  • SARS-CoV ORF 3a SEQ ID NO: 12 Fig. 19
  • ORF of gene 3b SEQ ID NO: 13 Fig. 20
  • ORF of gene 6 SEQ ID NO: 14 Fig. 21
  • ORF of gene 7a SEQ ID NO: 15 Fig. 22
  • ORF of gene 7b SEQ ID NO: 16 Fig. 23
  • ORF of gene 8a SEQ ID NO: 17 Fig. 24
  • ORF of gene 8b SEQ ID NO: 18 Fig. 25
  • sequence of the N protein is also designated sequence 9a.
  • the 9b sequence is included in the 9a sequence by a frame shift. Any amendment of the 9b sequence should preferably be carried out such that the sequence of 9a is not substantially amended, i.e. in a manner that still allows expression of a functional 9a or N protein.
  • the SARS-CoV sequences may be based on the Urbani strain genome (Genebank, accession number AY278741) .
  • the full-length cDNA clone two silent genetic markers at positions 10338 (C>T) and 11163 (T>A) were introduced.
  • the nomenclature for the genes outlined above is different to that used in the sequence deposited in the GeneBank.
  • the equivalences are:
  • Gene 3a is equivalent to Xl Gene 3b is equivalent to X2 Gene 6 is equivalent to X3 Gene 7a is equivalent to X4 Gene 8b is equivalent to X5
  • UTR means "untranslated sequence
  • sequence homology is determined using the Clustal computer program available from the European Bioinformatics Institute (EBI) , unless otherwise stated.
  • the following proteins encoded by the above sequences of the SARS-CoV may be characterized by functional features.
  • the Rep Ia and Ib sequences encode the viral replicase which is characterized in that it allows replication of the viral RNA.
  • the M protein is a membrane protein and is characterized in that it associates with other proteins to an envelope of a virus particle.
  • the N protein is a nucleocapsid protein and is characterized in that it associates with the viral RNA to a nucleocapsid of a virus particle.
  • the S or spike protein is characterized in that it binds to the cellular receptor.
  • the protein of 3a is characterized in that it is also present in the viral membrane .
  • the nucleic acids encoding an attenuated SARS-CoV may further include a nucleic acid sequence not derived from SARS-CoV, for example a foreign gene.
  • the foreign gene may be of any origin.
  • the nucleic acids of the present invention may be used as a vector for the expression of foreign genes of other pathological microorganisms, such as viruses, bacteria, mycoplasma, etc. A vaccination with a respective nucleic acid or a SARS-CoV encoded thereby may thus provide protection against both SARS-CoV and the further pathological microorganism.
  • the virus particles in the vaccines of the present invention are preferably prepared starting from a SARS-CoV replicon.
  • Methods to prepare the nucleic acids and the virus particles of the present invention may comprise the preparation of a SARS-CoV derived replicon in a bacterial artificial chromosome (BAC) .
  • BAC bacterial artificial chromosome
  • BAC bacterial artificial chromosome
  • vaccines are provided, which are preferably capable of inducing both a systemic immune response and a mucosal immune response against SARS-CoV.
  • the spike protein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, the spike protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies .
  • the nucleic acid further encodes co-stimulating molecules, for example CD80 or CD86 or immunostimulating cytokines, for example TNF-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2), IL-12 or IL-18.
  • co-stimulating molecules for example CD80 or CD86 or immunostimulating cytokines, for example TNF-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2), IL-12 or IL-18.
  • the vaccine of the present invention may be administered to a mammal to obtain an immune response that reduces or eliminates the disease symptoms caused by infection with the SARS-CoV virus.
  • the vaccine is preferably used for vaccinating a mammal, especially a human, a civet cat, a raccoon or a ferret. All of these animals are known to serve as a host for SARS-CoV.
  • the vaccine may be administered in accordance with methods routinely used in the art. Specifically vaccine may be administered by oral, intramuscular, intravenous, subcutaneous or intranasal administration. Oral administration is especially preferred.
  • the vaccines may also comprise pharmaceutically acceptable carriers, excipients and/or adjuvants.
  • Adjuvants and carriers suitable for administering genetic vaccines and immunogens via the mucosal route are known in the art. Conventional carriers and adjuvants are for example reviewed in Kiyono et al . , 1996.
  • chemokines that are used to modulate immune responses are also encompassed by the present invention. Respective compounds and their medical use has been reviewed in Toka et al . , 2004. It is specifically advantageous to use one of granulocyte/macrophage colony- stimulating factor, interleukin-2 (IL-2), IL-12, IL-18. Combinatorial approaches utilizing several cytokines and chemokines might also be applied.
  • a replicon can be generated using a set of overlapping cDNAs spanning the untranslated 5' and 3' ends of the genome, the entire replicase gene, and the nucleoprotein (N) gene.
  • the strategy for the construction of the SARS-CoV replicon as a bacterial artificial chromosome (BAC) can be summarized as follows :
  • SARS-CoV replicon cDNA The construction of a SARS-CoV replicon cDNA is illustrated with further details in Example 1.
