チャイナの嘘
中国カンシノバイオロジックのCOVIDウイルスベクターワクチンの特許は2020年3月18日に出願されています。記録上、第Ⅰ相試験の開始は20年3月ですが、それより前の20年2月29日に非公式に接種が開始されていたのがAP通信に報じられています。じゃあ、開発はいつ始まったんでしょうかね！？チャイナが世界中にまき散らしたんですから、いい加減、『チャイナ・ウィルス』とハッキリ言いましょう！！
https://t.co/7VOOlTTqJv?amp=1
Recombinant novel coronavirus vaccine taking human replication-defective adenovirus as vector
Abstract
The invention provides a novel coronavirus vaccine taking human type 5 replication-defective adenovirus as a vector. The vaccine takes replication-defective human type 5 adenovirus with combined deletion of E1 and E3 as a vector, HEK293 cells integrating adenovirus E1 genes as a packaging cell line, and carried protective antigen genes are 2019 novel coronavirus (SARS-CoV-2) S protein genes (Ad5-nCoV) which are subjected to optimization design. After the S protein gene is optimized, the expression level in the transfected cells is obviously increased. The vaccine has good immunogenicity on mouse and guinea pig models, and can induce organisms to generate strong cellular and humoral immune responses in a short time. The protection effect research on the hACE2 transgenic mouse shows that the virus load in lung tissues can be obviously reduced after a single immunization of Ad5-nCoV14 days, and the vaccine has a good immune protection effect on 2019 novel coronavirus. In addition, the vaccine is fast and simple to prepare, and can be produced in a large scale in a short time to cope with sudden epidemic situations.
Classifications
C07K14/005 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
View 7 more classifications
CN111218459A
China
Download PDF Find Prior Art Similar
Other languagesChineseInventor陈薇吴诗坡侯利华张哲�王步森郭强张金龙宋小红付玲张军陈旖赵拯浩朱涛李荩莘春林Current Assignee Institute of Pharmacology and Toxicology of AMMS Academy of Military Medical Sciences AMMS of PLA CanSino Biologics Inc
Worldwide applications
2020 CN
Application CN202010193587.8A events
2020-03-18
Application filed by Institute of Pharmacology and Toxicology of AMMS, CanSino Biologics Inc
2020-03-18
Priority to CN202010193587.8A
2020-06-02
Publication of CN111218459A
2020-09-11
Application granted
2020-09-11
Publication of CN111218459B
Status
Active
2040-03-18
Anticipated expiration
InfoPatent citations (2) Non-patent citations (4) Cited by (3) Legal events Similar documents Priority and Related ApplicationsExternal linksEspacenetGlobal DossierDiscuss
Description
Recombinant novel coronavirus vaccine taking human replication-defective adenovirus as vector
Technical Field
The invention relates to a recombinant novel coronavirus vaccine, and aims to prevent a novel coronavirus epidemic situation. The invention belongs to the technical field of biological engineering.
Background
2019A novel coronavirus, SARS-CoV-2 (also known as 2019-nCoV), was named by the world health organization on 12 months 1 in 2020. It is a new strain of coronavirus that has not previously been found in humans. The virus is the seventh coronavirus (CoV) that can infect humans. The incubation period of 2019-nCoV infection of a human is generally 1-14 days, and after 2019-nCoV infection, common signs comprise respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. By 3, 17 days in 2020, 81116 confirmed cases and 3231 dead cases are reported in China; 98486 cases were reported in other countries around the world, 3848 cumulative deaths, with an average mortality of 3.94%.
the novel coronavirus belongs to a β genus coronavirus, is enveloped, has circular or elliptical particles, is usually polymorphic, and has the diameter of 60-140 nm, and the phylogenetic analysis (29,903 nucleotides) of a virus complete gene group shows that the virus is most closely related to a group of SARS-like coronavirus (gentus Betacononavirus, subvenus Sarbecovirus) which is previously found in Chinese bat bodies (89.1 percent of nucleotide similarity). in an S gene evolutionary tree, 2019-nCoV has the closest relationship with bat coronavirus bat-SL-CoVZC45, the amino acid consistency is 82.3 percent, and the amino acid consistency with SARS-CoV is about 77.2 percent, receptors bound when the SARS-CoV enters human cells are the same as those of SARS-CoV, and are angiotensin converting enzyme 2(ACE 2).
