CN116200349A - Novel coronavirus vaccine based on improved vaccinia virus Tiantan strain - Google Patents

Novel coronavirus vaccine based on improved vaccinia virus Tiantan strain Download PDF

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CN116200349A
CN116200349A CN202310095863.0A CN202310095863A CN116200349A CN 116200349 A CN116200349 A CN 116200349A CN 202310095863 A CN202310095863 A CN 202310095863A CN 116200349 A CN116200349 A CN 116200349A
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recombinant
gene
vaccinia virus
virus
nucleotide sequence
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郭斐
许丰雯
范张玲
梅珊
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Institute of Pathogen Biology of CAMS
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Abstract

The present invention provides a recombinant vaccinia virus that lacks the TK region or both the TK region and the B2R gene encoding the poxin protein as compared to the wild-type vaccinia virus Tiantan strain and that comprises an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK region. Also provided are novel coronavirus vaccine compositions comprising the recombinant vaccinia virus and a pharmaceutically acceptable carrier, and the use of the recombinant vaccinia virus for preparing vaccine compositions for preventing novel coronavirus SARS-CoV-2 infection in a subject. The invention also provides methods of making recombinant vaccinia viruses.

Description

Novel coronavirus vaccine based on improved vaccinia virus Tiantan strain
Technical Field
The invention relates to the field of vaccine preparation, in particular to a novel coronavirus vaccine based on an improved vaccinia virus Tiantan strain.
Background
Vaccinia virus (vaccina virus, VACV) is a double stranded DNA virus, about 200kbp in genome, a vaccine strain of smallpox, used for the 20 th century for vaccination of smallpox, and eventually succeeded in eradicating smallpox. The vaccinia virus Tiantan strain independently developed in China plays an important role in the process of eliminating smallpox. The vaccinia virus has wide host range and large exogenous gene capacity, and the life cycle of the vaccinia virus is completed in host cytoplasm without the risk of integrating genes into host cells, and the vaccinia virus has been used as a vaccine strain for preventing infectious diseases and treating tumors at present.
There are a variety of pattern recognition receptors (pattern recognition receptor, PRR) that recognize pathogens in mammalian cells and activate downstream natural immune signaling pathways by binding to pathogen-associated pattern molecules (pathen-associated molecular patterns, PAMP) such as DNA, RNA, etc. of the pathogen, producing interferons and inflammatory factors, combating microbial infection and promoting adaptive immune responses. cGAS (Cyclic GMP-AMP Synthase) is an intracellular DNA receptor that, upon binding to viral DNA, activates its enzymatic activity, synthesizes 2',3' -cGAMP, which binds to STING (Stimulator of Interferon Genes, an interferon gene stimulator), down-transfers an activation signal, recruits TBK1 (TANK Binding Kinase 1), phosphorylates the latter to form p-TBK1, and further exerts kinase activity, phosphorylates IRF3 (Interferon regulatory Factor 3), forms p-IRF3, ultimately inducing expression of factors such as type I interferon. The vaccinia virus B2R gene encodes a protein poxin which specifically degrades intracellular 2',3' -cGAMP, thereby blocking the native immune activation signal induced by intracellular cGAS.
The novel coronavirus (SARS-CoV-2) is an enveloped single-stranded positive-stranded RNA virus. Novel coronaviruses belong to the genus β of the family coronaviridae and have so far remained pathogens threatening public health worldwide.
The spike protein (S) on the surface of the novel coronavirus binds to angiotensin converting enzyme 2 (ACE-2, angiotensin-converting enzyme) on the surface of the host cell, and mediates the virus to enter the host cell and then replicate in the host cell, thereby causing the disease. Thus, vaccines designed based on the novel coronavirus S protein are currently an important means of preventing infection by the novel coronavirus.
Disclosure of Invention
The invention aims to provide a novel coronavirus vaccine based on an improved vaccinia virus Tiantan strain.
In a first aspect, the invention provides a recombinant vaccinia virus that lacks the gene encoding thymidine kinase (thymidylate kinase, TK) or both the gene encoding TK and the B2R gene encoding poxin protein, and that comprises an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK encoding gene, as compared to the wild-type vaccinia virus Tiantan strain.
In a second aspect, the invention provides a novel coronavirus vaccine composition comprising the recombinant vaccinia virus of the first aspect and a pharmaceutically acceptable carrier.
