EP1893752A2 - Attenuated sars and use as a vaccine - Google Patents

Abstract

The present invention relates to nucleic acids encoding attenuated SARS-CoV viruses which are 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. According to a further aspect of the present invention, the nucleic acids encoding an attenuated SARS-CoV virus, 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. The present invention further relates to the viruses encoded by these nucleic acids as well as the medical use of the nucleic acids and of the viruses.

Description

Attenuated SARS and use as a Vaccine

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.

TECHNICAL BACKRGOUND

Therapy approaches that involve the insertion of a functional gene into a cell to achieve a therapeutic effect are also referred to as gene therapy approaches, as the gene serves as a drug. 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. Currently, 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.

In an alternative approach, 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.

Since the etiologic pathogen causing SARS was identified as a new coronavirus (SARS-CoV; see Drosten et al . , 2003; Holmes and Enjuanes, 2003; Marra et al . , 2003; Rota et al . , 2003), the study of coronavirus molecular biology was given a very high priority in order to develop effective strategies to prevent and control coronavirus infections.

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) . When a coronavirus infects a cell, the genomic RNA (gRNA) replicates in the cytoplasm and a set of subgenomic RNAs (sgRNA) of positive and negative polarity is produced (Sethna et al . , 1989; Sawicki & Sawicki, 1990; Van der Most & Spaan, 1995; and Enjuanes, 2005) .

Due to the fact that the coronaviruses replicate in the cytoplasm, use of coronaviruses as a vector for gene therapy and vaccination has been suggested. Specifically, defective interfering (DI) genomes of coronaviruses were produced. These DI genomes are deletion mutants which require the presence of a complementing or helper virus for replication and/or transcription (see Chang et al . , 1994; WO97/34008; Spanish patent application P9600620; Izeta et al . , 1999; and Sanchez et al . , 1999). 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).

The construction of 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.

Although SARS-CoV emerged only in 2002, genome sequences of SARS-CoV isolates have been published recently and provide important information on the organization, phylogeny and variability of the SARS-CoV genome (Marra et al . , 2003; Rota et al . , 2003). About two-thirds of the 29.7-kb Urbani strain genome encode the replicase gene that comprises open reading frames Rep Ia and Rep Ib, the latter one being expressed by ribosomal frameshifting (Thiel et al . , 2003) . Translation of both ORFs results in the synthesis of two large polyproteins that are processed by viral proteinases to yield the repli- case-transcriptase complex (Ziebuhr et al . , 2000).

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; Zύniga et al . , 2004). Synthesis of subgenomic negative sense RNA species is regulated by the transcription- regulating sequences (TRSs) , that include a highly conserved core sequence that is found preceding each gene and at the 3' end of the leader sequence (Thiel et al . , 2003).

The preparation of a full-length cDNA of the SARS-CoV Urbani strain has been described previously (Yount et al . , 2003) where it was shown that the recombinant virus replicated as efficiently as wild-type virus. The production of SARS-CoV- like particles, by expression of the viral structural proteins M, E and S in the baculovirus system was also described (Mor- tola & Roy, 2004) . It was shown that the simultaneous high level expression of S, E and M by a single recombinant virus allowed the assembly and release of virion- like particles (VLP) .

In the SARS-CoV virion envelope the four structural proteins, 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) . In fact, 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).

The effect of the deletion of group-specific genes in different coronaviruses has been studied previously. Reports using murine hepatitis virus (MHV) as a model have shown that deletion of ORFs 4, 5a, 2a, and HE are attenuating in the natural host (de Haan et al . , 2002). Similarly, studies deleting ORF 7 of TGEV (Ortego et al . , 2002) and ORFs 3abc and 7ab of feline infectious peritonitis virus (FIPV) (Haijema et al . , 2004) led to virus attenuation. However, other SARS-CoV deletion mutants wherein the ORFs 3a, 3b, 6, 7a, and 7b were deleted one at a time did not show significant changes in in vitro (no reduction in titers in tissue culture) and in vivo replication efficiency (no attenuation of the virus) in the mouse model (Yount et al . , 2005). These deletion mutants have not yet been evaluated in other animal models with regard to the presence of an attenuated phenotype .

Kuo et al. (2003) reported that E gene mutants of the mouse hepatitis virus (MHV) lacking the entirety of genes 4, 5a and E are able to assembly coronavirus-like particles. However, the virion assembly in the mutant occurs with orders of magnitude lower efficiency than that of the wild-type with the consequence that such kind of virus mutants will not be useful as vaccines. Another group, HUANG et al . (2004), examined the role of specific SARS-CoV genes on virus assembly by examining the formation of non- infectious VLPs consisting of only a few SARS-CoV viral genes but no genetic material. From their experiments the authors concluded that gene E is not essential for VLP formation as long as the M and N proteins are present.