Coronaviruses are a large family of viruses, and the main causes of outbreaks of infections in humans to date are SARS-CoV, MERS-CoV and SARS-CoV-2. The reports on the structure, function, infection key target, action mechanism and vaccine development of SARS-CoV-2 are not many, and many researches mainly focus on SARS-CoV and MERS-CoV vaccines, and the main research and development types include virus vector vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle vaccines, inactivated vaccines, attenuated live vaccines and the like. The target antigens for recombinant coronavirus vaccines are generally in the form of full-length S protein and truncated S protein (S1 and RBD). The research results of the research on the types of the novel coronavirus vaccines in the research include adenovirus vector vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines and inactivated vaccines, and the research results are not reported yet. The invention aims to provide a novel recombinant coronavirus vaccine.
Disclosure of Invention
Based on the above purposes, the invention firstly provides an S protein gene optimized polynucleotide for encoding a novel coronavirus vaccine, and the sequence of the polynucleotide is shown as SEQ ID NO. 1. The polynucleotide takes replication-defective human type 5 adenovirus with combined deletion of E1 and E3 as a vector, takes HEK293 cells integrated with adenovirus E1 genes as a packaging cell line, and is packaged to obtain the novel coronavirus vaccine of the recombinant adenovirus vector.
The present invention also provides a vector containing the polynucleotide.
In a preferred embodiment, the vector is pDC 316.
The present invention also provides a human replication-deficient recombinant adenovirus capable of expressing the polynucleotide.
In a preferred embodiment, the recombinant adenovirus is derived from the AdMax adenovirus system.
The invention also provides application of the recombinant adenovirus in preparation of a vaccine for preventing novel coronavirus pneumonia.
In a preferred embodiment, in the above application, the recombinant adenovirus is prepared as an injection, nasal drops or spray.
In a more preferred embodiment, the recombinant adenovirus is prepared as an intramuscular injection.
Finally, the present invention provides a method for preparing the recombinant adenovirus capable of expressing the novel coronavirus S protein, which comprises the following steps:
(1) constructing a shuttle plasmid vector comprising a polynucleotide encoding a 2019 novel coronavirus S protein;
(2) transfecting the shuttle plasmid vector of step (1) into a host cell together with a backbone plasmid;
(3) culturing the host cell of step (2);
(4) harvesting the human replication-deficient recombinant adenovirus released from the cells of step (3);
(5) carrying out amplification culture on the recombinant adenovirus in the step (4);
(6) purifying the culture product in the step (5).
Preferably, the shuttle plasmid vector of step (1) is pDC 316.
Preferably, the backbone plasmid in step (2) is pBHGlox _ E1,3Cre, both plasmids belonging to the AdMax adenovirus system and being used together for recombinant adenovirus packaging in a host cell comprising a polynucleotide encoding a novel coronavirus S protein.
Preferably, the cells in step (3) are HEK293 cells.
Preferably, the expansion culture method in step (5) is suspension culture.
Preferably, the purification method in step (6) is Source 30Q chromatography.
The recombinant adenovirus capable of expressing the novel coronavirus S protein provided by the invention is used as a novel coronavirus vaccine (Ad5-nCoV), has good immunogenicity on mouse and guinea pig models, and can induce an organism to generate strong cellular and humoral immune response in a short time. The protective effect research on hACE2 transgenic mice shows that the single immunization of Ad5-nCoV14 days can obviously reduce the virus load in lung tissues. The vaccine has a good immune protection effect on the 2019 novel coronavirus. The preparation method of the vaccine is quick, simple and convenient, and can realize large-scale production in a short time to cope with sudden epidemic situations.
Drawings
FIG. 1 shuttle plasmid pDC316-nCoV _ Sopt map;
FIG. 2 shows an identification map of Western blot expressed in S protein cells;
FIG. 3 shows a Western blot identification map of the primary virus target antigen expression of a recombinant novel coronavirus vaccine candidate strain;
FIG. 4 is a graph comparing serum IgG antibody levels at day 9 of intramuscular injection of mice with immune Ad 5-nCoV;
FIG. 5 is a graph comparing serum IgG antibody levels at 9 and 14 days of intramuscular injection of mice with immune Ad 5-nCoV;
FIG. 6 is a graph comparing serum IgG antibody levels of different recombinant adenovirus constructed by intramuscular injection of mice;
FIG. 7 is a graph comparing the levels of serum antibodies induced by different immunization regimens on day 14 of mice with recombinant adenovirus;
FIG. 8 Ad5-nCoV induced CD8+T cell immune response comparison plots;
FIG. 9 Ad5-nCoV induced CD4+T cell immune response comparison plots;
FIG. 10 is a representation of the cellular immune response in the intramuscular injection group;
FIG. 11 is a representation of cellular immune responses in nasal drops of the immune group;
FIG. 12 is a representation of cellular immune responses in a control group;
FIG. 13 is a graph comparing the levels of lung wash antibodies 14 days after Ad5-nCoV immunization;
FIG. 14 is a graph comparing the immunogenicity assays of Ad5-nCoV guinea pig models;
FIG. 15 is a graph comparing the percent weight loss of mice after SARS-CoV-2 challenge;
FIG. 16 is a graph comparing the lung tissue viral load of mice after SARS-CoV-2 challenge.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.