In a third aspect, the invention provides the use of the recombinant vaccinia virus of the first aspect for the preparation of a vaccine composition for preventing a novel coronavirus SARS-CoV-2 infection in a subject.
In a fourth aspect, the invention provides a method of making a recombinant vaccinia virus comprising:
(1) Constructing a recombinant plasmid containing an upstream and downstream homology arm of a poxin protein coding gene B2R;
(2) Homologous recombination is carried out on the wild vaccinia virus Tiantan strain and the recombinant plasmid constructed in the step (1), and recombinant virus is obtained after purification, and the recombinant virus lacks B2R genes;
(3) Constructing a recombinant plasmid containing an upstream and downstream homology arm of TK coding gene and a nucleotide sequence for coding SARS-CoV-2 spike protein S;
(4) Homologous recombination is carried out on the wild vaccinia virus Tiantan strain and the recombinant plasmid constructed in the step (3), and recombinant virus is obtained after purification, wherein the recombinant virus lacks TK gene and contains inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK gene; alternatively, the recombinant virus obtained in the step (2) and the recombinant plasmid constructed in the step (3) are subjected to homologous recombination, and purified to obtain a recombinant virus which lacks both the TK gene and the B2R gene and contains an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK gene.
In a fifth aspect, the invention provides a recombinant vaccinia virus prepared by the method of the fourth aspect.
In a sixth aspect, the invention provides a recombinant vaccinia virus that lacks the B2R gene encoding a poxin protein as compared to a wild-type vaccinia virus Tiantan strain.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses an optimized vaccinia virus vector, which is prepared by transfecting recombinant plasmids with homologous arms on the upstream and downstream of a poxin protein coding gene B2R on the basis of independently developed smallpox vaccine strain vaccinia virus Tiantan strain in China, and carrying out recombination and screening processes to obtain recombinant virus delB2R with B2R gene deletion, wherein the recombinant virus with B2R deletion can induce a higher level of natural immune response. The recombinant virus is used as a vaccine vector, recombinant virus TKB2-S with a novel coronal S protein coding sequence introduced at a TK gene position is obtained through the processes of recombination and screening by transfecting recombinant plasmids with novel coronal spike protein S coding genes and TK gene homology arms, and the expression of the novel coronal S protein can be detected after the recombinant virus infects cells. Meanwhile, by using wild vaccinia virus as a control and transfecting recombinant plasmid with novel coronal spike protein S coding gene and TK gene homology arm, recombinant virus TK-S is constructed. Subsequently, the mice were immunized with recombinant viruses dTK, dTKB2, TK-S, TKB2-S, respectively. Higher S-specific antibodies and neutralizing antibody titers were detected in the mouse serum by TKB2-S group than by TK-S. The invention optimizes the vaccine vector of the vaccinia virus Tiantan strain, and the constructed novel coronavirus vaccine based on the improved vaccinia virus vector can better induce the immune response of organisms to the novel coronavirus S protein carried by the virus vector.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below. It is apparent that the drawings in the following description are only specific examples or the results thereof recited in the present application. Other embodiments and their results will be apparent to those of ordinary skill in the art from the drawings.
FIG. 1 shows a schematic diagram of the homologous recombination between the recombinant plasmid pdelB2R and the wild vaccinia virus provided in example 1 of the present invention; HR: homologous recombination.
FIG. 2 shows the positions of the PCR primers B2Rup-F and B2Rdown-R binding to the recombinant virus delB2R and the wild-type virus template in example 1 of the present invention.
FIG. 3 shows an electropherogram after PCR using primers B2Rup-F and B2Rdown-R, where N is a negative control, WT is wild vaccinia Tiantan strain, delB2R is a recombinant virus.
FIG. 4 shows the level of the recombinant virus delB2R provided in example 1 of the present invention inducing a natural immune response, wherein Mock is a blank control and WT is a wild vaccinia virus Tiantan strain.
FIG. 5 shows a schematic representation of the recombinant plasmid pTK-S provided in example 2 of the invention and the homologous recombination with wild-type vaccinia virus; HR: homologous recombination.
FIG. 6 shows the positions of the PCR primers J1RJ3R-F and J1RJ3R-R binding to the recombinant virus TK-S and the wild-type virus template in example 2 of the present invention.
FIG. 7 shows an electrophoretogram after PCR using primers B2Rup-F and B2Rdown-R, and J1RJ3R-F and J1RJ3R-R, wherein A is dTK, B is TK-S, C is dTKB2, D is TKB2-S, and E is wild-type virus.