  • Methods for preparing the virus particles used in the vaccines of the present invention may use a replicon as described above. According to a preferred aspect of the present invention these methods are based on the use of the plasmid pBAC- SARS-CoV-REP. Bacteria containing this plasmid were deposited on September 1, 2004 with the Colecci ⁇ n Espa ⁇ ola de Cultivos (Tipo, CECT in Valencia, Spain) . The deposit was given the provisional accession number 7020.
  • the plasmid pBAC-SARS-CoV-REP comprises sequences of a replication competent, non- infectious SARS-CoV genome encoding the SARS-CoV replicase and the SARS-CoV N protein.
  • This vector provides a stable, safe and easy to handle basis for producing the virus particles and the vaccines of the present invention.
  • the virus particles and the vaccines may be produced by cloning the at least one further SARS-CoV gene into the replicon.
  • Genomic viral RNA may be used as a starting material to obtain the sequences encoding the further SARS-CoV proteins.
  • Genomic RNA of the SARS-CoV Urbani strain can be obtained from the Center for Disease Control in Atlanta, USA.
  • a cDNA library is prepared from the virus RNAs according to methods known in the art. The full length genes can be reconstructed from the cDNA library. These genes may then be combined with transcriptional regulatory sequences (TRS) and/or translation regulatory sequences (such as internal ribosome entry sites, IRES) and can be inserted into a cloning site of the replicon.
  • TRS transcriptional regulatory sequences
  • IRES internal ribosome entry sites
  • a full length clone encoding a full length copy of the genomic nucleic acid of SARS-CoV can be prepared and may subsequently be amended to produce a nucleic acid encoding an attenuated virus.
  • the nucleic acid may then be used to transfect a helper cell line. If the replicon is used, a cell line expressing the essential SARS-CoV proteins not encoded by the nucleic acid needs to be used (e.g. a packaging cell line). Alternatively, a host cell may be transfected with the replicon nucleic acid as described above and a helper virus or other nucleic acid expressing the essential SARS-CoV proteins. In this manner the nucleic acid encoding SARS-CoV replicase, SARS-CoV N protein and at least one further SARS-CoV protein (i.e. the replicon) will associate with the further essential SARS-CoV proteins to form the virus particle of the vaccines of the present invention.
  • the vaccines can be obtained by producing a virus particle as outlined above and formulating the same into a vaccine, for example by mixing the same with a carrier, adjuvants and/or excipients .
  • the testing of the vaccines preferably comprises a sequence of several steps.
  • the immunogenicity of attenuated SARS-CoV may be tested by producing them in tissue culture of Vero E6 cells.
  • the antigens obtained can be partially purified by conventional procedures including centrifugation and it can be tested whether these induce SARS-CoV neutralizing antibodies in mice and rabbits .
  • Selected vaccines may further be tested for protection in animal systems, such as BalB/c mice, ferrets and macaques (using conventional methods as described for example in Enserink, 2003; Yang et al . , 2004; ter Meulen et al . , 2004; Martina et al., 2003; and Kuiken et al . , 2004).
  • animal systems such as BalB/c mice, ferrets and macaques (using conventional methods as described for example in Enserink, 2003; Yang et al . , 2004; ter Meulen et al . , 2004; Martina et al., 2003; and Kuiken et al . , 2004).
  • the animal model systems can be sequentially used for vaccine efficacy and preliminary safety testing.
  • vaccine candidates can be tested in the mice model, then in the ferret SARS-CoV model (Martina et al . , 2003). Further decisions can be based on protection against SARS-CoV challenge, the selected vaccine candidates may then be tested in macaques .
  • the clinical, virological, gross pathological and immunological (neutralizing antibodies, cytokine profiles in plasma and cells by mRNA analyses) data may be collected systematically from these SARS-CoV-infected animals, according to protocols that have been established previously (Kuiken et al . , 2004 ) .
  • Figure 1 shows the genetic structure of SARS-CoV Urbani strain. Letters and number inside the boxes indicate the viral genes. L, leader sequence; UTR, untranslated region. Relevant restriction sites are indicated.
  • Figure 2 shows the construction of the intermediate plasmid pBAC-SARS-CoV 5' 3' .
  • CMV cytomegalovirus
  • Rz hepatitis delta virus ribozyme
  • BGH bovine growth hormone
  • FIG 3 shows the generation of the SARS-CoV derived replicon pBAC-SARS-CoV-REP.
  • the strategy for the construction of a SARS-CoV replicon is illustrated.
  • SARS-CoV replicon is assembled by sequential cloning of subgenomic overlapping cDNA fragments generated by RT-PCR, into plasmid pBAC-SARS-CoV 5' 3' of Figure 2.
  • the genetic map of SARS-CoV replicon is shown at the bottom. Relevant restriction sites used in the cloning step are indicated.
  • Rep Ia, Rep Ib, and N indicate the viral genes.
  • FIG. 4 shows the functional analysis of the SARS-CoV derived replicon of Figure 3.
  • the genetic structure of the SARS-CoV replicon is illustrated at the top.