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 . SUMMARY OF THE INVENTION

This problem is now solved by nucleic acids encoding attenuated SARS-CoV viruses. According to one aspect of the present invention, 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.

According to a further aspect 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. In the context of the present invention a functional E protein is a protein that:

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. In accordance with this embodiment, 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.

In one aspect of the present invention, 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:

any of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19; or or any sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19.

In an alternative embodiment, the nucleic acids preferably are not capable of expressing a protein, which protein is encoded by:

any of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19; or or any sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19.

According to this aspect of the invention, in one preferred embodiment the nucleic acid only encodes the viral proteins replicase and S and M and N.

According to a further aspect, 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. Again, 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. In a preferred embodiment of the invention 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. In the vaccine 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 .

In another aspect 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.

In a preferred embodiment 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.

In a further preferred embodiment 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.

In another preferred embodiment 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) .

In yet another embodiment of the present invention 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) .

In another preferred embodiment 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.

According to another embodiment 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) .

In yet another embodiment 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.

For preparing a SARS-CoV vaccine, 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 .

Further preferred embodiments of the present invention are described in the following detailed description, in the examples and in the claims .

DETAILED DESCRIPTION OF THE INVENTION

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 .

In a preferred embodiment 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.

In a further embodiment 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.

According to the most preferred embodiment of the present invention, 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.

The term "amending a sequence" is used to refer to the modification of a nucleic acid sequence with techniques well known in the art. When „amending 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^~„ or ,,ΔE" 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.

According to a preferred aspect of the present invention, 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. This has the specific advantage that the modifications of the SARS-CoV genome can be carried out very conveniently.

The present inventors have surprisingly found that 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.

Using the attenuated virus as a vaccine, infection of a cell will lead to replication of the virus and production of further infective virus particles. However, the titer of the attenuated virus will be significantly lower when compared to the wild-type SARS virus. In other words, 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.

In an alternative embodiment, 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.

In the present application 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) .

The use of 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. However, 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. 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.

Obviously these approaches may generate a 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.

The term "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.

The term "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.

The term "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.

In the enclosed sequence listing and Figures, sequences of the following SARS-CoV are provided:

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:

SEQ ID NO: 11 Fig. 18

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

ORF of gene 9b SEQ ID NO: 19 Fig. 26 The 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) . In 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

The sequences of the SARS genes and features of the SARS-CoV:

5' UTR 1-264

ORF Ia (Rep Ia) 264-13413

ORF Ib (Rep Ib) 13398-21485

5 (Spike protein) 21492-25259 3a 25268-26092

3b 25689-26153

E (Envelope protein) 26117-26347

M (membrane protein) 26398-27063

6 27074-27265 7a 27273-27641 7b 27638-27772 8a 27779-27898

8b 27864-28118

9b 28130-28426

N (Nucleoprotein) 28120-29388

3¹ UTR 29389-29727;

wherein UTR means "untranslated sequence".

For the purposes of the present application sequence homology is determined using the Clustal computer program available from the European Bioinformatics Institute (EBI) , unless otherwise stated.

In a further embodiment of the present invention, 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 .

In a further alternative embodiment of the present invention, 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. For example, 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) . Based on this BAC containing a SARS viral replicon, a full length copy of the genomic sequences of the SARS-CoV nucleic acid, can be prepared as described in the Examples. The full length clone can then be used to introduce point mutations, deletions or substitutions which will inactivate certain structural or non-structural genes. Alternatively, one might also start with the replicon as deposited and amend the same by introducing only those sequences which one wants to have in the nucleic acid and deleting sequences which should not be included. In other words it is not a necessity to prepare a full length clone as an intermediate product for the preparation of a nucleic acid encoding an attenuated virus.

In accordance with the present invention vaccines are provided, which are preferably capable of inducing both a systemic immune response and a mucosal immune response against SARS-CoV.

Similar to other coronaviruses, 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 .

In another embodiment of the invention 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.

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. The addition of 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. In addition, as more is discovered regarding the requirements for memory development of T cells, boosters involving key cytokines such as IL-15 and IL-23 may prove beneficial to long-term maintenance of the memory pool. 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 :

(i) Identification of appropriate restriction sites in the viral genome to be used in the engineering of the replicon.

(ii) Generation of an intermediate plasmid as the basis for construction of the SARS-CoV replicon.

(iii) Generation of overlapping SARS-CoV cDNA subclones by RT- PCR and replicon assembly using the restriction sites selected.

