HK1108115B - Sars vaccine based on replicative vaccinia virus vector - Google Patents

Description

SARS vaccine based on replicative vaccinia virus vector

Technical Field

The present invention relates to the field of antiviral immunology. More specifically, the invention relates to a vaccine against SARS-CoV based on a replicative vaccinia virus vector, a preparation method and uses thereof.

Background

Since the first infectious atypical pneumonia was found in Guangdong province in China in 11 months 2002, the infectious disease had once been widely prevalent worldwide. The world health organization warns the world as such in 3 months 2003 and named it as Severe Acute Respiratory Syndrome (SARS). SARS is spread mainly through respiratory tract, and has strong infectivity, high death rate up to 5-15%, and serious harm to the health and safety of people. The world health organization announced on 16.4.2003 that the pathogen causing SARS belongs to a new variant of coronavirus and was named "SARS-CoV" (Peiris J S M, Lai S T, point M L, et al. The sequencing of the SARS-CoV genome has been completed by the concerted efforts of several national scientists, including China, and sequence analysis has shown that the SARS-CoV genome contains 5 major Open Reading Frames (ORFs) encoding DNA polymerase protein, protuberant protein (S protein), envelope protein (E protein), membrane glycoprotein (M protein), and nucleocapsid protein (NC protein), respectively.

The protuberant protein is the main cell receptor binding protein of coronavirus, and the change of amino acid can significantly affect the virulence of the virus. In addition, the functional site prediction of the protuberant protein of SARS-CoV is carried out, and it is found that a plurality of antigenic determinants May exist in the protuberant protein, and the protuberant protein of SARS-CoV which is currently prevalent in the world has higher conservation, and can be used as an important target for vaccine research (Walgate R. SARS vaccine raw: US and European groups moving for ward, but WHO outer rate put SARS "back in the box", Available May2 at http:// www.Biomedcentral.Com/news/20030502/03).

The nucleocapsid protein is another important structural protein in coronavirus, is located in the core part of virus particles, and has important significance for the accurate assembly of the virus particles. Chinese researchers obtain lymphocytes from blood of SARS patients in recovery period, humanized Fab antibody obtained by genetic engineering means can be specifically combined with SARS-CoV nucleocapsid protein, which shows that nucleocapsid protein is also an important antigenic site of SARS-CoV (Du run bud, in building stone, Liang Mi Fang, etc. the primary research of humanized anti-Severe Acute Respiratory Syndrome (SARS) virus genetic engineering antibody, the report of virology 2003, 19 (2): 104-.

Although the epidemic situation of SARS is effectively controlled worldwide, the possibility of its reoccurrence is not excluded. The anti-SARS-CoV vaccine is the most effective way to prevent the SARS epidemic. The SARS whole virus inactivated vaccine developed in China at present enters into clinical experiment stage, and the vaccine carries all antigens of the virus and has good immunogenicity. However, from the viewpoint of SARS pathology, SARS whole virus inactivated vaccine has potential risk of causing autoimmune reaction of organism; in addition, the production condition of the vaccine is high, and hidden troubles also exist in the aspect of biological safety. Therefore, it is urgent to develop a new generation of safe and effective SARS genetic engineering vaccine.

The types of vaccines currently available for selection include the following: traditional vaccines (inactivated and live attenuated), synthetic peptide and protein subunit vaccines, DNA vaccines, and live vector vaccines. Live vector vaccines offer advantages over other types of vaccines in that: (1) can actively infect target tissues or cells, and improves the efficiency of foreign genes entering the cells; (2) the carrier has adjuvant effect and can induce the production of cytokines and chemokines; (3) most induce a long-term immune response. In the field of live vector vaccines against SARS, non-replicating vectors are currently used.

Disclosure of Invention

The object of the present invention is to provide a novel vaccine and immunization method against SARS-CoV infection, in order to provide a solution to the above mentioned problems of the prior art.

It is an object of the present invention to provide a vaccine against SARS-CoV based on a replicative vaccinia virus comprising a replicative vaccinia virus as a vector having Thymidine Kinase (TK) region of its genome inserted with polynucleotides encoding the nucleocapsid protein and the protuberant protein of SARS-CoV.

In a preferred embodiment of the invention, the replicative vaccinia virus is a vaccinia virus Tiantan strain and preferably the vaccine does not contain a selectable marker gene.

In a preferred embodiment of the invention, the polynucleotides encoding the nucleocapsid protein and the protuberant protein of SARS-CoV inserted into the TK region of the vaccinia virus Tiantan strain are adapted for high efficiency expression in mammalian cells by codon optimization. In a specific embodiment, the polynucleotide encoding the nucleocapsid protein of SARS-CoV inserted into the TK region of the vaccinia virus Tiantan strain has the sequence as shown in SEQ ID NO: 1 and/or the polynucleotide encoding the protuberant protein of SARS-CoV has the nucleotide sequence shown in SEQ ID NO: 2.

The vaccine of the present invention may further comprise a pharmaceutically acceptable suitable adjuvant and/or carrier.

Another object of the present invention is to provide a DNA vaccine against SARS-CoV comprising a vector comprising a polynucleotide encoding a nucleocapsid protein of SARS-CoV and/or a polynucleotide encoding a protuberant protein of SARS-CoV operably linked to a promoter. In one embodiment of the DNA vaccine of the present invention, the polynucleotide encoding the nucleocapsid protein of SARS-CoV has the sequence as set forth in SEQ ID NO: 1, and the polynucleotide encoding the protuberant protein of SARS-CoV has the nucleotide sequence shown in SEQ ID NO: 2.

Another object of the present invention is to provide a method of immunizing against SARS-CoV comprising administering to an individual an immunologically effective amount of the vaccine of the present invention against SARS-CoV using a replicating vaccinia virus, particularly a vaccinia virus Tiantan strain, as a vector. The immunization methods of the invention may further comprise administering to the individual one or more DNA vaccines against SARS-CoV, such as the DNA vaccines of the invention described herein, prior to administration of the vaccine.