Example 1 preparation of recombinant novel coronavirus vaccine with human replication-defective adenovirus as vector
S protein gene optimization and synthesis
The target antigen of the recombinant novel coronavirus vaccine is the S protein of a novel coronavirus strain (Genebank number: NC-045512.2). The S protein gene is optimized to improve the expression level of the S protein, so that the immunogenicity of the vaccine is improved.
First, most rare codons of the S protein gene were changed to high-usage-frequency codons by codon optimization using Upgene software (Gao, W.Rzewski, A.Sun, H.Robbins, P.D. & Gambottoto, A.UpGene: Application of a web-based DNA code optimization. Biotechnol Prog,2004.20(2): p.443-8.). Second, protein translation efficiency may not be significantly improved given that software optimization may mechanically alter codons to the highest usage frequency codons, subject to tRNA efficiency, mRNA secondary structure, etc. In this case, we used a method of replacing part of the high-frequency and low-frequency codons by artificial analysis while uniformly distributing the high-frequency codons and the low-frequency codons in the S protein gene. Also considering that increasing the GC content of mRNA helps to enhance the stability of mRNA, we suitably increased the GC content of the S protein gene and distributed G, C nucleotides as evenly as possible throughout the GP gene. Again, we changed the original signal peptide of the S protein (1aa-12aa) to the tissue plasminogen activator signal peptide, tPA, to further elevate the expression level of the S protein.
Before gene optimization, the content of rare codons (usage frequency < 70%) of the novel coronavirus S protein gene is 34%, the content of high-usage-frequency codons (usage frequency > 90%) is 23%, and the GC content is 36%. After gene optimization, the rare codon content is reduced to 3%, the high-frequency codon content is increased to 81%, the GC content is increased to 58%, and the homology with the original S protein gene sequence is 70.4%.
After S protein gene optimization, a Kozak sequence is added in front of a translation initiation codon, a restriction site EcoRI is inserted into the upstream of the whole sequence, and a restriction site HindIII is inserted into the downstream of the whole sequence, so that a gene sequence is synthesized. Meanwhile, as a control, the original sequence of the S protein gene was synthesized. The optimized sequence of the S protein gene (the restriction sites are EcoRI and HindIII) is shown in SEQ ID NO. 1, and the original gene sequence of the S protein (the restriction sites are SmaI and SalI) is shown in SEQ ID NO. 2.
2. Vector construction and S protein in vitro expression identification
2.1 vector construction
The synthesized gene sequences are subjected to double enzyme digestion by HindIII and SalI or SmaI and SalI respectively, and the target gene is recoveredfragment, ligated to shuttle plasmid pDC316 of AdMax adenovirus System (Microbix Biosystems Inc., Canada), transformed DH5- α competent, Amp-coatedrAnd (3) an LB plate, selecting a single clone for colony PCR identification, and performing sequencing verification on the clone which is positive in PCR identification. The plasmid with the non-optimized S protein gene sequence was designated as pDC316-nCoV _ S, and the plasmid with the optimized S protein gene was designated as pDC316-nCoV _ Sopt. The plasmid map of pDC316-nCoV _ Sopt is shown in FIG. 1.
Meanwhile, amplifying an original signal peptide S protein gene optimization sequence by using an overlap extension PCR method, connecting the sequence to a pDC316 vector, and marking as pDC316-nCoV _ oriSIP-Sopt; amplifying an unoptimized sequence of a tPA signal peptide S protein gene, connecting the unoptimized sequence to a pDC316 vector, and marking as pDC316-nCoV _ tPA-S; the tPA signal peptide S1 protein (S1 protein is a truncated protein of 12-685 amino acids of S protein) gene optimization sequence (the corresponding gene optimization sequence is 25 bp-2103 bp of SEQ ID NO: 1) is connected to the pDC316 carrier and is marked as pDC316-nCoV _ S1 opt; and the original sequence of the tPA signal peptide S1 protein gene, which is connected to the pDC316 vector and is marked as pDC316-nCoV _ S1.