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 Colecciόn Espaήola 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. As is shown in the examples, in this manner 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. For example 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).

The animal model systems can be sequentially used for vaccine efficacy and preliminary safety testing. First, 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 . To achieve this goal, a virus stock can be titrated in young adult cynomolgus macaques. In first experiments increasing logarithmic dilutions of this virus stock (10°, 10¹, 10², 10³, 10⁴, 10⁵ and 10⁶ TCID₅₀) should be used to infect macaques intra-tracheally (n= 2 per dilution) . 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 ) .

Brief description of the Figures:

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' . A PCR-fragment containing the 5' end 678 nt and the 3' end 973 nt of the genome of SARS-CoV Urbani strain, flanked at its left end by the cytomegalovirus (CMV) immediate-early promoter and at its right end by a poly (A) tail followed by the hepatitis delta virus ribozyme (Rz) and the bovine growth hormone (BGH) termination and polyadenyla- tion sequences, is cloned in BAC. To facilitate the assembly of SARS-CoV replicon, a multicloning site containing the restriction sites CIaI, Mluϊ, Pmel , and BarnHl, is cloned downstream of the 5' end viral sequence.

Figure 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. CMV, CMV immediate-early promoter; TRS N, natural TRS of N gene; pA, tail of 25 A residues; Rz, hepatitis delta virus ribozyme; BGH, bovine growth hormone termination and poly- adenylation sequences. Figure 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. Starting from the SARS-CoV replicon cloned in a BAC (see Figure 3 above) , the full-length clone was assembled by introducing the remaining SARS-CoV sequences in two steps. In a first step, 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. In a second step, 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-ΔE 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- ΔE or the recombinant wild-type virus. At different times post-infection, 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-ΔE. Both viruses, the parental and the E^" produced a significant cytopathic effect in Vero E6 cells (Fig. 8A) . The effect was observed earlier with the full length virus and the SARS-CoV-ΔE viral plaques were also smaller than plaques produced by the wild type virus (Fig. 8B) . An additional demonstration of the rescue of the recombinant E^" virus was the staining of the infected cells in immunofluorescence using SARS-CoV specific antibodies (Fig. 8C) .

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; ΔE, SARS-CoV-ΔE; 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-ΔE (ΔE) . S, N, and E viral proteins; M, mock infected cells .

Fig. 11 shows the effect of temperature and pH changes on rSARS-CoV-ΔE virus infectivity. Supernatants containing recombinant SARS-CoV and SARS-CoV-ΔE 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-ΔE- 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-ΔE-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- ΔE-infected cells are displayed on the left and right side respectively. Bars, 2 μm in panel A and 200 nm in panels B and C.

Fig. 13 shows the morphology of rSARS-CoV-ΔE 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-ΔE- 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-ΔE- 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 10³ TCID₅₀ of rSARS-CoV or rSARS-CoV-ΔE . 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-ΔE in hamsters after inoculation with 10³ TCID₅₀ of rSARS-CoV or rSARS-CoV-ΔE. (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-ΔE- 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 following examples illustrate but do not limit the embodiments of the invention.

EXAMPLES

The following cells, viruses, plasmids and bacteria strains were used:

Cells. 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) . All cells were maintained in DMEM supplemented with 25mM HEPES and 10% fetal bovine serum and antibiotics at 37⁰C in a CO₂ incubator. Viruses. The genomic 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. 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 μF, 2.5 kV, and 200 Ω 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.

EXAMPLE 1 Construction of a SARS-CoV replicon cDNA

A cDNA that contained the untranslated 5' and 3' end of the Urbani genome and the replicase and N genes, was cloned as a BAC under the control of a CMV promoter.

Additionally, 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.

In a first step the appropriate restriction sites in the viral genome that can be used in the engineering of the replicon and the full length cDNA clone of SARS-CoV were identified (Fig. D •

In a second step 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. Additionally, a multicloning site containing the restriction sites CIaI₁ MIuI, Pmel, and BamΑI , selected in the first step, was cloned in between the SARS-CoV sequences to allow the assembly of the SARS-CoV replicon.

In a third step the overlapping SARS-CoV cDNA subclones were generated by RT-PCR and the replicon was assembled using the restriction sites selected. Using the pBAC-SARS-CoV 5' 3' as starting point, 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 μg/ml chloramphenicol at 37°C. Cells from these primary cultures (considered passage 0) were propagated serially by diluting 10⁶-fold daily. Each passage was considered to represent about 20 generations .