The invention also provides an immunization kit, which comprises one or more DNA vaccines aiming at SARS-CoV, the DNA vaccine of the invention as described in the specification, and the vaccine aiming at SARS-CoV with replication type vaccinia virus as a vector. Optionally, the kit further comprises instructions for a vaccination protocol that directs a primary immunization with the one or more DNA vaccines against SARS-CoV followed by a booster immunization with the vaccine against SARS-CoV of the invention using a replicating vaccinia virus as the vector.

In addition, the invention also provides a universal transfer vector pVTT1.0 of the vaccinia virus, and the preservation number is CGMCC No. 1458.

Drawings

FIG. 1: the route of construction of the vaccinia virus universal transfer vector pVTT1.0 is shown.

FIG. 2: the route of construction of the transfer plasmid pVTT-NS is shown.

FIG. 3: shows the restriction analysis and identification result of the vaccinia virus universal transfer vector pVTT1.0. Lane M shows a DNA Marker DL15000 (available from Dalibao bioengineering, Inc.); lanes 1, 2 and 3 show the restriction of the vaccinia virus universal transfer vector pVTT1.0 with KpnI, NdeI or EcoRV, respectively.

FIG. 4: PCR amplifies the coding sequence of SARS-CoV nucleocapsid protein and PCR fusion amplifies promoter P E/L + P7.5 and the coding sequence of SARS-CoV nucleocapsid protein. Lane M is DNA Marker DL2000 (available from Dalibao bioengineering, Inc.); lane 1 shows the band of the coding sequence of the PCR amplified SARS-CoV nucleocapsid protein; lane 2 shows the band of the coding sequence of the PCR fusion amplification promoter P E/L + P7.5 and SARS-CoV nucleocapsid protein.

FIG. 5: and displaying the result of the T-NC plasmid enzyme digestion analysis and identification. Lane 1 shows the results of the double digestion of the T-NC plasmid with Spe I and Not I; lane 2 shows the result of Sal I single cleavage of the T-NC plasmid.

FIG. 6: and displaying the restriction analysis and identification result of the T-NC + P + S plasmid. Lane M is DNA Marker DL15000 (available from Dalibao bioengineering, Inc.); lanes 1-4 show the double digestion of the picked T-NC + P + S plasmid (Nos. 1-4) with Sac I and Not I.

FIG. 7: shows the restriction analysis and identification results of the vaccinia virus universal transfer vector pVTT1.0 and the vaccinia virus transfer vector pVTT-NS. Lane M shows a DNA Marker DL15000 (available from Dalibao bioengineering, Inc.); lane 1 shows the double digestion of the vaccinia virus universal transfer vector pVTT1.0 with Sac II and Spe I; lane 2 shows the double digestion of the vaccinia virus transfer vector pVTT-NS by Sac II and Spe I.

FIG. 8: and (3) performing result inspection on the obtained product by randomly picking 9 sixth-generation leucovirus clones after passage of the fifth-generation rVTT-NS, extracting a virus DNA template and amplifying the SARS-CoV nucleocapsid protein coding sequence by using NC primers 1 and 2 as primers. Wherein lane M shows a DNA Marker DL15000 (available from Dalibao bioengineering, Inc.); lane N shows PCR amplification of the wild-type virus negative control; lanes 1-9 show the results of PCR amplification of the NC protein coding sequence (1.2Kb) of SARS-CoV from randomly picked 9 sixth generation viral clone genomes.

FIG. 9: showing the result test of randomly picking 9 sixth generation leucovirus clones after passage of the fifth generation rVTT-NS, extracting virus DNA template, and amplifying the S protein coding sequence of SARS-CoV by using S primer 1 and S primer 2 as primers. Wherein lane M shows a DNA Marker DL15000 (available from Dalibao bioengineering, Inc.); lane P shows the positive control result with plasmid pSK-S as template; lane N shows PCR amplification of the wild-type virus negative control; lanes 1-9 show the results of PCR amplification of the S protein coding sequence (3.6Kb) of SARS-CoV by randomly picking 9 sixth generation viral clone genomes.

FIG. 10: it was shown that each generation of rVTT-NS infected CEF cells, and the cells and supernatant were harvested 48 hours later, and Western Blot analysis was performed with human multi-antiserum (provided by the center for prevention and control of viral diseases in the Chinese center for prevention and control of diseases). Lane N shows the results of the immunohybridization of the wild-type virus negative control; lane M shows a prestained protein Marker; lanes 1, 3, 5, 6 show the immunohybridization bands of rVTT-NS-infected CEF cells at passage 1, 3, 5, 6, respectively.

FIG. 11: shows that ELISPOT detects the T lymphocyte reaction of secreting IFN-gamma after in vitro stimulation of the N antigen epitope peptide (A) and the S antigen epitope peptide (B). 1X 10 per well using NC protein stimulating peptide and S protein stimulating peptide, respectively⁶The mouse spleen cells were stimulated for 30 hours and the number of IFN-. gamma.secreting T lymphocytes was examined.

FIG. 12: the titer levels of the NC protein (A) and S protein (B) -specific antibody IgG of the serum SARS-CoV of the experimental group animals are shown.

FIG. 13: the SARS-CoV DNA vaccine pDRVSV1.0-S and pDRVSV1.0-N structure diagram.

Preservation of

The transferred plasmid pVTT1.0 is preserved in China general microbiological culture Collection center (CGMCC) at 9/19 of 2005 with the preservation number of CGMCC No. 1458.

The plasmid pSC65 is preserved in China general microbiological culture Collection center (CGMCC) at 24.2.2004, and the preservation number is as follows: CGMCC No. 1097.

The plasmid pSK-N is preserved in China general microbiological culture Collection center (CGMCC) at 9/19 th 2005, and the preservation number is as follows: CGMCC No. 1459.

The plasmid pSK-S is preserved in China general microbiological culture Collection center (CGMCC) at 9/19 th 2005, and the preservation number is as follows: CGMCC No. 1457.

Detailed Description

The vaccine against SARS-CoV of the present invention is constructed based on a replicative vaccinia virus vector, which is different from non-replicative vaccinia virus vectors currently used conventionally, such as MVA (modified virus Ankara), NYVAC (New York Vaccinia), and ALVAC (avipoxy virus and arypox) (Paoletti E.applications of poxvirus vectors to vaccine: Andate. Proc. Natl. Acad. Sci. USA. Vol.93, pp.11349-11353). In the vaccine of the present invention, the TK region of the replicating vaccinia virus vector is inserted with polynucleotides encoding the nucleocapsid protein (NS) and the protuberant protein (S) of SARS-CoV, which upon administration is capable of eliciting a protective immune response against SARS-CoV in an individual.

