Patent Application
20230022109
The present invention relates to a recombinant novel coronavirus vaccine for preventing the novel coronavirus epidemics. The present invention belongs to the field of bioengineering technology
The 2019 novel coronavirus, that is, SARS-CoV-2 (also known as 2019-nCoV), was named by the World Health Organization on Jan. 12, 2020. SARS-CoV-2 is a new strain of coronavirus that has never been previously found in the human body, which is the seventh member of coronavirus (CoV) that can infect humans. The incubation period of 2019-nCoV after infected is generally 1 to 14 days, and the common signs after infected include respiratory symptoms, fever, cough, shortness of breath and dyspnea. In severe cases, infection may lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. As of Mar. 17, 2020, 81116 cases with confirmed diagnosis and 3231 deaths have been reported in China; and 98,486 cases and 3,848 cumulative deaths have been reported in the rest of the world, with an average mortality rate of 3.94%.
The novel coronavirus belongs to the Beta coronavirus genus, wrapped with an envelope, having round or elliptical, usually pleomorphic particles, with a diameter of 60-140 nm. Phylogenetic analysis of the whole genome of the virus (29,903 nucleotides) indicates that the coronavirus is most closely related to (89.1% nucleotide similarity) a group of SARS-like coronaviruses previously found in bats in China (genus Betacoronavirus, subgenus Sarbecovirus). In the evolutionary tree of S gene, 2019-nCoV is most closely related to the bat coronavirus bat-SL-CoVZC45, with an amino acid identity of 82.3%, while 2019-nCoV and SARS-CoV share approximately 77.2% amino acid identity. When 2019-nCoV entering human cell, the receptor that binds to 2019-nCoV is angiotensin converting enzyme 2 (ACE2), which is the same as that binds to SARS-CoV.
Coronaviruses are a large family of viruses. So far, SARS-CoV, MERS-CoV and SARS-CoV-2 are the main causes of human infection outbreaks. There are few reports on the structure, function, key targets of infection, mechanism of action, and vaccine development of SARS-CoV-2. Several studies mainly focus on SARS-CoV and MERS-CoV vaccines, with main types of research and development including viral vector vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle vaccines, inactivated vaccines, and live attenuated vaccines. The target antigen of the recombinant coronavirus vaccine is generally in the forms of full-length S protein and truncated S protein (51 and RBD). The types of novel coronavirus vaccines under development include adenovirus vector vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines and inactivated vaccines, and the results have not been reported yet. The purpose of the present invention is to provide a recombinant novel coronavirus vaccine.
For the above purposes, the present invention firstly provides an optimized polynucleotide whose sequence as shown in SEQ ID NO: 1 for encoding the S protein of the novel coronavirus used as vaccine. The recombinant adenovirus-vectored novel coronavirus vaccine is obtained after the said polynucleotide is packaged via a replication-deficient human type-5 adenovirus with deletion of E1 and E3 as a vector, and HEK293 cell integrated with adenovirus E1 gene as a packaging cell line.
The present invention also provides a vector containing the above polynucleotide.
In a preferred embodiment, the vector is pDC316.
The present invention also provides a human replication-deficient recombinant adenovirus expressing the above polynucleotide.
In a preferred embodiment, the recombinant human adenovirus is derived from the AdMax adenovirus system.
The present invention also provides the use of the above-mentioned recombinant adenovirus in preparation of the vaccine for preventing novel coronavirus disease.
In a preferred embodiment, the recombinant adenovirus is prepared as an injection, nasal drops or spray in the above use.
In a more preferred embodiment, the recombinant adenovirus is prepared as an intramuscular injection.
Finally, the present invention provides a method for preparing the above-mentioned recombinant adenovirus expressing the S protein of the novel coronavirus. The method includes the following steps:
(1) Construction of a shuttle plasmid vector containing the polynucleotide encoding the S protein of the 2019 novel coronavirus;
(2) Co-transfection of the shuttle plasmid vector of step (1) and backbone plasmid into a host cell;
(3) Cultivation of the host cells of step (2);
(4) Harvest of the human replication-deficient recombinant adenovirus released from the cells of step (3);
(5) Enlarge cultivation of the recombinant adenovirus of step (4);
(6) Purification of the culture product of step (5).