2.2 in vitro expression characterization of S protein
The 6 shuttle plasmids and pDC316 vector constructed above were transfected into HEK293 cells using the transfection Reagent TurboFectTransfection Reagent (Thermo Scientific, REF, R0531), and WB detection was performed 48 hours later by harvesting the cells. The experimental method is as follows:
transfection: the day before the experiment, HEK293 cells were treated at 8X 105Cells/well were seeded in 6-well plates at 37 ℃ with 5% CO2The cells were cultured in a cell incubator overnight. 1 hour before transfection, the medium was changed to fresh 2% FBS-containing DMEM medium, 2mL per well. During transfection, 2 μ g of the corresponding plasmid was taken from each transfection well, added to 200 μ L of FBS-free DMEM medium, mixed well, added with 3 μ L of transfection reagent, mixed well gently, and left at room temperature for 15 minutes. The mixture of plasmid and transfection reagent was added to the 6-well plate gently and mixed well with gentle shaking. Cells at 37 ℃ and 5% CO2Culturing in a cell culture box, changing the culture medium into a fresh DMEM culture medium containing 10% FBS after 5 hours, collecting cells after 48 hours, preparing a sample, and carrying out WB detection.
Sample preparation: 48 hours after transfection, the medium was carefully aspirated, the cells were resuspended in PBS, centrifuged at 500g for 5 minutes, and the supernatant discarded. The cells were resuspended in 200. mu.L of RIPA buffer (Thermo Scientific, Prod #89900, with the addition of appropriate amounts of protease inhibitors and nucleases), ice-washed for 15 minutes, centrifuged at 12000rpm for 5 minutes at 4 ℃ and the supernatant taken, added to 1/3 volume of 4 XSDS-PAGE loading buffer containing 200mmol/L DTT, heated at 95 ℃ for 5 minutes and frozen for WB detection.
Western blot detection: SDS-PAGE was performed using 4-20% SDS-PAGE gradient gels in 10 wells, loading 30. mu.L per well. Electrophoresis conditions: 80V, 15 min; 180V until bromophenol blue just comes out of the gel. Proteins on the SDS-PAGE gel were transferred to nitrocellulose membrane by an electrotransfer at 300mA for 1 hour. After completion of the electrotransfer, the nitrocellulose membrane was blocked with 5% skim milk powder for 1 hour, and then an anti-S protein rabbit polyclonal antibody (Casino, Cat. 40150-T52) was added at a dilution of 1:2000 and left overnight at 4 ℃. The membranes were washed 4 times with WB wash, each time on a shaker for 5 minutes. Then, HRP-labeled goat anti-rabbit IgG antibody (CST,7074S) diluted at 1:10000 in 5% skim milk powder was added and incubated at room temperature for 1 hour. The membrane was washed 4 times with WB wash and ImmobilonTMWestern Chemimelescent HRPSubsrate (MILLIPORE, Cat. No. WBKLS0500) was subjected to a Chemiluminescent reaction, and images of different exposure times were acquired using a Chemiluminescent imager.
the results of examining the internal control using β -actin as a sample are shown in FIG. 2, in which 1: pDC316-nCoV _ S cells were transfected, 2: pDC316-nCoV _ tPA-S cells were transfected, 3: pDC316-nCoV _ oriSIP-Sopt cells were transfected, 4: pDC316-nCoV _ Sopt cells were transfected, 5: pDC 316-transfected empty vector cells, 6: Marker was preimpregnated, 7: pDC316 empty vector cells were transfected, 8: pDC316-nCoV _ S1 cells were transfected, 9: pDC316-nCoV _ S1opt cells were transfected, and the results showed that no gene-optimized sequence was obtained, and that no expression of the relevant protein was detected in the transfected cells, and that expression of the S protein was detected after S protein gene optimization, and that the S protein was significantly expressed after S protein original signal peptide was changed to tPA signal peptide, the expression level of the S protein was further increased, and that expression of the S protein was also detected in S6335 after S protein optimization.
3. Recombinant adenovirus packaging, preparation and identification
3.1 recombinant adenovirus packaging
The constructed vectors pDC316-nCoV _ Sopt, pDC316-nCoV _ oriSIP-Sopt and pDC316-nCoV _ S1opt are co-transfected with the skeleton plasmids pBHGlox _ E1 and 3Cre of the AdMax adenovirus system to carry out packaging of the recombinant adenovirus. The process is as follows:
a) one day prior to transfection, HEK293 cells were seeded in six-well plates at 8 × 10 per well5The cells were cultured in MEM + 10% FBS at 37 ℃ with 5% CO2The cells were cultured in a cell incubator overnight.
b) The day of transfection, the medium was changed and the culture was continued in fresh 10% FBS-containing MEM medium. When the cells grew to 80-90% of the basal area, the backbone plasmid (pBHGlox _ E1,3Cre) and shuttle plasmid were taken and transfected with TurboFect transfection reagent (Thermo scientific, REF, R0531) according to the instructions attached thereto. The method comprises the following specific steps:
(1) taking 3.2 mu g of skeleton plasmid and 0.8 mu g of shuttle plasmid from each transfection hole, and mixing uniformly; the plasmid was diluted with 400. mu.L of Opti-MEM medium.