The term "replicative" refers to vaccinia virus vectors that can be replicated in humans. In a preferred embodiment of the invention, the replicating vaccinia virus vector is vaccinia virus Tiantan strain (VTT). The vaccinia virus Tiantan strain has made a great contribution to the elimination of smallpox in China, has active application in the research of live vector vaccines, and has the advantages of good safety, convenient inoculation, no need of adjuvant and the like.

Preferably, the polynucleotides encoding the nucleocapsid protein and the protuberant protein of SARS-CoV inserted into the TK region of the replicative vaccinia virus genome are codon-optimized for engineering. The term "codon-optimized modification" refers to the selection of the most preferred genetic codons in humans according to the frequency of their usage in the human genetic codon preference table, and the reverse translation of the amino acid sequence of a polypeptide back into a nucleotide sequence such that the nucleotide sequence is suitable for efficient expression in human and mammalian cells. In the present invention, nucleotide sequences encoding the nucleocapsid protein and the protuberant protein, which have been modified by codon optimization, can be obtained by reverse translation according to the known amino acid sequences of the nucleocapsid protein and the protuberant protein of SARS-CoV (see the amino acid sequence of the isolated strain HKU-39849 of the hong Kong strain of SARS-CoV published by Genebank). The nucleotide sequence obtained can be subjected to minor adjustments and modifications, and the degeneracy of the genetic code is utilized to eliminate redundant restriction enzyme recognition sites and eliminate potential sequences that are unfavorable for gene synthesis, such as secondary nucleic acid structures. In a specific embodiment, the polynucleotide encoding the nucleocapsid protein of SARS-CoV has the sequence as set forth in SEQ ID NO: 1, and the polynucleotide encoding the protuberant protein of SARS-CoV has the nucleotide sequence shown in SEQ ID NO: 2.

In the present invention, polynucleotides encoding the nucleocapsid protein and the protuberant protein of SARS-CoV are inserted into the Thymidine Kinase (TK) gene in the vaccinia virus genome by a suitable method, such as homologous recombination, to form a recombinant, replicating vaccinia virus carrying the gene of interest. Preferably, the vaccine of the invention, i.e. the recombinant replication competent vaccinia virus, does not contain a selectable marker gene.

To this end, the present invention provides a universal transfer vector for vaccinia virus to recombine a gene of interest into the TK region of vaccinia virus genomic DNA. In a specific embodiment, the vaccinia virus universal transfer vector of the invention is the transfer plasmid pVTT1.0 (CGMCC No.1458) containing a dual selection marker for neo gene and lacZ gene. The transfer plasmid vector contains the following elements: three selection markers: amp resistance gene, lacZ gene and neo gene. The lacZ gene promoted by the p7.5 promoter is used for blue-white spot screening of recombinant vaccinia virus, the neo gene promoted by the PE6 promoter is used for purifying recombinant vaccinia virus with a screening marker and a target gene (the proliferation of a wild strain of the vaccinia virus is inhibited under the action of G418), and in order to enable the neo gene to play a better role, the tail of the neo gene is provided with a poly (A) sequence of 200 bp. Sequence of homology arms tkL and tkR: tkL and tkR are partial fragments of Thymidine Kinase (TK) of vaccinia virus, and are homologous sequences for homologous recombination between vaccinia virus and the transfer plasmid. ③ lacZ' sequence: the 200bp sequence is completely homologous with the 200bp sequence at the tail part of the lacZ gene, so that the recombinant vaccinia virus with the selection marker and the target gene generates intramolecular homologous recombination, and the selection marker is lost. And fourthly, the early and late promoter pE/L of the vaccinia virus. The multiple cloning site is positioned at the downstream of the promoter pE/L.

By adopting the universal transfer vector pVTT1.0, the target gene can be recombined into the TK region of vaccinia virus genome DNA, and the recombined vaccinia virus genome does not contain a selective marker gene. Accordingly, the present invention also provides a method for constructing a vaccine against SARS-CoV based on replicative vaccinia virus, the method comprising: placing the polynucleotide encoding SARS-CoV nucleocapsid protein (NS) and protuberant protein (S) under promoter pE/L of pVTT1.0 using transfer plasmid pVTT1.0 (CGMCC No.1458) to construct recombinant plasmid pVTT-NS; pVTT-NS generates homologous recombination with the vaccinia virus Tiantan strain in chick embryo cells, so that the target gene of SARS-CoV, the neo gene and the lacZ gene of the double selection marker are recombined into the TK region of the vaccinia virus genome DNA; under the selection pressure of antibiotic G418, using low melting point agarose added with X-gal and neutral red to spread spots, picking blue recombinant vaccinia virus containing both target gene and selection marker, and carrying out three rounds of single-spot purification; then under the selection without G418 pressure, the blue recombinant vaccinia virus itself loses the neo gene and the lacZ gene due to intramolecular homologous recombination of a small fragment of lacZ' with about 200bp in the transfer plasmid and the complete lacZ gene, thereby obtaining the recombinant vaccinia virus Tiantan strain only containing the coding sequences of the nucleocapsid protein and the protuberant protein of SARS-CoV.

The vaccine of the present invention may further comprise pharmaceutically acceptable suitable adjuvants, carriers and/or excipients, suitable adjuvants, carriers and excipients being known in the art.

The invention also provides a DNA vaccine against SARS-CoV, the DNA vaccine comprising a vector comprising a polynucleotide encoding a nucleocapsid protein of SARS-CoV and/or a polynucleotide encoding a protuberant protein of SARS-CoV operably linked to a promoter. Vectors for use in the construction of DNA vaccines are known in the art, for example the eukaryotic expression vector pcDNA3.1. After the gene of interest is obtained, the DNA vaccine can be constructed by ligating the sequence of the gene of interest into an appropriate vector by a known method. In one embodiment of the DNA vaccine of the present invention, the polynucleotide encoding the nucleocapsid protein of SARS-CoV has the sequence as set forth in SEQ ID NO: 1, and the polynucleotide encoding the protuberant protein of SARS-CoV has the nucleotide sequence shown in SEQ ID NO: 2.