  • the N gene TRS , the core sequence (italic letters) , and the relevant restriction sites are indicated.
  • L leader sequence;
  • UTR untranslated region.
  • BHK-21 and human 293T cells are mock transfected (MOCK) or transfected with SARS-CoV replicon (SARS-CoV-REP) or a non- replicative cDNA clone (GFP-NR) using Lipofectamine 2000.
  • Total RNA is isolated at 24 hpt and analyzed by RT-PCR with specific oligonucleotides to detect gene N mRNA. Duplicate RT-PCR products amplified in parallel were resolved by electrophoresis in 1% agarose gels.
  • Fig. 5 shows the construction of a SARS-CoV full-length cDNA clone from the SARS-CoV replicon.
  • the full-length clone was assembled by introducing the remaining SARS-CoV sequences in two steps.
  • a BamHI-Nhel fragment was inserted into the replicon including the following genes: part of 3a, 3b, E, M, 7a, 7b, 8a, 8b, and part of N.
  • the full-length cDNA clone was completed by cloning the Pmel-BamHI fragment including the 3' end of Rep Ib, gene S, and the 5' fragment of 3a into the former plasmid.
  • Genes and relevant restriction sites used in the cloning steps are indicated. The following abbreviations are used: CMV, cytomegalovirus immediate-early promoter; pA, tail of 25 A residues; Rz, hepatitis delta virus ribozyme,- BGH, bovine growth hormone termination and polyadenylation sequences.
  • Fig. 6 shows the strategy used to generate a cDNA encoding SARS-CoV-E " .
  • Core sequence and gene E open reading frame are shown.
  • the point mutation to change initial ATG to GTG is shown in bold and italics.
  • Gene E core sequence (CS, a highly conserved sequence that is in the middle of the TRS) is shown in a box.
  • the two point mutations introduced in gene E CS (ACGAAC to ACCAAT) are shown in italics.
  • the deleted sequence within gene E ORF is underlined.
  • Fig. 7 shows the kinetic of SARS-CoV and SARS-CoV- ⁇ E virus production in Vero E6 (A) , Huh-7 (B) , and CaCo-2 (C) cells that were infected at a moi of 0.5 with either the rSARS-CoV- ⁇ E or the recombinant wild-type virus.
  • virus titers were determined by plaque assay on Vero E6 cells. Error bars represent standard deviations from the mean from three experiments. This Figure shows that the E " derivative is reduced in its growth and thus attenuated.
  • Fig. 8 shows (A) cytopathic effects produced by the defective and parental viruses in Vero E6 cells at 24 hours post infection (upper squares) ; and (B) plaque morphology of the indicated viruses on Vero E6 cells at 72 hours post infection (circles in the middle) ; (C) immunofluorescence microscopy of the indicated viruses with a SARS-CoV specific polivalent antibody (bottom squares) .
  • the left half of the Figure shows infection with SARS-CoV wild-type and the right part shows SARS- CoV- ⁇ E. Both viruses, the parental and the E " produced a significant cytopathic effect in Vero E6 cells (Fig. 8A) .
  • Fig. 9 shows the RT-PCR analysis of infected cells.
  • RT-PCR was carried out using a viral sense primer which hybridizes in the viral leader sequence and reverse sense primers which hybridize in open reading frames of genes S, E, M and N respectively.
  • M mock infected cells
  • ⁇ E SARS-CoV- ⁇ E
  • FL SARS-CoV wild-type .
  • Fig. 10 shows the analysis of SARS-CoV protein expression in Vero E6 cells after being infected with SARS-CoV (FL) or SARS- CoV- ⁇ E ( ⁇ E) .
  • Fig. 11 shows the effect of temperature and pH changes on rSARS-CoV- ⁇ E virus infectivity.
  • Supernatants containing recombinant SARS-CoV and SARS-CoV- ⁇ E viruses were incubated for 30 min at the indicated temperature (Fig. HA) or pH (Fig. HB) and virus infectivity was evaluated by titration of culture supernatants on Vero E6 cells. Error bars represent standard deviations from the mean from three experiments.
  • Fig. 12 shows an ultrastructural analysis of rSARS-CoV- ⁇ E- infected Vero E6 cells 24 hours post-infection after processing for electron microscopy of ultrathin sections.
  • A Cytoplasm of an infected cell filled with virions.
  • B Sites of budding of the nucleocapsid into the lumen of swollen Golgi sacs. Dense material in the cytoplasm of SARS-CoV- ⁇ E-infected cells is indicated with arrows.
  • C Mature virus particles found in swollen Golgi sacs that appeared as large vacuoles. Pictures representing rSARS-CoV-infected cells or rSARS-CoV- ⁇ E-infected cells are displayed on the left and right side respectively. Bars, 2 ⁇ m in panel A and 200 nm in panels B and C.
  • Fig. 13 shows the morphology of rSARS-CoV- ⁇ E virions released from infected Vero E6 cells.
  • A Electron micrographs of ul- trathin sections showing extracellular viruses lining the cell surface.