EXAMPLE 3 Sequence analysis

DNA was sequenced using an automatic 373 DNA sequencer (Applied Biosystem) using fluorochrome labeled dideoxynucleotides and temperature resistant DNA polymerase (Perkin Elmer) .

EXAMPLE 4 Transfection of SARS-CoV replicon cDNA

BHK-21 and 293T cells were grown to 95% confluence on 35 mm- diameter plates and transfected with 5 μg of the SARS-CoV replicon, using 6 μg of Lipofectamine 2000 (Invitrogen) according to the manufacturer's specifications.

EXAMPLE 5 RNA isolation and RT-PCR analysis

Total intracellular RNA was extracted at 24 h post- transfection (hpt) with the RNeasy Mini Kit (Qiagen) and used as template for RT-PCR analysis of gene N mRNA transcription. RT reactions were performed with Moloney murine leukaemia vi- rus reverse transcriptase (Ambion) and the antisense primer URB-28630RS (5'-TGCTTCCCTCTGCGTAGAAGCC-B') (SEQ ID N0:7), complementary to nucleotides 510 to 531 of gene N. 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. For the quantitative analysis by realtime RT-PCR of gene N mRNA, 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).

EXAMPLE 6 Analysis of -the SARS-CoV derived replicon

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.

The role of the N protein in SARS-CoV replicon activity was analyzed. To achieve this objective, a SARS-CoV replicon lacking the N gene was constructed and the replicon activity compared with that of the replicon expressing the N gene. A basal activity of the replicon lacking N gene was detected, but replicon activity increased more than 100-fold when N protein was present. These data indicated that 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 Colecciόn Espafiola de Cultivos (Tipo, CECT in Valencia, Spain) . The deposit was given the provisional accession number 7020.

EXAMPLE 7 Preparing a Full Length Clone

A SARS-CoV full-length cDNA clone (pBAC-SARS-CoV*^'1') was prepared according to the cloning strategy outlined in Fig. 5.

Starting from the SARS-CoV replicon cloned into a BAC (Figure 3; and Examples 1 to 6, above), the full-length clone was assembled by adding the rest of the genes in two steps. In a first step, 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. In a second step, 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.

The cDNA for these two fragments was obtained by RT-PCR cloning of RNA fragments which were obtained from the Center for Disease Control in Atlanta. The RNA corresponds to the genomic sequences of the Urbani strain of SARS-CoV.

Genes and relevant restriction sites used in the cloning steps are shown in Fig. 5. Functional analysis of the resulting full-length cDNA clone showed that the clone was fully stable when propagated in E. coli.

After transfection of Vero cells, 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.

EXAMPLE 8

Preparing Attenuated Viruses containing all genes of SARS-CoV except the gene encoding protein E, an structural protein

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-ΔE) :

(a) Two point mutations in gene E core sequence (CS) (Fig. 6) have been introduced. This part is the center of the sequences regulating gene transcription (TRS) and the introduction of the mutations will prevent expression of gene E. The original TRS sequence ACGAAC has been replaced by ACCAAT. (b) A point mutation in the initiation codon of E gene open reading frame has been introduced. Initial codon ATG has been change to GTG.

(c) A deletion of 142 nucleotides within ORF gene E has been introduced.

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. In addition, to avoid the possibility of genetic reversion of the recombinant virus, a 142 nt covering the majority of E gene were deleted. To maintain the wild-type transcription levels of the M gene, 48 nt upstream of the published transcription regulating sequence (TRS) of the M gene at the 3' end of the E gene (Thiel et al . , 2003) were not altered.

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. The oligonucleotides SARS-16501-VS

(5 ' -GGCTCATGTGGTTTATCATTAGTATTGTACAAATGGCACC-3 ' ) and SARS- 16973-ΔE-RS (5'-CCTTCAGAAGAGTTCAGATTTTTAACACGCTTAACGTACCTGTTT- CTTCCGAAACGAATGAGTACACAATGGTACTCACTTTCTTGTGCTTAC-S'), that includes a deletion between nucleotides (nt) 26153 and 26295 of the SARS-CoV genome, two point mutations in the core sequence

(CS) of the E gene, and one point mutation which abrogates the start ATG codon of this gene, were used to generate a PCR product from nt 25170 and 26326 of the SARS-CoV genome. The oligonucleotides SARS-16943-VS (5'-GCGTGTTAAAAATCTGAACCTCTGAA- GG-3') and SARS-N733-RS (5'-GGCCTTGTTGTTGTTGGCC-S') were used to generate a PCR product spanning nt 26296 to 28852 of the SARS-CoV genome. 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- ΔE. Both plasmids pBAC-SARS-5' -3 ' -DN and pBAC-SARS-5 ' -3 ' -DN-ΔE 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. Finally, the BamHI-Rsrll fragment of pBAC-SARS-CoV^FL containing the SARS-CoV full-length cDNA, corresponding to nt 26045 to 29783, was exchanged by that of plasmid pBAC-SARS-5' -3 ' -DN-ΔE to generate the plasmid pBAC-SARS-CoV-ΔE which lacks the E gene.