Preferably, the shuttle plasmid vector of step (1) is pDC316.
Preferably, the backbone plasmid of step (2) is pBHGloxΔE1,3Cre. Both plasmids belong to the AdMax adenovirus system, and are used together in host cell for packaging of recombinant adenovirus containing the above-mentioned polynucleotide encoding the S protein of the novel coronavirus.
Preferably, the cell of step (3) is HEK293 cells.
Preferably, the enlarge cultivation method of step (5) is suspension culture.
Preferably, the purification method of step (6) is Source 30 Q chromatography.
When applied in mouse and guinea pig models as the novel coronavirus vaccine (Ad5-nCoV), the recombinant adenovirus expressing the S protein of the novel coronavirus provided by the present invention has shown good immunogenicity, and can induce strong cellular and humoral immune response in a short time. The protective effect on hACE2 transgenic mice suggests that a single dose of Ad5-nCoV could significantly reduce the viral load in the lung tissue on Day 14 after immunization, indicating that the vaccine has a favorable immune protection effect against the 2019 novel coronavirus. The preparation of the vaccine is quick and easy, which can be produced on a large scale in a short period of time to respond to sudden outbreaks.
The present invention is furtherly described with specific examples below, and the advantages and features of the present invention will become clearer with the description. However, these examples are only exemplary, and do not constitute any limitations on the protection scope defined by the claims of the present invention.
1. Optimization and Synthesis of S Protein Gene
The target antigen of the recombinant novel coronavirus vaccine is the S protein of the novel coronavirus strain (Genebank number: NC_045512.2). The expression level of the S protein is increased through optimization of the S protein gene, thereby improving the immunogenicity of the vaccine.
Firstly, codon optimization is conducted by UpGene software (Gao, W. Rzewski, A. Sun, H. Robbins, P. D. & Gambotto, A. UpGene: Application of a web-based DNA codon optimization algorithm. Biotechnol Prog, 2004. 20 (2): p. 443-8.), to change most of the rare codons of the S protein gene to high-frequency codons. Then, considering that optimization through software may mechanically change the codons to those used at highest frequency, the protein translation efficiency may not be significantly improved due to the influence of the use efficiency of tRNA, mRNA secondary structure and other factors. In this case, manual analysis is used to replace part of the high-frequency and low-frequency codons, while the high-frequency and low-frequency codons are uniformly distributed in the S protein gene. Meanwhile, considering that increase the content of GC in mRNA may help to enhance the stability of mRNA, the content of GC in the S protein gene is appropriately increased, and G, C nucleotides are distributed in the entire S gene as evenly as possible. Also, the original signal peptide (1aa-12aa) of the S protein is changed to the tPA, i.e, tissue plasminogen activator signal peptide, to furtherly enhance the expression level of the S protein.
Before gene optimization, the content of rare codons (use frequency <70%) in the novel coronavirus S protein gene is 34%, high-frequency codons (use frequency>90%) is 23%, and GC is 36%. The optimized gene shares 70.4% of homology with the original S protein gene sequence. The content of rare codons drops to 3%, the content of high-frequency codons increases to 81%, and the content of GC increases to 58%.
After the optimization of S protein gene, Kozak sequence is added ahead of the initiation codon, and restriction enzyme EcoRI is inserted at the upstream of the entire sequence, and HindIII is inserted at the downstream. The gene sequence is synthesized. Meanwhile, the original sequence of the S protein gene is synthesized as a control. The sequence of S protein gene with optimization (EcoRI and HindIII as the restriction enzyme) is shown in SEQ ID NO: 1, and the original sequence of S protein gene (SmaI and SalI as the restriction enzyme) is shown in SEQ ID NO: 2.