  • B Supernatants of rSARS-CoV and rSARS-CoV- ⁇ E- infected cells were concentrated in an airfuge and analyzed by electron microscopy following negative staining with sodium phosphotungstate . Pictures on the left represent rSARS-CoV- infected cells, while on the right represent rSARS-CoV- ⁇ E- infected cells. Bars, 200 nm in panel A and 100 nm in panel B.
  • Fig. 14 shows the in vivo growth kinetics of the defective viruses in Hamsters after inoculation with 10 3 TCID 50 of rSARS-CoV or rSARS-CoV- ⁇ E .
  • Viral titers in lung (A) and nasal turbinates (B) were determined in Vero E6 cells monolayers.
  • the non- parametric Mann-Whitney U statistical method was used for ascertaining the significance of observed differences. Statistical significance was indicated by * (p value ⁇ 0.05) The dotted line indicates the lower limit of detection.
  • Fig. 15 shows the pathology of rSARS-CoV- ⁇ E in hamsters after inoculation with 10 3 TCID 50 of rSARS-CoV or rSARS-CoV- ⁇ E.
  • A Immunohistochemically stained sections of lungs at 2 days post- infection.
  • B Hematoxylin & eosin stained sections of lungs at 2 days post-infection.
  • C Immunohistochemically stained sections of lungs at 5 days post-infection.
  • D Hematoxylin & eosin stained sections of lungs at 5 days postinfection. Pictures on the left represent rSARS-CoV-infected lungs, while pictures on the right represent rSARS-CoV- ⁇ E- infected lungs. Bars, 100 nm in all panels.
  • Fig. 16 shows the mutations introduced to abolish expression of 9b gene.
  • the first 96 nucleotides of gene 9b ORF are shown.
  • Three ATG codons were mutated to ACG and are shown in bold characters.
  • the C mutated is shown in bold and in italics.
  • the stop codon introduced, TAA is shown in bold.
  • Fig. 17 shows the mutations introduced to abolish expression of gene 3b.
  • the first 75 nucleotides of gene 3b ORF are shown.
  • Three ATG codons mutated to ACG in phase are shown in bold and the C mutated is shown in bold and in italics.
  • the stop codon introduced by changing the codon TCA to TAA is shown in bold.
  • Fig. 18 to 26 show the sequences of the full lengths SARS-CoV Urbanis strain and of selected genes thereof .
  • the African Green monkey kidney-derived Vero E6 cells were kindly provided by Eric Snijder (Medical Center, University of Leiden, The Netherlands) .
  • the human colon carcinoma- derived CaCo-2 cells were obtained from the European Collection of Cell Cultures.
  • the human liver-derived Huh- 7 cells were kindly provided by R. Bartenschlager (Dept. of Molecular Virology, University of Heidelberg, Germany) and were referred to in Gillim-Ross et al . , 2003; Hattermann et al . , 2005; Mos- sel et al . , 2005.
  • Baby hamster kidney cells (BHK-21) and human 293T cells were purchased from the American Collection of Cell Cultures (ATCC) .
  • RNA of Urbani strain was obtained from the Center for Disease Control and Prevention (CDC) in Atlanta, GA, USA, after signature of a material transfer agreement .
  • Plasmids and bacteria strains Plasmids and bacteria strains. Plasmid pBeloBACll (Wang et al., 1997) was kindly provided by H. Shizuya and M. Simon (California Institute of Technology, Pasadena, Ca) . Escherichia coli DHlOB strain was obtained from GIBCO/BRL. DHlOB cells were transformed by electroporation at 25 ⁇ F, 2.5 kV, and 200 ⁇ with a Gene Pulser unit (Bio-Rad) according to the manufacturer's instructions. Plasmid DNA was isolated with the Qiagen (Chatsworth, CA) Large Construct Kit according to the manufacturer's specification.
  • a multicloning site containing unique restriction sites Pad, Ascl, and BamHl was cloned downstream of the replicase gene to allow cloning of heterologous genes (Fig. 3) .
  • This approach use a two-step amplification system that couples replicon RNA transcription in the nucleus from the CMV promoter with a second amplification step in the cytoplasm driven by the viral polymerase.
  • the plasmid pBAC-SARS-CoV-REP, encoding the SARS-CoV replicon was stable for at least 180 generations during its propagation in DHlOB cells, as determined by restriction endonuclease analysis.
  • the intermediate plasmid pBAC-SARS-CoV 5' 3' was generated as the basis to construct the SARS-CoV replicon and also the full length cDNA construction (Fig. 2) .
  • This plasmid contained the first 678 nt of the 5 ' end of the Urbani strain genome under the control of the cytomegalovirus (CMV) immediate-early promoter and the last 973 nt of the genome followed by a 25-bp synthetic poly (A) , the hepatitis delta virus ribozyme, and the bovine growth hormone termination and polyadenylation sequences to make an accurate 3' end.
  • CMV cytomegalovirus
  • a SARS-CoV replicon was engineered following the strategy described in Figure 3.