It is another object of the present invention to provide a method of immunization against SARS-CoV comprising administering to an individual an immunologically effective amount of a vaccine against SARS-CoV based on a replicative vaccinia virus of the invention, in particular based on the vaccinia virus Tiantan strain. The term "effective amount" refers to an amount of the vaccine of the present invention sufficient to stimulate the production of cellular and/or humoral immunity to a pathogen in an individual, and the specific amount administered, as well as the rate and time of administration, will depend on the condition of the individual and can be determined by a physician as the case may be. The immunization methods of the invention may further comprise administering to the individual one or more DNA vaccines against SARS-CoV, such as the DNA vaccines of the invention described herein, prior to administration of the vaccine. The inventor finds that the immune scheme (namely Prime-boost strategy) of performing initial immunization by using a DNA vaccine containing the coding sequence of SARS-CoV nucleocapsid protein and/or protuberant protein and then performing booster immunization by using a SARS-CoV Tiantan strain recombinant vaccinia virus vaccine can successfully induce a human body to generate high-level humoral and cellular immune response and high-titer neutralizing antibodies.

The invention also provides an immunization kit comprising one or more DNA vaccines against SARS-CoV, the DNA vaccines of the invention as described herein, and a vaccine against SARS-CoV based on replicative vaccinia virus of the invention. Optionally, the kit further comprises instructions for a vaccination protocol that directs a primary immunization with the one or more DNA vaccines against SARS-CoV followed by a booster immunization with a replication-competent vaccinia virus-based vaccine against SARS-CoV of the present invention.

The invention is further illustrated with reference to the following specific examples.

Examples

Example 1: construction of vaccinia Virus Universal transfer vector pVTT1.0

1. Construction of recombinant plasmid pSC-neo

The plasmid pIRESneo (purchased from Clontech) was digested with XhoI and then with SmaI (reaction temperature 25 ℃), and filled in with Klenow enzyme to recover a 1.2kb target fragment neo-polyA; the transition vector plasmid pSC65 (preservation number: CGMCC No.1097) is cut by BglII enzyme, filled by Klenow enzyme, treated by dephosphorylation enzyme (CIAP), and the vector is recovered; the two were ligated at 16 ℃ for 4h and transformed into E.coli TOP 10. Multiple single colonies were picked, plasmids were extracted in small amounts, identified with XbaI and PstI, and the correct recombinant clone was designated pSC-neo.

2. The vaccinia virus vector early promoter PE6 and lacZ fusion fragment were synthesized artificially.

The gene was synthesized by overlap PCR (overlaping PCR). Firstly, respectively listing the positive strand and the negative strand of the sequence PE6 and lacZ fusion polynucleotide fragment, then dividing the positive strand and the negative strand into oligonucleotides with the length of 50bp (the most 5' end of the two strands is about 25bp in length) according to the gene length, wherein except the sequences at the two ends, each sequence of the positive strand and the negative strand is complementary with the oligonucleotides of the two complementary strands by about 25 bp. All 8 oligonucleotides were mixed with the same number of complementary oligonucleotides to form an annealing system, which was slowly annealed after heating in the PCR tube. PCR amplification is carried out by taking the annealing product as a template and the matched upstream and downstream primers to obtain a 429bp PE6 and lacZ fusion polynucleotide fragment. The synthesized vaccinia vector early promoter PE6 and lacZ fusion fragment were ligated to T-easy universal sequencing vector to obtain plasmid pT-lacZ' -PE6, and the sequencing results were in accordance with the expected design (SEQ ID NO: 9).

3. The vaccinia virus transfer vector pVTT1.0 was obtained.

The plasmid pT-lacZ '-PE 6 prepared above was digested with SmaI + HindIII, and a 0.4kb lacZ' -PE6 fragment was recovered; was ligated to the plasmid pSC-neo to obtain the vaccinia virus transfer vector pVTT1.0 (see FIG. 1 for the construction process). The results of KpnI, NdeI and EcoRV are shown in FIG. 3.

Example 2: codon optimization of nucleocapsid protein and protuberant protein coding sequence of SARS-CoV and construction of DNA vaccine:

1. optimizing genetic codons. The most preferred genetic codon was selected based on the frequency of genetic codon usage in the human genetic codon preference table and reverse translated back into nucleotide sequences based on the amino acid sequences of the nucleocapsid protein and the protuberant protein of SARS-CoV (see amino acid sequence of isolate HKU-39849, a hong Kong strain of SARS-CoV published by Genebank).

2. Modification and adjustment of gene sequences. Minor modifications and modifications, use of the degeneracy of the genetic code to eliminate redundant restriction enzyme recognition sites, eliminate potential nucleic acid secondary structures, etc., sequences that are detrimental to gene synthesis.

3. Artificially synthesizing target genes. Genes were synthesized by overlap PCR. The nucleocapsid protein and protuberant protein gene sequences are firstly listed respectively as positive strand and negative strand, then the positive strand and negative strand sequences are divided into oligonucleotides with the length of 50bp (the most 5' -end of the two strands is about 25bp in length) according to the gene length, and except the sequences at the two ends, each positive strand and negative strand sequence are complementary with the oligonucleotides of the two complementary strands by about 25 bp. Every 10 or so oligonucleotides are mixed with the same number of complementary oligonucleotides to form an annealing system, and the mixture is heated in a PCR tube and then slowly annealed. And performing PCR amplification by using the annealing product as a template and paired upstream and downstream primers to obtain a gene fragment of about 500 bp. The multiple overlapped fragments are mixed, then heated and annealed, an annealing system is taken as a template, and primers at two ends are used for PCR amplification, so that a longer gene fragment can be obtained. The fragments with the length of more than 1Kb are spliced by PCR through a preset enzyme cutting site to obtain a full-length gene sequence.