FIG. 8 shows the detection of SARS-CoV-2S protein expression by Western Blot in example 2 of the present invention, wherein A is wild-type virus, B is dTK, C is TK-S, D is dTKB2, and E is TKB2-S.
FIG. 9 shows the results of detection of S antibodies in post-immunization sera of mice provided in example 3 of the present invention, wherein dTK-1st is the post-primary-immunization mice serum of control virus dTK, dTK-2nd is the post-secondary-immunization mice serum of control virus dTK, dTKB2-1st is the post-primary-immunization mice serum of control virus dTKB2, dTKB2-2nd is the post-secondary-immunization mice serum of control virus dTKB2, TK-S-1st is the post-primary-immunization mice serum of recombinant vaccinia virus TK-S, TKB2-S-1st is the post-primary-immunization mice serum of recombinant vaccinia virus TKB2-S, TKB2-S-2nd is the post-secondary-immunization mice serum of recombinant vaccinia virus TKB2-S.
FIG. 10 shows the results of the detection of neutralizing antibodies in serum after immunization of mice provided in example 3 of the present invention, wherein dTK is the serum of mice after secondary immunization with control virus dTK, dTKB2 is the serum of mice after secondary immunization with control virus dTKB2, TK-S is the serum of mice after secondary immunization with recombinant vaccinia virus TK-S, TKB2-S is the serum of mice after secondary immunization with recombinant vaccinia virus TKB2-S.
Detailed Description
The present invention provides a recombinant vaccinia virus which lacks the TK gene or both the TK gene and the B2R gene encoding the poxin protein, and which comprises an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deletion of the TK gene, as compared to the wild-type vaccinia virus Tiantan strain.
In a specific embodiment, the nucleotide sequence encoding SARS-CoV-2 spike protein S is as set forth in SEQ ID NO: 1.
In a specific embodiment, the deleted TK gene is the deleted SEQ ID NO: 9; deletion of B2R gene is deletion of SEQ ID NO:10, and a nucleotide sequence shown in seq id no.
The invention also provides a novel coronavirus vaccine composition comprising the recombinant vaccinia virus and a pharmaceutically acceptable carrier.
The invention also provides the use of said recombinant vaccinia virus or said cell for the preparation of a vaccine composition for preventing a novel coronavirus SARS-CoV-2 infection in a subject;
preferably, the subject is a human.
The invention also provides a method of making a recombinant vaccinia virus comprising:
(1) Constructing a recombinant plasmid containing an upstream and downstream homology arm of a poxin protein coding gene B2R;
(2) Homologous recombination is carried out on the wild vaccinia virus Tiantan strain and the recombinant plasmid constructed in the step (1), and recombinant virus is obtained after purification, and the recombinant virus lacks B2R genes;
(3) Constructing a recombinant plasmid containing an upstream and downstream homology arm of TK gene and a nucleotide sequence for encoding SARS-CoV-2 spike protein S;
(4) Homologous recombination is carried out on the wild vaccinia virus Tiantan strain and the recombinant plasmid constructed in the step (3), and recombinant virus is obtained after purification, wherein the recombinant virus lacks TK gene and contains inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK gene; alternatively, the recombinant virus obtained in the step (2) and the recombinant plasmid constructed in the step (3) are subjected to homologous recombination, and purified to obtain a recombinant virus which lacks both the TK gene and the B2R gene and contains an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK gene.
In a specific embodiment, the recombinant plasmid constructed in step (1) further comprises a vaccinia virus promoter; preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO: 2.
In a specific embodiment, wherein the recombinant plasmid constructed in step (3) further comprises a vaccinia virus promoter; preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO: 3.
The invention also provides recombinant vaccinia viruses prepared by the methods.
The invention also provides a novel coronavirus vaccine composition comprising the recombinant vaccinia virus prepared by the method and a pharmaceutically acceptable carrier.
The invention also provides the use of the recombinant vaccinia virus prepared by the method for preparing a vaccine composition for preventing novel coronavirus SARS-CoV-2 infection.
The invention also provides a recombinant vaccinia virus which lacks the B2R gene encoding the poxin protein compared to the wild-type vaccinia virus Tiantan strain.
In a specific embodiment, the deletion B2R gene is a deletion of SEQ ID NO: 1O.
Examples
In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in further detail with reference to examples.