  • the plasmid pBAC-SARS-CoV- REP contained the untranslated 5' and 3' ends of the Urbani genome, the replicase gene followed by a multicloning site containing unique restriction sites Pad, AscX, and BamHI, to allow cloning and expression of heterologous genes, and the nucleoprotein (N) gene under the control of its natural TRS.
  • EXAMPLE 2 Analysis of cloned cDNA stability
  • the stability of the viral sequences cloned into pBeloBACll was analyzed by studying the restriction endonuclease pattern at different passages. Bacteria transformed with recombinant plasmid were grown in 10 ml of LB containing 12.5 ⁇ g/ml chloramphenicol at 37°C. Cells from these primary cultures (considered passage 0) were propagated serially by diluting 10 6 -fold daily. Each passage was considered to represent about 20 generations .
  • BHK-21 and 293T cells were grown to 95% confluence on 35 mm- diameter plates and transfected with 5 ⁇ g of the SARS-CoV replicon, using 6 ⁇ g of Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications.
  • the cDNAs generated were amplified by PCR using the reverse primer URB- 28630RS and the forward primer URB-29VS (5'-GCCAACCAACCTCGAT- CTCTTG-3') (SEQ ID NO : 8 ) , spanning nucleotides 29 to 50 of the Urbani leader sequence.
  • the primers used for RT (URB- 28163RS, 5' -TGGGTCCACCAAATGTAATGC-3 ' (SEQ ID NO: 9), complementary to nucleotides 43 to 63 of gene N) and PCR (reverse primer URB-28163RS and the forward primer URB-27VS, 5'- AAGCCAACCAACCTCGATCTC-3' (SEQ ID NO: 10), spanning nucleotides 27 to 47 of the Urbani leader sequence) were designed using the Primer Express software (Applied Biosystems) .
  • the SYBR Green PCR master mix was used in the PCR step following the manufacturer's specifications (Applied Biosystems).
  • the SARS-CoV derived replicon was functional in several cell lines. Expression of gene N mRNA was used to study replicon activity by RT-PCR analysis. BHK-21 and human 293T cells were transfected with either SARS-CoV replicon or a non-replicative cDNA clone using Lipofectamine 2000. Total intracellular RNA was extracted at 24 hpt and used as template for RT-PCR analysis of gene N mRNA transcription using specific oligonucleotides. High levels of gene N mRNA were detected in both BHK-21 and 293T cells transfected with the SARS-CoV replicon (Fig. 4), showing that the replicon is active in these cell lines.
  • a quantitative analysis of gene N mRNA in transfected 293T and BHK-21 cells was performed by real-time RT-PCR using the primers described in Example 5.
  • the SARS-CoV replicon activity was 8-fold higher in 293T cells than in BHK-21 cells. This activ- ity increase could be explained by a higher replication level of SARS-CoV replicon in human 293T cells or simply because the transfection efficiency of 293T cells is twice higher than that of BHK-21 cells.
  • N protein plays an important role as an enhancer of coronavirus replicon activity.
  • Bacteria containing the plasmid pBAC-SARS-CoV-REP comprising sequences of a replication competent, non- infectious SARS-CoV genome encoding the SARS-CoV replicase and the SARS-CoV N protein were deposited on September 1, 2004 with the Colecci ⁇ n Espafiola de Cultivos (Tipo, CECT in Valencia, Spain) . The deposit was given the provisional accession number 7020.
  • a SARS-CoV full-length cDNA clone (pBAC-SARS-CoV* '1 ') was prepared according to the cloning strategy outlined in Fig. 5.
  • the full-length clone was assembled by adding the rest of the genes in two steps.
  • a BamHl-Nhel fragment including the following genes: part of 3a, 3b, E, M, 7a, 7b, 8a, 8b, and part of N was inserted into the BAC containing the replicon.
  • the full-length cDNA clone was completed by cloning the Pmel-BamRI fragment including genes 3' end of Rep Ib, S, and 5' fragment of 3a in the former plasmid.
  • RNA corresponds to the genomic sequences of the Urbani strain of SARS-CoV.
  • infectious virus was recovered from the cDNA clone, and was found to be identical to the parental virus in terms of plaque morphology, growth kinetics, as well as mRNA and protein patterns.
  • Protein E was considered to be essential for coronaviruses . To abolish gene E expression three different modifications were introduced into the SARS-CoV full-length cDNA clone of Example 7 to obtain a cDNA encoding SARS-CoV as a BAC wherein the E gene is deleted (SARS-CoV- ⁇ E) :
  • the expression of the E protein was abrogated by the introduction of point mutations within the start ATG codon of the coding sequence of the E gene.
  • the introduced mutations were silent for gene 3b that partially overlaps with the E gene.
  • a 142 nt covering the majority of E gene were deleted.
  • TRS transcription regulating sequence
  • the E gene deletion was introduced by overlap extension PCR using the plasmid pBAC-SARS-CoV FL of Example 7 containing the SARS-CoV full-length cDNA.
  • CS PCR-16943-VS
  • SARS-N733-RS SARS-N733-RS
  • Both overlapping products were used as templates for PCR amplification using primers SARS-16501-VS and SARS-N733-RS.