4. The synthesized target genes encoding the nucleocapsid protein (N) and the protuberant protein (S) of SARS-CoV were ligated into the pSK universal sequencing vector and the sequencing results were in accordance with the expected design (the target gene sequences of N and S of codon optimized SARS-CoV are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively). Obtaining the plasmid pSK-S, and storing the plasmid pSK-S in China general microbiological culture Collection center (CGMCC) at 9/19 th 2005, wherein the preservation number is as follows: CGMCC No. 1457. The plasmid pSK-N is preserved in China general microbiological culture Collection center (CGMCC) at 9/19 th 2005, and the preservation number is as follows: CGMCC No. 1459.

Then, the plasmids pSK-S and pSK-N were digested simultaneously with SmaI + SalI, and the resulting polynucleotides encoding the nucleocapsid protein (NC) and the protuberant protein (S) of SARS-CoV were ligated with the SmaI + SalI digested DNA vaccine vector pDRVSV1.0 (Chinese patent application: 200410028280.3), to obtain the DNA vaccines pDRVSV1.0-S and pDRVSV1.0-N of SARS-CoV (FIG. 13).

Example 3: construction of target gene expression element and vaccinia virus transfer vector

PCR amplification fusion promoter PE/L + P7.5 sequence

Designing a primer:

p7.5 primer 1: 5'-GAAGATCTGTCGACTTCGAGCTTATTT-3' (SEQ ID NO: 3);

PE/L primer 2: 5'-GAGAATTCGTTTAAACCGATGC-3' (SEQ ID NO: 4)

The pE/L + p7.5PCR amplification reaction adopts a kit of Dalianbao bioengineering GmbH, and the reaction system is as follows:

1. mu.l of plasmid pSC65 (preservation number of plasmid pSC 65: CGMCC No. 1097.), 1. mu.l of each of the forward and reverse primers (P7.5 primer 1, PE/L primer 2), 5. mu.l of 10 XPyrobest buffer, 5. mu.l of dNTP mix (2.5 mM each), 0.5. mu.l of Pyrobest DNA polymerase (5U/ml), ddH₂O37.5. mu.l. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30 s; 7min at 72 ℃; 4 ℃ is prepared.

The extension product of pE/L + p7.5PCR amplification reaction was purified and recovered using the E.Z.N.Acycle-Pure Kit from Omega.

PCR amplification of nucleocapsid protein (NC) coding sequence of SARS-CoV

Designing a primer:

NC primer 1: 5'-CATCGGTTTAAACGAATTCTCACCATGAGCGATAATGGCCC-3' (SEQ ID NO: 5);

NC primer 2: 5' -CCGGATCCTTATCAGGCCTGTGTAGAATC-3’(SEQ ID NO：6)

The NC gene PCR amplification reaction of SARS-CoV adopts the reagent box of Dalianbao biological engineering company Limited, and the reaction system is as follows:

1. mu.l of plasmid pSK-N (preservation number of plasmid pSK-N: CGMCC No.1459), 1. mu.l of each of the forward and reverse primers (NC primer 1, NC primer 2), 5. mu.l of 10 XPyrobest buffer, 5. mu.l of dNTP mix (2.5 mM each), 0.5. mu.l of Pyrobest DNA polymerase (5U/ml), ddH₂O 37.5μl。

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 1 min; 7min at 72 ℃; 4 ℃ is prepared.

The extension product of NC PCR amplification reaction was purified and recovered using the E.Z.N.A. Cycle-Pure Kit from Omega (nucleocapsid protein coding sequence is shown in SEQ ID NO: 1).

PCR fusion amplification promoter PE/L + P7.5 and nucleocapsid protein coding sequence of SARS-CoV

The reaction system is as follows:

mu.l of template recovered by pE/L + p7.5PCR reaction, 5. mu.l of template recovered by SARS-CoV nucleocapsid protein-encoding gene PCR reaction, 1. mu.l each of forward and reverse primers (P7.5 primer 1 and NC primer 2), 5. mu.l of 10 XPyrobest buffer, 5. mu.l of dNTP mix (2.5 mM each), 0.5. mu.l of Pyrobest DNA polymerase (5U/ml), ddH₂O 37.5μl。

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 1min for 30 s; 7min at 72 ℃; 4 ℃ is prepared.

The products of the fusion PCR amplification of the nucleocapsid coding sequences of PE/L + P7.5 and SARS-CoV were recovered by purification using the Omega E.Z.N.A. Cycle-Pure Kit, and the results are shown in FIG. 4. The product is connected with a T-easy universal vector of Promega company to obtain a T-NC plasmid, and the sequencing result is correct. The restriction enzyme digestion analysis and identification are carried out by using corresponding restriction enzymes, and the restriction enzyme digestion identification results are respectively shown in figure 5.

Obtaining S, NC Gene expression elements of SARS-CoV

The plasmid pSK-S (the preservation number of the plasmid pSK-S is CGMCC No.1457) vector artificially synthesizing the protuberant protein coding sequence (SEQ ID NO: 2) of SARS-CoV is connected by double digestion of SalI and SacI, the protuberant protein coding sequence of SARS-CoV obtained by digestion is connected to the T-NC plasmid treated by double digestion of SalI and SacI, and the plasmid T-NC + P + S with S, NC gene expression elements of SARS-CoV is obtained (the construction process is shown in figure 2). Extracting plasmid, and performing enzyme digestion analysis and identification with corresponding restriction enzymes, wherein the enzyme digestion identification results are shown in FIG. 6.

Construction of S, NC Gene vaccinia Virus transfer vector plasmid pVTT-NS of SARS-CoV

The plasmid T-NC + P + S carrying the expression element of the target gene is cut by SpeI enzyme to fill in and SacII enzyme to cut. The digested fragment of S, NC gene expression element of SARS-CoV was recovered by using Omega gel recovery kit, and then ligated to SmaI and SacII double digested vaccinia virus universal transfer vector pVTT1.0 to obtain vaccinia virus transfer vector plasmid pVTT-NS carrying S, NC gene expression element of SARS-CoV (see FIG. 2 for the construction process). Extracting plasmid, and performing enzyme digestion analysis and identification with corresponding restriction enzymes, wherein the enzyme digestion identification results are shown in FIG. 9.