2. Construction of the Vectors and Identification the Expression of S Protein In Vitro
2.1 Construction of the Vectors The above synthesized gene sequence is double-digested with EcoRI and HindIII, or SmaI and SalI, and the target gene fragment is extracted and connected to the shuttle plasmid pDC316 of the AdMax adenovirus system (Microbix Biosystems Inc., Canada), and then is transformed into DH5-α competent cells and spread on Ampr LB plate. Monoclones are selected for PCR identification, and sequencing verification is performed for those positive clones. Plasmid containing S protein gene sequence without optimization is marked as pDC316-nCoV_S, whereas plasmid containing S protein gene with optimization is marked as pDC316-nCoV_Sopt. The plasmid map of pDC316-nCoV_Sopt is shown in
Meanwhile, the optimized S protein gene sequence encoding the original signal peptide is amplified by overlap extension PCR, and is connected to the pDC316 vector, which is marked as pDC316-nCoV_oriSIP-Sopt; the non-optimized S protein gene sequence leading by tPA signal peptide is amplified and connected to the pDC316 vector, which is marked as pDC316-nCoV_tPA-S; the optimized gene sequence encoding S1 protein (S1 protein is the truncated S protein at amino acids 12-685) leading by tPA signal peptide (corresponding gene sequence with optimization is 25 bp to 2103 bp of SEQ ID NO: 1) is connected to the pDC316 vector, which is marked as pDC316-nCoV_S1opt; and the original gene sequence encoding S1 protein leading by tPA signal peptide is connected to the pDC316 vector, which is marked as pDC316-nCoV_S1.
2.2 Identification of the Expression of S Protein In Vitro
The 6 shuttle plasmids constructed above and the pDC316 vector are transfected into HEK293 cells by TurboFect Transfection Reagent (Thermo Scientific, REF, R0531), and the cells are collected 48 hours later for Western blot. The method is as follows:
Transfection: On the day before the experiment, HEK293 cells are inoculated into a 6-well plate with 8×105 cells/well and cultured overnight at 37° C. in 5% CO2 incubator. One hour prior to transfection, the medium is replaced with fresh DMEM medium containing 2% FBS, with 2 mL per well. During transfection, 2 μg of each plasmid is added to each 200 μL of FBS-free DMEM medium, mixed, and 3 μL of transfection reagent is added and mixed gently. The mixture is incubated at room temperature for 15 minutes, and gently dripped into the wells of the 6-well plate and mixed by gently shaking the plate. The plates are cultured at 37° C. in 5% CO2 incubator. 5 hours later, the medium is replaced with fresh DMEM medium containing 10% FBS. Cells are collected 48 hours later to prepare samples for Western blot.
Sample preparation: 48 hours after transfection, the medium is carefully aspirated and discarded, cells are resuspended in PBS and centrifuged at 500 g for 5 minutes, and the supernatant is discarded. Cells are resuspended with 200 μL of RIPA buffer (THERMO Scientific, Prod #89900, supplemented with appropriate amount of protease inhibitors and nucleases), placed on ice for 15 minutes, and centrifuged at 12000 rpm for 5 minutes at 4° C. The supernatant is collected, and ⅓ volume of 4×SDS-PAGE loading buffer containing 200 mmol/L DTT is added. The supernatant is heated at 95° C. for 5 minutes, and stored frozen for Western blot.
Western blot: 10-well 4%-20% SDS-PAGE gradient gel is used for SDS-PAGE, and the loading quantity of each sample is 30 μL per well. Electrophoresis conditions: 80 V, 15 min; 180V, until the bromophenol blue migrates out of the gel. The protein on the SDS-PAGE gel is transferred to nitrocellulose membrane by an electric rotating apparatus at 300 mA for 1 hour. After electroporation, the nitrocellulose membrane is blocked with 5% skim milk for 1 hour, and then the anti-S-protein rabbit polyclonal antibody (Sino Biological, Cat #40150-T52) at a dilution of 1:2000 is added, and incubates at 4° C. overnight. The membrane is washed 4 times with washing buffer and shaken on the shaker for 5 minutes each time. Then HRP-labeled goat anti-rabbit IgG antibody (CST, Cat #70745) at a dilution of 1:10000 in 5% skim milk is added. The membrane is incubated for 1 hour at room temperature, and washed 4 times with washing buffer. Immobilon™ Western Chemiluminescent HRP Substrate (MILLIPORE, Cat #WBKLS0500) is used for chemiluminescence reaction, and a chemiluminescence imager is used to capture images with different exposure times.