  • the final PCR product was digested with the enzymes BamRl and Nhel and cloned in the intermediate plasmid pBAC-SARS-5' -3' -DN to generate the plasmid pBAC-SARS-5 ' -3 ' -DN- ⁇ E.
  • Both plasmids pBAC-SARS-5' -3 ' -DN and pBAC-SARS-5 ' -3 ' -DN- ⁇ E contain the first 7452 nt of the SARS-CoV genome under the cytomegalovirus (CMV) promoter and the last 3683 nt followed by a 25-bp synthetic poly A, the hepatitis delta virus ribozyme (Rz) , and the bovine growth hormone (BGH) termination and polyadenylation sequences to make an accurate 3' end.
  • CMV cytomegalovirus
  • Rz hepatitis delta virus ribozyme
  • BGH bovine growth hormone
  • Vero E6 cells grown to 90% confluence in 12.5 cm 2 flasks were transfected with 6 ⁇ g of the plasmid pBAC-SARS-CoV- ⁇ E or with the parental plasmid pBAC- SARS-CoV FL as a control, using 12 ⁇ g of Lipofectamine 2000 (In- vitrogen) according to manufacturer's instructions. After an incubation period of 6 h at 37° C, the transfection media was replaced and incubated at 37 °C for 72 h.
  • the ⁇ E virus was rescued, indicating that gene E is not essential for the viral cycle.
  • the titer of the defective virus rSARS-CoV- ⁇ E was 20 to 50-fold lower than that of the parental virus (recombinant wild-type rSARS-CoV) .
  • Viral titers were around 5-x 10 7 pfu/ml for the parental virus and 3-x 10 6 pfu/ml for the defective virus, as determined in a plaque assay on Vero E6 cells (Fig.7A).
  • RNA from Vero E6 infected cells was extracted using the Qiagen RNeasy kit according to the manufacturer's instructions and used for RT-PCR analysis of S, E, and N gene mRNA transcription. Reactions were performed at 42°C for 1 h using Moloney leukemia virus reverse transcriptase (Ambion) and the antisense primers SARS-S613-RS (5'-CTACTATAGGTTGATAGCCC-S') , complementary to nt 594 to 613 of S gene,- SARS-E231-RS (5'- TTAGACCAGAAGATCAGGAACTCC-3' ) , complementary to nt 208 to 231 of E gene; and URB-28630-RS (5'-TGCTTCCCTCTGCGTAGAAGCC-S') , complementary to nt 511 to 532 of N gene.
  • SARS-S613-RS 5'-CTACTATAGGTTGATAGCCC-S'
  • SARS-E231-RS 5'-
  • the cDNAs were amplified by PCR using the virus sense primer URB-29-VS (5'- GCCAACCAACCTCGATCTCTTG-3' ) , spanning nucleotides 29 to 50 of the SARS-CoV leader sequence and the reverse primers described for the RT reactions.
  • the RT-PCR products were resolved by electrophoresis in 0.8 % agarose gels .
  • the identity of the E " virus was confirmed by analyzing the synthesis of viral sg mRNAs using an RT-PCR assay (Fig.9). As expected the 250 bp PCR product from the E gene sg mRNA was not detected in rSARS-CoV- ⁇ E- infected cells.
  • Membranes were blocked for 1 h with 5% dried skim milk in TBS (20 mM Tris-HCl, pH 7.5 , 150 mM NaCl) and incubated with polyclonal antibodies specific for N, S (Imgenex; dilution 1:500), and E proteins (kindly provided by Shen Shuo, Institute of Mo- lecular and Cellular Biology, Singapore; dilution 1:2000). Bound antibody was detected with horseradish peroxidase- conjugated goat anti-rabbit antibody (Cappel) and the ECL detection system (Amersham Pharmacia Biotech) .
  • Protein extracts of Vero E6 cells infected with the rSARS-CoV- ⁇ E virus contained S and N proteins but no E protein.
  • Western analysis using antibodies against S and N proteins revealed a 170 kDa band and a double band of approximately 46 kDa, respectively.
  • Western blot analysis using a specific E protein antibody failed to detect a band of approximately 10 kDa, corresponding to E protein that was observed in Western blot analysis of rSARS-CoV-infected Vero E6 cell extracts (Fig. 10) .
  • Subconfluent monolayers (90% confluency) of Vero E6, Huh- 7 and CaCo-2 cells were infected at a multiplicity of infection (moi) of 0.5 with the viruses rSARS-CoV- ⁇ E and rSARS-CoV.
  • Culture supernatants were collected at different times postinfection and virus titer was determined following standard procedures using closed flasks or sealed plates.
  • the titer of the rSARS-CoV- ⁇ E virus was -20 -fold lower than the recombinant wild-type virus.
  • Huh-7 cells maximal titers were reached 48 h postinfection ( ⁇ 5-x 10 5 pfu/ml for the recombinant wild-type virus) (Fig. 7B), whereas in CaCo-2 cells maximal titers were reached -72 h post- infection ( ⁇ 4-x-10 5 pfu/ml for the recombinant wild-type virus) (Fig. 7C) .