Example 4: construction and screening of Tiantan strain recombinant vaccinia virus vaccine rVTT-NS containing nucleotide sequence of nucleocapsid protein and protuberant protein of SARS-CoV

The vaccinia virus Tiantan strain is used for infecting 80 percent of chicken embryo cells CEF with the amount of 0.1-0.01 pfu/cell virus, after the cells are adsorbed for 1-1.5 h, a liposome transfection technology (Lipofectin kit of INVITROGEN) is adopted to transfect recombinant plasmid pVTT-NS into the CEF cells, so that the S, NC gene expression element of SARS-CoV, the neo gene and the lacZ gene double selection marker are homologously recombined into the TK region sequence of the vaccinia virus genome DNA. The first three rounds of recombinant vaccinia virus selection were performed by pressure screening at 400ug/ml G418, followed by spot-plating with X-gal and neutral red-loaded low melting agarose, to select blue recombinant vaccinia virus containing both the desired gene and the selection marker (the wild strain that did not undergo recombination was inhibited by the presence of G418). Then under the pressure selection without antibiotic G418, the blue recombinant vaccinia virus can generate intramolecular homologous recombination between a small lacZ' segment of about 200bp at the upstream of S, NC gene expression element of SARS-CoV of the transferred plasmid and the complete lacZ gene, thereby losing neo gene and lacZ gene to obtain the recombinant vaccinia virus only containing S, NC gene expression element of SARS-CoV, and the white recombinant virus only containing the target gene can be picked up by spreading the spot with low melting point agarose added with X-gal and neutral red. The white spot virus obtained by primary screening is purified by five rounds of single spots to obtain the single clone rVTT-NS of the recombinant vaccinia virus vaccine containing the S, NC gene expression element of SARS-CoV.

Example 5: PCR and Western blot detection of rVTT-NS passage stability

randomly picking 9 sixth-generation leucoviruses after downward passage of rVTT-NS, extracting a virus DNA template (virus genome extraction kit by Wettel Biotechnology of Hangzhou), and amplifying NC target genes by using NC primers 1 and 2 (the primer sequence and the method refer to example 3); the primer sequence is represented by S primer 1: 5'-CTCTACGTAGCGGCCGCTAACCATGTTTATCTTTCTGCTG-3' (SEQ ID NO: 7); and S primer 2: 5' -TCCCCCGGGTTATCAGGTGTAG-3' (SEQ ID NO: 8) is used as a primer to amplify the target gene of S, and the reaction system is as follows: 5. mu.l of rVTT-NS DNA; 1. mu.l of each of the forward and reverse primers (S primer 1 and S primer 2); 10X Pyrobest buffer 5. mu.l; dNTP mix (2.5 mM each) 5. mu.l; LA DNA polymerase (5U/ml) 0.5. mu.l; ddH₂O 32.5μl。

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 3 min; 7min at 72 ℃; 4 ℃ is prepared.

Agarose gel electrophoresis shows that positive bands are amplified in randomly picked virus genomes, and the NC gene is 1.2 kb; the S gene was 3.6 kb. As shown in fig. 6 and 7.

Infection of CEF cells by rVTT-NS in the fifth generation, harvesting of the cells and supernatant after 48 hours, Western Blot analysis by using human multi-antiserum (provided by the research of the first-pass science), and appearance of a specific positive reaction band, wherein the NC protein is 4.4 KDa; the S protein is 120KDa, which indicates that the constructed rVTT-NS vaccine can stably express the target gene, and is shown in figure 10.

Example 6: Prime-Boost immunization experiments using DNA vaccines containing NC and S coding sequences of SARS-CoV and vaccinia virus vaccine rVTT-NS

1. DNA vaccines containing NC and S coding sequences of SARS-CoV and Prime-Boost immunization strategy for the Tiantan vaccinia virus vaccine rVTT-NS.

In this example, BALB/c (H-2d) female mice (weighing 19-25 g, purchased from the Chinese drug biologicals institute) 6-8 weeks old were used to test the efficacy of the vaccines of the present invention. The DNA vaccine of example 2 containing NC and S target genes of SARS-CoV was prepared into 1mg/ml injection solution using 1 XPBS. Recombinant vaccinia virus rVTT-NS 1X 10⁸pfu/mL. Groups of 4 mice were immunized, 6 mice each. The vaccination strategy for each immunization group is shown in table 1. The DNA vaccine was injected into tibialis anterior at 100 ug/mouse/dose (50 ug per hind limb). The rVTT-NS recombinant vaccinia virus dose is 10⁷pfu/mouse/time. Immunodetection was performed at week 10. For the control, pCDNA empty vector and vaccinia virus Tiantan strain without the target gene was used.

TABLE 1 Prime-Boost immunization protocol against DNA vaccine of SARS-CoV and against the Tiantan vaccinia virus vaccine rVTT-NS:

ELISPOT detects the T lymphocyte reaction of secreting IFN-gamma after in vitro antigen epitope peptide stimulation.

The ELISPOT experiment for detecting IFN-gamma adopts a kit of U-CyTech company in the Netherlands, and the specific procedures refer to the use instruction of the U-CyTech company. The stimulating polypeptide is 16 peptides of S protein (S1, VFNATKFPSVYAWERKKI; S2, SVYAWERKKISNCVADY; S3, STFFSTFKCYGVSATKL; S4, KCYGVSATKLNDLCFSNV; S5, NIDDSTGNYNYKYRYLR; S6, NYNYKYRYLRHGKLRPF; S7, RASANLAATKMSECVL; S8, AATKMSECVLSKRVDF; S9, LMSFPQAAPHGVGVFLHV; S10, APHGVVFLHVTYVPSQER) and 1 peptide of NC protein (N1, QIGYYRRATRRVRGGDGK). The results show that initial immunization with single or double needle SARS-CoV (NC and S gene) DNA vaccine, followed by booster immunization of mice with the inventive vaccinia virus vaccine against SARS-CoV, induced a high level of T lymphocyte immune response in the body, and the specific results are shown in FIG. 11.