HEK293T, vero, heLa cells used in the present invention were cultured with DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin at 37℃with 5% CO 2 Culturing in incubator, and passaging every 2-3 days. THP1 cells used in the present invention were cultured with 1640 medium containing 10% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin at 37℃and 5% CO 2 Culturing in incubator, and passaging every 2-3 days.
The whole genome sequence of the wild vaccinia virus Tiantan strain is stored in an international gene bank, and the number is GenBank No. AF095689. The person skilled in the art can obtain wild-type vaccinia virus Tiantan strain by conventional means. In the following examples, wild type vaccinia virus Tiantan strain was offered by the institute of technology at the center for disease prevention and control center virus disease in China.
EXAMPLE 1 construction of recombinant vaccinia Virus delB2R with deletion of the B2R Gene
1. Method of
1.1 construction of recombinant Virus delB2R
1.1.1, recombination
The recombinant plasmid pdelB2R is synthesized by self and mainly comprises vaccinia virus P7.5, a P11 promoter and B2R gene homology arms (B2R left and B2R right), and the nucleotide sequence of the recombinant plasmid pdelB2R is shown as SEQ ID NO: 2.
HEK293T cells were seeded in six well plates at a cell density of 5X 10 5 Each well was followed by 2. Mu.g of recombinant plasmid after 16 hourspdelB2R transfected cells. After further incubation for 24 hours, the HEK293T cells were infected with the wild-type vaccinia virus Tiantan strain at a multiplicity of viral infection (MOI) of 0.1. After 24-48 hours of infection, the cells are repeatedly frozen and thawed for three times, and homologous recombination occurs between the recombinant plasmid pdelB2R and the wild type virus, so as to obtain the mixed virus of the recombinant virus delB2R and the wild type virus.
1.1.2 purification of recombinant viruses
The mixed virus obtained by the method is further purified by a plaque experiment. PCR was performed using the B2R specific primers B2Rup-F (SEQ ID NO: 5) and B2Rdown-R (SEQ ID NO: 6), with the PCR band size of 442bp for the recombinant virus delB2R being positive plaques and 711bp for the wild-type virus. After positive plaques were obtained, the plaque assay was repeated for multiple rounds of screening until pure recombinant virus delB2R was obtained.
1.1.3 identification of recombinant viruses
Extracting DNA of recombinant virus delB2R, and carrying out PCR identification. Amplification primers for PCR identification are as above. Meanwhile, sequencing the PCR product proves that the recombinant virus delB2R is obtained, and the poxin coding gene is accurately knocked out in the B2R region.
1.2 level of recombinant Virus delB2R induced innate immune response
THP1 cells are infected by wild type virus and recombinant virus delB2R (MOI is 1) respectively, and cells are harvested after 6 hours for Western Blot experiments to detect the expression of related proteins such as p-IRF3, p-TBK1 and the like in the cells.
2. Results
2.1 construction of recombinant plasmid pdelB2R
The constructed recombinant plasmid pdelB2R comprises vaccinia virus P7.5, P11 promoter and B2R region homology arms (B2R left and B2R right), and the structure and homologous recombination of the pdelB2R recombinant plasmid are schematically shown in FIG. 1.
2.2 screening, purification and identification of recombinant Virus delB2R
The positions of the primers B2Rup-F and B2Rdown-R in the PCR binding to the recombinant virus delB2R and the wild-type virus template are shown in FIG. 2. The gel electrophoresis pattern after PCR is shown in FIG. 3. The sizes of PCR products of the recombinant virus delB2R and the wild-type virus are different, so that the purified recombinant virus delB2R is obtained.
2.3 level of recombinant Virus delB2R induced innate immune response
As shown in FIG. 4, compared with the wild type virus, the recombinant virus delB2R can induce THP1 cells to express higher levels of p-IRF3 and p-TBK1, further induce interferon factor expression and activate natural immunity.
EXAMPLE 2 construction of recombinant vaccinia Virus TK-S and TKB2-S expressing the novel coronal S protein and control viruses dTK and dTKB2
1. Method of
1.1 construction of recombinant plasmid pTK-S and control plasmid pTK
Codon optimization is carried out on the coding nucleotide sequence of the novel coronavirus SARS-CoV-2 spike protein S, and the optimized nucleotide sequence is shown as SEQ ID NO: 1. Recombinant plasmid pTK-S was constructed, which included vaccinia virus P7.5, P11 promoter, S-encoding gene and TK gene homology arms (J1R, J3R). The nucleotide sequence of the recombinant plasmid pTK-S is shown as SEQ ID NO: 3.