β-actin is used as the internal reference, and results are shown in
3. Packaging, Preparation and Identification of Recombinant Adenovirus
3.1 Packaging of the Recombinant Adenovirus
The above constructed vectors pDC316-nCoV_Sopt, pDC316-nCoV_oriSIP-Sopt and pDC316-nCoV_S1opt are respectively co-transfected with the backbone plasmid pBHGloxΔE1,3Cre of the AdMax adenovirus system into HEK293 cells to package the recombinant adenovirus. The process is as follows:
a) On the day before transfection, HEK293 cells are inoculated into a 6-well plate with 8×105 cells/well, with MEM+10% FBS as the medium, and are cultured overnight at 37° C. in 5% CO2 incubator.
b) On the day of transfection, the medium is replaced, and the cells continue to be cultured in fresh MEM medium containing 10% FBS. When cells grow to cover 80%-90% of the bottom area, the backbone plasmid (pBHGloxΔE1, 3Cre) and shuttle plasmid are taken to transfect with TurboFect transfection reagent (Thermo scientific, REF, R0531) according to the manufacturer's instructions. The specific steps are as follows:
(1) 3.2 μg of backbone plasmid and 0.8 μg of shuttle plasmid are needed per transfection well, and mixed. The plasmids are diluted with 400 μL Opti-MEM medium.
(2) 6 μL of TurboFect transfection reagent is taken to added into the plasmids diluent in Opti-MEM medium, and gently mixed.
(3) The mixture of transfection reagent and plasmids are left at room temperature for 20 minutes, and then added to the cells.
c) On the day following transfection, the cells which cover the whole bottom are passaged into a 25 cm2 cell culture flask, and continue to be cultured in MEM medium containing 5% FBS. Daily observation is conducted, and cells are passaged into a 75 cm2 cell culture flask when they cover the bottom of the flask. Daily observation for signs of virus generation is performed. The phenomenon of virus generation is that the cells become large and round, in shape of grape, and obvious plaques begin to appear. Virus are collected when most of cells are diseased and detached from the bottom.
d) Cells with virus generation are resuspended, and centrifugated at 500 g for 10 minutes. The supernatant is discarded, and cells are resuspended in 2 mL PBS, and frozen in a refrigerator at −70° C. or lower and thawed in a water bath at 37° C. for three times. Cells are centrifugated at 12000 g for 10 minutes, the supernatant containing viruses is collected, and the precipitate is discarded.
The virus strain containing the optimized full-length S protein gene sequence leading by tPA signal peptide is marked as Ad5-nCoV_Sopt, the strain containing the optimized full-length S protein gene sequence leading by the original signal peptide is marked as Ad5-nCoV_oriSIP-Sopt; and the strain containing tPA signal peptide pulsing the optimized 51 protein gene sequence is marked as Ad5-nCoV_S1opt.
3.2 Identification of Recombinant Adenovirus
3.2.1 PCR Amplification of S and 51 Gene and Sequencing
The following universal primers of pDC316 vector are used to amplify the complete sequence of S or 51 protein:
2 μL of proteinase K is added to 50 μL of vaccine candidate solution, and digested at 50° C. for 30 minutes to release the genome of the virus, which is used as the template to amplify the gene sequences of S protein or S1 protein.
The results of agarose gel electrophoresis show that a single target band could be amplified in all three candidates with correct fragment size. The target bands are extracted and sequenced, and the alignment results indicate that the sequences tested are completely correct.