To rescue the engineered virus, Vero E6 cells grown to 90% confluence in 12.5 cm² flasks were transfected with 6 μg of the plasmid pBAC-SARS-CoV-ΔE or with the parental plasmid pBAC- SARS-CoV^FL as a control, using 12 μg 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. Cell supernatants were harvested, passaged twice on fresh Vero E6 cells and the propagation-competent viruses (rSARS-CoV-ΔE and recombinant wild-type rSARS-CoV) recovered from both plasmids were cloned by three rounds of plaque purification.

The ΔE virus was rescued, indicating that gene E is not essential for the viral cycle. The titer of the defective virus rSARS-CoV-ΔE was 20 to 50-fold lower than that of the parental virus (recombinant wild-type rSARS-CoV) . Viral titers were around 5-x 10⁷ pfu/ml for the parental virus and 3-x 10⁶ pfu/ml for the defective virus, as determined in a plaque assay on Vero E6 cells (Fig.7A).

Both viruses, the parental (SARS-CoV) and the E^" (SARS-CoV-ΔE) produced a significant cytopathic effect in Vero E6 cells

(Fig. 8A) , although with the parental virus this effect was observed earlier than with the E^" virus. SARS-CoV-ΔE viral plaques were smaller than plaques produced by the wild type virus (Fig. 8B) . An additional demonstration of the rescue of the recombinant E^" virus was the staining of the infected cells in immunofluorescence using SARS-CoV specific antibodies

(Fig.8C) .

EXAMPLE 9 RNA isolation and RT-PCR analysis of pBAC-SARS-CoV-ΔE

Total 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. 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-ΔE- infected cells. The results showed that while gene E mRNA was produced by the parental virus this mRNA was not observed in the E^" virus. In addition, PCR products of 1050 bp in the case of the rSARS-CoV-ΔE virus, and of 1200 bp in the case of the recombinant wild-type virus were identified. The sequence of these PCR products matched that of the sg mRNAs starting at the TRS of gene 3, and confirmed that the rSARS-CoV-ΔE virus maintained the mutations and deletion introduced to prevent E gene expression. In contrast, no differences in the PCR products derived from the mRNA encoding S and N proteins were detected.

EXAMPLE 10 Western Blot analysis

The absence of E protein production, resulting from disruption of E gene expression, was further shown in cells infected with the E^" virus in contrast to the presence of this protein in cells infected with the parental virus by Western blot assays with E specific antibodies.

Cell lysates were analyzed by sodium dodecylsulfate-polyacryl- amide gel electrophoresis (SDS-PAGE) . Proteins were transferred to a nitrocellulose membrane with a Bio-Rad mini protean II electroblotting apparatus at 150 mA for 2 h in 25 mM Tris-192 mM glycine buffer, pH 8.3, containing 20% methanol. 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- ΔE 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. In contrast, 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) .

EXAMPLE 11 Growth kinetics of rSARS-CoV-ΔE

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-ΔE 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.

Growth kinetics in Vero E6 cells of the rSARS-CoV-ΔE and recombinant wild-type viruses showed similar profiles (Fig. 7A). Cytopathic effect in Vero E6 cells was detected by 24 h postinfection. In contrast, in Huh-7 and CaCo-2 cells, cytopathic effect was not detected, even at 72 h post- infection. In Vero E6-infected cells, maximal virus titers were reached between 24 and 48 h post-infection (peak titer was ~8 x-10⁶ pfu/ml for the recombinant wild-type virus) . The titer of the rSARS-CoV- ΔE virus was -20 -fold lower than the recombinant wild-type virus. In Huh-7 cells, maximal titers were reached 48 h postinfection (~5-x 10⁵ 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-10⁵ pfu/ml for the recombinant wild-type virus) (Fig. 7C) . In both Huh-7 and CaCo-2 cell lines, rSARS-CoV-ΔE virus grew to titers -200-fold lower than the recombinant wild-type virus. These data indicate that although the E protein has a significant effect on SARS-CoV growth, it is not essential for SARS-CoV replication in cell culture. The same was confirmed in the mink lung cell line MvILu.