In addition, a control recombinant plasmid pTK was constructed which included vaccinia virus P7.5, P11 promoter and TK gene homology arms (J1R, J3R). The nucleotide sequence of the control recombinant plasmid pTK is shown as SEQ ID NO: 4.
1.2 construction of recombinant virus TK-S, TKB2-S and control recombinant virus dTK and dTKB2
1.2.1, recombination
HEK293T cells were seeded in six well plates at a cell density of 5X 10 5 Cells were transfected with 2. Mu.g of recombinant plasmid pTK-S at each well after 16 hours. After further culturing for 24 hours, HEK293T cells were infected with the wild-type vaccinia virus Tiantan strain and recombinant virus delB2R, respectively, at a multiplicity of viral infection of 0.1. Cells were repeatedly thawed three times 24-48 hours after infection. Homologous recombination occurs between the recombinant plasmid pTK-S and the wild type virus, and the S gene fragment is inserted into the TK gene of the wild type virus to obtain the recombinant virus TK-S with the TK gene deleted and the S gene inserted at the position of the deleted TK gene; in addition, homologous recombination occurs between the recombinant plasmid pTK-S and the recombinant virus delB2R, and the S gene fragment is inserted into the TK gene of the recombinant virus delB2R to obtain the B2R gene and the B2R geneThe TK gene is deleted at the same time and the S gene is inserted into the recombinant virus TKB2-S at the position of deleting the TK gene.
Control recombinant viruses dTK and dTKB2 were constructed simultaneously. HEK293T cells were seeded in six well plates at a cell density of 5X 10 5 Cells were transfected with 2. Mu.g of control recombinant plasmid pTK 16 hours later per well. After further culturing for 24 hours, the HEK293T cells were infected with the wild-type vaccinia virus Tiantan strain and the recombinant virus delB2R, respectively, at a multiplicity of viral infection of 0.1. Cells were repeatedly thawed three times 24-48 hours after infection. Homologous recombination occurs between the control recombinant plasmid pTK and the wild type virus to obtain a TK gene-deleted control recombinant virus dTK; homologous recombination occurs between the control recombinant plasmid pTK and the recombinant virus delB2R, and the control recombinant virus dTKB2 with the B2R gene and the TK gene deleted simultaneously is obtained.
1.2.2 purification of recombinant viruses
Recombinant viruses were screened and purified by the same method as in example 1, using specific primers J1RJ3R-F (SEQ ID NO: 7) and J1RJ3R-R (SEQ ID NO: 8) for the TK region.
1.2.3 identification of recombinant viruses
And carrying out PCR identification on the extracted recombinant viruses TK-S and TKB2-S and the genomic DNA of the control recombinant viruses dTK and dTKB2. Meanwhile, the PCR product is sequenced, and the fact that the recombinant virus is accurately inserted into the coding gene of the S protein in the TK region is confirmed. Recombinant viruses TK-S and TKB2-S were confirmed to be obtained.
Further, vero cells were infected with wild vaccinia virus Tiantan strain, dTK, TK-S, dTKB2 and TKB2-S recombinant viruses, respectively, at a multiplicity of infection of 0.01, for 48 hours, and after that, the cells were harvested and examined for expression of SARS-CoV-2S protein by Western Blot.
2. Results
2.1 construction of recombinant plasmid pTK-S
The constructed recombinant plasmid pTK-S comprises vaccinia virus P7.5, P11 promoter, S and TK region homology arms (J1R, J3R). A schematic representation of the homologous recombination between the recombinant plasmid pTK-S and the wild-type virus is shown in FIG. 5.
2.2 screening, purification and identification of recombinant viruses TK-S and TKB2-S
The positions of the primers J1RJ3R-F and J1RJ3R-R in the PCR binding to the recombinant virus TK-S and the wild-type virus template are shown in FIG. 6. post-PCR gel electrophoresis using primers B2Rup-F and B2Rdown-R and J1RJ3R-F and J1RJ3R-R is shown in FIG. 7: when amplified with primers B2Rup-F and B2Rdown-R, the amplified fragments (731 bp) obtained for the wild-type virus (E), the control recombinant virus dTK (A) and the recombinant virus TK-S (B) are larger than those obtained for the control recombinant virus dTKB2 (C) and the recombinant virus TKB2-S virus (D) (421 bp). When the primers J1RJ3R-F and J1RJ3R-R are used for amplification, the amplified fragments of the recombinant viruses TK-S (B) and TKB2-S (D) are larger than those of the wild-type virus (E), the control recombinant viruses dTK (A) and dTKB2 (C) due to the insertion of the S gene sequence.