(2) mu.L of TurboFect transfection reagent was added to the plasmid diluted in Opti-MEM medium and mixed gently.
(3) The transfection reagent and plasmid mixture was left at room temperature for 20min and then added to the cells.
c) The day after transfection, confluent cells were passaged at 25cm2Culturing in 5% FBS-containing MEM medium in cell culture flask, observing every day, and transferring 75cm when cell grows to the bottom2In the cell culture flasks, the cells were observed daily for signs of toxicity. The appearance of the virus is that the cells become bigger and round, become grape-shaped, and begin to appear obvious plaques. The cells are detoxified when most of them are diseased and fall off from the bottom.
d) The detoxified cells were resuspended, centrifuged at 500g for 10 minutes, the supernatant was discarded, the cells were resuspended in 2mL of PBS, and then placed in a refrigerator at-70 ℃ and a water bath at 37 ℃ for three repeated freeze thawing. The supernatant containing the virus was collected by centrifugation at 12000g for 10 minutes, and the pellet was discarded.
The virus strain expressing the optimized sequence of the full-length protein gene of the tPA signal peptide S is marked as Ad5-nCoV _ Sopt, the virus strain expressing the optimized sequence of the full-length protein gene of the original signal peptide S is marked as Ad5-nCoV _ oriSIP-Sopt, and the virus strain expressing the optimized sequence of the protein gene of the tPA signal peptide S1 is marked as Ad5-nCoV _ S1 opt.
3.2 identification of recombinant adenovirus
3.2.1 PCR amplification of S and S1 full sequences and sequencing identification
The full sequence of the S or S1 protein was amplified using the universal primers for pDC316 vector, the primer sequences are as follows:
pDC316-F:ACGTGGGTATAAGAGGCG
pDC316-R:CGATGCTAGACGATCCAG
taking 50 mu L of vaccine candidate strain virus liquid, adding 2 mu L of protease K, digesting for 30min at 50 ℃ to release virus genome, and taking the virus genome as a template to amplify gene sequences of S protein and S1 protein.
The PCR amplification conditions were:
the reaction procedure is as follows:
agarose gel electrophoresis results show that three virus species can be amplified to form a single target band, and the size of the fragment is correct. And (4) carrying out glue recovery and sequencing on the target band, wherein the comparison result shows that the sequencing result sequence is completely correct.
3.2.2 identification of target antigen expression
HEK293 cells are infected by different constructed recombinant adenoviruses, and the cells are collected after 48 hours to carry out Western blot detection on target antigens, wherein the obvious expression of target proteins can be detected by three virus seeds Ad5-nCoV _ Sopt, Ad5-nCoV _ oriSIP-Sopt and Ad5-nCoV _ S1 opt. The results are shown in FIG. 3, where 1: pre-dyeing a Marker; 2 and 3: ad5-nCoV _ oriSIP-Sopt infected cells; 4 and 5: ad5-nCoV _ Sopt; 6: ad5-nCoV _ S1opt infects cells. 7 and 8: and (4) blank cells.
3.2.3 recombinant adenovirus culture
HEK293 cells at 37 ℃ with 5% CO2Suspension culture was carried out at 120rpm under the conditions. When inoculating with virus, diluting cells with activity greater than 95% to 1.0 × 106cells/mL, final volume 300 mL. P5 generation recombinant adenovirus with MOI 10 infecting HEK293 cells at 37 deg.C, 5% CO2The culture was shaken at 130 rpm. Samples were taken every 24 hours and cell activity and density were measured.
After the inoculation of the virus seeds for 72 hours, when the cell activity is reduced to below 40 percent, the shake flask of the cell is repeatedly frozen and thawed twice in a refrigerator with the temperature of-70 ℃ and a water bath kettle with the temperature of 37 ℃. Adding Benzonase (20U/mL), and performing enzymolysis for 2 hours in a water bath at 34-36 ℃. The supernatant containing the virus was collected by centrifugation at 12000g for 10 minutes, and the pellet was discarded.
3.2.4 recombinant adenovirus purification
The virus supernatant was adjusted with 5 Xequilibration buffer to a conductance of 18mS/cm, pH 7.5. Separating and purifying adenovirus particles with Source 30Q, and purifying with chromatography column using solution A (20mmol/L Tris +150mmol/L NaCl +2 mmol/LMgCl)2pH 7.5. ) Balancing, loading, wherein the flow rate of loading is 5 mL/min; after the sample loading is finished, balancing to a Uv base line by using a solution A, eluting with a gradient of 10mL/min,50min, 0% B-20% B, collecting the elution peak by separating tubes, and finally eluting with 100% B, wherein the solution B is 20mmol/L Tris +2mol/L NaCl +2mmol/L MgCl2pH7.5。
3.3 identification and Titer determination of Ad5-nCoV
3.3.1PCR amplification of the complete sequence of the target protein Gene and sequencing identification
The experimental procedure and procedure were the same as 3.2.1. Agarose gel electrophoresis results show that three virus species can be amplified to form a single target band, and the size of the fragment is correct. And (4) carrying out glue recovery and sequencing on the target band, wherein the comparison result shows that the sequencing result sequence is completely correct.