3.2.2 Identification the Expression of Target Antigen
HEK293 cells are infected with the vaccine candidates constructed above, and are collected 48 hours later for Western blot detection of the target antigen. Significant expression of the target protein can be detected in all the three candidates, Ad5-nCoV_Sopt, Ad5-nCoV_oriSIP-Sopt and Ad5-nCoV_S1opt, as shown in
3.2.3 Culture of the Recombinant Adenovirus
HEK293 cells are suspension-cultured at 37° C. in 5% CO2, at 130 rpm. When inoculated with the virus strain, cells with a viability greater than 95% are diluted to 1.0×106 cells/mL, with 300 mL as the final volume. HEK293 cells are infected with the fifth generation of the recombinant adenovirus at MOI 10, and are cultured at 37° C. in 5% CO2, with shaken at 130 rpm. Samples are collected every 24 hours for the measurement of cell viability and density.
After about 72 hours, when the cell viability drops to below 40%, the cell shake flasks are frozen in a refrigerator at −70° C. or lower and thawed in a water bath at 37° C. for two times. Benzonase (20 U/mL) is added, and reacted in a water bath at 34° C.-36° C. for 2 hours. The samples are centrifuged at 12000 g for 10 minutes, the supernatant containing viruses is collected, and the precipitate is discarded.
3.2.4 Purification of the Recombinant Adenovirus
The virus supernatant is adjusted with 5× equilibration buffer to a conductivity value of 18 mS/cm and pH 7.5. Source 30Q is used to separate and purify the adenovirus particles. The column is equilibrated with solution A (20 mmol/L Tris+150 mmol/L NaCl+2 mmol/L MgCl2, pH7.5), and samples are loaded at a flow rate of 5 mL/min. After the completion of sample loading, solution A is used to equilibrate the column until the UV is decreased to the baseline. Solution B (20 mmol/L Tris+2 mol/L NaCl+2 mmol/L MgCl2, pH7.5) is used for gradient elution at 10 mL/min from 0% to 20% in 50 minutes, and the elution peaks are collected in separate tubes. Finally, 100% solution B is used for elution.
3.3 Identification and Titer Determination of Ad5-nCoV
3.3.1 PCR Amplification of the Target Gene and Gene Sequencing
The method and process are the same as those described in section 3.2.1. The results of agarose gel electrophoresis show that a single target band could be amplified in all three vaccine candidates, with correct fragment size. The target bands are extracted and sequenced, and the alignment results indicate that the sequences tested are completely correct.
3.3.2 Infectious Unit Determination
Clontech Adeno-V™ Rapid Titer Kit is used to measure the infectious unit of the recombinant adenovirus. The procedure is conducted according to the manufacturer's instructions, and the specific method is as follows:
a) HEK293 cells are inoculated into a 24-well plate, with cell density of 5×105 cells/mL, 0.5 mL per well, with MEM+10% FBS as the medium.
b) The virus to be detected are diluted 10-fold with medium, from 10−2 to 10−6, to prepare a series of diluted virus samples, and added to the cells with 50 μL per well.
c) Cells are cultured at 37° C. in 5% CO2 incubator for 48 hours.
d) Cell medium is aspirated and discarded to allow cells to dry slightly (do not over-dry). 0.5 mL of ice-cold methanol is gently added to each well, and left at −20° C. for 10 minutes to fix the cells.
e) Methanol is aspirated and discarded, and cells are gently washed 3 times with PBS+1% BSA. 0.25 mL of Anti-Hexon antibody diluent (1:1000 dilution) is added to each well, and incubates at 37° C. for 1 hour.
f) Anti-Hexon antibody is aspirated and discarded, and cells are gently washed 3 times with PBS+1% BSA. 0.25 mL of HRP-labeled Rat Anti-Mouse Antibody (1:500 dilution) is added to each well, and incubates at 37° C. for 1 hour.