The PCR result proves that the TK gene is deleted in the recombinant viruses TK-S and TKB2-S, the S coding gene is inserted, and the accurate insertion of the S coding gene in the TK gene of the vaccinia virus is confirmed according to the sequencing of the PCR product, so that the recombinant viruses TK-S and TKB2-S are obtained.
Western Blot detection results show that the S protein of the novel coronavirus SARS-CoV-2 can be detected after the recombinant viruses TK-S (C) and TKB2-S (E) infect cells, as shown in FIG. 8.
EXAMPLE 3 mouse immunization experiments with recombinant vaccinia Virus TK-S and TKB2-S expressing the novel coronavirus SARS-CoV-2S protein
1. Method of
1.1 mouse immunization protocol
BalB/C mice at 6 weeks of age were divided into 4 groups of 5: the first group is a virus vector control group, and mice are immunized by a recombinant virus dTK deleted in TK gene; the second group is a virus vector control group, and a recombinant virus dTKB2 which simultaneously deletes TK genes and B2R genes is used for immunizing mice; the third group is a recombinant virus vaccine group, and mice are immunized by recombinant virus TK-S deleted in TK gene and inserted in S gene; the fourth group is a recombinant virus vaccine group, and mice are immunized with a recombinant virus TKB2-S in which the TK gene and the B2R gene are deleted simultaneously and the S gene is inserted at the site of the deleted TK gene. Each virus was prepared as a PBS suspension for immunization. Immunization was performed by leg intramuscular injection on day 0, wherein the viral vector control groups were immunized with 100 μl (1×)10 7 PFU/ml) dTK or dTKB2 (note: PFU: plaque forming units), 100 μl (1×10) of recombinant viral vaccine each was immunized with 7 PFU/m 1) TK-S or TKB2-S. The same dose and the same route are adopted for boosting once (secondary immunization) at the 3 rd week, and blood is taken at the 12 th day after each immunization for antibody detection. The harvested mouse serum is inactivated at 56 ℃ for 30min and then stored in a refrigerator at-80 ℃.
1.2 detection of S antibodies in mouse serum
The S antibody titer in the mouse serum was detected by ELISA.
1.3 detection of neutralizing antibodies in mouse serum
1. Mu.g of plasmid pACE2 expressing human angiotensin converting enzyme 2 (ACE 2) was transfected into HeLa cells according to the procedures conventional in the art to construct a HeLa-ACE2 cell line.
According to the conventional operation method in the art, a pcDNA3.1 plasmid expressing SARS-CoV-2S protein and a psPAX2 plasmid expressing Gag-Pol, a pli-GFP plasmid carrying firefly luciferase were each co-transfected with 1. Mu.g of HEK293T cells, and after 48 hours, the supernatant was collected to obtain a novel coronavirus expressing S protein, which was used to infect HeLa-ACE2 cells, and the status of virus infection was indicated by detecting the level of firefly luciferase. Serial 4-fold gradient dilutions of the twice immunized mouse serum were performed, after incubation with the above-described novel coronavirus for 1h at 37 ℃, heLa-ACE2 cells were infected, and after 48h, expression of firefly luciferase was detected using Steady-Glo Luciferase Assay System (Promega) to indicate the condition of neutralizing antibodies in the mouse serum.
2. Results
2.1 detection of S antibodies in mouse serum
ELISA detection results of S antibody titer in mouse serum show that no S antibody is detected in mouse serum of the virus vector control group (dTK and dTKB 2); antibodies to S can be detected in serum of mice immunized with recombinant virus TK-S, and the level of S antibodies after secondary immunization (TK-S-2 nd) is higher than that of the primary immunization (TK-S-1 st); the S antibody titers detected in the sera of mice immunized with recombinant virus TKB2-S were higher than those of the TK-S group (as shown in FIG. 9).
2.2 detection of neutralizing antibodies in mouse serum
No neutralizing antibodies were detected in the mouse serum (dTK and dTKB 2) of the control group of viral vectors, and neutralizing antibodies were detected in the serum of the recombinant vaccinia virus TK-S immunized mice, whereas the neutralizing antibodies levels of the recombinant virus TKB2-S immunized mice were significantly better than those of the TK-S group (as shown in fig. 10).