EXAMPLE 12 Indirect immunofluorescence microscopy

Subconfluent Vero E6 cells grown in 9 cm² slide flasks were infected at a moi (multiplicity of infection) of 1. At 24 hpi (hours post- 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. Cells were then washed three times with PBS, incubated with Cy5- conjugated anti-human immunoglobulin G (Jackson Immunore- search) at 1:200 dilution in blocking solution for 30 min at room temperature and washed five times with PBS. The slides were removed, mounted with glass coverslips and analyzed with a Zeiss Axiophot fluorescence microscope. The pattern of immunofluorescence was similar for the two viruses and, in both cases, cell membrane and cytoplasmic vesicles were prominently stained (Fig. 8C) .

EXAMPLE 13 Stability of rSARS-CoV-ΔE

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-ΔE viruses were incubated at temperatures ranging from 4°C to 80⁰C for 30 min. Both viruses showed similar inactivation profiles, with a reduction of 10³-fold after incubation at 60⁰C. Heating at 80 °C or higher temperatures led to residual virus infectivity (Fig. HA) .

Incubation of recombinant wild-type and rSARS-CoV-ΔE virus at different pH for 30 min showed that both viruses were stable from pH 5 to 9 (Fig. HB) . These results indicate that the E protein has little influence on the stability of the virions under the different temperatures and pH tested.

EXAMPLE 14 Electron microscopy

E protein has been implicated in virus morphogenesis (Fischer et al., 1998; Kuo and Masters, 2003) . Therefore, the assembly of rSARS-CoV and rSARS-CoV-ΔE viruses was studied by electron microscopy (Fig. 12) .

For conventional electron microscopy, Vero E6 cells monolayers were infected with rSARS-CoV and rSARS-CoV-ΔE 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 4⁰C. 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 60⁰C 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 .

For negative staining electron-microscopy, supernatants of Vero E6 cells infected for 20 h were fixed with 10% formaldehyde and concentrated onto carbon-coated ionized copper grids in a Beckman airfuge at 21 psi using an Electron Microscopy Rotor (EM- 90, Beckman) . Grids were stained with 2% phospho- tungstic acid pH 7 for 1 min. Samples were 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-ΔE 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-ΔE than in the recombinant wild-type virus-infected cells, and an increase of dense material in the cytoplasm of rSARS-CoV-ΔE 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-ΔE- infected cells. In addition, vesicles observed in rSARS-CoV-ΔE-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.

The potential influence of E protein deletion on SARS-CoV virion morphology was studied by electron microscopic evaluation of ultra-thin sections of infected cells, and concentrated negative-stained viruses.

For negative staining electron-microscopy, supernatants of Vero E6 cells infected for 20 h were fixed with 10% formaldehyde and concentrated onto carbon-coated ionized copper grids in a Beckman airfuge at 21 psi using an Electron Microscopy Rotor (EM-90, Beckman) . Grids were stained with 2% phospho- tungstic acid pH 7 for 1 min. Samples were examined at 80 kV in a Jeol JEM-1010 (Tokyo, Japan) electron microscope (Fig. 13) . Extracellular virion morphology observed in ultrathin sections by electron microscopy was similar for rSARS-CoV-ΔE and recombinant wild-type viruses and abnormal structures were not seen (Fig. 13A) .

Negative staining of purified recombinant wild-type and rSARS- CoV-ΔE viruses showed particles surrounded by club-shaped projections, indicating that E protein expression apparently has little influence on virion morphology (Fig. 13B). Nevertheless, purified rSARS-CoV-ΔE virus showed a higher frequency of virus aggregation and amorphous structures than wild-type virus (Fig. 13B), suggesting that the defective virus was more sensitive to mechanical shearing forces.

EXAMPLE 15 Virus replication in hamsters

The in vivo growth of rSARS-CoV and rSARS-CoV-ΔE viruses was determined by infecting Golden Syrian hamsters (Roberts et al, 2005) .

The animal protocol employed in this example was approved by the National Institute of Allergy and Infectious Disease Animal Care and Use Committee. Male Golden Syrian hamsters, LVG (SYR) , were obtained from Charles River Laboratories (Wilmington, MA) and pair-housed in individually ventilated microiso- lator rodent cages. Hamsters were rested for at least three days before initiation of the following experiments.

Golden Syrian hamsters (44 days of age) were lightly anesthetized by isoflurane (USP-Baxter Healthcare, Deerfield, IL) inhalation and inoculated intranasally with 10³ TCID₅₀ of rSARS- CoV or rSARS-CoV-ΔE in 100 μl 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. For viral titer determination, 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 TCID₅₀/g of tissue, with a lower limit of detection of 10¹'⁵ TCID₅₀/g. It has been determined that three plaque forming units (pfu) is equivalent to one TCID₅₀.