3.3.2 infectious titer assay
Using Clontech Adeno-XTMThe Rapid Titer Titer Kit was used to determine the Titer of the recombinant adenovirus. The operation is carried out according to the instructions attached to the kit, and the specific method is as follows:
a) HEK293 cells were seeded in 24-well plates. CellsDensity of 5X 105cells/mL, 0.5mL per well in MEM + 10% FBS.
b) The virus to be detected is extracted from 10 using a culture medium-2To 10-6A10-fold dilution was performed to prepare a series of dilutions of the virus sample, 50. mu.L per well, added to the cells.
c) The cells were cultured at 37 ℃ in a 5% CO2 incubator for 48 hours.
d) The cell culture medium was aspirated and the cells were allowed to air dry slightly (without overdrying). 0.5mL of ice methanol was gently added to each well, and the mixture was left at-20 ℃ for 10 minutes to fix the cells.
e) Methanol was aspirated and cells were gently washed 3 times with PBS + 1% BSA. 0.25mL of anti-Hexon antibody dilution (1:1000 dilution) was added to each well and incubated at 37 ℃ for 1 hour.
f) The Anti-Hexon Antibody was aspirated off, the cells were gently washed 3 times with PBS + 1% BSA, 0.25mL of HRP-labeled Rat Anti-Mouse Antibody (1:500 dilution) was added to each well, and incubated at 37 ℃ for 1 hour.
g) Before 0.25mL of HRP-labeled Rat Anti-Mouse Antibody was aspirated, 10 × DAB substrate was diluted with 1 × Stable Peroxidase Buffer to 1 × DAB working solution and allowed to reach room temperature.
h) The Rat Anti-Mouse Antibody dilutions were aspirated and the cells were gently washed 3 times with PBS + 1% BSA. 0.25mL of DAB working solution was added to each well and allowed to stand at room temperature for 10 minutes.
i) DAB working solution was aspirated and cells were gently washed 2 times with PBS.
j) Brown/black positive cells were counted under a microscope. At least 3 fields were counted randomly per well and the average number of positive cells was calculated.
k) Infectious titer (ifu/mL) was calculated. The formula is as follows:
infectious titer (ifu/mL) field positive cell number x field per well/(virus volume (mL) × dilution)
The titer determination result shows that the infection titer of the purified recombinant adenovirus after concentration reaches 1.0 multiplied by 1010ifu/mL or more.
3.3.3 viral particle count assay
20mmol/L Tris-Cl, 2mmol/L EDTA (pH 7.5) solution and 2.0% SDS solution were mixed in equal volumes to prepare a virus lysate. And (3) taking a virus sample to be detected with a proper volume, adding 1/19 volume of virus lysate, repeatedly blowing and beating the virus sample by using a pipette for 10 times, uniformly mixing the mixture, and vortexing the mixture for 1 minute. The resulting mixture was digested in a thermostatic water bath at 56 ℃ for 10 minutes with shaking, centrifuged at 12000rpm for 5 minutes, and the supernatant was collected to determine OD values at 260nm and 280 nm. The number of adenovirus particles was calculated.
The measurement result of the number of virus particles shows that the purified recombinant adenovirus reaches 1.0 multiplied by 10 after being concentrated11VP/mL or more.
Example 2 immunological evaluation of different constructs of recombinant adenovirus in mouse models
1. Vaccine humoral immune response detection
100 female BALB/c mice (6-8 weeks old) of SPF grade were randomly divided into 10 groups of 10 mice each. Mice were immunized with Ad5-nCoV according to the grouping shown in Table 1. The intramuscular injection is 100 μ L injected into the inner side of the hind thigh, and the nasal drip immunization is performed by anesthetizing the mouse with isoflurane and dripping 20 μ L into the nasal cavity. The grouping is shown in table 1.
TABLE 1 vaccine humoral immune response test mouse cohort
Mice were bled at specific time points after immunization, sera were isolated and the titers of IgG antibodies against the novel coronavirus S protein in the sera were determined using ELISA. The results are shown in FIGS. 4 to 7 (ns, P ≧ 0.05;, P < 0.01;, P < 0.001;, P < 0.0001).