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that modifications may be made to the described embodiments in various different ways without departing from the spirit and scope of the invention. Accordingly, the foregoing description is illustrative in nature and is not to be construed as limiting the scope of the invention as claimed.
Sequence description
SEQ ID NO:1 represents a codon optimized nucleotide sequence encoding SARS-CoV-2 spike protein S;
SEQ ID NO:2 represents the nucleotide sequence of the recombinant plasmid pdelB 2R;
SEQ ID NO:3 represents the nucleotide sequence of the recombinant plasmid pTK-S;
SEQ ID NO:4 represents the nucleotide sequence of the control recombinant plasmid pTK;
SEQ ID NO:5 represents the nucleotide sequence of primer B2 Rup-F;
SEQ ID NO:6 represents the nucleotide sequence of primer B2 Rdown-R;
SEQ ID NO:7 denotes the nucleotide sequence of primer J1RJ 3R-F;
SEQ ID NO:8 denotes the nucleotide sequence of primer J1RJ 3R-R;
SEQ ID NO:9 shows the nucleotide sequence of the deleted vaccinia virus Tiantan strain TK gene in the recombinant vaccinia virus of the invention; and
SEQ ID NO:10 shows the nucleotide sequence of the deletion vaccinia virus Tiantan strain B2R gene in the recombinant vaccinia virus of the invention.

Claims (10)

1. A recombinant vaccinia virus that lacks the gene encoding thymidine kinase TK or both the gene encoding TK and the B2R gene encoding poxin protein, and that comprises an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK encoding gene, as compared to the wild-type vaccinia virus Tiantan strain.
2. The recombinant vaccinia virus of claim 1, wherein the nucleotide sequence encoding SARS-CoV-2 spike protein S is set forth in SEQ ID NO: 1.
3. The recombinant vaccinia virus of claim 1 or 2, wherein the deletion TK gene is the deletion of SEQ ID NO: 9; deletion of B2R gene is deletion of SEQ ID NO: 1O.
4. A novel coronavirus vaccine composition comprising the recombinant vaccinia virus of any one of claims 1-3 and a pharmaceutically acceptable carrier.
5. Use of the recombinant vaccinia virus of any of claims 1-3 for the preparation of a vaccine composition for preventing a novel coronavirus SARS-CoV-2 infection in a subject; preferably, the subject is a human.
6. A method of making a recombinant vaccinia virus comprising:
(1) Constructing a recombinant plasmid containing an upstream and downstream homology arm of a poxin protein coding gene B2R;
(2) Homologous recombination is carried out on the wild vaccinia virus Tiantan strain and the recombinant plasmid constructed in the step (1), and recombinant virus is obtained after purification, and the recombinant virus lacks B2R genes;
(3) Constructing a recombinant plasmid containing an upstream and downstream homology arm of TK gene and a nucleotide sequence for encoding SARS-CoV-2 spike protein S;
(4) Homologous recombination is carried out on the wild vaccinia virus Tiantan strain and the recombinant plasmid constructed in the step (3), and recombinant virus is obtained after purification, wherein the recombinant virus lacks TK gene and contains inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK gene; alternatively, the recombinant virus obtained in the step (2) and the recombinant plasmid constructed in the step (3) are subjected to homologous recombination, and purified to obtain a recombinant virus which lacks both the TK gene and the B2R gene and contains an inserted nucleotide sequence encoding SARS-CoV-2 spike protein S at the position of the deleted TK gene.
7. The method of claim 6, wherein the recombinant plasmid constructed in step (1) further comprises a vaccinia virus promoter; preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO: 2.
8. The method of claim 6 or 7, wherein the recombinant plasmid constructed in step (3) further comprises a vaccinia virus promoter; preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO: 3.
9. Recombinant vaccinia virus prepared by the method of any of claims 6-8.
10. A recombinant vaccinia virus that lacks the B2R gene encoding a poxin protein compared to a wild-type vaccinia virus Tiantan strain; preferably, the deletion B2R gene is a deletion of SEQ ID NO:10, and a nucleotide sequence shown in seq id no.
CN202310095863.0A 2023-01-18 2023-01-18 Novel coronavirus vaccine based on improved vaccinia virus Tiantan strain Pending CN116200349A (en)

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