3. Detection of specific anti-SARS-CoV nucleocapsid protein and protuberant protein IgG-binding antibodies in the serum of immunized mice

To test the specific humoral immunity levels induced by the individual immunization and booster immunization of SARS-CoV recombinant vaccinia virus, mouse sera were taken at week 10, the ELISA plates were coated with SARS-CoV nucleocapsid protein and protuberant protein antigen (the gift from the national institute of health and vaccine research center), and the levels of SARS-CoV nucleocapsid protein and protuberant protein specific IgG antibodies were tested by indirect ELISA. The SARS-CoV nucleocapsid protein and protuberin-specific IgG antibody titers for each of the immunization groups are presented in FIG. 12.

4.SARS-CoV neutralizing antibody titers in the sera of immunized mice.

Neutralizing antibody assays were performed on the sera of immunized mice using a plaque reduction neutralization assay to evaluate their immune efficacy. Vero-E6 cells were used to inoculate 2-well plastic cell culture plates, two layers of agar-containing medium were added. The plaque test is established with neutral red as staining agent. Neutralizing antibodies against SARS-CoV BJ 01 strain were determined using plaques that were 50% reduced. The results of the tests are shown in Table 2.

Table 2 experimental groups the sera of the animals neutralize the titer of SARS-CoV antibodies in vitro:

immunization test group	Neutralizing antibody titer
		Negative control group	＜1∶20
Two-needle SARS-CoV NC + S DNA vaccine group	1∶446
		Single-needle SARS-CoV NC + S vaccinia rVTT-NS group	1∶1280
Double-needle SARS-CoV NC + S vaccinia rVTT-NS group	1∶1807
		Single needle vaccinia rVTT-NS group	1∶181

The above examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or essential attributes thereof, and accordingly, such modifications and variations are also within the scope of the invention as defined in the appended claims.

Sequence listing

<110> Chinese disease prevention and control center

Prevention and control center for venereal disease and AIDS

<120> SARS vaccine based on replicative vaccinia virus vector

<130>I200501573CB

<160>9

<170>PatentIn version 3.2

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gccaccatga gcgataatgg cccccagagc aaccagagaa gcgcccccag aatcacattt 60

ggcggcccta ccgacagcac cgacaacaat cagaacggcg gcagaaatgg cgccagaccc 120

aagcagagga gacctcaggg cctgcccaat aataccgcca gctggttcac agccctgaca 180

cagcacggaa aggaggagct gagattccct agaggccagg gcgtgcccat caataccaac 240

agcggccctg acgatcagat cggctactac cggagggcca ccagaagagt gagaggcggc 300

gacggcaaga tgaaggagct gagcccccgg tggtactttt actacctggg caccggacct 360

gaagccagcc tgccttacgg cgccaataag gagggcattg tgtgggtggc cacagagggc 420

gccctgaaca cccctaagga ccacatcggc accaggaacc ccaacaacaa tgccgccacc 480

gtgctgcagc tgcctcaggg aaccacactg cccaagggct tttacgccga gggcagcaga 540

ggaggatctc aggccagcag caggagcagc agcagaagca ggggcaacag cagaaatagc 600

acccccggca gcagcagagg aaatagcccc gccagaatgg cctctggcgg aggagagaca 660

gccctggccc tgctgctgct ggacagactg aatcagctgg agagcaaggt gagcggaaag 720

ggacagcagc agcagggaca gaccgtgaca aagaagtctg ccgccgaggc ctctaagaag 780

ccccggcaga agagaacagc cacaaagcag tacaacgtga cccaggcctt tggcagaaga 840

ggccctgagc agacccaggg caacttcggc gaccaggacc tgatcagaca gggcaccgac 900

tacaagcact ggcctcagat cgcccagttt gccccttctg ccagcgcctt cttcggcatg 960

agccggatcg gcatggaggt gaccccttct ggcacctggc tgacatacca cggcgccatc 1020

aagctggacg acaaggaccc ccagttcaag gacaacgtga tcctgctgaa caagcacatc 1080

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gaggcccagc ctctgcctca gagacagaag aagcagccca ccgtgacact gctgcctgcc 1200

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tctacacagg cctgataacc cgggggattc ccg 1293

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cgatctggat cgctgtacca cctttgatga tgtgcaggcc cctaattaca cccagcacac 120

cagcagcatg cgcggcgtgt actaccctga tgaaatcttt cgcagcgata ccctgtacct 180

gacccaggat ctgtttctgc ctttttacag caatgtgacc ggctttcaca ccatcaatca 240

cacctttggc aatcctgtga tcccttttaa ggatggcatc tactttgccg ccaccgagaa 300

gagcaatgtg gtgcgcggct gggtgtttgg cagcaccatg aataataaga gccagagcgt 360

gatcatcatc aataatagca ccaatgtggt gatccgcgcc tgtaattttg agctgtgtga 420

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taatgccttt aattgtacct ttgagtacat cagcgatgcc tttagcctgg atgtgagcga 540

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cctgaccgcc tttagccctg cccaggatat ctggggcacc agcgccgccg cctactttgt 780