g) Before 0.25 mL of HRP-labeled Rat Anti-Mouse Antibody is aspirated and discarded, 10×DAB substrate is diluted to 1×DAB working solution with 1×Stable Peroxidase Buffer, and allow the solution to reach room temperature.
h) Diluent of Rat Anti-Mouse Antibody is aspirated and discarded, and cells are gently washed 3 times with PBS+1% BSA. 0.25 mL of DAB working solution is added to each well, and left at room temperature for 10 minutes.
i) DAB working solution is aspirated and discarded, and cells are gently washed 2 times with PBS.
j) Brown/black positive cells are counted under a microscope. At least 3 fields are randomly counted for each well, and the mean number of positive cells is calculated.
k) Infectious unit (ifu/mL) is calculated, with the following formula:
Infectious unit (ifu/mL)=Number of positive cells in the field×Number of fields in each well/(Virus volume (mL)×Dilution factor)
The results show that after concentration of the purified recombinant adenovirus, the infection titer can reach above 1.0×1010 ifu/mL.
3.3.3 Determination of the Virus Particle
Equal volume of 20 mmol/L Tris-Cl, 2 mmol/L EDTA (pH 7.5) and 2.0% SDS solution are mixed to prepare the virus lysate buffer. An appropriate volume of the virus sample to be tested is taken, 1/19 volume of virus lysate buffer is added, the mixture is mixed through repeated blow with pipette for 10 times, and vortexed for 1 minute. The mixture is shaken and digested in a constant temperature water bath at 56° C. for 10 minutes, centrifuged at 12000 rpm for 5 minutes. The supernatant is taken, and the OD values at 260 nm and 280 nm are measured. The number of adenovirus particles is calculated.
Results of the virus particle show that the purified recombinant adenoviruses can reach above 1.0×1011 VP/mL after concentration.
1. The Humoral Immune Response Induced by the Vaccine Candidates
100 SPF female BALB/c mice (age 6-8 weeks) are randomly divided into 10 groups, with 10 mice in each group. The mice are immunized with the vaccine candidates based on the grouping as shown in Table 1. For the intramuscular injection, the mouse are injected with 100 μL of the vaccine candidates at the inner side of posterior thigh; for the intranasal immunization, the mouse are anaesthetized with isoflurane, and 20 μL of the vaccine candidates is dripped into the nasal cavity of the mouse. The grouping is shown in Table 1.
Blood samples are collected from the mice at specific time points after immunization, and the serum is separated. The titer of IgG antibody against the S protein of novel coronavirus in the serum is detected by ELISA, and the results is shown in
High level of serum IgG antibody can be produced in mice on Day 9 after intramuscular injection with Ad5-nCoV_Sopt, and the mean titer of antibody in the high-dose group is over 105; the level of serum IgG antibody induced by intramuscular injection with Ad5-nCoV_Sopt shows a significant dose-dependent manner, with the higher level of serum IgG antibody response in the higher dose group (see
The results of serum IgG response induced by intramuscular injection with Ad5-nCoV_Sopt, Ad5-nCoV_oriSIP-Sopt and Ad5-nCoV_S1opt are shown in
The serum IgG response induced by intramuscular injection and intranasal immunization with Ad5-nCoV_Sopt on Day 14 after immunization are shown in
2. The Cellular Immune Response Induced by the Vaccine
30 SPF female BALB/c mice aged 6-8 weeks are randomly divided into three groups, with 10 mice in each group. 5×108 VP of Ad5-nCoV_Sopt is given through intramuscular injection (im.) or intranasal immunization (in.); and 1×107 ifu Ad5 vector is given through the same immunization routes as the control, with 5 mice per immunization route. For the intramuscular injection, the mouse are injected with 100 μL of the vaccine candidates at the inner side of posterior thigh; for the intranasal immunization, the mouse are anaesthetized with isoflurane, and 20 μL of the vaccine candidates is dripped into the nasal cavity of the mouse. The grouping is shown in Table 2.