  • rSARS-CoV- ⁇ E virus grew to titers -200-fold lower than the recombinant wild-type virus.
  • Subconfluent Vero E6 cells grown in 9 cm 2 slide flasks were infected at a moi (multiplicity of infection) of 1.
  • moi multiplicity of infection
  • cells were washed in ice-cold PBS and fixed with 8% paraformaldehyde for 30 min at room temperature.
  • the cells were then permeabilized with 0.2% saponin in blocking solution (phosphate-buffered saline [PBS], pH 7.4, containing 10% fetal bovine serum [FBS] ) for 1 h at room temperature and incubated with a polyclonal SARS-CoV specific antibody, kindly provided by Anlong Xu (Zhongshan Uninersity, Guangzhou, China) for 90 min at room temperature.
  • PBS phosphate-buffered saline
  • FBS fetal bovine serum
  • Fig. 11 To analyze whether the E protein influences the stability of SARS-CoV, the effect of temperature and pH on virus infectiv- ity was analyzed (Fig. 11) . To this end the recombinant wild- type and rSARS-CoV- ⁇ E viruses were incubated at temperatures ranging from 4°C to 80 0 C for 30 min. Both viruses showed similar inactivation profiles, with a reduction of 10 3 -fold after incubation at 60 0 C. Heating at 80 °C or higher temperatures led to residual virus infectivity (Fig. HA) .
  • Vero E6 cells monolayers were infected with rSARS-CoV and rSARS-CoV- ⁇ E viruses at a moi of 1.
  • the cells were fixed in situ 20 hpi with 2% glutar- aldehyde in phosphate Na/K buffer (pH 7.4) for 1 h at room temperature. Cells were removed from the dishes in the fixative and transferred to eppendorf tubes. After centrifugation, cells were washed three times in phosphate Na/K buffer (pH 7.4) and processed for embedding in Epoxy, TAAB 812 resin (TAAB Laboratories, Berkshire, England) according to standard procedures.
  • Postfixation of cells was done with a mixture of 1% osmium tetroxide and 0.8% potassium ferricyanide in distilled water for 1 h at 4 0 C. After five washes with distilled water, samples were incubated with 2% uranyl acetate in water for 1 h, washed three times, and dehydrated twice in increasing concentrations of acetone (50, 70, 90 and 100%) for 10 min each at room temperature. Infiltration in the resin was done in increasing concentrations of acetone/epon (3:1, 1:1, 1:3 and 100% epon) . Polymerization of infiltrated samples was done at 60 0 C during 2 days. Ultrathin sections of the samples were stained with saturated uranyl acetate and lead citrate and examined at 80 kV in a Jeol JEM-1010 (Tokyo, Japan) electron microscope .
  • the number of the intracellular mature virions present in the cytoplasm was lower in cells infected with rSARS-CoV- ⁇ E than with the recombinant wild-type virus (Fig. 12A) . This observation is consistent with the lower virus titer of the defective virus. Coronaviruses assemble by budding into the lumen of Golgi complexes (Ng et al . , 2003). Accordingly, the sites of nucleocapsid invagination into the lumen of the Golgi complexes were analyzed.
  • the number of mature virions in these sites was lower in the case of rSARS-CoV- ⁇ E than in the recombinant wild-type virus-infected cells, and an increase of dense material in the cytoplasm of rSARS-CoV- ⁇ E virus-infected cells was observed, probably corresponding to aberrant virions (Fig. 12B) .
  • Virus particles were seen in vesicles either as single particles (data not shown) or as groups of viruses in enlarged vesicles (Fig. 12C) . In this case, the number of mature virions was considerably higher in the rSARS-CoV-infected cells than in rSARS-CoV- ⁇ E- infected cells.
  • vesicles observed in rSARS-CoV- ⁇ E-infected cells contained dense, granular material interspersed between the virions that could correspond to aborted viral assembly processes. Overall, these data suggest that the E protein has an important role in viral trafficing and assembly.
  • Golden Syrian hamsters (44 days of age) were lightly anesthetized by isoflurane (USP-Baxter Healthcare, Deerfield, IL) inhalation and inoculated intranasally with 10 3 TCID 50 of rSARS- CoV or rSARS-CoV- ⁇ E in 100 ⁇ l total volume.
  • Hamsters were sac- rificed two, five, and eight days after virus inoculation (4 hamsters/group/day) and lungs and nasal turbinates were harvested and stored frozen.
  • tissue samples were thawed, weighed, and homogenized to a final 10% (w/v) suspension in Leibovitz's L-15 Medium (Invitrogen, Grand Island, NY) with gentamicin (Invitrogen) and amphotericin B (Quality Biological, Gaithersburg, MD), which were added to the tissue culture medium at final concentrations of 0.1 mg/L and 5 mg/L, respectively.
  • Tissue homogenates were clarified by low-speed centrifugation and virus titers were determined in Vero cell monolayers in 24 and 96-well plates as described previously by Subbarao et al . , 2004.
  • Virus titers are expressed as TCID 50 /g of tissue, with a lower limit of detection of 10 1 ' 5 TCID 50 /g. It has been determined that three plaque forming units (pfu) is equivalent to one TCID 50 .