The mice can generate higher serum IgG antibody by intramuscular injection of Ad5-nCoV _ Sopt on day 9, wherein the average value of the antibody titer of the high-dose group reaches 105The above; serum IgG antibody level induced by Ad5-nCoV _ Sopt intramuscular injection shows obvious dose dependence, and the higher the dose, the higher the serum IgG antibody level(FIG. 4). Serum antibody levels were further elevated from day 9 post-immunization to day 14 post-immunization, with serum IgG antibody titers in the high and low dose groups being significantly higher at day 14 post-immunization than at day 9 (fig. 5).
Serum IgG antibody levels induced by intramuscular injection of three recombinant adenoviruses, Ad5-nCoV _ Sopt, Ad5-nCoV _ oriSIP-Sopt and Ad5-nCoV _ S1opt, were analyzed, and the results are shown in FIG. 6, wherein A, serum IgG antibody titer at day 9 after immunization; b, serum IgG antibody titers at day 14 post immunization. The results showed that the immune response induced by Ad5-nCoV _ Sopt was most rapid among the three recombinant adenoviruses, and Ad5-nCoV _ Sopt produced significantly higher serum IgG antibody levels than Ad5-nCoV _ oriSIP-Sopt and Ad5-nCoV _ S1opt at day 9 post-immunization (FIG. 6, A). Serum antibody levels of Ad5-nCoV _ oriSIP-Sopt were elevated at day 14 post-immunization (fig. 6, B), but Ad5-nCoV _ S1opt induced serum IgG antibody levels significantly lower than the other two vaccine candidates at either day 9 or 14 post-immunization. The results show that among the three recombinant adenoviruses, serum IgG antibody titer induced by Ad5-nCoV _ Sopt is the most rapid, and the immunogenicity is the best.
Compared with serum antibody titers induced by intramuscular injection or nasal drop Ad5-nCoV _ Sopt at day 14 after immunization, the results are shown in FIG. 7, and the serum IgG antibody levels induced by the two immunizations have no significant difference no matter the high dose, the medium dose or the low dose.
2. Cellular immune response detection
30 SPF-grade female BALB/c mice, 6-8 weeks old, were randomly divided into three groups of 10 mice each. 5X 10 injections or injections by intramuscular (im.) or nasal drops (in.) were administered, respectively8Ad5-nCoV _ Sopt of VP; and inoculated with 1X 10 in the same immunization protocol7ifu Ad5 vector served as a control, 5 each for each immunization regimen. The intramuscular injection is 100 μ L injected into the inner side of the hind thigh, and the nasal drip immunization is performed by anesthetizing the mouse with isoflurane and dripping 20 μ L into the nasal cavity. Experimental grouping is shown in table 2.
TABLE 2 grouping of Ad5-nCoV _ Sopt mouse model cellular immune response assay experiments
mice were sacrificed 14 days post immunization, splenic lymphocytes were isolated, cultured for 6 hours with stimulation with the S protein contig pool, while cytokine secretion was blocked by addition of a protein secretion blocker 6 hours later, Fc receptors were blocked, dead cells and cell surface molecular markers were stained, intracellular cytokines were stained after cells were fixed and perforated, cell surface markers included CD3, CD4, CD8, and CD107a molecules, intracellular cytokines included IFN γ, TNF α, and il2. flow cytometry (BDFACS Canto) was usedTM) Analysis of CD4+T cells and CD8+T cells, upon stimulation with specific peptides, express levels of IFN γ, TNF α, IL2 and CD107 a.
Ad5-nCoV _ Sopt induced CD8+T cells and CD4+The T cell immune responses are shown in fig. 8 and 9, and representative results are shown in fig. 10 to 12, wherein fig. 10 is a representation of the cell immune response in the intramuscular injection group, fig. 11 is a representation of the cell immune response in the nasal drip immune group, and fig. 12 is a representation of the cell immune response in the control group. The results show that Ad5-nCoV _ Sopt can induce the immune mice to generate obvious cellular immune response whether the mice are immunized by intramuscular injection or nasal drip. 14 days after immunization of mice, splenocytes stimulated with the S protein overlapping peptide library, no matter CD8+T cells are also CD4+T cells expressing IFN gamma, TNF α and IL2 at levels significantly higher than those of Ad5 vector Control group (Control) (P)<0.05). At the same time, intramuscular injection (im.) induced a more intense cellular immune response than nasal drop immunization (in.). Intramuscular injection induced CD8+T cells and CD4+the levels of IFN γ, TNF α and IL2 produced by T cells were all significantly higher than nasal drops.