Virus titers in nasal turbinates and lungs of hamsters infected with the rSARS-CoV-ΔE virus were 100 and 1000-fold lower than those of the recombinant wild-type virus, suggesting that the rSARS-CoV-ΔE 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) .

EXiUIPLE 16 Histopathologic examination of the lungs of hamsters

Pulmonary pathology associated with the replication of the recombinant wild-type and rSARS-CoV-ΔE viruses were compared in Golden Syrian hamsters (Fig. 15) . Isoflurane-anesthetized golden Syrian hamsters (40 days of age) were inoculated intranasally with 10³ TCID₅₀ rSARS-CoV (one hamster/day) or rSARS-CoV-ΔE (two hamsters/day) in 100 μl 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. 15A and 15C) and immuno- histochemistry (Fig. 15B and 15D) . Lungs were fixed in 10% neutral buffered formalin for three days, routinely processed, and subsequently paraffin embedded. The lung was studied histopathologically using hematoxylin and eosin stained sections .

In agreement with the virus titer (see Example 15 above) , the immuhistochemistry analysis revealed higher antigen concentration in the lungs of rSARS-CoV infected animals than in rSARS- CoV-ΔE-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-ΔE virus (Fig. 15) . This indicates that the rSARS-CoV-ΔE virus is attenuated in growth and accompanying pathology in the hamster model.

EXAMPLE 17

Preparing Attenuated Viruses lacking one or more structural and non-structural genes

The following further mutants were prepared:

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 deletion.

To abolish expression of gene 9b:

(a) A point mutation in the initiation codon of this gene was introduced .

(b) The ATG present in the parental virus was mutated to ACG to prevent expression of the corresponding protein.

(c) A stop codon was introduced after this mutated ACG and another two ATG in phase within the ORF have been changed (see Fig. 16) .

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) .

Gene 3b deletion

To abolish expression of gene 3b the following strategy was used (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.

All these mutations have been designed to introduce silent changes in the genes 3a and E, which overlap with gene 3b.

Simultaneous deletion of genes 3a and 3b.

To abolish expression of genes 3a and 3b we have employed three strategies to engineer a cDNA encoding SARS-CoV with non- functional genes 3a and 3b (Fig. 17) :

(a) The core sequence present in the TRS that controls the expression of gene 3 mRNA was inactivated by changing the sequence ACGAAC to ACCAAT.

(b) Parts of these genes were partially deleted by introducing a deletion between nucleotides 25274 and 26015 of SARS-CoV sequence.

(c) The initiation codon ATG of gene 3a were replaced by ACG. In addition, a stop codon has been introduced close to the first.

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Claims

Claims

1. Nucleic acid encoding an attenuated SARS-CoV virus 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.

2. Nucleic acid encoding a SARS-CoV virus, wherein the nucleic acid is 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.

3. Nucleic acid encoding a SARS-CoV virus, wherein the nucleic acid does not encode a sequence of a functional protein E of the SARS-CoV virus.

4. Nucleic acid encoding a SARS-CoV virus, wherein the nucleic acid comprises the sequences encoding the viral proteins replicase, S, M and N and further comprising or not comprising the sequences encoding protein 3a.

5. Nucleic acid according to claim 4 , wherein the nucleic acid does not comprise the sequences encoding the viral proteins E, 6, 7a, 7b, 8a, 8b and 9b.

6. Nucleic acid encoding a SARS-CoV virus, wherein the nucleic acid comprises the sequences of the viral proteins replicase, S, M, N and E and further comprising or not comprising the sequences encoding protein 3a.

7. Nucleic acid according to claim 6, wherein the nucleic acid does not comprise the sequences encoding the viral proteins 6, 7a, 7b, 8a, 8b and 9b.

8. Nucleic acid according to any one of claims 2 to 7, wherein the virus is attenuated in vivo and may be attenuated in vitro.