The mice are sacrificed on Day 14 after immunization, and the splenic lymphocytes are separated. The overlapping peptide pool covering the S protein is used for stimulating the lymphocytes for 6 hours in vitro; at the same time, the protein secretion blockers are added to block the secretion of the cytokines. After 6 hours, Fc receptors are blocked, and dead cells and cell surface molecular markers are stained. After the cells are fixed and perforated, intracellular cytokines are stained. Cell surface markers include CD3, CD4, CD8 and CD107a molecules, and intracellular cytokines include IFNγ, TNFα and IL2. The expression level of IFNγ, TNFα, IL2 and CD107a in CD4+ T cells and CD8+ T cells after SARS-CoV-2 specific peptides stimulation is determined by Flow cytometry (BD FACS Canto™).
The immune responses of CD8+ T cells and CD4+ T cells induced by Ad5-nCoV_Sopt are shown in
3. The Antibody Level in Trachea-Lung Washes
Trachea-lung washes is collected when the mice undergoing test for cellular immune response, and the levels of IgG and IgA antibodies against the S1 protein of novel coronavirus in trachea-lung washes are detected by ELISA. Grouping is shown in Table 2, and the results are listed in
4. Summary of the Immunogenicity in Mouse Model
All the three vaccine candidates, Ad5-nCoV_Sopt, Ad5-nCoV_oriSIP-Sopt and Ad5-nCoV_S1opt, have a good immunogenicity and can induce high levels of serum IgG antibody in mice. Ad5-nCoV_Sopt is the fastest to induce the IgG antibody titer, with the best immunogenicity. Ad5-nCoV_Sopt is selected as the best candidate for recombinant novel coronavirus vaccine, which is marked as Ad5-nCoV. Results of cellular immune response indicate that Ad5-nCoV given by intramuscular injection and intranasal immunization both can induce specific cellular immune responses in the mice model, and intramuscular injection can induce stronger cellular immune responses. Results of the antibody response in trachea-lung washes suggest that intranasal immunization can induce higher levels of IgG and IgA antibodies in trachea-lung washes.
56 SPF guinea pigs, weighing 200 to 250 g, are randomly divided into 4 groups, with 14 guinea pigs in each group, half male and half female. The guinea pigs are immunized with Ad5-nCoV based on the grouping shown in Table 3. The route of immunization is intramuscular injection with 200 μL of Ad5-nCoV at the inner side of posterior thigh.
Blood samples are collected at specific time after immunization, and serum is separated. The titer of IgG antibody against the S protein of novel coronavirus in the serum is detected by ELISA. As shown in
Live virus challenge with SARS-CoV-2 (SARS-CoV-2/WH-09/human/2020/CHN) is conducted in a biosafety level 3 laboratory.
The mice are divided into 3 groups (see Table 4). 5×109 VP or 5×108 VP of Ad5-nCoV is injected intramuscularly into the muscle of posterior thigh, with an injection volume of 100 μL, or the same volume of PBS is injected in the control mice. Two weeks after immunization, the mice are transferred to a biosafety level 3 laboratory for SARS-CoV-2 challenge, with intranasal challenge as the route (105 TCID50/mouse). Observation for 3 consecutive days after challenge is conducted, and weight changes are recorded. All mice are euthanized on Day 3 after infection, and the viral load in lung tissue is tested.
Weight loss after infection is observed in the model group, with the highest mean percentage of weight loss is 3.36%. Compared to the model group, the weight in the high-dose group increases to a certain extent on Day 3 after infection, at 2.55% of the mean percentage of weight increase. The mean weight loss in the low-dose group is 4.72% on Day 3 after infection. Results show that mice in the high-dose group have no obvious symptoms after infection (see
The present invention discloses a recombinant 2019 novel coronavirus vaccine for preventing the 2019 novel coronavirus diseases. The said vaccine can be easily industrialized, having the industrial applicability.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010193587.8 | Mar 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/096024 | 6/15/2020 | WO |