  • Virus titers in nasal turbinates and lungs of hamsters infected with the rSARS-CoV- ⁇ E virus were 100 and 1000-fold lower than those of the recombinant wild-type virus, suggesting that the rSARS-CoV- ⁇ E virus was attenuated in growth in this species (Fig. 14) .
  • Infectious virus was detected in lung and nasal turbinates of animals infected with the recombinant wild-type virus at eight days post-infection, whereas the defective virus was already cleared at this time point (Fig. 14) .
  • Pulmonary pathology associated with the replication of the recombinant wild-type and rSARS-CoV- ⁇ E viruses were compared in Golden Syrian hamsters (Fig. 15) .
  • Isoflurane-anesthetized golden Syrian hamsters 40 days of age) were inoculated intranasally with 10 3 TCID 50 rSARS-CoV (one hamster/day) or rSARS-CoV- ⁇ E (two hamsters/day) in 100 ⁇ l total volume.
  • Hamsters were sacrificed two (Fig. 15A and 15B) and five days (Fig. 15C and 15D) after infection, lungs were inflated with and stored in 10% formalin and processed for histopathological examination (Fig.
  • the immuhistochemistry analysis revealed higher antigen concentration in the lungs of rSARS-CoV infected animals than in rSARS- CoV- ⁇ E-infected animals.
  • Animals infected with the recombinant wild-type virus showed more pulmonary disease (more bronchiolitis and interstitial pneumonitis) than those infected with the rSARS-CoV- ⁇ E virus (Fig. 15) . This indicates that the rSARS-CoV- ⁇ E virus is attenuated in growth and accompanying pathology in the hamster model.
  • a virus lacking all non structural genes of SARS-CoV (genes 3b, 6, 7a, 7b, 8a and 8b) .
  • a virus lacking all non structural genes (genes 3b, 6, 7a, 7b, 8a, 8b) and a structural gene (gene E) .
  • a virus lacking all non structural genes (genes 3b, 6, 7a, 7b, 8a, 8b) and structural gene 3a.
  • a virus lacking all non structural genes (genes 3b, 6, 7a, 7b, 8a, 8b) and the two structural genes 3a and E.
  • Viruses lacking structural genes (E, 3a, or both and any combination of non-structural genes 6, 7a, 7b, 8a, 8b and
  • 9b such as viruses lacking genes 6, 7a, 7b, 8a, 8b, and 9b and viruses lacking genes 6, 7a, 7b, 8a, 8b, 9b and E, respectively.
  • Gene 9b completely overlaps with gene N; all the N gene introduced mutations were therefore silent with respect to the sequence of the N gene (i.e. did not lead to an amino acid change) .
  • FIG. 17 A point mutation was introduced in the initiation codon of this gene.
  • the initiation codon ATG present in the parental SARS-CoV has been mutated to ACG.
  • the second and third ATGs in phase present in the gene were replaced by ACG.
  • the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host. Virology 296:177-189.
  • the small envelope protein E is not essential for murine coronavirus replication. J. Vi ⁇ rol. 77:4597-4608.
  • Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence. Virology 308, 13-22.
  • Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence. J. Virol. 73:7607-7618.
  • Coronavirus transcription subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis. J Virol. 64 (3) : 1050-6.
  • Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice. J. Virol. 79:14909-14922.

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  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Pulmonology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des acides nucléiques codant des virus SRAS-CoV atténués capables de réduire, dans une culture cellulaire, le titre viral maximum par un facteur de 2 par rapport au titre viral maximum du virus SRAS-CoV de type sauvage dans la même culture cellulaire. Selon un autre aspect de l'invention, les acides nucléiques codant un virus de SRAS-CoV atténué peuvent être obtenus par un procédé selon lequel le génome d'un virus de SRAS-CoV est modifié par transformation de la séquence du gène codant la protéine SARS-CoV E, de manière à empêcher l'acide nucléique d'exprimer une protéine E fonctionnelle. Par ailleurs, l'invention concerne des virus codés par ces acides nucléiques ainsi que l'utilisation médicale de ces acides nucléiques et de ces virus.
EP06762172A 2005-06-24 2006-06-23 Sras attenue: utilisation comme vaccin Withdrawn EP1893752A2 (fr)

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EP05013727A EP1736539A1 (fr) 2005-06-24 2005-06-24 Vaccins comprenant le SARS-CoV atténué
EP06762172A EP1893752A2 (fr) 2005-06-24 2006-06-23 Sras attenue: utilisation comme vaccin
PCT/EP2006/006091 WO2006136448A2 (fr) 2005-06-24 2006-06-23 Sras attenue: utilisation comme vaccin

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CN108383913A (zh) * 2018-02-12 2018-08-10 四川大学 重组猪il-23增强pcv2疫苗免疫佐剂的制备及应用
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WO2006136448A3 (fr) 2007-07-19
EP1736539A1 (fr) 2006-12-27
WO2006136448A2 (fr) 2006-12-28

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