9. Nucleic acid according to one of claims 1 to 8 , wherein the nucleic acid sequence encoding

(a) the replicase is SEQ ID Nos : 1 and 2 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NOs : 1 and 2 ; and/or

(b) the N or 9a protein is SEQ ID No: 3 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NO: 3; and/or

(c) the S protein is SEQ ID No: 4 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NO: 4; and/or

(d) the M protein is SEQ ID No: 5 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NO: 5; and/or

(e) the E protein is SEQ ID No: 6 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NO: 6; and/or

(f) the protein of gene 3a is SEQ ID No: 12 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID NO: 4; and/or

(g) the protein of gene 6 is SEQ ID No: 14 or a sequence havi least 85% or at least 95% to the sequence of SEQ ID

NO: 14; and/or

(h) the protein of gene 7a is SEQ ID No: 15 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID

NO: 15; and/or

(i) the protein of gene 7b is SEQ ID No: 16 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID

NO: 16; and/or

(j) the protein of gene 8a is SEQ ID No: 17 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID

NO: 17; and/or

(k) the protein of gene 8b is SEQ ID No: 18 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID

NO: 18; and/or

(1) the protein of gene 9b is SEQ ID No: 19 or a sequence having a homology of at least 70%, preferably at least 85% or at least 95% to the sequence of SEQ ID

NO: 19.

10. Nucleic acid according to one of claims 1 to 9, wherein 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 when compared to maximum viral titer of wild-type SARS-CoV virus in the same cell culture.

11. Nucleic acid according to one of claims 1 to 10, wherein the attenuated SARS-CoV virus is capable of producing a maximum viral titer in cell culture that is reduced reduced preferably by a factor between 2 and 100, more pre- ferred by a factor between 5 and 50 and most preferred by a factor between 10 and 25 when compared to maximum viral titer of wild-type SARS-CoV virus in the same cell culture .

12. Nucleic acid according to claim 1, wherein the cell culture is a culture of Vero E6, CaCo-2 or Huh7 cells trans- fected with the nucleic acid.

13. Nucleic acid according to one of claims 1 to 12, wherein the nucleic acid further comprises a sequence not derived from SARS-CoV, preferably a foreign gene.

14. Nucleic acid according to one of claims 1 to 13, wherein the nucleic acid further encodes co-stimulating molecules selected from the list consisting of CD80, CD86, immu- nostimulating cytokines such as TNF-alpha, IFN-gamma, che- mokines, granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2) , IL- 12 and IL-18.

15. Host cell comprising a nucleic acid according to any of claims 1 to 14.

16. SARS-CoV virus particle comprising the nucleic acid of any of claims 1 to 14.

17. SARS-CoV virus particle obtainable by transfecting a host cell with a nucleic acid of any of claims 1 to 14.

18. Vaccine comprising a SARS-CoV virus particle according to claim 16 or 17 or an inactivated SARS-CoV virus particle or a nucleic acid according to any of claims 1 to 14 or a host cell according to claim 15 and a pharmaceutically acceptable adjuvants, carrier or excipient.

19. Vaccine according to claim 18, wherein administration of the vaccine to a mammal gives rise to an immune response that reduces or eliminates the disease symptoms caused by- infection with the SARS-CoV virus.

20. Vaccine according to claim 18 or 19, wherein the vaccine is to be administered to a human or an animal, wherein the animal is preferably a civet cat, a raccoon or a ferret.

21. Vaccine according to one of claims 18 to 20, wherein the vaccine is to be administered orally, intramuscularly, intravenously, subcutaneousIy or intranasalIy .

22. Vaccine according to one of claims 18 to 21, wherein the adjuvant is selected from the list consisting of cytokines, TNF-alpha, IFN-gamma , chemokines granulocyte/ macrophage colony-stimulating factor, interleukin-2 (IL-2), IL- 12, IL-18, IL-15 and IL-23.

23. Use of a virus particle according to claim 16 or 17 or a nucleic acid according to claims 1 to 14 for the preparation of a vaccine for vaccinating a mammal, specifically a human, a civet cat, a raccoon or a ferret against SARS-CoV infection.

24. Method for preparing a SARS-CoV vaccine, comprising modifying a nucleic acid comprising the genome of a SARS-CoV virus by amending the nucleic acid according to claims 2 to 9.

25. Method for preparing a SARS-CoV vaccine according to claim 24, comprising amending the sequence of the gene encoding the SARS-CoV E protein so that the gene cannot express a functional E protein.

26. Method according to claim 25, wherein 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.

27. Method according to claim 26, wherein 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) .

28. Method according to claim 27, wherein 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 3a, 3b, 6, 7a, 7b, 8a and 8b (corresponding to SEQ ID NO: 12 to 18) .

29. Method according to claim 24, wherein 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.

30. Method according to claim 29, wherein the method further comprises amending the sequence of the genome of a SARS- CoV such that the nucleic acid is not capable of express- ing 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) .

31. Method according to one of claims 24 to 30, wherein 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.

32. Method according to claim 24 or claim 31, wherein 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.

33. Method according to one of claims 24 to 32, wherein the method further comprises the mixing of the nucleic acid or of the SARS-CoV virus particle with a pharmaceutically acceptable adjuvants, carrier or excipient.