gggctacctg aagcctacca cctttatgct gaagtacgat gagaatggca ccatcaccga 840

tgccgtggat tgtagccaga atcctctggc cgagctgaag tgtagcgtga agagctttga 900

gatcgataag ggcatctacc agaccagcaa ttttcgcgtg gtgcctagcg gcgatgtggt 960

gcgctttcct aatatcacca atctgtgtcc ttttggcgag gtgtttaatg ccaccaagtt 1020

tcctagcgtg tacgcctggg agcgcaagaa gatcagcaat tgtgtggccg attacagcgt 1080

gctgtacaat agcacctttt ttagcacctt taagtgttac ggcgtgagcg ccaccaagct 1140

gaatgatctg tgttttagca atgtgtacgc cgatagcttt gtggtgaagg gcgatgatgt 1200

gcgccagatc gcccctggcc agaccggcgt gatcgccgat tacaattaca agctgcctga 1260

tgattttatg ggctgtgtgc tggcctggaa tacccgcaat atcgacgcca ccagcaccgg 1320

caattacaat tacaagtacc gctacctgcg ccacggcaag ctgcgccctt ttgagcgcga 1380

tatcagcaat gtgcctttta gccctgatgg caagccttgt acccctcctg ccctgaattg 1440

ttactggcct ctgaatgatt acggctttta caccaccacc ggcatcggct accagcctta 1500

ccgcgtggtg gtgctgagct ttgagctgct gaatgcccct gccaccgtgt gtggccctaa 1560

gctgagcacc gatctgatca agaatcagtg tgtgaatttt aattttaatg gcctgaccgg 1620

caccggcgtg ctgaccccta gcagcaagcg ctttcagcct tttcagcagt ttggccgcga 1680

tgtgagcgat tttaccgata gcgtgcgcga tcctaagacc agcgagatcc tggatatcag 1740

cccttgtagc tttggcggcg tgagcgtgat cacccctggc accaatgcca gcagcgaggt 1800

ggccgtgctg taccaggatg tgaattgtac cgatgtgagc accgccatcc acgccgatca 1860

gctgacccct gcctggcgca tctacagcac cggcaataat gtgtttcaga cccaggccgg 1920

ctgtctgatc ggcgccgagc acgtggatac cagctacgag tgtgatatcc ctatcggcgc 1980

cggcatctgt gccagctacc acaccgtgag cctgctgcgc agcaccagcc agaagagcat 2040

cgtggcctac accatgagcc tgggcgccga tagcagcatc gcctacagca ataataccat 2100

cgccatccct accaatttta gcatcagcat caccaccgag gtgatgcctg tgagcatggc 2160

caagaccagc gtggattgta atatgtacat ctgtggcgat agcaccgagt gtgccaatct 2220

gctgctgcag tacggcagct tttgtaccca gctgaatcgc gccctgagcg gcatcgccgc 2280

cgagcaggat cgcaataccc gcgaggtgtt tgcccaggtg aagcagatgt acaagacccc 2340

taccctgaag tactttggcg gctttaattt tagccagatc ctgcctgatc ctctgaagcc 2400

taccaagcgc agctttatcg aggatctgct gtttaataag gtgaccctgg ccgatgccgg 2460

ctttatgaag cagtacggcg agtgtctggg cgatatcaat gcccgcgatc tgatctgtgc 2520

ccagaagttt aatggcctga ccgtgctgcc tcctctgctg accgatgata tgatcgccgc 2580

ctacaccgcc gccctggtga gcggcaccgc caccgccggc tggacctttg gcgccggcgc 2640

cgccctgcag atcccttttg ccatgcagat ggcctaccgc tttaatggca tcggcgtgac 2700

ccagaatgtg ctgtacgaga atcagaagca gatcgccaat cagtttaata aggccatcag 2760

ccagatccag gagagcctga ccaccaccag caccgccctg ggcaagctgc aggatgtggt 2820

gaatcagaat gcccaggccc tgaataccct ggtgaagcag ctgagcagca attttggcgc 2880

catcagcagc gtgctgaatg atatcctgag ccgcctggat aaggtggagg ccgaggtgca 2940

gatcgatcgc ctgatcaccg gccgcctgca gagcctgcag acctacgtga cccagcagct 3000

gatccgcgcc gccgagatcc gcgccagcgc caatctggcc gccaccaaga tgagcgagtg 3060

tgtgctgggc cagagcaagc gcgtggattt ctgtggcaag ggctaccacc tgatgagctt 3120

tcctcaggcc gcccctcacg gcgtggtgtt tctgcacgtg acctacgtgc ctagccagga 3180

gcgcaatttt accaccgccc ctgccatctg tcacgagggc aaggcctact ttcctcgcga 3240

gggcgtgttt gtgtttaatg gcaccagctg gtttatcacc cagcgcaact tctttagccc 3300

tcagatcatc accaccgata atacctttgt gagcggcaat tgtgatgtgg tgatcggcat 3360

catcaataat accgtgtacg atcctctgca gcctgagctg gatagcttta aggaggagct 3420

ggataagtac tttaagaatc acaccagccc tgatgtggat ctgggcgata tcagcggcat 3480

caatgccagc gtggtgaata tccagaagga gatcgatcgc ctgaatgagg tggccaagaa 3540

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gccttggtac gtgtggctgg gctttatcgc cggcctgatc gccatcgtga tggtgaccat 3660

cctgctgtgt tgtatgacca gctgttgtag ctgtctgaag ggcgcctgta gctgtggcag 3720

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agcgggtggg tttggaatga tgtaaagctt aaaaattgaa attttatttt ttttttttgg 180

aatataaata agctcgaagt cgacgcccaa ctggtaatgg tagcgaccgg cgctcagctg 240

gaattccgcc gatactgacg ggctccagga gtcgtcgcca ccaatcccca tatggaaacc 300

gtcgatattc agccatgtgc cttcttccgc gtgcagcaga tggcgatggc tggtttccat 360

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gccatgttt 429

Claims (9)

1. A vaccine against SARS-CoV based on a replicative vaccinia virus comprising a replicative vaccinia virus as vector wherein the Thymidine Kinase (TK) region of the vaccinia virus genome is inserted with a polynucleotide encoding the SARS-CoV nucleocapsid protein and the protuberant protein and the replicative vaccinia virus is a vaccinia virus day jar strain.

2. The vaccine of claim 1, wherein said vaccine does not contain a selectable marker gene.

3. The vaccine of claim 1 or 2, wherein said polynucleotide is codon-optimized for high efficiency expression in mammalian cells.

4. The vaccine of claim 3, wherein the polynucleotide encoding SARS-CoV nucleocapsid protein has the amino acid sequence as set forth in SEQ ID NO: 1 and/or the polynucleotide encoding the SARS-CoV protuberant protein has the nucleotide sequence shown in SEQ ID NO: 2.

5. The vaccine of any one of claims 1-4, further comprising a pharmaceutically acceptable adjuvant and/or carrier.

6. Use of a vaccine according to any one of claims 1 to 5 in the preparation of a vaccine composition for the prevention of SARS-CoV infection.

7. An immunization kit comprising the vaccine of any one of claims 1-5.

8. The kit of claim 7, further comprising one or more DNA vaccines against SARS-CoV.

9. A universal transfer plasmid pVTT1.0 of vaccinia virus, which is preserved in China general microbiological culture Collection center (CGMCC) at 9/19/2005 with the preservation number of CGMCC No. 1458.