3. Detection of lung wash antibody levels
The mice subjected to cellular immune reaction detection are sacrificed while collecting lung washing liquid, and the levels of IgG and IgA antibodies against the novel coronavirus S1 protein in the lung washing liquid are detected by an ELISA method. Experimental groups are shown in table 2 and results are shown in figure 13 (×, P <0.0001), where a, lung wash IgG antibody levels; b, lung wash IgA antibody levels. The results show that 14 days after Ad5-nCoV _ Sopt immunization, nasal drip immunized mice can induce higher levels of lung wash IgG antibody and IgA antibody titer, which is obviously higher than that of the intramuscular injection group and the control group. Whereas the intramuscular injection group could detect only low levels of lung wash IgG antibody titers and failed to detect lung wash IgA antibodies.
4.4. Mouse model immunogenicity evaluation summary
The three recombinant adenoviruses of Ad5-nCoV _ Sopt, Ad5-nCoV _ oriSIP-Sopt and Ad5-nCoV _ S1opt have good immunogenicity and can induce mice to generate high-level serum IgG antibodies. Among them, serum IgG antibody titer induced by Ad5-nCoV _ Sopt was the most rapid, and immunogenicity thereof was the best. Ad5-nCoV _ Sopt was selected as the best candidate for recombinant novel coronavirus vaccine and was designated as Ad 5-nCoV. The cellular immune response detection result shows that both intramuscular injection and nasal drip immunization Ad5-nCoV can induce the mice immunized by the cells to generate specific cellular immune response, wherein the intramuscular injection immune mode can induce strong specific cellular immune response. The detection result of the lung washing liquid antibody shows that the nasal drip immunity can induce the generation of high-level lung washing liquid IgG and IgA antibodies.
Example 3 immunological evaluation of Ad5-nCoV on a Guinea pig model
56 SPF-rated guinea pigs weighing 200 to 250 grams were randomly divided into 4 groups of 14 animals each, hermaphrodite. Guinea pigs were immunized against Ad5-nCoV according to the grouping shown in Table 3. The immunization was performed by intramuscular injection of 200. mu.L into the medial thigh.
TABLE 3 grouping of Ad5-nCoV guinea pig immunogenicity assays
Guinea pigs were bled at specific time points after immunization, sera were isolated, and IgG antibody titers against the novel coronavirus S protein in the sera were detected using ELISA. The results are shown in FIG. 14 (ns, P.gtoreq.0.05;. P < 0.05;. P, P < 0.01;. P < 0.001;. P, P < 0.0001). The results show that high levels of serum IgG antibody titers were detected 14 days after Ad5-nCoV immunization in guinea pigs. The antibody titer between the medium-dose group and the high-dose group has no significant difference, and the serum IgG antibody level of the low-dose group is lower and is significantly lower than that of the high-dose group and the low-dose group. The detection result shows that Ad5-nCoV has good immunogenicity on a guinea pig model.
Example 4 evaluation of the protective Effect of Ad5-nCoV on hACE2 transgenic mouse model
A SARS-CoV-2(SARS-CoV-2/WH-09/human/2020/CHN) live virus challenge test was performed in a biosafety level 3 laboratory.
The experiment was divided into 3 groups (Table 4), each of which was injected intramuscularly in the hind leg with Ad5-nCoV 5X 109VP or 5X 108VP, injected in a volume of 100. mu.L, or an equivalent volume of PBS injected intramuscularly. 2 weeks after immunization, mice were transferred to biosafety level 3 laboratories for SARS-CoV-2 challenge in the form of nasal drops (10)5TCID50A/only. Mice were continuously observed for 3 days after challenge and body weight changes were recorded. All mice were euthanized on day 3 post-infection and lung tissue viral load was examined.
Table 4 Ad5-nCoV animal protection test grouping.
The mice in the model group showed weight loss after infection, with an average percentage of loss of up to 3.36%. Compared with the model group, the body weight of the mice in the high-dose group is increased to a certain extent at 3 days after infection, and the average increase percentage is 2.55%. The mean percentage reduction in body weight was 4.72% on day 3 after infection in the low dose group mice. Mice given the high dose group showed no obvious symptoms after infection (figure 15). In terms of viral load, as shown in FIG. 16, the lung tissue viral load test result 3 days after infection of the model group mice was 106.18copies/mL. The lung tissue virus load 3 days after the infection of the high-dose group mice is 103.11copies/mL, significantly lower than model group (p)<0.001). The lung tissue virus load 3 days after infection of the low dose group mice was 103.90copies/mL, significantly lower than that of the model group (p)<0.001). Shows that the lung tissue virus load is reduced by 3.07lg value after the high-dose Ad5-nCoV inoculation,the lung tissue viral load decreased by a value of 2.28lg following low dose Ad5-nCoV immunization. The research result indicates that Ad5-nCoV has obvious protective effect on infected mice.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
Kangxinuo biological shares Co
<120> a recombinant coronavirus vaccine using human replication-defective adenovirus as vector
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>3876
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
0コメント